Abstract
Invited Speakers
Lessons learned for therapy development for Duchenne muscular dystrophy
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Duchenne muscular dystrophy is a severe muscle wasting disorder that affects ∼1 in 5000 new-born boys. Patients generally loose ambulation at around 12 years of age, need assisted ventilation before their twenties and die in the 2–4th decade. The disease is caused by mutations in the DMD gene that abolish production of functional dystrophin. The causative gene was identified in 1986, but the first drug for Duchenne (Translarna, previously Ataluren/PTC124) received conditional marketing approval in Europe only in August 2014. This drug only applies to a subset of patients and many other therapeutic approaches are in clinical and preclinical development. Several challenges impeded development of drugs for a rare disease like Duchenne muscular dystrophy. Scientific challenges included the size of the gene and protein involved, the abundance of skeletal muscle (∼30–40% of the body), and the relatively rapid decrease in muscle quality. Clinical challenges included the lack of standardized care and access to good care. Translational challenges involved the lack of expertise with conducting clinical trials and lack of infrastructure (patient registries, trial site registries, outcome measures, natural history data etc). Regulatory challenges included the limited knowledge of regulators on specific rare diseases, such as Duchenne, but also lack of expertise from the Duchenne field in dealing with regulators and requirements for drug approval (functional or surrogate outcome measures that measure clinical benefit). Many of these challenges have now been overcome through networking of researchers, clinicians, Industry and patient representatives. In fact patient organisations have been involved in each step of the therapy development process, through funding of basic research and clinical trials, crucial input into development of outcome measures that correlate with clinical benefit and care standards, participation of patients in trials and meetings with regulators and are actively involved in identification and development of surrogate endpoints. In this session a scientist and a patient representative will give their perspective on the lessons learnt and success factors for drug development for Duchenne muscular dystrophy.
Viruses with Good Intentions: Developing Oncolytic Agents for Therapy of Cancer
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Viruses have two faces. On the one hand they can cause harm and illness, and the virus may manifest directly as a contagious disease. A viral infection can also have delayed consequences, and in rare cases, may cause cellular transformation and cancer. On the other hand viruses may provide hope for an efficacious treatment of serious disease. Examples of the latter are the use of viral vaccines, viral gene-transfer vectors in experimental gene-therapy, or as therapeutic anticancer agent in an oncolytic-virus therapy setting. Already there is evidence for antitumor activity of oncolytic viruses, although in many studies therapeutic efficacy is relatively rarely seen. This demands for more efficacious viruses. There are several options for improving the anticancer efficacy. We can use genetic modification (reverse genetics) strategies to enhance their oncolytic activity, but should do so without compromising their safety. Likewise we can increase their specificity but should take care not to thwart their antitumor efficacy. Alternatively, we can use classical bioselection (forward-genetics) strategies to isolate mutants with new phenotypes. Finally we may exploit the existing viral diversity and evaluate panels of new viruses for their antitumor efficacy in human–tumor models, to identify candidates with improved potency. In this presentation I will illustrate the various approaches by some salient examples.
Evolutional approaches AAV
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Redirecting T cells by chimeric antigen receptors
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Adoptive cell therapy is aiming at targeting defined cells in the patient by antigen specific T cells. To treat cancer, patient's T cells are ex vivo engineered with pre-defined specificity for cancer cells, amplified and re-administered to the patient by transfusion. The targeting specificity is provided by a recombinant receptor molecule, chimeric antigen receptor (CAR), which is composed in the extracellular part of an antibody-derived binding domain for target recognition, and in the intracellular part of a T cell receptor (TCR) derived signaling domain for T cell activation upon target engagement. The CAR initiates a downstream signaling cascade resulting in pro-inflammatory cytokine release, T cell amplification and lytic degranulation leading to the elimination of target cells. Due to the particular structure the CAR exhibits several properties different to the TCR, including target recognition independently of the major histocompatibility complex, high affinity binding, and recognition of targets which are not physiologically recognized by T cells like carbohydrates. The TCR CD3 signaling moiety in a CAR can be combined with costimulatory signaling chains like the CD28, OX40 or 4-1BB endodomain, giving rise to the so-called 2nd generation CAR; combined costimulatory moieties build up a 3rd generation CAR. Such CAR T cells can substantially reduce the tumor burden as long as the targeted antigen is present on the cancer cells. Adoptive cell therapy with engineered 2nd generation CAR T cells) is achieving impressive efficacy in early phase trials, in particular in hematologic malignancies, strongly supporting the notion that redirected immune cells can control cancer. We discuss the pros and cons of the CAR T cell strategy, recent developments of TRUCKs, which release a recombinant payload upon CAR engagement into the targeted tumor lesion, and the challenges in translating the strategy to clinical practise.
Genetic info exchange by vesicles
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Clinical advances in gene therapy from bench to bedside: a patients' perspective
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I am 42 and live with severe haemophilia B. Therapeutic strategies for haemophilia has altered drastically during my life, and promises to advance even further in the near future. Though there are many intriguing scientific and clinical questions that remain to be answered, one question, above all else, is often rationalised and thereby overlooked, or assumed. That question is simple - “What do the patients think?” I may be only one person living with haemophilia, but my experiences and concerns are echoed, and sometimes overshadowed, by others within the haemophilia community. As scientists, clinicians, and patients strive to work ever closer together, the promise of future treatment strategies to make a more personal and meaningful difference for haemophiliacs is attractive indeed.
Gene therapy for Haemophilia
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Haemophilia A or B, characterized by a deficiency of respectively coagulation factor VIII or IX, is characterized by severe spontaneous bleedings, especially located in joints, which can result in hemophilia arthropathy and disability and reduced quality of life. In order to reduce bleeding symptoms haemophilia patients are prophylactically treated with intravenously administered coagulation factor concentrates 2 to 3 times a week, starting in early childhood. A moderate increase of factor levels results in a dramatic improvement of bleeding phenotype, as patients with moderate or mild haemophilia (Factor levels above 1 or 5% of normal, respectively) have no spontaneous bleeding. Therefore gene therapy resulting in expression levels that would allow patients with severe hemophilia B to reduce or eliminate their need for prophylactic treatment through endogenous expression of Factor VIII or IX may be of enormous benefit. Gene therapy has been successfully used in the academic research setting in a limited number of patients with severe haemophilia B, primarily using vectors based on adeno-associated virus (AAV) serotype 2 and 8. During the lecture a summary of recent studies on gene therapy for haemophilia will be presented. Several research groups and companies are now exploring new developments to improve current treatment strategies to eventually make gene therapy commercially available for a larger number of patients. Also the background and scientific rationale of a planned gene therapy trial in Europe with AAV-FIX for haemophilia B will be presented.
Towards a cure for Crigler-Najjar syndrome a collaborate effort of researchers and patients
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Crigler-Najjar (CN) syndrome is very rare metabolic liver disorder characterized by the accumulation of unconjugated bilirubin, the yellow colored degradation product of heme. This main risk associated with this inherited severe form of jaundice is irreversible brain damage. This can be caused by a high concentration of the neurotoxicity of unconjugated bilirubin. In the absence to lower bilirubin levels severely affected subjects die during their first year of life. As for other rare diseases funding to support research to understand the cause of this disease and to develop a therapy is limited. The Dutch Crigler Najjar Foundation, founded in 1988, raised money to initiate research and to improve the phototherapy devices to optimize this life saving but cumbersome treatment. Importantly, and typical for the culture of the foundation, they organized a yearly meeting between the researchers and patients. This allowed the researchers to explain what they were doing, progress problems etc. and was very important for patients to explain to the researcher their specific needs. In 1991 the Najjar Foundation already funded research to clarify the genetic background of CN syndrome. This resulted in the identification of the gene responsible for CN syndrome and the disease causing mutations during the years to follow. Knowledge on the genetic background stimulated the development of gene therapy approaches to overcome the need for a liver transplantation, the only curative option presently available. Several vectors have been tested in the relevant animal model but most were not suitable for use in the clinic due to low efficacy and/or immunogenicity. The recent clinical benefit with Adeno Associated Viral Vectors for inherited liver diseases helped to design an efficient vector to treat CN disease. A European network of patient organizations from France, Italy, Germany and The Netherlands with expert clinicians and researchers is now determined to make this treatment option available for these patients. The close collaboration with the group of patients was important for the design of an acceptable clinical protocol and to use clinical endpoints that are relevant for patients in the future.
Targeted genome engineering
Gene expression switches
Technology for the regulation of gene expression in mammalian cells and tissues is essential for both functional genomic research and applications in biotechnology and medicine, e.g. to allow the careful titration of the therapeutic protein. Several artificial gene expression systems have been developed that are controlled by clinically approved small molecules. The most widely used regulatory circuit is the so-called Tet-On system that is based on the Escherichia coli tetracycline repressor protein (TetR) and doxycycline (Dox) as small molecule inducer. The ins and outs of this Tet-On system and its optimization by means of evolution methods will be described.
Opening-up the stem cell niche for human hematopoietic stem cells in the mouse – an optimal model to study human hematopoiesis in vivo
Cellular and molecular mechanisms regulating human hematopoietic stem cell (HSC) function are largely unknown and their study requires a surrogate environment that supports HSC maintenance in vivo. Immune-deficient mouse strains are attractive candidates for providing such a surrogate microenvironment because they are easy to maintain and stem cell biology has been studied extensively in mice. However, in currently available mouse models maintenance of human HSCs is limited. To improve engraftment of human HSCs we generated a series of novel immune deficient recipient mouse strains that have functionally impaired endogenous mouse HSCs. We find that a mutant Kit receptor opens up stem cell niches across species barriers and allows for robust, uniform and sustained engraftment of human HSCs after transfer into adult mice without necessity for irradiation conditioning prior transplantation. Human HSCs give rise to multi-lineage repopulation over long periods of time in consecutive recipients and the transplantation of limited numbers of donor HSCs proofed expansion of the human HSC cell pool in the recipient mice. Moreover we show that improved engraftment of human HSCs in the recipients results in increased repopulation of cells of the myeloid lineage independent of human cytokines and growth factors. Our data suggests that Kit-signaling regulates the competition between mouse and human HSCs. The recipients were instrumental for in vivo analysis that revealed two functionally distinct subpopulations in the human HSC pool and efficient xenogenic transplantation under steady-state conditions has more and broad applicability for research focusing on stem cell biology and for translational approaches.
Retro- and lentiviral vectors for gene therapy: From basic biology to clinical application
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Retroviral and lentiviral vectors have shown promising results with respect to genetic modifications in experimental systems and more importantly in clinical studies. However, since the initial description of retroviral gene transfer systems, many improvements have been developed to increase their efficacy and safety. In this educational talk, we will cover the developments of improved retro- and lentiviral gene therapy approaches and also discuss potential ways to improve the risk-benefit assessment of retro- and lentiviral vectors. Specifically, we will address the following topics: (I) Retroviruses as evolutionary optimized delivery vehicles for genetic information. (II) From the infectious virus to the retro- or lentiviral vector. (III) A plethora of ways to improve efficiency and safety of retro- and lentiviral vectors. (IV) How to influence integration site preferences. (V) A brief overview of past and current clinical trials using retro- and lentiviral vectors. In summary, the educational overview talk will construct a bridge from the basic biology of retro- and lentiviral vectors to their promising applications in clinical trials.
Oncolytic adenoviral therapy to enhance the cure rate for patients with localized prostate cancer
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(Arthrogen) Gene therapy for arthritis
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CF patients taking the lead
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Cystic Fibrosis (1:4.750) is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. CF patients suffer from chronic lung infections, reducing median life expectancy to 40 years. Clinical treatment of patients with CF has been merely symptomatic. Emerging knowledge on the CFTR mechanisms has led to the development of drugs targeting key intracellular processes, with new opportunities for curative treatment. However, large variability in clinical phenotype of patients with similar genetic mutations severely hamper the prediction of responses to new drugs targeting the basic defect. The Dutch CF Foundation (NCFS) has a research agenda from the patients' perspective, with a top 5 of research topics that is leading for the funding policy. A patient/parent group has been trained to participate in the reviewing of research proposals from a patients' perspective and patients are now also participating in “user groups” per project. NCFS is coordinating the Dutch CF Research Network, which is a fruitful platform for facilitating (inter)national studies. With forthcoming mutation specific therapies/trials, this network will make it possible for all Dutch patients to participate in new trials. With support of the Dutch CF Registry (also funded and coordinated by NCFS) eligible patients can easily be identified. In 2012 NCFS started, together with a group of basic scientists and clinical researchers, a 5 years translational research programme, called “HIT CF”: a break through research program aiming towards personalized medicine in CF. It consists of four tracks with 16 projects, with a total budget of 4 million euro.
Organoid cultures for developing cystic fibrosis therapies
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Cystic fibrosis is caused by approximately 2000 mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacotherapy has been successfully developed for approximately 10 mutations, but the efficacy of existing CFTR-targeting drugs remains unknown or unclear for most patients, and is variable for patients with identical CF-causing mutations. We recently established methodology to quantify CFTR function using intestinal stem cell organoid cultures that can be efficiently cultured, expanded and biobanked from 1–4 rectal biopsies of individual CF patients. Intestinal organoids form closed multicellular 3D-structures of a single polarized epithelial layer with a central lumen. We found that forskolin induces rapid fluid secretion into the lumen, causing organoid swelling that is fully CFTR-dependent. Using organoid cultures from over 100 individuals, we observed CFTR genotype-specific profiles of residual and drug-repaired CFTR function that quantitatively reflected published outcome data of clinical trials with these drugs. Organoids with identical CF-causing mutations demonstrate differential responses to drugs. We also developed a bioassay to measure the activity of drugs in patient plasma samples using organoids, allowing the further personalization of therapy by functionally modeling pharmacokinetics. We are now initiating clinical trials to assess the predictive capacity of CFTR function measurement in organoids for in vivo disease severity and response to CFTR therapy. These studies point out that functional assays using patient-specific organoid cultures can play an important role for identifying novel CFTR-modulating drugs and patients that respond to these drugs.
European Union support to gene and cell therapy research in Horizon 2020 (2014–2020)
The European Union, through its Framework Programmes for Research and Technological Development, has financially supported collaborative efforts in gene and cell therapy research to bring the technology closer to the clinic and to the market. Most of the latest projects involve (often multicentre, international) clinical trials and reflect the European leading position in the field. The new European programme for research and innovation, Horizon 2020 (2014–2020), will continue to support gene and cell therapies as innovative/advanced technologies for tackling health challenges. An overview of some of the funding opportunities will be presented during the Education Day, 23 October 2014 (collaborative research projects, Marie Sklodowska-Curie training actions, European Research Council frontier research grants) as well as a selective overview of some ongoing FP7 projects.
D. Gancberg et al., Mol Ther 2012, 20: 2191. D. Gancberg et al., Hum Gene Ther 2014, 25: 1. D. Gancberg et al., Hum Gene Ther Clin Dev 2014, 25: 51–71.
Writing a Horizon 2020 application: do's and don'ts
Horizon 2020 is the European Research and Innovation funding programme that runs from 2014 to 2020 with a budget of nearly €80 billion. This programme offers various possibilities of funding for gene and cell therapy research, for instance via the European Research Council, the Marie Sklodowska-Curie actions and the Grand Societal Challenge “Health, Demographic Change and Wellbeing”. However, the first calls in Horizon 2020 have learned that the competition is fierce and success rates are limited. As such, a Horizon 2020 application needs to be excellent at all levels. This lecture will address the key factors for a successful proposal.
Clinical Trials workshop abstracts provided separately to those who attended.
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Immunological and metabolic correction after lentiviral vector mediated haematopoietic stem cell gene therapy for ADA deficiency
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Highlights of clinical progress
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Gene therapy for Wiskott-Aldrich Syndrome
The Wiskott-Aldrich Syndrome (WAS) is a monogenic X-linked immunodeficiency also characterized by thrombocytopenia, eczema, and a high susceptibility to develop tumors and multiple autoimmune manifestations. The life expectancy of patients affected by WAS is severely reduced, unless they are successfully cured by HLA-matched hematopoietic stem cell (HSC) transplantation. However, several WAS patients lack a compatible bone marrow donor and complications, such as autoimmunity, can arise in a significant fraction of HSC transplanted patients. Administration of gene-corrected autologous HSC represents an alternative therapeutic approach, potentially applicable to all WAS patients. A clinical trial based on HSC transduced with retroviral vectors showed correction of WAS disease phenotype but was associated with a high incidence of leukemias. We developed an approach using a lentiviral vector (LV) encoding for WAS under the control of the homologous 1.6 kb WAS promoter. Six patients were treated in a phase I/II clinical trial with LV transduced CD34+ cells, following reduced intensity conditioning. Results indicate high level of gene transfer obtained with LV-WAS, resulting in stable engraftment of transduced HSC and restored WAS expression. All patients are currently clinically well, independent from platelet transfusions. Insertions analyses confirm that hematopoiesis remains highly polyclonal, in the absence of aberrant clonal expansion. Although a longer observation is required to establish the long-term safety, lentiviral GT represents a promising treatment for WAS.
Targeted oncolytic and immunotherapeutic viruses: Emerging multi-mechanistic biologics for cancer
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Targeted oncolytic and immunotherapeutic viruses are an emerging multi-mechanistic therapeutic platform designed to induce both acute tumor debulking as well as chronic suppression of tumor outgrowth. Cancer-specific viral replication and immunostimulatory transgene expression (e.g. GM-CSF) result in direct cytolysis followed by tumor-specific humoral and cellular immunity. Product activation is driven by commonly activated genetic pathways in cancer. JX-594 (SillaJen Inc) is an oncolytic vaccinia virus derived from the Wyeth vaccine strain and has been engineered for 1) enhanced cancer targeting by TK disruption and 2) has been “armed” with the transgene of granulocyte-macrophage colony stimulating factor (GM-CSF) to augment oncolysis-induced anti-tumoral immunity (Nature Rev Cancer 2009). JX-594 replication within tumors, coupled with tumor-specific expression of GM-CSF, creates a pro-inflammatory microenvironment and exposes tumor antigens resulting in immune response induction to the patient's endogenous tumor antigens. Recent clinical results demonstrate convincingly that products from this therapeutic class can achieve highly selective and potent cancer destruction systemically through a multi-pronged MOA, including tumor-specific immunity (Nature 2011; Nature Med 2013; Science Translat Med 2013). Recent preclinical and clinical trial results demonstrated potential synergy with checkpoint inhibitor antibodies. “Directed Evolution” vector discovery principles can be applied to diverse viral species to create and isolate optimized, proprietary vectors targeting specific cancer cells or immune cells (Nature Reviews Genetics 2014). Given recent clinical validation, we expect this therapeutic class of “personalized” yet “off-the-shelf” active immunotherapeutics to expand rapidly.
Enadenotucirev, a group B oncolytic adenovirus: assessment of potency, safety and selectivity
Enadenotucirev (EnAd) is a bio-selected chimeric Ad11p/Ad3 oncolytic adenovirus active in a broad range of cancer cell lines, in vivo models and ex-vivo biopsies inducing lysis more rapidly than wild type Ad11p, Ad3 or Ad5. In normal cells, EnAd is attenuated and shows little or no genome replication or cytotoxicity. The virus capsid is derived from Ad11p for which seroprevalence is low and tumour cell infection can be achieved in the presence of human blood. The combined pre-clinical data from a number of laboratories opened up the possibility of initiating early phase trials. In cancer patients EnAd has been well tolerated at doses up to 6×10^12 following intravenous delivery, with transient inflammatory side effects consistent with an innate immune response to virus particles. Acute responses were decreased over time with each administration suggesting that innate-response-conditioning can be achieved. At 1×10^13, acute inflammation was found to be dose limiting while doses of 1×10^11 or below were without major side effects. To date the profile of side effects is not suggestive of any off target replication. Pharmacokinetic measurements were used to adjust the infusion rate in order to manage toxicities and maximise delivery. Viable particles were readily isolated from the blood stream and active virus was found in all tumour samples evaluated, together with high levels of infiltrating CD8+ cells. The clinical data gathered to date indicate that EnAd is well-tolerated and further trials are warranted. Successful intravenous delivery of EnAd also provides a strong rational for the development of a pipeline of variants expressing therapeutic proteins.
What T cells see on human cancer
With the aim to develop more specific approaches to adoptive T cell therapy and other immunotherapies, we have set out to explore how the T cell-based immune system of individual patients can recognize autologous tumor cells. Most human tumors contain large numbers of mutations, of which hundreds can be present within expressed genes. As the resulting altered protein sequences are foreign to the immune system, T cell recognition of these ‘neo-antigens’ is likely to be of importance. However, the vast majority of the mutations in human cancers are unique to individual patients and, because of this, broadly applicable approaches to link the consequences of DNA damage to tumor-specific T cell activity have long been lacking. Using in-house developed technologies, we have recently demonstrated the feasibility of cancer exome-driven analysis of tumor-specific CD8+ T cell reactivity. The data obtained demonstrate that CD8+ T cell recognition of neo-antigens is a common feature in human melanoma. Furthermore, based on the distribution of mutation loads in other major human cancer types, we propose that also in many other tumors, the repertoire of neo-antigens should suffice to allow CD8+ T cell recognition. In ongoing projects, we are exploring 1). whether the development of neo-antigen specific CD4+ T cell reactivity is also common in melanoma; 2). Whether neo-antigen specific CD8+ T cell reactivity is observed in other tumor types; and 3). whether T cell responses against neo-antigens are also qualitatively superior as compared to T cell responses against non-mutant antigens. Based on the data obtained, the development of ‘personalized immunotherapies’ that exploit cancer genome information to target patient-specific antigens should be explored.
Directed Evolution of New Adeno-Associated Viruses for Therapeutic Gene Delivery
Viral vectors offer a number of advantageous properties for therapeutic gene delivery that have enabled increasing clinical success. However, they also pose a number of challenges – including prior immunological exposure to viruses such as adeno-associated virus, inefficient delivery to some therapeutically valuable cell types, difficulty with targeted delivery, and an inability to overcome some tissue transport barriers in vivo – all which arguably stem from the fact that they did not naturally evolve to be utilized as human therapeutics. In many cases there is insufficient knowledge of viral infection mechanisms and structure-function relationships that underlie gene transfer challenges to empower rational design efforts to improve the gene delivery properties of viral proteins. As a complementary approach, we have been developing directed evolution – the iterative generation of large libraries genetic mutants and selection for enhanced properties – to create new viruses with valuable properties for gene therapy. For example, adeno-associated virus (AAV) vectors have enjoyed recent success in several clinical trials, and we have evolved AAV for the ability to transduce nonpermissive cells in vitro and in vivo, cross tissue barriers to gene delivery, target delivery to specific cell types, and evade pre-existing immunity. Directed evolution thus represents a powerful approach for engineering ‘designer’ viruses with enhanced gene delivery properties and therapeutic potential.
Pre-existing immunity to AAV overcome by in silico ancestral capsid design
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AAV is emerging as a potent and safe gene transfer vector, with demonstrated efficacy and safety in early clinical studies in hemophilia and inherited forms of blindness. AAV serotypes are endemic in humans. This leads to extensive pre-existing immunity (PEI) complicating broad applications of AAV gene therapy. PEI blocks transduction due to neutralizing antibodies, and may negatively impact safety due to capsid T-cells. Several approaches to mitigate this problem are considered however all further add complexity to the clinical protocol and only modestly reduce PEI. Subjects with PEI are therefore often excluded from trial participation. Here, we aimed to in silico design an AAV without shared epitopes with AAVs endemic in humans. If AAV primarily evolved by immune escape mechanisms, we speculated an ancestral AAV would not be recognized by PEI of humans currently. Through ancestral sequence reconstruction we inferred the ancestral state of the common ancestor of AAV1, 2, and 8, called Anc80 which is over 8% distinct from its closest homologue. Using maximum-likelihood methods, a probabilistic space of putative Anc80 variants was generated to account for ambiguity in the prediction algorithm. One variant demonstrated high yield in production, in vitro infectivity, and liver, muscle and retina targeting up to levels of AAV8. Importantly, PEI to Anc80 was approximately 10-fold reduced by measures of IVIG and individual sera from the US and EU. Long term expression, murine safety and particle stability data further justify the use of Anc80 as a potent, low seroprevalent in vivo gene transfer vector.
The great escape: immune evasion by Epstein-Barr virus
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Herpesviruses stand out for their capacity to establish lifelong infections of immunocompetent hosts, generally without causing overt symptoms. These viruses, including the oncogenic Epstein-Barr virus, encode a wide variety of gene products to allow infection, replication, and persistence in the face of a functional host immune system. Viral evasion tactics appear to target virtually any stage of the innate and adaptive host immune response. Detailed knowledge is available on the molecular mechanisms underlying herpesvirus obstruction of HLA class I-restricted antigen presentation to T cells. More recently, herpesvirus-encoded functions that interference with activation of the innate immune response are being elucidated. This opens the way for clinical applications, including exploitation of human herpesvirus evasion strategies for the rational design of novel strategies in gene therapy.
Adeno-associated viral (AAV) vectors are the most promising platform for in vivo gene transfer
Adeno-associated viral (AAV) vectors are the most promising platform for in vivo gene transfer. Clinical trials of AAV gene transfer let to some of the most exciting results for the field of gene therapy, however, because AAV vectors are administered directly to patients, they interact with the host immune system at many levels, as shown in preclinical and clinical studies. In particular, immune responses directed against the virus capsid or the transgene product encoded by the vector may result in lack or loss of therapeutic efficacy. Studies conducted so far enabled investigators to better understand the underlying mechanisms driving these responses and to develop strategies to manage them to achieve long-term expression of the donated gene. However, a more comprehensive understanding of the determinants of immunogenicity of AAV vectors, and of potential associated toxicities, is needed. Additionally, neutralizing antibodies to AAV still represent and significant limitation to the scope of in vivo gene transfer. Immunosurveillance studies together with the use of experimental models of immunity to AAV will be crucial to overcome the limitations posed by the immune system on the safety and efficacy of in vivo gene transfer.
The Role of Stem Cell Exhaustion in Aging and Disease
Aging is characterized by the progressive erosion of tissue homeostasis and functional reserve in all organ systems. Although controversy remains as to the molecular mechanism(s) underlying the process of aging, accumulated cellular damage, including DNA damage, appears to be a major determinant of lifespan as well as age-related pathologies. Moreover, there is evidence that the accumulation of damage in stem cells renders them defective for self-renewing and regenerating damaged tissues. We have demonstrated that a population of muscle progenitor cells (MPCs) isolated from the ERCC1-deficient mouse model of accelerated aging, are defective in their proliferation abilities, differentiation capacity and resistance to oxidative stress. We have observed that intraperitoneal (IP) injections of wild-type (WT)-MPCs into Ercc1 knockout (Ercc1-/-) mice resulted in an improvement in age related pathologies. Although the mechanisms by which the transplantation of WT-MPCs extend the lifespan of these progeria mice is still under investigation, we have obtained evidence that the beneficial effect imparted by the injected cells occur through a paracrine effect that involve angiogenesis. In an attempt to determine whether the defect observed in ERCC deficient MPCs was not exclusive to this progeria model, we have isolated and characterized MPCs from another progeroid mouse models, the zinc metalloproteinase (Zmpste24) knock-out mouse, an animal model of the Hutchinson-Gilford progeria syndrome (HGPS). Similar to ERCC deficient MPCs, we have observed that Zmpste24-/- MPCs have proliferation and differentiation defects, characteristics also observed in MPCs isolated from naturally aged mice. These results suggest that the defect in MPCs is not specific to a particular model of progeria and can also be observed in naturally aged animals. Finally, we have investigated whether a defect in MPCs can also be observed in skeletal muscle disease such as Duchenne muscular dystrophy (DMD), which is a degenerative muscle disorder characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. Interestingly, DMD patients lack dystrophin from the time of birth; however, the onset of muscle weakness only becomes apparent at 4–7 years of age, which happens to coincide with the exhaustion of the MPC pool. There are several lines of evidence that support this concept including the gradual impairment of the myogenic potential of MPCs isolated from DMD patients during aging, which results in a reduction of muscle regeneration in older DMD patients. In addition to muscle weakness, DMD patients acquire osteopenia, fragility fractures, and scoliosis indicating that DMD may represent a model of premature musculoskeletal aging with a potential dysfunction in MPCs. Here, we report that dystrophin–utrophin double knockout (dko) mice, an animal model of DMD, exhibit a spectrum of degenerative changes in various musculoskeletal tissues including skeletal muscle, bone, articular cartilage, and intervertebral discs. In contrast to that observed with MPCs isolated from the mdx mice (dystrophin deficient and mild phenotype), we have recently shown a defect in the MPCs isolated from dKO mouse. We have observed that the MPC defect from the dKO mouse model appears to be age dependent and not specific to MPC since other stem cell population also appears to be affected. These results taken together support the concept that stem cell exhaustion plays a role in aging and disease.
Gene replacement therapy for myotubular myopathy
Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a severe congenital disease that affects the entire skeletal musculature. Previous local studies in Mtm1-mutant mice demonstrated potential efficacy of gene therapy to treat the disease. More recently, we showed long-term survival data in XLMTM mice and dogs following intravenous delivery of recombinant adeno-associated virus serotype 8 (AAV8) vectors expressing myotubularin under the muscle-specific desmin promoter. Our progress on dose-finding experiments in animal models by systemic vector administration and product development for clinical application will be presented.
Cardiac regeneration: Stem cells and beyond
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Myocardial infarction (MI), blockage of a coronary artery, leads to deprivation of oxygen in a part of the heart muscle, and irreversible loss of cardiomyocytes. Since cardiomyocytes are unable to proliferate sufficiently, the damaged contractile tissue is replaced by a rigid scar, thereby diminishing the pump function of the heart. This will further attenuate cardiac contractility and ultimately result in heart failure. Novel approaches to ameliorate or even reverse the progression of heart failure, include the use of progenitor or stem cells with the ability to differentiate into new cardiac tissue. We and others have shown that multipotent cardiac stem/progenitor cells reside in the heart that can differentiate into cardiac myocytes, smooth muscle cells, and vascular endothelial cells after transplantation into the injured myocardium, but due to the low retention of cells, this strategy has thus far had limited impact on cardiac function. Endogenous regeneration of the mammalian neonatal heart, and the discovery that it may still persist in adulthood sparked hope for novel cardioregenerative therapies. Therefore we are exploring new options to restore the contractile force of the heart: the different stem cell sources combined with biomaterials as therapeutic agents in cardiac repair as well as more novel approaches like the activation of endogenous cell populations, and the use of paracrine factors and exosomes to repair the injured myocardium.
Cardiovascular and Pulmonary Diseases
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Targeted Genome and Epigenome Editing Using Engineered CRISPR-Cas and TALE Technologies
Targeted genome and epigenome editing technologies have recently emerged as important tools for biomedical research and as potential reagents for therapies of gene-based diseases. We have recently developed improvements to the clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nuclease platform that substantially reduce off-target effects. These two “next-generation” CRISPR platforms utilize: truncated guide RNAs and fusions of dimerization-dependent FokI nuclease domains to catalytically inactive Cas9 (dCas9). We have also begun to engineer and validate new transcription activator-like effector (TALE)-based technologies for modifying specific histone marks or methylated CpGs in DNA. These epigenome editing technologies can be used to induce targeted alterations in endogenous human gene expression. Taken together, these methodologies provide transformative tools for understanding human biology and offer promising pathways forward for developing therapies based on targeted alterations of gene sequence and expression.
Development and Applications of CRISPR-Cas9 for Genome Editing
The Cas9 endonuclease from the microbial adaptive immune system CRISPR can be easily programmed to bind or cleave specific DNA sequence using a short RNA guide. Cas9 is enabling the generation of more realistic disease models and is broadening the number of genetically-tractable organisms that can be used to study a variety of biological processes. Previously we have demonstrated that the Cas9 nuclease can be successfully engineered to facilitated genome editing by targeting DNA double strand breaks or single strand nicking in mammalian cells. The Cas9 nuclease can also be modified to modulate transcription, and alter epigenetic states in living cells. In this presentation we will look at the latest developments including structure-function analysis of Cas9 as well as assessing the targeting specificity. We will also describe applications of the Cas9 nuclease for understanding the function of the mammalian genome through genome-scale functional screening as well as in vivo genome editing.
Prosthetic Networks – Synthetic Biology-Inspired Treatment Strategies For Metabolic Disorders
Since Paracelsus' (1493–1541) definition that the dosing makes the drug the basic treatment strategies have lagely remained unchanged. We continue to use a precise prescribed dose of a small-molecule drug, a protein therapeutic or a therapeutic transgene to constitutively modulate or complement the activity of a disease-relevant target. However, this treatment concept does neither consider the metabolic dynamics nor the interdependence of the most important pathophysiologies of the 21st century such as obesity, diabetes and cardiovascular disorders. Synthetic biology-inspired prosthetic networks may act as metabolic prostheses that provide the dynamic interventions, the immediate pre-disease action and the multi-target capacity required to meet with the treatment challenges of the future. Prosthetic networks consist of synthetic sensor-effector gene circuits that (i) seamlessly operate in implanted designer cells, (ii) constantly sense, monitor and score metabolic disturbanes in peripheral circulation, (iii) process OFF-level concentrations of pathologic metabolites, and (iv) coordinate an adjusted therapeutic response in an (v) automatic and self-sufficient manner. We will present our latest generation of synthetic mammalian gene circuits and provide a few examples of prosthetic networks operating in animal models of prominent human diseases to highlight the challenges and impact of synthetic biology on future biomedical applications.
The use of AAV vectors to express antibodies to prevent and/or treat infectious diseases
Advances in the isolation and engineering of antibody (Ab) molecules have yielded novel insights into host defense mechanisms. Some of these Abs have very broad and potent antiviral activities. However, use of these proteins as therapeutic proteins is challenging in terms of cost of production and administration protocols especially in the context of prophylaxis. It simply is not practical in many situations to repeatedly deliver the Ab to prevent an infection; this is especially challenging in developing countries. We have used vectors based on adeno-associated viruses (AAV) to deliver Abs against a variety of virus pathogens including HIV, pandemic influenza, respiratory syncytial virus and Ebola. Our approach is to deliver the vector to the airway to prevent the transmission of respiratory pathogens and inject the vector IM to achieve systemic levels of the Ab for use in therapeutic applications and for arthropod borne infectious agents and viruses transmitted by direct contact.
In vivo barriers and advances in adenovirus vector technology
Viral vectors are perceived as very efficient gene delivery vehicles, not only for ex vivo modification of different mammalian cell types but also for in vivo gene delivery and therapy. For many of the in vivo applications large amounts of vector particles have to be injected either locally or systemically to achieve a therapeutic effect. In many cases the production of the required vector doses have been found to be quite challenging requiring a significant investment in the development of improved production technologies. Examples are the production of adeno-associated virus (AAV) vectors being under clinical development for the treatment of coagulation disorders and for diseases of lipid metabolism, respectively, or oncolytic viruses such as measles virus (MV) currently used in clinical studies for virotherapy of cancers. When having a closer look at the vector doses required to achieve a therapeutic effect it frequently becomes clear that only a very small fraction of vector particles is delivered to the target cell or the target tissue. This inefficiency can be explained, at least in part, by in vivo barriers, which the different viral vectors encounter when injected either systemically or locally. Using adenovirus as an example for a frequently used vector type with significant clinical potential and solid tumours as a therapeutic target recognized barriers will be discussed and their impact on vector delivery and vector activity. An improved molecular understanding of these barriers may lead to the development of improved technologies with increased potency at lower doses thus facilitating clinical application in the future.
Architecture and dynamics of genome - nuclear lamina interactions
Evidence is mounting that the spatial organization of the genome inside the cell nucleus is non-random and linked to the regulation of nuclear processes such as trancription and genome maintenance. We proviously found that the nuclear lamina (NL) interacts with hundreds of large genomic regions termed lamina associated domains (LADs). LADs are 0.1–10 Mb in size and together cover ∼35% of the genome, indicating that they are a major architectural component of the genome. Most genes in LADs are repressed, suggesting a role for NL contacts in gene regulation. The dynamics of LAD-NL interactions and the underlying molecular mechanisms are poorly understood. We previously developed a 'molecular contact memory' approach to to track LADs in living cells. In each nucleus, only a fraction of all LADs is positioned at the periphery; these LADs are in intermittent molecular contact with the NL but remain constrained to the periphery. Upon mitosis, LAD positioning is not detectably inherited but instead is stochastically reshuffled. Thus, interactions with the NL are highly dynamic. We now report a 96-well version of our DamID method that can be used to map LAD-NL interactions genome-wide in a large number of single cells. Analysis of these maps from >100 single cells confirms the apparently stochastic nature of NL interactions. In general, LAD-NL interactions on large chromosomes appear to be more robust than on small chromosomes. Additional results of computational analyses of these datasets will be presented and discussed in the broader context of genome organization and gene regulation.
Transposable elements and their epigenetic control mechanisms are key regulators of transcriptional networks in pluripotent stem cells
Endogenous retroelements (EREs) account for more than half of the human genome. These mutagens constitute essential motors of evolution, which are subjected to tight epigenetic control notably during the waves of genome reprogramming that take place during early embryogenesis. We demonstrated that this process is in part mediated by a large family of transcriptional repressors, the tetrapod-specific KRAB-containing zinc finger proteins (KRAB-ZFPs), and by their cofactor KAP1 (KRAB-associated protein 1). KRAB/KAP1-mediated regulation is responsible for silencing a very broad range of EREs in human embryonic stem (ES) cells, in an evolutionally dynamic fashion. As a consequence, it exerts a marked influence on the transcriptional landscape of these cells. During the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs), we observed that the transcriptional activity of EREs is altered, which likely both perturbs and facilitates the establishment of pluripotency. New light is shed on these events by our determination of the genomic binding sites of hundreds of human KRAB-ZFPs, which opens the door to deciphering stem cell transcriptional networks and, more generally, to cracking the code of DNA recognition by poly-zinc finger proteins.
WNT7A and PAX6 Define Corneal Epithelium Homeostasis and Pathogenesis
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The surface of the cornea consists of a unique type of non-keratinized epithelial cells arranged in an orderly fashion, and this is essential for vision by maintaining transparency for light transmission. Cornea epithelial cells (CECs) undergo continuous renewal from limbal stem or progenitor cells (LSCs)1,2 and deficiency in LSCs or corneal epithelium—which turns cornea into a non-transparent, keratinized skin-like epithelium—causes corneal surface disease that leads to blindness in millions of people worldwide3. How LSCs are maintained and differentiated into corneal epithelium in healthy individuals and which key molecular events are defective in patients have been largely unknown. Here we report establishment of an in vitro feeder-cell-free LSC expansion and three-dimensional corneal differentiation protocol in which we found that the transcription factors p63 (tumour protein 63) and PAX6 (paired box proteinPAX6) act together to specify LSCs, and WNT7A controls corneal epithelium differentiation through PAX6. Loss of WNT7A or PAX6 induces LSCs into skin-like epithelium, a critical defect tightly linked to common human corneal diseases. Notably, transduction of PAX6 in skin epithelial stem cells is sufficient to convert them to LSC-like cells, and upon transplantation onto eyes in a rabbit corneal injury model, these reprogrammed cells are able to replenish CECs and repair damaged corneal surface. These findings suggest a central role of the WNT7A–PAX6 axis in corneal epithelial cell fate determination, and point to a new strategy for treating corneal surface diseases.
Engineering light responses and outer segments
No Abstract Available
Mutatis mutandis: why a cell makes mutations and how this can benefit our fitness
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Mutagenesis in the germ line originates inherited disease and the evolution of species while somatic mutagenesis is essential for the development of our immune system, but also for the evolution of cancer. Mutations invariably require the inadvertent misincorporation by a DNA polymerase during replication. For instance, inadvertent misincorporations are induced by the replicative DNA polymerases during replication of the genome, and these are corrected by DNA mismatch repair. Somatic effects in DNA mismatch repair underlie many sporadic cancers while inherited DNA mismatch repair defects cause the most common cancer predisposition, Lynch syndrome. I will describe how the mutator phenotype of DNA mismatch repair-deficient cancer can be exploited to develop selective suicide gene therapy. Replication of DNA damaged by genotoxic compounds, including chemotherapy of cancer, requires specialized, mutagenic, translesion synthesis (TLS) DNA polymerases. Inhibition of TLS appears a promising adjuvant to chemotherapy, or to prevent cancer induced by environmental mutagenic agents. To investigate feasibility of the latter, we have investigated the induction of sunlight-induced skin cancer in partially TLS-deficient mice. Despite the reduction of mutagenesis, the development of skin cancer was accelerated in these mice, resulting from the induction of strong hyperplasia of the skin in response to sunlight. Moreover, complete deficiency of mutagenic TLS results in a progeroid phenotype caused by the inability of stem cells to replicate their genomes, damaged by metabolic byproducts. In conclusion, the inherent induction of mutations at damaged DNA is a small price to pay for the fitness benefits provided by TLS.
Towards the lentiviral gene therapy of mobilized CD34+ Cells from Fanconi anemia patients
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Aiming to prevent the severe hematopoietic syndromes characteristic of Fanconi anemia (FA) patients we have proposed a new gene therapy trial of FA-A patients based on the transduction of HSCs with lentiviral vectors. Since the collection of a significant number of CD34+ from FA patients constituted one of the difficulties in previous FA gene therapy trials, we have proposed to mobilize CD34+ cells from these patients with G-CSF and the CXCR4 antagonist, plerixafor. The first two FA-A patients that were treated with this mobilization regimen did not reach the required number of CD34+ cells in PB, and therefore were not subjected to CD34+ cell mobilization. A third patient, with higher PB cell counts and CD34+ cell numbers in BM, was also mobilized. In this patient, two apheresis were conducted allowing the collection of 4 million CD34+ cells/Kg, which have been cryopreserved for patient's gene therapy when a therapeutic intervention of the bone marrow failure is recommended. The gene therapy trial will be performed with a LV carrying the FANCA gene driven by the PGK promoter. To optimize the viability of FA HSCs during the transduction process, short CD34+ pre-stimulations (8–10 h), followed by a single transduction cycle of 12–14 h will be used. Under these conditions transduction efficacies of 35–80%, measured by qPCR in individual colonies, have been obtained. The validation of the transduction conditions is currently being conducted prior to open patient's recruitment for the gene therapy trial. Presented on behalf of EUROFANCOLEN.
TRUCKs: the new generation of CARs
Adoptive therapy with engineered T cells with an antigen-specific chimeric antigen receptor (CAR) is achieving impressive efficacy in early phase trials, in particular in hematologic malignancies, strongly supporting the notion that the immune system can control cancer. Such CAR T cells can substantially reduce the tumor burden as long as the targeted antigen is present on the cancer cells, however, loss of MHC or immunogenic antigens make them invisible to cytotoxic T cells and may contribute to deadly tumor relapses. We discuss a new generation of CAR T cells, so called TRUCKs, which release inducible IL-12 upon CAR engagement in the targeted tumor lesion. Locally accumulating IL-12 in turn attracts an innate immune cell response towards those cancer cells that are invisible to CAR T cells. CAR IL-12 T cells produced accumulation of activated macrophages in the targeted lesion that was critical to the anti-tumor response. Secondly, we discuss the strategy to engineer T cells with two CARs recognizing two different antigens in order to improve selectivity in tumor recognition. CAR T cells with two CARs, one providing the primary signal, the other the costimulation, exhibit an altered antigen-dependent activation threshold in favor to recognize target cells with two antigens than cells with one antigen only.
Development of Chimeric Antigens Receptors for the treatment of cancer
Recent clinical success in the treatment of B-cell cancers using chimeric antigen receptor (CAR)-transduced T cells targeting CD19 has sparked great interest. Genetic manipulation to “re-educate” the patient's own immune cells to detect and destroy tumors opens a plethora of new possibilities for the clinical management of incurable diseases. A major difference between the CD19 and other potential tumor antigens is the pattern of antigen expression. Whereas CD19 is expressed exclusively in malignant cells and non-essential tissues, most other tumor antigens studied, particularly in the case of solid organ tumors, demonstrate expression in both tumor and normal tissues. Thus, treatment with a potent CAR may be too risky for immediate clinical application as a single agent due to the potential of life-threating toxicity. Combination of a CAR with safety modules, such as inducible suicide systems, would potentially protect patients from life-treating toxicities but would not prevent them. Potential strategies to restrict the cytotoxicity of CAR-transduced cells to tumor tissue include; splitting T cell activation and co-stimulation signals and combining the CAR with an inhibitory CAR directed to an antigen expressed in critical normal tissues. Combination of CARs with more sophisticated modulatory receptors (mCARs) may constitute the next generation of immune receptors, designed to ‘fine tune’ the biological activity of CAR-transduced lymphocytes in terms of intensity and/or duration. Finally, genome engineering affords another level of synthetic biology to potentially reprogram T cells with new properties to improve both safety and efficacy.
Systemic virotherapy for multiple myeloma
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Multiple myeloma is a highly pro-angiogenic disseminated malignancy of terminally differentiated plasma cells that initially responds to therapy with proteasome inhibitors, imids, steroids and alkylators, but eventually becomes refractory to these drug classes. Because of their professional role as immunoglobulin synthesis “factories” myeloma plasma cells are metabolically active, making them a good substrate for virus infection. Suppression of antiviral antibody titers due to disease-associated immune paresis is frequently observed in myeloma patients and worsens after long term immunosuppressive antimyeloma drug exposure. In light of the above features, and particularly the low antiviral antibody titers, multiple myeloma is an ideal target for systemic oncolytic virotherapy. We have tested several oncolytic viruses in preclinical multiple myeloma models and are developing strategies to facilitate systemic virus delivery in the presence of neutralizing antiviral antibodies. MV-NIS, an attenuated measles virus coding for the thyroidal sodium iodide symporter, has recently completed phase I clinical testing with encouraging outcomes and other viruses in the translational pipeline will soon follow suit. Several aspects of our ongoing antimyeloma program will be highlighted.
Beyond clinical trials for antisense-mediated exon skipping for Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disorder, while Becker muscular dystrophy (BMD) is milder muscle disease. Both are caused by mutations in dystrophin, a protein, which stabilizes muscle fibers during contraction by linking muscle actin to the extracellular matrix. In DMD patients mutations disrupt the open reading frame, generating prematurely truncated, nonfunctional dystrophins. In BMD patients, mutations maintain the reading frame allowing production of internally deleted, partly functional dystrophins. The exon skipping approach uses antisense oligonucleotides (AONs) to induce skipping of targeted exons during pre-mRNA splicing, with the aim of reading frame restoration, converting of the severe DMD into the milder BMD phenotype. This approach is mutation specific. However, as mutations cluster in a few hotspots, skipping of some exons applies to larger groups of patients (e.g. exon 51 skipping applies to 13%). Proof of concept (dystrophin restoration) has been obtained in patient-derived cultured cells, animal models and a phase 1 trial where patients were treated locally with AONs. Multiple phase 2 and 3 trials have recently been completed for exon 51 skipping. In the phase 2 trials AON treated patients outperformed placebo treated patients in the 6 minute walk test, while no significant difference was observed in the phase 3 trial. Further analysis of the phase 3 results is ongoing, as well as phase 2 clinical trials for exon 44, 45 and 53 skipping and efforts to streamline clinical development of antisense-mediated exon skipping (COST funded network, see
Pip peptide conjugates of exon skipping PMO for therapy of Duchenne muscular dystrophy
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Antisense oligonucleotide analogues are used to redirect splicing by intra-nuclear targeting of pre-mRNA towards therapeutic applications. In collaboration with the group of Matthew Wood (Oxford, UK), we have applied peptide conjugates of PMO oligonucleotides (P-PMO) to target dystrophin pre-mRNA in an mdx mouse model of Duchenne muscular dystrophy (DMD), a severe and fatal muscle degenerative disease of young boys. In the mdx mouse there is a point mutation in exon 23 of dystrophin pre-mRNA that leads to premature protein termination. The mutation is skipped through targeting by the PMO, leading to restoration of a shorter but functional dystrophin. In particular, we have developed a class of Arginine-rich peptides, called Pip, which when conjugated to PMO leads to substantially higher exon skipping in cultured mouse mdx myotubes than for naked PMO. Intravenous injection of Pip6a-PMO (the current paradigm) into the mdx mouse leads to enhanced exon skipping and dystrophin production in skeletal muscle as well as in heart, a tissue less accessible to other P-PMOs. Both heart and muscle function is improved by repeat low dose injections. Pip-PMO is being developed for clinical use in DMD patients. We describe also how linking two PMOs via a single Pip6a peptide can enable double simultaneous pre-mRNA targeting in muscle cells and in vivo. Further we describe new chemistry that allows P-PMO to be fluorescently labelled for mouse distributive imaging use. Finally, we describe a new method of parallel P-PMO conjugate synthesis on suitable scale for cell screening.
Perinatal gene therapy ameliorates a mouse model of neuronopathic Gaucher disease
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Toward a curative single intervention therapy in Pompe disease
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Lentiviral hematopoietic stem cell gene therapy has entered initial clinical trial for lysosomal storage disorders (Biffi et al., 2013; Cartier et al., 2009). We have focused on Pompe disease, the only disorder so far developed for stem cell gene therapy (van Til et al., 2010) in which allogeneic stem cell transplantation has not been attempted as potential treatment due to lack of enzyme expression in the hematopoietic system. Briefly, Pompe disease is a rare autosomal recessive disorder caused by mutations in the gene encoding acid alpha-glucosidase (GAA). Glycogen accumulation in skeletal, cardiac and smooth muscles results in progressive muscle weakness, cardiomyopathy and respiratory problems. Enzyme replacement therapy (ERT) is the only effective treatment, requiring biweekly administration. Although of benefit to many patients, ERT may result in immune responses and the costs are high. A curative intervention represents an unmet medical need. Using self-inactivating (SIN) third generation lentiviral vectors to transduce hematopoietic stem cells and a codon-optimized, modified therapeutic transgene, approximately 30% successfully transduced stem cells resulted in high levels of GAA and correction of the phenotype in the Pompe mouse model at low viral copy numbers. Of particular interest was robust immune tolerance to the recombinant transgene product. Also brain glycogen levels normalized entirely, with all astrocytes, which play a key role in glycogen storage and glycogenolysis, showing active a-glucosidase activity. We are currently further developing efficacy and finalizing the safety evaluation to prepare for an initial clinical trial in the most severely affected CRIM-negative Pompe patients.
Long term safety and efficacy of a novel self-complementary adeno-associated viral vector encoding human FIX in patients with severe haemophilia B
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We previously demonstrated therapeutic expression of coagulation factor IX (FIX) following a single systemic administration of a self-complementary adeno-associated virus (AAV) vector (scAAV2/8-LP1-hFIXco) in six patients with severe haemophilia B (HB). Transient hepatocellular toxicity observed in some subjects at the high dose level mandating expansion of this dose cohort. We report data from the expanded high dose cohort and longer follow-up of the initial subjects, thus better defining the safety and durability of transgene expression. Four additional severe HB subjects (FIX=1%) were sequentially enrolled to the high dose level (2×1012 vector genome[vg]/kg) and monitored using an array of clinical and laboratory parameters. Stability of transgene expression and long-term safety was assessed in the six subjects recruited to the initial dose-escalation arm. Plasma transgenic FIX levels have remained stable at 1–6% for >3 years without evidence of late toxicity in the dose-escalation cohort. Highest FIX expression (5.1±1.7%) was observed in the high-dose cohort resulting in a >90% reduction in bleeding frequency and FIX usage (confidence interval of 95%). Transient subclinical increase in alanine aminotransferase was observed between weeks 7–10 in four of six high-dose subjects but resolved promptly following treatment with prednisolone. Over two-thirds of the patients on prophylaxis prior to gene transfer have discontinued prophylactic FIX treatment long-term without suffering spontaneous bleeding. The others have increased interval between prophylactic infusions. Systemic administration of scAAV2/8-LP1-hFIXco results in sustained therapeutic FIX expression without prolonged or late toxicity, supporting further evaluation of this approach for haemophilia and other monogenetic disorders affecting the liver.
Platelet-Mediated Gene Therapy of Hemophilia
Hemophilia A is monogenic bleeding disorder that was considered to be ideal target for gene therapy. However, classical approaches which rely on correction of plasma FVIII levels have yet to be proven beneficial in human trials. We use platelets as a target for gene therapy of hemophilia A by utilizing its unique characteristics of containing the bioactive proteins and serving as both “storage” and trafficking “vehicle” in blood circulation. When FVIII is driven by the platelet-specific alphaIIb promoter (2bF8), FVIII expresses and stores together with its carrier protein VWF in platelet alpha-granules and that platelet-derived FVIII corrects murine hemophilia A phenotype even in the presence of high titer of anti-FVIII inhibitory antibodies (inhibitors). To apply this promising approach to clinical translatable gene therapy model, we use lentivirus transduction and transplantation to introduce platelet-FVIII expression. Our studies have demonstrated that 2bF8 lentiviral gene delivery to HSCs can introduce sustained therapeutic levels of platelet-FVIII in hemophilia A mice even with pre-existing anti-FVIII immunity. 2bF8 lentiviral gene delivery to human HSCs can introduce functional FVIII expression in human platelets and rescues the hemophilic phenotype in humanized hemophilia A mice. In addition, we have developed an effective in vivo selection system to enhance plt-F8 expression. We found that 2bF8 gene therapy can not only restore hemostasis but also induces immune tolerance in hemophilia A mice. In summary, our results strongly suggest that platelet-mediated gene therapy may be a promising strategy for gene therapy of hemophilia A even in the high-risk setting of pre-existing inhibitors.
PK/PD modeling of CAR-T cell immunotherapy targeted at the cancer-initiating antigen CD44v6
T cells genetically modified with tumor-reactive chimeric antigen receptors are living medicines with their own laws of distribution, persistence and therapeutic activity. As clinical experience with CAR-T cells is increasing, new knowledge is becoming available on their complex pharmacokinetics (PK)/pharmacodynamics (PD) relationships. We are currently developing a CAR-T cell based strategy for targeting the cancer-initiating antigen CD44v6 on multiple hematological and solid tumors. In doing so, it is crucial to pre-clinically model the PK/PD of CD44v6 CAR-T cells for enhancing their anti-tumor activity, while mitigating potential toxicities.
High affinity T-cell receptors for targeting B-cell malignancies
Therapeutic reactivity of antibody-mediated immunotherapy and chimeric antigen receptor (CAR) engineered T-cells is based on targeting of extracellular target antigens. These immunotherapeutic strategies demonstrate great clinical benefit. However, recent clinical studies have demonstrated that escape variants, not expressing the targeted antigen, result in relapse of the malignancy. In addition, various different B-cell malignancies do not express the thus far targeted antigens. Therefore, new immunotherapeutic strategies targeting other B-cell malignancy associated target antigens have to be explored. Since T cell receptors (TCR) are able to recognize both antigens derived from extracellular and intracellular proteins in the context of HLA, the potential target antigen array useful for TCR gene therapy is much broader. This research focusses on defining high affinity TCRs specific for B-cell malignancy associated genes to create TCR modified T-cells with potent anti-B-cell tumor reactivity. A novel attractive strategy will be exploited that uses the immunogenicity of foreign HLA molecules, leading to the generation of T-cells directed against B-cell malignancy associated antigens in the context of allogeneic HLA. By linking the HLA-peptidome of B-cell malignancies with microarray expression analysis and high throughput T-cell enrichment and analysis techniques potentially useful high affinity TCRs are identified that exhibit potent anti-B-cell tumor reactivity and exert a stringent safety profile.
Derivatives of Human Pluripotent Stem Cells: the new patient?
Derivation of many different cell types from human pluripotent stem cells (embryonic stem cells or HESCs and induced pluripotent stem cells or hiPS cells) is an area of growing interest both for potential cell therapy and as a platform for drug discovery and toxicity. Most particularly, the recent availability of methods to introduce specific disease mutations into human pluripotent stem cells and/or to derive these cells as hiPS cells by reprogramming from any patient of choice, are creating unprecedented opportunities to create disease models “ in a dish” and study ways to treat it or slow down its rate of development. Understanding the underlying developmental mechanisms that control differentiation of pluripotent cells to their derivatives and mimicking these in defined culture conditions in vitro is now essential for moving the field forward. We have used these methods to produce cardiomyocytes and vascular endothelial cells from diseased hESC- and hiPSC and have examined drug responses of hESC-derived cardiomyocytes to a variety of cardiac and non-cardiac drugs and an hiPSC model for vascular disease in which Thalidomide has a therapeutic effect, will be shown. In addition, we show that iPSC derived cardiomyocytes with mutations in ion channel genes can accurately predict changes in cardiac electrical properties observed in primary cardiomyocytes despite being relatively immature. Examples will be shown of how metabolic diseases are beginning to be modelled in similar ways, stepping towards therapies for aspects of these complex conditions based on treating stem cells and their derivatives as “patients”.
Genome Editing with Zinc Finger Nucleases
The ability to engineer precise genetic modification of human stem cells would both accelerate research and extend the range of their potential therapeutic application. This possibility is now being realized via the use of zinc finger nucleases (ZFNs). ZFNs are customizable, sequence-specific endonucleases that can be designed to introduce a discrete cleavage event at any user-chosen location within the stem cell genome. We have previously shown that ZFNs targeting CCR5, an obligate co-receptor for entry of R5-tropic HIV, render modified CD4 T cells resistant to HIV analogous to cells from subjects homozygous for a naturally occurring CCR5 mutation (CCR5Δ32). This talk will describe the recent clinical results with ZFN-modified CD4 T cells as well as preclinical proof-of-concept studies towards the development of autologous, CCR5-disrupted CD34 stem cells as potential treatments for HIV.
Novel rAAV vectors for episomal and integration based gene transfer
Recombinant AAV vectors (rAAV) have provided some successes in early clinical trials. However, when moving from animals to humans a number of unanticipated responses have occurred that resulted in lower than expected efficacy. As more human data is obtained it has become clear that the large differences in vector transduction profiles between various animal species makes it difficult to select an appropriate preclinical model and extrapolate results into humans. We provide an argument for the use of a chimeric murine-human liver xenograft mouse model as a more reliable surrogate to establish clinical outcomes. In addition, we have used human xenotransplant models in combination with multi-species shuffled AAV capsid libraries to select for chimeric capsids that when vectorized provide novel transduction properties. Recent capsid isolates as well as new methods for screening will be discussed. Finally, the novel AAV capsids for both episomal and new integration based gene transfer studies will be compared.
Primary Immune Deficiencies
No Abstract Available
Update on gene therapy trials for severe combined immunodeficiency and Wiskott-Aldrich Syndrome
Previous clinical trials with a first generation MLV-based gamma (γ) retrovirus vector (RVV) have led to leukemia in trials of X-linked severe combined immunodeficiency (SCID-X1) and Wiskott-Aldrich Syndrome (WAS). In an international effort through the TAGTC we developed a self-inactivating (SIN-RVV) (SIN-γc) containing deletions of enhancer sequences to improve safety in SCID-X1. We enrolled 9 boys with SCID-X1 on parallel trials in Europe and United States. All subjects received bone marrow derived CD34+ cells transduced with the SIN-γc vector (van der Loo et al., 2012) without conditioning. Overall survival was 88%; of 8 evaluable patients, 7 recovered peripheral blood (PB) T cells with normalization of T cell proliferation responses in vitro. Comparison of vector insertion sites in PB revealed significantly less clustering of insertions near lymphoid proto-oncogenes in patients in the current vs. previous trials. These data show that a modified SINγ-RVV vector retains efficacy and suggests that insertional mutagenesis is significantly abrogated using this vector. In the WAS trial, we have enrolled 2 patients for gene therapy with ex vivo gene-modified HSCs using a SIN lentiviral vector produced by Genethon (Evry, France) with the WASP cDNA expressed from WAS gene cis-regulatory elements. Both patients engrafted with gene modified cells after conditioning. With short follow-up both patients have improvement in immune and hematologic parameters, and have had no genotoxicity to date. These data show that cis-regulatory elements of the WAS gene within a SIN lentivirus vector lead to improvement of WAS symptoms.
Gene and Cell Therapy for Neural Diseases
No Abstract Available
Efficient lentiviral vector-mediated delivery of hARSA in the brain of juvenile non-human primates
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Metachromatic leukodystrophy (MLD) is a severe demyelinating disease caused by a genetic deficiency in the lysosomal arylsulfatase A (ARSA). We have shown that a single injection of lentiviral vector (LV) coding for the ARSA gene into the external capsule of MLD mice results in rapid, widespread and long-lasting ARSA activity in CNS tissues, highlighting a major rationale for LV application to intracerebral gene therapy (GT). However, scaling-up issues should be addressed in larger brains before considering a clinical application. Since a large animal model of MLD is not available, we tested effectiveness and tolerability of intracerebral LV GT in normal juvenile non-human primates (NHP). We applied convection-enhanced delivery to inject LV.GFP (n=2) or LV.hARSA (n=6) in white matter regions and in the thalamus (2 unilateral injections; 5×107 TU/site). One and 3 months after treatment we assessed gene transfer efficiency, transgene expression/distribution, histopathology, immune response and risk of insertional mutagenesis related to the GT strategy. The surgical procedure was well tolerated. Histopathology revealed mild inflammation limited to the injection areas. We documented efficient transduction of neurons and glia and transgene overexpression at the injection sites. Importantly, we measured supraphysiological ARSA activity in the injected and contralateral hemispheres of LV.hARSA-injected NHP, suggesting robust production and widespread distribution of hARSA along axons and through CSF circulation. Integration site analysis indicated a polyclonal pattern of LV integration in juvenile NHP brain tissues. These data support the rationale for clinical application of the proposed intracerebral LV GT platform to address CNS pathology in MLD.
HIV-1 Capsid controls tropism through evasion of innate sensors
HIV-1 capsid forms a conical shaped core containing the viral genome and the enzymes required for reverse transcription (RT) and integration. Textbook models suggest that the HIV-1 capsid comes off before RT is completed. We hypothesise that the capsid's role is to shield the reverse transcription complex from being detected by host pattern recognition receptors that would otherwise detect viral replication complexes and activate a defensive innate immune response. To test this hypothesis, we examined the role of HIV-1 capsid binding cofactors in protecting the virus from innate immune DNA sensors, a process we call cloaking. We found that by disturbing recruitment of these cofactors we could reveal HIV-1 to cytoplasmic DNA sensors including cGAS. Preventing capsid interaction with Cyclophilin A, or the capsid binding 3′ end processing factor CPSF6, caused HIV-1 to trigger innate immune sensing and type 1 interferon (IFN) secretion in human monocyte derived macrophages. The importance of IFN in suppressing HIV-1 infectivity was illustrated by rescue of replication by blockading the IFN receptor with antibody. We hypothesise that RT and uncoating occur in complex with nuclear pore associated factors, which control this process through sequential interaction with capsid. In cell lines, where sensing pathways are defective, altering cofactor recruitment has less impact on infectivity but integration targeting is altered. Importantly, HIV-1 CA interactions can be drugged and such inhibitors make potent antivirals. Thus, lentiviral capsid is a druggable target that links innate evasion and integration targeting. The implications for HIV-1 vector use will be discussed.
Defining the integration landscape of viral vectors in the era of next generation sequencing
Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, technologies such as linear amplification-mediated (LAM) PCR have been developed. Subsequent deep sequencing and bioinformatical analyses of integration site sequences enable qualitative and quantitative measurements of the clonal kinetics of tissue (hematopoiesis) regeneration in gene transfer studies, and uncover the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. Clonality data on various oncoretroviral and lentiviral clinical gene therapy trials show us that integration site distribution is non-random and vector type dependent, potentially influencing cellular gene expression. The enormous power of cost-effective next generation sequencing devices may open a further level of precision, allowing in principle unbiased genome wide insertion and vector persistence studies based on validated and standardized target enrichment sequencing assays.
A New Downstream Process for Large Scale Manufacturing of AAV9 Vectors
Serotype 9 of Adeno Associated Virus displays high affinity for muscular and neuronal tissues, thus making AAV9 vectors promising tools to treat neuromuscular disorders like Spinal Muscular Atrophy, or Lysosomal Storage Diseases affecting the brain like Sanfilippo syndrome. However, such genetic diseases require enormous amounts of vectors to treat patients, then raising the need for large scale manufacturing process. With the development of Baculovirus expression system and transfection of suspension cells in bioreactors, upstream process seems to be no longer a limitation. This is not the case for downstream processing of large volumes. While one single immunoaffinity chromatography resin released a significant bottleneck for the purification of a broad range of serotypes (AAV1, 2, 5, 6 and 8), no convenient tool was available for AAV9 vectors. The presentation will show the development of a new AAV9-specific immunoaffinity matrix exhibiting unique characteristics and how this step integrates in a complete industrial process.
High titer production of GMP grade SIN gamma-retroviral vectors
Over the last years it has become clear, that the use of retroviral and lentiviral vectors in a self-inactivating (SIN) configuration has a highly reduced risk for insertional mutagenesis and is therefore advantageous in a clinical setting. While lentiviral vectors to date are mainly produced in transient production systems, we have developed a stable system for the manufacturing of high titer SIN gamma-retroviral vectors, making use of the recombinase mediated cassette exchange technology (RMCE) developed earlier by Verhoeyen et al. (2001) and Schucht et al. (2006). Here we present HEK293-based packaging cells (Ghani et al., 2007 and 2009) successfully modified to contain a targetable locus. Our tagging vector employed to introduce the tag into the host cell genome, combines Flp- and Cre-recombinase recognition sites. This combinatorial approach allowes RMCE and, once the producer clone is established, the removal of residual tag-vector sequences eliminating the possibility for accidental recombination. The resulting producer cells are highly productive and the titers achieved with the new packaging cells range from about 5×10e6 to 5×10e7 infectious particles IP/ml for ampho, GALV- or RD114-pseudotyped SIN-vectors. This system allows the GMP production of SIN gamma retroviral vectors at reproducibly high titers from molecularly defined cell clones, which simplifies the GMP production of batch sizes large enough to support later phase clinical trials or marketed therapies.
Generation of dopamine neurons for cell therapy in Parkinson's disease
Parkinson's Disease (PD) is a particularly interesting target for stem cell based therapy. The central pathology is confined to a small group of neurons in the midbrain, the nigral dopamine (DA) neurons and their projection to the striatum. Transplants of DA neurons could be used to restore DA neurotransmission in the striatum, substitute for the lost neurons, and bring back normal motor behavior. Proof-of-principle that this can work has been obtained in trials where fetal DA neuroblasts, have been transplanted to the putamen in patients with advanced PD. Despite these encouraging results, work with human fetal tissue presents a number of ethical and logistical problems and therefore does not represent a realistic therapeutic option in the future. Further progress in this field is critically dependent on the development of a bankable and renewable source of transplantable DA neurons. We have developed a method to generate human neural progenitors and neurons from human embryonic stem cells (hESCs), which recapitulates human fetal brain development. By addition of a small molecule to activate canonical WNT signalling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. The DA neurons obtained via our protocol closely resembled their fetal counterparts, making them useful as a model system for studies of human fetal brain development and also for developing transplantable therapeutic cells.
Gene therapy for neurotrophic factors to promote regeneration of injured peripheral nerves
1. Mason MR, Tannemaat MR, Malessy MJ and Verhaagen J (2011). Gene therapy for the peripheral nervous system: a strategy to repair the injure nerve? Curr Gene Ther 11:75–89.
2. Hoyng, S.A., Gnavi, S., De Winter, F., Eggers, R., Ozawa, T., Zaldumbide, A., Hoeben, R.C., Malessy, M.J., Verhaagen, J. (2014). Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve. Gene Therapy 21: 549–557.
Stem cells based technologies as non-viral transfection approaches
Our lab is engaged in developing novel targeted non-viral gene delivery approaches, which are based on stem cells as a whole cell and as a nano ghost. We have designed unique nano vehicles which are termed nano ghosts (NGs) as they are produced from the intact cell membrane of mesenchymal stem cells (MSC) and are loaded with the therapeutic of interest during the production of the NG. The rational for using MSC as the cell source out from which we isolated the cell membrane, is the MSC homing capability towards cancer cells and its microenvironment. These MSC-NGs benefit the surface molecules of the MSC and thus preserve the targeting mechanism of the intact cells themself. To use these NGs as a non-viral vehicle, we load into this “Trojan horse” NG system therapeutic encoding DNA, which can be expressed at the tumor site. We currently have strong evidence that such unique NG system can interact with tumor cells and traverse the cDNA into the cells nuclei leading to its expression and biological activity. In a different approach, still using MSC with their unique inherent ability to target different tumor niche, we demonstrate for the first time that therapeutic ultrasound (TUS) can be used as a non viral approach to transfect these MSC with cDNA that encode for a therapeutic gene such as hemopexin-like domain fragment (PEX). The therapeutic effect of these TUS transfected MSC was demonstrated on mice baring prostate tumors leading to a significant inhibition of the tumor growth in only two weeks.
Adult stem cells organoids and regenerative medicine
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In biological and medical research stem cells have been a major focus. However, apart from the embryonic stem cell, the identity of stem cells in adult organism and organs remained elusive for a long time. In 2007 we discovered a unique marker (LGR5) for epithelial stem cells of the intestine that finally allowed the study adult stem cells (Barker, Nature 2007). The characterization and isolation of these cells let to a great increase in our understanding of stem cells. Furthermore, LGR5 has been shown to be a marker of adult stem cells of multiple other tissues (Huch, Nature 2013; Barker, Cell Stem Cell 2010; Jaks, Nature Genetics 2009). With the identification of these novel types of stem cells and the tools to isolate them, we were able to develop a culture system that allowed for the virtually unlimited expansion of these cells from several animals models including human samples. The essential common component of the culture media is the Wnt-agonist Rspondin. Recently, we have realized that our stem cell marker Lgr5 is the receptor for Rspondin (Lau, Nature 2012). This has opened up new avenues for adult stem cell therapies.
The culture system that we developed allows for genetically and phenotypically stable expansion of human stem cell from a variety of organs such as intestine, pancreas, lung, and liver (Sato, Nature 2009, Gastroenterology 2011; Huch, Nature 2013, EMBO J, 2013). The organoids faithfully represent the in vivo cells after prolonged expansion in vitro. Importantly, we were able to functionally transplant them back into mice colon and liver (Yiu, Nature Med, 2013; Huch Nature 2013). Currently, we are optimizing the method to be able to translate these finding to humans. In order to develop the organoid technology for clinical applications a foundation was created (Hubrecht Organoid Technology (HUB)) which is responsible for the translational organoid research and development.
Generation of regulatory T cells by FOXP3 gene transfer: different approaches towards CD4FOXP3 T cell-based therapy for autoimmune diseases
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Thymic-derived (t) regulatory T cells (Tregs) are a unique cell subset characterized by high, persistent and tightly regulated expression of FOXP3, able to confer stable suppressive function to these cells. Although other genes contribute to the functional maintenance and peripheral fitness of tTreg cells, the mutation of FOXP3 alone is sufficient to cause a severe, life-threatening disease with early onset systemic autoimmune manifestations, known as Immune-Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. The IPEX syndrome is a prototype autoimmune disease with an inborn break of peripheral tolerance mainly due to the dysfunction of FOXP3-mut Treg. We have shown that pathogenic CD4+ effector T (Teff) cells isolated from IPEX patients can be converted into stable Tregs (CD4FOXP3) by LV-mediated gene transfer and overexpression of FOXP3. The resulting CD4FOXP3 cells become anergic, express Treg markers and Treg signature, display reduced cytokine production and potent in vitro/vivo suppressive activity, stable even in an inflammatory environment. These data strongly suggest that CD4FOXP3 could be adoptively transferred in vivo to reestablish immune regulation, not only in IPEX patients but also in other autoimmune diseases with impaired peripheral immune tolerance. In addition, in the context of autoimmunity against known tissue antigens (Ag), generation of Ag-specific CD4FOXP3 could provide a higher therapeutic efficacy and safety with suppression restricted against the target Ags. For IPEX patients, we are currently exploring the feasibility of FOXP3 gene correction by site-specific gene editing in autologous hematopoietic stem cells, which should ultimately represent the most specific treatment for them, with long-lasting multi-lineage recovery.
Armed oncolytic adenovirus can overcome critical obstacles in adoptive T-cell therapy of solid tumors
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Adoptive cell transfer (ACT) with tumor infiltrating cells (TIL) grown ex vivo has shown promising results in clinical trials for metastatic melanoma, with up to half of patients benefiting and a significant proportion alive and without disease even a decade later. However, these results have come at the cost of significant toxicity, including mortality, due to the high dose chemotherapy used as preconditioning and high dose IL2 used to sustain the graft after therapy. Moreover, in non-melanoma solid tumors ACT has not provided impressive results heretofore. In fact, in non-melanoma tumors efficacy has been mostly lacking but toxicity and mortality have been present, especially with T-cell receptor (TCR) and chimeric antigen receptor (CAR) modified ACT. Based on clinical and preclinical work done at CGTG, including treatment of >300 patients with oncolytic adenoviruses, and the data emerging from clinical substrates, we proposed that oncolytic adenovirus could overcome the 4 obstacles identified for ACT in the past decade: i) anergy of the graft, and ii) lack of graft propagation mediated by tumor immunosuppression in the patient, iii) pooor homing of the graft to the tumor, iv) lack of HLA/MHC on tumor cells. Adenovirus per se was able to dramatically enhance efficacy of ACT in a preclinical model of melanoma but mice were not completely cured. Hypotheses i-ii proved to be valid in this model but iii-iv were not. To enhance efficacy further, theoretically promising cytokine arming devices were compared and the best ones were incorporated into viruses for preclinical development. A bicistronic oncolytic adenovirus coding for tumor necrosis factor alpha (for counteracting immunosuppression) and interleukin 2 (for graft stimulation) was identified as the optimal candidate. Clinical trials using this virus for enhancing tumor TIL and CAR therapy are planned to start in 2016.
Preliminary results of the phase 1 trial with the Delta24RGD oncolytic adenovirus, administered by CED in patients with recurrent Glioblastoma
This phase 1 trial started accrual in 2010 and since 20 patients have been treated with escalating doses of the oncolytic adenovirus Delta24 RGD. This is a double mutated serotype 5 adenovirus, with a deletion of 24 bp prohibiting cell cycle initiation via E2F and with an expanded tropism by insertion of RGD in the fiberknob, enabling binding to integrins, as well as to the native receptor CAR. This virus is administered in patients with recurrent Glioblastoma after failure of standard treatment and often second line treatments. The delivery method is “Convection Enhanced Delivery” (CED) which consists of prolonged microinfusion through up to 4 catheters in and around the tumor over a period of 2 to 3 days. Doses of 1×10e7 to 1×10e 11 were scheduled in 6 cohorts of 3 patients each. Data on toxicity, adverse events, and survival were collected. The MTD was reached at a dose of 1×10e 10 viral particles, due to environmental risk issues and not because of patient toxicity. Dose limiting toxicities with respect to patient safety were not encountered. Viral titers in CSF support the occurrence of prolonged viral replication in the tumor.
Human translation of Sleeping Beauty system and next-generation clinical trials
The administration of designer T cells is championed by academia and being adopted by industry. I will reveal how human T cells can be engineered ex vivo using a transposon and transposase for in vivo applications. We will discuss how this non-viral approach to gene therapy can be combined immunotherapy to redirect specificity and improve the effector functions of T cells manufactured for clinical trials. For example, T cells can be genetically modified to express a chimeric antigen receptor (CAR) to redirect specificity for cell-surface tumor-associated antigens. We will discuss how the Sleeping Beauty (SB) system can be adapted and used to stably express CARs to improve the therapeutic potential of clinical-grade T cells. These clinical data serve as a foundation for additional genetic engineering to co-express transgenes to improve persistence as well as provide an opportunity for genetic editing to eliminate undesired endogenous genes to improve T-cell potency and broaden their distribution and application.
Clinical potential of cell-derived vesicles: Challenges to analyse the molecular composition of the extracellular vesicle pool
Cell-derived vesicles have been implicated to play a role in many (patho)physiological processes. The release of vesicles is tightly regulated. Both the quantity and vesicle content (proteins, lipids, RNAs) is dependent on cell type and cellular activation state. Consequently, the pool of extracellular vesicles (EVs) is very heterogeneous. Importantly, EVs have a wide clinical potential as therapeutics and biomarker. Albeit this promise, the heterogeneity of the EV-pool, their small size (vast majority <200 nm) and the elaborate EV-isolation procedures hampers their proper analysis. In the past years proteomics, lipidomics and deep sequencing studies did reveal a wealth of information regarding the molecular EV cargo. However, single particle-based high-throughput multi-parameter techniques are needed to identify and analyse different EV subsets. For this purpose we developed a high-resolution flow cytometric method which enables integrated analysis of multiple parameters (quantity, light scattering and fluorescence) of individual nano-sized particles, e.g. EVs, liposomes and viruses. Using this technology we were able to identify the activation-dependent release of EV subsets by immune cells (Dendritic cells, T cells and mast cells) that differ in physical properties (buoyant (velocity) gradient centrifugation), RNA and/or protein content. Currently, we are exploiting high resolution flow cytometry for EV-based biomarker discovery in blood and milk. Our recent achievement to physically sort EV subsets will further allow comprehensive molecular and functional analysis EV subsets. We expect that such a single EV-based analysis approach will largely contribute to unravel the physiological function of EV-subsets and to define EV-based biomarkers and therapeutics.
Extracellular vesicles for therapeutic RNA delivery: Promises and pitfalls
Extracellular vesicles (EVs), including exosomes and microvesicles, form an endogenous system for information transfer between cells. Since the recent discovery that EVs are also capable of functionally transferring RNA molecules, they are increasingly being considered as therapeutic RNA delivery systems. However, in order to advance EV-based delivery technology, there are major hurdles to overcome, including development of reproducible and scalable methods for EV isolation, and protocols for loading EVs with the desired therapeutic molecules. In this talk, the promises as well as challenges in the development of EV-based delivery systems will be discussed. Subsequently, our recent work on novel EV isolation methods and efforts to load EVs with therapeutic cargo will be highlighted.
Preclinical studies on Newcastle disease virus for the treatment of pancreatic adenocarcinoma
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Oncolytic Newcastle disease virus (NDV) is a promising therapeutic agent for the treatment of pancreatic cancer. Recently, we showed that human pancreatic adenocarcinoma cell lines (HPACs) were susceptible to NDV. However, NDV infection resulted in variable replication kinetics and cytotoxic effects. These early findings suggested that not all HPACs have functional defects in the innate immune pathways, possibly resulting in resistance to oncolytic virus treatment. Using reverse genetic techniques, oncolytic efficacy could possibly be enhanced. To test this, we generated rNDVs that either expressed human IFNß as an IFN-stimulating protein or NS1 of influenza A virus as an IFN–blocking protein (rNDV-hIFNß F0 and rNDV NS1 F0). We also generated a rNDV with alterations in the Fusion protein F, improving multicycle replication (rNDV F3aa). We evaluated these viruses for immunomodulating and oncolytic efficacy in vitro in representative HPACs and in vivo in immune- deficient and immune-competent mouse models for pancreatic cancer. We further performed initial safety studies with these viruses in macaques. We conclude that although non-replicating rNDVs harboring immunomodulating proteins do have an oncolytic effect, the F protein of rNDV is more important as a target to increase oncolytic efficacy. Initial safety studies with these rNDVs are encouraging for further research on the application of rNDV for oncolytic therapy.
RNAi -based gene therapy for HIV-1
We are developing a gene therapy that should provide a durable therapeutic effect against HIV-1 replication upon a single treatment. Several groups focus on silencing of the CCR5 receptor, but this may select for the more pathogenic CXCR4-using virus variants. Thus, we designed an antiviral therapy based on the mechanism of RNA interference (RNAi). We propose to modify the hematopoietic stem cells of an HIV-infected individual with a lentiviral vector that stably integrates to provide a sustained effect. The vector encodes several small RNA molecules to activate the RNAi pathway, leading to destruction of the HIV-1 RNA genome inside infected cells. The stem cells will continue to produce new T cells that should inherit the HIV-non-susceptible phenotype. These modified cells are expected to preferentially survive because non-modified are infected by HIV-1 and cleared by the immune system on a daily basis. A collection of 4 potent short hairpin RNAs (shRNAs) were selected that were combined into a single lentiviral vector. Such a combinatorial RNA interference approach is required to prevent HIV-1 escape. More recently, we have mimicked the patient situation by testing the gene therapy against many HIV-1 variants and even different subtypes. We also tested the impact of ongoing virus replication in non-modified cells, again a situation that will occur in patients. The safety of this genetic treatment was tested in the humanized mouse model, where one of the 4 shRNAs was shown to have a mild impact on T cell development. The therapeutic impact of the triple-shRNA combination is currently being tested in this in vivo model for HIV-1 infection.
Gene editing in pluripotent stem cells to model primary immune deficiencies
In vitro disease modeling based on induced pluripotent stem cells (iPSCs) is a powerful method to study molecular and cellular pathogenesis and pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into cell types affected by disease. In my lab, we recapitulated T lymphopoiesis and myelopoiesis in vitro using iPSCs as a starting material. As paradigms we have chosen immunodeficiency syndromes that either affect differentiation or function of immune cells. Data will be shown that provide proof that the in vitro generation of T cells, monocytes and granulocytes can be used to investigate maturation and function of these immune cells as well as to validate designer nuclease based gene therapy strategies for immune deficiencies.
Gene Editing
No abstract available
Outcomes of Gene Therapy for ß Thalassemia Major via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral ßA T87Q Globin Vector
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Keynote Lecture; Nobel Laureate for Medicine
Induced pluripotent stem cells (iPSCs) were originally generated from mouse and human fibroblasts by the retroviral transduction of four genes, Oct3/4, Sox2, c-Myc and Klf4. The iPSCs have the ability to proliferate almost indefinitely, and to differentiate into multiple lineages. iPSCs can be made using various somatic cells, which may come from genetically characterized individuals, providing better opportunities for versatile medical applications. As a result, cell-based therapies, disease mechanisms and new drug development are being studied worldwide using iPSCs, and the iPSC technology has evolved at an accelerating pace.
We are currently trying to establish optimally safe and efficient technologies for iPSC generation, which could be made the world standard to realize medical applications in accordance with GMP. In terms of the safety of iPSC derivation, we have reported an integration-free method that does not result in any chromosomal damage using episomal vectors. In extended studies of iPSC-inducing factors, we proposed the use of L-Myc as an alternative to the oncogenic c-Myc in order to reduce the risk of tumorigenicity, while keeping the high efficiency. In order to avoid the need for conventional feeder cells or culture materials from different species, and to make them more suitable for the GMP setting, feeder cells were replaced with a recombinant laminin-based matrix and a culture medium free of animal-derived constituents (xeno-free) was developed. Regarding the quality control, some marker genes for neural differentiation-defective clones were identified, indicating that there may be a possibility of screening out the low-quality iPSCs before use, such as prior to their application for regenerative medicine. Thus, many improvements have been achieved in iPSC production in terms of both safety and efficiency.
This year, the world's first clinical trial using iPSCs was initiated to study the transplantation of iPSC-derived RPE (retinal pigment epithelium) sheets for age-related macular degeneration. In addition, iPSC studies have recently shown major progress for other conditions, such as corneal diseases, blood diseases and Parkinson's disease, suggesting that these human conditions may also be treated using iPSC-based regenerative medicine in the near future. From a broad perspective, we are proceeding with an iPSC stock project in which iPSC clones are being established from donors with a homologous HLA haplotype, which is associated with a decreased immune response, in order to provide quality-assured iPSCs for future cell transplantation.
Another application of iPSCs is to provide more effective systems for drug screening, toxicity studies and the elucidation of disease mechanisms using disease-specific iPSCs from patients with intractable diseases. In addition, using individual iPSCs may make it possible to predict the patient condition and provide a preemptive therapeutic approach to protect against the onset of the disease or personalized medicine. Moreover, it is expected that the repositioning of drug candidates which used to be categorized as false-positive or false-negative with conventional testing may be feasible.
StemBANCC – Patient iPSCs to Study a Wide Range of Diseases
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The StemBANCC project is an Innovative Medicine Initiative (IMI) project supported by the European Union. It is a collaborative effort between 11 pharmaceutical companies, 22 academic institutions and 2 small and medium sized enterprises (SMEs). The project has been started at the end of 2012 and will be completed in 2017. The aim of StemBANCC is to generate and characterize induced pluripotent stem (iPSC) cell lines from 500 patients. These iPSCs can be used by researchers to study a wide range of diseases, including central nervous system disorders, peripheral nervous system disorders, and diabetes. These stem cell lines will help to improve and speed up the drug development process, and ensure that patients benefit from more effective and safer drugs. The cells will be accessible to the entire scientific community and distributed in partnership with another IMI project, the European Bank for induced pluripotent Stem Cells (EBiSC). The main scientific goal of StemBANCC is to try to identify cellular phenotypes of disease – this will enable us to study the disease mechanism in human cells and to screen for compounds that “heal the disease in a dish”. We also seek to overcome the many challenges of incorporating stem cells in primary drug screens such as designing protocols for high throughput screening.
EBiSC: the European Bank for induced pluripotent stem cells
EBiSC (European Bank for induced pluripotent Stem Cells) is a large European public private partnership project supported jointly by the Innovative Medicine Initiative and members of the European Federation of Pharmaceutical Industries and Associations (EFPIA). EBiSC is designed to address the increasing demand by iPSC researchers for quality-controlled, disease-relevant research grade iPSC lines, data and cell services. Its goal is to demonstrate an operational banking and distribution service of iPSC lines after 3 years and to establish subsequently for Europe a centralised, not-for-profit bank providing all qualified users with access to scalable, cost-efficient and customised products. Led by Pfizer Ltd and managed by Roslin Cells, the EBiSC Consortium including EFPIA partners boasts the leadership, scientific expertise, facilities, networks and experience to achieve these goals and, being representative of all stakeholders from tissue donors to clinical and academic iPSC researchers and industrial users, to respond appropriately to advances in science and society.
MiRNAs 182 and 183 are necessary to maintain adult cone photoreceptor outer segments and visual function
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The outer segments of cones serve as light detectors for daylight color vision, and their dysfunction leads to several human blindness conditions. Here we show that the cone-specific disruption of the DGCR8 locus in adult mice led to the loss of miRNAs and the loss of outer segments, resulting in photoreceptors with significantly reduced light responses. However, the number of cones remained unchanged. The loss of the outer segments occurred gradually over one month, and during this time the genetic signature of cones decreased. Re-expression of the sensory-cell-specific miR-182 and miR-183 prevented the loss of outer segments. These miRNAs were also necessary and sufficient for the formation of inner segments, connecting cilia and short outer segments, as well as light responses in stem-cell-derived retinal cultures. Our results show that miR-182- and miR-183-regulated pathways are necessary for cone outer segment maintenance in vivo and functional outer segment formation in vitro.
Gene Therapy of Chronic Granulomatous Disease: Lessons Learned and Future Perspectives
Gene therapy of Chronic Granulomatous Disease (CGD) is a promising therapeutic treatment, as demonstrated by us and others in recent phase I/II clinical trials. Despite the undisputed clinical benefit experienced by all GT-treated CGD subjects, vector-related genotoxic effects, silencing of transgene expression and lack of long-term engraftment have all contributed to mitigate the outcome of the treatment. Within a collaborative network, we developed a new safety-improved lentiviral vector (G1XCGD). Extensive pre-clinical studies have demonstrated that G1XCGD has an improved safety profile when compared to the vectors used previously in clinical trials, and efficiently rescue the CGD phenotype in vitro and in vivo. We analyzed the hematopoietic compartment in mice with the X-linked form of the disease (X-CGD) to elucidate the mechanisms underlying the lack of long-term engraftment of gene modified cells. We found that HSCs from X-CGD animals were impaired in their long-term engraftment capacity and were outcompeted by wild-type HSCs in competitive repopulation assays. We found increased levels of IL-1b and other proinflammatory cytokines in the bone marrow of X-CGD mice, reflecting the hyperinflammatory conditions prevailing in CGD mice and patients. Thus, persistent hyperinflammation in CGD triggers proliferative stress leading to exhaustion of repopulating HSCs that can be reversed with inhibitors of IL-1b signaling. In summary, successful treatment of CGD patients with GT will include the pre-treatment of patients with anti-inflammatory drugs and the use of lentiviral vectors targeted to CD133+ cells for long-term engraftment of gene corrected cells and sustained expression of the therapeutic gene.
Selected Oral Presentations
A CRISPR way to eradicate latent HIV: Targeted excision of integrated HIV-1 DNA from human cells through vector-based genome engineering
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HIV is highly successful as a viral pathogen due to its ability to persist in cellular reservoirs. Unfortunately, current antiretroviral combination therapy can only control but not eradicate HIV, raising a need for alternative therapies. We thus combined two powerful technologies, synthetic AAV vectors and CRISPR-mediated gene engineering, with the ultimate aim to eliminate integrated HIV from infected human cells. Therefore, we first screened a library of 84 AAV capsid mutants generated through insertion of 6 different retargeting peptides into 12 natural AAV isolates. We identified novel peptide-capsid combinations that mediate potent transduction of typical HIV targets, including human T-cells, PBMCs, macrophages or dendritic cells. We concurrently created AAV vector genomes that express - alone or in combination - the Cas9 protein as well as one of 9 different g(uide)RNAs directed against the HIV-1 LTRs, with 8 simultaneously binding both LTRs. Transfection of cell lines carrying a stably integrated HIV-1 genome followed by T7 endonuclease-based analysis of cleavage events confirmed that 7 gRNAs cut the HIV-1 LTRs as expected. These findings were validated in experiments where the best gRNAs were delivered into the stable HIV-1 cells via our selected AAV capsid mutants. In addition, we created and tested a second generation of AAV vectors expressing concatemerized gRNAs against multiple HIV target regions. Our results provide the first evidence for the possibility to cut and destroy integrated HIV DNA with AAV/CRISPR vectors, raising hopes that this approach could be translated into a novel clinical modality capable of purging latent HIV reservoirs.
An AAV vector toolbox for CRISPR/Cas9-mediated genome engineering
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Its versatility and efficiency make the CRISPR DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from Adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA which dictates the targeting - either alone or in combination. All our constructs share a modular design that enables simple and stringent gRNA cloning as well as rapid exchange of promoters driving Cas9 or gRNAs. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. We adapted three different Cas9 orthologues, demonstrating varying Protospacer Adjacent Motif (PAM) recognition specificities, to function in an AAV vector context. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than three hours. Finally, we provide evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified by targeting the mir-122 locus in mouse livers. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans. We are currently exploring the power of our vectors to generate models of human monogenic diseases in mice.
Adenoviral vector DNA is a preferred homologous recombination substrate for accurate genome editing using engineered nucleases
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Sequence-specific nucleases and donor DNA can be customized to edit mammalian genomes through the engagement of the homologous recombination (HR) pathway. We report that the nature of the donor DNA greatly impacts the specificity and accuracy of the chromosomal editing process following site-specific genomic DNA cleavage by TALEN and CRISPR/Cas9 nucleases. After applying these designer nucleases together with donor DNA delivered in the form of either protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector (IDLV) or non-viral vector templates, we found that the vast majority of AdV-modified cells underwent “scarless” homology-directed genome editing, whilst a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. For instance, in contrast to myoblasts subjected to AAVS1-specific TALENs and AdV donor DNA, myoblasts exposed to IDLV donor DNA instead, displayed heterogeneous transgene expression levels indicating substantial positional effects on transgene activity. Indeed, clonal analyses by PCR screening revealed that a sizable amount of IDLV-modified cells underwent illegitimate recombination events involving random chromosomal DNA insertions (13.4%), non-HR-derived junctions between exogenous and target site sequences (10.6%) and concatemeric arrays of the donor DNA at the target site (38.5%). Importantly, these complex and random exogenous DNA integrants were absent from AdV-modified populations. Similar data were obtained by combining, in AAVS1-targeting experiments, the CRISPR/Cas9 platform with AdV-mediated delivery of HR substrates. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells. M.H. and I.M. contributed equally to this work.
Engineering human models of tumor-associated chromosomal translocations with the RNA-guided CRISPR–Cas9 system
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Cancer-related human chromosomal translocations are generated through the illegitimate joining of two non-homologous chromosomes affected by double-strand breaks (DSB). Effective methodologies to reproduce precise reciprocal tumour-associated chromosomal translocations are required to gain insight into the initiation of leukaemia and sarcomas. We present a strategy for generating cancer-related human chromosomal translocations in vitro based on the CRISPR/Cas system to induce DSBs at defined positions. Using this approach we have generated human cell lines and primary cells bearing chromosomal translocations resembling those described in acute myeloid leukaemia and Ewing's sarcoma with unprecedented high frequencies. FISH and molecular analysis at mRNA and protein levels of the involved fusion genes in those engineered cells revealed the reliability and accurately of this nuclease system approach providing a powerful tool for cancer studies.
Stimulated granulocyte differentiation in an induced pluripotent stem cell model of severe congenital neutropenia by vitamin B3
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The generation of induced pluripotent stem cells (iPSC) from patients with genetic disorders provides new opportunities for basic research, drug development and future combined gene and cell therapy. Glucose-6-phosphatase 3 (G6PC3) deficiency has been described to cause severe congenital neutropenia (SCN) associated with a defect in granulopoiesis. The current lack of suitable disease models has hampered the investigation of molecular mechanisms and the evaluation of therapeutic molecules. We generated “factor-free” iPSC from an SCN patient with a nonsense-mutation in the G6PC3 gene. The generated iPSC clones exhibited the capacity to differentiate into hematopoietic cells of the myeloid lineage and we identified cytokine conditions for disease modeling of SCN in vitro. Here, SCN iPSC produced highly reduced numbers of CD66b-positive granulocytes (0.1–4%) compared to control cells (10–35%) as analyzed by flow cytometry. These results reflect the phenotype in patients, who show a paucity of mature neutrophils. Additionally, we observed a 2–3 fold increase of CD14-positive monocytes (up to 85%) from the SCN iPSC. Most importantly, the addition of vitamin B3 (nicotinamide) clearly induced granulocytic differentiation and increased the number of granulocytes from SCN iPSC to similar levels as obtained from healthy control iPSC. In summary, we established a promising iPSC-derived in vitro disease model for SCN. This model will serve as a tool for evaluating the potency of therapeutic options, such as small molecules and gene therapeutic vectors, and may be useful for development of alternative treatment options for SCN patients.
Dual-Regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatous Disease
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Chronic Granulomatous Disease (CGD) is caused by defective NADPH oxidase function in phagocytes leading to increased susceptibility to fungal and bacterial infections. Gene therapy with hematopoietic stem cells (HSC) may represents a good alternative to conventional transplantation. Past clinical trials for X-linked CGD with gammaretroviral vectors resulted in low or short term engraftment and insertional mutagenesis. Our strategy is based on regulated lentiviral vectors (LVs) expressing gp91phox transgene only in differentiated myeloid cells while sparing HSC to reduce the risk of genotoxicity and perturbation of reactive oxygen species. Targeting was obtained using a myeloid-specific promoter (MSP) and a miRNA post-transcriptional regulation system. We designed different LVs combining either the PGK or MSP promoters with miRNA 126 target sequences and we tested them on human and murine HSC and in the mouse model of X-CGD. All vectors restored gp91phox expression and function in human X-CGD primary monocytes and differentiated myeloid cells. While unregulated LVs induced a transgene ectopic expression in CD34+ cells, transcriptionally and post-transcriptionally regulated LVs dramatically reduced this off-target expression while guaranteeing a good level of expression in differentiated cells. By combining transcriptional and post-transcriptional targeting in the dual regulated vector, we achieved high levels of myeloid-specific transgene expression, entirely sparing the most primitive CD34+CD38-CD90+HSC compartment (5-fold reduction). X-CGD mice transplanted with all vectors engrafted and restored gp91phox expression, with 20–70% of granulocytes and monocytes expressing human gp91phox. The MSP transcriptional control combined to miR126 detargeting represents a promising approach for further clinical development of gp91phox therapeutic vectors.
Ectopic gp91phox expression is detrimental to XCGD iPS cell-derived neutrophils
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X-linked chronic granulomatous disease (XCGD) is caused by gp91phox deficiency. This compromises neutrophil (NEU) killing of phagocytosed pathogens due to impaired production of reactive oxygen species (ROS). In previous XCGD gene therapy clinical trials, the sustained persistence of gene marked cells could not be achieved without adversely triggering insertional mutagenesis. Additionally cellular recovery (gp91phox and ROS) had been incomplete on a per cell basis in comparison with healthy controls. This led to the hypothesis that the ectopic expression of gp91phox in developing NEUs could impede further differentiation into mature NEUs. To investigate this theory, a modeling system was established by generating patient autologous XCGD-iPSCs and inducing NEU differentiation. In this culture system, the hierarchical transition of NEU differentiation could be demonstrated from developing (CD64dullCD15dull) to mature (CD64highCD15high). Alpharetroviral vectors were used to transduce XCGD-iPSCs with the expression of codon optimized gp91phox cDNA driven by the ubiquitous EF1a short promoter. In healthy iPSC-derived NEUs, gp91phox expression and ROS production could only be detected in the mature fraction. In NEUs derived from transduced XCGD-iPSCs, functional recovery in the mature fraction was incomplete. Through intracellular staining, ectopic gp91phox expression was detected in the developing fraction. Most critically this appeared to correlate with more prominent cell death. Mechanistic studies are under way to investigate the relationship between non-physiological ROS production and the induction of endoplasmic reticulum (ER) stress resulting in cell apoptosis. Therefore, affording cellular protection from the detrimental effects of non-physiological ROS production may improve XCGD clinical outcomes.
Large-scale hematopoietic differentiation of human induced pluripotent stem cells provides granulocytes or macrophages for cell and gene therapies
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Hematopoietic differentiation of pluripotent stem cells (PSC) holds great promise for cell replacement and gene-therapy strategies. Whereas in the past, interest has been directed primarily at reconstituting stem cells, more recently also long-lived mature myeloid cells have been described, transplantation of which may open new therapeutic scenarios. To prove this concept, we subjected hiPSC to an embryoid body (EB)-based differentiation protocol employing IL-3 in combination with M-CSF or G-SCF generating so-called “myeloid cell forming complexes (MCFC)” which continuously produced >95% pure monocyte/macrophages (iPSC-mac) and/or granulocytes (iPSC-gra) at a quantity of 0.4–2.0 million cells/week and 3.5 cm well over a period of 3–5 months. Of note, generation of myeloid cells was driven by a MCFC-resident CD34+ population of clonogenic progenitor/stem cells. Detailed characterization of iPSC-mac demonstrated typical monocyte/macrophage morphology, a CD45+CD11b+CD14+CD163+CD68+ surface marker profile, the ability to phagocytose latex-coated beads similar to peripheral blood (PB) macrophages polarized to M2, and secretion of MCP1, IL6, IL8, and IL10 upon LPS stimulation. IPSC-gra were CD45+CD11b+CD16+CD15+CD66b+ and morphologically included pro-myelocyte (3%), myelocyte (5%), meta-myelocyte (30%), bands (22%), eosinophils (2%), basophils (1%), and segmented-neutrophils (37%). Moreover, iPSC-gra migrated towards an IL8 or fMLP gradient, formed neutrophil extracellular traps, and up-regulate NADPH activity and ROS production upon PMA stimulation. In summary, we provide a novel hematopoietic differentiation protocol for iPSCs recapitulating key events of physiologic hematopoiesis and allowing for the prolonged large-scale production of myeloid lineage cells. Thus, this protocol appears particularly suited for cell-replacement strategies or to study hematopoietic development.
Adeno-associated virus vector (AAV) microdystrophin gene therapy for Duchenne muscular dystrophy
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Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there are no strongly effective treatments but new gene-based therapies are currently being developed. In the case of DMD, a number of groups are testing gene therapy with adeno-associated virus vectors expressing engineered micro-dystrophins (AAV-MDs). In our hands, highly sequence optimised AAV-MDs are available for use in mouse, dog and ultimately humans, expressed using a strong synthetic promoter specific for skeletal and cardiac muscle cells have been tested in detail in mdx mice, and in the GRMD dog. Here we report the outcome of a detail study of AAV-canine MD delivery by isolated limb perfusion. No immunosuppression was applied. In skeletal muscles of the treated limb we report widespread vector biodistribution, and sustained high level MD expression. In addition the treated limb exhibited very significantly improved parameters of muscle turnover (regenerative fibres), fibrosis (Collagen type I), 1H-MRI and 31P-NMR spectroscopy, and muscle strength. In the context of immune parameters, AAV administration elicited anti-AAV2/8 antibodies and a transient elevation of serum cytokines, but no evidence of cellular immunity to microdystrophin or to the AAV vector was observed. The current optimised microdystrophin is thus highly functional, not only in mice, but also in a large animal model, and that AAV2/8 vector system yields sustained microdystrophin expression without adverse immune responses. Current studies are focused on systemic delivery of AAV-canine MD vectors body-wide in the GRMD model.
MMP-9 serum levels increase over time in Duchenne muscular dystrophy patients and decrease upon treatment with drisapersen
Duchenne Muscular Dystrophy (DMD) is a severe progressive muscle disorder caused by the lack of dystrophin protein. Several therapeutic approaches are in clinical development, but biomarkers to evaluate patients' response in clinical trial settings are lacking. We here report a mean 6-fold increase of Matrix Metalloproteinase-9 (MMP-9) serum levels in DMD patients compared to healthy boys. Analysis of 168 longitudinal samples belonging to 66 patients from two independent DMD natural history cohorts collected over a period of up to 5.5 years shows that serum MMP-9 levels significantly increase in both cohorts by 1.5 and 3.5 ng/ml per week. Based on these data we monitored MMP-9 levels in 12 DMD boys treated over a period of 120 weeks with subcutaneous injections of drisapersen, a 2′-O-methyl-phosphorothioate antisense oligonucleotide able to skip exon 51 in the dystrophin pre-mRNA. At baseline, MMP-9 serum levels were comparable to non-treated DMD patients, while during treatment, MMP-9 levels decreased. These data, together with the extensive assay validation results, support the inclusion of serum MMP-9 levels as the first biomarker to be used in clinical trials in patients affected by Duchenne muscular dystrophy.
DYSTROPHIN deficient rats: generation and characterization of a new model for Duchenne muscular dystrophy
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Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. For pre-clinical evaluation of therapeutic approaches, few animal models are available. Large animal models of DMD such as dogs or pigs are expensive, difficult to handle and show important clinical heterogeneity, while mdx mice exhibit only limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations for some analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better mimic the human disease. Two lines of Dmd mutated-rats (Dmdmdx) were generated using TALENs. Animals of both lines showed undetectable levels of dystrophin by western-blot and less than 5% of dystrophin positive fibers by immunohistochemistry in muscles analyzed. At 3 months, limb and diaphragm muscles displayed intense necrosis and regeneration. At 7 months, these muscles showed severe fibrosis and adipose tissue infiltration. At both time points, Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, echocardiography showed significant concentric remodeling and alteration of diastolic function at 3 months. Subsequently, the heart morphology evolved into a dilated cardiomyopathy with necrotic and fibrotic tissue. In conclusion, Dmdmdx rats represent a promising small animal model that can now be used for pre-clinical evaluation of therapeutic approaches of DMD, in particular for testing effects on disease progression and cardiac anomalies that were difficult to assess using the current DMD animal models.
Molecular Mechanisms of Vascular Hyperpermeability in VEGF Therapy
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Vascular permeability reflects changes in the barrier function of the endothelium and its interendothelial junctions. Known common mediators of vascular hyperpermeability are growth factors, such as vascular endothelium growth factors (VEGFs), and inflammation mediators, such as histamine. Previously, pro-angiogenic therapy, utilizing VEGF induced vascular growth, has shown to induce blood vessel growth and skeletal muscle perfusion. At present, the main hurdle in VEGF therapy is the increased plasma protein extravasation and vascular hyperpermeability, leading to tissue oedema followed by instability of newly formed vessels. In this study, permeability profiles of various vascular permeabilizing agents were studied from intact vasculature of C57BL/6 mice by laser scanning microscopy. Extravasation of fluorescent microspheres, used as a detection marker for vascular permeability, was shown to be dependent on the VEGF type used and its corresponding receptor binding profile. By next-generation RNA sequencing, transcriptional alteration of proteins known to be involved in vascular permeability was monitored. Fold changes of several genes known to regulate calcium signalling, cell-cell and cell-ECM adhesion, ion flux and inflammation were compared between different permeabilizing agents. Since the exact cellular mechanisms of vascular hyperpermeability are largely unknown, the knowledge of the cellular mediators is needed for further optimization of VEGF therapy and for prevention of oedema formation in various pathophysiological conditions.
“First in human” clinical trial using allogeneic Cardiac Stem/Progenitor Cell (CSCs) for Acute Myocardial Infarction (AMI) treatment
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The high prevalence of ischemic heart disease and the absence of effective curative treatments encourage regenerative medicine as an alternative for the treatment of this disease. Several cell types were proposed as therapeutic agents and tested in clinical trials. These studies showed the safety of cell therapy however the efficacy observed so far has been modest. The type of cells used, the variability between different cell productions and the heterogeneity of patients assayed could be behind the poor efficacy of the results obtained. CTPX has developed a robust and reproducible GMP manufacturing process for the production of bioequivalent CSCs batches, with a strong cardioregenerative capability. The allogeneic nature of the process allows producing off-the-shelf cellular material to be administered in the acute phase of the disease, before the scar is established. We already initiated a “First in human” clinical trial to study safety of this product and to obtain efficacy data of intracoronary administration of allogeneic CSCs after an AMI. This clinical trial has a dose escalation phase in which safety of the procedure is being assayed with different cell dose. Once this phase is completed, a randomized phase double blinded and placebo controlled will be initiated. AMI patients will be MRI selected and administered with placebo or 35 million of CSCs. Safety and efficacy will be evaluated by MRI after 12 months. Immunological studies will be performed to study the response initiated after allogeneic cell administration. Preliminary safety results of the escalation phase will be presented.
A PET based approach for highly sensitive monitoring of teratoma formation from pluripotent stem cells
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Pulmonary transplantation of multipotent or pluripotent-stem cell derived macrophage progenitors as a novel treatment option for Pulmonary Alveolar Proteinosis
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Hereditary pulmonary alveolar proteinosis (herPAP) constitutes a rare lung disease caused by mutations in the granulocyte/macrophage-colony-stimulating-factor (GM-CSF) receptor genes (CSF2RA or CSF2RB) and is characterised by defective alveolar macrophages (AM), massive alveolar proteinosis, and life-threatening respiratory insufficiency. While established treatment is symptomatic only, we here introduce pulmonary cell transplantation (PCT) of multipotent or pluripotent stem cell derived macrophage-progenitors (MPs) as a novel, cause directed, and long-lasting therapy for herPAP. In Csf2rb-/- mice, PCT resulted in selective pulmonary engraftment of bone marrow-derived donor MPs as verified by flow- and chipcytometry, profound reduction of alveolar-protein levels, and significantly improved disease parameters such as lung function and lung densities on CT scan for more than nine months. In situ analysis revealed in vivo differentiation of MPs towards AM-like cells displaying a characteristic surface phenotype, poor antigen presentation, and high phagocytic activity. Moreover, in a humanized herPAP mouse model, PCT of human CD34+-derived MPs led to long-term human cell engraftment, 50–70% reduced alveolar protein levels, and significantly improved herPAP related CT signs for at least six months. Furthermore, when we investigated PCT of “healthy” human iPSC-derived MPs in the humanized PAP model we again observed strictly pulmonary long-term engraftment going hand in hand with significantly improved alveolar protein levels for at least two months. Thus, we here describe a novel, cause directed treatment approach to herPAP employing PCT of multi- or pluripotent stem cell-derived MPs, which may serve as a proof-of-principle to extend current HSC-based transplant concepts to strategies employing mature, long-lived cells.
Oncolytic adenovirus loaded with MHC-I restricted peptide as platform for oncolytic vaccine
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Cancer is a lethal disease still in need of novel therapies. Oncolytic adenoviruses have been particularly successful in the last decade in different tumour types. Several groups, including ours, have highlighted that oncolytic adenoviruses are capable to trigger some degree of tumour-specific immunity as “side-effect” of the anti-viral immunity. To exploit this natural characteristic we have developed a strategy to generate immune privileged oncolytic adenoviruses that converts the anti-capsid immune response into anti-tumour immune response; we have called our system PeptiCrad. We first have developed and evaluated a system to conjugate peptides on viral capsid based on electrostatic interaction. By dynamic light scattering (DLS) and Surface Plasma Resonance (SPR) we have assessed the efficacy of our strategy and quantified the amount of peptides loaded on every single particle. Following we studied the mechanism of cross-presentation by antigen-presenting cells pulsed with PeptiCrad vs peptide alone or the combination of peptides and virus. Then we assessed the biological function of PeptiCrad with regard of cell entry (CAR positive and CAR negative cells), and infectivity in presence of neutralizing antibodies as well as the oncolytic activity. More importantly, using the murine melanoma model overexpressing chicken ovalbumin (B16-OVA) we have demonstrated that SIINFEKL-PeptiCrad (oncolytic virus loaded with OVA derived peptide) was able to completely eradicate tumors from B16-OVA bearing mice producing one log higher OVA specific immunity compared with mice treated with the same virus in combination with the same amount of OVA peptides but not loaded on the capsid. In conclusion we have developed a novel and rapid system to produce tumor-specific immune-privileged oncolytic viruses. The system is versatile, efficient and easy to adapt to different other strategies than only cancer oncolytic vaccines.
Lentiviral-based anti-HIV therapeutic vaccine: design, preclinical studies and phaseI/II clinical trial preliminary results
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THERAVECTYS, a spin off the Pasteur Institute, develops a new generation of therapeutic vaccines using optimized lentiviral vectors. Its most advanced product, a therapeutic anti-HIV vaccine treatment, has entered clinical Phase I/II end of 2012. This vaccination should allow seropositive patients to gain an immunological status identical to the so-called “Sustainable Cured” patients who develop an efficient immunological response capable of controlling the infection without therapy. Vaccine candidates are integrative and self-inactivated live-recombinant lentiviral vectors. They have been classified as “Live recombinant vectored vaccines” (EMA, 2011). Preclinical studies demonstrated i) the generation of a strong, specific and very long lasting T-cell immune response (up to 2 years in murine animal models), ii) the restricted diffusion of the vaccine candidates after injection and iii) their fast disappearance within few weeks, correlated with an absence of macroscopic and microscopic toxicity. These data allowed the settlement of the anti-HIV therapeutic Phase I/II clinical trial that is held in France and Belgium and that has ended the enrollment of the 36 HIV-1 infected patients. THERAVECTYS' anti-HIV vaccine treatment is assessed at three doses and safety, tolerability and immunogenicity compared to a placebo group. Furthermore, vaccine efficiency is be evaluated by the interruption of the HAART treatment in all patients, including placebo. Final results are expected by 2014 and intermediary results will be presented.
Development of a next generation Semliki Forest virus-based DNA vaccine against cervical cancer
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Cervical cancer is the second most prevalent cancer among women worldwide. The disease develops as a result of infection with high-risk human papillomavirus (HPV) through persistent expression of early proteins E6 and E7 with transforming capacities in cervical epithelial cells. Our group pioneered the application of a replication-defective recombinant viral vector system based on Semliki Forest virus (SFV) for vaccination against cervical cancer. In preclinical studies, we demonstrated that recombinant SFV (rSFV) encoding HPV E6 and E7 (rSFVeE6,7), induces robust HPV-specific cellular immune and memory responses upon intramuscular (i.m.) administration in mice resulting in excellent therapeutic anti-tumour efficacy. Despite the clear potency of the SFV vector system, there are a number of inherent challenges. These include manufacturing costs, shelf-life and anti-vector responses. DNA vaccination is an alternative method with the potential to be inexpensive and safe. In this project, the drawbacks associated with both SFV-based vaccines and DNA vaccines are circumvented with the development of a DNA vaccine based on the SFV replicase (DREP). This next generation vaccine will exploit the advantages of both strategies for the development of an immunotherapeutic response against cervical neoplasia by encoding the fusion protein eE6,7 (DREPeE6,7). Our initial results show that upon intradermal delivery via electroporation of mice with DREP-eE6,7, similar E7-specific responses were elicited as compared to i.m. administration of rSFVeE6,7. These results will be further confirmed for assessment of therapeutic efficacy using a TC-1 tumor model.
ORCA-010, a novel potency enhanced oncolytic adenovirus, exerts strong antitumor activity in preclinical models
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Oncolytic adenoviruses represent a novel class of anticancer agents, designed to selectively replicate in and destroy tumor cells by lysis. One major challenge in the development of these viruses for clinical use is the improvement the anti-tumor potency. Using state of the art technology we have generated ORCA-010, an oncolytic adenovirus carrying the safety-enhancing E1AΔ24 deletion, the potency-enhancing T1 mutation and the infectivity-enhancing fiber-RGD modification. The oncolytic potency of ORCA-010 was evaluated in in vitro cytotoxicity assays using a panel of 15 cancer cell lines derived from a variety of tissue origins. The assays showed that ORCA-010 is more potent than Ad5-Δ 24RGD or ONYX-015. As ORCA-010 will initially be developed for the treatment of prostate cancer, selectivity experiments were performed using primary human prostate cells. In both primary prostate fibroblasts and epithelial cells ORCA-010 was shown to be as safe as Ad5-Δ 24RGD, an oncolytic adenovirus currently being tested in clinical trials. Evaluation of ORCA-010 in in vivo xenograft tumor models showed that ORCA-010 significantly inhibited growth of prostate, lung and ovarian tumors and conferred prolonged survival of tumor-bearing animals. Furthermore, analyses of PC-3 tumors showed that a substantial increase in infectious ORCA-010 viral particles could be found up to at least 4 weeks post treatment. Intratumoral virus replication was associated with substantial necrosis and fibrosis. These promising preclinical data support further development of ORCA-010 for clinical testing in a Phase I/II clinical trial in patients with prostate cancer.
Targeted Genome Editing in Human Long-Term Repopulating Hematopoietic Stem Cells for Correction of SCID-X1
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Targeted genome editing by artificial endonucleases has brought the goal of gene correction within the reach of gene therapy. Here we show that gene targeting in the most primitive hematopoietic stem/progenitor cells (HSPC) is constrained by poor gene transfer efficiency and a bias against the use of the homology directed DNA repair pathway in these cells. By combining Integrase Defective Lentiviral Vectors (IDLV) for donor template delivery and mRNA transfection to drive a transient spike of ZFN expression and using culture conditions tailored to expand HSPC, we have overcome these barriers and provide stringent evidence of TI in human HSPC. We enabled TI of a corrective cDNA into a mutational hotspot of IL2RG gene with high efficiency (∼6%) and specificity (>95%) in HSPC capable of long term multilineage engraftment in NSG mice. IL2RG edited cells generated polyclonal functional lymphoid cells that expanded in the mice in presence of human cytokines and were functionally indistinguishable from wild-type, proving that the corrective cDNA functionally restored g-chain receptors. Deep sequencing analysis performed on ZFN off-target sites proved the high specificity of ZFNs on HSPC. Importantly, we corrected the IL2RG gene in HSPC from a genotyped SCID-X1 patient, in which we found similar TI efficiency and confirmed restoration of g-chain expression in targeted cells. Finally, in order to model disease correction we optimized a similar protocol for efficient TI (∼6%) into murine lin- HSPC of a humanized SCID-X1 mouse and demonstrated the selective advantage of the lymphoid corrected cells upon transplant.
Targeted genome editing by lentiviral protein transduction of ZFN and Cas9 proteins
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Programmable nucleases including ZFN, TALEN and CRISPR/Cas9 are game changers in gene editing and gene therapy. Their therapeutic application, however, requires safe and efficient means of delivery. We have engineered lentiviral particles as effective carriers of ZFN proteins fused to Gag/GagPol polyproteins. By exposing 293T cells to VSV-G pseudotyped ‘all-in-one’ IDLVs containing both ZFN proteins and viral RNA carrying the donor sequence for homology-directed repair/insertion, we were able to integrate reporter gene expression cassettes (eGFP, Fluc, puro) into a safe genomic locus. Also, repair of genomic mutations was obtained in more than 8% of cells treated with ‘all-in-one’ IDLVs, whereas up to 24% of targeted alleles (CCR5 and AAVS1) were disrupted in primary cells, including normal human dermal fibroblasts and primary keratinocytes, exposed to ZFN-loaded viral particles. To establish lentiviral protein transduction for delivery of CRISPR/Cas9, we incorporated the Staphylococcus aureas Cas9 (about 3.2 bp) in lentiviral particles. To identify a position within the GagPol polyprotein with higher tolerance of a large foreign protein, SaCas9 was inserted into various positions and tested for cleavage activity upon lentiviral transduction. For one of these positions (SaCas9 fused to C-terminus of Gag), successful gene disruption was demonstrated in preliminary studies of lentiviral Cas9 protein delivery in 293T cells. Ongoing analyses are addressing targeted cleavage and gene insertion by virally delivered ZFN and Cas9 in induced pluripotent stem cells and hematopoietic stem cells exploiting non-VSV-G pseudotypes.
CCR5-Uco-TALEN: A novel highly active TAL effector nuclease (TALEN) for the targeted knock-out of the HIV-co-receptor CCR5
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The chemokine receptor CCR5 is an essential co-receptor of CCR5-tropic HIV strains playing a critical in the course of natural infection. Based on the unique case of HIV cure after transplantation of allogeneic, CCR5d32 homozygous stem cells (“Berlin patient”), genetic disruption of CCR5 using designer nucleases has been proposed as a promising HIV gene therapy. We introduce CCR5-Uco-TALEN, a novel TAL-effector nuclease that mediates high-level CCR5 knockout (>40%) in PM1 and primary T cells after mRNA electroporation. To test whether CCR5 editing effectively protected T cells from HIV infection, we first used LeGO vectors packaged either with a natural CCR5-tropic HIV envelope protein (BaL) or with an alternative envelope originated from Gibbon Ape Leukaemia Virus (GALV). Whereas treatment with CCR5-Uco-TALEN did not affect gene-transfer rates for GALV-env lentiviral vectors, transduction with the HIV-env vector was strongly (>50%, p<0.001) suppressed in CCR5-Uco-TALEN treated T cells. In another approach, we applied CCR5-Uco-TALEN to protect primary human T lymphocytes from infection with replication-competent CCR5-tropic HIV (modified to express luciferase). Again, more than 50% of CCR5-Uco-TALEN treated, unsorted T lymphocytes were resistant to HIV infection. In summary, we have developed the novel CCR5-Uco-TALEN facilitating high-level protection of primary T lymphocytes from infection by CCR5-tropic HIV strains. CCR5-Uco-TALEN is characterised by excellent specificity with low likelihood for off-target binding (closest off-target CCR2 has <85% identity). Moreover, the mRNA-electroporation protocol used for CCR5-Uco-TALEN delivery is well compatible with GMP conditions. In conclusion, we propose that CCR5-Uco-TALEN should be perfectly suited for clinical translation.
Towards TALEN-mediated targeting of FANCA in the AAVS1-homolog safe harbor locus in FA-A mice
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Fanconi anemia (FA) is a rare inherited disease associated with bone marrow failure and cancer predisposition. Mutations in any of the 16 FA genes discovered so far can disrupt a common DNA repair pathway known as the FA pathway. Within the FA patients, mutations in FANCA are the most frequent (>60%), justifying that most FA gene therapy studies have been developed for this complementation group. Because of the relevance of FA mouse models in the development of new FA therapies, and given the advantages of the safe harbor AAVS1 locus for gene editing strategies in human cells, we aimed to specifically insert the therapeutic FANCA gene into the murine Mbs85 locus – homolog of the human AAVS1 locus – in Fanca-/- hematopoietic stem cells (HSCs). To this end, two TALEN pairs targeted to two different regions of the murine Mbs85 locus were generated. The allelic cleavage activity of these two TALEN pairs was determined by T7-1 or Cel-1 endonuclease assay, was 44% and 25% in FA-A MEFs and in hematopoietic progenitors (lin- cells) respectively. For gene targeting experiments we generated a donor construct that harbors Mbs85 homology arms flanking the FANCA cDNA driven by PGK promoter and a selection cassette including the puromycin resistance gene. The correction of the FA-A phenotype upon homologous recombination of the designed donor at the Mbs85 locus both in FA-A MEFs and hematopoietic progenitor cells is currently under evaluation.
Evaluation of an optimised injection system for retinal gene therapy
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Preventing visual loss after corneal trauma by targeting miR-145
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MicroRNA (miRNA) gene therapies are of particular interest for complex processes such as fibrosis and scarring. This study focussed on the role of miR-145 in corneal scarring. The inhibition of myofibroblast activity after wound closure may prevent scar formation. MiRNA profiling was performed in healthy human cornea and corneal scar tissue. Levels of the miR-145 were confirmed using miRNA expression assays. Fibroblasts were isolated from human corneas and differentiated into myofibroblasts in presence of TGF-ß1. Real-time PCR and western blots were used to determine the effect of miR-145 and a miR-145 antimiR on profibrotic genes such as a-smooth muscle actin (aSMA). Cell contractility and migration were analysed using collagen contraction assays and wound scratch assays, respectively. Levels of miR-145 were 10-fold higher in corneal scar tissue compared to healthy cornea and 6-fold higher in TGF-ß1-treated myofibroblasts compared to non-treated fibroblasts (p<0.001). Inhibition of miR-145 using antimiR technology significantly decreased miR-145 levels (by 94%) and aSMA expression (by 75%) in myofibroblasts (p<0.05). Furthermore, miR-145 inhibition significantly reduced contractility, migration, and TGF-ß1 secretion of myofibroblasts compared to cells treated with a control antimiR. Hence, targeting miRNA-145 reversed the scarring myofibroblast phenotype. Our data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of corneal myofibroblasts. We show that inhibition of miR-145 significantly reduces myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce corneal scarring and subsequent visual loss.
Effective delivery of large genes to the retina by dual AAV vectors
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Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt disease (STGD). Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We compared their transduction efficiency in vitro and in the retina. Finally, we assessed the efficacy of the best performing strategies in the retina of the Abca4-/- mouse model of STGD. We found that dual AAV outperform oversized vectors, both in vitro and in the murine retina. While dual AAV overlapping vectors efficiently transduce only RPE, dual AAV trans-splicing and hybrid approaches transduce also mouse and pig photoreceptors. Although the levels of murine photoreceptor transduction mediated by dual AAV trans-splicing and hybrid vectors are lower than those achieved with a single AAV, they resulted in significant improvement of the retinal phenotype of Abca4-/- mice. Interestingly, the transduction efficiency of dual AAV vectors is significantly higher in the large cone-enriched pig retina than in mice, potentially due to the higher levels of dual AAV co-transduction. In conclusion we found that dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.
Development of EncorStat®, donor corneal tissue genetically engineered to prevent graft rejection
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Corneal transplantation is one of the most successful transplant procedures because of the relatively immune-privileged status of the eye and the fact that the cornea is largely avascular. However in high risk patients, which account for >20% of the 100,000 transplants carried out each year, the rejection rate is very high (50–90%) due to vascularisation of the recipient corneal bed. The prognosis in these patients is poor; some are no longer considered for replacement transplant and are left blind. The main reason for graft failure is irreversible immunological rejection and it is therefore unsurprising that neovascularisation (both pre- and post-grafting) is a significant risk factor for subsequent graft failure. Neovascularisation thus is a logical target to prevent graft rejection. EncorStat® is a human donor cornea modified prior to transplant by the ex vivo delivery of the genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin, which then prevent rejection by suppressing neovascularisation. The ex vivo gene therapy product used is a non-replicating, recombinant lentiviral vector product derived from the Equine Infectious Anaemia Virus (EIAV). Here we present data demonstrating the optimisation of the process and efficacy of EncorStat® corneas in two rabbit models of corneal rejection. The non-clinical data presented supports the evaluation of EncorStat® corneas in a First-in-Man trial. With support from the UK Technology Strategy Board, this trial will be conducted in 2015, following completion of nonclinical safety studies and GMP vector manufacture this year.
Comparative AAV wild-type and rAAV vector-mediated genomic integration profiles in human diploid fibroblasts analyzed by 3rd generation PacBio DNA sequencing
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Genome-wide analysis of AAV2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic hotspots near GAGY/C repeats, resembling consensus AAV Rep-binding sites (Hüser et al. PLoSPathog 6, 2010). Hotspots include the previously defined AAVS1 site on chr. 19q13.42. In contrast, rep-deficient AAV vectors (rAAV) show random integration profiles. Here we present the first genome-wide analysis of wild-type AAV integration in diploid human fibroblasts and the first to compare wild-type to rAAV integration, side by side under identical experimental conditions. High-throughput, 3rd generation PacBio-based DNASeq reveals that the pattern of AAV integration hotspots in human fibroblast differs from that in HeLa cells. Furthermore, DNase-Seq analysis of human fibroblasts and tissues including liver, muscle, heart, brain, skin and human embryonic stem cells compared to aneuploid HeLa cells reveals variant chromatin accessibilities at preferred AAV integration hotspots that correlate with cell type-dependent hotspot preferences (Hüser et al, in press). In summary, AAV integration patterns are dependent on cell-type-specific, variant chromatin accessibility, leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy.
A new bioinformatics tool to improve precision and quality of vector integration site identification after genomic alignment
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Vector integration sites (IS) are key tools for the monitoring of safety and efficacy of gene therapy treatments and for clonal dynamics assessments due to the capability of a transduced cell to stably transmit the proviral insertion to its progeny. State-of-the-art wet and bioinformatics techniques for IS retrieval may introduce some artifacts, such as PCR amplifications and sequencing errors or sequencing read parsing and mapping on reference genome. These biases mislead actual methods for IS identification (based on a rigid sliding window (SW) of 4 bp) in terms of precision in genomic location and of potential overestimation of integration sites. We designed a new bioinformatics algorithm to identify IS from mapped sequencing reads based on a Gaussian density profile. To validate our method, we performed 40,000 simulations on single IS and on couples of close IS and we measured precision and recall observing improvements in >90% of the simulations. Moreover, we performed 3 ad-hoc in vitro experiments on a cell clone with 6 known IS in which we measured the precision of IS identification obtaining an average of 100% with our new method whereas <30% using the SW one. We finally applied our new approach to our two published hematopoietic gene therapy clinical trials obtaining improvements in IS genomic placement and in decoupling of multiple IS in close proximity, with a IS reduction of potential false positive of 3%. We implemented our approach in a Python program that scales linearly in computational time and space.
High throughput, high nucleotide resolution integration site analysis of wtAAV reveals viral instability during the process of integration
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Wildtype adeno-associated virus (wtAAV) integrates its DNA into the human genome in order to establish latency. Large-scale integration analyses using high-throughput next generation sequencing have revealed that Rep-mediated wtAAV integration preferentially occurs in AAVS1, but that additional integration hotspots associated with Rep binding sites can be found as well. Until now most integration sites(IS) reported for wtAAV were derived from vector-genome fusion sequences, where the viral sequence fragment was mapped near the ITRs or near the p5 region of the viral genome. Most state-of-the-art methods for IS pattern analysis rely on amplification of the vector-genome junction with primers that bind closely to the ends of viral genomes. Therefore knowledge about the structure of the integrated wtAAV genome remains limited. We investigated 2,829,267 wtAAV concatemeric structures to analyze the stability of the wtAAV genome and to identify potential alternative breakpoints throughout the wtAAV genome. We found that breakpoints can occur at any position of the wtAAV genome. Apart from the ITRs and the p5 promoter there are a number of other preferred breakpoints throughout the wtAAV genome. Using targeted HiSeq sequencing of wtAAV and AAVS1 we determined 681 exact IS and found that junctions at a distance from the AAV ITRs and p5 region, don't only occur in wtAAV concatemeric structures but can also be observed in wtAAV integration events in the human genome. This suggests that comprehensive analysis of the wtAAV IS profile should include the analysis of viral-genome junctions throughout the whole viral genome.
Targeted Sequencing for Detection of Vector Integration Sites
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The determination of the exact position and distribution of viral integration sites in the host genome is crucial for biosafety assessment and for the understanding of viral integration mechanisms. Current mapping methods are based on amplification of the vector-genome junctions and subsequent PCRs (LAM PCR). Thus, these methods not only depend on the completeness of the chosen viral genome for PCR primer binding, but also introduce a bias to exact quantitative measurements of individual clonal contributions. Here, we present results from a targeted sequencing approach (SureSelect, Agilent) in which we enriched for genomic regions including vector sequences. This approach has major advantages over primer based approaches like LAM PCR: • As less PCR cycles are needed, a more quantitative estimation of cell clonality (clones with the sample integration site) should be possible. • Genomic regions that are captured together with the vector allow for relative quantification of vector copies per genome. • Integrations of incomplete vectors (common for adeno-associated vectors) can be detected without the need of specific primers. • As the entire vector is sequenced, mutations within the vector (and transgene) can be detected. Our preliminary results suggest a detection limit of 100–1000 copies of vector per 1 μg of initial DNA. Integration site distribution relative to gene elements and clonality are comparable to LAM-PCR. A four-fold dilution of the baits (120 nt RNA probes used for capturing the target sequence) did not change the sensitivity of the assay. In summary, targeted sequencing provides an important complementary tool to primer based approaches (like LAM-PCR) for mapping of vector/viral integration sites. To improve the efficiency and specificity, we are currently designing new sets of baits with less homologies to the host genome.
Synthetic mRNA Encoding Vaccinia virus PKR Inhibitors Improves Immunization with Semliki Forest Virus Replicons
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Self-replicating RNA vectors (replicons) based on the Semliki Forest virus (SFV) genome can elicit powerful immune responses directed against replicon-encoded proteins. Immune responses are driven by self-adjuvant activity of replicating RNA. However, protein kinase R (PKR) which is upregulated during innate immune response and activated by double stranded RNA replication intermediates, inhibits translation of replicon encoded transgenes. Although PKR inhibitors were shown to enhance translation of replicon RNA, they have not yet been used to improve vaccination with replicons. Therefore we aimed to integrate PKR inhibition into an RNA vaccine vector platform composed of antigen encoding replicon RNA and non-replicating synthetic mRNA encoding Vaccinia virus PKR inhibitors K3L and E3L. When co-transfected simultaneously with replicons, K3L and E3L mRNA increased translation of replicase, an enzyme complex encoded on the 5′ part of replicons that drives replication and subgenomic transcription. Increased replicase expression subsequently drove increased RNA replication. The translation of transgenes encoded on subgenomic transcripts increased approximately 4-fold upon K3L and more than 10-fold upon E3L mRNA cotransfection. In vivo, mRNA-encoded PKR inhibitors not only increased transgene expression and but also significantly improved immune responses to Influenza HA encoding replicons. We conclude that removal of PKR-mediated blocking of translation improves effects of vaccination with replicons. Intracellular translation of PKR inhibitor mRNA might preclude side-effects potentially related to systemic PKR inhibition with small molecules. Furthermore, the “RNA only” design of our vector platform facilitates formulation development required for the clinical translation of our concept.
Messenger RNA in cancer immunotherapy: TriMix delivering an antitumor message
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Messenger RNA (mRNA) is frequently applied in the field of cancer immunotherapy. In this regard mRNA has been mainly used for the ex vivo modification of dendritic cells (DCs). In the pursuit of an optimal DC-vaccine, we studied mRNA as a tool to deliver tumor antigens and immunomodulatory proteins. This research has culminated in a well-defined protocol for the preparation of a potent DC-vaccine. Herein DCs are electroporated with tumor antigen mRNA and TriMix, a mix of three mRNA molecules encoding the co-stimulatory molecule CD70 and two DC activation stimuli, CD40 ligand and active TLR4. Vaccination of stage III/IV melanoma patients with these so-called TriMix-DCs has proven to be safe and well tolerated, to induce tumor antigen-specific immune responses and more importantly to induce objective clinical responses in over a quarter of patients. More recently, we studied intranodal administration of tumor antigen and TriMix mRNA for the treatment of cancer in various mouse tumor models. In situ modification of DCs and subsequent induction of T cell-mediated antitumor responses was demonstrated. Moreover mRNA immunization was shown to be as effective as DC immunization. We further demonstrated that the TriMix platform can be used to activate T cell responses without co-delivery of tumor antigen mRNA when delivered into the tumor. Preliminary data further demonstrate that the therapy outcome can be further improved when combined with immune checkpoint blockade. Therefore, we believe that TriMix mRNA as an off-the-shelf therapeutic represents an important step in the development of future cancer immunotherapeutic strategies.
Best of both worlds - a novel versatile AAV vector toolbox for combinatorial CRISPR and RNAi expression
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The CRISPR/Cas9 system currently revolutionizes the gene therapy field due to the unique ease and efficiency with which it allows gene engineering in human cells. It only requires a small g(uide)RNA which will direct a co-expressed Cas9 protein to a target DNA for cleavage and gene disruption (or repair/modification, if a recombination template is also provided). However, targets prone to rapid mutation, such as human viruses, will require co-expression of multiple gRNAs to thwart adverse mutational escape. To fill in this gap, we created novel highly customizable AAV vectors for simultaneous expression of up to three gRNAs in a single self-complementary backbone. These vectors are genetically stable and robustly knock-out up to three targets in human cells, as demonstrated with various cellular and viral genes, including miR-122 and HIV-1. Moreover, by targeting three different genes involved in apoptosis, we exemplify the value of our vectors for dissection of cellular pathways and for gene annotation. Notably, our constructs stably accommodate and deliver minimal gRNA expression cassettes down to 200 bp, which minimizes the amount of ectopic DNA and improves vector safety. We also engineered a second battery of AAV vectors allowing juxtaposition of multiple gRNAs and shRNAs under three different promoters. Using hepatitis B virus as example, we demonstrate their power to concomitantly tackle a clinically relevant human pathogen on the DNA (CRISPR) and RNA (RNAi) level. Our robust and versatile AAV vectors for combinatorial gRNA/shRNA expression should substantially foster the (co-)application of powerful gene knock-out/-down technologies in human gene therapies.
In vivo mRNA introduction using polyplex nanomicelles to treat neurological disorders
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Neurological disorders are difficult to cure completely considering poor nerve regeneration capacity and difficulties in accurately targeting neural tissues. Administering mRNA is a promising approach for treating neurological disorders because mRNA can provide proteins and peptides in their native forms for mature non-dividing neural cells, without the need of entering their nuclei. However, direct mRNA administration into neural tissues in vivo has been challenging due to too unstable manner of mRNA and its strong immunogenicity. Thus, using a suitable carrier is essential for effective mRNA administration. We established a novel carrier based on the self-assembly of polyethylene glycol (PEG)-polyamino acid block copolymer, i.e. polyplex nanomicelles. The core-shell structure surrounded by PEG layer allows stable retention of mRNA in the inner core. The functional polyamino acids have capacity of smooth endosome escape, providing efficient and sustained protein expression in CNS (PLoS One 8:e56220, 2013). To confirm the effectiveness for treating nerve disorders, we used a mouse model of olfactory dysfunction. Intranasal administration of mRNA-loaded nanomicelles provided efficient protein expression in nasal tissues, particularly in the lamina propria which contains olfactory nerve endings. The immunogenicity of mRNA was well suppressed by incorporating mRNA into nanomicelles, allowing repeat administration of mRNA into nose. For mice with olfactory dysfunction, administering BDNF-expressing mRNA remarkably enhanced the recovery of olfactory function along with repairing the olfactory epithelium to a nearly normal architecture. These results show the therapeutic potential of introducing exogenous mRNA for the treatment of neurological disorders, providing a new strategy of mRNA-based therapy.
Phase 1 clinical trial of liver directed gene therapy with rAAV5/2-PBGD in acute intermittent porphyria: safety data
Acute intermittent porphyria (AIP) is a rare genetic disease due to mutations in the gene encoding porphobilinogen deaminase (PBGD). The aim of this trial is to explore the safety of gene therapy in AIP patients. In this phase 1 clinical trial, 4 increasing doses of a recombinant adeno-associated serotype 2/5 vector expressing a codon-optimized human-PBGD under the control of a liver-specific promoter (AAV2/5-PBGD) were administered to 8 AIP patients by intravenous infusion. Besides safety, inflammatory cytokine production, humoral and T-cell response, as well as viral shedding were analyzed. Furthermore, at the end of the follow up a liver biopsy were obtained in 6 patients. AAV2/5-PBGD was well tolerated. No treatment-related severe adverse events were observed. No changes in blood cell count, liver and renal tests, inflammatory cytokine production were observed during the 12 months follow-up. One patient at the highest dose experienced an AIP attack 3 days post-injection. The transient, slight increase in transaminases detected in this patient was likely related to the attack and not to the vector. All patients developed neutralizing antibodies against AAV5. No T-cell responses were detected. Viral clearance from all biological fluids was completed after 2 weeks in the lowest dose and 4 weeks in the highest dose cohorts. Liver biopsy analysis revealed the presence of vector genomes in all patients analyzed 12 months after vector administration. The treatment with AAV2/5-PBGD and is safe in patients with AIP. AAV2/5-PBGD transduces human liver and no cellular immune response against the vector or the transgene was detected.
Full correction of neurologic and somatic lysosomal pathology in Mucopolysaccharidosis type IIIB by AAV9-based gene therapy
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Mucopolysaccharidosis type IIIB (MPSIIIB) is a Lysosomal Storage Disease (LSD) caused by deficiency in alpha-N-acetylglucosaminidase (NAGLU), an enzyme involved in the degradation of the glycosaminoglycan (GAG) heparan sulfate. Accumulation of undegraded HS leads to lysosomal pathology, resulting in severe progressive neurodegeneration and mild somatic disease. There is no cure for MPSIIIB. Here we demonstrate that delivery of AAV9 vectors enconding NAGLU to the CSF can reverse the pathological phenotype of MPSIIIB. Transduction was detected in the whole encephalon, even in areas that are not easily reached by vectors following direct injection in the CNS parenchyma. Therapeutic levels of NAGLU were documented throughout the encephalon and mediated correction of lysosomal pathology and neuroinflammation in MPSIIIB mice. Furthermore, NAGLU gene transfer resulted in a striking normalization of CNS gene expression profile, which was in untreated animals enriched in genes associated with microglial activation. Additionally, vector leaked to the circulation and transduced the liver, which efficiently produced and secreted NAGLU leading to normalization of GAG content and lysosomal function in somatic organs. As a result of whole-body disease correction, treated MPSIIIB mice had normal behavior and lifespan. Intra-CSF delivery of vectors encoding canine NAGLU to naïve and anti-AAV9 antibody positive dogs was safe and resulted in detection of active enzyme in the CSF despite the presence of pre-existing immunity against the vector. This work provides evidence of the therapeutic potential of intra-CSF AAV9-mediated gene transfer for the treatment of MPSIIIB and other LSDs characterized by CNS and peripheral disease.
Development of ex vivo gene therapy for familial LCAT deficiency syndrome by self-transplantation of therapeutic-enzyme secreting adipocytes
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Familial lecithin:cholesterol acyltransferase (LCAT) deficiency syndrome is an autosomal recessive disease characterized by severe dysfunction of HDL and subsequent generation of abnormal LDL leading to life-threatening complications such as renal failure and corneal opacity. Gene therapy-based LCAT protein replacement is suggested to be effective; however, no approach for permanent correction of the symptoms and/or preventing the patients from the complications has been reported. Our previous basic experimental data together with clinical experience in transplantation therapy strongly suggested that autologous adipocyte transplantation is an effective maneuver to perform ex vivo gene therapy enabling sustained secretion of therapeutic enzymes. We have recently reported that ceiling culture-derived proliferative adipocytes (ccdPAs) are useful for an effective LCAT production using retroviral gene transduction. LCAT protein secreted by the lcat gene-transduced ccdPAs caused maturation of HDL derived from LCAT-deficiency patients and reduced renal-damaging LDL poor in esterified-cholesterol, in vitro. Furthermore, subcutaneous transplantation of the ccdPAs ameliorated the circulating abnormal lipoproteins in lcat-deficient mice. In addition, fibrin glue, which is clinically available scaffold, increased the survival of transplanted lcat-gene transduced ccdPAs in mice. Based on the accumulated pre-clinical data in animal models, the ex vivo gene therapy protocol for clinical research (first in human) has been approved by Ministry of Health, Labour and Welfare followed by initiation of patient enrollment in Japan. Thus, ccdPAs would provide an excellent platform for developing a protein replacement therapy not only for LCAT deficiency but also other disorders caused by serum protein deficiency requiring long-term therapeutic protein supplementation.
Lentiviral vector based hematopoietic stem cell gene therapy mediates sustained expression of functional thymidine phosphorylase in a mouse model for mitochondrial neurogastrointestinal encephalomyopathy
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Deficiency in the enzyme thymidine phosphorylase (TP) results in systemic thymidine (Thd) and deoxyuridine (dUrd) accumulation, affecting mtDNA replication and causing mitochondrial dysfunction. Symptoms in MNGIE patients include gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogeneic hematopoietic stem cell (HSC) transplantation reduces disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Therefore, we examined the option of ex vivo HSC gene therapy in Tp-/-Upp-/- mice, which mimic human MNGIE including brain abnormalities in white matter as demonstrated by T1 and T2 weighted MRI scans. Syngeneic lentiviral vector (LV) gene modified HSC overexpressing the TP cDNA sequence, and driven by either the phosphoglycerate kinase (PGK) or the spleen focus forming virus (SFFV) promoter, were transplanted in Tp-/-Upp-/- mice following sublethal irradiation. Enzyme activity in treated mice rapidly increased after transplantation. 14 months post treatment LV-PGK-TP recipients showed an average 1.2-fold wild type TP activity and LV-SFFV-TPco mice a 36-fold increase, resulting in sustained reduction of plasma and urine Thd and dUrd levels, with a vector copy number of on average 1/donor cell and 75% donor cell chimerism (N=12). Currently, we are investigating whether the treatment prevents the mitochondrial deoxyribonucleoside triphosphate pool imbalances and the subsequent reduction in mtDNA replication rate. Overall, stem cell gene therapy provide stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, and will be further optimized for optimal efficacy and safety to develop a clinical protocol to treat MNGIE patients.
Safety and efficacy of AAV5-hFIX in non-human primates
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We have developed AAV5-hFIX, adeno-associated virus serotype 5-based vector containing the human coagulation factor IX gene, in our scalable and GMP compliant baculovirus-based production platform. To evaluate the safety and efficacy of AAV5-hFIX in a relevant model, 4 groups of each 3 cynomolgus macaques were dosed intravenously with respectively 5×1011, 5×1012, 2.5×1013 and 0.93×1014 genome copies (gc) per kg BW. Blood was sampled at least weekly, from 4 weeks pre-trial until sacrifice 26 weeks after dosing. The resulting hFIX protein expression correlated with the dose and was sustained for the duration of the 6 months follow-up. In the highest dose group hFIX levels peaked around 30% of normal and stabilized around 10–15% whereas in the animals dosed the lowest dose, hFIX levels never increased above 1% of normal. Based on the observed expressed hFIX and by extrapolating the dose one-to-one between non-human primates and humans, the MABEL in man would be 5×1012 gc/kg. In the highest dose group, one animal presented with fast-decreasing levels of expressed hFIX. No changes in clinical chemistry and haematology were observed and liver enzymes were not elevated. (RT)-QPCR demonstrated homogeneous vector DNA delivery and transgene expression in the liver. No loss of transduced hepatocytes that would be resulting from a T cell reaction was detected. Xenogeneic antibodies against human FIX were detected this in animal. No signs of adverse reactions or significant macroscopic or microscopic abnormalities were observed in any animal. Administration of AAV5-hFIXco vector resulted in therapeutically relevant hFIX levels and was well tolerated.
Baboon envelope pseudotyped LVs mediate high level gene transfer in human B cells allowing secretion of FIX at therapeutic levels in humanized NSG mice
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B-lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B-cells are potent specialized protein secreting cells. B-cells, though, are poorly transduced by VSV-G-pseudotyped lentiviral vectors (LVs). Since we recently showed that Baboon envelope pseudotyped LVs (BaEV-LVs) outperformed VSV-G-LVs for gene transfer into HSCs, we evaluated them here for gene transfer into human (h) B-cells. Upon B-cell receptor stimulation, BaEV-LVs transduced up to 70% of the B-cells while VSV-G-LV transduction did no exceed 5%. Remarkably, BaEV-LVs permitted efficient transduction of 30% of resting B cells while VSV-G-LVs were inefficient. Importantly, BaEV-LVs transduced memory and naive B cells. and permitted up to 80% transduction of plasmocytes. Adaptive transfer of mature hB cells transduced with BaEV-LVs into NSG mice allowed differentiation into plasmablasts and plasma B cells confirming normal differentiation in vivo. Of note, high-level gene marking in mature, plasmablast and plasma B-cells was revealed in these mice. These results encouraged us to evaluate BaEV-LV transfer of Factor IX (FIX) into hB cells for treatment of hemophilia, a severe congenital blood disorder. We produced high-titer BaEV-LVs carrying a codon optimized FIX with a hyperactivating FIX-R338L mutation resulting in a 15-fold gain in FIX activity. These BaEV-FIX-LVs efficiently transduced hB-cells and upon adoptive transfer into NSG mice we obtained therapeutic levels of FIX in the serum at 3 to 4 weeks post-engraftment. Concluding, the BaEV-LVs might represent valuable tools for therapeutic protein secretion in treatment of hemophilia from autologous B-cells in vivo.
Targeting FVIII expression to specific cell-types to overcome immunological responses for Hemophilia A gene therapy
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Hemophilia A (HA), a X-linked bleeding disorder, due to mutations of clotting FVIII gene. HA patients are treated with recombinant or plasma-derived FVIII with high probability to develop inhibitors. Antigen-presentation occuring in the liver by sinusoidal endothelial cells and/or Kupffer cells (KC) induces tolerance rather than immunity towards antigens presented to T-cells. We investigated the role of these cell types in HA gene transfer using Lentiviral vectors (LV)-expressing FVIII driven by cell-specific promoters and microRNA target sequences (miRTs). We prepared LVs containing GFP or BDD-FVIII under the control of the ubiquitous PGK promoter, the CD11b (monocyte/macrophage-specific) and the VEC (endothelial-specific) promoter containing several combinations of microRNA target sequences such as miRT142.3p (silenced in hematopoietic cells) or miRT126 (silenced in endothelial cells) or miRT122 (silenced in hepatocytes) and injected HA mice. After a characterization of the described LV using GFP as transgene expressed in vivo, we injected HA mice with LV.PGK.FVIII-miRT142, LV.CD11.FVIII-miRT126 and LV.VEC.FVIII-miRT122-142.3p. In the first group anti-FVIII-Abs were detected 2 w after vector delivery, however the presence of miRT142 halved inhibitors titer in comparison with LV-injected mice without miRT142.3p. Mice injected with LV.CD11b.FVIII-miRT126 and LV.VEC.FVIII-miRT122-142.3p, reached long-term phenotypic correction up to 1 y with an average of 6–8% FVIII activity and absence of anti-FVIII-Abs. Moreover, tolerance was obtained because FVIII was expressed and functional in FVIII-immunized mice producing FVIII-Abs. In conclusion, LV expressing FVIII under control of cell-specific promoters combined with miRT-combinations were able to overcome FVIII off-target expression limiting immune responses and providing phenotypic correction in treated HA mice.
Redirection of CD4+ T cells with ICOS-based chimeric antigen receptors
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T cell persistence is likely to promote long-term antitumor effects after adoptive T cell transfer; however, T cells expressing chimeric antigen receptors (CARs) have not persisted well in patients with solid tumors. We hypothesized that CD4+ and CD8+ T cells may need distinct costimulation signals to persist, and may therefore need to be redirected with CARs constructed using different costimulatory domains. To test this hypothesis, we compared the in vivo antitumor effects and persistence of combined CD4+ and CD8+ T cells redirected with CARs containing CD28, 4-1BB or ICOS-based costimulatory domains. Using multiple mouse tumor models, we demonstrate that the ICOS intracellular domain enhanced the in vivo persistence of CAR-expressing CD4+ T cells, an effect that was independent of the CAR intracellular domain used to redirect CD8+ T cells. In contrast, persistence of CD8+ T cells was highly dependent on the intracellular domain used to redirect CD4+ T cells; specifically, CD4+ T cells expressing an ICOS-based CAR significantly increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. We further demonstrate that the antitumor effect of CAR-expressing CD8+ T cells was enhanced when co-injected with redirected CD4+ T cells. Collectively, our data suggest that combining CD4+ T cells redirected with an ICOS-based CAR with CD8+CAR-T cells will enhance persistence and antitumor efficacy of CAR T cells.
NY-ESO-1-specific Single Edited T cells efficiently eliminate multiple myeloma without inducing xenogeneic GvHD
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T cell receptor (TCR) gene transfer has yielded promising results in cancer patients. To permanently remove the expression of the endogenous TCR and the risk of TCR chains mispairing, we developed TCR gene editing, based on the combination of: knockout of the endogenous TCR genes by transient exposure to -a and -B chain specific Zinc Finger Nucleases, and introduction of tumor-specific TCR genes by lentiviral vectors. While the complete editing (CE) procedure requires multiple manipulation steps requiring repeated cell activation cycles, ‘single TCR editing’ (SE), based on the disruption of a single endogenous TCR chain, followed by transfer of the tumor specific TCR, generates redirected T cells devoid of their natural TCR repertoire, in a single round of cell activation. We exploited a HLA-A2 restricted TCR specific for NY-ESO-1 to compare safety and efficacy of unedited TCR transferred (TR), SE and CE T cells. We generated TCR edited T cells with an early differentiated memory phenotype. We observed significantly higher levels of the tumor-specific TCR expression in edited versus transferred T cells. Edited lymphocytes were more efficient than unedited-TCR transferred cells in killing NY-ESO-1+ cancer cells. In NSG mice engrafted with the U266 cell line, SE and CE T cells completely eliminated the disease, without inducing xenogeneic GvHD. Thus TCR single editing is effective in eliminating the risk of graft versus host disease associated with the infusion of donor-derived lymphocytes while improving their anti-tumor activity. The relative simplicity of the SE protocol should facilitate its clinical application to patients with hematological malignancies.
A traceless selection system that allows efficient generation of auxiliary vector-free Transposon and CRISPR-modified T cell products
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Recent years have seen major breakthroughs in genome engineering systems, such as the transposon-mediated gene delivery systems and CRISPR-Cas9-mediated genome editing tools. For clinical use of both systems it would be valuable to develop strategies that allow both the enrichment of cells undergoing the intended stable genetic modification and depletion of cells that by random episome integration display continued expression of the auxiliary gene. Here we demonstrate how the inclusion of a single selection marker in modified Sleeping Beauty gene transfer and hCas9-mediated genome editing systems can be used to achieve these goals. We demonstrate that this strategy allows the ‘traceless’ generation of cell products with high frequencies of stably gene-modified primary human T cells (>70% using the Sleeping beauty transposon, against an average efficiency of 24% without enrichment; >40% hCas9 genome edited primary T cells, against an average of 12% without enrichment). Furthermore, we show that the same selection marker can be used to remove cells harboring unwanted integration of the auxiliary vector with an efficiency of >95%. The dual purpose selection system described here should facilitate the use of genome editing tools for the generation of clinically used cell products.
Donor T-cells for hematopoietic-restricted minor histocompatibility antigens are induced in patients with combined Graft-versus-Leukemia and Graft-versus-Host disease
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Patients with hematological malignancies can be successfully treated with allogeneic stem cell transplantation (alloSCT) and donor lymphocyte infusion (DLI). In HLA-matched alloSCT, donor T-cells can mediate beneficial Graft-versus-Leukemia (GvL) and undesired Graft-versus-Host Disease (GvHD) by recognition of minor histocompatibility antigens (MiHA). Development of T-cell therapies to selectively stimulate GvL without GvHD requires identification of multiple hematopoietic-restricted MiHA. We isolated 170 MiHA-specific CD8+ T-cell clones from two patients who entered complete remission and suffered from GvHD following DLI after HLA-matched alloSCT. All T-cell clones were analyzed for reactivity against a peptide mixture of known MiHA. T-cell clones specific for LB-GEMIN4-1V, ZAPHIR, LB-ADIR-IF and LRH-1 were isolated, of which LRH-1 has therapeutic relevance based on hematopoietic-restricted expression. In addition, all MiHA specific T-cell clones were tested against patient derived fibroblasts (FB). The majority of T-cell clones showed reactivity against FB after pre-treatment with IFN-γ. Finally, we identified 7 novel MiHA by WGAs, of which 6 MiHA are ubiquitously expressed as demonstrated by microarray gene expression analysis (CLYBL, PFAS, GLE1, PNO1, YIPF1, FBXO7). The remaining T-cell clone recognized a novel MiHA encoded by the hematopoietic-restricted ITGB2 gene, and strongly recognized primary leukemic cells, but lacked reactivity towards FB, illustrating its therapeutic relevance. In conclusion, we isolated T-cells for 4 known and identified 7 novel MiHA by WGAs in two patients with combined GvL and GvHD. One novel MiHA is hematopoietic-restricted and has potential therapeutic relevance. Our data show that WGAs followed by microarray gene expression analysis allows rapid identification of therapeutic MiHA.
Genetic correction of induced pluripotent stem cells from a Wiskott-Aldrich Syndrome patient normalizes the immune defects
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Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease. It is caused by mutations in the WAS gene (WAS) which encodes for the WAS protein (WASp), exclusively expressed in hematopoietic cells and required for proper lymphoid cell function. We investigated restoration of T- and NK-cell functionality following a zinc-finger nuclease (ZFN)- mediated genome editing strategy for correction of WAS. We generated induced pluripotent stem cells (iPSC) from skin fibroblasts of a WAS patient carrying an insertional frame-shift mutation. Subsequently, a WAS-2A-eGFP transgene was targeted at the endogenous chromosomal location by homology-directed repair using ZFN, thereby correcting the gene defect and creating a GFP reporter for WASp expression. Hematopoietic progenitor cells were generated from WAS iPSC and gene-corrected iPSC (cWAS). GFP expression was pronounced in all CD43+ hematopoietic lineages. Hematopoietic precursors were cultured on OP9-DL1 to generate T and NK cells. NK cells were readily obtained from cWAS and hESC (WA01/WA09) progenitors, but to a far more limited extent from WAS progenitors. WAS-derived NK cells were unable to generate IFN-g or TNF-a upon stimulation with K562. Cytokine production was restored in cWAS- derived NK cells. Interestingly, in T cell generation, although WAS, cWAS, and WA01 lines were able to generate CD5+CD7+ T cell precursors, only low numbers of CD3+TCR-a-b and TCR-g-d cells were obtained with WAS. Significantly, T cell generation was restored in cWAS. Taken together these results indicate that targeted correction of the WAS gene results in restoration of the lymphoid developmental and functional defects observed for WAS-iPSC.
Ex vivo Gene Therapy in a Mouse Model of Leukocyte Adhesion Deficiency Type I
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Leukocyte Adhesion Deficiency Type I (LAD-I) is a primary immunodeficiency characterized by recurrent and life-threatening bacterial infections. It is caused by mutations in the ITGB2 gene, encoding the integrin ß2 common subunit (CD18). These mutations lead generally to defective or absent expression of ß2 integrins in the leukocytes surface, implying that leukocytes are unable to adhere to the endothelium and extravasate to infection sites. As a mouse model for the ex vivo gene therapy of LAD-I we have selected CD18HYP mice, which resemble LAD-I moderate phenotype with reduced expression of ß2 integrins. All four hCD18-LVs that expressed human CD18 from ubiquitous (PGK and UCOE) or myeloid (Chim and MIM) promoters succeed to express hCD18 in lin- cells from CD18HYP mice. After transplantation of hCD18-LV-transduced lin- cells into lethally irradiated CD18HYP, hCD18+ cells were detected both in PB and BM. hCD18+ cells expressed higher levels of mCD11a, indicating that hCD18 is able to heterodimerize with mCD11 subunits and increase the expression of ß2 integrins. After secondary transplantation, hCD18+ cells could be still detected even at the BM LSK subpopulation, and typical LAD-I leukocytosis was corrected. Transplanted animals were evaluated for in vivo neutrophil migration, showing that corrected hCD18+ neutrophils extravasate in response to inflammatory stimulus as WT neutrophils. All these results allow us to propose new CD18-LVs that could be good candidates for future clinical applications in LAD-I patients.
Finding levels of RAG1 expression that will correct RAG1 Severe Combined Immunodeficiency
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RAG1-SCID patients lack B- and T lymphocytes due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment alternative for RAG1-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy for RAG1 has proven challenging. We have previously shown with an SFFV-driven vector that sufficient RAG1 expression could be achieved for correction at clinically acceptable vector copy numbers (VCN). In our current study, we tested clinically relevant lentiviral SIN vectors with different internal elements; UCOE, PGK, MND, and UCOE-MND in tandem to deliver human RAG1 sequences. We used Rag1-/- mice as a preclinical model for RAG-SCID to assess the strength of the various vectors at low to very low VCNs (0.01–0.2). We observed that peripheral B and T cell reconstitution levels directly correlated to the Rag1 expression levels in the transplant. And although most treated mice reconstituted their peripheral T cell compartment, in animals that received a transplant with low RAG1 expression this effect was merely transient. This was confirmed by the data from the thymus; only mice that received a transplant with high RAG1 expression, showed a subset composition comparable to mice receiving WT cells, including a high percentage of double positive cells, assuring a continuous supply of T cells. Mice receiving a transplant with low RAG1 expression, were mostly lacking the double positive population. We conclude that functional restoration of RAG1-deficiency at low VCN can be achieved with clinically acceptable vectors.
Transplantation of human SCID stem cells in NSG mice gives new insights into human T-cell development and reveals where SCID mutations act
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Severe combined immunodeficiency (SCID) is characterized by a deficiency of T cells, which can be accompanied by deficiencies in B cells and/or NK cells depending on the genetic defect. Until recently, it was not possible to study the effect of the genetic defect on the T-cell developmental arrest, because thymus biopsies are difficult to obtain from SCID patients. In this study, we have characterized the blocks in T-cell development for different types of SCID, caused by mutations in ADA, Artemis, IL7RA and IL2RG by transplanting CD34+ hematopoietic stem cells (HSC) from bone marrow into the NSG xenograft model. We observed blocks in T-cell development for IL7RA- and IL2RG-SCID at the CD4-CD8- DN stage, which is much earlier than anticipated based on data from knockout mice and human gene expression studies. Furthermore, it appears that TCRß rearrangements are initiated much earlier than previously thought, as determined from the developmental block and the extent of rearrangement observed in mice transplanted with Artemis-SCID HSC. In contrast, the block in B-cell development in the xenograft model was identical to the block that was present in the bone marrow of the Artemis-SCID patient. These data provide previously unattainable insight into human T-cell development using SCID as human loss-of-function models. Finally, this shows that the NSG xenotransplantation model is a valuable tool to study human T- and B cell development and function, which might even have diagnostic implications for yet undiagnosed SCID patients.
New model for Ubiquilin2-linked ALS: investigation of pathological mechanisms and therapeutic perspectives
Ubiquilin2 is a protein involved in degradation pathways via the ubiquitin proteasome system and autophagy. Mutations in Ubiquilin2 have been identified in inherited forms of amyotrophic lateral sclerosis (ALS) and represent the first reported mutations in a protein that is directly linked to the protein degradation pathways. Ubiquilin2-positive inclusions have been observed in the spinal cord and brain of both familial and sporadic ALS patients, but the pathogenetic role of this protein is still unclear. Disease modelling in animals is essential for physiopathological investigations and identification of new therapeutic targets. The objective of our study was to develop a mouse model of Ubiquilin2-related-ALS using AAV to overexpress either the wild-type (AAV-Ubiwt) or mutant (AAV-UbiPro497His) human Ubiquilin2. After intracerebroventricular injection of the vectors in naïve FVB mice, western blot and immunofluorescence analyses revealed high levels of human Ubiquilin2 in the brain and the spinal cord of both AAV-Ubiwt and AAV-UbiPro497His injected animals. Ubiquilin2-positive inclusions were identified in AAV-UbiPro497His injected animals, similarly to ALS patients. The injected animals displayed a reduced brain size compared to controls, a severe astrogliosis in both the spinal cord and the brain, and a reduced number of ChAT+ motor neurons in the whole spinal cord. Ubiquilin2-injected mice had a shortened life span and a body weight loss phenotype; they developed muscle weakness, with loss of muscle mass and strength. This innovative and successful application of AAV for ALS modelling will be useful to further dissect the molecular mechanisms of this complex pathology and to envision therapeutic strategies.
High level expression of human iduronidase throughout the brain in a murine model of mucopolysaccharidosis type I (MPS I) after non-invasive AAV-mediated gene delivery to the CNS
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Mucopolysaccharidosis Type I (MPS I) is an inherited metabolic disease, caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). Individuals with the most severe form of the disease (Hurler syndrome) suffer from systemic illness including neurodegeneration, mental retardation, and early death. Current treatments for MPS I include hematopoietic cell transplantation and enzyme replacement therapy. However, these treatments do not effectively treat all central nervous system (CNS) manifestations of the disease. To investigate novel approaches to treat neuronopathic manifestations of severe MPS I, AAV9 encoding IDUA was infused intrathecally (IT), intracerebroventricularly (ICV), or administered intranasally to adult IDUA-deficient mice. Some groups were either immunosuppressed with cyclophosphamide or immunotolerized with commercially available IDUA to modulate anti-IDUA immune responses. Animals were sacrificed at 8–11 weeks after AAV9-IDUA administration, evaluating all areas of the brain for IDUA enzyme activity, tissue glycosaminoglycans (GAG), and IDUA gene copies. IT and ICV injected animals had elevated levels of IDUA ranging from 10- to 1000-fold that of wild type in all brain areas. Levels were highest in the cerebellum in IT-injected mice, with high levels broadly distributed throughout the brain in ICV-injected mice. In animals treated intranasally, wild-type levels of IDUA were restored in all parts of the brain, with higher levels observed in the olfactory bulb (100-fold). Tissue GAG storage was reduced to normal levels in all treatment groups. Results from these studies provide evidence for biochemical correction and thus effective treatment of MPS I using clinically applicable methods of delivering AAV9-IDUA to the CNS.
OXB-102: An Enhanced Gene Therapy for Parkinson's Disease
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Oral dopaminergic treatments are the primary standard of care for Parkinson's disease (PD); although these are highly efficacious early on, over time they lead to debilitating long term side effects that seriously impact on the quality of life and restrict the longevity of such treatments. OXB-102 is a lentiviral vector derived from the equine infectious anaemia virus (EIAV) that delivers the genes encoding the three key enzymes in the dopamine (DA) biosynthetic pathway, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and GTP-cyclohydrolase (CH1), to non-dopaminergic striatal neurons of the sensorimotor putamen, thus providing these cells with the ability to synthesise and release their own DA. The effectiveness of this strategy has already been demonstrated in rodents, non-human primates and Parkinson's (PD) patients (Palfi et al, Lancet 2014) with a precursor gene therapy vector called ProSavin®. OXB-102 is an improved version of ProSavin® that expresses the same enzymes but with an increased DA production per integrated genome. In non-clinical studies the efficacy of a full-strength (FD) and a 1/5th (LD) dose of OXB-102 has been compared to the efficacy of full-strength ProSavin® in the ‘gold standard’ MPTP NHP model of PD; a vector expressing the LacZ reporter gene (LacZ) was used as a negative control. The longitudinal follow-up consisted of recording Clinical Rating Scores (CRS) and video-based quantification of locomotor activity before and after vector injection. The full-strength and the 1/5th dose of OXB-102 were as efficacious as the full-strength dose of ProSavin®, whereas the control LacZ treated macaques still maintained a significant PD phenotype. Positron emission tomography (PET) was also carried out at baseline and at 3 and 6 months following vector administration using 18F-FMT, a presynaptic biomarker that acts as a substrate of AADC. There was a significant increase in the FMT signal of both of the OXB-102 treatment groups that was greater than in the ProSavin® treated animals whilst there was no significant change in the FMT signal in the control treatment group. These results indicate that OXB-102 is more potent than ProSavin® and supports the clinical development OXB-102.
AAV-mediated delivery of SMN1 in a mouse model of spinal muscular atrophy
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Spinal muscular atrophy (SMA) is a neuromuscular autosomal recessive disease that represents the most common genetic cause of infant death. SMA is caused by homozygous loss of the telomeric copy of the Survival of Motor-Neuron gene (SMN1), with retention of the homologous centromeric SMN2. Deficiencies in the ubiquitous SMN function affect multiple tissues and organs, however the neural tissue is primarily affected, resulting in a-motor neuron degeneration in the spinal cord and proximal muscles weakness. Gene replacement therapy with rAAV vectors has shown therapeutic efficacy in the SMNΔ7 severe mouse model of SMA. We report a study aimed at optimizing design, dose and route of administration of rAAV vectors encoding a codon-optimized hSMN1 cDNA under the control of a constitutive PGK promoter. Single-stranded (ss) or self-complementary (sc) AAV9 vectors were produced by triple plasmid transfection in 293 cells and injected in the intracerebroventricular space (ICV) alone or in combination with intravenous (IV) administration (ICV+IV) in a dose-escalating mode in neonatal SMNΔ7 mice. Vector biodistribution and SMN protein expression in the CNS and peripheral tissues were analyzed 90 days post-injection in all experimental conditions. Mice treated with the scAAV9 vector showed a significant, dose-dependent rescue of lifespan, growth and motile functions, while the ssAAV9 vector failed to provide a therapeutic benefit. This study confirms the potential of scAAV-SMN delivery for gene therapy of SMA.
Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter
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Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2 kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter and some enhancers of UBC, as well as the first exon, first intron, and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, experiments were carried out to investigate whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. Expression analysis revealed that this intron loss led to a two-fold reduction in transgene expression levels. Movement of the entire expression cassette to the opposite strand prevented intron loss, thus restoring full expression from vector forms. This increase in expression was mostly due to context-dependent enhancer activity within the intron, as movement of intronic enhancer sequences to multiple promoter-proximal sites did not result in equivalent expression when compared to the naturally occurring form of the promoter. Reversal of the expression cassette prevented intron loss and restored full expression in uni- and bidirectional lentiviral vectors.
Development of F/HN pseudotyped Lentivirus for airway gene transfer
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We are developing a simian immunodeficiency virus (SIV) pseudotyped with F/HN proteins from Sendai virus (rSIV.F/HN) for efficient airway cell transduction for cystic fibrosis (CF). In mice, a single dose of rSIV.F/HN produces reporter gene expression for 2 yrs and three monthly lung administrations is feasible without loss of efficacy. We constructed third-generation rSIV.F/HN vectors for clinical use and evaluated a range of enhancer/promoters configurations in airway models. High-level, stable reporter expression was observed in vivo (1–7 months) with the most robust gene expression directed by the hCEF promoter (Hyde 2008; PMID:18438402). In the mouse lung rSIV.F/HN-hCEF.EGFP directed expression in 14% of lung epithelial cells on day 14 (P<0.0001 versus controls). Insertion site profiling indicated integration patterns similar to other lentiviral vectors in clinical development. Importantly, when rSIV.F/HN stability was tested in clinically relevant bronchoscope and aerosol delivery devices, there was no significant loss of transduction (P=0.64). Delivery of rSIV.F/HN expressing CFTR to the lung lobes of sheep via bronchoscopic instillation resulted in vector-derived CFTR mRNA at approximately 30% of levels of endogenous ovine CFTR (P<0.0001 versus non-treated lobes; day 7). To catalyse the development of large-scale cGMP-compatible virus production, we developed scalable, serum-free, suspension cell culture in rocking bioreactors, producing median purified vector titres >2e9TU/mL. The process is animal-free, utilises cGMP-compliant virus capture and purification reagents and is approved by the UK MHRA for regulatory studies. Our future plans include large-scale manufacturing of vector for toxicology studies and first-in-man CF clinical trials.
BET-independent MLV-based Vectors Target Away from Promoters and Regulatory Elements
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Stable integration of the viral DNA in the host cell genome makes retrovirus-derived vector particles attractive tools for gene therapy. Retroviral integration is not random; whereas lentiviruses prefer integration in active transcription units, gammaretroviruses, like the Moloney Murine Leukemia Virus (MLV) favour strong enhancers, transcription start sites (TSS) and CpG islands. Adverse events in clinical trials employing MLV-based vectors occurred, in which vector integration activated proto-oncogenes, leading to clonal expansion and leukemogenesis. This integration bias is driven by cellular proteins that are co-opted by the viral integrase (IN). We unveiled the bromodomain and extra-terminal (BET) family proteins (BRD2, -3, -4) as cellular binding partners of MLV integrase (IN) that direct MLV integration via interacting with the unstructured C-terminal tail. We engineered third generation gammaretroviral vectors that are no longer tethered by BET-proteins, yet integrate with wild-type efficiency, by introducing a single mutation W390A or deleting the MLV IN C-terminal tail. MLV integration detargeted from RefSeq and oncogene TSS, CpG islands and DNaseI-hypersensitive sites, thereby reducing the risk of insertional mutagenesis. In addition, we used synthetic peptides to engineer MLV IN-fusions that support efficient integration with a more random distribution pattern. These results suggest that third generation MLV vectors have superior properties with respect to the current MLV-derived vectors used in clinical trials and open perspectives for engineered retroviral vectors with a safer integration site profile. We are currently evaluating the genotoxic profile of these viral vectors.
Measles virus glycoprotein pseudotyped lentiviral vectors transduce HSC SCID repopulation cells at efficiencies reaching up to 100%
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Hematopoietic stem cell (HSC) based gene therapy holds promise for the cure of many inherited and acquired diseases. The field is now moving towards the use of lentiviral vectors (LVs) evidenced by recent clinical trials. In these trials, VSV-G-LVs were applied at high doses in combination with strong cytokine-cocktails, which might compromise the 'HSC' character of the cells. Thus, there is room for improvement. Previously, we have shown that measles virus glycoprotein displaying LVs (MV-LVs) were able to transduce efficiently resting T- and B-cells. Now, we evaluated these MV-LVs for hCD34+-cell transduction under mild cytokine prestimulation (TPO, SCF or SCF+TPO) in order to better preserve the 'HSC' characteristics. After a single application, MV-LVs transduced 100% of TPO+SCF stimulated hCD34+-cells. Even more striking was that these MV-LVs allowed efficient transduction of up to 70% of quiescent hCD34+-cells, while VSV-G-LVs only reached 5% transduction. Importantly, reconstitution of primary and secondary recipient NSG mice with MV-LV transduced pre-stimulated or resting hCD34+-cells demonstrated that this high transduction levels in all analyzed myeloid and lymphoid engrafted lineages in all hematopoietic tissues. We now confirmed that only the CD46 receptor is responsible for MV-LV entry into hCD34+-cells. Interestingly, a different integration pattern for MV-LVs was revealed in resting and cytokine-pre-stimulated hCD34+-cells. Together. these MV-LVs paves the way to HSC-based gene therapy of multiple diseases including diseases characterized by low numbers of HSCs such as Fanconi Anemia, for which high level HSC correction is needed to be successful in the clinic.
Development of the manufacturing process for the ex vivo gene therapy for ADA-SCID (GSK2696273): process design, validation and comparability
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GSK2696273 is a novel medicinal product which contains genetically modified cells. It has been developed for the treatment of severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. GSK2696273 contains autologous CD34+ cells transduced ex vivo with a replication deficient retroviral vector containing the human adenosine deaminase (ADA) cDNA sequence. The product has been administered to 18 paediatric patients, aged approximately 6 months to 6 years, 12 of which were enrolled in the pivotal clinical study. With the exception of the very first patient, all vector, drug substance and drug product has been manufactured at MolMed, Milan, and all patients have been treated at Hospital San Raffaele, Milan. A summary from a systematic review of the data from the manufacturing campaigns of vector produced to date and the 12 batches of drug product produced for the pivotal clinical study, as well as links between the manufacturing process, batch properties and clinical safety and efficacy will be presented. In addition, efforts to further develop the manufacturing process to ensure security of supply and delivery of a robust, safe and efficacious product whilst minimising changes to reduce the comparability risk will be presented. More specifically, process development, validation and comparability data will be presented.
GLP preclinical studies for gene therapy medicinal products combine highest regulatory standards with outstanding scientific significance
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Gene therapy clinical trials using hematopoietic stem cells have shown therapeutic efficacy for multiple diseases. Despite promising results in patients, few have reached more advanced regulatory milestones. In the EU, Advanced therapy medicinal products including gene therapy (GTMP), go through a centralized marketing authorization procedure. However, due to the novelty and complexity of these products, the evaluation process and final opinion have been controversial. Particularly, the quality of preclinical and clinical studies was not considered to meet regulatory expectations. The fact that GTMP trials are often sponsored by academia, charities and small companies with limited financial resources or regulatory experience further complicates this process. As this therapeutic strategy is applied to an increasing number of monogenic diseases, rigorous studies in appropriate non-clinical models are needed to fulfill the requirements for future market registration. We have recently certified the first academic GLP Test Facility within the San Raffaele Telethon Institute for Gene Therapy. This Test Facility aims at performing validation, biodistribution and tumorigenicity/toxicity studies to support the regulatory acceptance process for GTMPs. Fit-for-purpose study designs for GTMP safety assessment were developed and adopted to GLP conditions. Several parameters of tumorigenicity/toxicity or biodistribution studies such as appropriate animal models, dosing, study duration and analyses at termination were evaluated on a science-based approach. We have successfully completed several preclinical safety studies for gene therapy of immunodeficiencies, metabolic and blood disorders. The obtained information supports the discussion with regulatory bodies, allows efficient early collection of proof-of-concept data and minimizes the use of animals (3Rs).
Automated Magnetic Isolation and Lentiviral Vector Modification of Human CD34+ Cells in a Functionally Closed System
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Hereditary monogenic genetic disorders can effectively be treated by overexpression of the wild-type gene product via viral modification of isolated CD34+ progenitor cells. However, current manufacturing processes are performed manually or at best by using semi-automated procedures. If cellular and gene therapies are to move from their current translational setting into routine clinical use, a standardized production of cellular therapeutic agents and their genetic modification is required. A closed and highly automated manufacturing procedure would lead to large improvements in product performance, product safety and operator safety. We have recently developed a functionally closed and fully automated cell processing device, the CliniMACS Prodigy®, which enables complex cellular products to be manufactured. In this functionally closed system, the following steps have been automated: concentration and washing of blood products, magnetic labeling and enrichment of CD34+ cells, cultivation and pre-activation of the enriched CD34+ cells, transduction with lentiviral vectors and resuspension of the final cell product in buffer for infusion. Automated cell preparation, cell labeling and magnetic CD34+ cell separation from mobilized apheresis samples achieved purities of over 90%. A newly developed automated transduction process with a single transduction cycle resulted in transduction efficiencies of up to 90% even with low numbers of target cells. These preliminary data demonstrate that automated cell processing and genetic manipulation of haematopoetic progenitor cells can be performed in a closed system. Work in progress aims to further optimize this procedure and improve the efficiency of CD34+ cell transduction to the levels achieved by current manual protocols.
High yield adenovirus production in fixed-bed bioreactor
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The production of viral based gene therapy vectors or vaccines often utilizes anchor-dependent cells. In small scale, viruses can easily be produced in cell culture flasks. However, controlled and cost effective production of viruses requires disposable bioreactors. Novel iCELLis fixed-bed bioreactor, composed from medical-grade polyester (PET) microfiber carriers, is an option for upscaling adherent viral production system from laboratory into commercial scale. The aim of this study was to evaluate iCELLis Nano for adenovirus type 5 production and optimize process parameters. 3D PET carriers enabled HEK293 cell attachment and logarithmic growth. Dissolved oxygen, pH, and temperature were controlled during the process and cell growth was monitored by nuclei count and can be measured using capacitance by biomassprobe. Glucose and lactate values were monitored and controlled by re-circulation or perfusion of cell growth medium. During the harvest, cells were chemically lysed inside the bioreactor. Viral particle productivity was high, reaching values up to 100 000 vp/cell (HPLC titer). However, in 10 cm high compaction bed height it was noticed that if the cells were unevenly distributed into carriers, productivity could be decreased most probably due to changes in cell confluency and MOI. Downstream purification of the product was efficient with good recovery and viral purity. As a conclusion, iCELLis Nano proved to be efficient for adenovirus production. Next goal is to upscale the process from Nano scale (0.8–4 m2) into large 100–500 m2 iCELLis.
Phase 1–2 clinical trial in patients with decompensated liver cirrhosis treated with bone-marrow derived endotelial progenitor cells: preliminary safety and efficacy analysis
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Advanced tuning of Notch signaling to regulate in vivo myogenic repair of murine and human mesoangioblasts
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Somatic stem cells hold attractive potential for the treatment of muscular dystrophies (MDs). Mesoangioblasts (MABs) are alkaline phosphatase+ muscle-derived pericytes and have been shown to efficiently regenerate dystrophic muscles in mice and dogs. In addition, HLA-matched MABs are currently being tested in a phase 1 clinical study on Duchenne MD patients (EudraCT #2011-000176-33). The Notch pathway regulates muscle regeneration and satellite cell commitment. However, little is known about Notch-mediated effects on other resident myogenic cells. We therefore asked whether Notch signaling played a pivotal role in regulating MAB myogenic capacity. Through different approaches of loss- and gain-of-function in murine and human MABs, we determined that the interplay between Delta-like ligand 1 (Dll1)-activated Notch1, Maml1 and Mef2C supports MAB commitment in vitro. Also, adenoviral-based priming of homing MABs with Dll1 and Mef2C ameliorates engraftment and functional outcome after intra-arterial delivery in dystrophic mice. Furthermore, using a transgenic mouse model of conditional Dll1 deletion, we demonstrated that Dll1 ablation, either on the injected cells, or on the receiving muscle fibers, impairs MAB regenerative potential. Our data corroborate the perspective of advanced combinations of cell therapy and signaling tuning to enhance therapeutic efficaciousness of somatic stem cells.
Antigen-specific myeloid-derived suppressor cells ameliorate experimental autoimmune encephalomyelitis
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We previously reported that the transfer of bone marrow cells (BMCs) transduced with MOG40-55 autoantigen improved murine experimental autoimmune encephalomyelitis (EAE). We also showed that the majority of cells generated in retroviral transduction cultures of BMCs consist of transgene-expressing myeloid-derived suppressor cells (MDSCs). The present work aimed to characterize these MDSCs and to investigate their contribution to the therapeutic effect observed. To this end, we transduced BMCs with either the retroviral vector encoding the autoantigen (Ii-MOG) or a control vector (Ii). A single infusion of transduced total BMCs or purified MDSCs was administered seven days before (preventive arm) or 13 days after (therapeutic arm) EAE induction. Mice were daily weighted and assessed for clinical signs using a 6-point scale. In the preventive approach, the infusion of Ii-MOG-BMCs and -MDSCs but not their controls ameliorated the disease, indicating that antigen-specific MDSCs likely contribute to the therapeutic effect. Furthermore, the spleens of Ii-MOG-MDSC treated mice contained less activated T cells (CD3+CD4+CD25+FoxP3-) and more B regulatory cells (CD45+B220+CD5+CD1d+) than controls. Therapeutic infusion of Ii-MOG-BMCs was associated with a significant improvement of maximum and accumulated clinical scores, and with lower percentage of activated splenic T cells in comparison with controls. Mice treated with BMCs or MDSCs expressing Ii-MOG showed milder inflammatory infiltration and demyelination and a significant reduction in CD3+ cells in the spinal cord in comparison with controls. Immunological and pathological studies are currently ongoing. In conclusion, retroviral transduction of murine hematopoietic cells generates antigen-specific MDSCs that have a therapeutic effect in EAE.
Modulation of the ATP: adenosine balance by AAV-5 mediated gene delivery of CD39-CD73 in fibroblast-like synoviocytes from rheumatoid arthritis patients
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The conversion of extracellular ATP to adenosine is an important mechanism of immune suppression by Tregs, and this is done by expression of ENTPD1 (CD39) and 5NTE1 (CD73). CD39 converts pro-inflammatory extracellular ATP to ADP and AMP, whereas CD73 converts AMP to anti-inflammatory adenosine. We have found evidence suggesting that the ATP/adenosine balance is skewed in the synovial compartment of rheumatoid arthritis (RA) patients. Therefore, we aim to ameliorate this balance by AAV mediated expression of CD39 and CD73. To test this approach in vitro, primary fibroblast-like synoviocytes (FLS) isolated from synovial biopsies of RA patients, were transduced with an AAV5 vector expressing CD39 and CD73. LPS activated THP-1 cells (monocyte cell line) were added in the presence of ATP and after an overnight incubation cytokines were measured in the supernatant. Production of IL-1ß, TNF and CCL2 was significantly inhibited (68–88%) when FLS expressed CD39 and CD73. We further investigated adenosine receptor (ADORA) expression and effects of adenosine on FLS and primary monocytes. Both cell types showed a distinct ADORA expression profile, with ADORA1/2B predominant on FLS, and ADORA3/2B predominant on monocytes. Interestingly, both cell types showed a striking upregulation of ADORA2A, but not of ADORA-1, -2B or -3, after stimulation with LPS or TNF/IL-1ß. When adenosine was added to stimulated primary monocytes and FLS, cytokine production was significantly reduced. Concluding, these data show that the modulation of ATP:adenosine levels by CD39 and CD73 may be an effective approach for the treatment of inflammatory disease such as RA.
Modulation of mouse models of rheumatoid arthritis by systemic delivery of immunoregulatory bovine milk derived exosomes
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Exosomes contain immunoregulatory miRNAs, mRNA and proteins. In this study we use bovine milk-derived exosomes (BMEs) for treatment of collagen-induced arthritis (CIA) model and the spontaneous polyarthritis model in IL1Ra-/- mice. BMEs were isolated from commercial milk by ultracentrifugation and their size was ±120 nm as characterized by Nanosight and electron microscopy. BME express the exosome specific marker CD63, miR-let-7a and miR-124a, and milk specific beta-casein mRNA. Uptake of PKH-67 labeled BMEs by RAW264.7 macrophages and murine splenic adherent cells was visualized by confocal microscopy. In RAW cells, BMEs significantly increased (+65%) LPS-induced IL-6 production, while monocyte chemotactic protein (MCP)-1 (a confirmed miR-124a target) was significantly reduced (-11%). Exposure of lymphocytes to BMEs facilitate T cell differentiation towards the pathogenic Th17 lineage in vitro, as determined by increased expression of ROR-γT and IL-17 mRNA. Mice receiving two i.v. BME injections, one day before immunization and at booster injection (day 21), showed a delayed onset of CIA but later on disease markedly aggravated. Delivery of BMEs to IL1Ra-/- mice by daily oral gavages between 5 and 15 weeks of age also showed an initial delay followed by more severe disease thereafter. In the CIA model, oral delivery of BMEs also delayed onset of disease and in these mice BME-treatment reduced splenic expression of T-helper cell specific transcription factors T-bet (Th1) and RORgammaT (Th17), and of Th2 related genes MCP-1 and IL-10. In this study we showed that BMEs are immunoregulatory and can be used for exosome-mediated (gene) therapy in arthritis.
Immune checkpoint blockade enhances oncolytic Measles virus therapy
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We hypothesized that combining oncolytic Measles virus (MV) with immune checkpoint blockade using antibodies against CTLA-4 and PD-L1 can provide synergistic anti-tumor effects. Targeted immunomodulatory MV vectors encoding anti-CTLA-4 and anti-PD-L1 antibodies were cloned and characterized in vitro. Oncolytic efficacy was addressed in human melanoma xenografts. Immunotherapeutic efficacy was evaluated in a fully immunocompetent murine model of subcutaneous syngeneic malignant melanoma (B16-CD20) and compared to parental MV with or without systemic administration of anti-CTLA-4 and anti-PD-L1. Viral replication and oncolysis of MV anti-CTLA-4 and MV anti-PD-L1 were not impaired. In the B16-CD20 model, treatment with MV anti-CTLA-4 and MV anti-PD-L1 led to an increase in tumor-infiltrating cytotoxic T cells as well as a decrease in regulatory T cells. Therapeutic benefits in terms of delayed tumor growth and prolonged median overall survival were observed for animals treated with MV anti-CTLA-4 and MV anti-PD-L1, respectively. Further investigations in preparation of a Phase I trial are currently ongoing. Combining oncolytic MV with immune checkpoint modulation is a promising approach with direct implications for future clinical application.
Decapitated reoviruses with a little iLOV
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To create armed oncolytic reoviruses it is mandatory to identify a suitable location for insertion of a transgene in the reovirus' segmented dsRNA genome. We explored a strategy based on our jin-3 mutant reovirus. This mutant can infect cells independent of the reovirus receptor JAM-A. Jin-3 harbors one mutation in the S1 segment resulting in a G196R substitution in the tail of the spike protein s1. The use of the S1 segment of jin-3 allows replacement of the codons for the JAM-A-binding head domain without compromising its capacity to infect cells. This provides a location for inserting up to 648 nt of foreign sequences without exceeding the size of the wild-type S1 segment. To prevent inclusion of the foreign protein into the capsid we inserted the codons for the porcine teschovirus-1 2A element between those encoding the tail of the spike and the codons for the small fluorescent protein iLOV. Modified reoviruses were generated by co-transfection of expression plasmids for all reovirus segments. This yielded replication-competent reoviruses containing the S1His-2A-iLOV segment. Density-gradient centrifugation confirmed that the s1-tail was associated with the virus capsid. Exposure of both JAM-A positive and JAM-A negative cells to the rS1His-2A-iLOV reoviruses yielded iLOV-expression in both cell types, confirming retention of the jin-3-derived expanded-tropism phenotype. Our data demonstrate that the S1 head-domain is a suitable location for inserting heterologous transgenes. Taken together this strategy paves the way for generating decapitated and armed replication-competent reoviruses carrying a small heterologous transgene.
Insertion of an albumin-binding domain in adenovirus hexon improves the pharmacokinetics and antitumor efficacy of oncolytic adenoviruses
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Recombinant adenoviruses have applications as vaccines, gene therapy vectors, and oncolytic viruses. When multiple tissues or tumor metastases need to be efficiently transduced with these recombinant adenoviruses, the administration of the virus in the bloodstream is preferred compared to the direct intratissular or intratumoral administration. However, adenoviruses interact with blood cells and proteins that contribute to clear the virus from the bloodstream, limiting systemic efficacy. Albumin, the most abundant serum protein, has been bound to proteins to extend their half live in blood. Here we have inserted an albumin-binding domain (ABD) in the outer surface of the main adenovirus capsid protein, the hexon, of oncolytic adenovirus ICOVIR15 to promote albumin binding. The resulting virus, ICOVIR15-ABD, shows positive binding for both human and mouse albumin by ELISA. Binding to human albumin was confirmed by immuno-electron microscopy. This albumin binding is able to shield the virus against neutralizing antibodies in vitro, maintaining the infectivity of the virus in their presence. In vivo, this ABD modification extends the virus half live in blood and improves its antitumor efficacy compared to a non-modified adenovirus.
Immunotherapy against pancreatic cancer using sequential administration of antigenically distinct oncolytic viruses expressing oncostatin M
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Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard protocols do not take full advantage of OV as cancer vaccines. Repeated viral administrations may polarize immune responses against strong viral antigens. In addition, the rapid onset of neutralizing antibodies limits the efficacy of tumor transduction as early as one week after the first viral dose. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed. In the present work we have evaluated a protocol consisting on sequential intratumoral administrations of Adenovirus (Ad) and Newcastle Disease Virus (NDV)-based OV encoding the cytokine oncostatin M (OSM). Using an aggressive orthotopic pancreatic cancer model in Syrian hamsters, we found that Ad-OSM or NDV-OSM caused only a marginal antitumor effect. In addition, strong secretion of OSM in serum limited the maximal tolerated dose of Ad-OSM. In contrast, moderate doses of Ad-OSM followed one week later by NDV-OSM were safe and showed significant antitumor effect. Similar efficacy was observed when the order of virus administrations was reversed. Investigation of the impact on tumor microenvironment and immune response against cancer cells is underway. We conclude that sequential administration of oncolytic Ad and NDV encoding OSM is a promising approach against pancreatic cancer.
An innovative CAR-T cell spacer allowing selection/tracking and enabling superior antitumor effects in vivo
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Chimeric antigen receptors (CARs) frequently include CH2CH3 spacers conferring the adequate flexibility for target antigen engagement. A serious drawback of CH2CH3-spaced CARs is however their interaction with Fc receptors (FcRs), leading to non-specific recognition of and by phagocytes. To circumvent this problem, we substituted the CH2CH3 spacer with extracellular domains from the low-affinity nerve-growth-factor receptor (LNGFR) having different lengths. As a model, we used our recently developed CD44v6-specific CAR, which is active against acute myeloid leukemia and multiple myeloma (Casucci et al, Blood 2013). LNGFR-based spacers allowed selecting CAR-T cells with immunomagnetic beads coupled to anti-NGFR mAbs, without interfering with their in vitro expansion and preserving their TSCM/TCM functional phenotype, imprinted by CD3/CD28-bead stimulation and IL-7/IL-15. Most importantly, LNGFR-spaced CAR-T cells maintained potent cytotoxic, proliferative and cytokine-release abilities in response to CD44v6-expressing tumor cells in vitro, while losing CD44v6-independent recognition mediated by FcRs. Noticeably, soluble NGF did not activate LNGFR-spaced CAR-T cells, ruling out potential toxicities from the physiological LNGFR ligand. In vivo, LNGFR-spaced CAR-T cells outperformed CH2CH3-spaced CAR-T cells in terms of expansion and persistence, mediating significantly superior antitumor effects in different xenograft mouse models. Interestingly, the premature disappearance of CH2CH3-spaced CAR-T cells was due to their engulfment by mouse phagocytes, as showed by experiments with clodronate. In conclusion, we demonstrated that directly incorporating NGFR sequences in the CAR allows for a single molecule to function both as a therapeutic and a selection/tracking gene, resulting in an optimal toxicity/efficacy profile.
TAP-independent presentation of exogenous epitopes by oncolytic adenoviruses enhances specific immune responses and antitumor activity
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Defects in MHC-I antigen presentation pathway are frequently associated to immune evasion of tumor cells. Among defects at different levels, most common is the downregulation of transporter associated protein (TAP) that translocates peptides generated by the proteosome into the endoplasmatic reticulum (ER) to be loaded into MHC-I molecules. To solve this problem, immunogenic peptides have been connected to ER-targeting signals to generate TAP-independent presentation. Our group and others are interested in using oncolytic adenoviruses to promote immune responses against tumors by expressing and/or displaying tumor antigens. However, the strong immunodominance of virus antigens mask responses against tumor epitopes. In this work, we propose the insertion of epitopes in adenovirus protein E3-19K after the ER-targeting signal that traffic this protein to the ER. We selected OVA257 model epitope and gp10025 tumor epitope, which were separated by linkers cleavable by the ER protease furin. The TAP-indepency of such ER-targeted epitopes may enhance their immunogenicity in TAP-defective tumor cells. In vitro, OVA257 was presented by MHC-I in TAP-defective tumor cells infected with the virus containing the epitopes in E3-19K, but not in the fiber HI-loop. This presentation was unaffected by proteasome inhibition but abrogated when ER-targeting signal was removed and linkers replaced by non-relevant sequences. In vivo, this virus generated stronger specific immune responses and delayed tumor growth more efficiently than the virus displaying the epitopes in the HI-loop. We conclude that the ER-targeted adenovirus E3-19K protein can be used to promote exogenous epitope immunodominance in TAP-defective tumor cells.
Alpharetroviral vectors for the transduction of primary human natural killer cells: selective enhancement of tumor cytotoxic activity by chimeric antigen receptors
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With their unique capability of killing transformed cells without MHC restriction and without the need for prior sensitization, natural killer (NK) cells are an attractive cell type for tumor immunotherapy. However, tumor cells can escape NK cell killing activity by either upregulating inhibitory or downregulating activating receptor ligands. To overcome such tumor escape mechanisms, NK cells can be equipped with chimeric antigen receptor (CAR) expression cassettes, thereby enhancing their tumor-specific cytotoxicity. To determine the most efficacious method to transfer transgenes to human NK cells, we compared different transduction protocols, retroviral vector systems, and retroviral pseudotypes with one another. Using our optimized transduction conditions in primary human NK cells, we demonstrated that alpharetroviral vectors produced the highest transduction levels (>50%). In a CD107a degranulation assay, we showed that transduction of primary human NK cells with a control alpharetroviral vector did not impair degranulation activity and thus did not negatively affect NK cell cytotoxicity. Therefore, we designed an alpharetroviral self-inactivating vector with a CD19CAR expression cassette. After transduction, NK cells showed 20–80% selective enhancement of NK cell cytotoxicity towards CD19-positive leukemia cells in contrast to untransduced NK cells. In summary, we demonstrated that alpharetroviral vectors were most efficacious for transducing primary human NK cells, and that these modified NK cells retained their killing capacity. Furthermore, alpharetroviral transduction of NK cells with a CD19CAR expression cassette allowed for retargeting of NK cells towards CD19-positive leukemia cells. Taken together, alpharetroviral vectors are promising tools for NK cell-mediated cancer immunotherapy applications.
Targeted in vivo TCR gene transfer into mouse T cell subsets for immunotherapy of cancer
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The possibility to deliver genes in vivo to specific cells by targetable, injectable vectors would greatly improve gene therapy. Especially in adoptive T cell therapy (ATT) with T cell receptor (TCR) gene-modified T cells, ex vivo transduction protocols require antibody- and cytokine-mediated activation of T cells and thereby alter their phenotype. Therefore, targeted gene transfer into T cell subsets would be an alternative option to overcome these problems. We developed gamma-retroviral vectors, which allow targeted gene transfer into murine (m) CD8+ and CD4+ T cells, the most important T cell subsets for ATT. Vectors were pseudotyped with the measles virus (MV) envelope glycoproteins hemagglutinin (H) and fusion (F). Binding specificity was redirected by addition of gene sequences encoding CD4- or CD8-specific single-chain antibody fragments to a mutated H protein, unable to recognize its native receptor. Using this approach, we developed two MV envelopes targeting either murine CD8+ or CD4+ T cell subsets (MVm8, MVm4). The initially low virus titer was increased to 106– 107 IP/ml by modifications of the H- and F-protein. Vectors encoding marker genes that were pseudotyped with either MVm8 or MVm4 selectively infected CD8+ or CD4+ murine T cells. Important, both targeting vectors transduced specifically CD8+ and CD4+ murine T cells after i.v. injection into mice. At present, we are developing a protocol for the in vivo transduction of T cell subsets with tumor-reactive TCR genes to study tumor rejection.
Re-engineering of human CYP4B1 for optimal catalytic processing of 4-ipomeanol and use as a suicide gene in adoptive cell therapy
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The in vivo control of cellular therapies can be achieved by genetically modifying the infused cells to express a suicide gene that causes the cells to kill itself through apoptosis. We developed a novel human suicide gene by systematically optimizing the inactive human cytochrome P450 family 4, subfamily B, polypeptide 1 (CYP4B1) to efficiently process the pro-drug 4-Ipomeanol (4-IPO) into a highly toxic DNA alkylating agent and thereby inducing cell death. Using lentivirus-mediated expression in human liver and primary T-cells, we demonstrate that a proline residue at position 427 in human CYP4B1 and 422 in the rabbit CYP4B1 are critically important for both protein stability and catalytic activity towards 4-IPO. Systematic substitution of human CYP4B1 with peptide regions from the highly active rabbit enzyme revealed that the introduction of 12 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein resulted in a mutant human enzyme (P+12) that was as stable and as active as the rabbit wild-type CYP4B1 protein. The pro-drug 4-IPO has already been used in three clinical phase I/II studies, wherein serum levels have been achieved of up to 90 μM, a concentration at which effective killing of human transduced primary T-cells expressing the P+12 mutant protein occurs already at 24 h and by >90% after 72 h. These studies showed a potential novel human suicide gene system for adoptive cellular therapies in humans without major bystander effects for neighboring cells and proliferation-independent elimination of cells.
Long-term episomal gene transfer for safe engineering of T-cells for adoptive cell therapy of cancer
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Immunotherapy with T-cells engineered ex vivo with a novel T-cell receptor (TCR) or chimeric antigen receptor (CAR) is currently facing major breakthroughs in the treatment of cancer. Integrating retroviral (RV) or lentiviral (LV) vectors are commonly used for T-cell engineering. However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal and long-term cell engineering method using non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element for either over-expression or down-regulation of genes. T-cells engineered with a NILV-S/MAR(CD19CAR) vector have similar levels of CAR expression as T-cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV-S/MAR-engineered CD19CAR-T-cells exhibited similar cytotoxic capacity upon CD19+ Target cells recognition as LV-engineered T-cells. We propose that NILV-S/MAR vectors are superior to current options for enabling long-term gene expression without the risk of insertional mutagenesis.
Gene therapy based on cytidine deaminase-targeting overcomes pancreatic cancer resistance to chemotherapy
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Pancreatic ductal adenocarcinoma (PDAC) is anticipated to move to the second leading cause of cancer death worldwide by 2020 in the absence of improvements in treatment. In this dismal context, we recently performed the first phase Ib clinical trial of non-viral vector-mediated therapeutic gene delivery to sensitize cancer cells to chemotherapy (Thergap trial). From this study, we learned that intratumoral gene therapy was feasible and safe, and resulted in survival benefit for a subset of patients; we also learned that such beneficial effect wasn't correlated to the dose administered, as enforced expression of gemcitabine-converting genes reaches a “therapeutic plateau”. This strongly suggests that alternative mechanisms must also be targeted to achieve improved precision therapy. Accordingly, we speculate that cytidine deaminase (CDA) is a key target in PDAC resistance to gemcitabine as CDA is involved in the deamination of the vast majority of the gemcitabine entering cells, thus evading drug metabolizing genes. We found that CDA levels are elevated in PDAC patients. We transduced human PDAC-derived cells with shRNAs targeting CDA delivered by lentiviral vectors and found that cell proliferation was strongly impaired with massive induction of cell death by apoptosis. In addition, CDA silencing sensitizes PDAC cells to gemcitabine. Using proteomics, we demonstrate that targeting CDA strongly alters mitochondrial metabolism. In vivo, silencing CDA inhibits tumor growth in very aggressive PDAC experimental models, as monitored by non-invasive imaging. This study paves the way for the targeting of CDA using gene therapy to overcome pancreatic tumors resistance to standard-of-care treatment.
Results of a Phase IIb non-viral gene therapy trial from the UK CF Gene Therapy Consortium
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The UK Cystic Fibrosis Gene Therapy Consortium has conducted a double-blinded placebo-controlled multi-dose Phase IIb study to assess the efficacy of non-viral CFTR gene transfer at improving CF lung disease. Our optimized non-viral formulation consists of the cationic lipid GL67A and a codon-optimised, CpG-depleted CFTR expression plasmid (pGM169). Expression is regulated by a hCEFI enhancer/promoter shown capable of directing persistent expression in the lung. The intention-to-treat aimed to have all subjects receive 12 doses of nebulised gene therapy or placebo (0.9% saline) at intervals of 4 weeks over a 48 weeks period (136 patients received at least one dose and 115 completed 9 or more doses). Subgroups of patients (∼20/group) were enrolled for molecular assays (mRNA and chloride transport) in both nose and lower airway. The primary endpoint was FEV1, an outcome acceptable to regulatory agencies. In our longitudinal study of outcome measures, FEV1 was shown to be the assay with the most statistical power. Secondary outcomes included lung clearance index (a sensitive measure of small airway disease), quality of life questionnaires and CT scans as well as a number of exploratory assays. An adaptive design allowed the early identification of cumulative side effects: 17 subjects received 3 doses of gene therapy or placebo at 4-weekly intervals before any further subjects were dosed. No safety issues were identified by the Data Monitoring and Ethics Committee (DMEC) in this cohort. Dosing has now been completed, with no safety issues identified following three DSMB reviews. Results from these studies will be reported.
Virus-free delivery of microRNA into freshly isolated patient derived CD105+ MSCs using a novel magnet-bead based vector system
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A therapeutic anti-Hepatitis C virus shmiRNA integrated into the miR-122 genomic locus mediates a potent anti-viral response
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The success of RNAi therapies critically depends on shRNA expression strategies that allow persistent, safe and specific target knockdown without cytotoxicity from RNAi overexpression. Here, we propose a novel approach which is integration of a promoter-less miRNA-adapted shRNA (shmiRNA) into a genomic miRNA locus. For proof-of-concept, we demonstrate the feasibility to stably protect Huh7 liver cells against infection with Hepatitis C virus (HCV), by engineering an anti-HCV shmiRNA into the liver-specific miR-122 (hcr) locus. Towards this aim, we exploited TALEN- and CRISPR/Cas9-mediated homologous recombination to integrate the anti-HCV shmiRNA upstream of the miR-122 sequence, into a pre-validated position that allows co-expression of both hairpins from the cellular miR-122 promoter. We indeed obtained several cell clones that exhibited proper mono- or bi-allelic shmiRNA integration as detected by PCR and sequencing. Notably, luciferase reporter assays and qRT-PCR confirmed anti-HCV shmiRNA and unaltered miR-122 expression and activity in one clone that we selected for in-depth analysis. Remarkably, replication of a subgenomic HCV replicon and an infectious full-length genome were significantly impaired in these cells. A Dengue virus control was unaffected, proving that our engineered cells specifically impair HCV replication. Intriguingly, albeit our recombination template was designed to preserve endogenous miR-122, we also obtained clones with miR-122 mutations. Congruent with the role of miR-122 as essential HCV host factor, these showed an even greater inhibition of HCV. This exemplifies the power of our new shRNA expression strategy to induce synergistic effects and highlights its particular usefulness for other miRNA-dependent human diseases.
Integration driven HIV-1/STAT5B chimeric transcripts confer a selective advantage to blood cells in patients under anti-retroviral therapy
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HIV-1 has evolved several strategies to escape innate and acquired immunity and to persist in long lived cellular reservoirs of infected patients. Enrichment of HIV-1 integrations in expanded and long lived T-cells in patients under Anti-Retroviral Therapy (ART) has suggested that insertions in cancer associated genes such as BACH2 promote cell growth by insertional mutagenesis. Here, we found that in the peripheral-blood-mononuclear-cells (PBMC) from 54 HIV-1 infected patients under ART, BACH2 and STAT5B were targeted by a significant number of integrations with respect to the other lentiviral based datasets (p<0.0001) and with the same orientation of gene transcription. Moreover, we identified aberrant chimeric transcripts starting from the HIV LTR fused by aberrant splicing with STAT5B in total PBMC of 3 HIV-1 patients under ART, indicating that the HIV-1 long terminal repeat regulates the expression of this gene. Forced expression of both genes in naïve CD4+ T-cells from healthy donors significantly skewed their differentiation towards T-regulatory cells with a marked immunosuppressive potential. Moreover, STAT5B expressing Treg cells proliferated in an IL-2 independent fashion. Overall, our data suggest that in infected patients under ART HIV-1 mediated insertional activation of BACH2 and STAT5B in T-cells has occurred and induced their trans-differentiation towards a long lived immunosuppressive phenotype. Therefore, like many other retroviruses, also HIV-1 takes advantage of insertional mutagenesis to increase its persistence, however not by inducing cell transformation, but rather by creating a long lived cellular reservoir potentially able to diminish the immune surveillance against infected cells and escape the immune system.
Cross-Clade Inhibition of HIV on Primary Cells by CXCR4 or CCR5 Fused to the C34 Peptide from gp41 HR2
HIV-1 entry into CD4+ T cells requires binding to CD4 and either the CCR5 (R5) or CXCR4 (X4) co-receptor. Thus, strategies that disable productive co-receptor (CoR) engagement should provide potent protection from HIV infection. Previously we described a 34 amino acid peptide from the C-terminal heptad repeat-2 domain of gp41 (C34) which, when fused to the amino terminus (NT) of either R5 or X4, inhibits HIV-1 infection in transformed cells in vitro. Moreover, our initial studies suggested that C34-R5 or C34-X4 fusions provided trans-dominant resistance to infection irrespective of viral tropism (i.e. either C34-R5 or C34-X4 could inhibit entry of R5, X4 or dual-tropic isolates). Here we demonstrate that C34-R5 or C34-X4 expression by lentiviral transduction in primary CD4 T-cells from multiple donors results in almost complete inhibition (>98%) of HIV-1 infection based on intracellular p24 levels and RT activities. GFP-only and C34-CD4 expressing cells were infected at levels similar to untransduced T-cells. C34/CoR expression was >90% on Day 0 and stable during the 14 days of culture (>85%). Trans-dominant inhibition by C34-R5 or C34-X4 occurred for X4, R5 and dual-tropic primary isolates from clades B and A/E. Remarkably, when C34-CoR transduced and untransduced cells were mixed (1:4, respectively) and challenged with diverse HIV isolates, a condition that provides a more sustained exposure to HIV, selective enrichment of C34-CoR expressing cells occurred from the expected starting levels of ∼25% up to 60% C34-CoR+ cells during viral replication. Lastly, PMA/ionomycin and anti-CD3/CD28 stimulation of C34-R5 and C34-X4 expressing T-cells resulted in the expected robust expansion of these T cells which produced levels of intracellular IFNg, MIP-1b, TNFa, and IL-2 that were indistinguishable from untransduced cells. This novel method of engineering HIV-resistant, functional CD4 T-cells that can be expanded ex vivo and adoptively reinfused represents a promising and innovative approach with the potential to control HIV infection in humans.
Off-target-free gene delivery to clinically relevant cell types by receptor-targeted adeno-associated viral vectors
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Adeno-associated virus (AAV) derived viral vectors have recently received increasing attention due to substantial therapeutic benefit. However, their broad tropism prevents applications where cell-specific gene delivery is crucial. Here we describe the first adeno-associated viral (AAV) vector that allows genetic modification of rare cell types ex vivo and in vivo, while showing no detectable off-targeting. Using a designed ankyrin repeat protein (DARPin) as targeting module and carefully purifying the DARPin-displaying particles, AAV vectors specific for Her2/neu, EpCAM and CD4 were generated. When targeted to the tumor antigen Her2/neu, a single intravenous (i.v.) injection of vector was sufficient to track 75% of all tumor sites in a metastatic mouse model and to extend survival longer than the cytostatic antibody trastuzumab (Herceptin). CD4-targeted AAV vectors specifically transduced human CD4+ cells present in spleen of a humanized mouse model after systemic injection while CD8+cells remained unmodified. Mimicking conditions of circulating tumor cells revealed that EpCAM-targeted AAV efficiently detects tumor cells highly diluted in human blood opening the avenue for tumor stem cell tracking. Thus, by the approach developed here it has now become possible to deliver genes to the target cell type of choice with antibody-like specificity.
Capsid-engineering overcomes barriers toward endothelial cell transduction
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Endothelial cells (EC) are highly relevant targets for genetic modification in translational and basic research. Thus, the development of a vector that efficiently transduces EC and that possesses the ability to evoke long-term transgene expression would have broad implications. Adeno-associated viral (AAV) vectors are characterized by an excellent safety profile and low immunogenicity, but demonstrated relatively poor transduction efficiencies for EC in comparison with other cell types. Aiming to develop an optimized AAV variant for EC transduction and to identify the barrier/s hampering efficient EC transduction by natural occurring serotypes, we conducted AAV peptide display selection, a technology that enables screening for viral capsid variants that overcome both pre- and post-entry barriers. Using an improved selection and monitoring protocol, we identified two capsid variants, which were delivered efficiently and fast to the cell nucleus. These variants transduced EC with a significantly higher efficiency than the parental serotype, AAV2. The most efficient variant AAV-V reached a transduction efficiency of up to 65% on primary human umbilical vein EC with a particle-per-cell ratio as low as 1000. Using these variants as tools, we then performed infection biology analysis that allowed identifying multiple barriers toward AAV-mediated transduction of EC. These barriers were efficiently overcome by AAV-V, which successfully transduced EC in less than 4 hours as indicated by onset of transgene expression. In conclusion, we here describe novel AAV capsid variants identified by a directed evolution approach and a comprehensive screening assay for improved EC transduction and their contribution to deciphering AAV-EC interactions.
Successful repeated hepatic gene delivery in mice and non-human primates achieved by sequential administration of AAV2/5 and AAV1 vector serotypes
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The major challenge in AAV-based gene therapy is the presence of circulating neutralizing antibodies (NAB) against AAV vector capsids. NAB can be present in patient's blood prior to AAV treatment due to naturally acquired infections with the wild type AAV virus (pre-existing NAB). Anti-AAV NAB are also raised after first administration of AAV in the course of gene therapy treatment. There is a need to develop strategies that would permit a repeated AAV gene delivery not only to be able to treat the patient's that have pre-existing NAB but also for the patients that might experience overtime a decrease in therapeutic protein expression due to the natural turnover of transduced cells. To address those issues, we explored the feasibility of using the AAV2/5 and AAV1 serotypes for repeated, liver-targeted gene delivery in murine (C57BL/6 mice) and non-human primates (M. fasicularis) animal models. Sequential AAV based gene delivery with AAV2/5 and AAV1 proved to be successful in both models as therapeutic levels of expression were achieved for the two reporter transgenes (hSEAP and hFIX) used in the study. In contrast, the re-administration of the same serotype (AAV2/5-hSEAP followed by AAV2/5-hFIX) was unsuccessful due to the total inhibition of secondary AAV2/5 transduction by anti-AAV2/5 NAB. Our data demonstrates that a successful re-administration can be achieved in AAV-based gene therapy when combining AAV2/5 and AAV1 in both mice and non-human primates. Additionally, the levels of protein expression achieved with AAV1 illustrate the potential of AAV1 serotype for liver targeting.
Exhaustive characterization of DNA contaminants in rAAV productions by next generation sequencing
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The recent positive outcomes of clinical trials with rAAV vectors have led to a new interest in gene therapy for the treatment of rare and acquired diseases. While these new therapeutic agents head toward the pharmaceutical industry, an exhaustive quality control has to be performed to ensure the efficiency and biosafety of vector batches. Although various developments were made during the past years to improve rAAV manufacturing, DNA contaminants remain a major concern. Quantitative PCR analyses have already shown that rAAV productions contain DNA fragments derived from antibiotic resistance genes, rAAV helper factors or packaging cell genome. This technological approach is however based on the quantification of limited candidate sequences, extrapolating that all other DNA sequences would be encapsidated with a similar efficiency. To address this issue, we developed a method based on next generation sequencing that allows the deep characterization of encapsidated DNA contaminants and provides an advanced genomic identity of the vector genome. We validated our protocol by comparing GMP grade vector batches produced via transfection of HEK293 cells and purified either by CsCl gradient, affinity column or ion exchange chromatography. Among these purification processes, CsCl led to cleaner rAAV productions regarding DNA contamination. Furthermore, the total of the 4.7 kb rAAV vector genome was equally represented in the NGS data, confirming its homogeneous encapsidation. Finally, in addition to provide new insights in rAAV vectorology, we believe that our protocol could be of great interest to compare rAAV production systems and purification methods for “quality by design” process development.
A Non Human Primate Model For Autologous Transplantation Of iPSC-Derived Hematopoietic Cells
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iPSC-based therapy is hardly conceivable in humans without prior validation in large preclinical animal models closely related to humans. We generated iPSCs from Macaca cynomolgus to evaluate the engraftment capacity of cy-iPSC-derived hematopoietic cells after autologous transplantation. We first optimized a hematopoietic differentiation protocol based on an EB approach and followed the differentiation process over 3 weeks by time-lapse FACS and CFC assays to identify emerging cells with hemangioblastic and hematopoietic phenotypes. Up to 20% of CD34+ cells emerged around Day 12 to 15 of differentiation; however, fewer than 1/200 CD34+ cells were able to form CFCs. We analyzed the expression of genes associated with mesodermal and hemato/endothelial differentiation during the 3 weeks of differentiation. Expression of hematopoietic genes was consistent with the emergence of hematopoietic cells as determined by FACS and CFC assays. To evaluate the engraftment potential of cy-iPSC-derived hematopoietic cells, we injected 106 cells into either the femur (12 mice) or the retro-orbital sinus (10 mice) of sublethally irradiated NSG mice. At 5–7 weeks post-injection, hematopoietic engraftment was observed in 9/12 mice that received an intra-femoral injection and in none of the mice that received a retro-orbital injection. Up to 0.5% of mouse BM cells stained positive for a macaca CD45+ antibody. CD45+CD14+, CD45+CD11b+ myeloid and CD45+CD20+ lymphoid macaca populations were detected. Large-scale production of hematopoietic cells is currently underway to enable the autologous transplantation of cells to donor monkeys in order to evaluate the capacity of these cells for short and long-term hematopoietic engraftment.
Exploring bone marrow microenvironment in a murine model of ß-thalassemia
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Over the last two decades many steps were made in the field of allogenic bone marrow (BM) transplantation to definitively cure ß-thalassemia. In parallel, experimental autologous transplantation protocols were developed to permanently correct by gene therapy also patients lacking an HLA-identical donor. Both in allogenic and in autologous setting, thalassemic HSCs and thalassemic BM niche represent the central elements. Thalassemic BM is a stress environment, characterized by the expansion of erythroid progenitors. It is still ignored whether other hematopoietic subpopulations might be affected by the altered BM. To this purpose, we investigated the frequency of hematopoietic primitive and committed progenitors within BM Lin- cells of th3/+mice, a murine model of ß-thalassemia. We observed a significantly lower frequency of HSCs in adult th3/ +mice as compared to age-matched wt animals (4.93±0.22 and 8.52±0.59% of LSK cells, respectively). In order to evaluate functional characteristics of thalassemic HSCs and BM niche, we performed transplantation experiments in a competitive setting. We detected a lower engraftment and repopulating capacity of th3/+Lin- cells compared to the wt ones. These preliminary results revealed a never investigated defect both in frequency and in function of thalassemic murine HSCs. Our hypothesis is that peculiar aspects of thalassemic BM, as stressed condition of marrow overstimulation, abnormal regulation of bone metabolism, iron overload and hormonal factors may interfere with the maintenance of HSCs. Investigation of these elements will unravel new molecular mechanisms in the pathophysiology of the disease to be translated to allogenic and autologous BM transplantation experiences.
Mesenchymal stromal cells enhance the engraftment of low numbers of hematopoietic stem cells in a mouse model of autologous transplantation
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Mesenchymal stromal cells (MSCs) have been reported to enhance engraftment of human hematopoietic stem cells (HSC) in NOD/SCID mice. Also some beneficial engraftment effects have been associated to MSC transplantation in patients subjected to allogeneic HSC transplantation. In these transplantation modalities, the HSC engraftment effects mediated by MSCs could result from the immunosuppressive properties of MSCs. The question of whether or not the hematopoietic engraftment role of MSCs is maintained in autologous transplantations is currently unknown. Using a congenic mouse transplantation model (CD45.1/ CD45.2) we have observed that the co-infusion of MSC with low numbers of purified HSCs (LSK cells) significantly improved the short- and long-term hematopoietic engraftment in sublethally irradiated recipients. This improvement was MSC dose-dependent and due to an increased homing of the LSK cells in the recipient's bone marrow. Additionally, we found that the increased HSC engraftment mediated by MSCs was not due to paracrine effects, but required the co-infusion of the HSCs with the MSCs. This result, and the observation that LSK cells rapidly interacted with MSCs prior to transplantation, strongly suggest that the reported engraftment effects of MSCs require the direct contact of these cells with the HSCs, and therefore that MSCs act as HSC carriers improving their homing into the BM HSC niches. These results show for the first time in an autologous transplantation model the hematopoietic engraftment beneficial effects of MSCs.
Retroviral expression of dominant-negative Mpl disrupts THPO/MPL signaling and HSC maintenance
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Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, HSC maintenance and post-transplant expansion. MPL deficiency in patients results in thrombocytopenia and aplastic anemia. In the Mpl-/- mouse model HSC defects and thrombocytopenia could be corrected by lentiviral overexpression of Mpl. Expression of an intracellular-truncated, signaling-deficient Mpl receptor on transplanted Lin- cells in wildtype mice induced thrombocytopenia and HSC defects, therefore, termed dominant-negative (dn)Mpl. dnMpl mice had 4-fold reduced lin-, Sca-1+ and c-Kit+ (LSK) cells and bone marrow cells did not engraft in secondary recipients. In vitro experiments revealed systemic competition for Thpo binding and intracellular inhibition of wtMpl-signaling as the underlying mechanisms of dominant-negative inhibition. dnMpl expression allowed the engraftment of BM cells without further conditioning. Transcriptome analysis was performed to better understand molecular changes in Mpl-signaling inhibited HSC. The expression profile of dnMpl LSK cells negatively correlated with known HSC stemness signatures and the Wnt-, Jak/Stat-, and PI3K/Akt-signaling pathways. Whereas, expression of genes involved in cell cycle progression were positively correlated. Cell cycle status of HSC was confirmed by flow cytometry, with significantly less quiescent cells. Protein expression of typical mouse HSC markers (Tie2, EPCR (CD201) and Esam1) was significantly reduced (**p<0.005) in dnMpl mice compared to control mice and similar to expression in Mpl-/- and Thpo-/- mice. In summary, we demonstrated the induction of HSC defects by dominant-negative inhibition of Thpo/wtMpl-interaction in vivo in the adult mouse. Further, we could identify Thpo/Mpl-induced genes and pathways that are potential therapeutic targets for the regeneration of HSC.
CD34+ cells isolated from different sources: exploring their biology for future clinical perspectives
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Successful gene therapy of inherited blood diseases relies on transplantation and engraftment of a significant dose of autologous genetically engineered hematopoietic stem/progenitor cells (HSPCs). Gene therapy trials in pediatric patients are performed isolating CD34+ cells from bone marrow (BM), while in adults mobilized peripheral blood stem cells (PBSC) represent the favorite target due to the higher yields of CD34+ cells. Currently, G-CSF alone is the most used agent for stem cell mobilization, while Plerixafor is a new mobilizing drug that antagonizes the binding of SDF1 to CXCR4. The availability of new sources of HSPCs, potentially superior in terms of CD34+ cell yield, transduction efficiency and biological features, have a significant impact on the feasibility and efficacy of gene therapy. To this aim, we performed a deep characterization of HSPCs from different cell sources (BM, Plerixafor and G-CSF PBSC) including studies of biological, functional properties and subpopulations composition. The phenotypic analysis revealed that Plerixafor mobilizes preferentially HSCs and LT-HSCs, with a percentage of CD34+CD38-/low CD90+CD45RA- CD49f+ cells higher than that found in G-CSF PBSCs. This result mirrors the enrichment of SRCs found in the CD34+ cell population mobilized by Plerixafor. We are now extending all these analyses to HSPCs derived from Plerixafor+G-CSF mobilized cells. In order to define the molecular features of HSPCs from different sources, we studied gene expression by microarrays analysis focusing on self-renewal, homing and engraftment processes. Bioinformatic analysis revealed the molecular machinery underlying 'stemness' properties of cells derived from different cell sources.
Defining regulatory elements in human embryonic and somatic stem cells by MLV integration profiling
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The investigation of structural and functional characteristics of the human genome improves the understanding of the molecular circuitry wiring the genetic and epigenetic programs of stem cells. In this study, we present a genome-wide map of functional regions of chromatin in pluripotent and multipotent human stem cells, based on the integration properties of the Moloney murine leukemia virus (MLV). By using high-throughput sequencing technology, we recovered and mapped several thousand of MLV integration sites (ISs) in the genome of embryonic (ESCs) and somatic stem and progenitor cells: ESC-derived neural stem cells (NESCs), keratinocyte stem cells (KSCs), CD34+ hematopoietic stem/progenitor cells (HSPCs), erythroid and myeloid progenitors. Then, these “integromes” were compared by rigorous statistical analysis to discover shared or lineage-specific regulatory regions and target genes, and integrated with publicly-available maps of histone modifications (in ESCs and HSCs) typical of active and poised promoters and enhancers, determining the peculiar epigenetic pattern at ISs loci. As a result, MLV ISs targeted genes and enhancers related to the peculiar stem cell identity (by GREAT analysis), and were differentially associated to epigenetic marks of active and poised regulatory regions (H3K4me1, H3K4me2, H3K4me3, H3K27Ac, H3K27me3, H3K9me3) in pluripotent and somatic stem cells. In conclusion, functional genome mapping of human stem and progenitor cells through retroviral “scanning”, associated to epigenetic profiling, may be used as a tool to identify regulatory regions involved in the execution of stem cell genetic programs, providing a substantial contribution to a comprehensive description of the molecular circuitry associated with stemness and commitment.
Poster Presentations
Anti-tumor efficacy of the novel non-viral gene therapeutic system on the basis of herpes simplex virus thymidine kinase and GM-CSF
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A non-viral gene therapeutic system (GTC) designed for anti-tumor suicide gene therapy has been developed and characterized It is based on the herpes simplex virus thymidine kinase (HSVtk) and murine granulocyte-macrophage colony stimulating factor (mGM-CSF) genes carried by non-viral copolymer PEI-PEG-TAT, with gancyclovir (GCV) as a pro-drug. In vitro transduction of S37 cells with CMV-HSVtk-mGMCSF or CMV-HSVtk constructs causes production of HSVtk enzyme. The CMV-HSVtk-mGMCSF+GCV and CMV-HSVtk+GCV systems exhibit dose-depending cytotoxic activity ([GTC]=1 μg DNA/ml, [GC]=50 μM). The efficacy also depends on the incubation time (48 h for transfection and 48–72 h for incubation with GCV). Maximum cytotoxic activity achieved in vivo was 70–75%. It has been shown in vivo in the S37 sarcoma model that the therapeutic efficacy of the non-viral delivered bicistronic construct correlates with its single dose and multiplicity of injections. The efficient scheme of gene therapy was developed: 3-fold (with five-day intervals) intra-tumor injection of GTS in 0.04 μg DNA/mm3 single dose against the background of a fifteen-day intra-peritoneal infusion of GCV. It has been revealed that the bicistronic construct with HSVtk and mGM-CSF genes more efficiently inhibits metastasis than the monogenic one with a HSVtk gene. The novel GTS developed on the basis of HSVtk and mGM-CSF genes included into non-viral polymeric carrier is efficient for suicide therapy in the model of transplanted S37 murine sarcoma: it inhibits the growth of primary and metastatic nodes by 80–90% and increases the life span of animals by 70–75%.
Combined delivery of Temozolomide and suicide gene for glioblastoma therapy
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Glioblastoma multiforme (GBM) is the most malignant form of brain tumor in human. GBM usually causes death within 1–2 years after conventional therapies consisting of surgery, radiation therapy, and chemotherapy. In this research, the combined delivery of suicide gene and Temozolomide (TMZ) to GBM was studied as an effective therapeutic method. The R7L10 peptides, which are composed of 7-arginine and 10-leucine, can form self-assembled micelles in aqueous solution. In this study, We confirm combined delivery of TMZ and herpes simplex virus thymidine kinase gene (HSV-TK) into glioblastoma cells by using R7L10 might be an effective treatment of GBM. TMZ was loaded into R7L10 by the oil-in-water (O/W) emulsion/solvent evaporation method. To confirm the formation of the pDNA/R7L10-TMZ complex, a gel retardation assay was performed. Stability of the pDNA/R7L10-TMZ complex was evaluated by heparin competition assay. The transfection efficiency of R7L10-TMZ against C6 rat glioblastoma cells in vitro was higher than R7L10. Also, TK/R7L10-TMZ complexes had the highest anti-tumor effect than other complexes. In the xenograft nude mouse model and intracranial glioblastoma model, the TK/R7L10-TMZ complexes injected group most efficiently suppress tumor growth. These results suggest that combined delivery of HSV-TK gene and TMZ by R7L10 carrier be useful for treatment of glioblastoma.
MicroRNA (miR)-155 is associated with the leukemogenic potential of class IV granulocyte-colony stimulating factor receptor (G-CSFR) in CD34+ progenitor cells
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miR-155 contributes to myeloproliferative disorders. The mechanisms involved in the leukemogenic potential of the class IV G-CSFR+CD34+ cells remain unclear. We investigated the role of G-CSFRIV in the regulation miR-155 levels and in the leukemogenic potential of CD34+ cells. G-CSFRI or IV isoforms were overexpressed in CD34+ cells followed by G-CSF stimulation. Proliferation rates of G-CSFRI+ and G-CSFRIV+CD34+ cells were analyzed in colony forming unit assays. Ki67/AnnexinV/PI cell staining was used in cell cycle and apoptosis analyses. Stat5 activation was determined by phosphflow. miR-155 levels and its candidate target genes were quantified by real time-PCR. Upon G-CSF stimulation, higher proliferation rates (14.1%, p>0.01) and colony numbers (52.0±20.2 vs.102.3±6.7, p<0.01) were observed when G-CSFRIV+ progenitor cells were used in comparison to G-CSFRI+ cells. An increase in S phase (18.60±1.67% vs. 24.53±2.85%) in G-CSFRIV+ progenitor cells, whereas apoptosis was decreased. G-CSF induced sustained Stat5 activity in G-CSFRIV+ cells. Upregulation of miR-155 was observed in G-CSFRIV+ cells in the presence of G-CSF (RQ: 0.70±0.11 vs. 1.41±0.36, p<0.01). Stat5 inhibitor and antimiR technology prevented G-CSF-induced upregulation of miR-155 in G-CSFRIV+CD34+ cells (RQ: 1.76±0.36 vs. 1.05±0.08 with and without Stat5 inhibitor respectively, p<0.05). Hyperproliferation of G-CSFRIV+CD34+ cells was decreased by the Stat5 inhibitor (4336±2457 vs 6151±2559, p<0.05). This study shows G-CSFRIV is associated with a Stat5-dependent deregulation of miR-155, but can be inhibited by Stat5 inhibitors and antimiR technology. This may represent a novel therapeutic approach to decrease the risk of leukemia in patients showing an elevated G-CSFRIV/G-CSFRI ratio.
Reovirus: inducer of autophagy – balancing life and death
The mammalian orthoreovirus Type 3 Dearing (T3D) has been evaluated extensively as oncolytic agent. Despite evidence for anti-tumor efficacy, the mechanisms by which reovirus T3D induces cell death are poorly understood. Reovirus-induced apoptosis is regarded as the primary cell-death mechanism. However, blocking the apoptotic pathway does not completely block reovirus-induced cell death. Recently, autophagy has been implicated in reovirus-induced cytolysis in multiple myeloma cells. Here, we show that reovirus T3D induces autophagy in the immortalized retinoblast cell line 911 and in the glioblastoma cell lines U87-MG and U251-MG. This was evident from the formation of Atg5-Atg12 complex, LC3-I/LC3-II conversion, and the appearance of acidic vesicular organelles. Furthermore, electron microscopy revealed the presence of cytoplasmic material engulfed in double-membraned vesicles. The precise role of the autophagocytic process in reovirus infection is still unclear. Autophagy possibly functions as a cellular survival mechanism that inhibits or delays reovirus-induced cell death and thereby enhances reovirus replication. Alternatively, autophagy may be required for the redistribution of cellular organelles and formation of the so-called viral factories in the cytoplasm. We demonstrate that autophagosomes are abundant in U251-MG cells persistently infected with reovirus T3D. Stimulation of autophagy with rapamycin induced cytolysis in these cells. This suggests that even though autophagy may function as a cell survival mechanism, it is only tolerated to a certain extent. Altogether, our data show that reovirus T3D induces autophagy in a series of cell lines and highlight the central role for autophagy in reovirus T3D infection.
Combining the oncolytic adenovirus Delta24-RGD with temozolomide improves survival and influences immune cell subsets in a murine malignant glioma model
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The conditionally replicating oncolytic adenovirus Delta24-RGD specifically lyses tumor cells and is currently tested in clinical phase I/II trials for recurrent malignant glioma, recently also combined with the chemotherapeutic agent temozolomide (TMZ). We previously showed that the efficacy of Delta24-RGD is mainly dependent on an induced anti-tumor immune response. As known, TMZ has pronounced effects on the immune system, which could hamper the viral-induced anti-tumor immune response. To assess the combined effects, we tested different treatment regimens in an immune competent glioma mouse model. Therefore, C57BL/6 mice were injected intracranially with murine GL261 cells. TMZ was given for 5 days before (pre-treatment regimen) or after (post-treatment) intratumoral injection of Delta24-RGD. Mice were followed for survival or sacrificed at earlier time-points to access intratumoral influx of immune cells. Splenocytes were isolated and ex vivo stimulated with GL261 to assess INFy production as a measure of anti-tumor immune response. The post-treatment regimen significantly improved survival compared to single treatments alone. The pre-treatment regimen is improving survival, however does not differ significantly from the virus mono treatment. In this group, influx of dendritic cells is diminished, followed by a diminished influx of CD8+ T-cells. Splenocytes from viral treated and combination treated mice recognize GL261 cells. The addition of TMZ to Delta24-RGD treatment does not hamper the therapeutic efficacy of the virus, moreover the post-treatment regimen improves survival significantly compared to Delta24-RGD or TMZ alone. This regimen does not hamper the intratumoral influx of immune cells and elicits an anti-tumor immune response.
Oncolytic adenovirus sensitive to Notch-activated pathway actively eliminate pancreatic cancer stem cells
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Notch signaling pathway is activated in pancreatic cancer showing upregulation of Notch receptors and the surrogate marker of Notch activation Hes1 in neoplastic cells compared to normal epithelial tissue. Furthermore, the Notch signaling pathway participates in the maintenance of the pancreatic cancer stem cell (CSC) population, contributing to tumor aggressiveness and resistance to current therapies. We hypothesized that a dual-regulated oncolytic adenovirus sensitive to Notch pathway activation controlled by the uPAR promoter could be an efficient therapeutic agent to act against pancreatic tumors, leading to the elimination of both CSCs and the bulk of the tumor. In the present work we engineered an oncolytic adenovirus bearing regulatory sequences recognized by the transcriptional factor CSL upstream a minimal uPAR promoter regulating the E1A gene (AdNuPARmE1A). First, we tested for several CSL-binding site combinations and demonstrate that optimized-engineered sequences respond to Notch activation in a reporter adenovirus (AdNuPARmLuc). Transfection of increasing amounts of the Notch intracellular domain (NICD) led to the upregulation of luciferase expression in AdNuPARmLuc infected cultures. Moreover, incubation of infected cultures with a gamma secretase inhibitor reduced the activation of the reporter adenovirus, demonstrating high sensitivity to Notch pathway activation. AdNuPARmLuc displayed enhanced activity than AduPARLuc, lacking Notch signaling-responsive sequences, in pancreatic cancer cell lines and in tumorspheres. The oncolytic virus, AdNuPARmE1A demonstrated enhanced selectivity showing good cytotoxic response in cancer cells, and limited activity in non-tumor epithelial HPDE cells. All together, we present AdNuPARmE1A as a highly efficient virus targeting both CSCs and the bulk of pancreatic tumors.
Antisense CTLA4 and Foxp3 oligonucleotides increase antitumor response of therapeutic vaccination with GM-CSF-modified tumor cells
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The lack of antitumor efficacy of therapeutic vaccines is proposed to be partially due to the establishment of an immunosuppressive response (tolerance) mainly mediated by regulatory T cells (CD4+CD25+Foxp3+). We propose combining therapeutic vaccination with GM-CSF-modified tumor cells plus the silencing of immunosuppressive molecules Foxp3 and CTLA4 as a strategy for effective antitumor immune response. Methodology. In vitro efficacy of CTLA4 and Foxp3 gene silencing was assessed using ASO (antisense oligonucleotides) and PPRH (Polipurine Reverse Hoogsteen). ASO-FAM's and PPRH-FAM's entry into the cell was confirmed by flow cytometry. C57BL6 mice were injected with B16 tumor cells and then were treated with 2×105 irradiated B16 cells genetically modified to produce GM-CSF and with/without anti-CTLA4 and/or anti-Foxp3 ASO (500 μg/mouse). Blood samples were collected at different times. The CTLA4 and Foxp3 expression was measured by RT-qPCR and flow cytometry. Tumor development and survival were monitored. Results. In vitro CTLA4 and Foxp3 gene silencing was more efficient with ASO rather than PPRH. Higher PPRH concentrations than ASO were needed to achieve the same effect. The animals vaccinated with GM-CSF gene-modified tumor cells that were cotreated with anti-CTLA4 or anti-Foxp3 ASO delayed tumor growth compared to the control and achieved 20 and 40% survival, respectively. Conclusions. The combination of therapeutic vaccination with the GM-CSF-modified tumor cells and CTLA4 and Foxp3 gene silencing enhances antitumor response, achieving tumor growth delay or even preventing tumor development in some cases, achieving overall animal survival. Partially supported by SAF 2011-27002.
The efficiency of gene therapy mediated by genetically modified mesenchymal stem cells on human ovarian carcinoma cell line SKOV-3 in vitro
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Human ovarian carcinomas represent the leading cause of death among all the gynecological malignancies due to late detection of tumor development, formation of metastases, chemoresistance and recurrence of the disease. Drug resistance to therapy remains one of the major obstacles in the treatment. Mesenchymal stem cells are heterogenic population of cells and they can serve as vectors in so called gene directed enzyme prodrug therapy (GDEPT). They can carry genes which code for special enzyme and this enzyme is able to convert non-toxic prodrug to a highly toxic chemotherapeutic exclusively in the vicinity of malignant cells. This therapeutic approach prevents systemic toxicity caused by normally used chemotherapeutics. We genetically modified mesenchymal stem cells derived from adipose tissue (AT-MSC) to express gene for: 1. Herpes simplex virus thymidine kinase (HSV-tk), which phosphorylates ganciclovir (GCV) to its toxic derivates; 2. bacterial (BCD) or yeast cytosine deaminase::uracil phosphoribosyltransferase (BCD; CD::UPRT), which catalyzes conversion of 5-fluorocytosine (5-FC) to highly toxic 5-fluorouracil (5-FU). Synthesized toxic compounds induce apoptotic processes and together with bystander effect increase the eradication of tumor cells. We evaluated the cytotoxicity of cisplatin and 5-FU on a model of chemoresistant human ovarian carcinoma cell line SKOV-3. We examined two different therapeutic approaches to eradicate SKOV-3 cells – HSV-tk/MSC+GCV and BCD or CD::UPRT-MSC+5-FC in vitro and we reached remarkable efficiency in elimination of tumor cells using CD::UPRT-MSC treatment in vitro.
Novel ex vivo gene manipulation technology of mature adipocytes for sustained protein replacement therapy of variety of intractable diseases
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Gene therapy-based protein replacement is one of absolute needs for treatment of inherited diseases. Based on much experiences of clinical transplantation therapy in cosmetic and reconstructive surgery, adipose tissue is now recognized as a source of proliferative cells for cell-based therapy. Most commonly used adipose tissue-derived cells are stromal vascular fractions (SVF), which is sediment after collagenase digestion followed by centrifugation. Many studies demonstrated that SVF exhibits differentiation potential for multi-lineage, suggesting suitability for regenerative medicine. However, accumulated data indicate that SVF are heterogeneous, and therefore imply that SVF may not result in stable therapeutic gene vehicle for protein replacement purpose. To solve this problem, we have developed manipulation procedure of mature adipocytes through ceiling culture with subsequent retroviral/lentiviral gene transduction. The propagated cells, designated as ceiling culture-derived proliferative adipocytes (ccdPAs), provide efficient gene transduction efficiency with stable secretion of the transduced-gene products. GMP production procedure, by which the gene-transduced ccdPAs could be expanded up to nearly 10^12 cells from 1 g of fat tissue within one month after fat tissue preparation, has been developed. Our platform technology has been applied for ex vivo gene therapy of familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Clinical translational research (first in human) protocol has been approved by Ministry of Health, Labour and Welfare followed by initiation of patient enrollment in Japan. We further continue our research and development to obtain the approval as gene therapy medicine. We are also developing protein replacement therapy of hemophilia and lysosomal storage diseases.
Integration Profile of Lentiviral Vectors in Clinical Gene Therapy for X-Adrenoleukodystrophy
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Here, we present data on a large scale integration site (IS) analysis of patient samples collected from 4 X-Adrenoleukodystrophy (X-ALD) patients enrolled in the first clinical trial to treat a monogenetic cerebral disease using autologous hematopoietic stem cell transplantation using a HIV-1 based lentiviral SIN-vector. The first patient has been treated already more than 6 years ago. So far no severe adverse events have been reported from this and other clinical trials using HIV-1 based SIN vectors. Linear amplification mediated PCR (LAM-PCR) followed by next generation sequencing performed on ex vivo transduced cells prior to reinfusion and on engrafted cells revealed a polyclonal hematopoietic reconstitution in all patients. In total, more than 38,000 unique IS have been retrieved from distinct time points and sorted cell fractions. The characteristic insertion profile for lentiviral vectors with a preference for gene coding regions (>70%) could be confirmed. Moreover, individual chromosomes showed high targeting of IS, affected genomic regions, close to KDM2A, PACS1 and HLA genes, are described for lentiviral clustering. A very similar landscape of ALD vector integration has been revealed in all four patients. The detection of identical IS identified in myeloid and lymphoid lineages as well as in CD34+ cells suggests a successful ex vivo transduction of early hematopoietic progenitors. Lentiviral gene therapy shows to be safe and effective as the cerebral disease has been stabilized in 4 out of 4 patients.
Pre-clinical assessment of AAV-mediated gene therapy for Crigler-Najjar syndrome (UGT1A1 deficiency)
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Crigler-Najjar syndrome is an autosomal recessive rare disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene, resulting in the accumulation of high levels of bilirubin in serum, which, if not rapidly treated, is associated with neonatal jaundice, brain damage, and death. Phototherapy for 10–12 hours per day is used for the treatment of Crigler-Najjar syndrome. However, the treatment has several shortcomings, leaving liver transplantation the only curative option for severely affected individuals. Here we developed a series of single-stranded (ss) and self-complementary (sc) AAV vector constructs expressing the UGT1A1 transgene under the transcriptional control of liver-specific promoters. Constructs were pseudotyped into AAV8 serotype vectors, characterized for purity and potency, and tested in vitro in hepatocyte cell lines and in vivo in Gunn rats. ssAAV vectors were produced with considerably higher yields by triple transfection of adherent HEK293 cells and exhibited superior homogeneity of vector genome content when characterized by analytical ultracentrifugation and denaturing gel electrophoresis. An ssAAV-UGT1A1 vector construct was selected for further characterization based on superior product homogeneity and high potency in vitro and in vivo. In Gunn rats, following the administration of the ssAAV8-UGT1A1 vector, partial correction of levels of bilirubin was achieved at vector doses as low as 5×1010 vector genome (vg)/kg, with complete correction of the disease phenotype starting at doses of 5×1011 vg/kg. Current efforts are towards the scale up of vector production to support the clinical translation of results obtained.
Human placental ex vivo studies to support an adenovirus-mediated vascular endothelial growth factor (VEGF) gene medicine for the treatment of severe early onset fetal growth restriction (FGR)
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Comparative efficacy of diet induced obesity in C57BL/6J versus Balb/C mice for gene therapy applications
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Because diabetes is the one of the most prevelant diseases and leading cause of death in the world, the development of effective antidiabetic drugs requires constitution of appropritate animal models of diabetes prior to clinical trials.1 Despite obese/obese (ob/ob), diabetes/diabetes (db/db) mice or Zucker Diabetic Fatty (ZDF) rats have been extensively used in experimental gene therapy applications, these models carry mutations in genes affecting appepite such as leptin (ob/ob), leptin receptors (db/db, ZDF) making them unsuitable to follow up long term metabolic changes. In addition these models lack the cause of diabetes but only display the features of T2DM such as insulin resistance, glucose intolerance and hyperglycemia due to evantual beta cell loss. Although, C57BL/6J mice have been considered to be genetically predisposed to diet induced obesity (DIO), a gold model of DIO, most of the experimental manipulations are complicated due to their small size. Thus, we have evaluated Spraque Dawley (SD) rats for their potential to display main features of T2DM following a special diatery regimen. Our results suggested that either two months of feeding with high fat diet (HFD) or a low dose of spreptozotocin (STZ) injection was needed to induce DIO in SD rats. Nonetheless, induction of hyperglycemia still required a moderate dose of STZ injection.
1. Tasyurek MH, Altunbas HA, Canatan H, Griffith TS, Sanlioglu S: GLP-1-mediated gene therapy approaches for diabetes treatment. Expert Rev Mol Med 2014;16:e7 Grant support: TUBITAK-112S114.
Induction of Diet Induced Obesity in Sprague Dawley Rats as a Model for Type 2 Diabetes
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Because diabetes is the one of the most prevelant diseases and leading cause of death in the world, the development of effective antidiabetic drugs requires constitution of appropritate animal models of diabetes prior to clinical trials.1 Despite obese/obese (ob/ob), diabetes/diabetes (db/db) mice or Zucker Diabetic Fatty (ZDF) rats have been extensively used in experimental gene therapy applications, these models carry mutations in genes affecting appepite such as leptin (ob/ob), leptin receptors (db/db, ZDF) making them unsuitable to follow up long term metabolic changes. In addition these models lack the cause of diabetes but only display the features of T2DM such as insulin resistance, glucose intolerance and hyperglycemia due to evantual beta cell loss. Although, C57BL/6J mice, a gold model of DIO, have been considered to be genetically predisposed to diet induced obesity (DIO), most of the experimental manipulations are complicated due to their small size. Thus, we have evaluated Spraque Dawley (SD) rats for their potential to display main features of T2DM following a special diatery regimen. Our results suggested that feeding with high fat diet (HFD) for prolonged period of time was needed to induce DIO in SD rats. In addition, a low dose of spreptozotocin (STZ) injection shortened this time period facilitating appearance of DIO related features. Nonetheless, induction of hyperglycemia still required a moderate dose of STZ injection.
1. Tasyurek MH, Altunbas HA, Canatan H, Griffith TS, Sanlioglu S: GLP-1-mediated gene therapy approaches for diabetes treatment. Expert Rev Mol Med 2014;16:e7 Grant support: TUBITAK-112S114.
Gene therapy overcomes the need for multiple infusions of enzyme replacement therapy in a mouse model of lysosomal storage disease
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Enzyme replacement therapy (ERT) has become the standard-of-care for several lysosomal storage disorders (LSDs). Despite ERT's undisputed efficacy, the requirement for multiple and costly administrations as well as ERT's limited improvement of some LSDs manifestations prompts the search for better therapies. Using a mouse model of mucopolysaccharidosis VI we compared the efficacy of a single intravascular administration of an adeno-associated viral (AAV) vector targeting liver to weekly infusions of human recombinant enzyme at the same doses used in MPS VI patients. While gene therapy results in increased and stable levels of circulating enzyme up to one year after vector administration, ERT has typical peak-and-drop serum kinetics. We found that urinary GAGs were reduced to approximately normal levels both in mice treated with gene therapy and those treated with ERT. Similarly, normalization of GAGs occurred in all tissues analyzed, including heart valves and myocardium, independently of treatment. We observed that gene therapy improved skeletal skull abnormalities slightly better, albeit not significantly, than ERT. Both therapies seemed to similarly improve animal motor performance with gene therapy possibly associated with less animal distress. Thus, a single vector administration that converts liver into a factory organ for systemic secretion of therapeutic proteins is at least as effective as ERT in a mouse model of LSD, possibly eliminating problems with compliance and costs. Only testing in humans will prove whether this holds true in a clinical setting.
Prevalence of anti-AAV8 neutralizing antibodies and ARSB cross-reactive immunologic material in MPS VI patient candidates for a gene therapy trial
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Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Preexisting immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for Mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder due to arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 missense (62.5%) and 27 null (37.5%) ARSB mutations. The detection of ARSB protein in twenty-four patients out of 34 (71%) was predicted by the type of mutations. Pre-existing Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. The seroprevalence to AAV8 was different depending on the location of the referral center. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8.
Protection of pancreatic beta cells by delivery of curcumin using peptide micelles under hypoxia
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Department of Bioengineering, college of Engineering Hanyang Univ. Seoul 133-791, Korea Islet transplantation is one of the therapeutic approach for diabetic patients. For an average-size person (70 kg), a typical transplant requires about one million islets, isolated from two donor pancreases. After islets are isolated from a donor, pancreatic beta cells go into apoptosis because of the oxidative stress. To enhance the viability of pancreatic beta cells, we treated pancreatic beta cells line (INS-1) to curcumin, as an anti-oxidant agent. However it is a hydrophobic drug and has low bioavailability. A non-toxic and efficient carrier is required for the curcumin delivery. In this study, curcumin is loaded into amphiphilic peptides composed of 3 arginines and 6 valines. A zeta size potential measurement and scanning electron microscope (SEM) shows that they formed nanoparticles. The size is about 250 nm and the surface charge is 17.49 mV. R3V6 could efficiently load curcumin at 1:0.3 weight ratio (R3V6:cur cumin). In contrast the only curcumin group, curcumin loaded in amphiphilic peptide (R3V6-Cur) effectively was delivered into INS-1 cells. In order to evaluate the viability of the pancreatic beta cells under hypoxic condition, we conducted MTT assay. It showed that the R3V6-cur group had the highest viability. It is due to the anti-oxidative effect of curcumin. Therefore, the RV-curcumin complex may be useful for improving viability of pancreatic beta cells after islet transplantation.
Non-viral introduction of microRNA into bone marrow derived CD133+ stem cells
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Stem cell derivatives and fibroblasts – Acute retention and biodistribution in the mouse model
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Lentiviral stem cell gene therapy for Pompe disease
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Pompe disease is a rare autosomal recessive metabolic disorder caused by deficiency of lysosomal hydrolase acid a-glucosidase (GAA). GAA degrades glycogen to glucose, and deficiency results in generalized tissue glycogen accumulation leading to cardiorespiratory failure in the early-onset patients within the first year of life. Enzyme replacement therapy (Alglucosidase alfa, Myozyme) is currently the only effective treatment requiring high dose biweekly administrations with many patients developing antibody responses compromising long-term therapy. Previously, we overexpressed codon-optimized GAA in the hematopoietic system at high vector copy number per cell (VCN - 7) resulting in complete correction of the disease phenotype in Pompe mice, including restoration of motor-function and induced immune tolerance to the transgene product. Although vector related adverse events were not observed during long-term follow-up, and preliminary vector integration profiles are benign, the vector design was further optimized. For this purpose, we developed an in vitro assay using Hepa-1-6 or C2C12 Gaa deficient cells through CRISPR/Cas9 genomic engineering. These Gaa KO cell lines were applied in a transwell system to test chimeric GAA variants, based on signal peptides from human alpha-1-antitrypsin and insulin-like growth factor II (IGFII). These variants provided increased excretion/uptake rate of GAA in vitro, and at low VCN (1-2), IGFII-GAAco also resulted in significant glycogen reduction in heart, skeletal muscles and brain, reduced heart mass, and improved rotarod performance. Currently, additional modifications to GAA are evaluated to improve efficacy, reduce the number of transplanted HSCs and VCN needed further to the minimum required for optimal clinical efficacy.
Up-regulation of Glucagon Like Peptide-1 Expression in pancreases of C57BL6J Mice with Diabetes
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Incretins are gut-derived factors that enhance glucose-induced insulin secretion from islet beta-cells, one of which is the glucagon-like peptide-1 (GLP-1), a novel medication used against Type 2 Diabetes (T2DM). Since reduced incretin response to food ingestion is one of the primary defects associated with glucose intolerance and hyperglycemia in T2DM patients, we decided to analyze alteration in GLP-1 expression pattern in pancreases of diabetic animals. In order generate mice model of obesity induced T2DM, C57BL6J mice were fed with high-fat diet (HFD) for two months prior to streptozotocin (STZ) injection. Obesity associated insulin resistance and glucose intolerance were clearly observed in mice fed HFD compared to mice fed standard-diet. Then, diabetes was successfully induced via STZ injections. Insulin, glucagon and GLP-1 content of pancreatic islets were revealed by immunohistochemistry. Our results indicated that pancreatic islets of diabetic animals displayed higher GLP-1 expression compared to non-diabetics without any change in GLP-1 receptor (GLP-1R) expression profile. In conclusion, we believe that up-regulation of GLP-1 expression might be a protective mechanism to recover from pancreatic beta cell damage induced by STZ. As a result, GLP-1 mediated gene therapy approaches might be needed to restore insulin sensitivity, glucose intolerance and pancreatic beta cell mass in T2DM.
1. Tasyurek MH, Altunbas HA, Canatan H, Griffith TS, Sanlioglu S: GLP-1-mediated gene therapy approaches for diabetes treatment. Expert Rev Mol Med 2014;16:e7 Grant Support:TUBITAK-112S114.
Gene therapy of Type I mucopolysaccharidosis by liver organ stem cells in the mouse model
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Type I mucopolysaccharidosis (MPS-I) is a lysosomal storage disorder (LSD) resulting from a deficiency in the enzyme a-L-iduronidase (Idua). Accumulation of its substrates causes tissue damage with subsequent organ dysfunctions and mental retardation. The current treatments, namely enzyme replacement therapy and hematopoietic stem cell (HSC) transplantation bear significant limitations. Several studies in mouse models have shown that HSC transplantation could be more successful when coupled to gene therapy by which higher levels of the enzyme could be achieved. However, as HSCs cannot be cultured for a long time, it is impossible to apply targeted modifications and select clones to eliminate the risk of insertional mutagenesis, which might be induced with the current delivery methods. With the recent advances in establishment of long-term culture of adult liver stem cells, it is now possible to make targeted genetic modifications and select genetically stable clones. Here, by setting-up a proof-of-principle study in Idua-deficient (Idua-/-) mouse model we decided to explore whether liver stem cell-mediated gene therapy could be an alternative treatment of MPS-I. We established culture of liver stem cells from Idua-/- mice and transduced them with a lentiviral vector carrying the functional IDUA gene under the control of SFFV-promoter (LV-IDUA). The LV-IDUA transduced liver organoids both expressed and secreted supranormal levels of the functional enzyme. We are currently transplanting the modified liver stem cells into Idua-deficient mice and monitoring the efficacy of the treatment. Given it is successful, liver stem cell-mediated gene therapy could be applicable to other forms of LSDs.
Codon optimization for viral gene therapy of Crigler-Najjar syndrome
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Crigler-Najjar (CN) syndrome is an inherited liver disorder which results in severe hyperbilirubinemia due to UGT1A1 deficiency. Previously we showed that viral gene therapy using AAV8 containing the UGT1A1 transgene and a liver-specific promoter resulted in hepatocellular UGT1A1 expression and reduction in serum bilirubin levels in the Gunn rat, the animal model for CN-syndrome. Notably, viral gene therapy was more effective in male than in female rats. Codon optimization (co) of a transgene results in a more efficient translation of the transgene product. Therefore we compared the efficacy of UGT1A1-co over wild-type UGT1A1. SsAAV8 and scAAV8 vectors containing the human UGT1A1 transgene with a liver-specific promoter were injected via the tailvein in male and female Gunn rats. The UGT1A1 transgene was wild-type or codon-optimized with different algorithms. A low or high dose viral vector (1.5×10e12 vg/kg resp. 5×10e12 vg/kg for ssAAV8 and 5×10e11 vg/kg resp. 1×10e12 vg/kg for scAAV8) was administered. A high viral dose of ssAAV8-LP1-UGT1A1 resulted in >90% correction of serum bilirubin in male and female rats. Partial correction was observed with a low viral dose, which was lost over time. No difference was observed between UGT1A1-wt and UGT1A1-co. Serum bilirubin levels were >95% corrected in rats injected with high dose scAAV8-HLP-UGT1A1-wt/co. A difference was observed in female Gunn rats injected with a low viral dose, scAAV8-HLP-UGT1A1-co appeared somewhat better in comparison to scAAV8-HLP-UGT1A1-wt, 93 vs 83% reduction. In conclusion, UGT1A1 codon-optimization was not beneficial in ssAAV8 treated Gunn rats. However, a low dose scAAV8-HLP-UGT1A1-co seemed superior to scAAV8-HLP-UGT1A1-wt in female rats.
Lentiviral ex vivo gene therapy to the murine model of MPSII
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Targeted elimination of activated Hepatic Stellate Cells by anti-EGF-receptor scFv-sTRAIL fusion protein
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Efficient Gene Targeting in the Safe Harbor AAVS1 Site of Human Hematopoietic Repopulating Cells
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Gene targeting constitutes a new step in the development of safe and efficient gene therapy in patients with inherited or acquired diseases. To assess the feasibility of targeting therapeutic transgenes in the safe harbor AAVS1 locus of human hematopoietic stem cells, we have designed a donor IDLV that carries the EGFP reporter gene under the control of the PGK promoter, flanked by AAVS1-homology sequences. Cord blood CD34+ cells were transduced with the donor IDLV and then transfected with ZFNs-mRNAs specific for AAVS1 locus. These cells were cultured in methylcellulose and also transplanted into NSG-SGM3 mice to assess the efficacy of gene targeting. Mean data corresponding to five independent experiments showed that 10.14±3.27% of the CD34+ cells were EGFP positive. BFU-E and CFU-GM colonies showed the specific integration of EGFP in the AAVS1 locus. Analyses of the BM of transplanted NSG-SGM3 mice showed that 16.27±3.35% of these cells were positive for the human CD45 marker 3 months post-transplantation. Among these cells, 14.31±11.82% expressed the EGFP marker gene. Transplants into secondary recipients showed low but stable levels of human hematopoietic engraftment. Importantly, a similar proportion of human hematopoietic cells were EGFP+, indicating targeted integration into long-term repopulating cells. Taken together, our data show the feasibility of targeting exogenous genes into the AAVS1 safe harbor locus of human hematopoietic progenitors and repopulating cells at efficacies compatible with a therapeutic effect in different monogenic diseases affecting the immune-hematopoietic system. Current studies aim the insertion of FANCA in hematopoietic progenitor cells from FA-A patients.
Hypoxia specific Heme oxigenase-1 expression for gene therapy of ischemic brain
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Hypoxia-inducible Heme oxigenase-1(HO-1) expression was used in gene therapy for ischemic stroke. We constructed the HO-1 plasmid DNA with the epo enhancer and SV-40 promoter. In order to investigate the effect of the plasmid DNA containing the epo enhancer, a plasmid DNA without the epo enhancer is used as a control. In vitro gene expression efficiency of each vector type was evaluated in the N2A cells under both hypoxic and normoxic conditions. Heme oxygenases-1 catabolizes free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. Through measuring the in vitro HO-1 ELISA, heme oxigenase-1 is overexpressed by the epo enhancer under the hypoxia condition. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. Increase of the cell viability is caused by the effect of HO-1, and we determine viability using the cytotoxicity assay. Plasmid DNA is injected to 9 week-SD rat's brain by the stereotaxic equipment, some increasing amount of HO-1 is detected in vivo experiment. Both experimentation In vitro and in vivo, decrease of the infarct volume by the epo enhancer is observed under the hypoxia situation. In conclusion, the HO-1 plasmid DNA including the epo enhancer is useful to overexpressing the heme oxigenase, and it is expected to take advantage of treatment of the ischemic brain disease.
Structure function studies of Vectofusin lentiviral transduction enhancers in human hematopoietic stem/progenitor cells
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Gene transfer into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using HIV-1-based lentiviral vectors (LVs) has several therapeutic applications ranging from rare diseases to cancers. In such therapeutic context, efficiency, safety and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and by reducing the amount of LV used on the cells. We recently described a new transduction enhancer called Vectofusin-1, a short histidine-rich amphipathic peptide derived from the LAH4 family of DNA transfection agents. Vectofusin-1 promotes transduction with a broad range of retroviral pseudotypes, but the critical determinants of the peptide activity are still unknown. In this structure-function study, we compared a family of twelve Vectofusins-1 isomers and showed that Vectofusin-1 is the lead peptide for the transduction enhancement of hCD34+ HSPCs with VSV-G but also GALVTR pseudotypes. Using the Ala-scanning strategy, we designed numerous mutants of Vectofusin-1 and evaluated their capacity to promote GALVTR-LV infectivity. We showed that the transduction improvement of HSPCs is requiring: Lysine residues on the N-terminal extremity of Vectofusin-1; Hydrophobic residues consisting of leucine (instead of Valine or Isoleucine); An hydrophilic angle of 140° formed by the Histidine residues in the Schiffer-Edmundson helical wheel representation. In conclusion, these results help to decipher the mechanism of action of Vectofusins, especially Vectofusin-1, in the context of hCD34+ cell-based gene therapy.
Editing the human AAVS1 locus with CRISPR/Cas9
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One approach of gene therapy is gene editing with engineered nucleases such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) or the CRISPR/Cas9 system. The latter uses small guide RNA (sgRNA) to direct specific DNA double-stranded breaks by the Cas9 endonuclease. When DNA breaks are repaired by the natural process of homologous recombination (HR), delivery of an HR donor template allows replacement of a defective gene with a normal allele at its natural location. In order to improve CRISPR/Cas9 activity and specificity for a target sequence, we modified the structure of the sgRNA by decreasing the length of the sequence complementary to the target site and altering the binding sequence of Cas9. We tested the efficacy of these newly designed sgRNAs on the adeno-associated virus integration site 1 (AAVS1) in HEK293T cells transduced by lentiviral vectors. We used an Integration-deficient Lentiviral vector (IDLV) carrying a cassette with Cas9 and a modified sgRNA targeting AAVS1, together with another IDLV carrying a GFP donor cassette integrating at AAVS1 site after HR. We will present our results with the newly designed sgRNAs on Cas9 nuclease activity and homologous recombination at the AAVS1 target site. The improvement of the sgRNA structure tested on the AAVS1 locus can potentially be applied to other genome sequences targeted by the CRISPR/Cas9 system.
Towards the Targeted Gene Therapy of Fanconi Anemia Cells in an Intergenic Safe Harbor Locus
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The specific insertion of therapeutic genes in safe harbor (SH) loci constitutes a relevant strategy that may further improve the safety of gene therapy in the future. Because ideal SH loci should be located far away from genes and microRNA coding regions, we aimed here for the specific insertion of the EGFP transgene in a human SH locus (termed SH6) located in an intergenic region where the closest ORF is more than 350 kb away. A donor vector carrying the EGFP gene driven by the long EF1alfa promoter and flanked by two homology arms of 1,500 base pairs each was constructed. Two different nucleases, SH6-meganuclease (SH6-MN) and SH6-TALEN, were used to facilitate the integration of the EGFP in the SH6 genomic site of human cord blood (CB) CD34+ cells. Nucleofection of CB CD34+ cells with either SH6-MN or SH6-TALEN encoding plasmids showed similar amounts of EGFP+ cells after 4 days of in vitro culture. At this time, around 3–5% of the primitive hematopoietic progenitors (CD34+/CD38-/CD90-) were EGFP+. In these samples, and also in derived hematopoietic colonies, integration at the SH6 site was confirmed by PCR. Aiming to improve the viability of gene-edited HSCs, cells were nucleofected with nuclease mRNAs, increasing their viability around 3-fold. While one of the drawbacks of intergenic SH sites is the limited expression of inserted genes, in the case of Fanconi anemia, a very weak expression of FANCA showed therapeutic benefits, suggesting the relevance of the SH6 site in HR-based FA gene editing.
Improving gene edition tools for Wiskott-Aldrich Syndrome gene therapy
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Specific nucleases, including ZFNs, TALENs and CRISPR (Clustered Regulatory Interspaced Palindromic Repeats) are powerful tools for genome editing. Gene therapist can now aim for genetic rescue instead of gene addition to treat diseases. However, their efficiency and safety should be analyzed in detail before translation into clinic. Primary inmunodeficiencies (PID) are a main target for gene editing strategies since a small number of corrected cells could cure the patient. Our final aim is to developed gene edition tools for efficient genetic rescue of human hematopoietic stem cells (hHSCs) from WAS patients. We designed different CRISPR-based gene editing tools in a Lentiviral vector (LV) backbone. To study efficiency and safety of our systems we developed a WAS-specific GFP-reporter cellular model harboring GFP coding sequence disrupted by WASP sequences (K562-GF-WASP-P). Integrative-deficient LVs (IDLVs) were generated harboring Cas9 or Cas9 and gRNA. The generation of Indels on the WASP locus of K562 cells using IDLV was similar to that obtained with nucleofection (up to 80%), but the viability of the gene-edited cells was superior in IDLV. Efficiency of genetic rescue was determined in K562-GF-WASP-P model using a donor containing the wt eGFP. We obtained up to 4% of eGFP positive cells upon nucleofection of the different components. A similar efficiency was obtained for gene addition using a donor harboring an eGFP expression cassette. In summary we have developed new CRISPR based tools for efficient gene edition of the WASP locus and new cellular models to study efficiency and safety of WASP gene edition.
Genetic modification of platelets using lineage specific lentiviral vectors for gene therapy
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The characteristics of platelets as natural carriers of proteins and factors involved in biological processes such a coagulation, inflammation, immune response and angiogenesis, offers a new alternative of delivering therapeutic proteins by gene therapy. To avoid transgene-related toxicities, we developed third generation self-inactivating lentiviral vectors for megakaryocytes and platelet-specific expression by using lineage-specific promoters. We introduced promoter fragments of the murine platelet factor 4 (PF4–1219 bp), human glycoprotein 6 (hGP6−348 bp), human glycoprotein 9 (hGP9–793 bp), murine GP9 (676b), human glycoprotein 1b alpha (GP1ba-594 bp) and as a control, the phosphoglycerate promoter (PGK). The vectors expressing eGFP as marker protein were studied in a murine BM transplantation model. High transgene expression in platelets from all lineage-specific promoters was observed (PF4>HGP6>GP1ba>mGP9) and all vectors conferred good specificity with low expression in leukocytes compared to the PGK promoter. In in vitro differentiation assays, transcriptional targeting was possible with high and specific transgene expression in megakaryocytes (specificity: GP1ba>mGP9>PF4>HGP9>HGP6). To target proteins into the platelet granules, protein domains of known granule-specific proteins will be used. Furthermore, we demonstrate efficient platelet-specific knockdown by lineage-specific miR expression. This system could be used for specific delivery of soluble factors to modulate coagulation process (hemophilia), angiogenesis and/or for the development of mouse models for thrombopathias and the modification of platelet functions.
CRISPR/Cas9-mediated gene editing for type 1 myotonic dystrophy
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Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disease caused by a CTG triplet expansion in the 3′ non-coding region of the DM protein kinase (DMPK) gene. Expanded DMPK transcripts are sequestered in the nucleus in small foci and interfere with the splicing function of CUG-binding proteins, leading to misregulation of the alternative splicing of numerous transcripts. Currently, no effective treatment is available for patients. We aim at developing a strategy to excise the CTG expansion from the human DMPK locus in DM1 muscle cells. For that purpose, we are using the recently developed RNA-guided CRISPR-Cas9 system that introduces targeted double-stranded DNA breaks in the genome. We have generated several constructs that express a small size Cas9 nuclease under either an ubiquitous or a muscle-specific promoter and various expression cassettes for guide-RNAs (gRNAs) targeting the 3′UTR of the DMPK gene. Transfection studies show that Cas9 is well expressed in various cell lines, including myoblasts. We will present our results on DNA excision at the targeted locus.
Engineered nucleases-mediated in situ correction of a genetic defect by homologous recombination into the native locus
Engineered nucleases specific for genomic targets are extensively used to generate DSBs that increase the rate and efficiency of homologous recombination (HR). We seek to determine the efficacy of nucleases in a clinical relevant genetic defect. The genetic defect we are addressing is the junctional epidermolysis bullosa (JEB), a family of severe skin adhesion disorders due to autosomal recessive mutations in the LAMB3 gene coding for the laminin-332 heterotrimer, a key component of the dermal-epidermal junction. Recently, we provided proof of principle that ZFN-mediated, AAVS1-targeted GFP addition can be achieved in human keratinocytes and in long-term repopulating epithelial stem cells in a preclinical model of xenotransplantation of human skin equivalents on immunodeficient mice. This project aims at in situ correction of the LAMB3 gene in primary keratinocytes from Herlitz JEB patients. Recently TALEN-based gene correction for dystrophic EB has been reported. Similarly, we have developed a genome editing approach for JEB. In particular we have designed TALENs specific for the second intron of LAMB3 gene and a HR cassette including a splicible LAMB3 cDNA (from exon 3 to the end of the gene). In particular immortalized JEB keratinocytes were nucleofected with TALEN mRNAs and infected with an IDLV vector carrying the HR cassette. The in situ gene correction has been evaluated by site-specific PCR, knock-in expression of the corrected LAMB3 gene and by functional restoration of the laminin-332 production. In parallel, CRISPR-Cas9 nuclease has been designed on the same locus to compare the transduction efficiency and cleavage activity.
Generation of novel RAG1 mouse models using CRISPR/Cas9
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Gene Editing of the PKLR locus in Human Hematopoietic Progenitors by TALE nucleases
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Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in PKLR gene that encodes the erythroid specific Pyruvate Kinase (PK) enzyme. Erythrocytes from PKD patients show an energetic imbalance since defective pyruvate kinase (PK) enzyme fails to produce ATP, causing chronic non-spherocytic hemolytic anemia. Gene therapy of autologous hematopoietic stem cells (HSC) is proposed as definitive treatment for PKD patients. Towards PKD gene therapy, we have already demonstrated that the co-transfection of TALENs targeting the second intron of the PKLR locus with a matrix carrying an exon 3–11 partial codon optimized version of PKLR and the puromycin selection gene mediates the specific insertion of the therapeutic gene in the second intron of the endogenous PKLR in human cell lines and human induced pluripotent stem cells (iPSC). Because of the difficulties of generating iPSC-derived hematopoietic repopulating cells, we aimed to implement our gene editing system in cord blood CD34+ cells. Purified CD34+ cells were electroporated with the RPK TALENs and the therapeutic matrix. After 96 hours of puromycin selection the number of surviving cells decreased to 0.8%. Interestingly, PCR analyses showed that up to 96% of myeloid and erythroid colonies derived from puromycin resistant cells had been gene edited by the specific integration of therapeutic matrix in the second intron of PKLR. Modifications in the procedure, including alternative delivery methods, introduction of TALENs as mRNAs and optimization of the selection period, are currently being evaluated to improve the gene editing of CD34+ cells in the PKLR locus.
Correction of haemoglobin levels in a humanized mouse model of thalassemia after fetal gene therapy
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Towards the development of engineered immune cells broadly applicable to cancer patients
Engineering aßT cells with receptors to re-direct the immune system against cancer has most recently been described as one of the scientific breakthroughs. However, a more careful clinical translation is clearly needed given the many challenges and pitfalls during the implementation resulting frequently in an inefficient clinical product or even failure of clinical efforts. Therefore, we took advantage of selected highly tumor-reactive γδTCR genes, recently proposed as a very intriguing alternative strategy to CARs and aßTCRs, developed a novel stepwise evaluation process and assessed efficacy of the GMP-grade cell product. By studying the optimal expression cassette we found transgene expression is depending on TCR-chain orientation and different 2A elements. In addition, suicide mechanisms strongly suppressed expression of introduced receptors and the introduced suicide gene itself, questioning safety and clinical applicability of suicide cassettes in the context of engineered immune cells. Most importantly, we developed a novel selection method by depleting non and poorly engineered cells with clinical-grade anti-aßTCR-beads. This selection method translated into highly purified, but untouched engineered immune cells with strong anti-tumor reactivity both in vitro but also in two different humanized mouse models. Furthermore, this strategy eliminated residual allo-reactivity. All together, we generated strong evidence for the need of a careful and step-wise evaluation of newly designed engineered immune cells. Finally, we provide for the first time a GMP-grade enrichment procedure for the generation of untouched engineered immune cells with improved anti-tumor and reduced allo-reactivity suitable for the application in both an autologous and allogeneic clinical scenario.
Study of the function and stability of LYP in Jurkat T cells deficient in Csk
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Efficient method for targeted non-viral delivery of microRNAs to endothelial cells
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Treating bone defect by transplantation of genetically modified MSC spheroids
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Cell transplantation is promising for treating various diseases. However, the effects are not always sufficient because of the limited survival rate and functions of the cells after transplantation. To improve the therapeutic efficacy, we recently established a new system of genetically modified spheroid transplantation (Biomaterials 35:2499,2014). Micropatterned plates possessing cell adhesion areas that are arrayed with 100-μm diameter allows preparation of a number of regularly-sized spheroids. In addition, genetic modification is done using our original gene carrier based on biocompatible polycations, which can introduce gene without disrupting the spheroid structure. In this study, we applied this system for MSC spheroid transplantation to enhance bone regeneration. In vitro analyses revealed that MSC spheroids receiving transfection of Runx2-expressing gene led to increased capacity of secreting osteogenic factors such as BMP as well as the enhanced differentiation efficiency into osteoblasts. The spheroid capacity was well maintained even under culture conditions mimicking pathological microenvironments of inflammation and ischemia. Transplanting MSC spheroids that received Runx2 transfection into bone defect on rat femur induced significantly rapid bone regeneration compared with controls of MSC spheroids w/o Runx2 transfection, or MSCs obtained from conventional monolayer culture. Histological analyses using stably GFP-expressing MSCs revealed that a larger number of GFP-positive cells was observed in the defect compared with the controls, suggesting that MSCs in the spheroids were likely to have higher chemotactic activity. In conclusion, this spheroid system combined with gene introduction effectively maintained the MSC activity after transplanted into living tissue, leading to the enhanced bone regeneration.
Development of regulated derivatives of group I-based trans-splicing ribozyme for cancer gene therapy
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Human telomerase, which is regulated mainly by the hTERT expression, has been an important target for cancer therapies. Previously, we developed hTERT-targeting trans-splicing ribozyme based on Tetrahymena group I intron for cancer therapy. The ribozymes were shown as useful anti-cancer agents through reducing target RNA level and simultaneously triggering anti-cancer gene activity selectively in cancer cells expressing the target RNA. We have used adenovirus for in vivo delivery of the ribozyme and tissue-specific promoter to express ribozyme for increase in specificity. We needed high dose of the adenoviruses for anti-cancer efficacy because of lower level of ribozyme than constitutive strong promoters, which has limitations for clinical usage because of potential toxicity and immunogenicity. In this study, we tried to use less amount of adenovirus for in vivo delivery of the ribozyme, while maintaining the efficacy and specificity. To this end, we improved intracellular efficacy of the ribozyme by modification of ribozyme expression cassette through incorporation of factors known to increase RNA level such as splice site and WPRE (woodchuck hepatitis virus post-transcriptional regulatory element). Then, we found that the expression level of the ribozyme and anti-cancer effect were efficiently increased by adenovirus vector harboring the modified ribozyme cassette. Importantly, systemic delivery of ten-fold lower level of the modified adenoviral vector induced anti-cancer efficacy as similar as unmodified adenoviral vector without liver toxicity in orthotopic HCC model. The results indicate that trans-splicing ribozyme can be optimized as efficient and safe anti-cancer modality through control of its intracellular expression.
Targeting transgene expression under the control of FVIII promoter: determining the identity of FVIII producing cells for gene therapy of Hemophilia A
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To assess the FVIII-producing cells, we performed in silico analysis of transcriptional factors (TF) consensus sequences on FVIII promoter (pF8) sequence. The results predicted the presence of several myeloid-specific TF, in addition to hepatocytes- and endothelial-specific TF. To evaluate pF8 activity in cells and tissues, we inserted in a lentiviral construct the human pF8 sequences (short:1175 bp; long:2350 bp) driving GFP or FVIII expression. In vitro LV-transduction showed GFP expression in several myeloid, hepatic and endothelial cells. Since no differences in GFP expression between long and short pF8, we used the short pF8 for further studies. We injected 5×108TU of LV.short-pF8.GFP in C57Bl/6 mice and evaluated GFP expression by FACS and immunofluorescence analysis in several organs at different time-points. GFP expression was restricted to hepatic non-parenchymal cells (22±3%), meanwhile hepatocytes were barely detected (<2%). In particular, by costaining of GFP and liver sinusoidal endothelial (LSEC) or Kupffer cell-specific markers revealed pF8 was predominantly active in LSEC. Instead, in hematopoietic organs, such as spleen (5–6%) and bone marrow (10–22%, BM), GFP expression was virtually restricted to myeloid cells. Interestingly, GFP expression remained constant in liver up to 6 months after LV-injection decreasing by that time in spleen and BM. We then injected 109TU LV.shortpF8.hFVIII in hemophilia A mice (n=6). aPTT assay demonstrated FVIII activity between 5 and 10% of normal FVIII in treated mice up to 8 weeks later, the longest time tested. Our results demonstrate that pF8 is differentially active in cell-subpopulations of several organs contributing to identify the FVIII producing cells.
PhiC31 integrase fused to designer DNA binding domains for sequence-specific genome engineering
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Genome engineering relies on proteins and enzymes which allow custom designed DNA sequence-specificity. Here we describe reprogramming of the DNA targeting specificity of the PhiC31 integrase, which was previously shown to mediate sequence-specific DNA recombination in the prokaryotic and the eukaryotic genome. However, the large number of pseudo sites in the human genome and occurrence of chromosomal translocations limit its use for therapeutic applications. To further improve the integration specificity, we designed novel enzymes in which the PhiC31 protein is fused to different designer DNA binding domains (DBD) for sequence-specific genome engineering. To validate the design principle of the fusion proteins we tested their activity in mammalian cells and we evaluated excision and binding activities to the specific DBD. Based on fusion with DBD derived from the synthetic polydactyl zinc finger E2C and the adeno-associated virus Rep 68/78 protein, we found that excision and integration activities retained up to 80% of the wild type integrase. Moreover, we show enhanced binding activity and recombination activity at the intended DNA target site. We further assembled transcription activator-like effector (TALE) proteins targeting a pseudo attP` site on human chromosome 19 (Chr19q13.31) which represents one natural hot spot of PhiC31 integrase. The evaluation of these novel chimeric TALE/integrase fusion proteins with respect to activity and specificity are ongoing. In summary, we show reprogramming of a large serine recombinase (LSR) with enhanced specificity representing a powerful tool for bioengineering in genetic studies, stem cell engineering and gene therapies.
Dependence of gene transfer efficiency on the composition of PDMAEMA polyplexes
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Ocular diseases are among the most debilitating conditions affecting all segments of the population. Gene therapy is an alternative to traditional treatments for several diseases, and adequate carriers for delivery are in high demand. Methacrylates bearing amino groups are versatile polymers whose cationic nature enables the formation of a complex with DNA (polyplex). In recent years, several studies have demonstrated the potential of poly(2-(N,N′-dimethylamino)ethylmethacrylate) (PDMAEMA) as a gene therapy vehicle. To improve the internalization and transfection efficiency of non-viral vectors, such as PDMAEMA, reports suggest the incorporation of the anionic polymer hyaluronic acid (HA). Therefore, we synthetized PDMAEMA and prepared different formulations with HA and DNA (N/P ratios of 12.5:1 and 20:1). The polyplexes were in the nanometer range, had positive charge, as desired for cell membrane penetration, and efficiently complexed DNA. Cytotoxicity, evaluated in HEK293 cells and a retinal pigment epithelium cell line (D407), was absent for all polyplex formulations. Transfection efficiency, evaluated by fluorescence microscopy and flow cytometry, showed that both cell lines were efficiently transfected by polyplexes, but we observed that the transfection efficiency is more dependent on the PDMAEMA:DNA ratio than on the presence or absence of HA.
Fundação para a Ciência e Tecnologia (PTDC/SAU-BEB/098475/2008, to G.A. Silva; PEst-OE/EQB/LA0023/2013; PEst-OE_QUI_UI4023_2011; SFRH/BD/52424/2013 to D. Bitoque; SFRH/BD/70318/2010 A.V. Oliveira) and Marie Curie Reintegration Grant (PIRG-GA-2009-249314) under the FP7 program.
The effects of body fluids on the growth of bacterial vectors for gene therapy in vitro
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Bacteria-mediated gene therapy has several specific advantages in comparison to the use of viral and non-viral vectors. The main applications of bacterial vectors are diseases of the gut and urogenital system. These sites are colonized by microflora under healthy conditions and are, thus, especially suitable for bactofection or alternative gene therapy. Tumors can be accessed via intravenous administration of bacteria. There are no published data on the effects of the relevant body fluids on the survival of bacterial vectors for gene therapy. The aim of our study was to analyze the effects of saliva, plasma and urine on the growth kinetics of the widely used bacterial vector – Salmonella typhimurium SL7207. An overnight culture of Salmonella typhimurium 7207 was mixed with saline, saliva, plasma or urine. Body fluids were collected before application from one male and one female volunteer after overnight fasting. Serial dilutions of the body fluids were tested and the growth kinetics was compared to positive and negative controls. The absorbance of saline, plasma and urine did not change. In saliva a subtle bacterial growth was observed. The growth of the bacterial vector was inhibited by 31% in saliva (p=0,02) and by 62% in plasma (p=0,01) after 6 hours. No growth inhibition was seen in urine. The effects of saliva and plasma were partially dose dependent. The results indicate that this vector is suitable for the treatment of urinary tract diseases, but not for intravenous administration. The application in gut diseases might be complicated not only by the negative effects of saliva, but also by gastric and duodenal juices that has not been tested in this study. The obtained results should be confirmed in vivo and potentially be taken into account in animal experiments as well as human studies using bacteria-mediated gene therapy. Research on the underlying mechanisms might help to design improved bacterial vectors.
Transgenic exosomes: delivery platform for membrane bound therapeutic enzymes
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Exosomes are small (<100 nM) vesicles that play specialized roles in immunity, intracellular communication and waste disposal. Recently there has been a resurgence of interest in exosomes for a wide range of applications, including diagnostics and therapeutics. Our group is investigating the use of exosomes as a delivery platform for membrane bound proteins. Specifically, we using exosomes to deliver ENTPD1 (CD39) and 5NTE1 (CD73) as a novel treatment for inflammatory disease. Our recent data has demonstrated that the balance between pro-inflammatory extracellular ATP and anti-inflammatory adenosine is skewed in the synovial compartment of rheumatoid arthritis (RA) patients, likely contributing to ongoing inflammation. CD39 is a membrane bound ATPase that converts extracellular ATP and ADP into AMP, while CD73 is a GPI-anchored protein that converts AMP into adenosine. The combination of these two enzymes is predicted to tilt the ATP:adenosine balance from pro-to anti-inflammatory. We have developed a protocol for the production and purification (10L scale) of transgenic exosomes containing murine CD39-CD73 from stably transfected HEK293 cells. Interestingly we found that exosomal CD39 has an unexpected advantage when compared with soluble CD39, in that exoCD39 showed >10 fold higher specific activity. This is likely due to maintaining the native structure of CD39. Exosomes containing CD39 and CD73 were very potent in reducing pro-inflammatory cytokine production in in vitro inflammation assays (IC50 in pM range). Transgenic exosomes are a promising approach for the delivery of membrane bound therapeutic enzymes and may have potential for the treatment of disease, including rheumatoid arthritis.
Bacterial vectors for gene therapy have beneficial effects in a murine model of colitis
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The non-specific effects of bacterial vectors for gene therapy are largely unknown. Inflammatory bowel diseases are associated with changes in gut microflora. Bacteria-mediated gene therapy might be suitable for this disease as the target tissue is accessible for the bacterial vectors. The aim of our study was to prove whether a bacterial vector for gene therapy has any effects on dextran sodium sulfate (DSS) colitis in mice. Colitis was induced in male mice using 2,5% DSS in the drinking water for 9 days with one day recovery. Live or heat-inactivated attenuated Salmonella typhimurium strain SL7207 (1×10E9) were administered via gavage every other day. Weight and stool consistency were monitored daily. After sacrifice colon weight/length and spleen length/weight was measured and samples were taken for histology, biochemistry and gene expression analysis. DSS induced diarrhea, weight loss, increased spleen weight and reduced colon length in comparison to the control group. Live, but not inactivated Salmonella partially but significantly prevented colitis-associated changes in the observed parameters. Although biochemical and histological outcomes are yet to be determined, based on the clinical variables it is clear that the severity of colitis was lower in the live Salmonella-treated groups in comparison to the DSS group receiving inactivated Salmonella and DSS group without treatment. These results point towards a possible bias in experiments using bacterial vectors for gene transfer in vivo. Although the mechanism is unclear, the design of the experiments should include appropriate control groups to distinguish gene-specific and non-specific effects of bacteria-mediated gene therapy.
Enhancement of osteogenic vector
The work was focused on the improvement of osteoinductive vectors to compensate low transfection efficiencies. Here we describe improvements of an osteogenic vector for significantly enhanced mediation of osteogenic differentiation of target cells and tissues. Codon optimization, addition and mutation of cis and trans acting elements, as well as screening for more potent secretion signal in order to improve BMP2 secretion were under investigation of this study. Furthermore, we describe a space saving hybrid vector capable of overexpressing bone morphogenic protein 2 (BMP2) while simultaneously down regulating several previously elucidated inhibitory genes by inserted shRNAs (either alone or in clusters) in order to enhance therapeutical effect. Various enhancements showed several fold higher impact in osteogenic differentiation of target cell lines and mesenchymal stem cells. By combining the most promising improvements, even higher differentiation rates could be achieved even with low transfection efficiencies. Finally, combinations of BMP heteromers (BMP2 to BMP7 vector ratio) revealed ideal ratio for the mediation of enhanced osteogenic differentiation. Altogether, we present several improved and highly efficient vectors and their impact in osteogenic differentiation which can be used in viral as well as non-viral gene therapeutical applications, and furthermore can easily be transfered to other fields of gene therapy.
Efficient and non-invasive plasmid-DNA administration into the tibialis cranialis muscle of dwarf mice
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Our group investigated an alternative treatment for growth hormone deficiency based on hGH-DNA plasmid administration followed by electrotransfer. Aiming at improving this strategy, hGH plasmid-DNA (50 μg) were administered into the exposed quadriceps or non-exposed tibialis cranialis muscle of immunodeficient dwarf (lit/scid) mice. For the quadriceps, we utilized our previous optimized electrotranfer conditions (eight 90 V/cm pulses of 20 ms with 0.5 s intervals) while for the tibialis a new combination of high/low (H/L) voltage pulses (one 800 V/cm pulse of 100 μs and one 100 V/cm pulse of 400 ms) was used. After 3 days, blood was withdrawn and hGH determined, both groups showing similar results: 5.0±2.2 and 3.5±0.9 ng/mL (P>0.05) of serum for quadriceps and tibialis treatment, respectively. A second experiment (28-day assay) was carried out in order to compare the growth parameters relative to the two groups: the slopes of the body weight variation curves were thus found similar (0.066 and 0.063 g/mouse/day) while the final body weight increase was 16.1% for the quadriceps and 18.9% for the tibialis group, respectively. Tail and nose-to tail length increase was 4.5% and 7.1% for the quadriceps and 4.8 and 4.6% for the tibialis group. Circulating mIGF-1 levels were also determined, being 126±46.5 and 106±93.2 ng/ml (P>0.05) for quadriceps and tibialis treatment, respectively. Based on these results, hGH-DNA administration into the tibialis muscle followed by HV/LV electrotransfer proved to be an efficient and less aggressive treatment, much more suitable for pre-clinical testing since it avoids muscle exposition.
Development and Therapeutic Efficacy of the DNA Complex-Releasing Systems Comprising Injectable Auto-Forming Alginate Gel
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For in vivo plasmid transfection, we have developed a novel gene transfection system comprising very small (<70 nm) plasmid complexes with negative surface charge (Biomaterials 31 (2010) 2912–2918). It has already achieved the high extragene expression in tumor tissues. Multiple transfection with certain cytokine genes effectively induced tumor regression, and the small tumors completely disappeared. However, single injection often leads to unsatisfactory efficacy owing to the short duration of the gene expression. On the other hand, an alginate gel is known to degrade by dissociation of the ionic crosslinking under acidic conditions. It is expected that the alginate gel would be gradually degraded under the low-pH environment in tumor tissue. We have recently reported that the alginate gel could be automatically formed in a living body by injection of alginate solution, probably due to the interaction with divalent cations such as Ca2+ ion in body fluids. Moreover, we found that the degradation rate of alginate gel could be controlled by addition of amorphous calcium phosphate (ACP) as buffering agent and supplier of Ca2+. In this study, we prepared injectable auto-forming alginate gel including pDNA-GM-CSF complexes and ACP as a durable gene transfection system, which would be slowly degraded, and release DNA complex in the body. The slow-releasing behavior of the system, and therapeutic effect were examined in tumor model mice.
Muscle gene transfer mediated by mesoangioblasts and non-viral vectors
Duchenne muscular dystrophy (DMD) is an X-linked disease in which dysfunctional dystrophin leads to muscle wasting. Transplantation of myogenic stem cells, genetically modified to express a functional dystrophin is an attractive option for treatment of the disease. There has been very modest success in DMD cell therapy due to several factors; one of them being the difficulty to achieve efficient gene transfer due to the large size of the dystrophin gene and the transgene silencing. The present study investigates the potential use of non-viral gene transfer in mesoangioblasts for cell therapy for DMD. Mesoangioblasts are muscle progenitor cells in the embryonic dorsal aorta wall of mice and humans, with reported ability to fuse with myofibers. This study focuses on two approaches for muscle gene expression with mesoangioblasts. The first approach utilizes non-replicating episomal plasmid vectors, which are hypothesized to remain in the post-mitotic muscle tissue. The second approach involves genomic integration of the transgene by the PiggyBac transposon system. Intramuscular transplantation of mesoangioblasts containing the integrating transposons or episomal plasmid in mouse tibialis anterior muscles led to widespread eGFP expression in myofibres. In case of the transposon based approach, transposition in mesoangioblasts allowed persistent expression after transplantation in mouse muscle and their fusion with resident myofibers. These findings provide proof-of-principle that both non-viral vector systems used may have potential to improve cell-based therapies for DMD. These approaches are currently being used with dystrophin-containing vectors to assess functionality of treated murine muscles and confirm potential therapeutic use.
Alveolar bone regeneration by BMP gene transfer to periodontal tissue using in vivo electroporation
Alveolar bone defects are generally reconstructed by bone grafts or artificial bone grafts. We aim to develop non - surgical methods for alveolar bone regeneration therapy. We constructed non-viral gene transfer system using in vivo electroporation and succeeded to induce bone in the muscles by bone morphogenetic protein (BMP) gene transfer. In this study, we tried to apply our gene transfer system on periodontal tissues for alveolar bone regeneration. We injected GFP, lacZ or BMP gene expression non-viral vector to periodontal tissues in the palatal region of 9 weeks male wistar rats and immediately electroporated. For histological analyses, we made time course samples and HE staining and immunohistochemical staining. To investigate how the gene transfer to periodontal tissues affects bone remodeling, we performed double bone staining using calcein and tetracycline. One day after gene transfer to periodontal tissues, there were many inflammatory cells. But, seven days after gene transfer, no differences were observed comparing with control sites. Double bone staining also did not show any differences between gene transferred tissue and control site for 7 days. On the other hand, 21 days after BMP gen transfer, we found new bone like tissues were added on the alveolar bone. We succeeded to non-viral gene transfer to the periodontal tissues of rats safely and efficiently. And, we found new bone like tissue in the BMP gene transferred periodontal tissues. Therefore, our results make it possible to develop a new non-surgical therapy for alveolar bone regeneration.
High and prolonged sulfamidase secretion by liver of MPS-IIIA mice following hydrodynamic tail vein delivery of antibiotic-free pFAR4 plasmid vector
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Mucopolysaccharidosis type IIIA (MPS-IIIA) or Sanfilippo A syndrome is a lysosomal storage genetic disease which results from the deficiency of the N-sulfoglucosamine sulfohydrolase (SGSH) protein, a sulfamidase required for the degradation of heparan sulfate glycosaminoglycans (GAGs). The accumulation of these macromolecules leads to somatic organ pathologies, severe neurodegeneration and patients' death. To asses a novel gene therapy approach based on prolonged secretion of the missing enzyme by the liver, mediated by hydrodynamic gene delivery, we first compared a kanamycin and an antibiotic-free expression plasmid vector, called pFAR4. Thanks to their reduced vector size, pFAR4 derivatives containing either a ubiquitous (CAG) or a liver-specific (hAAT) promoter promoted a higher reporter gene expression level than the control plasmid. With the pFAR4 vector combined to the hAAT promoter, transgene expression was found prolonged, for more than 6 months. Substitution of the reporter gene by the SGSH cDNA similarly led to high and prolonged serum levels of sulfamidase protein that was efficiently taken-up by neighboring organs after its endocytocis by mannose 6-phosphate receptors-expressing cells, leading to a correction of GAG accumulation in somatic organs. A similar reduction in GAG content was also observed in the brain, at early stages of the disease. Thus, this study contributes to the effort towards the development of novel biosafe non-viral gene vectors for therapeutic protein expression in liver, and represents a first step towards an alternative gene therapy approach for the MPS-IIIA disease.
Modified chitosan biopolymers for efficient nanoparticle formation and transfection of splice switching oligos
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Antisense oligonucleotide (ON)-mediated gene therapy through the use of splice switching oligonucleotides (SSO) holds great therapeutic potential. Although the use of “naked” ONs for cell delivery in vivo is possible, development of delivery vectors for this specific class of molecules could improve their efficiency, achieve a specific cell targeting and decrease toxicity levels. To this end, chitosan, a biodegradable and biocompatible biomaterial-based vector, associated with negligible toxicity effects, holds great potential as a nucleic acid carrier. Yet, its ability to deliver SSOs has not been duly explored. In this work we use a trymethylated chitosan (TMC), possessing permanent positive charges at physiological pH, and further modified it with stearic acid groups for improved SSO complexation and condensation into nanoparticles. The modified TMC was found to induce formation of nanoparticles under 200 nm in size with narrow polidispersity index, with SSO binding efficiencies above 90%. Importantly, hydrophobic chain conjugation was found critical for stability of the TMC/SSO nanoparticles in presence of serum proteins. Cell binding, internalization mechanisms and dynamics were analyzed by flow cytometry and confocal imaging. We further report on the delivery efficiency of SSOs in vitro in a luciferase splice-correction model cell line and in differentiated C2C12 mouse myotubes for dystrophin exon skipping. At optimal concentration and N/P molar ratio of 80 (positive Nitrogen/negative Phosphate) the chitosan nanoparticles were found to mediate above 50% of the correction levels attained with Lipofectamine. Our results highlight the potential and warrant further development of this vector for SSO delivery.
Toward delivery of mRNA to cells with lithotripter shock waves
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mRNA transfection is conceptually more efficient than the commonly delivered plasmid DNA because it does not need to impart damage to the nuclear envelope thereby increasing the chances of the cell remaining viable. Furthermore, shock waves (SWs) have been shown to induce cellular uptake by transiently altering the permeability of the plasma membrane, thereby overcoming a critical step in gene therapy. However, accompanied shock wave bioeffects include dose dependent irreversible cell injury and cytotoxicity. In this work, we investigated the effect of lithotripter SWs on the viability and permeabilisation of mouse colorectal carcinoma (CT26.WT) cells in vitro. Viability was assessed by MTS assay and permeabilisation by the internalisation of propidium iodide and 250-kDA FITC-dextran. SWs were delivered by means of a clinical lithotripter (SLX-F2, Storz Modulith) and cell response assessed as shock wave amplitude, number and pulse repetition frequency. Optimal parameters were determined which resulted in 20.5% of live cells being permeabilised whilst maintain cell survival rates at 81%. Post shock wave stability of eGFP mRNA was evaluated using the rabbit reticulocyte lysate cell-free translation system. For optimum shock wave parameters no statistical significance (p<0.05) was detected between shock wave-exposed and sham samples suggesting the SWs did not damage the mRNA. Subsequent studies demonstrate response to SWs was sensitive to cell and uptake molecule concentrations. One promising result was the SWs propagated in agar instigated increased FITC-dextran distribution and depth with increasing concentration. These results encourage enhanced transfection efficiencies, laying the foundation for non-invasive and high safety RNA-based gene therapy.
Chitosan-based vectors mediate retinal delivery and long-term gene expression
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One of the major hindrances to a successful non-viral gene therapy has been the low gene transfer efficiency and short transgene expression. Our goal is to develop chitosan-based non-viral vectors optimized for retinal gene therapy with continued gene expression. Chitosan-pDNA polyplexes were formulated and characterized regarding their physical properties, gene transfer efficiency on both retinal pigment epithelial and HEK293 cells, and in vivo biocompatibility. Our results show that chitosan polyplexes have size and surface charge consistent with gene delivery. When considering off the shelf applications, vector stability is crucial. Our vectors are stable both in storage (4°C) and physiological conditions (37°C), and remain stable after several freeze-thaw cycles. Following moderate in vitro efficiency, in vivo, upon sub-retinal injection, there is an absence of inflammatory response and sustained transgene expression in RPE cells at least 6-month post-injection. These results show that chitosan-based vectors can successfully transfer genes to the retina. The authors acknowledge the financial support of Fundação para a Ciência e Tecnologia (PTDC/SAU-BEB/ 098475/2008 to G.A. Silva, SFRH/BD/70318/2010 individual fellowship to A.V. Oliveira) and to IBB/LA under the project PEst-OE/EQB/LA0023/2013 and the Marie Curie Reintegration Grant (PIRG-GA-2009-249314 to G.A. Silva) under the FP7 program.
Bio-inspired approaches for siRNA delivery
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The major unresolved challenge in the field of RNAi therapeutics remains to reconcile both safe and effective drug delivery. This abstract describes several bio-inspired approaches that are currently under investigation in our group in the context of small interfering RNA (siRNA) delivery. Following the broad application of hydrogels as controlled drug release matrices, we designed polysaccharide hydrogel nanoparticles (nanogels) based on dextran as nanocarriers for siRNA. The biological performance of our nanogel formulation was investigated both in vitro and in vivo. Combining the strengths of in vivo SPECT/CT imaging in mice with ex vivo advanced microscopy tools, we were able to demonstrate the size-selective disassembly of the nanogels at the kidney glomerular basement membrane. In addition, we designed hybrid lipid-polymer nanocomposites, based on dextran nanogels and clinically approved pulmonary surfactant, significantly improving colloidal stability and cellular siRNA delivery. A second approach describes the reversible coupling of nanoparticles to the surface of tumoritropic cytotoxic CD8+ T cells; a strategy that we are exploring to allow a better penetration of nanomedicines into tumors and to obtain a synergistic anti-tumor effect in combination with adoptive T cell therapy. Finally, we are investigating the possibility to exploit extracellular vesicles (EVs) as bio-inspired siRNA delivery tools. We were able to demonstrate that aggregation artifacts during electroporation obscure the actual encapsulation efficiency of siRNA into isolated extracellular vesicles, emphasizing the need for alternative methodologies to improve siRNA loading of EVs to extend their therapeutic application.
Nanoparticles with Tuneable Surface Properties Fabricated with Oligopeptide-terminated Poly(ß-amino ester)s for Highly Efficient Gene Delivery
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Non-viral vectors offer potential advantages, including infinite packing capacity, ease of production and low immunogenicity, but are less efficient than viral vectors. We have recently developed a new family of oligopeptide-modified poly(ß-aminoester)s (pBAEs) as promising nucleic acid delivery vectors with improved features in terms of particle formation, transfection efficiency and biocompatibility. Interestingly, DNA particles obtained from oligopeptide-modified pBAEs showed different intracellular distribution – i.e. perinuclear or cytoplasm – depending on their oligopeptide composition. Here we present an extended family of pBAEs that incorporate terminal oligopeptide moieties synthesized from both positive and negative amino acids. Polymer formulations of mixtures of negative and positive oligopeptide-modified pBAEs are capable of condensing both DNA and siRNA into discrete nanoparticles. We have demonstrated that efficient delivery of nucleic acids in a cell-type dependent manner can be achieved by careful control of the pBAE formulation. In addition, our approach of adding differently charged oligopeptides to the termini of poly(ß-amino ester)s is of great interest for the design of tailored complexes having specific features, such as tuneable zeta potential, which may also result in improved nanoparticle stability. This is of great interest to develop vectors that are able to withstand biological barriers, which are known to be incompatible with the highly positive charge of conventional polyplexes.
(1) Segovia N, Dosta P, Cascante A, Ramos V, Borrós S, Oligopeptide-terminated poly(ß-amino ester)s for highly efficient gene delivery and intracellular localization, Acta Biomater. 2014 May;10(5):2147–58.
Tissue hAAT gene expression but low secretion after pig liver hydrofection “in vivo”
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Biodistribution of anthrax DNA vaccine delivered by intramuscular injection and electroporation Yeonsu Kim, Na Young Kim, Jai Myung Yang, Sungho Shin (shshin@sogang.ac.kr ) Department of Life Science, Sogang University, Seoul, 121-742, Korea
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DNA vaccination is novel method for prophylactic vaccination against infectious pathogens. Anthrax, the most probable bioweapon-induced disease, is caused by the bacterium Bacillus anthracis. Since DNA vaccines have the potential to provide protection against biological agents, in present study, we have studied anthrax DNA vaccine biodistribution after intramuscular injection with or without electroporation. The 20 kDa domain IV of PA protein gene was used for antigen. After administration into Balb/c mice with 3.5×1013 copies of purified DNA vaccine plasmids, antigen gene was extracted and assayed by absolute quantification method of real-time polymerase chain reaction. Our results demonstrated that plasmids were detected in all tissues and organs such as brain, heart, lung, kidney, spleen, and liver, the tissue of injection site, and blood, 48 hours post administration without specific tissue tropism. In addition, the dramatic increase in delivery efficacy provided by electroporation. For example, electroporation increased levels of plasmid the injection site by approximately 103–104 times. Furthermore, with the two-needle electrode, electroporation could deliver about 10 times more plasmid DNA into tissues than using the tweezer electrode. Taken together, our results suggest that this DNA vaccine provides long-lasting effectivity and the lack of tropism ensures the safety, which represents an attractive candidate for further development.
Efficient non-viral gene delivery by minicircle Sleeping Beauty transposon system into hematopoietic stem cells for gene therapy applications
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The Sleeping Beauty (SB) transposon system is a non-viral, plasmid-based gene delivery platform that combines simplicity, inexpensive manufacture and advantageous, close-to-random profile of genomic integration. SB has been widely applied in preclinical settings to successfully treat a variety of inherited and acquired disorders, and is currently in clinical use in the context of human T cell therapy. However, efficient correction of hematopoietic stem cells (HSCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. Our goal was to decrease excessive cell toxicity associated with electroporation of HSCs, and to increase gene delivery efficiency – both largely dependent on the amount and size of the plasmid vector to be delivered. Our attempts to improve SB gene transfer into hard-to-transfect HSCs were performed utilizing the minicircle technology devoid of bacterial plasmid backbone sequences. Minicircle SB vectors generated from parental plasmids by recombination event are reduced in size, which facilitates their easier penetration through cellular and nuclear membranes and therefore introduction of higher numbers of molecules per cell. We established greatly improved SB transposition and stable gene delivery in HSCs by delivering the SB transposon components in minicircles. Minicircles allowed decreasing the overall dose of DNA without compromising the efficiency of gene delivery, which also resulted in a reduced electroporation toxicity. Implementation of minicircle technology offers a possibility to further refine the SB transposon system for clinical translation in the context of HSC-based gene therapy to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.
Experimental gene therapy for carbohydrate and lipid metabolism disorders in rabbit
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Effects of Platelet Derived Growth Factor (PDGF) on proliferation, apoptosis and PDGF-R expression by MSCs
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Optimization of Genetic Barcode Design
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Marking of cells with genetic barcodes (BCs) is an expression-independent method which has been applied to analyse clonal composition and kinetics during reconstitution of the haematopoietic system. Genetic barcodes are short sequences introduced into cells by viral vectors. Individual barcodes are quantified after PCR amplification from genomic DNA by next-generation sequencing (NGS) and sophisticated bioinformatic analysis. In order to investigate susceptibility of barcode marking for errors generated during analysis, we used our recently introduced lentiviral BC16 system. We mixed four cell clones with known BC16-sequences and integration sites at equal proportions to generate specific “minibulks”. After NGS analysis, we observed an unexpected high frequency of “false” (descendent) barcodes, which could not be explained by NGS errors. We reasoned that they were due to PCR-based errors and supported this by computer simulations. Modelling of individual barcodes and their deterioration during PCR amplification allowed us to derive predictions for experimental optimisations. Based thereon, we systematically changed PCR conditions, using, e.g., high-fidelity polymerase, prolonged elongation times to get rid of unfinished PCR products, and lower cycle numbers. Only reduction of PCR-cycle numbers resulted in a clear decrease in the frequencies of descendent barcodes. We conclude that the PCR-amplification step is critical for NGS-based analysis of barcode frequencies. Incidences of artificially generated barcodes can be reduced by optimising vector design and by decreasing PCR-cycle numbers required for barcode retrieval. We have developed optimised barcode constructs by including adaptor sequences for NGS and increasing barcode length for better discrimination.
Effect of inflammatory environment on equine bone marrow derived mesenchymal stem cell (BM-MSCs) proliferation, differentiation and immunomodulatory gene expression profile
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BM-MSCs are being investigated as a new treatment for equine osteoarthritis because of their immunomodulatory ability. Inflammatory conditions can induce an enhancement of this property. The aim of this work is to analyze the response of BM-MSCs to three different inflammatory environments. BM-MSCs from three animals were exposed to: a) 20% allogeneic inflammatory synovial fluid (SF); b) 20 ng/ml of TNFα and IFNγ CkC20) and c) 50 ng/ml of these pro-inflammatory cytokines (CkC50). After 72 hours, proliferation and differentiation abilities, and expression of 19 immunomodulatory-related genes were analyzed. BM-MSC proliferation and tri-lineage differentiation potentials kept unaltered when SF exposure, however they were diminished after both CkC20 and CkC50 exposure. A significant higher gene expression of IL6, IDO and VCAM, and a decrease of COX1 and CyclinD2 were observed in both CkC conditions. A significant overexpression of COX2, MHCII and CD40, and a decrease of CD40L were observed in CkC50 but not in CkC20 conditions. On the other hand, a significant higher IFNγ and a lower IL10 expression were observed in CkC20 but not in CkC50. Under SF conditions, ALCAM expression was found significant higher. No expression for IFNγ and IDO was found. These outcomes suggest that BM-MSCs change their immune and inflammatory-phenotype according to the surrounding environment, and may be dose-dependent of the stimulus. This provides a better understanding about the behavior of BM-MSCs inside the inflamed joint and constitutes a previous step for the usage of MSCs as equine osteoarthritis treatment.
Development and characterization of systems for the in vivo delivery of FGF-2
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A variety of human pathologies such as critical limb ischemia, stroke, etc. are a consequences of severe blood flow violation resulted in the tissue ischemia. Various studies have proven that the administration of several pro-angiogenic growth factors could induce neovascularization in injured tissues. FGF-2 appears to be a candidate as a potentiating agent for therapeutic angiogenesis. However, FGF-2 application is limited by the need to maintain its prolonged local release at levels sufficient for the angiogenesis stimulation. Thus, the present study focuses on the development of systems for a delivery of FGF-2 and on their characterization. The scaffolds were prepared by freeze-drying collagen I solutions containing the polymer developed on the basis of cross-linked modified heparin. The scaffolds structure was analyzed by SEM, AFM and CLSM. The non-viral gene transfer method, RT-PCR, Western blot analysis were used in the study. It was determined by SEM and CLSM that the scaffolds mainly had a layered structure with pores (from 76 to 150 μm) forming a connections between the layers. Scaffolds containing the developed polymer were able to incorporate FGF-2. The expression vector pC1-F contained the fgf-2 gene had been constructed. This vector was used for the CHO-K1 cells transfection. As a result, a stable transgenic cell line expressing FGF-2 was obtained. Proposed systems promoted angiogenesis in CAM assay. The developed porous functionalized collagen scaffolds incorporating FGF-2 and the transgenic mammalian cell line, expressing FGF-2, can be used as a vehicles for the sustained delivery of the growth factor.
Optimizing megakaryocytic differentiation protocols from hPSCs
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Megakaryocytes (MKs) are bone marrow cells originating from hematopoietic stem cells (HSC) by a differentiation process known as megakaryopoiesis. Subsequently, these cells mature to produce of platelets, a process denominated thrombopoiesis. Different labs have generated MKs and platelets in vitro from HSCs purified from bone marrow, umbilical cord or fetal liver tissues. However, culture and expansion capacities of these cells are limited. In contrast, human pluripotent stem cells (hPSCs) represent a more advantageous system with an unlimited growth and the potential to differentiate into any cellular lineage, including MKs. During megakaryopoiesis more immature MKs grow under very low oxygen tensions (pO2=1–2%) and THPO is required. Then, mature MKs move to the bone marrow sinusoidal space where oxygen tension increases (pO2=5–6%) and THPO concentration goes down. Currently; megakaryocytic differentiation protocols from hPSCs are inefficient. We hypothesized that these protocols would improve under hypoxic conditions or reducing thrombopoietin (THPO) throughout thrombopoiesis. To test this hypothesis we generated mature MKs and platelets from hPSCs under different hypoxic conditions or reducing THPO at final differentiation stage. Our results showed that hypoxia reduces cell viability and number of megakaryocytic progenitors. Importantly, those progenitors selected under hypoxic conditions have a higher megakaryopoyetic potential, increasing both the number of mature MKs and platelets, especially under more physiological conditions. In addition, THPO reduction after day 20 increased platelet production and the ratio Platelet/MK. These results demonstrate that hypoxia and THPO are important factors to consider for the development of ex vivo systems to generate human platelets from hPSCs.
Effect of contact lens surface on canine Mesenchymal Stem Cell (MSCs) gene expression profile
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Mesenchymal Stem Cells (MSCs) represent an excellent tool to improve the healing of corneal ulcers. MSCs are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide array of range of therapies. The main difficulty when using MSCs to treat corneal ulcers is their application and maintenance in the injured area. To solve this handicap organic or inorganic scaffolds can be used. However organic scaffolds such as amniotic membrane need to be sewn to the injured area. These surgical procedures require general anaesthesia in usually aged patients in which the anaesthesia is an additional risk. Instead of exposing our animal to a surgical procedure, the contact lens is a non-invasive alternative that can be applied without surgery. In this study we observed the effect of a common commercialised contact lens surface on canine MSCs gene expression profile. The positive expression of specific MSCs markers such as CD90, CD105 and CD73 was confirmed by RT-qPCR assay confirming their mesenchymal origin. Additionally the absence of expression in hematopoietic markers CD184, CD29, CD34 and CD45 was also confirmed by RT-qPCR. Moreover, the expression of cell surface adherence markers ICAM1 and VCAM1 where significant upregulated in contact lenses cultured MSCs when compared with control MSCs. This may indicate a higher ability to adherence and migration to the lesion, improving the results of the treatment.
Differential gene and miRNA profiling in dystrophic dog and impact of MuStem cell-based therapy
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Duchenne Muscular Dystrophy (DMD), the most common form of inherited neuromuscular disorder, is caused by mutations in the dystrophin gene leading to the protein lack. The Golden Retriever Muscular Dystrophy (GRMD) dog model has increasingly been used to assess efficacy of therapeutic approaches. The aim of the study was to establish a molecular characterization of this clinically relevant model and the consequences of a systemic delivery of MuStem cells, muscle-derived stem cells generating dog clinical benefit (Rouger et al., 2011). Interestingly, we report for the first time the GRMD dog muscle transcriptome using gene microarrays. Disease-related deregulation is observed for 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. We also define the expression levels of six miRNAs including myomiRs in GRMD dog muscle by RT-qPCR and present for their distribution by in situ hybridization. Importantly, we reveal the involvement of several biological pathways following MuStem cell transplantation with the identification of 31 genes highly modulated. Ubiquitin-mediated protein degradation as well as muscle regeneration are enhanced in MuStem cell-transplanted GRMD dog. Concomitantly, we show a down-regulation of a set of genes having metabolic functions associated with lipid homeostasis and energy. In addition, we demonstrate a corrective effect of MuStem cell therapy on the muscle expression of miR-1, miR-133 and miR-486. Here, we precise the pathophysiological events that trigger the muscle fiber degeneration in DMD model and pave the way to the understanding of MuStem cell action modalities.
A Protocol for Cell Transfection with Nanomagnetic Formulations of mRNA Therapeutics
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Recently, stabilized non-immunogenic messenger RNAs (SNIM RNAs) encoding therapeutically relevant proteins have attracted increasing attention worldwide as a substitute for gene therapy. Unlike DNA, SNIM RNAs do not harbor the biological risk of inserting into the patient's genome; hence they are conceptually safer than gene therapy agents. Furthermore, SNIM RNAs only need to reach the cytoplasm to become active while DNA needs to reach the nucleus. In our experiments, three different SNIM RNAs encoding for reporter genes (Luciferase, RFP and GFP) were synthesized. Then Primary Mouse Embryonic Fibroblast (PMEF) and Porcine Fetal Fibroblast (PFF) cells were transfected in mono-and-co-culture with single or combinations of SNIM RNAs using polifection, lipofection and magnetofection methods. Toxicity was investigated with MTT test. According to the results, co-transfection of different SNIM RNAs is not only feasible, but also can improve the transfection efficacy. Expectedly, magnetofection improves co-transfection and transfection efficacy as well. It was noticed that same magnetic nanoparticles behave differently in transfection on mono-culture and co-culture of cells. Finally, MTT results indicated that co-transfection does not significantly increase the toxicity, and cells can tolerate this higher amount of nucleotides and enhancers. In conclusion, we established a well optimized protocol for co-transfection of different SNIM RNAs on mono-and-co-culture of different cell lines. This is relevant in research and therapeutic applications when simultaneous translation of several mRNA-encoded proteins is required.
The Sleeping-Beauty Transposon Technology for the generation of cellular and animal models of Haemophilia B and the assessment of RNA-based therapeutic approaches
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Gene replacement therapy strategies are potentially limited in scope by the lack of physiologic gene regulation and the size of the transgene expression cassette. Variants of the U1 small nuclear RNA (U1snRNAs) were shown to rescue gene expression impaired by splicing mutations, in an approach that may circumvent the pitfalls of gene addition strategies. However, the evaluation of the U1snRNAs efficacy and safety requires cellular and animal models specific for a given mutation. Here we exploited the hyperactive Sleeping Beauty transposon technology (SB-100X) to create models of Haemophilia B (HB) caused by the IVS5-2a/c or IVS5-8t/g splicing mutations. By transfecting Hek293 cells with expression plasmids encoding for human splicing-competent FIX minigenes (pSC-hFIX) together with the SB-100X, we generated stable cell lines harboring the SC-hFIX cassette. Cells expressing the mutant minigenes displayed an aberrant splicing pattern characterized by exon 5 skipping, which was partially rescued (∼45%) with U1snRNAs. Correction of the RNA defect resulted in the detection of secreted hFIX protein in medium, providing the rationale for the creation of transposon-based mouse models. Preliminary experiments were conducted through hydrodynamic injection of plasmids carrying SC-hFIX variants together with the SB-100X in wild-type C57BL/6 mice. Measurable levels of FIX protein were detected only in animals injected with the wild-type pSC-hFIX. Rescue of mutant hFIX minigenes in vivo with U1snRNAs is ongoing, together with experiments in HB mice to validate the model and define the efficacy and safety of AAV-mediated delivery of U1snRNAs to correct the bleeding phenotype caused by FIX splicing mutations.
AAV-mediated antisense oligonucleotide delivery is an effective therapeutic approach for CEP290-associated LCA
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Leber congenital amaurosis (LCA) is a genetically heterogeneous disorder characterized by severe visual impairment starting in the first year of life. The most frequent genetic cause of LCA, present in up to 15% of all LCA cases in some European and North American populations, is an intronic mutation in CEP290 (c.2991+1655A>G) that creates a cryptic splice donor site that results in the insertion of an aberrant exon into CEP290 mRNA. We previously showed that antisense oligonucleotides (AONs) effectively restore normal CEP290 splicing in patient-derived lymphoblastoid cells. We aimed to combine AON technology together with adeno-associated virus (AAV) delivery to transduce patient-derived fibroblast cells and assess the AON-containing AAVs therapeutic effect. AONs cloned into a modified U7snRNA construct and packaged into AAV2/2 fully restored normal CEP290 pre-mRNA splicing and significantly increased CEP290 protein levels. Moreover, a ciliary phenotype present in these fibroblasts was completely rescued upon transduction of AON-containing AAVs. Together, our data show that AAVs are an excellent therapeutic vector for the delivery of AONs to redirect splice defects, and highlight the tremendous potential that AONs hold for the treatment of CEP290-associated LCA.
Enhanced microRNA suppression activity of RNA pol II-transcribed Tough decoy inhibitors fused to WPRE
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Dysregulation of microRNA (miRNA) expression and thus changed expression of miRNA target genes is associated with development of a range of diseases. To achieve efficient suppression of miRNA activity in experimental and therapeutic contexts different miRNA inhibitor designs have been studied. Previously, we showed high potency of vector-encoded hairpin-shaped Tough Decoy (TuD) miRNA inhibitors and improved the design to obtain increased and synchronized inhibition of two or more miRNAs. To further refine the guidelines for production of efficacious RNA pol II-transcribed TuDs, we studied here the importance of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) for function of vector-encoded TuDs. For a panel of four TuDs, each targeting a specific miRNA, we consistently observed levels of suppression that were twofold higher when the TuD was fused to the WPRE RNA element relative to TuDs without WPRE. These findings indicate that optimized nucleocytoplasmic transport is crucial for TuD function and that the inclusion of WPRE is essential for miRNA suppression by RNA pol II-transcribed TuDs. Based on the discovery of naturally occurring circular RNAs with miRNA sponge activity, we hypothesized that TuD function could be further improved in the context of RNA circles. Notably, miR-7-targeting TuDs suppressed miR-7 activity with more than four-fold higher efficiency than an expressed circular RNA sponge containing 73 seed-targets for miR-7. We have initiated the production of circular RNAs containing TuDs targeting a panel of miRNAs and are currently investigating the potential anti-miR activity of such engineered miRNA suppressors.
Development of cancer gene therapy based on cancer-specific RNA replacement through microRNA regulation
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We have previously presented that trans-splicing ribozymes based on Tetrahymena group I intron could be potent anti-cancer devices through diminishing target RNA level and simultaneously triggering anti-cancer gene activity only in cancer cells expressing the target RNA. Particularly, hTERT-targeting ribozyme was shown to specifically and efficiently retard tumors in various xenograft carcinomatosis nude mouse models. However, concerns for the hTERT-targeting approach include potential side effects to proliferating hTERT+ normal cells. In this study, to improve cancer-specificity of the ribozyme, we developed cancer-specific ribozyme expression construct via microRNA regulation. To circumvent hepatocellular carcinoma (HCC) as target models, we created tissue-specific microRNA-regulated ribozyme system by incorporating perfect antisense sequence against normal hepatocyte-selective miRNA-122a that is down-regulated in HCC to the 3′-UTR region of ribozyme. Adenovirus encoding the modified hTERT-targeting ribozyme specifically and efficiently induced transgene and cytotoxicity in liver cancers through cancer-specific RNA replacement. Moreover, specificity of cancer regression was highly increased without compromising its anti-cancer efficacy in xenograft orthotopic HCC model. Furthermore, to assess the effect of this system in syngeneic model, we constructed mTERT-targeting ribozyme harboring miRNA-122a target sites. Adenoviral vector encoding the modified ribozyme selectively and efficiently regressed mTERT+ HCC with minimal liver toxicity due to least ribozyme expression in the normal livers in syngeneic orthotopic mouse model. Noticeably, cytotoxicity caused by the adenovirus induced specific immunogenic cancer cell death in the syngeneic model, generating specific immunity to the cancers. Therefore, specifically targeted transgene induction through microRNA-regulated RNA replacement provides novel strategies for efficient and safe cancer gene therapy.
Elimination of cervical cancer in vivo by a short-hairpin RNA targeting human papillomavirus type 16 E6/E7
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Human papillomavirus (HPV) is the major causative agent of cervical cancer. The HPV oncoproteins E6 and E7 induce carcinogenesis by inactivating host tumor suppressor genes. Therefore, stable expression of specific inhibitors of E6 and E7 in cancer cells could provide effective treatment without affecting normal tissue. Here, we propose a novel therapeutic approach that uses an adeno-associated virus (AAV) vector encoding a short-hairpin (sh) RNA to target E6 and E7 (shE6E7) of HPV type 16 (HPV-16). Three different HPV-16-positive cervical cancer cell lines (BOKU, SiHa, and SKG-IIIa) were tested for gene transfer efficiency using AAV vectors of different serotypes. In all three cervical cancer cell lines, the highest gene transfer efficiency was obtained using AAV2 vectors. The proportions of GFP-positive cells at the dose of 1×105 vg/cell were 87.3%, 98.3%, and 87.9%, respectively. Transduction of cervical cancer cells by AAV2-shE6E7, an shRNA-encoding AAV vector that targets both E6 and E7, resulted in apoptosis, G1 arrest, and cell growth inhibition. In transduced cells, E6, E7, and p16 expression was reduced, whereas p53, p21, and pRb expression was enhanced. Next, AAV2-shE6E7 was directly injected into cervical cancer cell-derived subcutaneous tumors in mice. Tumor growth was markedly inhibited by a single AAV2-shE6E7 administration, and most tumors showed complete regression without any adverse effects. These results suggest the utility of AAV2-shE6E7 as a novel therapeutic approach for cervical cancer.
Magnetic microbubbles and ultrasound enhance adenoviral transduction of HUVEC in an in vitro flow system
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Increased uptake by Kupffer cells and reduced liver transduction and toxicity following serotype 5 adenovirus pseudotyping with serotype 3 fiber
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The use of adenovirus 5 (Ad5) in gene therapy has been hampered by the strong liver tropism of the virus and associated hepatotoxicity. Mutations of hexon protein ablating Ad interaction with blood coagulation factor X were previously shown to dramatically reduce hepatocyte transduction. Interestingly, pseudotyping Ad5 fiber with a fiber from Ad3 also led to a strong reduction in hepatocyte transduction. We report reduced liver and spleen transduction 2 days after systemic administration of Ad bearing either whole or only the shaft of the Ad3 fiber. Liver transduction by these vectors was further reduced after FX depletion, demonstrating their efficient use of FX for hepatocyte transduction in vivo. While both Ad did not show significant difference in initial liver uptake, they were cleared from the liver more rapidly. This phenotype was attributed to an intrinsic property of the Ad3 fiber, since Ad5 pseudotyped with Ad3 fiber as well as Ad3 were strongly taken up by Kupffer cells. Finally, an Ad pseudotyped with Ad3 fiber was shown to efficiently transduce tumors while avoiding hepatocyte transduction after dissemination from tumor site of administration. Taken together, our data demonstrate that the nature of the Ad fiber has a strong impact on in vivo Ad behavior.
Compartmentalized liver transduction: a new paradigm in adenoviral gene therapy
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Still unresolved complications associated with adenoviral transduction of the liver are a strong immune response followed by tissue damage and loss of transgene expression. To stringently control and assess two critical variables at the core of these adverse events, viremia and high quantities of vector dose, we implemented a novel mode of vector administration: compartmentalized liver transduction (CLT). CLT is achieved by arresting blood circulation to a portion of the liver, delivering the adenovirus through an intra-parenchymal injection, allowing viral endocytosis to be completed and reestablishing blood flow. Implementing CLT in a rat model, reporter protein was detected with unprecedented low doses of vector (10^2), gene expression was confined to the site of injection, viremia was reduced to a minimum, no vector genomes were detected in distal tissues/organs, linear dose response curves were obtained, no adverse effects were observed including transaminase elevation, apoptosis and tissue damage, and long-term gene expression was documented (12 months). Results were reproduced with E1a-E1b deleted and E1-E3 deleted adenoviral vectors. Our results conclude that CLT induces in situ tolerance enabling long term protein synthesis and secretion. We further conclude that CLT is a safe and effective mode of vector delivery to the liver with a promising future in the field of gene therapy.
Testing the safety of novel lentiviral vectors with a reduced splicing interference potential
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The excellent therapeutic potential of self-inactivating (SIN) lentiviral vectors (LV) has been demonstrated in pre-clinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could endanger their clinical applications. Systemic vector injection into newborn tumor-prone Cdkn2a-/- and Cdkn2a+/- mice, conducted in our previous work, demonstrated that SINLVs harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset with respect to control mice. Integration sites analyses of vector-induced tumor showed that oncogene activations or tumor suppressor inactivations by LV integrations occur by combining mechanisms of transcript truncation, induction of aberrant splicing and/or enhancer-mediated overexpression of cellular transcription units. To reduce genotoxic splicing-capture events, we designed LVs harboring sequences complementary to microRNAs (mirT sequence), which are active in hematopoietic cells (mir223 and mir142-3p) within the SIN LTR or in the vector backbone and outside the gene expression cassette that allow selective degradation of vector-mediated aberrantly spliced transcripts. Transduction of mirT SINLVs in human CD34+ cells induced a fivefold reduction of the amount of vector-mediated aberrant splicing events versus control vector. We are currently performing RNA sequencing on CD34+ cells transduced with mirT SINLVs in order to quantitatively and qualitatively characterize the vector-mediated aberrant transcript events. Furthermore, we are testing these new mirT-vectors in our tumor prone mouse models to evaluate their genotoxicity and to dissect the modality through which the new mirT integrated proviral genome interacts with the surrounding cellular genome. These studies are fundamental to inform current and future choices of vector design with improved biosafety.
Presynaptic NMJ aCAR-targeted Lentiviral Vector for Neuroprotective Gene Therapy in ALS
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Aiming to overcome the limitations of existing viral vectors for non-invasive CNS delivery, we have engineered novel lentiviral vectors with tropism to Motor Neurons (MNs) delivered via the Neuromuscular Junction (NMJ). We have described the generation of coxsackievirus and adenovirus receptor (aCAR)-targeted vector, which exhibits binding specificity and efficient transduction of target cells in vitro. We have shown that in vivo intramuscular (i.m.) delivery of aCAR-targeted vector in mouse leg muscles results in specific retrograde transduction of MNs (and not DRGs). Utilizing the aCAR-targeted vector, the neuroprotective effects of lentivirally expressed IGF-1 are currently being investigated in vivo for inducing neuronal survival and delaying or ameliorating neuropathology and behavioural phenotypes associated with the SOD1G93A mouse model of ALS. To achieve this we cloned IGF-1 into HIV-1 based vectors under the CMV promoter and assessed expression of these proteins via Western Blot analysis. Utilizing cell factories, we produced high titer lentiviral preparations, which we subsequently delivered i.m. to key muscle groups prior to onset of disease (28d). Motor performance, coordination and gait analysis were assessed weekly from 70d onwards, for both treated and control groups. Thus far we have observed therapeutic efficacy in vivo with IGF-1 pre-treatment with up to 15% extension of survival compared to eGFP treated controls, which was linked to improved motor performance. More cohorts of male and female mice are being treated and MN integrity at endstage is assessed. aCAR-targeted vector appears to be a good candidate for minimally invasive neuroprotective gene therapy of ALS.
Clonality estimation in transuced cell populations using a generalized measure of diversity
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The location of viral vector integration can be taken as unique cellular marker for each transduced cell and its clonal progeny. Current gene therapy (GT) studies use classical diversity indices such as Shannon and Simpson for estimating a sample's clonality, which may be biased towards richness or evenness, leading to contradictory results. These shortcomings in clonality analysis demands a framework and a proper clonality index, which allows a robust and reliable strict determination of the actual clonal diversity present in a sample. Starting from Rényi numbers, a generalized form of entropy used in information theory, we have designed a new Polyclonal Monoclonal Diversity index (PMD), which estimates clonality/diversity of transduced cell populations without a preference for evenness or richness. PMD results on artificial datasets showed the superior clonal diversity determination compared to the Shannon and Simpson index. Applying the PMD index on preclinical GT data, the analysis showed no induction of selection. A sequential decrease in clonal diversity is observed from pre-transplant culture to primary and to secondary animals. Transduced cell clones present in higher abundance were not shared between pre-transplant, primary and secondary samples. We were able to show that technical factors, i.e. restriction enzymes used during LAM-PCR, vector type and animal sex did not affected the study clonality. To conclude, the development and implementation of PMD index circumvent previous restrictions in clonal diversity measurements. It shows its value mainly in high-throughput insertion-site analyses where it is central to compare clonality studies and to detect clonal distortions in samples.
Engineering of synthetic AAV capsids via rational assembly of “Virobytes”
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Vectors derived from Adeno-associated viral (AAV) serotypes represent powerful tools for therapeutic DNA delivery because of numerous assets, including their small genome which makes AAV highly amenable to genetic engineering. Consequently, we and others have previously created libraries of chimeric AAVs through homology-based shuffling of capsid genes from multiple wildtype isolates. Here, we report a new strategy which no longer relies on natural homologies but rather permits rational and directed assembly of synthetic AAV capsids from pre-designed “Virobyte” fragments. To establish this methodology, we segregated each capsid gene of AAV serotypes 2, 8 and 9 into five Virobytes that comprised different hypervariable regions and that are flanked by type IIS DNA endonuclease restriction sites. The latter were designed such that after cleavage, each Virobyte carries unique overhangs permitting their directional, potent and rapid reassembly in a combined endonuclease/ligase reaction. Unlike conventional AAV shuffling, this allows for the rational recombination of hypervariable capsid parts from different serotypes, while preserving the integrity of the assembly-activating protein AAP which is easily disrupted during homology-based shuffling. Thereby, our Virobyte approach is not only useful to create libraries for selection of desired candidates, but also to dissect the function of specific capsid parts in juxtaposition with others. Moreover, we present a software package called “Salanto” that we developed for the analysis of synthetic AAV sequences and that is now publically available. As a whole, our new method and tools should substantially advance the fields of fundamental AAV research and of AAV vector engineering and application.
Evaluating biodistribution of replication-competent retroviral vector (RRV) delivered via tracheal instillation and intravenous injection in lung cancer models
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As a tool for cancer treatment, oncolytic virotherapy is an emerging field targeting rapidly proliferating cancer cells using replicative capabilities of viruses. Replication-competent retroviral vector (RRV) is one of most promising candidates, which uses intrinsic tumor selectivity of murine leukemia virus (MuLV) for cancer targeting strategy. MuLV is known to have high selectivity and stability in gene transfer throughout solid tumors. Application of RRV in vivo, however, brings questions with safety concerns and biodistributions when introduced both locally and systemically. Therefore development of method for evaluating RRV in vivo is critical. Primer set for quantitative PCR (qPCR), specifically targeting env region of MuLV was designed and confirmed that it has detection limit of 102 copies in 100 ng tissue genomic DNA. Standard curves were generated at 7 different concentrations and showed linearity (R2>0.99). Two types of lung cancer model mice, CT-26-induced lung cancer metastasis model and K-rasLA1 genetically engineered spontaneous lung cancer model, were used to check biodistribution. RRV, encoding eGFP gene, was prepared with additional buffer change to minimize serum content and 100 ul (1×108 TU/ml) was introduced to lungs via tracheal instillation (local) and tail vein injection (systemic). Mice were monitored and sacrificed at regular intervals. Genomic DNAs were isolated from tissues including lung (normal/tumor) and bone marrow. Based on the standard curve generated, biodistribution of RCR was evaluated with isolated genomic DNA from each tissue and confirmed that qPCR primer set designed to detect env region of RCR can be applied for safety evaluation in in vivo.
Adenoviral vector system inhibiting VA RNA for pri-miRNA delivery
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Recombinant adenoviral vector has been generally used to deliver and express tumor suppressive primary miRNA (pri-miRNA) in cells for cancer gene therapy. Pri-miRNA expression through transfection with plasmid vector is normally processed into mature miRNA. However, here we found that adenovirus-mediated pri-miRNA expression impaired miRNA maturation. Previous studies demonstrated that adenoviruses express two non-coding virus-associated (VA) RNA, VAI and VAII RNA. VAI RNA is known to inhibit the activation of PKR, interferon-inducible double-stranded RNA dependent protein kinase, and plays an important role in avoiding innate immune response. VAI and VAII RNAs are processed into mivaRNAI and mivaRNAII, respectively, which can be incorporated into RISC and thus act similarly to miRNAs. Therefore, the VA RNAs interfere with the cellular RNAi pathway through competition with precursor miRNAs (pre-miRNAs). In this study, we observed whether adenovirus VA RNAs are the main factors to influence the processing of pri-miRNA when delivered using adenovirus. Then, pri-miRNA processing was specifically blocked by VA expression and efficiently recovered by anti-VA expression. These results indicate that expression of adenovirus VA RNAs should be suppressed when using adenovirus to deliver and express not only pri-miRNA but also shRNA. Now, we are developing more efficient and safe adenoviral pri-miRNA delivery system through inhibiting or deleting VA RNAs from adenoviral vector backbone.
Virus production with the ICELLIS® single-use bioreactor
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Viruses are used in several medical applications including vaccination and gene therapy. Many viruses are produced in anchorage dependent cell lines (e.g. HEK293, VERO, MDBK, etc). The iCELLis bioreactor from Pall LifeSciences is particularly effective for virus production. Adherent cells grow in a microfiber fixed-bed providing up to 500 m2 of growth surface area in a small reactor volume. Environmental conditions, combined with the large growth surface area in the iCELLis yields high cell productivity. A number of viruses, including AAV, Lenti, Adeno, Vaccinia, Bovine Herpes, Paramyxo and Influenza, were produced in iCELLis bioreactors using either transient transfection or infection. Processes were tested using identical parameters in small scale iCELLis bioreactors as used for previous standard processes (e.g. pH, DO, T, seeding density, etc). MDBK, HEK 293 and Vero cells were scaled up to 66, 133 m2 and 660 m2 (prototype) bioreactors respectively. Production of influenza, BHV and an undisclosed lytic virus was scaled up to 20, 66 and 660 m2, respectively. Intracellular viruses were harvested in situ using lysis buffer. Transfection efficiency was evaluated by flow cytometry. Virus productivity was evaluated by various quantitation methods. Good regulation and biomass growth was achieved for all cell lines tested. Lenti and AAV transfection efficiency was comparable in iCELLis compared to control vessels. Specific virus productivities in the small and large scale iCELLis bioreactors were also similar or better than that achieved in standard processes. The technology can be considered as an efficient tool for the industrial scale production of viral vectors.
Investigating a replicative mechanism for the establishment and maintenance of recombinant adeno-associated viral vector genomes
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Over the last 30 years, adeno-associated virus (AAV) has been developed as a recombinant vector for gene transfer. Recombinant AAV (rAAV) vectors consist of a transgene cassette flanked by the viral inverted terminal repeats encapsidated in a non-enveloped icosahedral capsid. rAAV vectors have been used successfully in multiple clinical trials, but there are still aspects of the biology of the vector that remain unknown. Upon infection with rAAV, the vector is trafficked through the cell and delivered to the nucleus, where the vector genome persists. It is known that these vector genomes can persist in vivo and maintain stable transgene expression for at least 10 years in large animal models. It was previously thought that integration of the vector genome into the host cell chromosome was responsible for this long-term persistence. However, it has now been demonstrated in muscle and other tissues that rAAV vector genomes persist in the host cell nucleus as episomal circular monomers and concatemers, and it is these forms that are most likely responsible for the observed long-term expression of the transgene. While there is sufficient evidence for the presence of these episomal circular forms, it is unknown if replication is required to establish and maintain them over time. To elucidate this aspect of the vector life cycle, we are investigating the role of DNA replication in the establishment and maintenance of episomal circular rAAV monomers and concatemers.
RD3-MolPack technology is an efficient platform for stable production of lentiviral vectors for human hematopoietic stem cells and primary T lymphocytes genetic manipulation
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Several recent clinical trials have shown the successful application of lentiviral vectors (LVs) for the genetic modification of human hematopoietic stem cells (HSCs) and T lymphocytes, which have been described as innovative treatments for rare genetic diseases and tumors, respectively. However, the clinical production of LVs is cumbersome from a safety, cost and reproducibility standpoint. In this regard, development of LV producing packaging cells represents an obliged goal in LV-gene therapy, allowing reduction of the manufacturing cost and increase of the overall quality of the vectors. We exploited our RD-MolPack technology, previously described for the stable and constitutive generation of LTR-driven and self-inactivating (SIN) Tat-dependent LV (RD2-MolPack), to develop the RD3-MolPack packaging cells, suitable for producing Tat-independent SIN LV. This technology is based on the RD114-TR envelope glycoprotein that, in contrast to the most utilized VSV-G envelope, permits generation of constitutive packaging cells. The RD3-MolPack-SIN-GFP producer cells were obtained by integration of the linearized plasmid carrying the transfer vector SIN-GFP. The selected RD3-MolPack-GFP clone grows in continuous culture in disposable two-compartment bioreactor for longer than 3 months producing, on average, not concentrated supernatant with a titer of 1–5×106 TU/ml when calculated on CEM A3.01 cells. RD3-MolPack-SIN-GFP LV transduced both human HSC and primary T lymphocytes at levels comparable to that of VSV-G SIN-GFP LV although it has a 30-fold less infectivity. Overall, our data show that RD-MolPack system might be a promising tool to simplify and widen a cost-effective clinical applicability of LV-gene therapy.
Evaluation of the production of EIAV lentiviral vectors in suspension cells
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Equine infectious anaemia virus (EIAV) lentiviral vectors are currently generated by transient co-transfection of adherent Human Embryonic Kidney 293T (HEK293T) cells with three plasmids that encode the necessary components for vector production (Vector genome, EIAV Gag/Pol, and a heterologous envelope, VSV-G). Manufacturing scale up is restricted by the adherent growth mode and therefore production would benefit from the use of a suspension cell line. The ultimate aim is to use producer cell lines to produce vector at manufacturing scale, in at least 200L bioreactors. Oxford BioMedica has a HEK293T based EIAV packaging suspension cell line (PC120.2S). Other cell lines are being explored to determine if they could be a suitable alternative to a HEK293T based suspension cell line. CEVEC's CAP-T cells are a human amniocyte cell line, which grow in serum-free media, in suspension. The CAP-T cells were evaluated by transient co- transfection to assess their ability to produce EIAV lentiviral vectors. A protocol for the production of EIAV vector from CAP-T cells was developed by testing an array of transfection conditions using Lipofectamine 2000CD. Transfection efficiency was assessed during vector production and the resulting vector was quantified using the vector titration GFP FACS assay. The process that produced the highest yield of vector from the CAP-T cells was further optimised and adapted for use in 500 mL bioreactors. Data will be presented on the optimisation and relative productivity of the CAP-T cells.
Pre-clinical evaluation of helper-dependent canine adenovirus in a human CNS 3D in vitro model
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Neurodegenerative diseases represent a burden in terms of patient suffering and economic cost. Viral vectors derived from canine adenovirus serotype 2 (CAV-2), due to their ability to bypass the clinical disadvantages associated with the use of human adenoviruses, presenting a preferential neuronal tropism and high level of axonal retrograde transport, are considered potential tools for the treatment of neurodegenerative disorders. However, accurate pre-clinical evaluation of efficacy and safety is critical for clinical translation. Human stem cells have great potential as complementary tools, bridging the gap between animal models and clinical trials. Herein we describe the assessment of helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human 3D neural in vitro model. Human midbrain-derived neural progenitor cells (hmNPC) were cultured as neurospheres in dynamic culture systems. Differentiated neurospheres enriched in neurons, astrocytes and oligodendrocytes, presented increased expression of mature neuronal markers, along with synaptic functionality, neurotransmitter synthesis/release and were able to elicit voltage- and ligand-activated currents. Assessment of hd-CAV-2 transduction was performed by evaluation of transgene expression and toxicity (cell viability and impact on neuronal gene expression), leading to stable long-term (up to 30 days) transgene expression with low toxicity. The evaluation of vector specific tropism showed that hd-CAV-2 has an increased neuronal tropism in contrast to hAd5 that preferentially transduced the glial cell population. This work demonstrates in a physiologically relevant 3D human cell model, that hd-CAV-2 vectors represent an efficient vehicle for gene therapy of human neurodegenerative diseases, with stable long-term transgene expression and minimal cytotoxicity.
Albumin-binding domain fusion to the Vesicular Stomatitis Virus Glycoprotein protects lentiviral vector from serum inactivation
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Vesicular stomatitis virus G glycoprotein (VSV-G), the most widely used envelope protein for retroviral and lentiviral vector pseudotyping, offers several advantages including effective delivery to a broad range of cell types, enhanced vector stability and high-titer production. However, serum inactivation of VSV-G pseudotyped vectors is a significant barrier for in vivo gene delivery. Several strategies have been employed to prevent this inactivation, including chemical coupling of polyethylene glycol chains (PEGylation). This approach extended the circulation half-life of active vector and reduced the rate of vector inactivation in the serum. As an alternative to chemical modifications, there are other half-life extension strategies including the fusion of therapeutic proteins to an albumin-binding domain (ABD). Given this information, we hypothesized that genetic fusion of the ABD to VSV-G might effectively protect the vector from serum inactivation. Here we have employed the ABD from streptococcal protein G to generate a VSV-ABD envelope fusion protein. Lentivectors pseudotyped with VSV-ABD glycoprotein are able to bind human serum albumin (HSA) in vitro. We found that ABD fusion to VSV-G did not affect to lentiviral vector titer production or transduction efficiency. Intravenous administration of VSV-ABD pseudotyped lentivector showed in vivo blood half-life extension and reduced lentivector sequestration in mouse liver, spleen and bone marrow. More importantly, we demonstrated that VSV-ABD pseudotyped lentiviral vectors are protected from human complement inactivation. Therefore, VSV-ABD pseudotyping represents an improvement for in vivo gene therapy applications as it increases the vector circulation time in the bloodstream and protects it from complement inactivation.
Therapeutic efficacy of an HIV-based lentivirus mediated vasoactive intestinal peptide gene delivery for diabetes
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Type 2 Diabetes (T2DM) is characterized by insulin resistance, glucose intolerance and beta cell loss leading to hyperglycemia. Because vasoactive intestinal peptide (VIP) displayed insulinotropic and anti-inflammatory properties, it has been regarded as a novel therapeutic agent for the treatment of diabetes.1 Despite all these beneficial properties, VIP is extremely sensitive to peptidases (DPP-4) requiring constant infusions or multiple injections to observe any therapeutic effect. Thus, an HIV-based lentiviral vector encoding human VIP (LentihVIP) was constructed to test the therapeutic efficacy of hVIP in diet induced obesity (DIO) animal model of T2DM. Similarly, a lentiviral vector carrying beta-galactosidase (LentiLacZ) gene was also generated and used as a control vector in gene delivery studies. hVIP gene expression was shown by immunocytochemistry (ICC) and peptide secretion was confirmed by ELISA in HepG2 liver and MIN6 pancreatic beta cell lines. Functional properties of the hVIP peptide were demonstrated by cAMP production assay and Glucose Stimulated Insulin Secretion (GSIS). Intraperitoneal (IP) delivery of LentihVIP vectors into C57BL/6J mice significantly increased serum VIP concentrations compared to control animals. Consequently, LentihVIP delivery in DIO animal model of T2DM resulted in improved insulin sensitivity, glucose tolerance, and protection against STZ-induced diabetes in addition to reduction in serum triglyceride/cholesterol levels. All these beneficial results suggested that LentihVIP delivery should be evaluated as a novel therapeutic approach for the treatment of T2DM. 1- Sanlioglu AD, Karacay B, Balci MK, Griffith TS, Sanlioglu S: Therapeutic potential of VIP vs PACAP in diabetes. J Mol Endocrinol 2012;49:R157-R167 Grant Support: TUBITAK-111S157
Minicircle DNA derived AAV
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Especially as part of a DNA drug, certain sequence motifs contained in plasmid DNA are not desirable. Such undesired sequences are either used for controlling the bacterial replication of the plasmid or to select for the plasmid during cloning or during production. An improved product safety, meeting actual and future regulatory requirements for clinical applications, was obtained by use of minicircles, i.e. circular and ccc-supercoiled expression cassettes. Besides the enhanced safety profile, initial results proofed their extremely high expression efficacy. But also for a viral, e.g. AAV mediated gene transfer, the bacterial sequence motifs may be an issue. AAV are produced by co-transfection of HEK293 producer cells. Here, it has been shown that by so-called reverse packaging backbone sequences of the ITR containing vector plasmid may be encapsidated. As a result of such events, an AAV mediated transfer of the antibiotics resistance gene instead of the therapeutic gene into the target cells appears to be a potential risk of plasmid derived AAV vectors. This can be overcome by usnig minicircle DNA for AAV production. Here we present first results on manufacturing of AAV Helper & Packaging minicircles and transfer minicircles as well as their use in AAV production.
Use of XPansion bioreactor for the manufacturing of viral vectors midsize batches
Despite progresses made in R&D in the field of gene therapy, viral vector batches for clinical trials are still largely manufactured by scaling-out the laboratory methods in classical multi-tray stacks (MTS). In a more restrictive GMP environment this technology presents a number of disadvantages and risks due to the huge amount of manipulations during the upstream process steps. It also rises the problem of FTE costs when considering increase of batch size. Xpansion is a compact 2D, low-shear stress and controlled bioreactor, fully operated in closed system offering an alternative for production of viral vectors production. Its similarities with classical 2D multi-tray stacks allow for an easy transfer of methods for growing and transfecting cells at larger quantity. In addition, control of culture variables such as pH and D.O. offers the possibility of further optimizing transfection process. The application described here demonstrates the successful transfer of HEK 293 culture, transfection and viral vector production, from MTS to Xpansion technology. Comparable cell growth and viral vector titers were obtained compared to MTS. The scalability of the process from XP10 (R&D scale) to XP200 scale (production scale) has also been demonstrated. Finally, considerations on production costs are also exposed.
Use of XPansion as midsize bioreactor for the manufacture of viral vectors
Most of batches produced for clinical trials coming from progress made in R & D are still manufactured with 2D multi-tray stacks technology. In a more restrictive GMP environment this technology presents a number of disadvantages and risks due to the huge amount of manipulations during the process steps. Xpansion is a compact 2D, closed and controlled bioreactor system for which we can align multiple units to increase the batch size; offering an alternative for growing and transfecting HEK 293 cells at higher quantities. The application described here demonstrates the satisfactory passage and promising utilization of the Xpansion bioreactor for the production of viral vectors by transfection.
Biodistribution of semi-replication-competent competent retroviral vector (RRV) delivered to knee and ankle joints in collagen-induced arthritis model
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Even murine leukemia virus (MuLV) has intrinsic tumor selectivity and stability in solid tumors, it still has limitation of transgene capacity and concern about risk of dissemination and insertional mutagenesis. Therefore we developed semi-replication competent retroviral vector (sRRV) in which MuLV gag-pol and env genes were separated into two transcomplementing defective retroviral vectors. As there has been a report that immunosuppressed Rhesus monkeys have developed lymphomas after exposed to high titer replication-competent retrovirus (RCR), safety concern with use of RCR in vivo is not negligible. Primer set for quantitative PCR, specifically targeting env region of MuLV was designed and confirmed that it has detection limit of 101 copies in 100 ng genomic DNA. Rheumatoid arthritis (RA) is a typical autoimmune disease, which there is no cure for complete recovery. Application of viral vectors carrying therapeutic genes is now in use for clinical trials, we generated RA model by immunizing and boosting bovine type II collagen into ten-weeks old male CBA1/J mice to check biodistribution of sRRV which can deliver a therapeutic gene to fibroblast-like synoviocytes (FLS) in vivo. sRRV, encoding eGFP gene, was prepared and 10 ul (1×108 TU/ml) was locally introduced to ankle and knee joints. Mice were sacrificed after 21 days, and genomic DNAs from all tissues including joints, bone marrow and buffy coat were isolated. Based on the condition of standard curve generated, Ct values were evaluated. Use of qPCR primers targeting env region of MuLV is promising for screening sRRV residues in vivo.
Lentiviral protein transduction of Pol-fused piggyBac transposase promotes rapid and efficient DNA transposition
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Gene transfer by conventional DNA transposon-based vectors relies on co-delivery of the DNA transposon, typically on plasmid DNA, and genetic material encoding the gene-inserting transposase protein. The need for intracellular production of the transposase, however, raises concerns related to cytotoxicity due to sustained transposase expression and insertional mutagenesis. By fusing the hyperactive Piggybac (PB) transposase protein (hyPBase) to the N-terminus of the lentiviral Gag polyprotein, we have recently engineered integrase-defective lentiviral vectors (IDLVs) as a tool for efficient delivery of transposase protein. However, IDLVs consisting of hyPBase-containing Gag-Pol polyproteins are incapable of transfering gene vectors, and unmodified Gag-Pol needs to be included in the virus particles to create ‘all-in-one' IDLVs carrying transposase subunits that efficiently catalyze transposition from reverse-trancribed vector RNA. Here, we present an alternative approach for IDLV-based transposase protein delivery. By fusing hyPBase to the C-terminus of the Gag-Pol polyprotein we show robust incorporation and subsequent release of the transposase in lentiviral particles. Notably, these vectors support efficient vector transfer and provide a small window of transposase activity, facilitating robust DNA transposition in cells treated with ‘all-in-one' IDLVs. Interestingly, DNA transposition by such IDLVs promotes generation of single-copy clones. In contrast, conventional plasmid-based systems result in multi-copy clones and give rise to prolonged transposase activity detectable in cells as long as two weeks after plasmid transfection. We believe that transposase delivery by lentiviral protein transduction may increase the applicability and safety of transposon-based gene vehicles in particular in hard-to-transfection cell types where low copy numbers are crucial.
Generation of A Lentiviral Vector Encoding Human Glucagon Like Peptide-1 (GLP-1) for Diabetes Treatment
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Glucagon-like-peptide-1(GLP-1) is an incretin hormone with multiple effects that make it an attractive candidate for type 2 diabetes therapy (T2DM)1. However, GLP-1 exhibited a limited clinical benefit due to its short half-life. A lentiviral construct encoding human GLP-1 has been developed using Gateway Cloning Strategy to provide constant GLP-1 expression in vivo without the risk of developing hypoglycemia. To accomplish this, human GLP-1 gene sequence with a furin cleavage site preceding the insulin signal peptide was synthetically generated by a DNA synthesizer. An addition of CACC site was necessary to generate an entry vector using Directional TOPO Cloning Strategy. GLP-1 was cloned into pLenti6.3/V5-DEST Gateway Vector using Virapower Hiperform Lentiviral Gateway Expression Kit from Invitrogen. Transient transfection employing four seperate plasmids resulted in production of lentiviral vectors in roller bottles. Viral supernatants were exposed to ultra-concentration and further purification processes prior to analysis. Physical titers were determined by p24Elisa while real time RT-PCR assays were employed to assess functional titers. The presence of GLP-1 expression was confirmed by immunocytochemistry and protein secretion by Elisa. cAMP production and glucose stimulated insulin secretion assays demonstrated that GLP-1 peptide encoded by the LentiGLP-1 was functional and displayed insulinotropic activity, respectively. 1-Tasyurek MH, Altunbas HA, Canatan H, Griffith TS, Sanlioglu S: GLP-1-mediated gene therapy approaches for diabetes treatment. Expert Rev Mol Med 2014;16:e7 Grant Support:TUBITAK-112S114
Scavenger receptor class B type I promotes adeno-associated virus type 8 transduction
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Recombinant adeno-associated viral vectors type 8 (AAV8) are non-pathogenic vectors for gene therapy that have shown early promise in clinical trials. AAV8 transduction allows a robust long-term transgene expression in a wide variety of tissues. The receptor 37/67-kilodalton laminin receptor is the main receptor for AAV8. However, adeno-associated viral vectors use a variety of cellular receptors and co-receptors for efficient cell transduction and most of them remains unknown. Here, we show that AAV8-mediated transduction is reduced in scavenger receptor class B type I (SR-BI) knockout mice. Both luciferase expression and viral DNA in the liver were lower in SR-BI deficient mice than in wild type mice. SR-BI deficiency also reduced AAV8 transfection in vitro both in mouse embryonic fibroblasts from knockout mice or in cells expressing a shRNA against SR-BI. Finally, we show that SR-BI-mediated lipid transfer is required for optimal AAV8 transduction as specific inhibitors were able to reduce both the level of transgene and the number of infected cells. Taken together, these results suggest that SR-BI plays a role in vector internalization and probably functions as a co-receptor for AAV8.
Mystery solved: VSV-G-lentivectors do not assure efficient gene transfer into unstimulated T, B cells and HSCs because they lack the LDL-receptor
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Vesicular stomatitis virus G protein pseudotyped lentiviral vectors (VSV-G-LVs) signify a major advancement in the gene and immunotherapy field as illustrated recently by the successful clinical trials. Although VSV-G-LVs allow efficient transduction of non-dividing cells, they do not govern efficient stable transduction of quiescent T, B cells and hematopoietic stem cells (HSCs), which hampers their application in gene and immune therapy areas where conservation of cell phenotype is essential. Nevertheless, the reason why VSV-G-LVs did not allow gene transfer into these resting cells remains unclear. Along-kept secret of VSV was revealed by the identification of its receptor, the low-density-lipid-receptor (LDL-R), explaining its broad tropism. Here, we evaluated LDL-R expression in unstimulated T, B and CD34+ cells and confirmed a very low expression of LDL, perfectly coinciding with VSV-G-LV-mediated poor transduction in these target cells. Stimulation of T cells through the T-cell receptor or of hCD34+-cell with ‘early-acting-cytokines’ remarkably up-regulated the LDL-R surface expression and permitted high level VSV-G-LV transduction. In contrast, B-cell receptor stimulated B cells augmented their LDL-R expression only marginally, in agreement with poor VSV-G-LV transduction levels. Finally, we confirmed specific VSVG-LV entry and transduction through the LDL-R using an anti-LDL-R antibody or by competition with soluble LDL-R resulting in reduction or almost complete inhibition of transduction, respectively. In conclusion, although post-entry blocks may still play a role in VSV-G-LV transduction of resting T, B and HSCs to some extent, we confirmed here that entry is already compromised by low expression of the VSV-receptor, LDL-R.
Large-Scale Clinical Grade Retroviral Vector Production using a Fixed-bed Bioreactor
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The successful genetic engineering of patient cells with retroviral vectors for phase I/II clinical trials depends on the large-scale manufacture of high titer, clinical grade vector stocks. Current methodologies to produce retroviral vectors from stable packaging cell lines using roller bottles and 10-layer cell factories are limited by a narrow “harvest window”, labor intensity, open-system operation, and requirement of large incubator space. To circumvent these shortcomings, we tested and optimized the scalable production of clinical grade vector stocks in a disposable iCELLis Nano bioreactor using our 293GP and PG13 GMP-grade packaging cell lines. Both 293Vec and PG13 cell lines demonstrated sustained cell growth for up to 12 days. High titer vector stocks could be harvested for up to 10 days, a much broader “harvest window” compared to the 3-day harvest afforded by the CF-10s. For PG13 and 293Vec packaging cells, the average vector titer and the total vector stocks' yield in the bioreactor were 3.2 to 7.6 fold, and 7.3 to 17.8 fold higher than those obtained in CF-10s, respectively. High titer serum-free vector supernatants were generated in the last phase of the production. All vector harvests can efficiently primary T cells and showed linear relationship between transduction efficiency and MOI. Titers for serum-containing vector remain the same, although dropped 50% in serum-free vector harvests after one year −80° C storage. The vectors produced from the iCELLis bioreactors passed the biosafety testing for clinical applications. These findings demonstrate for the first time that the fixed-bed bioreactor can efficiently produce large-scale, clinical grade retroviral vector stocks, which will greatly benefit clinical trials using gene transfer approaches.
Endhanced levels of Zinc Finger Nuclease CCR5 Modified CD4 cells (SB-728-T) Reduce Viral Load (VL) in HIV Subjects Treatment Interruption (TI)
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Wharton's jelly mesenchymal stem cells differentiated into hepatocyte-like cells show expression of immunomodulatory molecules
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Mesenchymal stem cells derived from Wharton's jelly (WJ-MSCs) are promising tools for cellular therapy due to their ability to differentiate into diverse cell types and their immunomodulatory activity. We aimed to characterize the extent of maintenance of the naive traits of these cells also in a highly specialized differentiated counterpart. WJ-MSCs were differentiated into hepatocyte-like cells (HLCs) with a multistep protocol. RT-PCR, flow cytometry, IHC and ICC were performed to assess expression of key markers in both naïve and differentiated WJ-MSCs. HLCs were able to store glycogen (PAS staining), incorporate specific live cells stains, perform enzymatic reactions (CYP3A4 induction and activity) and metabolic activities (G6Pase activity) which are usually featured by mature hepatocytes. We demonstrated for the first time that key immunomodulatory molecules as HLA-E and B7-H3 are also expressed by HLCs derived from WJ-MSCs. Data showed that also after hepatogenic differentiation protocol, HLCs mostly maintain the expression of immunomodulatory molecules, apart expressing new markers and functional features of mature cells. It is noteworthy that in different pathologic settings this intrinsic immunomodulatory ability of differentiated cells, may help survive the interaction with the host immune system, even in the absence of a specific immunosuppressive therapy. This, together with the acquisition of key mature hepatocyte functions, may render WJ-MSCs promising in cell therapy applications for liver diseases.
Pre-clinical evaluation of biodistribution of ART-I02 after a single intra-articular or intravenous dose in rats with a 90 day recovery period
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ART-I02 (Recombinant AAV5.NFkB.hIFN-ß) is an adeno-associated type 5 vector expressing human interferon-ß, under development for local treatment of rheumatoid arthritis (RA). A GLP biodistribution study was performed in Sprague Dawley rats, with ART-I02 administered intra-articularly in the knee joint at maximum feasible dose (MFD) (1.625E11 vg/ml) or intermediate dose (1.625E10 vg/ml). To study maximum exposure, a group was injected intravenously. Animals were euthanized 7, 30, and 90 days after injection. Fluids and tissues were collected to assess biodistribution of ART-I02 vector genomes, using a validated qPCR. Highest vector copy numbers were found in the injected joint, adrenal glands, liver and spleen, with detection of vector DNA at lower numbers in a wide range of tissues. Vector DNA was detected in blood and urine at early timepoints, but not at 30 and 90 days after injection. A dose dependent effect was seen as the highest copy number was found in the MFD group when compared to the intermediate dose group. After i.v. injection, the distribution of vector between organs was more homogenous. For both routes of administration the number of copies decreased over the 90 day observation period. Low ART-I02 copy numbers were detected in the reproductive organs. Although available literature indicates that there is no risk for germline transmission when using AAV5, a germline transmission study with ART-I02 has been planned as required by regulatory guidelines. In conclusion, administration of ART-I02 resulted in dose-dependent biodistribution, with highest copy numbers found at the site of injection.
A single dose toxicity study of ART-I02 by intra-articular and intravenous injection in rats with a 90 day recovery period
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ART-I02 (Recombinant AAV5.NFkB.hIFN-ß) is an adeno-associated type 5 vector expressing human interferon-ß, under development for local treatment of rheumatoid arthritis (RA). The safety and tolerability of ART-I02 were assessed in a GLP toxicology study in Sprague Dawley rats injected intra-articular (i.a.) in one knee joint with a single dose of ART-I02. The rats received either a maximal feasible dose (MFD) of 2.5E11 viral genomes (vg)/joint, intermediate dose of 2.5E10 vg/joint, low dose of 2.5E9 vg/joint, or placebo in their left knee. For maximal systemic exposure, 1 group of rats was i.v. injected with the same dose as the i.a. MFD group. The rats were sacrificed either 1, 7, 30 or 90 days after injection. Haematology, clinical chemistry, and necropsy including histopathological analyses were performed. There were no adverse clinical signs that were related to the treatment with ART-I02. An histopathological assessment was done of the full regulatory list for animals in the placebo, ART-I02 i.a. highest dose group, and ART-I02 i.v. group sacrificed after 1, 7 or 90 days. Importantly, no ART-I02 related findings were observed after i.v. administration. ART-I02 related microscopic findings were only observed in the injected knee joint. A mild to moderate local reaction to the virus was observed, which decreased in severity over time indicating recovery. In conclusion, administration of up to 2.5E11 vg/dose of ART-I02 was well tolerated in Sprague Dawley rats. There was no evidence of systemic toxicity.
Validation of the Disease-Inducible CXCL10 Promoter for Gene Therapy in Rheumatoid Arthritis
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We have previously shown the feasibility of local disease-inducible gene therapy in a mouse model of arthritis (Vermeij, Broeren et al, ARD, 2014). Our current efforts focus on the translation to the human rheumatoid arthritis (RA) patient. A microarray analysis on joint tissue of 20 RA patients and 7 patients without joint disease showed a 10-fold upregulation of the CXCL10 gene in RA synovium. CXCL10 is an inflammation-associated chemokine, which was not found to be upregulated in microarrays performed on synovium from human osteoarthritis or from mouse models of RA. The CXCL10 promoter was isolated from human cDNA and cloned into a SIN-lentiviral luciferase reporter vector. Transduced THP-1 cells stimulated with TNFa, the most important catabolic cytokine in human RA, resulted in a 15.3-fold upregulation of the luciferase signal. The induction could be counteracted dose-dependently with Etanercept, showing sensitivity to an effective biological drug in the clinic. The CXCL10 promoter also responded to serum from RA patients, an important source of TNFa and could significantly distinguish between RA serum and healthy donor serum (P=0.017). The regulation of the CXCL10 promoter was also shown in RA synovial fibroblasts, which confirms that the CXCL10 promoter construct is functional in the most prevalent cell type in the joint capsule. The selected proximal promoter from the CXCL10 gene, responds to inflammatory mediators (e.g. TNF-alpha) present in serum of RA patients, and is functional in both macrophages and synovial fibroblasts. The CXCL10 promoter can thus provide local and inflammation-inducible human gene therapy suitable for RA.
Disease-regulated Local Interleukin-10 Gene Therapy Diminishes Synovitis and Articular Cartilage Damage in Experimental Arthritis
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Rheumatoid arthritis (RA) is a chronic destructive autoimmune disease with periods of exacerbation and remission. A disease-regulated gene therapy that offers flexible drug delivery would be an attractive approach. To test this, the anti-inflammatory interleukin-10 (IL-10) gene was put under control of inflammation-dependent promoters and delivered intraarticularly in a mouse model of RA. The proximal promoters of Saa3 and Mmp13 were selected as both genes were differentially regulated in the inflamed synovium during experimental arthritis. Mouse knee joints were injected intraarticularly with SIN-lentiviral vectors expressing a luciferase reporter under control of these two promoters. The Saa3 and Mmp13 promoters showed induction at day 1 and day 4 after arthritis induction respectively. Next the luciferase gene was replaced by the IL-10 gene. Expression of IL-10 was higher in arthritic joints, compared to healthy joints. Overexpression of IL-10 resulted in less synovitis and cartilage proteoglycan depletion and correlates with upregulation of IL-1Ra and SOCS3 expression. IL-1Ra counteracts the detrimental effects of IL-1 on cartilage damage and SOCS3 inhibits the JAK/STAT pathway and subsequent inflammation which can explain the diminished synovitis and PG depletion. The treatments using the Saa3 and Mmp13 promoter were as effective as with constitutive expression using the PGK promoter. Probably because IL-10 is expressed at day 1 of arthritis without any treatment, major therapeutic differences between the Mmp13 and Saa3 promoter were not evident in our study. Yet we can conclude that local inflammation-dependent IL-10 gene therapy suppresses experimental arthritis and is a promising strategy for treatment of inflamed joints in RA patients.
IL-10 impairs induction of anti-hepatitis C virus T-cell responses in patients vaccinated with transduced dendritic cells in a phase 1–2 clinical trial
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IL-12 in antitumor immunotherapy: to target or not to target?
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Potent and long-lasting antitumor immunity is induced upon proper activation of antigen presenting cells (APCs), the subsequent stimulation of TH1-polarized CD4+ T cells and activation of tumor specific cytotoxic T cells (CTLs). One of the most prominent TH1-dictating cytokines is the pro-inflammatory cytokine IL-12. However, its systemic delivery can lead to toxic side effects. On that account we hypothesized that targeted delivery of both IL-12 and ovalbumin could enhance a lentiviral vector (LV)-based antitumor vaccine at both the safety and efficacy level. To test this hypothesis, we compared targeted with untargeted delivery of IL-12 and ovalbumin after subcutaneous injection of C57BL/6 mice with APC-targeted or broad tropism LVs respectively, encoding ovalbumin together with eGFP or IL-12. When the induction of ovalbumin-specific CD4+ T cells was evaluated, we demonstrated more proliferation of T-bet+ TH1 cells secreting IL-2 and IFNgamma after targeted delivery of IL-12. However, this only increased the proliferation of ovalbumin-specific effector cells without improvement of their functionality, which was reflected in a lack of increased IFNgamma secretion by CTLs and NK cells, no increased ovalbumin-specific target cell lysis nor therapeutic efficiency in the MO4 tumor model. Therefore we conclude that targeted delivery of IL-12 does not improve the potency of antitumor vaccination. However, it does outperform untargeted delivery in terms of TH1 polarization, which could open new prospects for vaccination in the fields of autoimmunity and allergy.
Helper-dependent adenoviral vector expressing Il-1Ra treats osteoarthritis efficiently in small and large animal models
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Osteoarthritis (OA) is the most common joint disorder and leading cause of disability in the aged population. Current treatment options for OA are limited to life style modifications, exercise, analgesics and invasive procedures such as joint replacement in severe cases. Interleukin 1 has been identified as a key player in OA, which promotes inflammation and cartilage degradation by inhibiting matrix synthesis and triggering cartilage degrading enzymes such as metalloproteinases and aggrecanases. Here we evaluated helper-dependent adenoviral vector (HDAd)-mediated intraarticular gene therapy using interleukin-1 receptor antagonist (Il-1Ra) in small and large animal models. In mice we showed that HDAd transduces synoviocytes efficiently after intraarticular injection and mediates gene expression for more than one year. In a mouse OA model we showed efficient treatment of the disease by histologic evaluation and significantly increased cartilage volume and cartilage surface area measured by micro-CT. In a horse model of OA, functional parameters (lameness, range of motion, lameness after flexion) were markedly improved and gross cartilage lesions as well as synovial membrane thickening were significantly reduced by a single intraarticular injection of HDAd-Il-1Ra. Neither systemic nor local adverse effects were observed in treated horses. These studies show that an HDAd expressing Il-1Ra can safely and efficiently treat OA in small and large animals. Importantly, data in mice and horses suggest that HDAd-Il-1Ra is disease-modifying. These results indicate that HDAds may be ideally suited for intraarticular gene therapy to treat joint diseases, especially when large genes or combinations of genes need to be transferred.
Risk reduction in T cell Adoptive Cellular Immunotherapy with the XuriTM Cellbag
The successful manufacture of cellular immunotherapeutics requires a system that can minimise the risk of failure. Xuri Cellbag single use bioreactors have evolved to a design where the user need only execute a single step, the addition of a supply of medium. This addition can be performed using established techniques such sterile welding, negating the requirement for a laminar flow cabinet or similar device. The single use bioreactors are constructed with all components attached and simplify the overall set up procedure. User feedback was analysed to design-in features and functions that aid implementation and set-up of the single use bioreactor. Data on the expansion of T cells in the Xuri Cell Expansion System W25 shows equivalent performance to the existing product, confirms a reduction in the number of step up steps and is ready-to-go. Cell viability, yield and phenotype are comparable to the current Cellbag design.
High-affinity CD20-specific TCRs suitable for adoptive immunotherapy in the treatment of CD20low B cell malignancies
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Therapeutic monoclonal antibodies (mAb) such as Rituximab have demonstrated the clinical efficacy of targeting the B-cell antigen CD20 for the treatment of B-cell malignancies. Although CD20 is also expressed on healthy B-cells which are depleted in the course of therapy, long-term B-cell aplasia is well manageable. However, refractory disease to CD20-targeted mAb treatment has been reported with various resistance mechanisms: downregulation of CD20 expression, internalization of CD20:mAb complex, inhibition of complement-dependent cytotoxicity and absence of an effector-cell repertoire in patients treated with chemotherapy prior to mAb infusion. Therefore, additional therapeutic strategies are required. T-cell receptor (TCR) gene transfer is an attractive strategy to equip T-cells with TCRs of defined antigen-specificity. Due to their high sensitivity TCRs can induce T-cell activation even when antigen expression is very low. To isolate CD20-specific T-cells, we used peptide-MHC-tetramers composed of CD20 peptide bound to HLA-A2. Since negative thymic selection depletes T-cells recognizing CD20 in self-HLA from the naïve T-cell repertoire, we isolated CD20-specific high-avidity T-cells from HLA-A2-negative individuals. The isolated tetramer-positive T-cell clones efficiently recognized primary chronic lymphocytic leukemia, mantle cell lymphoma and acute lymphoblastic leukemia (ALL), whereas no recognition of a panel of CD20-negative hematopoietic and non-hematopoietic cells was observed. The T-cell clones more efficiently recognized ALL cell-lines with low CD20 expression than did CD20-targeted mAbs. Transduction of these CD20-specific TCRs resulted in efficient expression of the introduced TCRs and conferred CD20-specificity onto recipient cells. These CD20-specific TCRs could provide additional therapies for B-cell malignancies by administering TCR-engineered T-cells with potent effector functions.
Cord blood-derived mesenchymal stem cells inhibit IL-23-induced psoriasis-like skin inflammation in mice
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Mesenchymal stem cells (MSCs) have activity to inhibit the proliferation or activation of lymphocytes, and their inhibitory effects do not require HLA-matching because MSCs express low level of HLA molecule. Therefore, it seems that MSCs can regulate immune responses. In this study, we determined whether MSCs could inhibit IL-23-induced psoriasis-like skin inflammation. MSCs were injected subcutaneously on day −1 and day 7, and IL-23 was injected intradermaly every other day until day 14. Mouse ears were collected on day 15, and H&E staining and real-time PCR were performed to investigate whether MSCs could inhibit IL-23-induced skin inflammation. IL-23-induced skin inflammation was inhibited when MSCs were injected. The expression of proinflammatory cytokines, such as IL-1 beta, IL-6, IL-17 and TNF-alpha was inhibited by MSC injection and the expression of chemokines, such as CCL17, CCL20, and CCL27, was also decreased in the mouse skin. We also determined whether MSC could not only prevent but also treat psoriasis-like skin inflammation in mice. Furthermore, in vitro experiments also showed anti-inflammatory effect of MSCs. Co-culture of MSC with keratinocytes suppressed IL-20 and CCL20 expression. Finally, we showed that MSC could inhibit Th17 cell differentiation, which is important for the pathogenesis of psoriasis. These results suggest that MSCs could be useful for cell therapy in treating psoriasis.
Lack of extracellular superoxide dismutase induces severe psoriasis-like skin inflammation in mice
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Psoriasis is a common chronic and complex autoimmune inflammatory skin disorder. The histological characteristics of psoriasis are epidermal hyperplasia, mononuclear leukocyte infiltration into the dermis, and increased angiogenesis. The number of psoriasis patients has increased, but the mechanisms involved in the pathogenesis of psoriasis remain unclear. Extracellular superoxide dismutase (EC-SOD) has anti-chemotactic activities. Since it is well known that immune cell infiltration is seen in psoriatic lesions and psoriasis patients express low level of EC-SOD, we hypothesized that lack of EC-SOD induces IL-23-mediated psoriasis-like skin inflammation. In this study, we determined whether loss of EC-SOD causes more severe IL-23-induced skin inflammation. Ear skin after IL-23 administration was thicker in EC-SOD KO mice compared to wild type mice. Additionally, infiltration of CD4+ T cells, macrophages, and dendritic cells into IL-23 injection sites was more significantly increased in EC-SOD KO mice. The expression levels of pro-inflammatory cytokines and chemokines were also more elevated in EC-SOD KO mice. In vitro experiment also showed that treatment with recombinant EC-SOD inhibited IFN-g/TNF-a- or IL-22-induced chemokine expression in HaCaT cell line. Additionally, EC-SOD KO dendritic cells expressed high level of MHCII and treatment with recombinant EC-SOD decreased the high level of MHCII. Therefore, EC-SOD may inhibit IL-23-induced psoriasis-like skin inflammation through inhibition of immune cell infiltration and immune responses. These results suggest that EC-SOD over-expressing cells for cell therapy, and EC-SOD expressing plasmid or viral vector for gene therapy could be used for management of psoriasis.
Modifying TCR specificity by transcription activator-like effector nucleases and RNA-guided nucleases
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The introduction of high avidity T-cell receptor (TCR) genes into T-cells for adoptive T-cell therapy holds a lot of promises for the clinics, but still comprises certain challenges. The parallel expression of endogenous and exogenous TCR-chains provokes competition for surface expression and may result in mispairing, potentially leading to autoimmunity. Designernucleases like transcription activator-like effector nucleases (TALEN) and RNA-guided nucleases (RGN) can be used for targeted genome editing. They introduce specific DNA double strand breaks (DSB) at their target site, potentially resulting in permanent gene knockout during DSB repair. To disrupt endogenous TCR expression we assembled seven TALEN monomers and two guide RNAs targeting the constant regions of the TCR a-chain (TRAC) and the TCR ß-chain (TRBC1 and TRBC2), respectively. TALEN and RGN activity at the target sites was examined in 293T, K562, Jurkat and T-cells using Cel1-assay and deep sequencing. Here we show the specific DSB induction by homodimeric and obligate heterodimeric TALEN and RGN leading to mutation frequencies of up to 24% and 40%, respectively. TALEN and RGN specificity was examined by the identification of integrase-deficient lentiviral vector (IDLV)-marked DSB using LAM-PCR and deep sequencing. In order to evaluate the TALEN for targeted gene addition through homology directed repair (HDR), K562 cells were nucleofected with TALEN and a donor template containing 800 bp homologous sequences to the TRAC locus. TALEN that show high efficiency and specificity to their target sequence will be used for generation of T-cells with high avidity TCR.
Oncolytic Plasmid System; a Novel Antitumor Strategy by Plasmids Encoding Pathogenic Antigens Blocking the Immune Escape of Tumor
Since ancient Egypt, a number of clinical trials were conducted using microorganisms to treat tumors. It has remarkably advanced after genetically engineered oncolytic virus was developed in 1991. Oncolytic virus is expected to proliferate in and destroy tumor cells. The virus is also thought to make the infected cells present the virus antigen on their surfaces. Those antigen on the cells or their fragments would effectively interact with APCs, and be recognized as a “danger signal”. It would enhance the immune system against simultaneously captured tumor-associated antigen, and block the immune escape of tumor. We thought that expression of the pathogenic protein on the tumor cell surface could be induced not only by virus infection but also by the transfection of the pathogenic antigen-encoding plasmid. For in vivo gene transfection, we have developed a highly effective synthetic gene transfection system comprising very small (<70 nm) plasmid complexes with negative surface charge (Biomaterials 31 (2010) 2912–2918). In this study, the small complexes were made of the plasmids harboring myco-bacterium tuberculosis-antigen genes, ESAT-6 and Ag85B, and explored for therapeutic efficacy in tumor-bearing mice. Animal clinical studies were also performed to examine the effect on primary tumor-bearing dogs.
The role of perfusion in maintaining high density T-cell cultures
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T-cell therapies are a rapidly growing field of personalised medicine, attracting the interest of venture capitalists and pharmaceutical companies alike. They exploit the T-cell's innate ability to protect against pathogens, as well as seek and destroy cancerous cells. Although there are many different forms of T-cell therapies currently being trialled, they follow a common protocol. T-cells are isolated from the patient, modified and expanded in the laboratory and then infused back into the patient ready to fight disease [1]. Autologous T-cell therapies require large numbers of T-cells to be activated and expanded ex vivo before being infused back into the patient. This is commonly achieved by using the XuriTM Cell Expansion System, a rocking bioreactor, where the combination of rocking agitation and perfusion media exchange allows high cell concentrations to be reached. This study explores the role of perfusion in supporting high density T-cell cultures. The impact of perfusion on cell growth and viability was analysed as was the role of perfusion in controlling the key metabolites and growth factors. 1. June, C.H., Adoptive T cell therapy for cancer in the clinic. J Clin Invest, 2007. 117(6): p. 1466–76.
Long term persistence of TK-cells and dynamics of memory T cells in patients treated with suicide gene therapy for hematological malignancies
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Suicide gene therapy applied to allogeneic hematopoietic stem cell transplantation is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease, with complete resolution of the adverse reaction. In the present work we studied the long-term fate of TK-cells, and exploited TK gene marking to shed light on memory T cell dynamics. We analyzed 9 patients in complete remission after receiving post-transplant TK-cell infusions for hematological malignancies. At a median follow-up of 7.4 years (range 3.2–12.3), TK-cells were detected in all patients, at low levels (median=4 cells/uL) and ganciclovir sensitivity was preserved. Long-term persisting TK-cells displayed a memory phenotype comprising effector memory, central memory and stem memory T cells (TSCM). The number of TK-cells circulating at the longest follow-up did not correlate with the number of infused cells, but instead with the peak of TK-cells observed within the first months after infusion. The number of infused TSCM cells positively correlated with early TK-cell expansion and with their long-term persistence, suggesting that TSCM might play a privileged role in the generation of a long-lasting immunological memory. Further studies on TK-cell repertoire and vector integrations are ongoing to elucidate the in vivo dynamics of infused memory T cells.
High-level co-expression of a novel human suicide gene, CYP4B1, with CD19 or CD22 CARs in SIN lentiviral vectors
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Increased safety of donor lymphocyte infusion (DLI), genetic engineering of T-cells with either enhanced T-cell receptors or chimeric antigen receptors (CARs) can be achieved by equipping T-cells prior to re-infusion with a suicide gene, which allows in vivo control of the transduced cells. We recently developed a novel human suicide gene system by rendering the inactive human cytochrome P450 enzyme CYP4B1 capable of efficiently converting the inert natural 4-ipomeanol into highly toxic DNA-alkylating metabolites. The aim of the present study was to establish an optimal lentiviral co-expression system for CYP4B1 with MACS selection markers (tNFGR or tCD34) for DLI or with CARs against CD19 and CD22. To this end, we analyzed the expression levels of two transgenes in a standard lentiviral vector achieved by using 2A-like peptides, the EMCV IRES site or a 2-promoter system. The results revealed that T2A was optimally suited to achieve high-level expression of both transgenes. Expression by the MPSV promoter in primary human T-cells was as high as achieved with the splice active EF1a promoter, however with 10X higher titers. We next demonstrated that the position of CYP4B1 and also tCD34 or tNGFR either 5′ or 3′ of the T2A site was uncritical for both expression level and functional activity. However, analysis of the cytotoxic activity of T-cells against leukemia revealed that the CAR activity is higher when expressed 3′ of T2A. These studies establish a clinically suitable lentiviral vector for expressing two transgenes at high expression levels that is currently tested in murine models.
Both Soluble TRAIL and Adenovirus-Mediated Endogenous Expression of TRAIL Induces Proliferation in Min6 Mouse Pancreatic Beta Cells
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Diabetes is related to impairment in beta cell function/beta cell loss. TRAIL (TNF-Related Apoptosis-Inducing Ligand) was recently correlated with induction of cell survival and proliferation in vascular smooth muscle cells and human vascular endothelial cells. TRAIL also protected pancreatic beta cells against damage induced by TNF-alpha and FasL, and generally does not induce apoptosis in these cells. We aimed to test a possible proliferative effect of TRAIL on Min6 cells. TRAIL receptor expressions were detected by Western blotting. Cells were treated with differing doses of (0, 0.1, 1, 10, 100 ng/ml) soluble TRAIL (sTRAIL) for 24, 48, or 72 hours. Survival and proliferative status were tested by WST-1 and Ki67, respectively. Phosphorylation levels of ERK, Akt, and p38 molecules were detected by Western blotting. Min6 cells were also infected with various doses of adenoviral vectors encoding TRAIL. DcR2 was expressed at the highest level, followed by DR5 and DcR1. sTRAIL treatment did not decrease viable cell numbers. Proliferation rate increased most significantly with 10 ng/ml sTRAIL and at 48 hours. ERK and p38 phosphorylation levels significantly increased at 5 and 15 minutes of TRAIL application, while Akt phosphorylation was slightly induced at 15 and 30 minutes. Adenovirus-mediated TRAIL transfer also increased proliferation. According to our results, TRAIL does not induce apoptosis in Min6 cells, but induces proliferation at certain doses, presumably activating ERK and p38 pathways. Although further investigation is required, these findings may support a possible therapeutic role for TRAIL in diabetes (Supported by: TUBITAK 112S450, Akdeniz University 2012.03.0122.003).
Construction of A Lentiviral Vector Encoding Human Vasoactive Intestinal Peptide for Diabetes Treatment
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Type 2 Diabetes Mellitus (T2DM) is characterized by insulin resistance in association with beta cell dysfunction followed by beta cell loss. Since vasoactive intestinal peptide (VIP) is a neuropeptide with insulinotropic and anti-inflammatory functions, it has been regarded as a therapeutic agent for diabetes treatment.1 3rd generation lentiviral vectors encoding human VIP long isoform (LentihVIP) were constructed using Gateway cloning strategy. In addition, beta-galactosidase (LentiLacZ) and red fluorescent protein (LentiRFP) encoding cDNAs were also cloned into lentiviral vectors in order to visualize gene expression in various tissues. The lentiviral particle titers were assessed using QuickTiter HIV Lentivirus Quantitation (Cell Biolabs). While LacZ staining was used to reveal beta galactosidase expression, fluorescent microscopy was employed to visualize RFP signal in transduced tissues. As VIP expression from LentihVIP construct was demonstrated using immunocytochemistry of LentihVIP infected HepG2 cell line, the functionality of LentihVIP vectors was confirmed by cAMP production and glucose stimulated insulin secretion assays. Intravenous (IV) and intraperitoneal delivery (IP) of LentiRFP constructs revealed similar tissue distribution pattern of transgene expression in C57BL/6J mice four weeks after gene delivery. LentihVIP vector was also injected at different doses into C57BL/6J mice to determine tissue distribution of VIP expression in vivo including its safety. 1- Sanlioglu AD, Karacay B, Balci MK, Griffith TS, Sanlioglu S: Therapeutic potential of VIP vs PACAP in diabetes. J Mol Endocrinol 2012;49:R157-R167 Financial support: TUBITAK-111S157
Inhibition of Ad Infection by use of AAV Vectors expressing Anti-Adenoviral Artificial microRNAs
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Immunosuppressed patients can develop severe disseminated adenovirus (Ad) infections, which often end fatally. As the efficacy of anti-Ad drugs is limited, new approaches to treat Ad infections are desirable. Artificial microRNAs (amiRs) represent a type of small regulatory RNAs that is suitable to silence target genes by RNA interference. Thus, we hypothesized that amiRs can also inhibit Ad infection. AmiRs directed against five adenoviral genes playing a key role in the adenoviral replication cycle were developed and inserted into plasmids. Each amiR recognized its target sequence and significantly inhibited gene expression in reporter assays. Moreover, self-complementary AAV2 vectors expressing these amiRs inhibited the replication of Ad5 in HeLa cells. Based on different efficiency of the amiRs and on the fact that multimerization of amiRs can improve silencing, two AAV2 vectors with optimized amiR expression cassettes were developed: one bearing six identical copies of an amiR directed against the adenoviral pTP, the other contains two amiRs directed against different target sites of E1A, one amiR directed against IVa2 and three identical amiR copies directed against pTP. Both vectors significantly inhibited Ad5 replication and improved cell survival and viability of infected HeLa cells. However, the amiR-pTP vector seems to have higher efficiency. In conclusion here we show that AAV vector-mediated expression of anti-Ad amiRs is a powerful new approach to inhibit Ad replication in vitro. Studies are underway to verify the data in vivo.
Non-viral brain-derived neurotrophic factor gene delivery in animal model of urinary tract infections
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Urinary tract infections (UTI) represent a serious health-care problem. Due to high resistance rates against conventional agents, an ongoing need remains for new treatment strategies. Brain-derived neurotrophic factor (BDNF) is the most abundant neurotrophin with certain functions in nervous system and brain. Only little data exist on BDNF function in bladder and kidneys, either under physiological or pathological circumstances. Increased production of BDNF in the bladder was observed in chronic cystitis. The aim of this study was to elucidate the effects of non-viral gene therapy in a murine model of UTI. Twenty six female mice (12 weeks old) were divided into four groups: CTRL, BDNF, UTI/CTRL and UTI/BDNF. BDNF groups received a murine BDNF expression plasmid via transurethral application. One day later mice received uropathogenic Escherichia coli (108 CFU) or saline by transurethral application. After 24 hours mice were sacrificed and kidneys were collected under aseptic conditions. Mice in the control group had a sterile kidney. UTI was successfully induced in most of the mice receiving uropathogenic E. coli. Preventive BDNF gene therapy did not decrease bacterial colonization of the kidney. BDNF gene therapy does not prevent UTI in mice. Further analyses of the transgene expression should be conducted later. Dosing and timing of the plasmid administration might not be ideal and should be tested further.
Detection and Characterisation of Human anti-AAV CD8+ T Cells using MHC class I Multimer-based Magnetic Enrichment
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Recombinant Adeno-Associated Virus vectors (rAAV) represent the most largely used platform for in vivo gene therapy. Despite promising results in large animal models and clinical trials, pre-existing antibody and memory CD8+ T cell responses directed against the capsid of rAAV remain one of the major hurdles to the safety and efficiency of in vivo rAAV-mediated gene transfer. Particularly, the impact of pre-existing capsid-specific CD8+ T lymphocytes on gene transfer remains elusive, all the more so since their detection and subsequent evaluation have proven to be a challenge with currently available tools (such as ELISpot or intracellular cytokine staining assays) because of their scarcity. Combining major histocompatibility complex class I (MHC I) multimer staining with magnetic enrichment, we were able to detect AAV2 and AAV8 capsid-specific CD8+ T cells ex vivo, among peripheral blood mononuclear cells from healthy donors (at frequencies ranging from 1e-5 to 7e-4 among CD8+ T cells). Using flow cytometry, we could further sort AAV8 capsid-specific CD8+ T cells and generate their corresponding clones. Undergoing characterisation and functional studies of the clones generated suggest a variety of activation profiles in terms of degranulation activity, IFN-γ and TNF-a expression. Evaluation of AAV8-specific T cell clones' killing activity on rAAV8-transduced myoblasts is currently under investigation, as is a most comprehensive study of their cytokine secretion profiles. Elucidating the different activation patterns of AAV capsid-specific CD8+ T lymphocytes will be important for the understanding of the onset of pre-existing anti-AAV immunity on rAAV-based gene transfer and its impact on clinical outcome.
Au/galectin-1 nanocomplex acts as a broad-spectrum inhibitor of influenza virus through multivalent interactions
Influenza poses pandemic threats to human health. The envelope glycoproteins, hemagglutinin and neuraminidase, of influenza virus can continuously undergo antigenic drift and shift to escape the host immunity. Currently available anti-influenza drugs include M2 ion channel blockers (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir). However, the continued emergence of drug-resistant influenza virus has become a major health concern. Considerable efforts are being made to develop broad-spectrum therapeutic agents against influenza virus. We have reported previously that galectin-1, which belongs to S-type lectins displaying specificity for ß-galactosyl-containing glycoconjugates, can bind to the surface glycoproteins of influenza virus through its carbohydrate recognition domain and inhibit viral infection. Therefore, galectin-1 may be a potential antiviral agent for influenza. In this study, we proposed a multivalent strategy for orientated immobilization of galectin-1 on the gold nanoparticle (AuNP) surface to enhance its binding affinity for influenza virus. We show that galectin-1 is well orientated on AuNP, which exposes its carbohydrate-recognition domain for interaction with influenza virus. Compared to unconjugated galectin-1, Au/galectin-1 nanocomplex (AuNP/Gal-1) greatly enhances the binding affinity and increases the antiviral activity against different subtypes of influenza A virus. We also verify that the improved antiviral activity of AuNP/Gal-1 is attributed to multivalent interactions of AuNP/Gal-1 with the virus. Moreover, AuNP/Gal-1 ameliorates influenza pathogenesis in mice. Our results implicate for the first time that protein-based AuNP/Gal-1 nanocomplex has promise as a broad-spectrum inhibitor of influenza virus. This study provides an innovative strategy for drug design targeting surface glycans of various pathogens.
Lentiviral DNA barcoding allows reliable tracking of gene modified hematopoiesis
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As more and more gene therapy trials make their way from preclinical phases into the clinic, the need for surveillance of gene modified cells increases. Initial problems with deregulated hematopoiesis led to a surge in research on virus integration behavior and new vector designs to reduce genotoxicity. As a result, we and others have developed methods to identify insertion sites. While application of deep sequencing technology allows to study the repertoire of clones in repopulating animals and patients, these technologies were hampered by a lack of calibration data and methodological difficulties related to the amplification of virus-genome boundaries or sheared DNA of variable lengths, as we have previously demonstrated (Brugman, Human Gene Therapy Methods 2013). We therefore developed a novel technique that combines DNA barcoding, deep sequencing and an error correction methodology to allow reliable quantification of the repopulating clones in a transplanted mice. We performed transplantations in NSG mice using lentivirally barcoded UCB CD34+ (n=8) cells or phenotypically defined human HSC (n=9) or in C57Bl6 mice using lentivirally barcoded LSK cells (n=6). In addition, repeated sampling and spike-in calibration experiments were performed. With this DNA barcoding method samples can be rapidly processed, which allows repopulation data to be available during repopulation rather than in retrospect. These experiments demonstrated that DNA barcoding of viral vectors provides a method to reliably quantify clonal repopulation of gene marked cells, which would also be suitable for clonal surveillance in a clinical gene therapy setting.
Genotoxicity after adeno-associated virus (AAV) gene therapy is dependent upon dose, treatment age and enhancer-promoter selection
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AAV gene therapy has recently been approved for clinical use and shown to be efficacious in a growing number of clinical trials. However, the safety of AAV as a vector has been challenged by studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile because numerous AAV studies have not described genotoxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of proximal microRNAs and Rtl1 were associated with HCC. In addition, we identify a number of genes with differential expression that may be useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are critical factors in AAV-mediated insertional mutagenesis. Our results help define a mechanism underlying AAV-mediated genotoxicity and have important implications for the design of both safer AAV vectors and gene therapy studies.
Lentiviral integration profiles are stable across multiple species, disease phenotypes and tissue types
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The potential of ex vivo hematopoietic stem cell (HSC) gene therapy to treat monogenic inherited disorders has been convincingly demonstrated in immune deficiencies and storage disorders. The initial trials have also underlined the inherent genotoxicity risks of gammaretroviral vectors and the role of the disease phenotype on genotoxicity. To understand the integration patterns of third generation self-inactivating lentiviral vectors, a broad range of vectors were created and used to transduce HSC for transplantation into murine models for X-linked, RAG1 and RAG2 immune deficiencies and Pompe disease. Vector-genome boundary sequences obtained from murine tissues and cultured HSC were pooled with those of rhesus and human origin in a large database of over 20,000 lentiviral integration sites (LVIS), stratified by disease phenotype, tissue, transgene, promoter and in vitro vs in vivo samples to compare the patterns under a wide variety of conditions. The resulting analysis demonstrates that the integration profiles are consistent among the three species studied. Neither promoter choice nor transgene affected the integration pattern. LVIS flanking oncogenes occurred at a frequency consistent with random distribution. Importantly, the integration pattern of vectors containing the viral spleen focus forming virus (SFFV) promoter did not differ from those with the cellular PGK, UCOE or IL2RG promoters. LVIS within 10 kb of a gene were more frequent in murine cultured cells than in hematopoietic cells following transplantation, suggesting that either the repopulating HSC differ in integration pattern from actively proliferating progenitors or a negative selection of those sites occurred during hematopoietic reconstitution in vivo.
The cell cycle status of the CD34+ stem cells determines the pattern of lentiviral integration in actively transcribed genes and in development-related genes
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Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer can lead to insertional mutagenesis. Although such risks had been originally estimated as extremely low, the onset of leukemia due to insertional activation of the LMO2 gene, the development of myelodysplasia following EVI1 activation and the HMGA clonal dominance observed in several clinical trials, led to a reevaluation of the mechanisms operating in insertional mutagenesis. Therefore, unravelling the principles of retroviral integration is mandatory towards safer gene therapy. In the present study, we undertook an experimental approach which enabled to directly correlate the cell cycle stage of the target cell with the integration profile of LVs. Specifically, CD34+ cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate the transcribed genes with the integration sites. Our results show that approximately 10% of integration events took place in actively transcribed genes and that the cell cycle stage of the target cell population affects the integration pattern. More specifically, the use of thymine promoted a safer profile, since it significantly reduced vector integration within cell cycle-related genes, while there was an increased possibility for integration into genes related to development, metabolism and the immune system, and decreased possibility within cell cycle and cancer-related genes, when transduction takes place during mitosis. Thus, these novel findings provide important insights into the biology of retroviruses towards safer gene therapy applications.
Assessing the safety of SIN LVs harboring chromatin insulators in a stringent in-vivo genotoxicity assay
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Systemic vector-administration in newborn tumor-prone Cdkn2a-/- mice enables to assess the genotoxic potential of Self-Inactivating (SIN) Lentiviral-Vectors (LVs) and the underlying molecular mechanisms of insertional mutagenesis. We showed that a SIN LV harboring CAAT box-binding transcription factor/nuclear factor-1 (CTF/NF1)-based chromatin insulator (CTF/NF1.SIN.LV) was able to block the interaction between cellular and viral enhancer/promoters in vivo. Indeed our integration site analysis in tumors obtained from these mice revealed that CTF/NF1.SIN.LV induced genotoxicity by inactivating tumor-suppressors such as Pten (11 integrations), while the non-insulated SIN.LV induced tumors by activating mainly Map3k8 oncogene (14 integrations). Our data showed that CTF/NF1.SIN.LV-induced genotoxicity was not due to inefficient insulation but rather to an escape mechanism involving inactivation of tumor-suppressors. Here we tested the genotoxic potential of a SIN LV containing a different insulating cassette based on the CCCTC-binding factor (CTCF) cloned within the LTRs, and the strong Spleen-Focus-Forming-Virus enhancer-promoter in internal position. Cdkn2a-/- mice injected with LVs harboring CTCF- or CTF/NF1-based chromatin insulators displayed a similar median survival time (196 and 203.5 days respectively) that was slightly longer than the one of the uninsulated SIN.LV group (186 days). A preliminary integration sites analysis on tumors arising from mice treated with the CTCF-insulated SIN LV (206 integrations) showed a strong targeting of Map3k8 (5 integrations), indicating the inefficient insulating activity of this cassette in-vivo. Additional integration site analysis is ongoing and will reveal the enhancer-blocking activity of CTCF insulators in-vivo. These results will be instrumental to understand the safety-profile of insulated-SIN.LVs and their enhancer-blocking ability in-vivo.
Evaluation of the ethics and social acceptability of a proposed clinical trial using maternal gene therapy to treat severe early-onset fetal growth restriction in pregnant women
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Mildly acidic culture conditions improve the production of highly infectious and stable lentiviral vectors
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Lentiviral vectors (LVs) clinical applications are increasing. Therefore, improving industrial production of clinical-grade LVs becomes an important challenge. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process like transfection, cell culture or media optimizations. Yet, the effects of physico-chemical parameters such as pH remain poorly studied. Mammalian cell cultures are generally performed at neutral pH, which may not be the optimal condition to produce high quantities of LVs with optimal infectious properties. In this study, we show that lentiviral transient production in HEK293T cells is inversely dependent on the pH value of the harvesting medium. Infectious and physical titers of LVs pseudotyped with GALVTR or VSV-G glycoproteins are enhanced by two to three-fold at pH6 compared to neutral conditions. pH6-produced LVs are highly infectious on cell lines but also on relevant primary target cells like human hCD34+ cells. pH6-produced LV particles are highly stable at 37°C (closed and open environment) and are resistant to multiple freeze-thaw cycles. Higher levels of intracellular pr55gag polyproteins are observed within HEK293T producer cells cultured at pH6. The positive effect of pH6 conditions is also observed for moloney-derived retroviral vectors, arguing that the pH6 effect is not limited to the lentivirus genus and is reproducible in various producer cell lines. Thus, mildly acidic culture conditions augment the output of particles with otherwise equivalent stability and infectivity as pH7-produced particles. This observation may help us in the design of more effective LV production protocols for clinical applications.
The impact of Cre activity and co-infection on adenovirus propagation: optimizing canine helper-dependent vector manufacturing
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The production of adenoviral helper-dependent vectors (HDVs) is commonly based in the Cre/loxP system to minimize helper vector (HV) contamination. HDV producer cells express Cre-recombinase and are generally maintained in culture for longer periods of time as they are needed for the serial amplification steps of the vector until sufficient titers are generated. However, Cre-recombinase induces toxicity which impairs cell-line performance. Therefore, the evaluation of cell-line stability in the scope of HDV production becomes critical. In this work, Cre-expressing cells stability, infection conditions and their relation to adenovirus amplification and HV contamination were evaluated during the development of a production protocol for HD canine adenovirus type 2 (CAV-2) vectors in serum-free medium. Despite presenting similar cell growth profile with increasing culture passages, the long-term Cre expression in MDCK-E1-Cre cells after 20 passages reduced the adenovirus production up to 7-fold. Furthermore, a slight increase in non-infectious particles was obtained with MDCK-E1-Cre cells. The best conditions to produce HD CAV-2 followed a high HDV/HV MOI ratio (5:0.1), where HV contamination levels were the lowest. Curiously, we found that such MOI ratio can effectively reduce HV contamination even when using cells without Cre recombinase. This raises the possibility of producing HDVs without Cre-expressing cells when optimal infection conditions are used. This work elucidates the impact of Cre and MOI ratio on the optimization of HD CAV-2 production in serum-free medium, identifying key aspects involved in vector yields and quality that need to be considered when designing a production for a gutless adenoviral vector.
Towards automated manufacturing of clinical scale gene-modified T cells
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Adoptive cancer immunotherapy using gene-modified T cells has proven clinical efficacy and tremendous potential. However, such personalized medicine faces several challenges in the complexity associated with the current clinical manufacturing methods, which hampers dissemination. Conventionally, the preparation of autologous gene-modified T cells comprises many (open) handling steps, is labour intensive and is not adapted to treat large numbers of patients or for commercial manufacturing. Moreover, an extensive training of personnel and a dedicated infrastructure is required, restricting these clinical procedures to very few institutions worldwide. In order to face these challenges, we have developed a unique cell processing platform, the CliniMACS Prodigy, which enables the automated manufacturing of clinical grade gene-modified T cells in a closed single use tubing set. Starting from blood products, the automated process enables magnetic labeling and enrichment of T cells and their subsequent stimulation, gene-modification with lentiviral vectors, expansion and final formulation with minimal user interaction. Within the process a novel stimulatory reagent has been implemented: MACS GMP TransActTM. TransAct is a colloidal reagent developed for polyclonal T cell stimulation that is soluble, biodegradable, sterile filtered and can be removed by washing. It is suitable for potent T cell activation, gene-modification and expansion. Clinically relevant numbers of functional gene-modified T cells have been generated within 10–14 days. The flexibility and ease of use associated with this device and the developed process for clinical scale production of engineered T cells creates a solution for the treatment of large patient groups and makes economic commercial-scale manufacturing possible.
A Serum-Free method for Cultivation of Human Mesenchymal Stem Cells
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There is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. However, most of the current cultivation systems rely heavily on using batch validated foetal bovine serum (FBS) for optimal cell proliferation. Moreover, FBS-based cultures have the potential to spread transmissible microbes to MSCs and then to their recipients. We tested the effect of the serum-free XuriTM MSC Medium and Attachment Solution on a number of MSC types. Compared to FBS-based cultivation, Xuri MSC Medium achieved a higher cell yield of multipotent cells from small volume culture up to bioreactor scales. Here we show that hMSCs cultured in Xuri MSC Medium showed homogeneous expression of the ISCT markers, and maintained their tri-lineage differentiation capabilities. More importantly, MSCs cultivated in Xuri MSC Medium maintain their capabilities to suppress the proliferation of activated T lymphocytes in vitro, indicating the functionality of these cells were not compromised by the replacement of serum. Collectively, the combination of Xuri MSC Medium and Xuri MSC Attachment Solution offers a better cultivation process for MSC in vitro expansion, thus providind an improved MSC cultivation solution to the field.
Vectofusin-1® shows an efficacy similar to that of Retronectin® in promoting lentiviral vector transduction of human hematopoietic stem cells
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Current gene therapy trials with lentiviral vectors (LVs)-genetically modified hematopoietic stem cells (HSCs) rely on the use of the transduction enhancer, Retronectin®, to get optimal transduction rates. Vectofusin-1® is an histidine-rich cationic amphipathic peptide that has been recently shown to increase LV gene transfer in HSCs through improvement of adhesion and fusion of LVs with the cellular membrane, without inducing toxicity. Vectofusin-1® might have some advantages over Retronectin®. In particular, being a short peptide, it is easily synthetized and purified; moreover its use requires less manipulation, since it can be added directly to the viral supernatant without a pre-coating step as in case of Retronectin®. Here we investigated the effect of Vectofusin-1® in promoting LV-transduction of HSCs. Human HSCs were transduced in the presence of either Retronectin® or Vectofusin-1® with VSV-G-pseudotyped SIN-GFP highly purified LV produced by transient transfection or RD114-TR-pseudotypedSIN-GFP not purified LV derived from MolMed's proprietary RD3-MolPack-SIN-GFP producer cells. Similar VSV-G-pseudotyped SIN-GFP LV transduction rates (>80% GFP+) of HSCs and colony-forming cells (CFC) can be achieved with either Retronectin® or Vectofusin-1®. When RD3-MolPack-SIN-GFP-derived LV was used, the addition of Vectofusin-1® resulted in a slightly lower transduction efficiency (97% vs 84% GFP+) compared to Retronectin®, that was also confirmed in the CFC. No major differences in the immunophenotype of HSCs and expansion rate were observed between cells transduced in the presence of Retronectin® and Vectofusin-1®. In summary, we conclude that Vectofusin-1® is as good as Retronectin® for clinical manufacturing of LV-transduced HSCs.
Implementation of sample and data collection systems through regional transplant coordintaion of Andalusia (RTCA) and its provision by the Andalusian public health system biobank (APHS BB)
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There is a growing demand of tissue and cells from “healthy subjects” for use in research and clinical trials, aimed particularly by the Andalusian Initiative for Advanced Therapies (AIAT) in the case of clinical trials. One of the main sources to obtain such samples are living or cadaveric donors, managed by de Regional Transplant Coordination of Andalusia (RTCA). Therefore, the organization of a coordinated circuit of samples and data collection would be useful to satisfy this demand. Moreover, obtaining samples and data through this circuit has a great advantage, since it is possible to obtain samples from donors who are clinically validated, which gives them a very special value. To achieve this objective, a procedure between RTCA and APHS BB has been performed. This procedure includes both the initial proceedings to resolve the request and the actions of mutual information to enable the continuous process improvement. Since 2013 until now, this procedure has allowed the collection of 638 samples of different types, mainly fresh samples for immediate delivery as fat, blood, eye, pancreas… Also, samples of different organs such as lung, spleen, muscle, artery…have been frozen. With these samples we have been able to attend the requirements of eight research projects which have been provided with 595 samples. The implementation of this collaborative protocol between RTCA and APHS BB enables the provision of samples from donors “clinically validated” necessary for the development of specific research projects and clinical trial, ensuring quality and safety.
Optimization of a Replication-Competent Lentivirus (RCL) detection cultural GMP assay for release of Lentiviral Vectors
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MolMed, a medical biotechnology company, develops innovative technologies in oncology and gene therapies and manufactures large-scale lentiviral (LV) vectors transduced hematopoietic stem cells. LV vectors are currently released with a complete panel of analytical methods to verify the identity, potency, purity and safety. Among safety issues, a challenge for moving any viral vector into the clinic is the ability to screen the products for the presence of Replication-Competent Lentivirus (RCL). A highly sensitive cultural RCL assay based on amplification in the permissive C8166-45 T-cell line followed by p24 protein and VSV-G DNA detection is currently used in MolMed, but a successful optimization was made in order to reduce timing and test cost following recent upgrade of process scale and yield and to improve operator safety. The new qualified assay, always based on C8166-45 cells, involves the analysis of 300 mL of initial not-purified vector preparation, instead of the 5% of final purified and concentrated product considering that RCL could be generated only during the vector cultural production phase. The infection procedure has been optimized and the reference positive control R8.7 produced without VSV-G-pseudotype to improve operator safety. 1 TCID50 of positive control is now able to infect 150×106 C8166-45 cells in presence of 1.3×109 vector transducing unit. This improvement allows scaling down the number of flasks to manipulate during the assay by 7 times achieving a considerable reduction of time and costs, maintaining the same test sensitivity (1 TCID50/300 ml or 5% of product) in compliance with Guidelines.
Development of a GMP analytical method for the phenotype analysis and absolute count of samples for release and in process control of LV transduced CD34+ cells
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MolMed, a medical biotechnology company, develops innovative technologies in oncology and gene therapies and manufactures large-scale lentiviral (LV) vectors transduced hematopoietic stem cells. LV vectors transduced cells are currently released with a complete panel of analytical methods to verify the identity, potency, purity and safety. Moreover the transduction processes are monitored with IPC (In Process Control) in order to control the performance of the CD34 selection process starting from bone marrow or mobilized peripheral blood to the final CD34+ purified cells and the impact of the transduction and culturing conditions on CD34 phenotype. A GMP FACS analytical method has been developed in compliance with European Pharmacopoeia (PhEu 2.7.23) and permits to perform the absolute count of CD34+ cells present in the samples using beads of a known and certified concentration. Moreover, exploiting 7AAD-exclusion, the method returns the viability of samples. Matching antigen expression (by means of fluorocrome conjugated monoclonal antibodies use) and physical parameters, the method is able to verify the identity of CD34+ cells and their purity identifying the main cell population present in the samples. The populations recognized by the method are: hematopoietic stem cells, granulocytes, monocytes, lymphocytes B, Lymphocytes T and Natural Killer cells. An advantage of this method is to characterize CD34+ cells in a rapid way and with small sampling volumes. The suitability of the method, including instruments, reagents and analysts is periodically monitored through the participation in the inter-laboratory controls system promoted by UK-NEQAS, an international External Quality Assessment (EQA)/Proficiency Testing (PT) provider.
A clinical-grade constitutive packaging cell line for the production of self-inactivating lentiviral vectors
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Gene therapy has proven to be a promising approach to treat previously intractable medical disorders, and self-inactivating (SIN) lentiviral vectors (LVs) are becoming preferred vectors for ex vivo hematopoietic stem cell- and T cell-based therapies. However, the field is hindered by the paucity of cGMP-compliant packaging cell lines (PCLs) that can produce large batches of therapeutic vectors with consistent efficacy and safety characteristics. We have developed a constitutive LV PCL, STAR, as a proof of principle (Ikeda et al. Nat Biotechnol, 569-572(2003)). Based on this experience, we constructed a modified cGMP-compliant PCL with favourable safety characteristics that constitutively produces SIN LVs. Initially, active genomic loci in traceable 293FT cells were tagged with mutant LoxP sites using an MLV-based vector. A clone with a single, stable, high-expressor site was isolated, and Cre recombinase-mediated cassette exchange was used to insert a codon-optimised Gag-Pol cassette. Then plasmids encoding Rev, RDpro (derived from RD114 envelope), and GFP-expressing SIN LV were sequentially stably transfected. Four clones, which produce high-titre vectors long term, were established following selection with antibiotics that select cells actively expressing vector components. Producer cell culture was scaled up to yield multiple 24-hour harvests with titres up to 9×10^6 293T IU/ml in a volume of >0.5 L/harvest even under serum-reduced conditions. The stably-produced RDpro-pseudotyped vectors efficiently transduced human T-cells and CD34+ cells. We are currently constructing stable PCLs for large-scale production of therapeutic SIN LVs to treat X-SCID and B-cell malignancies. Moreover, alternative envelope glycoproteins are being investigated for construction of WinPac-derived PCLs.
Implementation of sample and data collection systems through regional transplant coordintaion of Andalusia (RTCA) and its provision by the Andalusian public health system biobank (APHS BB)
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There is a growing demand of tissue and cells from “healthy subjects” for use in research and clinical trials, aimed particularly by the Andalusian Initiative for Advanced Therapies (AIAT) in the case of clinical trials. One of the main sources to obtain such samples are living or cadaveric donors, managed by de Regional Transplant Coordination of Andalusia (RTCA). Therefore, the organization of a coordinated circuit of samples and data collection would be useful to satisfy this demand. Moreover, obtaining samples and data through this circuit has a great advantage, since it is possible to obtain samples from donors who are clinically validated, which gives them a very special value. To achieve this objective, a procedure between RTCA and APHS BB has been performed. This procedure includes both the initial proceedings to resolve the request and the actions of mutual information to enable the continuous process improvement. Since 2013 until now, this procedure has allowed the collection of 638 samples of different types, mainly fresh samples for immediate delivery as fat, blood, eye, pancreas… Also, samples of different organs such as lung, spleen, muscle, artery…have been frozen. With these samples we have been able to attend the requirements of eight research projects which have been provided with 595 samples. The implementation of this collaborative protocol between RTCA and APHS BB enables the provision of samples from donors “clinically validated” necessary for the development of specific research projects and clinical trial, ensuring quality and safety.
AGORA survey: ATMP regulatory differences identified in Europe
Academic GMP facilities play a pivotal role in the development of advanced therapy medicinal products (ATMPs). Until now, only a few ATMPs have received marketing authorization but many ATMPs are currently tested in clinical trials (CT) or are administered under the hospital exemption clause (HEC) of the ATMP-regulation. AGORA (ATMP-GMP-Open-Access-Research-Alliance), a FP-7 supported project, performed a survey among European facilities on the impact of ATMP-regulation within countries. By collecting this information, we hope to gather more information on the position of academic centers. Using an election questionnaire, we gathered information on the activities of the respondents (type and number of products regulated in CT or HEC, number of patients, the requirements (e.g. GMP license) and time lines set by their regulating body). These data are correlated with opinions on the current legislation and necessary steps to improve the availability of ATMPs for patients. Until now, we do have preliminary data from 12 countries showing that for CTs and HECs requests, NMAs are the regulating bodies in most countries. However, for some respondents the position (e.g. NMA or NA) of the regulating body is unclear. Since the HEC is not always implemented in national legislation, other regulations (e.g. Tissue-and-Cell Directive 2004/23/EC) are also applied to ATMPs. We found that 70% of ATMPs are produced for CT compared with 20% under the HEC and 10% under other regulations (e.g. compassionate use, named patient use). More detailed information will be available and presented at the meeting.
Auxotrophy complementation for antibiotic-free plasmid stabilization
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The segregational stability of plasmids in a recombinant bioprocess is of extreme significance. Although this is commonly achieved by the selection pressure from antibiotics, their application for the production of therapeutic DNA for gene therapy or for DNA vaccines is undesirable. Similarly, the presence of antibiotic-resistance genes in the final product is to be avoided in order to be approved by regulatory authorities. The gene tpiA is responsible for the connection of the glycerol metabolic pathway with the essential glycolytic pathway in Escherichia coli. The knockout of genomic tpiA rendered cells completely auxotrophic in minimal medium with glycerol as sole carbon source while allowing growth at a reduced rate with glucose or in complex medium. This was advantageous for optimizing antibiotic-free cloning and selection of recombinant plasmid. Complementation of the auxotrophy by plasmid-borne tpiA led to high segregational and structural plasmid stability, resulting in stable production of a model recombinant enzyme under antibiotic-free conditions in a continuous cultivation. Thus, the complementation of tpiA represents a significant alternative to antibiotics as a selection principle and is therefore of potential interest for the recombinant production of biotherapeutics.
Optimization of AAV9-SMN gene therapy for Spinal Muscular Atrophy
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We recently set up an efficient gene therapy strategy for Spinal Muscular Atrophy (SMA) in the SMNΔ7 mouse model through intravenous (IV) and intramuscular (IM) neonatal administration of a self-complementary AAV of serotype 9 carrying a codon-optimized version of the human «Survival of Motor Neuron 1» gene (scAAV9-SMN1opti) (Dominguez et al., 2011; Benkhelifa-Ziyyat et al., 2013). Despite its dramatic efficacy, this gene therapy approach was not complete (median survival 121 days and only partial restoration of the body weight loss phenotype) and was only efficient in pre-symptomatic mice. Our objectives were (1) to optimize this first study by a comparative analysis of alternative delivery routes (intracerebroventricular [ICV] and IV plus ICV combination [Co-IV/ICV] (2) to extend these findings to gene therapy of post-symptomatic SMNΔ7and severe [Smn−/−; hSmn2+/+] mice, and (3) to investigate the contribution of central and peripheral tissues to the mouse rescue using a neuron-specific AAV9 vector encoding SMN under control of the Synapsin promoter (AAV9-SYN-SMNopti). Our results showed the crucial role of peripheral SMN on the SMA phenotype, and the dramatic efficiency of Co-IV/ICV administration for rescuing post-symptomatic SMA both in P5 SMNΔ7mice (increased lifespan up to 250 days instead of 14 days) and in P0-P2 [Smn−/−; hSmn2+/+] mice (up to 417 days days instead of 4 days). These results represent the highest increase in survival obtained to date in post-symptomatic SMA mice and provide Co-IV/ICV as an optimal delivery route for further clinical developments.
Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing in cell-lines expressing normal or mutant rhodopsin
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The most frequent cause of retinitis pigmentosa (RP), a group of hereditary retinal dystrophies leading to blindness, is the occurrence of point mutations in the Rhodopsin (RHO) gene causing a gain of function or dominant negative effect deleterious for photoreceptors. Moreover, variations in RHO expression level are also deleterious for the retina. Therapeutic strategy should thus both lead to suppression of mutant protein expression and restoration of the normal one at a physiological level. Spliceosome-mediated RNA trans-splicing allow respecting these constraints by repairing mutant RHO pre-mRNA. This approach consist in introducing by gene transfer an exogenous RNA – called PTM, for Pre-Trans-splicing Molecule – able to bind the pre-mRNA and promote splicing in trans, leading to replacement of the mutated part of the RHO pre-mRNA. We engineered fourteen different RHO-PTMs able to repair any mutation in RHO exons 2, 3, 4 and 5, which differ only on their binding sequence to RHO pre-mRNA. To determine the efficiency of each PTM, we transiently co-transfected HEK293T cells with a plasmid encoding a PTM and another encoding the wild-type (WT) or mutant RHO. The maximum trans-splicing efficiency observed at the mRNA level was about 25%. We improved this efficiency to 40% by refining PTM sequence. We then tested PTM at the protein level in a cellular model expressing RHO stably. While WT RHO was localized to the plasma membrane, mutated RHO was retained inside the cell. We showed that trans-splicing is able to partially restore a correct localization of RHO in vitro.
Systemic AAV9-SMN delivery is crucial for Spinal Muscular Atrophy (SMA) in mice
We have recently demonstrated that intravenous (IV) injection of self-complementary adeno-associated-virus vector of serotype 9 (scAAV9) allowed the unprecedented widespread transduction of motor neurons (MNs) in both neonatal and adult animals, including large animals (Barkats 2007, PCT/EP2008/063297; Duqué 2009). We then successfully translated this approach to SMA by injecting intravenously SMNΔ7 mice with an scAAV9-PGK-SMNopti (Dominguez 2011). In this study we investigated whether the efficacy of this first study could be improved by SMN delivery close to the MNs through intracerebroventricular (ICV) vector injection. The therapeutic effect mediated by ICV scAAV9-PGK-SMNopti injection overpassed that of IV, suggesting a crucial role of SMN in the nervous cells, in particular MNs. Unexpectedly, we found that ICV scAAV9-PGK-SMNopti injection mediated a high and widespread SMN expression not only into the central nervous system cells but also in peripheral organs such as the heart and the liver. To further investigate whether the mouse rescue resulted from central and/or peripheral SMN gene transfer, the mice were injected into the brain ventricles with an AAV9 vector expressing SMN under control of the neuron-specific Synapsin promoter scAAV9-SYN-SMNopti. Interestingly, a weak or lacking survival was observed after neonatal scAAV9-SYN-SMNopti injection although more than 70% MNs were transduced. Furthermore, co-injection of the scAAV9-PGK-SMNopti vectors into both the temporal vein and the brain ventricles (Co-IV/ICV) improved the efficacy of the treatment. Together, these results argue the crucial role of peripheral SMN expression for the treatment of SMA mice and provide Co-IV/ICV delivery as an optimal route for SMN gene therapy.
Magnetofection: a feasible approach for the treatment of age-related macular degeneration
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Age-related macular degeneration (AMD) is the main cause of vision loss among the elderly in Canada and it is the third leading cause of blindness worldwide. The current treatment for AMD is intravitreal injection of an anti-VEGF drug every 4–6 weeks for 1–2 years. The therapy is effective but frequent intravitreal injections are inconvenient for the patients and are also associated with risks of retinal detachment and intraocular hemorrhage. Gene therapy as an effective method for the treatment of AMD or other retinal diseases has been proposed by several research groups. We propose here magnetofection, which is the delivery of nucleic acids associated with magnetic nanoparticles under the influence of an external magnetic field, as an effective method for the treatment of AMD. Here, we report pilot in vitro results on magnetofection of ARPE-19 cells with luciferase mRNA. The luciferase expression increased significantly in magnetically transfected cells compared to lipofection. Additionally, the onset of enzyme expression in magnetofected cells was earlier: it was detected 3 hours after magnetofection, while in non-magnetically transfected cells the enzyme became detectable only 6 hours after transfection. Metabolic activities of the magnetically transfected cells, as determined by an MTT assay, showed no changes in activity. The results were promising and showed that magnetofection increases protein expression from ARPE-19 cells. Furthermore, we are working on optimizing the magnetofection method for siRNA delivery to ARPE-19 cells with the aim of down-regulating VEGF expression.
The role of ATR in retinal cilia formation and elongation
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ATR (Ataxia-Telangiectasia and rad3-related), a serine-threonine protein kinase, is activated in response to DNA damage. We have recently characterized a Seckel syndrome mouse model (ATR+/s) regarding its retinal phenotype and found a new role for ATR in photoreceptors (PRs). ATR+/s mice display massive destruction of PRs and accumulation of pigment in the retina, suggesting a Retinitis Pigmentosa-like degeneration. In WT mice, ATR is present in the primary cilium of the PRs and ATR+/s mice display shortened cilia, suggesting that the PR degeneration results from a ciliary defect in those animals. Our hypothesis is that ATR has a role in cilium formation and elongation. RPE-1 cells were cultured under serum-starvation conditions to induce primary cilia formation. ATR inhibition was performed by adding different concentrations of caffeine to the culture medium. Our results show that under serum-starvation cultures, ATR is located in the base of the primary cilia and co-localizes with the centrosome of the ciliated cells. Moreover we found that ATR co-immunoprecipitates with the centrosome marker gamma-tubulin in the cells under serum-starvation conditions. After caffeine treatment ATR expression decreases inducing a decrease in the ciliary length of 80%, showing a direct effect of ATR in cilia elongation in a caffeine-dependent manner. Our results show that ATR is interacting with the centrosome of the ciliated cells and its inhibition is associated with shortened cilia, indicating a different role of ATR that might be involved in retinal ciliopathies. Support: FCT Portugal (SFRH/BD/76873/2011), PEst-OE/EQB/LA0023/2013. IBB–Institute Biotechnology Bioengineering (Associated Laboratory).
Combined cell-cytokine therapy for EAE treatment
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Experimental allergic encephalomyelitis (EAE) is an adequate animal model of the human CNS inflammatory demyelinating diseases, so it is widely and intensively studied. Besides the fundamental problems, such as molecular understanding of inflammation, T-cell activation, and MHC restriction during this disease, searching for approaches to its treatment is the question of prime importance. The work presented is devoted to the development of cell and combined cell-cytokine therapies of EAE. EAE has been established in rats by standard method, at 17th day animals were suboccipital injected human UC-MSC alone, or these cells in combination with human recombinant Il-10, or Il-10 alone. Il-10 was chosen as the main anti-inflammatory cytokine. They were monitored for 60 days considering their physical state and behavior peculiarities comparing with untreated control group. It has been shown that dynamics of rats' clinical state changed similarly in all groups during first 30 days of study. Short deterioration (for 2–3 days) was determined in all experimental groups comparing with control with untreated EAE, presumably due to effect of anesthesia. After this period, control untreated animals demonstrated synchronization of the disease with periodical aggravation. On the contrary, animals, which received cells or anti-inflammatory cytokine Il-10, or both, showed complete recovery without relapses. The best result was obtained with combined cell-cytokine therapy – improvement was observed statistically earlier. Data obtained evidence about the prospects of combined cell-cytokine therapy in EAE treatment.
Viral vector approach to study the potential toxicity of mutant LRRK2 kinase fragments in the rat substantia nigra
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Mutations in the LRRK2 gene are the most common genetic causes of autosomal dominant Parkinson's disease (PD). Genetic models of LRRK2 mutations have provided new insights in the possible mechanisms underlying mutant LRRK2 toxicity, pointing to a key role of its kinase domain. However, transgenic models in rodents show limited neurodegeneration of dopaminergic striatal neurones in the substantia nigra pars compacta (SNc). As an alternative to transgenic models, we evaluated the potential neurotoxicity of lentiviral (LV) and adeno-associated viral (AAV) vectors expressing three different fragments of the C-terminal part of wild type (WT) or G2019S mutant LRRK2: 1) the kinase domain (K), 2) the kinase domain plus its regulatory ROC-COR domain (RCK) and 3) the RCK domain with the C-terminal WD40 domain of LRRK2 (RCK-WD40). LV and AAV vectors coding the different constructs were injected in the rat SNc. Histological evaluation showed that 20–60% of the tyrosine-hydroxylase-(TH)-positive dopaminergic neurons were transduced using these vectors. No major loss of TH-positive cells could be evidenced at 10 weeks and 25 weeks post transduction with LVs coding fragments with the G2019S mutation. Longitudinal behavioral evaluation of the rats injected with LVs and AAVs coding RCK-WD40 fragments showed no change in motor performance. However, transcriptomic studies of the rat striatum over-expressing RCK-WD40 showed that the G2019S mutation produced cellular disturbances as compared to controls (sham-operated, wild type and “dead kinase” G2019S/D1994A). Newly identified genes selectively deregulated by RCK-WD40 are under investigation as they could constitute novel biomarkers for LRRK2 mutations.
Mitochondrial gene therapy: AAV2-mediated direct delivery of nucleic acids to the mitochondrial matrix as a treatment for Lebers Hereditary Optic Neuropathy
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Leber's Hereditary Optic Neuropathy (LHON) is form of inherited blindness caused by point mutations in Complex I subunit genes ND1, ND4 or ND6, with the m.11778G>A mutation in ND4 being most common. Due to the location of these genes on the mitochondrial chromosome, conventional gene therapy tools specialized for delivering therapeutic genes to the nucleus must rely on allotopic expression and transport of the ND proteins. To facilitate transcription and translation of the ND genes in their natural environment, i.e. the mitochondrial matrix, an AAV2 vector was modified by introducing a mitochondrial targeting sequence (MTS) into the reading frame of capsid protein VP2, creating an AAV-MTS vector. Fluorescent proteins markers RFP or GFP were also integrated into the viral capsid in order to monitor re-trafficking of the AAV2 viral particles. We recoded GFP for mitochondrial-exclusive translation (mitoGFP) to unambiguously identify transduced cells where transcription and translation of the AAV-MTS delivered transgene was occurring in mitochondria. LHON cybrid cells containing the m.11778G>A mutation in 100% of their mitochondria were created from LHON patient fibroblasts. Complex I activity in these cybrids was measured via Seahorse analysis and found to be deficient compared to cells containing wild type mitochondria. Evaluation of the efficacy of AAV-MTS vectors containing wild type ND4 in improving Complex I activity and survival of LHON cybrid cells, as well as preventing or reversing vision loss in a LHON animal model is in progress.
Phase I clinical study for patients with retinitis pigmentosa: interim report of initial 5 subjects (low-titer group)
RNAi-based gene therapy of Huntington's disease
Gene therapy is one of the most advanced approaches investigated for the treatment of Huntington's disease (HD). The most upstream therapeutic target in HD is the mutated huntingtin (Htt) and the goal is gene silencing with artificial miRNAs (miHtt) delivered with adeno-associated viral vector (AAV). Three major approaches have been undertaken for the development of RNAi-based HD gene therapy: (1) total Htt silencing by targeting exon 1, (2) targeting the CAG repeats in Htt and (3) allele-specific inhibition of mutant Htt expression by targeting SNP rs362331 in exon 50 and SNP rs362307 in exon 67. In the total silencing approach, sequences in Htt exon 1 have been targeted and highly active therapeutic miHtt candidates have been selected. In the allele-specific approach, SNPs linked to the expanded CAG repeat allele have been targeted allowing specific silencing only of the mutant Htt. Murine striata were transduced with AAV5-miHtt12, and AAV5-miSNP67 and a strong knockdown of Htt reporter gene was observed within 8 weeks post injection. Ongoing research aims to assess the neuroprotective effect of the non-selective and allele-specific miHtt and expressed from AAV5 vectors in rodent HD models. Different viral delivery routes by direct intrastriatal injection or in the cerebrospinal fluid are being evaluated. Furthermore, the Htt silencing efficacy, safety and off-target potential of the different therapeutic candidates are determined. AAV5-miHtt delivery provides a novel approach for HD therapy as it might allow specific inhibition of the mutant Htt gene expression and hence reduce severity of neuropathology and slow down disease progression.
Transfection of freshly isolated pigment epithelial cells with pFAR4 miniplasmids using the Sleeping Beauty (SB100X) transposon system
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Efficient transfection of cells mediated by viral vectors or physical methods requires cell numbers well above the number that can be obtained from human iris biopsies. However, for gene therapeutic treatment requiring transplantation of genetically modified autologous cells, it will be advantageous to be able to efficiently transfect freshly isolated cells obtained immediately after isolation from a patient's biopsy. Here, we report the transfection of as few as 5×103 retinal (RPE) and iris (IPE) pigment epithelial cells from 2.25 mm2 small iris biopsies with the gene for pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB100X) transposon system and the free of antibiotic resistance (pFAR4) miniplasmid system. Freshly isolated RPE and IPE cells were transfected with the pFAR4-CAGGS-Venus or the pFAR4-CMV-PEDF transposon and the pFAR4-CMV-SB100X transposase using a ratio of 16:1 and a total plasmid concentration of 0.5 μg. Efficiency of transfection was analyzed by fluorescence microscopy and Western blot analysis for the Venus and the PEDF protein, respectively. The efficiency of transfection was 30.02±23.69% (cells isolated from whole iris), 17.91±11.83% (9 mm2 biopsy), and 13±12.8% (2.25 mm2 biopsy), as determined in 4 independent experiments. The transfected cells secreted 2261±1711 ng (cells isolated from whole iris), 608.7±356.8 ng (9 mm2), and 143.2±96.7 ng PEDF/mL (2.25 mm2) and secretion is stable for 140 days up to now. Here, we have shown that the SB100X transposon system in combination with the pFAR4 miniplasmid system allows for efficient integration of target genes into freshly isolated cells.
Combined IV and ICV scAAV9 and scAAV10-SMNopti delivery dramatically rescues symptomatic SMA mice
Spinal Muscular Atrophy (SMA) is a neuromuscular disorder, caused by SMN (“Survival of Motor Neuron”) gene, and characterized by motor neuron (MN) degeneration, progressive muscle weakness, and premature death in severe cases. We and others demonstrated that intravenous (IV) neonatal administration of self-complementary adeno-associated virus vector of serotype 9 (scAAV9), carrying an optimized human SMN1 expression cassette (SMNopti), mediated high and widespread SMN expression into the MNs, and prevented disease progression in the SMNΔ7 mouse model of SMA. Our objective was to investigate whether scAAV9 or scAAV10-SMNopti delivery could halt or reverse the disease in the severe (Smn−/− ; hSmn2+/+) symptomatic mouse model of SMA (hSmn2). We first investigated whether the transgene product could be expressed within the therapeutic window (mean survival 4 days) and found that both vectors allowed protein synthesis from 24 hours post-injection. The SMN-expressing vectors were then co-injected into the brain ventricle and the facial vein of P0-P2 mice and clinical monitoring was then daily performed. Both AAV serotypes allowed to increase survival up to 417 days (104-fold increase), representing the highest increase in survival obtained to date in hSmn2 mice. High SMN expression levels were found in the brain and spinal cord at final endstage, and the neuropathological analyses (MN survival, neuromuscular junction size, muscle atrophy) are in progress. This study demonstrates the efficacy of SMN-expressing AAV9 and AAV10 vectors for gene therapy of post-symptomatic SMA mice, and highlights the therapeutic potential of the co-IV/ICV route of delivery.
Generation of a zebrafish Eys knock-out model for inherited retinal degeneration
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Retinitis pigmentosa (RP) is a clinically diverse and genetically heterogeneous group of inherited retinal disorders with a prevalence of approximately 1 in 4,000 individuals. Mutations in Eyes shut homolog (EYS), a gene expressed uniquely in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar)RP, accounting for approximately 5–10% of arRP cases. In order to develop a treatment strategy for EYS-associated RP, a suitable model system is needed. Since the Eys gene is not present in the mouse genome, we are currently cloning and characterizing Eys in zebrafish, via RT-PCR analysis on zebrafish eyes cDNA, which revealed a high degree of similarity between the orthologous human and zebrafish genes. Furthermore, we are generating a stable Eys knock-out in zebrafish via CRISPR/Cas9 technology. The effect of EYS knock-out on retinal structure and function will be studied using immunohistochemistry and behavioral assays. Subsequently, this model will be used to assess the effect of administering rescue constructs that are being developed. In conclusion, our work will serve as the basis for understanding the function of EYS, and to initiate pre-clinical therapeutic intervention studies.
Therapeutic pri-miRNA expression for gene therapy of Huntington's disease
Huntington's disease (HD) is an incurable neurodegenerative disorder caused by cytotoxicity of expanded polyglutamine (polyQ) repeats at the N-terminal of the huntingtin (HTT) protein. Functional silencing of the aberrant HTT transcript by RNA interference (RNAi) using pri-microRNA (pri-miRNA) scaffolds is an attractive approach for HD therapy as it targets the root of the disease. We have developed several highly potent miRNAs (miHTT) targeting HTT exon 1 or mutant HTT-associated single-nucleotide polymorphism (SNP) and their knockdown efficacy has been verified in vitro and in vivo. To ensure the best miRNA safety profile, miRNA processing has been further optimized using 10 different cellular pri-miRNA scaffolds. The guide and passenger strand of each cellular pri-miRNA scaffold were replaced with HTT-targeting sequences while preserving the original hairpin structures. Moreover, pri-miHTT transcription was set under control of various promoters and HTT knockdown efficacy was characterized. We identified several pri-miRNA scaffolds that enhance miHTT activity and are currently performing NGS analysis to determine their processing patterns. The potential off-targeting activity of the miHtt* passenger strand was verified on specific Luc-Htt* reporters. The miHTT scaffolds regulated by the strong promoters induced stronger HTT knockdown in comparison with the weaker equivalents, which reflects the differences in the promoter activity. Rodent HD models will be injected with the adeno-associated virus (AAV) carrying the pri-miHTT constructs with the highest activity and lowest off-targeting effect of the passenger strand to evaluate their therapeutic and safety potential. Support: uniQure, EU-FP7 E!7900
An evaluation of viral vector-mediated gene delivery to the murine neonatal brain
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The brain is the most complex organ of the body. It contains a range of cells such as neurons, astrocytes, oligodendrocytes, resident microglia and ependymal cells. These cells are arranged into a large number of discrete regions that vary in size, cellular density and function. Efficient viral vector-mediated gene delivery to the neonatal murine brain is essential in pre-clinical studies of gene therapy for numerous early lethal neurological disorders. Furthermore, gene delivery to the developing brain is a powerful biological tool e.g. gene function or microRNA studies. More information is required on the biodistribution, cellular tropism and immunological response of commonly used vectors in the developing brain. We have evaluated a range of viral vectors including AAV and lentiviral vectors that contain the GFP reporter gene driven by the CMV promoter. These vectors were administered via intracranial injection into the brains of newborn (P1) pups. The mice were left for 30 days for the brains to reach their maximum size before being harvested and analysed to assess the above parameters. We demonstrate that different viral vectors mediate varying levels of biodistribution in the brain and differential cell tropisms. We also demonstrate that the neonatal brain is immunologically tolerant of relatively high doses of vector and extensive GFP expression. This data is essential in selecting the optimal vector for the desired application in the developing brain, whether this is for therapeutic purposes of biological investigative studies.
Therapeutic potential of erythropoietin in a mouse model of huntington's disease
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotid repeat expansion leading to an elongated polyglutamine stretch in the huntingtin protein. Huntington is expressed ubiquitously in both neuronal and glial cells of the brain, including regions of adult neurogenesis like the hippocampus. In recent years, the hematopoietic cytokine, erythropoietin, was found to mediate neuroprotection and enhance neurogenesis in various neurodegenerative disorders. The purpose of our study was to evaluate the therapeutic potential of human erythropoietin (hEPO) in the R6/2 transgenic mouse model of HD. R6/2 mice exhibit motor and cognitive deficits, decreased brain size and reduced hippocampal neurogenesis. We performed a single injection of a lentivirus encoding hEPO into the lateral ventricle of R6/2 mice at disease onset. The control group received lentivirus encoding the green fluorescent protein (GFP). We observed stable expression of hEPO or GFP in the brain for 4 weeks after virus administration. Notably, R6/2 mice injected with hEPO-lentivirus showed improved spatial working memory and reduced hippocampal atrophy on MRI compared with controls. Moreover, the total number of neuroblasts and the number of migrating neuroblasts in the dentate gyrus of the hippocampus was significantly increased in R6/2 mice injected with hEPO-lentivirus compared with controls. In conclusion, our results suggest that hEPO may be a promising cytokine for treatment of cognitive impairment in HD.
Anti-angiogenic VEFG isoform is markedly decreased in the retina of diabetic mice
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Alternative splicing of the vascular endothelial growth factor (VEGF) gene generates two families of VEGF isoforms. The VEGFxxx isoforms are the most potent endothelial pro-angiogenic growth factors. In contrast, the anti-angiogenic VEGFxxxb isoforms are highly expressed in normal tissues but down-regulated in pathologies associated with abnormal angiogenesis, such as some retinal diseases. The aim of this study was to evaluate the expression of VEGF isoforms in the retina of the Ins2Akita mice, a diabetic model that develops retinal complications. Upon comparison with normoglycaemic mice, we have found that the expression of VEGF remained unchanged in the retina of 6-month old diabetic mice but was slightly increased at 12 months of age. On the contrary, the expression of the anti-angiogenic VEGF165b isoform was highly reduced in the retina of diabetic mice at 6 and 12 months of age. Additionally, we also found increased levels of reactive oxygen species in the retinas of diabetic mice. Altogether these results show an imbalance between the pro- and the anti-angiogenic VEGF isoforms in the retina of diabetic mice and also show that these retinas are in a condition of oxidative stress. The changes observed in the retinas of diabetic mice were evident at a time-point were these mice show retinal complications, suggesting that both the balance between the VEGF isoforms and the oxidative stress status of the retina might contribute to the process of retinal disease. SFRH/BPD/78404/2011, Foundation for Science and Technology (S. Simao), SFRH/BD/52424/2013 (D. Bitoque), SFRH/BD/76873/2011 (S. Calado) EXPL-BIM-MEC-1433-2013. PIRG05-GA-2009-249314–EyeSee. PEst-OE/EQB/LA0023/2013.
Insights on the role of the Renin-Angiotensin System in diabetic retinopathy
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The Renin-Angiotensin System (RAS) is well described in the regulation of systemic blood pressure. All components of the RAS are expressed in the retina and can lead to retinal diseases, such as diabetic retinopathy. The aim of this study is to identify the presence of RAS in retinal pigment epithelial cells (RPE) and the effects of inhibition of the renin in the pathophysiology of diabetic retinopathy. We used D407 cells, a human RPE cell line, and assessed the expression of renin by immunocytochemistry and Western blot. The cells were exposed to different concentrations of a renin inhibitor, or angiotensin II, at different time points. We used calcein-am assays to evaluate the cellular viability and ELISA to assess activity of the renin. The production of reactive oxygen species (ROS) was measured by the presence of an oxidative stress marker. We observed that renin is expressed in D407 cells and secreted in the presence of different concentrations of glucose. Also, treatment with a renin inhibitor or angiotensin II does not affect the viability of these cells up to 8 hours of stimulation. The activity of renin in D407 cells is compromised by increasing concentrations of the renin inhibitor at different time points. We also observed that the treatment with angiotensin II influences the production of ROS in the retina. This work allows to conclude that renin is expressed in D407 cells and its activity can be affected by glucose, which may signal a role in diabetic retinopathy. SFRH/BPD/78404/2011, EXPL-BIM-MEC-1433-2013. PIRG05-GA-2009-249314–EyeSee. PestOE/EQB/LA0023/2013
Exon-skipping as a promising therapeutic approach for treatment of USH2A-associated retina degeneration
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Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants but for the loss of vision currently no treatment is available. Mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The USH2A transcript is built up by 72 exons that together encode Usherin, a large transmembrane protein consisting of 5,202 amino acids. Recently, we reported the identification of the first deep intronic mutation in the USH2A gene leading to the inclusion of a pseudoexon (PE40). Insertion of PE40 in the USH2A transcript subsequently results in a truncated protein lacking the C-terminal half including the transmembrane and intracellular region, if translated. In this study we explored the therapeutic potential of antisense oligonucleotides (AONs) to restore the native USH2A transcript. Our strategy is to induce skipping of PE40 by interfering with the splicing machinery in the PE40 region. For this purpose we used engineered AONs with complementary chemical backbones (phosphorothioate and morpholino) directed against intron-exon bounderies and exonic splice enhancer (ESE) regions of PE40. In this way we were able to induce the skipping of PE40 from the majority of the mature mRNA transcripts, predicting the translation of fully functional wild-type Usherin. Following this approach we expect to be able to stop the progression of this devastating blinding disorder and provide USH2A PE40 patients a prospect on vision in the future.
Effectiveness of G-CSF+ plerixafor mobilization in ß-thalassemia patients and whole gene expression analysis of the harvested CD34+ cells
Hematopoietic Stem Cells (HSC) mobilization is eligible source to obtain an high yield of CD34+ cells for ß-thalassemia gene therapy. We investigated on the safety and effectiveness of HSC mobilization by granulocyte-colony stimulating factor (G-CSF)+plerixafor, in four patients with ß-thalassemia major. The patients were treated according to the following scheme: G-CSF (10 mcg/kg/day) for four days and plerixafor (0,24 mg/Kg/day) on the fifth and eventually on the sixth day. The protocol resulted well tolerated with minimum side effects. Three of four patients reached the target cell dose of 8×106 CD34+/Kg with a single apheresis even one patient where a significant dose reduction of G-CSF was imposed due to early hyperleukocytosis. The CD34+ yields were higher than those of five patients previously mobilized with G-CSF alone (clinicalTrials.gov NCT00658385) and comparable to those of 12 healthy donors. After plerixafor administration, we found a mean value of 8-fold increase of peripheral blood CD34+ cells with higher frequency of cells that displayed primitive phenotype and ability to develop immature hematopoietic colonies. To further confirm the stemness of these cells we performed whole genome analysis on purified CD34+ cells from the same patients mobilized with the two protocols, G-CSF vs G-CSF+plerixafor. We highlighted that CD34+ cells mobilizied with G-CSF+plerixafor expressed higher level of genes involved in homeostasis, homing and engraftment. Therefore, our data suggest that HSCs mobilized with G-CSF+plerixafor are an optimal source for ß-thalassemia gene therapy. Work supported by Fondazione F. e P. Cutino (project: RiMedRi CUPG73F12000150004)
Validation of CRISPR/Cas9 nickase pairs in the generation of WASP-deficient Jurkat T cell
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Development of a diverse human T cell repertoire despite severe restriction of hematopoietic clonality in the thymus
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Hematopoietic gene therapy for immunodeficiencies relies on the generation of functional T cells from the transplanted gene modified HSC. However, exactly how individual HSC clones contribute to the developing T cell lineage after transplantation is currently unknown. To address this question, we made use of the NSG xenograft model and lentiviral cellular barcoding of human UCB-derived HSCs with the aim to study hematopoiesis and T cell development at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 weeks after transplantation. A very limited number of HSC clones (<10) repopulated the thymus, with clear restriction of the number of clones at the double negative stages. Yet, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T lymphocyte clones can develop from a single HSC clone. Our data imply that intra-thymic events, rather than HSC numbers determine T cell reconstitution after HSC transplantation. These data show that a very stringent selection takes place within the thymus of a transplanted individual, which suggests that factors affecting fitness of the thymus seeding cells might cause fitter cells to outcompete less qualified cells. Thereby, strong selective pressure is enforced on clones, such as those in which vector insertion has deregulated their gene expression program, resulting in such an increased fitness.
Optimisation of the manufacturing procedure for transduced autologous CD34+ cells for gene therapy of Chronic Granulomatous Disorder (CGD)
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Gamma retroviral vectors (gRV) have been used for proof of principle studies aiming to correct Chronic Granulomatous Disorder (CGD) by gene therapy. Lentiviral vectors (LV) are proposed to enhance safety of treatment for haematopoietic disorders. A multi-centre clinical trial has been designed to test a novel LV called G1XCGD containing the gp91phox gene and a myeloid specific promoter in CGD patients. The technologies for the production of autologous CD34+ cells transduced with LV are not well established. The process is based upon protocols for transduction with gRV. Integration with LV is not reliant on cell division and thus could be significantly modified. The study objective was to optimise the procedure for manufacturing of autologous CD34+ cells transduced with the G1XCGD vector. We have tested several key parameters for transduction, including culture containers, inclusion of Retronectin, protamine sulphate and cell density. We have also tested product stability for 0–30 hours in several alternative final formulations. A final protocol has been established for the effective transduction of cells with the G1XCGD vector using protamine sulphate and 2 rounds of transduction. This protocol has been tested in a variety of cell culture containers and efficiently transduces CD34+ cells with a VCN of 0.5–5; viability >70% and all microbiological testing meets the specification. Cell viability and CFU potency was remarkably stable in all the formulations tested, with viability of 84, 78 and 82% after 12 hours at 4°C in excipients saline 2% HAS, saline 5% HAS, plasmalyte 2% HAS, respectively.
Life-long gene expression in mouse airways after rAAV2/5 based gene therapy
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Assessment of hematopoietic and neurologic pathophysiology of Hax1 deficiency in a Hax1-ko mouse model
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Kostmann syndrome (KS) is the most common recessive form of Severe Congenital Neutropenias (SCN). SCN are characterized by marked decrease or absence of mature granulocytes in peripheral blood leading to recurrent life-threatening bacterial infections. KS is caused by HAX1 deficiency and is treated by life-long Granulocyte-Colony Stimulating Factor (G-CSF), which unfortunately leads in 25% of the patients to development of acute myeloblastic leukemia. Additionally, about 10% of KS patients present a very severe neuro-psychological impairment, which does not improve under G-CSF treatment. To develop alternative cellular therapies for both hematologic and neurologic manifestations of KS, we assessed the hematopoietic and neurologic systems of a Hax1-ko mouse model on clinical, cellular and molecular levels. The Hax1-ko mouse dies within 8 weeks of life presenting a foudroyant Parkinson-like phenotype due to post-natal neuronal apoptosis, astrocytosis and microgliosis in the striatum and an increasing motor impairment since the 4th week after birth. Quantitative PCR analysis on Hax1-ko striatum cell lysate showed increased levels of Casp8 and FAS mRNAs. Decreased hematopoietic stem and progenitor cells (p<0.005) and reduced spleen B cell pool (p<0.0005) characterized the Hax1-ko hematopoietic system. Finally, aiming an autologous HSC-based therapy for KS we developed a codon-optimized HAX1 expressing SIN lentiviral vector and surveyed the coHAX1-expressing wt hematopoietic cells over 20 weeks without observing any signs of hematological malignancies or phenotoxicity. Taken together, we used the Hax1-ko mouse to enlarge our view on KS pathophysiology and to start developing alternative cellular therapies for KS.
Assessment of immune responses to single and repeat dose aeroslisation of the non-viral formualtion PGM169/GL67A in cystic fibrosis patients
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We have previously shown that approximately 10% of CF and non-CF subjects carry self-reactive CFTR-specific T cells. It is unclear whether being positive for self-reactive T-cells affects disease severity or increases the risk of further T-cell activation after gene therapy. As part of the UKCFGTC Pilot study [patients received a single dose of pGM169/GL67A] peripheral blood mononuclear cells (PBMC) were collected prior to dosing and ∼4 weeks after nebulisation of 5 ml (n=2), 10 ml (n=6) or 20 ml (n=17). IFN-gamma ELISPOT assays were performed. Approximately 25% of samples failed assay quality controls. CFTR-specific T cells were detectable in one patient pre- and post-dosing. In the remaining 18 patients we did not detect CFTR-specific T cells. In addition we quantified anti-DNA antibodies (anti-nuclear and anti-cytoplasmic) in blood samples taken pre- and ∼4 weeks post-dosing (n=7 (5 ml), n=10 (10 ml) and n=17 (20 ml) to assess potential immune responses to the plasmid DNA. We did not observe any evidence for induction of anti-DNA antibodies after a single dose of pGM169/GL67A. The UKCFGTC has now completed a Phase IIb multi-dose clinical trial. CF patients (115 completed 9 or more doses) received 12 monthly doses of pGM169/GL67A or placebo by aerosol. PBMC were collected on two occasions prior to dose 1 to establish baseline levels for CFTR-specific T cells, approximately 4 weeks after Dose 4 or 5, and 2 to 4 weeks after Dose 12 and the ELISPOT was performed. In addition anti-DNA were quantified. Results from these studies will be reported.
Results of a Phase IIb non-viral gene therapy trial from the UK CF Gene Therapy Consortium
The UK Cystic Fibrosis Gene Therapy Consortium has conducted a double-blinded placebo-controlled multi-dose Phase IIb study to assess the efficacy of non-viral CFTR gene transfer at improving CF lung disease. Our optimized non-viral formulation consists of the cationic lipid GL67A and a codon-optimised, CpG-depleted CFTR expression plasmid (pGM169). Expression is regulated by a hCEFI enhancer/promoter shown capable of directing persistent expression in the lung. The intention-to-treat aimed to have all subjects receive 12 doses of nebulised gene therapy or placebo (0.9% saline) at intervals of 4 weeks over a 48 weeks period (136 patients received at least one dose and 115 completed 9 or more doses). Subgroups of patients (∼20/group) were enrolled for molecular assays (mRNA and chloride transport) in both nose and lower airway. The primary endpoint was FEV1, an outcome acceptable to regulatory agencies. In our longitudinal study of outcome measures, FEV1 was shown to be the assay with the most statistical power. Secondary outcomes included lung clearance index (a sensitive measure of small airway disease), quality of life questionnaires and CT scans as well as a number of exploratory assays. An adaptive design allowed the early identification of cumulative side effects: 17 subjects received 3 doses of gene therapy or placebo at 4-weekly intervals before any further subjects were dosed. No safety issues were identified by the Data Monitoring and Ethics Committee (DMEC) in this cohort. Dosing has now been completed, with no safety issues identified following three DSMB reviews. Results from these studies will be reported.
New Lentiviral Vectors for the Gene Therapy of Human Leucocyte Adhesion Deficiency Type I
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Leukocyte Adhesion Deficiency Type I (LAD-I) is a primary immunodeficiency characterized by recurrent and life-threatening bacterial infections. It is caused by mutations in the ITGB2 gene, encoding the integrin ß2 common subunit (CD18). These mutations lead generally to defective or absent expression of ß2 integrins in the leukocytes surface, implying that leukocytes are unable to adhere to the endothelium and extravasate to infection sites. As other monogenic immunodeficiencies, LAD-I is a good candidate for ex vivo gene therapy. Therefore, we have developed four SIN lentiviral vectors, expressing human CD18 from ubiquitous (PGK and UCOE) or myeloid (Chim and MIM) promoters. All CD18-LVs succeed to restore CD18 and CD11a expression in a LAD-I patient-derived lymphoblastoid cell line (ZJ). Corrected ZJ cells recovered the ability to aggregate and to bind to soluble ICAM-1 after proper stimulation. Due to the difficulty to access to patient samples, we have developed a shRNA-LV capable of down-regulating CD18 expression in CB CD34+ cells from healthy donors, thus mimicking HSCs from LAD-I patients. These cells can be differentiated in vitro into neutrophils, which recapitulated the LAD-I phenotype, with reduced adhesion capacity and diminished ability to mount a respiratory burst. When LAD-I like HSCs were corrected with LV.Chim.CD18 and then differentiated, they recovered a 45% of the WT CD18 expression. Despite of the moderate CD18 recovery, corrected neutrophils showed significant improvement in three different CD18-related functional assays. All these results allow us to propose new CD18-LVs that could be good candidates for future clinical applications for LAD-I patients.
Ectopic gp91phox expression is detrimental to XCGD iPS cell-derived neutrophils
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X-linked chronic granulomatous disease (XCGD) is caused by gp91phox deficiency. This compromises neutrophil (NEU) killing of phagocytosed pathogens due to impaired production of reactive oxygen species (ROS). In previous XCGD gene therapy clinical trials, the sustained persistence of gene marked cells could not be achieved without adversely triggering insertional mutagenesis. Additionally cellular recovery (gp91phox and ROS) had been incomplete on a per cell basis in comparison with healthy controls. This led to the hypothesis that the ectopic expression of gp91phox in developing NEUs could impede further differentiation into mature NEUs. To investigate this theory, a modeling system was established by generating patient autologous XCGD-iPSCs and inducing NEU differentiation. In this culture system, the hierarchical transition of NEU differentiation could be demonstrated from developing (CD64dullCD15dull) to mature (CD64highCD15high). Alpharetroviral vectors were used to transduce XCGD-iPSCs with the expression of codon optimized gp91phox cDNA driven by the ubiquitous EF1a short promoter. In healthy iPSC-derived NEUs, gp91phox expression and ROS production could only be detected in the mature fraction. In NEUs derived from transduced XCGD-iPSCs, functional recovery in the mature fraction was incomplete. Through intracellular staining, ectopic gp91phox expression was detected in the developing fraction. Most critically this appeared to correlate with more prominent cell death. Mechanistic studies are under way to investigate the relationship between non-physiological ROS production and the induction of endoplasmic reticulum (ER) stress resulting in cell apoptosis. Therefore, affording cellular protection from the detrimental effects of non-physiological ROS production may improve XCGD clinical outcomes.
Establishment of an in vitro model of the “Royal disease” by the use of patient-specific induced pluripotent stem cells
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The Royal Disease (RD), transmitted from Queen Victoria to European royal families, was unraveled as a severe form of hemophilia B (HB) caused by the mutation IVS3-3A>G in the Factor IX gene (F9). We identified an HB patient with bleeding symptoms exclusively attributable to RD mutation, which would represent excellent mimics of the disease. The present work aims to develop an in vitro model of RD, therefore we generated a patient-specific induced pluripotent stem cells (iPSCs) and differentiated them into hepatocyte-like cells (HLCs) where we performed a detailed analysis of F9 mRNA. The results from the patient's leukocytes were inconclusive because of the well-documented difficulty of studying F9 expression in these cells, while the use of the derived HLCs showed detectable levels of F9 mRNA by RT-PCR concomitantly with hepatocyte differentiation. Sanger sequencing showed the generation of an aberrant splicing site, in agreement with the previously reported results using an in vitro minigene-approach. Conversely, deep analysis by next generation sequencing, demonstrated the presence of a significant proportion (∼1,5–2%) of correctly processed F9 transcripts that could explain the residual FIX activity (∼0,5%) detected in the patient, leading a milder phenotype than expected and might also justify the relative long life of some of the European royal families' affected members, in a time without an effective therapy. In conclusion, the use of patient-specific iPSCs allows the establishment of a bona fide in vitro model of RD as well as an effective way for universal HB modeling, particularly to conduct F9 expression studies.
Mapping genes and pathways to preserve the hematopoietic stem cell activity upon ex vivo engineering
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New generation viral vectors and optimized transduction protocols had allowed applications of gene therapy for a number of diseases. To date the major achievements have been reached in pediatric patients, where transduced hematopoietic stem/progenitor cells (HSPCs) have demonstrated their therapeutic potential. Nevertheless, little is known about the response of HSPCs to the cytokines used in the transduction protocol in a disease context, and in pediatric vs adult cells. To answer this key question, we are investigating, by microarray analysis (n=50), the stem cell signature of cytokines stimulated bone marrow HSPCs from adult and pediatric healthy donor, and thalassemic patients. Both PCA and HCL show that the stimulation is the main variable that distinguishes the samples. SAM, Limma and ANOVA analyses indicate that there is no difference between thalassemic and healthy HSPCs, but there is a relevant “age effect”, both in the healthy and thalassemic context. More then 1,000 genes are strongly differentially regulated upon manipulation. GO and GSEA analyses show enrichment in terms belonging to cell cycle regulation, metabolic processes, and lymphoid differentiation. Analysis of custom gene sets identifies a decreased expression of negative regulators of cell cycle associated with self-renewal and of master genes of B-cell differentiation, with a corresponding increase in myeloid-specific genes. Collectively these data indicate, upon stimulation, an expansion of hematopoietic progenitors with multilineage potential, but reduced B-lymphoid priming. Validation and functional studies on selected genes are in progress and will help to develop strategies of gene transfer preserving the “stemness” features of HSPCs.
Finding levels of RAG1 expression that will correct RAG1 Severe Combined Immunodeficiency
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RAG1-SCID patients lack B- and T lymphocytes due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment alternative for RAG1-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy for RAG1 has proven challenging. We have previously shown with an SFFV-driven vector that sufficient RAG1 expression could be achieved for correction at clinically acceptable vector copy numbers (VCN). In our current study, we tested clinically relevant lentiviral SIN vectors with different internal elements; UCOE(short form), PGK, MND, and UCOE(short form)-MND in tandem to deliver human RAG1 sequences. We used Rag1−/− mice as a preclinical model for RAG-SCID to assessed the strength of the various vectors at low to very low VCNs (0.01–0.2). We observed that peripheral B and T cell reconstitution levels directly correlated to the RAG1 expression levels in the transplant. And although most treated mice reconstituted their peripheral T cell compartment, in animals that received a transplant with low RAG1 expression this effect was merely transient. This was confirmed by the data from the thymus; only mice that received a transplant with high RAG1 expression, showed a subset composition comparable to mice receiving WT cells, including a high percentage of double positive cells, assuring a continuous supply of T cells. Mice receiving a transplant with low RAG1, were mostly lacking the double positive population. We conclude that functional restoration of RAG1-defciency at low VCN can be achieved with clinically acceptable vectors.
Efficient transduction of PB-CD34+ hematopoietic progenitors from Pyruvate Kinase deficiency patients with a therapeutic lentiviral vector
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Pyruvate kinase deficiency (PKD) is an inherited enzymatic deficiency that affects erythrocyte energy metabolism. Most patients show chronic nonspherocytic hemolytic anemia (CNSHA), high reticulocytosis and acute splenomegaly, leading to intense iron overload, being life-threatening in severe patients. Currently there is no specific treatment for severely affected patients, and allogeneic bone marrow transplantation remains the only curative option. We have previously demonstrated the efficacy and safety of a PGK-coRPK therapeutic lentiviral vector correcting PKD phenotype in a mouse model of PKD. To validate the efficacy and potential toxicity of this LV vector in human target cells, CD34+ progenitors were isolated from the peripheral blood of PKD patients, expanded for 24 h in the presence of cytokines and transduced with pre-GMP viral supernatants. Transductions were performed with three different concentrations and functionality was evaluated by CFU assays before and after transduction. Proviral copy number was quantified in both, individual CFUs and CD34+ cells maintained in culture for 14 days, to determine the most suitable viral dosage. Our results show that the PGK-coRPK therapeutic vector is able to efficiently transduce over 90% of deficient CD34+ cells, providing 1–5 proviral copies per cell and with no significant alteration of proliferation capacity in vitro, supporting the potential use of the PGK-coRPK LV in a clinical setting. Metabolic profiling studies to determine whether glycolysis pathway is recovered after genetic correction are ongoing. These studies will provide an indirect demonstration of PGK-coRPK functionality in the target cells that would be used in a potential clinical trial for PKD.
Transcription factor Snail regulates TNF-beta-mediated synovial fibroblast activation in rheumatoid joint: therapeutic implications for rheumatoid arthritis
In rheumatoid arthritis (RA), abnormal expansion of pannus tissue containing synovial fibroblasts (SFs) is a crucial feature responsible for disease progression. Previous studies have suggested that transcription factor Snail may be a critical regulator of SF. In this study, we hypothesized that Snail can regulate SF activation in rheumatoid joint mediated by TNF-alpha which plays a pivotal role in the pathogenesis of RA. Using synovial samples from patients with RA and rats with collagen-induced arthritis (CIA), we examined the roles of Snail in the expression of cadherin-11 (Cad-11) and the myofibroblast marker a-SMA, invasive ability, and IL-6 production. Our results show that Snail and Cad-11 were expressed at higher levels in the synovium and SFs of RA patients and CIA rats. TNF-beta stimulation or Wnt signaling activation upregulated the expression of Snail, Cad-11, and alpha-SMA in SFs, whereas anti-TNF-beta therapy down-regulated their expression in CIA joints. While Snail-overexpressing SFs had increased expression levels of Cad-11 and alpha-SMA, as well as enhanced TNF-beta-mediated invasive capacity and IL-6 production, shRNA knockdown of Snail in CIASF led to opposite effects. Snail-overexpressing normal joints had hyperplastic synovium with increased Cad-11, alpha-SMA, and IL-6 expression. Notably, silencing of Snail expression ameliorated arthritis with reduced Cad-11 expression and extracellular matrix (ECM) deposition, whereas overexpression of Snail exacerbated arthritis with increased Cad-11 expression and ECM deposition in CIA joints. Collectively, our results demonstrate that Snail regulates TNF-beta-mediated SF activation in rheumatoid joint. These findings might contribute to the pharmacological development of therapeutics targeting SF in RA patients.
Personalized therapy for X-linked agammaglobulinemia (XLA) by oligonucleotide-induced splice-correction
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X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency resulting from mutations in the BTK gene severely impairing B cell development. Many XLA-causing mutations affect splicing of BTK pre-mRNA. Here, we assess the treatment-potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts. Both the SCO structural design and chemical properties were optimized using 2′-O-methyl, locked nucleic acid (LNA) or phosphorodiamidate morpholino (PMO) backbones. In order to have access to an animal model, we engineered a novel, Bacterial Artificial Chromosome (BAC)-transgenic mouse carrying an authentic, mutated, splice-defective human BTK gene. To avoid interference of the orthologous mouse protein, mice were bred onto a Btk knockout background. This represents a unique model to study treatment strategies for diseases resulting from splice-site mutations. For the first time the capacity of SCOs to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells, is demonstrated. The corrected BTK mRNA restored expression of functional protein as shown both by enhanced lymphocyte survival and re-established BTK activation upon B cell receptor stimulation. As final proof-of-concept BTK restoration in vivo in mice and ex vivo in primary patient cells was obtained. Thus, our approach may represent a future personalized medicine for XLA
Recombinant high mobility group box 1 A box and heparin for the treatment of acute lung injury
Acute lung injury accompanied a significant elevation in the levels of pro-inflammatory cytokines and leukocyte infiltration in the lungs. Also, Acute lung injury is a severe illness with high rate mortality. Therefore, we used HMGB1A and Heparin which have anti-inflammatory activities. High mobility group box 1 (HMGB1) is an abundant nuclear and cytoplasmic protein. Besides, HMGB1 is released from necrotic cells as well as macrophages and pro-inflammatory cytokine. Here, we used HMGB1 A box (HMGB1A) as an antagonist fragment of HMGB1. Also, HMGB1A have anti-inflammatory activity and heparin binding site (Amino acid 6∼12). Together with HMGB1A, we used heparin, which possesses anti-inflammatory activity, and inhibits NF-ϰB activation. For this reason, we examined HMGB1A and Heparin would reduce cytokine in in vitro and in vivo in mice. HMGB1A bound to heparin strongly by charge interaction. The HMGB1A and Heparin complex was non-toxic and decreases cytokine level, such as tumor necrosis factor-a (TNF-a) in lipopolysaccharide(LPS)-induced acute lung injury in mice. In conclusion, HMGB1A and Heparin complex would be better than only HMGB1A and only Heparin for anti-inflammatory activity to treat acute lung injury.
Expression of human FIX in mice after administration of AAV5-hFIX
AAV5-hFIX, adeno-associated virus serotype 5-based vector containing the human coagulation factor IX gene, has been developed in our scalable and GMP compliant baculovirus-based production platform to treat haemophilia B. Expression of human FIX has been investigated in wild-type mice and murine FIX knock-out mice (HB mice) that display elongated clotting times and spontaneous bleeds. Wild-type mice received AAV5-hFIX at doses ranging from 5×1011 to 2.3×1014 gc/kg. One group received AAV5-hFIX (2.3 ×1014 gc/kg) concomitantly with prednisone (1 mg/kg) orally thrice a week. Blood was sampled regularly from pre-trial until sacrifice 26 weeks after dosing. HB mice were injected once with AAV5-hFIX at 5×1011, 5×1013, or 2.3×1014 gc/kg. Blood samples were taken four weeks post-injection. In HB mice, ELISA revealed vector-dose dependent concentrations of hFIX protein, up to 3600% of normal in the high dose group. The aPTT test revealed vector-dose dependent FIX activity. FIX activity levels and protein levels corresponded one-on-one demonstrating that hFIX expressed in HB mice as the result of transduction with AAV5-hFIX displayed normal biological activity. In wild-type mice, intravenous administration of AAV5-hFIX resulted in dose-dependent levels of circulating hFIX up to 6000% of normal in the highest dose group after 4 weeks, followed by stabilization at a lower level that was sustained for the duration of the 6 months follow-up but the circulating hFIX levels in the prednisone group were significantly lower at all tested time points. The reason for this effect is not completely clear at present but seems related to the mechanism of action of corticosteroids.
Stable supply of functional reconstituted neutrophils achieved by transplantation of allogeneic umbilical cord blood induced remission of inflammatory state in X-CGD mice
Induced Pluripotent Stem Cell- (iPSC) based Strategy to Correct the Bleeding Phenotype in Hemophilia A
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Hemophilia A (HA) is a bleeding disorder caused by factor VIII (FVIII) gene mutations. Our aim is to develop a novel HA treatment strategy generating patient-specific iPSCs from peripheral blood cells anddifferentiating them into functional endothelial cells (ECs) secreting FVIII. CD34+ cells were isolated from healthy and hemophilic donors and reprogrammed with a cre-excisable polycistronic LV carrying OCT4, SOX2 and KLF4. iPSCs were obtained, characterized and the differentiation potential was assessed by adipogenic, osteogenic and chondrogenic differentiation. iPSCs were differentiated in ECs using VEGF/BMP4-supplemented EB medium. Endothelial markers expression was evaluated by FACS and RT-PCR. ECs, transduced with LV carrying GFP under the control of endothelial-specific promoters: Flk-1, Tie2 and VEC, were transplantedin NSG-HA mice. Reprogrammed CD34+ cells gave rise to ESC-like-iPSCs coloniespositivesfor AP staining and stem cell markers. EBs differentiated in osteogenic, chondrogenic and adipose tissues, and expressed three germ layers markers. Increased iPSCstelomeres length suggested telomerase reactivation and no abnormalitieswere detected after karyotype analysis. RT-PCR on HA-iPSCs showed hBDD-FVIII expression, confirming genetic correction by LV transduction.iPSCs differentiated into ECs, acquiring endothelial-like morphology, expressing ECs markers and formed tubules when cultured in matrigel.VEC-GFP-LV transduced ECs were transplanted in NSG-HA mice conditioned by monocrotaline and GFP+ cells were detected by immunofluorescence of liver sections up to 1 monthpost transplantation. These data will be instrumental to assess engraftment, proliferation and the FVIII expression levels from differentiated, gene corrected and reprogramming factor-free iPSCs to confirm the suitability of this approach for HA gene-cell-therapy.
rAAV2/5 encoding truncated CFTR corrects CF phenotype in mouse nasal epithelium
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Hematopoietic cells as a novel strategy for hemophilia A cell therapy
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Identification of cells capable of synthesizing and releasing factor VIII (FVIII) is critical for developing therapeutic approaches in hemophilia A (HA). Endothelial cells (EC), particularly liver sinusoidal EC (LSEC), express FVIII most in the body. However, recent studies of bone marrow (BM) transplantation suggested additional cell types could synthesize and release FVIII, correcting bleeding phenotype in HA mice. Therefore, to establish the ability of hematopoietic cells (HCs) in expressing FVIII, we analyzed several murine and human HC types. We found by immunostaining that FVIII was present in HCs isolated from peripheral blood, BM and human cord blood (hCB). The identity of these cell types was verified by costaining for FVIII and cell markers for monocytes, macrophages, dendritic cells and megakaryocytes. Moreover, FVIII expression was verified by RT-PCR and WB. We observed FVIII expression in mononuclear cells or EC in other organs, e.g., spleen, lungs and kidneys. Finally, we performed transplantation studies in immunocompromized? Null-HA mice (n=12) with wild-type CD34+ from hCB. FACS analysis showed engraftment higher than 40% in most of the mice up to 3 month after transplantation. aPTT performed on plasma of treated mice at 12 w showed FVIII activity levels between 2% and 5% of normal hFVIII, sufficient to ameliorate the bleeding phenotype; indeed 8 out 12 (66%) mice survived to the tail clip assay. Similar results were obtained in short term experiments injecting hCD11b+ cells in NOD-SCID.HA mice.
Gene expression of Notch receptors and ligands by plastic adherent and prospectively isolated MSCs
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Isolation, culture and characterization of the biological properties of murine amniotic membrane stem cells for regenerative cell therapy studies
Isolation and characterization of murine tympanic membrane stem cells
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Modeling Platelet Abnormality in Wiskott Aldrich Syndrome using Patient-derived induced Pluripotent Stem Cells
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Wiskott Aldrich syndrome (WAS) is an X-linked disease, which is caused by mutations in the gene encoding the WAS protein (WASp). As thrombocytopenia is the most typical feature of this disease, often exposing patients to a significant risk of life-threatening hemorrhage, it has been the main subject of study for many researchers. Because of limitations in disease modeling, precise mechanisms of the platelet abnormality remain to be elucidated. Here we established induced pluripotent stem cell (iPSC) lines from two XLT and one WAS patients as disease models to address the issues. We first confirmed that these disease-specific iPSCs retained gene mutations characteristic to each patient. Using our differentiation culture system, we demonstrated that numbers of both megakaryocyte (MK)s and platelets obtainable from both XLT- and WAS-iPSCs were significantly smaller than those from healthy iPSCs. Detailed analysis revealed that the observed defects were mainly due to insufficient production of proplatelet-bearing cells, but not to impaired platelet production per MK. Lentiviral-mediated gene transfer led to appearance of WASp expression in patient iPSC-derived MKs. The expression of WASp, however, did not reach the normal level that was seen in control-iPSC-MKs; yields of platelets showed some increase after gene transfer, but only marginally. Although further investigation is necessary, these results indicate the utility of iPSC-based disease modeling for WAS. We are now in the process of addressing how critically expression levels of WASp will affect the efficacy in platelet number recovery after gene transfer.
Mesenchymal stem cells regulate inflammatory mediators, proliferation and calcium-induced differentiation of keratinocytes
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Skin homeostasis and defenses are maintained by cross-talks between keratinocytes and various immune cells, which reside or are recruited in the skin through the production of various cytokines, chemokines, and growth factors. Therefore, the modulation of immune responses among cells is important when skin tissue is damaged during disease pathogenesis. It has been reported that MSC has a regulatory effect on immune responses. Thus, we determined whether MSC could modulate pro-inflammatory responses in keratinocyte stimulated with a combination of cytokines (IL-22, TNF-a, and IL-17), which can mimic in vitro psoriasis-like status in keratinocytes. We co-cultured the normal human keratinocytes with umbilical cord blood derived mesenchymal stem cells (MSC) in the presence of a cytokines mixture (IL-22, TNF-a, and IL-17) and evaluated the paracrine effects of MSC on keratinocytes. We found that MSC suppresses the production of pro-inflammatory cytokines in keratinocytes and the terminal differentiation of keratinocytes induced by increased intracellular calcium level. Furthermore, we also determined for the first time that MSC regulates the differentiation and proliferation fates of keratinocytes by regulating the RhoA-RockII and PI3K-AKT signaling pathways. Therefore, MSC could be used to prevent keratinocytes from chronic skin inflammation, such as psoriasis, and more detailed understanding of MSC's regulatory effects on immune responses might open a way to develop new therapeutics.
In vitro and in vivo characterization of immunomodulatory properties of MuStem cells
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Duchenne Muscular Dystrophy (DMD) is a genetic muscle disease caused by mutations in the dystrophin gene. To date, no curative treatment is available but adult stem cell-based therapy is one promising therapeutic strategy. We have already demonstrated that allogeneic muscle-derived delayed adherent stem cells (that we called MuStem) are able to phenotypically correct at clinical and tissue levels the Golden Retriever Muscular Dystrophy (GRMD) dog model (Rouger et al., 2011). Recently, some tissue-specific stem cell populations were shown to exhibit immunomodulatory properties that could increase their ability to engraft in an allogeneic recipient despite the absence of strong immunosuppression and improve their regenerative potential. To explore whether MuStem cells also exhibit such immunoregulatory properties, we transplanted dog leukocyte antigen-identical MuStem cells from healthy donors in GRMD dogs, under an immunosuppressive regimen, which was arrested two weeks after intra-arterial cell delivery. Monitoring of host humoral and cellular immune responses against transplanted MuStem cells and neosynthetized dystrophin protein are in progress. Concomitantly and from a clinical perspective, we have generated human MuStem cells. Interestingly, our preliminary data show the ability of MuStem cells from different donors to modulate allogeneic T cell proliferation and to express immunomodulatory molecules such as prostaglandin-E2 and indoleamin-2,3-deoxygenase-1. In conclusion, our study is critical for the understanding of the crosstalk between MuStem cells and the immune system, as well as the design of safe and efficient allogeneic stem cell-based therapy for the treatment of DMD patients.
Silk fibroin patches cellularized with Wharton's jelly mesenchymal stem cells improve wound healing in an experimental murine excisional wound splinting model
Pilot study of tolerance to intravenous infusion of increasing doses of mesenchymal stem cells in rabbits
Osteoblast-secreted extracellular vesicles stimulate the expansion of CD34+ human umbilical cord blood cells
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Umbilical cord blood (UCB) is increasingly used in hematopoietic stem cell (HSC) transplantations; however, the low cell numbers are still remaining as a limiting factor for proper engraftment. Osteoblasts play important roles in regulating HSC self-renewal and differentiation. Recently, extracellular vesicles (EVs) have been implicated in stem cell fate regulation via horizontal transfer of proteins and nucleic acids between cells. In this study, we focused on the characterization of osteoblast EVs and investigated their potential in ex vivo expansion of UCB-HSCs for clinical use. We used human pre-osteoblasts (SV-HFO cells) to isolate EVs, and characterized EVs by electron microscopy, proteomics, and RNA sequencing, and investigated their functional effect on human CD34+ UCB cells by qPCR and flow cytometry. Characterization analyses demonstrated that osteoblast EVs are heterogenic in size, contain novel osteoblast EV proteins primarily linked to ribosomal activity and RNA processing, and are enriched with small RNAs. Treatment of CD34+ UCB cells with osteoblast EVs led to donor-dependent 3–5-fold expansion of the CD34+ expressing progenitors in 10 days. miRNA profiling demonstrated that osteoblast EVs contain abundant amounts of miR-29a, one of the key regulators of early hematopoiesis. Interestingly, EVs treatment led to the down-regulation of HBP1 in CD34+ UCB cells. HBP1, a miR-29a target, has been shown to be a cell cycle inhibitor, which may explain the mechanism by which EVs stimulate UCB cell expansion. In conclusion, we demonstrated that osteoblasts secrete EVs that expand UCB cells ex vivo, and uncovered the first clues that contributed to the understanding of EV function.
Direct reprogramming of human fibroblasts to induced hepatocytes by defined factors
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Liver disease is a major cause of morbidity and mortality worldwide. The treatment of choice for these patients is organ transplantation, an invasive procedure associated with frequent post-transplant complications. Moreover, organ availability is very limited due to the scarce number of donors. Allogeneic hepatocyte transplantation constitutes a less invasive alternative to organ transplantation. However, it shows also some disadvantages as the need of serial transplants to get the necessary graft to revert the disease, increasing the risk of rejection due to the use of different donors and to the loss of function of transplanted cells in the long-term. Cellular reprogramming appears as a new therapeutic approach in regenerative medicine field for the treatment of diseases in which partial replacement of liver parenchyma with functional cells allows the reversion of disease phenotype. In this work we have identified combinations of liver transcription factors that after lentiviral transduction and in vitro culture in specific medium are able to reprogram human fibroblasts to hepatocyte-like cells (induced hepatocytes, iHeps). These cells show downregulation of fibroblast specific genes, morphological changes that resemble hepatocytes, expression of hepatic specific genes and acquisition of hepatic-specific functions. To test their in vivo functionality, 15 days after transduction the iHeps have been transplanted in immunodeficient mice that have been previously undergone a partial hepatectomy and local hepatic irradiation. In vivo iHep functionality data will be presented. Overall, we propose iHeps as an alternative for the treatment of liver disease which would avoid the problems associated with organ transplantation.
Additional Abstracts
Investigation of gene silencing effect of chitosan nanoparticles containing shRNA PDGF-D in breast cancer cell lines
Breast cancer is the most common highly malignant and fatal disease of woman. Angiogenesis plays a critical role in cancer. PDGF-D is known to regulate many cellular processes including angiogenesis and invasion. Over-expression of PDGF-D promoted tumor growth and metastasis in breast cancer. Inhibition of PDGF-D expression by shRNA technology decreased tumor growth and metastasis. On the other hand success of the shRNA therapy depends on suitable carrier system. RNAi (RNA interference) is a sequence–specific, post-transcriptional gene silencing mechanism. The three types of RNAi are siRNA, shRNA and microRNA, RNAi can be induced by endogenous expression of short hairpin RNAs (shRNAs), and this strategy holds the promise to knock down expression of cancer gene.
Use of chitosan as shRNA delivery system, shRNA loaded chitosan nanoparticles targeting to PDGF-D inhibition were prepared and the characterization and transfection efficency of chitosan particles in breast cancer cells such as MCF-7, MDA-MB 231, MDA-MB 435 were investigated in this project.
Single and multiple shRNA insert sequences were used as shRNA. The effects of single and multiple shRNA sequences, molecular weight of chitosan (150 kDa and 400 kDa) and the amount of shRNA on the characterization and transfection efficiencies of nanoparticles have been studied.
Size of nanoparticles changed between 200–400 nm and approximately 95–100% encapsulation efficiency were obtained. Release of shRNA changed with the molecular weight of chitosan.
PDGF-D silencing effect was observed in all 3 cell lines. Nanoparticles indicated time-dependent PDGF-D inhibition. The highest gene inhibition effect was measured in single shRNA contained nanoparticles (%90).
In conclusion, chitosan nanoparticle is suitable gene delivery vector for shRNA targeting to PDGF-D. Data obtained in this project can be used in in vivo studies of PDGF-D inhibiton and therapy in breast cancer model.
This study was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK), grant no:113S003.
Combined therapy of colon carcinomas with an oncolytic adenovirus and valproic acid
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The anti-tumor potential of oncolytic adenoviruses (CRAds) has been demonstrated in preclinical and clinical studies. While these agents failed to eradicate tumors when used as a monotherapy, they may be more effective if combined with conventional treatments such as radiotherapy or chemotherapy. This study seeks to evaluate the combination of a CRAd bearing a □24 deletion in E1A with valproic acid (VPA), a histone deacetylase inhibitor for the treatment of human colon carcinomas. This combination led to a strong inhibition of cell growth both in vitro and in vivo compared to treatment with CRAd or VPA alone. This effect did not stem from a better CRAd replication and production in the presence of VPA. Inhibition of cell proliferation and a non-apoptotic cell death were shown to be two mechanisms mediating the effects of the combined treatment. Moreover, whereas cells treated only with CRAd displayed a polyploidy (>4N population), this phenotype was strongly increased in cells treated with both CRAd and VPA. In addition, the increase in polyploidy triggered by combined treatment with CRAd and VPA was associated with the enhancement of H2AX phosphorylation (γH2AX), a hallmark of DNA damage. Finally, E1 and/or viral replication were shown to play a key role in the observed effects since no enhancement of polyploidy nor increase in □H2AX were found following cell treatment with a replication-deficient Ad and VPA. Taken together, our results suggest that CRAd and VPA could be used in combination for the treatment of colon carcinomas.
Dimeric CRISPR/Cas-based nucleases for high-precision genome editing in human cells
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The CRISPR/Cas9 system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. However, the discovery of high-frequency off-target mutagenesis induced by monomeric Cas9 suggests potential limitations over its use for human therapeutic applications. Here, we describe a novel RNA-guided FokI Nuclease (RFN) platform that, like Zinc Finger Nuclease (ZFN) and Transcription Activator-Like Effector Nuclease (TALEN) technologies, relies on binding and dimerization of two FokI-dCas9 proteins at the intended target site. In contrast to Cas9 nickases, RFNs are truly dimerization-dependent and induce little to no detectable unwanted mutagenesis while generating robust on-target alteration frequencies. In addition, we have increased the targeting range of RFNs by developing a novel expression system of multiple gRNAs bearing any 5′ nucleotide. RNA-guided FokI nucleases combine the ease of CRISPR-based gene targeting with the specificity of a truly dimeric genome editing system and will likely be a useful tool for high-precision genome editing applications.
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Neontimal hyperplasia surrounding the suture line remains the major cause of graft failure after coronary artery bypass graft surgery and peripheral vascular bypass surgery. Immuno suppressive drugs, such as tacrolimus, have been used to inhibit the development of neointimal hyperplasia(NIH). Preventing intimal hyperplasia is an important strategy includes manipulation of cellular response may be available.
The purpose of this study was to determine the efficacy of the sutures coated with siRNA and shRNA targeted to PDGF–D, which have not been studied before, in preventing intimal hyperplasia. The surface of suture Trofilen® (non–absorbable monofilament polyvinylideneflouride) was coated respectively with siRNAand shRNAPDGF–D using chitosan for controlled release of siRNA and shRNAto inhibiting cell proliferation.
Sutures were dip–coated with siRNA by immersion in aqueous solution of chitosan containing siRNAand evaporotion of solvents. After drying sutures were characterized by weight increase. Release time of coating sutures was studied in vitro. HUVEC (Human Umbilical Vein Endothelial Cell) cell line was used for cell culture studies. We determined to inhibition of cell proliferation with MTT assay, and PDGF-D inhibitionobserved with ELİSA assay.
In conclusion, chitosan complex is a suitable gene delivery vehicle for targeting siPDGF-D and shPDGF-D in NIH. Also data were shown both si and shPDGF-D can be used in vivo studies for NIH.