Abstract
Stem Cells, Pluripotency and Nuclear Reprogramming
One of the key issues raised by nuclear cloning is the question of genomic reprogramming, i.e. the mechanism of resetting the epigenetic modifications that are characteristics of the adult donor nucleus to ones that are appropriate for an embryonic cell. The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Major progress has been achieved to understand the molecular circuitry of pluripotency and self-renewal. This information provides crucial insights into mechanisms by which pluripotent cells may be stimulated to differentiate into different cell types or by which somatic cells might be reprogrammed back to the pluripotent state by exposure of the somatic nucleus to the egg cytoplasm.
The recent demonstration of in vitro reprogramming using transduction of 4 transcription factors by Yamanaka and colleagues represents a major advance in the field. Major questions regarding the mechanism of in vitro reprogramming need to be understood and will be one focus of the talk. Also, our progress in using iPS cells for therapy and for the study of complex human will be summarized.
Early Primate Multipotent Cardiovascular Progenitors Derived from Pluripotent Stem Cells Safely Engraft in Postmyocardial Infarcted Primate: A Proof of Principle in Regenerative Medicine
During embryogenesis, the cardiac cell fate is acquired as early as gastrulation. There is compelling evidence that embryonic stem cells (ESC) can recapitulate early steps in cardiogenesis. Identification from human pluripotent stem cells of early cardiovascular cell progenitors, at the origin of both the primary and secondary heart fields, would shed light on human normal and pathological cardiogenesis and would pave the way toward cell therapy for cardiac degenerative diseases. Here, we report the isolation, and a phenotypic characterisation of an early Oct-4+, SSEA-1+, Mesp1+, Nkx2.5+ and Isl1+ population of cardiovascular progenitors derived from human pluripotent stem cells. This multipotential progeny feature the capability to generate cardiomyocytes as well as smooth muscle and endothelial cells. We further bring a proof of concept that these progenitors can be used safely in cardiac regenerative medicine as allografted in infarcted primate myocardium, they differentiated in ventricular myocytes without any adverse effect.
Biopathologie, Centre de Recherche en Cancérologie de Marseille , Marseille, France
Inflammatory breast cancer (IBC) is associated with an extremely poor prognosis and a high propensity for early metastasis. Moreover, it displays a complete lack of valuable prognostic markers. To investigate the role of cancer stem cell population in IBC, we utilized SUM 149 IBC cell line and MARY-X IBC primary tumor xenograft.
Using an enzymatic assay based on the Aldehyde dehydrogenase activity, the ALDEFLUOR assay, we isolated an ALDEFLUOR-positive component cfor cell line and xenograft and for both models, limiting dilutions of cells were injected into mammary fat pads of NOD/SCID mice. Only ALDEFLUOR-positive cells generated tumors whereas the ALDEFLUOR-negative cells failed to do so.
To determine whether invasive and metastasis properties of IBC were mediated by the cancer stem cell component, we utilized an in vitro Matrigel invasion assay and intracardiac injection of lentivirus luciferase infected cells sorted for ALDFLUOR. We demonstrated that ALDEFLUOR-positive cells were three-fold more capable of invasion through Matrigel than ALDEFLUOR-negative population and were able to form metastasis as assessed by bioluminescent imaging quantified by photon flux.
To determine the clinical relevance of expression of stem/progenitor cells, we examined the expression of ALDH1 in a series of 109 IBC patients treated at our institution between 1976 and 2003. ALDH1 was expressed in 34% of tumors and ALDH1 expression strongly correlated with metastasis-free survival (MFS) (p = 0.0152) as well as tumor-specific survival (SS) (p = 0.0337). Multivariate analysis using Cox proportional hazard models demonstrated that ALDH1 is the only prognostic marker of MFS (p = 0.0055, HR = 2.81, 95%CI [1.355–5.815]) and the most powerful prognostic marker of SS (ALDH1: p = 0.0012, HR = 2.7, 95%CI [1.48–4.93]). Utilizing in vitro and mouse models, we tested the role of stem cell component for in IBCs and show that the aggressive nature of IBC may be mediated by a cancer stem cell component.
Characterization of Insulators and Barrier Elements for Gene Therapy
Gene transfer that relies on integrating vectors often suffers from epigenetic or regulatory effects that influence the expression of the therapeutic gene and/or of cellular genes located near the vector integration site in the chromosome. Insulator elements act to block gene activation by enhancers, while chromatin domain boundary or barrier sequences prevent gene-silencing effects. At present, the modes of action of insulator and barriers are poorly understood, and their use in the context of gene therapies remains to be documented. Using combinations of reporter genes coding for indicator fluorescent proteins, we constructed assay systems that allow the quantification of the insulator or barrier activities of genetic elements in individual cells. This presentation will illustrate how these assay systems were used to identify short DNA elements that insulate nearby genes from activation by viral vector elements, and/or that block the propagation of a silent chromatin structure that leads to gene silencing. We will show that some barrier elements do not merely block repressive effects, but that they can act to stabilize and sustain transgene expression. We will illustrate that this may be beneficial when transgenes are introduced into stem or precursor cells using non-viral vectors, where later differentiation may lead to the silencing of the therapeutic gene. We will show that these elements can be used to maintain efficient transgene expression upon the differentiation of murine precursor cells towards myofibers, in a model of cell therapy for muscle dystrophies.
Development of Foamy Virus Vectors
The first vectors derived from foamy viruses were established over ten years ago. Until now only used and further developed by a handful of investigators these vectors have been shown to be promising tools for the gene transfer into haematopoietic stem cells. Several inherent features of foamy virus-derived vectors, such as the high efficiency in targeting CD34-positive stem cells, a favourable integration profile, and the non-pathogenic nature of the parental virus argue for their wider use. The effectiveness in different preclinical and large animal models suggests the exploration of foamy virus vectors in human trials.
Of Treg and Therapy
Since their (re-)discovery, regulatory/suppressor T cells have moved from being politically incorrect to being of strategic importance for immunotherapy.
Indeed, it is now clear that Tregs play a role in the development/control of every immune response, and even in every inflammatory process. This is why Tregs are endowed with a considerable potential for immunotherapy.
On the one hand, the possibilities to differentiate, generate, or activate Tregs ex vivo and in vivo could help control auto-immune processes as well inflammatory diseases such as arteriosclerosis. On the other hand, the possibility to block or deplete Tregs is most probably a requisite for the development of efficient immunotherapies of cancer.
The manipulation of Tregs for therapeutic purposes is in its infancy. It requires the development of efficient methods to either produce or eliminate them, the development of novel drugs able to selectively act on Tregs and finally a better understanding of how these cells or molecules may be used in the clinic.
We are currently pursuing clinical development of ex vivo produced Tregs for treatment of autoimmune diseases (inclusion body myositis and uveitis), as well as ex vivo depletion of Tregs for treatment of cancer (leukemia and solid tumors).
We will present hurdles and potentials of these approaches, from bench studies to clinical trials.
Lentiviral Vectors as Vaccines
The aim of my group is to engineer viral vectors to be used as effective vaccines. Lentiviral vectors based on human immuno-deficiency virus type 1 (HIV-1) transduce dendritic cells in draining lymph nodes after subcutaneous injection. Using vectors encoding ovalbumin, or the tumour antigen NY-ESO-1 we have shown that lentiviral vector injection initiates potent antigen-specific CD4+ and CD8+ T cell responses. Protective and therapeutic anti-tumour immunity can be induced. To improve safety and efficacy, we have developed non-integrating lentiviral vectors, and also vectors with uptake and antigen expression targeted to antigen presenting cells. We have also expressed constitutive activators of NFkappaB or mitogen-activated protein kinase pathways. Triggering of NFkappaB or p38 led to activated DC, and substantially enhanced the anti-tumor immune response. Activation of ERK increased TGF-β expression, suppressed the immune response and stimulated expansion of regulatory T cells. These results provide a toolkit to regulate immune responses to immunization; vaccine responses to foreign or tumor antigens can be enhanced and harmful responses to self-antigens or introduced transgenes can be reduced.
Treatment of Large Established Tumors by Combined Immunotherapy Targeting Adaptive and Regulatory Responses
The observation that (i) tumors develop more frequently in immunocompromised patient and (ii) tumors develop spontaneously in several immunodeficient transgenic mice, demonstrates that the immune system can recognize tumor cells and plays a critical role in the prevention and elimination of these “transformed self” tissues. Several therapeutic vaccines that induce strong anti-cancer immune responses have thus been developed and have shown impressive results in murine models. However, translation of these approaches to the bedside has been so far disappointing except some remarkable successful cases on a minority of patients. Increasing evidences support the hypothesis that tumor tissues actively create a suppressive microenvironment that impairs the tumor specific responses, resulting in tumor escape and the failure of elimination. This has also demonstrated recently by our team, by using a transplanted TC1 tumor model, expressing E6 and E7 oncoproteins of human papillomavirus (HPV) type 16. Indeed, we have shown that a HPV16 E7-based tumor vaccine (CyaA-E7) targeted to dendritic cells induces efficient and specific anti-tumor immune responses, which were sufficient to eliminate established palpable tumor. However, this vaccine was incapable to eliminate the tumor if applied at a later stage when the tumor size is larger. Various treatment combinations were used to restore the anti-tumor activity of the CyaA-E7 vaccine in large tumors-bearing mice. A combination based on the CyaA-E7 vaccine injected with CpG and cyclophosphamide (CTX) was able to induce full eradication of large, established tumors in around 90% of treated animals.
Engineering and Vectorization of Virus-Like Particles (VLPs) as Novel Vaccines to Combat Acute and Chronic Infectious Diseases: Application to Avian Influenza
The manipulation of viral genomes and the techniques of viral engineering offer fascinating perspectives in different fields of the bio-medical research, notably for designing gene delivery strategies and for formulating and/or vectorizing novel vaccines. Viral-like particles (VLPs) can be derived from several viruses and are very promising platforms that combine the advantages of sub-unit vaccines, i.e. high bio-safety features, and those of inactivated or attenuated viruses, i.e. strong immunogenicity. The unravelling of the mechanisms driving assembly of viral particles and governing replication of viral genomes has allowed designing highly safe VLPs that can display virtually any type of antigen. Furthermore, several gene delivery vectors are available and allow sustained expression of antigenic formulations for the necessary time frames and within the appropriate tissues and/or intracellular localisations, as needed to raise appropriate immune responses. Finally, the assembly of VLPs does not require viral replication and propagation, as opposed to true viruses. This offers many possibilities for structural modifications of the displayed antigens through genetic manipulation, aiming to present epitopes that otherwise would be difficult to reveal in conventional attenuated/inactivated vaccines and that could trigger immune responses that may eventually be poorly or not induced naturally.
We will discuss novel vaccine candidates against highly pathogenic avian influenza viruses (HPAIV). Defective VLPs associating retroviral capsids and HPAIV hemagglutinin induced specific, high-titer neutralising antibody responses in mice. Furthermore, through appropriate engineering of HPAIV hemagglutins VLPs incorporating induced cross-reactive neutralising antibodies. Finally, vectorization of these novel immunogens induced strong and potent humoral responses that were protective against lethal HPAIV challenge in mice. These results indicate that delivery of gene constructs encoding VLP in vivo is a promising strategy to induce immune responses against infectious pathogens.
Gene Therapy for Inherited Retinal Degeneration: From Mouse to Man
Early-onset severe retinal dystrophy caused by defects in the gene encoding the retinal isomerase RPE65 is associated with poor vision at birth and complete loss of vision in early adulthood. In February 2007 we started a phase I/II clinical trial of gene therapy in 3 young adult subjects. We administered subretinally a rAAV-2/2 vector expressing RPE65 cDNA under the control of a human RPE65 promoter. Examination of systemic vector dissemination, immune responses, electrophysiology, retinal imaging and detailed psychophysical assessments of visual function suggest that subretinal adminstration of rAAV vector is safe in humans and can lead to improved visual function. These findings support further clinical studies in children with RPE65 deficiency and the development of gene therapy for other inherited retinal disorders.
UMR S 974, Inserm-UPMC-Institut de Myologie , UMR 7215 CNRS, Paris, France
Oculopharyngeal muscular dystrophy (OPMD) is a late onset hereditary muscle disease which is characterised by the selective affection of the pharyngeal muscles resulting in swallowing disorders, and by a ptosis from the dysfunction of the levator palpabrae superioris muscles. Swallowing disorders are determinant in the prognosis of the disease, and potentially life-threatening deglutition, due to aspiration and denutrition. At later stages of the disease, voluntary muscles, especially those of the proximal limbs, may be affected. The only available treatment, cricopharyngeal myotomy, may be performed to improve swallowing but has a long term effect in only a fraction of the patients.
In order to elucidate the potential physiopathological mechanisms involved in the development of this disease we isolated satellite cells from both the affected and unaffected muscles of OPMD patients and age matched control subjects. We observed defects in the proliferation of myoblasts isolated from affected CPM. However these defects were not observed in myoblasts isolated from unaffected muscles of OPMD patients, this strongly suggested to us that grafts of autologous myoblasts isolated from unaffected muscles could represent a new therapeutic approach which could be used to restore contractility to the affected pharyngeal muscles. The use of autologous myoblast transplantation as a therapeutic strategy for limited targets has been well described for heart infarction. A preclinical study was carried out in the dog in order to evaluate the tolerance and feasibility of such autografts and a phase 1 clinical trial is ongoing with 12 patients having already received autologous myoblast transplantation.
Inserm , U844, UM1, CHU Lapeyronie, Montpellier, France
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that principally attacks the joints. Numerous studies indicate that cytokines are critical in the processes that cause inflammation and joint destruction, with TNF-α playing a prominent role. Currently, when disease activity cannot be controlled with conventional drugs, patients are switched to biotherapies inhibiting TNF-α. Such biologics, together with an improved timing and dosing of conventional therapy, have largely improved the outcome of established arthritis in many patients, but not all, and still pose significant problems. We aimed at developing alternative approaches for targeted treatments in RA by evaluating the effect of RNA interference (RNAi)-based gene therapies in experimental models of RA.
RNAi is a powerful endogenous gene silencing mechanism used by mammalian cells to control posttranscriptionally gene products in sequence-specific. Adopted as a standard experimental tool, RNAi is of great interest for developing therapies that are based on inhibition of function. Important tasks however still remain for in vivo applications, such as delivery. We developed an innovative cationic liposome formulation and demonstrated that weekly intravenous administration of an anti-TNF siRNA efficiently decreased disease severity and restored immunological balance in collagen-induced arthritic mice. Importantly, in both preventive and curative settings, the most striking clinical effect was observed when combining 3 siRNAs targeting IL-1β, IL-6, IL-18 pro-inflammatory cytokines. Overall parameters were improved compared with the TNF siRNA lipoplex-based treatment. These data show that cytokines other than TNF can be targeted to improve symptoms of RA and reveal novel potential drug development targets.
Department of Human Genetics, Leiden University Medical Center , Leiden, the Netherlands
Antisense-mediated reading frame restoration is presently a very promising therapeutic approach for Duchenne muscular dystrophy (DMD). In this approach, antisense oligoribonucleotides (AONs) induce specific exon skipping during pre-mRNA splicing of mutated dystrophin transcripts to restore the disrupted open reading frame and allow synthesis of internally deleted, partly functional Becker-like dystrophin proteins. The approach is theoretically applicable to over 70% of all patients. Proof of concept has been achieved in cultured muscle cells from patients and the mdx mouse model. Recently we achieved the first proof of applicability to humans in vivo in a local-administration clinical trial in 4 patients, in which exon 51 skipping and dystrophin restoration was confirmed after a single intramuscular dose of AON, while no adverse effects were seen. Current research focuses on optimization of bioavailability, biodistribution, exon skipping efficiencies and functional benefit after systemic delivery of AONs in mouse models. We find that after intravenous or subcutaneous injections, AONs are more easily taken up by dystrophic fibers when compared to healthy, intact fibers. In the mdx mouse model, we are able to induce exon skipping and dystrophin restoration in all muscles, including the heart after long term systemic AON treatment, which is accompanied by improvement of muscle integrity and function and in the absence of toxicity. In parallel, we are assessing the specificity and bio-distribution of different AON chemistries after local and systemic treatment in various mouse models.
A Phase I/II safety/dose-finding trial (coordinated by Prosensa Therapeutics BV, Leiden, the Netherlands) where patients are treated with AONs systemically is currently ongoing.
Immunology and Hematology Unit—Hospital Necker -Enfants Malades
SCID X1 gene therapy was initiated 10 years ago, based on LTR-competent retrovirally-mediated IL2RG gene transfer into CD34 (+) bone marrow progenitors. Ten patients were treated. Sustained efficacy has been observed in eight of them including detection of polyclonal naïve and memory T cells as well as re-emergence of polyclonal T cells following chemotherapy that was required to treat secondary leukaemia (see below). Correction of the T cell- and (to some extents) B-cell immunodeficiency is associated with a sustained clinical benefit as patients are no longer susceptible to infections 7 to 10 years post gene therapy. A severe adverse event (SAZ), i.e. a T cell leukaemia, occurred 30 to 68 months post gene therapy in four patients. Chemotherapy led to complete and sustained remission in 3. Retrovirus insertions within oncogenes associated with LTR-driven expression of oncogenes such as LMO-2 (n = 3), BMI-1 (n = 1) or CCND 2 (n = 1) were the initiating factor of this process. Secondary genomic alterations were also found. It is remarkable to observe that the SAE occurred altogether in 5 out of 20 patients with SCID-X1 treated in both the French and UK trials while it was not observed in patients treated for Adenosine deaminase deficiency despite the fact that a similar pattern of provirus integration were observed in both cases. This observation indicates that a direct and/or indirect disease-associated factor is also involved in this oncogenetic process. The remarkable and sustained efficacy of IL2RG gene transfer is an incentive to develop a safer methodology. Utilization of self inactivated (SIN) vectors with an EF1α promoter as now envisaged in an international trial could provide both a safe and efficient approach.
Experimental Hematology, Hannover Medical School , Hannover, Germany
Retroviral vector-mediated gene transfer into hematopoietic stem cells (HSC) may be used to treat a great variety of genetic and acquired disorders. However, adverse reactions related to insertional mutagenesis have created substantial uncertainty. We have developed murine models to address the impact of target cell type, cell culture conditions and vector technology on the induction of insertional mutants. To this end, we compare results obtained using a cell-culture based assay with insights from studies of vector-modified HSC performed in serial transplantation with long-term follow-up (up to 20 months). In the worst case, we found that a single insertion of a vector with a strong viral enhancer-promoter next to a proto-oncogene such as Evi1 or Prdm16 may be sufficient to induce leukemia. Further experiments identified a hierarchy of factors governing the emergence of insertional mutants. The progeny of multipotent cells with a priori HSC potential were considerable more sensitive to insertional mutations than multipotent progenitor cells, more mature myeloid cells and T cells. The second most important factor in the induction of insertional mutants is the presence of a strong enhancer-promoter in the vector backbone. The third factor in this hierarchy is the insertion pattern of retroviral vectors, with the gammaretroviral insertion pattern showing a greater risk to induce insertional mutants than the lentiviral pattern. However, lentiviral vectors harbouring strong internal enhancer-promoter sequences with high activity in HSC were still able to trigger insertional transformation. These studies provide the basis for rational approaches to improve the therapeutic index of integrating vectors.
Which Model to Measure Vector-Induced Integration Risk? A Regulatory Perspective
While enumerating the different points to consider in the evaluation of a clinical trial, toxicity arrives ahead in the list. For gene therapy vectors the specific toxicity relates to: the transgene expression, within or outside the target tissue; the chemical or biochemical nature of the vector; the molecular status of the correcting genetic information, episomal or integrated; and the vector-transgene combination.
For the specific case of vectors integrated within the genome of the target cells, including oncoretrovirus- and lentivirus-derived vectors, the site of integration and a variegation effect resulting from the presence of foreign DNA, are the main concern. In the past few years, high throughput techniques have allowed the drawing of integration maps. Data analysis also showed integration hot spots and a marked tendency for integration within expressed-genes. As these data are statistical, they are poorly efficient to predict the consequences of this position effect in the case of a defined clinical trial. Therefore, the definition of a relevant system to evaluate integration during therapeutic gene transfer is still a matter of controversy.
Using data from recently published clinical trials, guidelines from European and American agencies and my experience as an expert in the gene therapy group within the AFSSAPS, I will try to synthesise our current understanding of the problem and propose options for the design of experimental procedures to address the question.
Etablissement Français du Sang (EFS) & Groupe d'Etude des Cellules Souches Mésenchymateuses , Tours, France
On behalf of the Société Française de Greffe de Moelle et Thérapie Cellulaire (SFGM-TC)
Due to their multi/pluripotency and immunosuppressive properties Mesenchymal Stem/Stromal Cells (MSC) are important tools for treatment of immune disorders and tissue repair. The increasing uses of MSC lead to the development of production processes that need to be in accordance with good manufacturing practices (GMP). In cellular therapy, safety remains one of the main concerns, and to avoid major side effects, accurate production controls have to be implemented.
In France, a simple and efficient process was developed and allows producing hundred millions of MSC in a short period of time using medium supplemented with either FCS + FGF2 or platelet lysate. Due to the observance of transformation in another process, the regulatory authority required a survey of karyotype for cultivated MSC. Five out 20 different productions showed the presence of aneuploidy with an extra chromosome 5 sometimes associated with extra chromosomes 8 or 20. On these five productions with aneuploidy, 3 were from the same donor. The abnormalities did not persist along time. Presenting or not aneuploidy, all the cultivated MSC reached senescence, and they did not exhibit any transforming events.
Our data demonstrate that aneuploidy can occur during the culture of clinical grade MSC whatever the process used, and suggest there is no occurrence of transformation of cultivated MSC.
Reprogramming Somatic Nuclei into Embryonic Nuclei Through Nuclear Transfer and the Stem Cell Potential of the Derived Cell Lines
Reprogramming refers to the multistep process that allows a cell nucleus to change its fate and adopt another one. This process can be induced either by a direct exposure of cells to a combination of specific factors or by reconstructing an embryo after the introduction of a cell nucleus into an enucleated oocyte. While the former approach has shown its compatibility with the production of pluripotent cells, only the later, also referred to as cloning, has resulted in a full reprogramming of nuclear functions into live adults. This demonstrates that genome-contained and time-dependent controlled sequences of pre-determined events can be made fully functionally reversible. Recent results obtained from different laboratories including our own lab have identified two key periods for the resetting of the functional activities of a donor nucleus. The first period is bound to nuclear de-differentiation and involves not only cell cycle related nuclear reorganization but also nuclear features such as the peculiar distribution of centromeric and pericentric heterochromatin of the embryonic one cell stage. This period is amenable to transient chromatin manipulation which can markedly improve developmental rates to the early blastocyst stage while keeping intact the potential of its inner cell mass cells to be grown in vitro into fully functional embryonic stem cells similar to those obtained from normal embryos.
The second period is related to the functional interactions established between the embryonic and the extraembryonic lineages during blastocyst differentiation and implantation. This period has a major impact on later developmental processes including postnatal life, this mainly through placental dysfunctions related to epigenetic defects. Although the in vitro derivation of stem cell lines from the reprogrammed trophoblast or epiblast lineages is not affected, some differences are observed in term of cell proliferation and pattern of gene expression with their normal counterparts obtained from fertilized blastocysts.
Taken together, these data suggests that the epigenetic errors associated with the reprogramming process induced after nuclear transfer are becoming only partially reversible in vitro between the two above defined embryonic periods. This differential situation offers a mean for analyzing the mechanisms associated with the resetting of a pluripotent-like state and for comparing the stability of cell lines derived from a same batch of cultured somatic cells used either as a source of nuclei in cloning experiments or transfected with transcription factors under conditions compatible with the induction of pluripotency.
Chromosomal Instability and Mesenchymal Stem Cells
Many clinical trials are currently on going with mesenchymal stems cells in various therapeutic indications around the world. In France, clinical trial using cells need to be authorised by Afssaps (the competent authority for clinical trials) prior to their beginning. Some of these clinical trials have been put on hold in France due to the occurrence of recurrent abnormal karyotypes in cell culture. Many hypotheses are assumed and further investigations are ongoing. Before these clinical trials will start up again, the sponsors will have to propose to Afssaps sufficient controls on the cells and an adapted follow-up for the patients to be confident on the safety of the patients enrolled in clinical trials.
Immune Reactions to Adenovirus and AAV (Natural Infection and Viral Vectors)
Vectors based on viruses are important technology platforms for gene therapy and genetic vaccines. Our laboratory has contributed to the development of vectors based adenoviruses and adeno-associated viruses (AAV) of which both have emerged as leading contenders for in vivo gene transfer. There is an emerging base of data in the use of these vectors in mice, large animal models (e.g., dogs and monkeys) and humans. Two themes have emerged from these studies: 1) animal models including monkeys do not always predict outcomes in humans especially in terms of vector toxicity and 2) there exists significant subject-to-subject variation in human studies in terms of efficacy and safety of in vivo gene transfer. Our hypothesis is that prior exposure to the virus used to make the vector through a natural infection and the ensuing immune memory response substantially influences vector performance. Our work therefore has focused on the study of natural infections in humans with adenoviruses and AAV. We learned that both viruses establish latent/persistent infections in humans with complicated associated immune responses. The influence of these events on in vivo gene transfer will be discussed.
INSERM UMR 649, CHU Nantes, Université de Nantes , Faculté de Médecine, Nantes, France .
In the absence of an immune response from the host, intramuscular (IM) injection of recombinant Adeno-Associated Virus (rAAV) results in the permanent expression of the transgene from the mouse to the primate models. However, recent gene transfer studies into animal models and humans indicate that the risk of transgene and/or capsid-specific immune responses occurs and depends on multiple factors. Among them, the route of delivery and the vector tropism are important ones although poorly addressed in large animal models. Here, we compared: (i) the IM and the regional intravenous (RI) deliveries of rAAV in the nonhuman primate (NHP) skeletal muscle and monitored the host immune response toward the transgene; (ii) the AAV8 cell tropism in the mouse and NHP models. This has important implications for the design of clinical trials for gene transfer in the skeletal muscle.
AAV-Mediated Gene Transfer in Limb-Girdle Muscular Dystrophies: Complex Strategies and Biosafety Issues
Limb-girdle muscular dystrophies (LGMD) are a group of genetically heterogeneous muscular dystrophies grouped together on the basis of common clinical features: they all primarily and predominantly affect proximal muscles. They are caused by mutations in genes coding for a variety of proteins such as calpain 3, dysferlin, sarcoglycans or titin. Gene transfer to the muscle in animal models using recombinant adeno-associated virus (rAAV) vectors has proven to be an effective option for the treatment of several LGMD. However, hurdles linked to the particularities of the transgene can be observed in some instance. For example, whereas for most of the LGMD, the size of the corresponding cDNA is compatible with the encapsidation capacity of rAAV, there are two notable exceptions: dysferlin and titin. To obtain correction of the genetic defects, we are applying strategies based on the capacity of AAV to concatemerize to transfer the dysferlin gene using head and tail vectors and spliceosome-mediated RNA trans-splicing to exchange exons in the titin gene. In addition, biosafety issues related to the generation of an immune response against the transgene in α-sarcoglycan gene transfer or to the toxicity of ectopic expression in calpain 3 gene transfer were identified. To circumvent these problems, restriction of transcription of transgenes to the target tissue by constructing cassettes carrying regulatory elements such as endogenous promoters and/or specific miRNA target are explored. In this presentation, results obtained with these different strategies will be presented.
Footnotes
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Presentors are underlined for all abstracts.