BrennerMalcolmCenter for cell and gene therapy, Baylor college of Medicine, United States of America, E-mail: mbrenner@bcm.edu
Bringing cell and gene therapy into mainstream medicine
Session: Keynote lecture 1
It is now twenty years since the first legal gene transfer studies were approved, and there has been considerable disappointment in the slow rate of progress that followed the initial studies. Gradually, however, as the limitations of available vectors are acknowledged and overcome, and with advances in our understanding of the molecular and cell biology of genetic diseases and of cancer, unequivocal successes are now being reported. This presentation will outline some of the major remaining scientific and structural roadblocks to successful gene therapy and outline approaches to overcome them, using genetically modified immune system cells as examples. The presentation will also show how increasingly successful clinical outcomes are leading encouraging investigators to devise ways in which gene transfer and genetically modified cell therapies can be more widely introduced into clinical practice and become closer to an economically viable standard of care.
NishikawaShinichiStem Cell Research Group, Riken Center for Developmental Biology, Japan, E-mail: nishikawa@cdb.riken.jp
The pathway of hematopoietic stem cell development explained
Session: Keynote lecture 2
Hematopoietic stem cell (HSC) is the most extensively studied stem cell, but yet its developmental pathway in mammals has not been fully explained. While it is established that the definitive HSC that maintains a life-long maintenance of hematopoietic system appears around E10-11 during embryogenesis, none of intermediate stages in the course of HSC differentiation from mesoderm has not been specified until recent years. In& this talk, I will introduce our attempts to define this course. According to our model updated by the latest results, 4 distinct stages exist between mesoderm & dHSC. The first stage is Flk1+Etv/ER71+ population appearing in the yolk sac of E7.0-7.5 embryo. As null mutation of etv2/er71 gene results in complete block of differentiation of endothelial and hematopoietic lineages, this is the major diverging point of those two lineages. The next stage in the HSC pathway is defined as Flk1+Runx1+GATA1+VECAD+. Etv2 expression is also maintained in this stage. While the major population derived from this stage is primitive hematopoietic cells, a small fraction of cells proceeds to the dHSC pathway. We have evidence that VECAD & GATA1 are downregulated in this stage, but definition of this stage requires further study. Subsequently, VECAD is reinduced in the embryo and resulting VECAD+Runx1+ hemogenic endothelial cells are integrated into the luminal wall of developing vascular system such as dorsal aorta & umbilical artery. Finally, CD45+ cells bud out from this Runx1+ endothelial cells by the downregulation of molecules involved in maintaining cell-cell junction of endothelial cells. With this new definition of intermediate stages in dHSC differentiation pathway, we are investigating the molecules that regulate this stage shift.
PetryHaraldAMT, Amsterdam, The Netherlands
AAV gene therapy and clinical applications: an update
Session: Educational Session 1a
Adeno-associated virus (AAV) a non-enveloped small virus is one of the most promising DNA vectors for clinical application in gene therapy. AAV has never been shown to cause a disease in humans, even though the majority of the population is been exposed. The recombinant AAV used for gene delivery is depleted of the two genes of the wild-type virus which are replaced by a therapeutic transgene-specific expression cassette. The presentation will cover the progress made over the last several years by using recombinant AAV vectors in the clinic to treat diseases in the muscle, liver, brain and the eye.
The progress made in this field of gene therapy will be illustrated in more detail by presenting the latest data of clinical trials in which AAV is used to cure lipoprotein lipase deficiency (LPLD) an orphan disease for which no treatment exist today. The product GlyberaTM consists of copies of the human lipoprotein lipase gene variant LPLS447X in an adeno-associated type 1 virus vector.
The LPLS447X variant has a deletion of two amino acids at the C-terminus of the protein. It is found in 20% of Caucasians, and is a so-called ‘gain-of-function’ mutation, being associated with lower plasma triglyceride (TG) levels, higher high density lipoprotein (HDL) cholesterol concentrations and lower rates of cardiovascular disease, when compared to the general population.
Long-term follow-up data from two clinical trials show that one administration with GlyberaTM brings significant and clinically important reduction in acute pancreatitis in lipoprotein lipase deficient patients. Recurrent acute pancreatitis is the most debilitating complication of lipoprotein lipase deficiency and is associated with significant morbidity and mortality. The data from both trials also confirm that the treatment is well-tolerated and safe.
BüningHildegardDepartment I of Internal Medicine and ZMMK, University of Cologne, Germany, E-mail: hildegard.buening@uk-koeln.de
The adeno-associated viral vector system
Session: Educational Session 1a
Adeno-associated viral vectors (rAAV) are emerging as one of the leading gene transfer systems owing to their lack of pathogenicity, low immunogenicity, high stability, longevity of transgene expression and the potential to integrate site-specifically. rAAV have been applied in over 60 clinical trials, and clinical benefit of rAAV based therapies has recently been reported for the treatment of rare diseases. Traditionally most rAAV vectors were based on AAV serotype 2. However, due to the high prevalence of anti-AAV2 antibodies in the human population, and low transduction efficiency of clinically relevant target cell types such as hepatocytes or endothelial cells, alternative serotypes and variants have recently been developed as vectors. A common feature of all AAV serotypes is the lack of cell-type specificity, making the use of targeting vectors mandatory if cell-specific gene transfer is required. Various targeting strategies have been developed where a selective binding domain is “displayed” on the capsid surface mediating stringent interaction with target cell receptors. rAAV featuring new combinations of capsid attributes can be engineered by mixing capsid domains or subunits from different serotypes or mutants. Furthermore, combinatorial libraries and high-throughput selections, initially developed for the selection of cell-type specific AAV targeting vectors, are now frequently used to generate specifically tailored AAV vectors. The application of this portfolio of AAV vector types has been greatly improved by the development of self-complementary AAV vector genomes, and the possibility to package vector genomes flanked by AAV2 packaging signals into capsids from different serotypes, from mosaic/chimeric or targeting vectors.
Viruses belonging to the genus Parvovirus, within the Parvoviridae family, are able to replicate autonomously in the absence of helper viruses, in contrast to the adeno-associated virus members. The experimental infectivity and excellent tolerance of some rodent autonomous parvoviruses in humans, together with their oncosuppressive effects in preclinical models, speak for the inclusion of these agents in the arsenal of oncolytic viruses under consideration for cancer therapy. In particular, wild-type parvovirus H-1PV can achieve a complete cure of various tumors in animal models and kill tumor cells that resist conventional anticancer treatments. There is growing evidence that parvoviral oncosuppression involves an immune component in addition to the direct viral oncolytic effect. This talk will summarize the recent assessment of H-1PV antineoplastic activity in glioma, pancreatic ductal adenocarcinoma, and non-Hodgkin lymphoma models, laying the foundation for the present launch of a first phase I/IIa clinical trial on glioma patients.
OgrisManfredGroupleader VectorologyDepartment of Pharmacy, Ludwig Maximilians Universität München, Germany
Physicochemical methods for gene delivery (Part 1)
Several physicochemical methods can be applied for the delivery of transgenes into cells ex vivo and in vivo, and some of them have already been used in advanced clinical trials for the treatment of monogenetic diseases, cancer and others. Major drawbacks compared to viral delivery methods are low transfection efficiencies in certain cell types, low level and short term gene expression and loss or inactivation of transgenes. As the latter problems can be solved by proper design of plasmids, transfection efficiency depends mostly on the physicochemical method used for gene delivery, which will be presented in this lecture. For ex vivo gene delivery a plethora of chemical transfection reagents are commercially available, most of them based on cationic lipids or polymers, which condense or aggregate nucleic acids into lipoplexes or polyplexes, respectively. Cellular binding and internalization usually occurs via electrostatic interaction with negatively charged cell membrane components. Solely physical methods include ballistic gene delivery with DNA coated metal spheres or electroporation, were an electric field lead to cellular gene delivery. Both methods can be applied ex vivo or localized in vivo. In vivo, intramuscular injection of naked DNA leads to efficient transgene expression, high expression in liver tissue can be achieved by the hydrodynamic delivery method. Further on, limitations and pitfalls in physicochemical gene delivery will be discussed.
RudolphCarstenGroupleader Experimental Gene TherapyDepartment of Pediatrics, Ludwig Maximilians Universität München, Germany
Physicochemical methods for gene delivery (Part 2)
Session: Educational Session 1b
Physicochemical methods for gene delivery have been intensively investigated during the last decades and first products have been approved for veterinary use. Although transfection rates achieved with nonviral delivery systems are frequently lower and gene expression is of only short duration when compared with viral vectors, they offer the advantage of being less immunogenic, less restricted to the size of the delivered transgene and being less expensive with respect to production. The currently used repertoire of physicochemical gene delivery methods have evolved from various basic concept using either naked DNA or complexed with gene transfer agents. Moreover, a variety of methods have been established which make use of different physical phenomena to introduce DNA into cells. In this lecture, basic concepts of various physicochemical gene delivery methods making use of physical forces for transfection will be discussed. These methods include electroporation, sonoporation, magnetofection and gene gun. Further on, data on aerosol gene delivery will be presented. In addition, advantages and restrictions of the physicochemical gene delivery methods will be discussed.
Vectors employed for gene and immune therapy are based on recombinant nucleic acids. Most gene vectors include viral or metazoan genetic components such as promoters and enhancers, which provide the necessary cis-acting elements for expression of one or more genes of therapeutic interest. In addition, certain virus-based gene vectors also carry essential elements involved in packaging of genetic information into virus-like structures. In the recipient cell, gene vectors can be transiently present or maintained for a long period of time. In these cases, the genetic information is either rapidly lost through spontaneous degradation by cellular nucleases or maintained by integrating it into the chromosome of the recipient cell. Because a prolonged effect of the therapeutic gene product is often preferred, gene vectors are commonly employed, which promote their chromosomal integration. Unfortunately, all integrating vectors can act as insertional mutagens, which might be destructive to the transduced cell. This problem can be avoided with gene vectors, which are stably maintained as extrachromosomal units in the recipient cell. This talk will concentrate on two extrachromosomal vector systems, which work in metazoen cells: oriP-based plasmids and non-viral vectors called pEPI. I will address the molecular mechanics involved in their extrachromosomal maintenance, their advantages and disadvantages, and possible applications.
KochanekStefanDivision of Gene Therapy, Ulm University, Ulm, Germany, E-mail: stefan.kochanek@uni-ulm.de
Adenovirus: biology and vectors
Session: Educational Session 2a
Adenovirus vectors belong to the most frequently used vectors for somatic gene therapy and for genetic vaccination. In this presentation, fundamental aspects of adenovirus as a common pathogen are discussed and knowledge of design, production and application of different adenovirus vector types is summarized. The advantages/disadvantages and applications of E1-deleted vectors (also called first-generation vectors), second generation vectors, helper-dependent or “gutless” vectors, and of replicating vectors are discussed. In situations, in which only short-term gene expression is required to achieve an effect, such as in prophylactic vaccination or in tumor therapy, first-generation adenovirus vectors are attractive choices, also because they can be produced to very high titers. When long-term gene expression is required, “gutless” adenovirus vectors have conceptual advantages, their main disadvantage being cumbersome production. Replicating (oncolytic) vectors are of interest for the treatment of malignancies, although clinical trials so far have been relatively disappointing. A current focus of research of several laboratories is the development of strategies that aim to improve specific gene transfer into target cells and/or reduce gene transfer failures due to pre-existing immunity, a topic which will be discussed in the following presentation.
KreppelFlorianDivision of Gene Therapy, University of Ulm, Helmholtzstraße 8/1, D-89081 Ulm, Germany, E-mail: florian.kreppel@uni-ulm.de
Adenovirus vector shielding and targeting
Session: Educational Session 2a
Recombinant adenovirus (Ad) vectors are highly potent gene delivery vehicles that may be used for a remarkably wide variety of different gene therapy approaches ranging from genetic vaccination to the treatment of tumor diseases. However, the use of adenovirus vectors for local or systemic gene delivery in vivo is severely hampered by numerous biological barriers, which limit gene transfer efficiency and thus vector efficacy. Therefore, the successful use of adenovirus vectors in vivo requires in-depth understanding of these biological barriers and the development of technologies that allow to overcome them.
In this session the specific barriers for in vivo gene delivery with adenovirus vectors including the interaction of Ad vectors with cellular and non-cellular blood components, the activation of innate immunity, and recently identified barriers that arise even after successful vector uptake into target cells will be discussed. Up-to-date shielding and targeting technologies based on chemical capsid modifications with synthetic polymers as well as genetic capsid modification approaches will be presented and critically evaluated for their performance to overcome the biological barriers for successful in vivo gene delivery with adenovirus vectors.
EhrhardtAnjaMax von Pettenkofer-Institut, Department of Virology, Ludwig-Maximilians-University Munich, Munich, Germany, E-mail: ehrhardt@mvp.uni-munchen.de
Adenoviral vectors for stable gene transfer – are they the better solution?
Session: Educational Session 2a
Recombinant adenoviral vectors (rAdV) have been extensively evaluated in vitro, in small and large animals (rodents, dogs, nonhuman primates), and in various animal models covering different diseases. Today, 24% of all gene therapeutic clinical trials world wide are based on rAdV. Over the recent years major improvements have been achieved in adenovirus vector design including the development of adenoviral hybrid vectors for maintenance of the therapeutic DNA. The goal of this educational session is to provide a state-of-the-art overview of the most advanced rAdVs which were explored in clinically relevant settings.
This tutorial first discusses relevant preclinical studies utilizing non-integrative gutless rAdV. As representative examples, studies applying clinically relevant rAdV doses for stabilized liver transduction (up to 964 days) in nonhuman primates and a canine model for hemophilia B will be discussed.
Moreover, this tutorial provides a state-of-the-art overview of available hybrid vectors utilizing adenoviruses for high transduction efficiencies in concert with various genetic elements for long-term transgene expression. Especially in cells with a high turnover rate (e.g. stem cells) these vectors may provide an important alternative to other integrating viral vector systems. Herein, the molecular design of adenoviral hybrid vectors and strategies for achieving persistence of therapeutic DNA will be explained. Towards this end, rAdVs were generated to deliver viral integration systems (AAV, retrovirus), non-viral systems (Sleeping Beauty transposase, integrase phiC31, retrotransposons), and episomally maintained DNA elements (EBV episomes). Advantages but also limitations of each adenoviral hybrid vector system will be discussed and potential clinical applications mentioned.
von LaerDorothee1NewrzelaSebastian2Al'GhailiNabil2HeinrichTim2BaumChristopher3CornilsKerstin4LiZhixiong3BrugmanMartijn3HansmannMartin-Leo5HartmannSylvia5JäckHans-Martin6FehseBoris4
Applied Virology and Gene Therapy, Georg-Speyer-Haus, Germany
Frankfurter Stiftung für krebskranke Kinder, Goethe-University, Germany
Abteilung für Experimentelle Hämatologie, Medizinische Hochschule Hannover, GermanyUniversity Medical Centre, Hamburg-Eppendorf, Germany
Pathology Medical Falculty, Goethe University Frankfurt, Germany
Nikolaus Fiebiger Center, University of Erlangen-Nürnberg, Germany, E-mail: laer@em.uni-frankfurt.de
T cell receptor monoclonality of T lymphocytes predisposes to transformation
Session: Educational Session 2b
Retroviral insertional mutagenesis in hematopoietic progenitor cells can activate neighboring proto-oncogenes and thus contribute to leukaemia development. Here, we analysed whether mature T cells bear the same risk of insertional mutagenesis. To address this issue, we highly transduce mature murine T cells and haematopoietic stem cells with gammaretroviral vectors expressing LMO2, TCL1, NPM-ALK or ΔTrkA. After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphoma/leukemia for all investigated oncogenes after characteristic periods, mostly of a CD8+ CD4+ double positive phenotype. None of the control vector-transduced mice developed malignancies. T cell-transplanted animals showed no signs of leukaemia development during a follow-up of more than 1 year. In contrast, TCR transgenic mice (OT-1, F14), which harbour a monoclonal T cell population, readily developed T cell lymphomas. This was seen for the T cell oncogenes dTrkA, TCL1 and NPM-ALK. The lymphomas had a dedifferentiated phenotype with a down regulation of T-cell markers and up-regulation of B-cell markers, but showed no B-cell receptor rearrangements. Co-transplantation of wild-type polyclonal T cells with dTrkA-transduced OT-1 T cells suppressed lymphoma development. The mechanisms that control transformation of polyclonal mature T cells are currently under investigation. We propose that clonal competition may control leukemogenesis in mature T cell populations.
SchambachAxelDepartment of Experimental Hematology, Hannover Medical School, Germany, E-mail: schambach.axel@mh-hannover.de
Retro- and Lentiviral Vectors: Vector design, concept and novelties
Human Immunodeficiency Virus (HIV-1) and Murine Leukemia Virus are evolutionally optimized to transfer and integrate their genome into the host cell chromatin (see talk by D. von Laer). The topic of this educational talk is focussed on how we can convert these pathogens into efficient and safe gene delivery tools for the correction of inherited or acquired diseases, and for advanced cell modification such as reprogramming. Recombinant retroviral vectors based on lentiviruses (e.g. HIV-1) and gammaretroviruses (e.g. MLV) are meanwhile relatively well characterized and efficient tools for gene therapy. Furthermore, promising vector systems have also been established on the basis of other retroviruses such as Equine Infectious Anemia Virus, Foamyvirus, and more recently alfaretrovirus. As integrating vectors with semi-random insertion profiles can be mutagenic, an important area of current research is to develop retrovirus-based vectors which are less likely to activate or disrupt neighbouring genes. In addition, an increasing number of investigators explores how intermediate steps of the retroviral life cycle can be used for transient delivery of nucleic acids or proteins and how the transduction with retro- and lentiviral vectors can be visualized. An overview of recent and future clinical applications of retroviral vectors will conclude the presentation.
Lentiviral vectors allow stable long-term transgene expression in nondividing cells and in tissues. These properties have made them ideal gene delivery vehicles for research and therapeutic applications, including clinical trials. However, further efforts in vector design are required to improve safety and efficacy of lentiviral mediated gene transfer. Special attention has to be given to measures that i) restrict gene transfer to the cell type relevant for a particular therapeutic application and ii) allow the transduction of quiescent cells of the hematopoietic system. Recent improvements have come from combining lentiviral vectors with engineered envelope proteins, as those of measles virus, by destroying the natural receptor tropism and adding a targeting domain that will mediate attachment to the cell surface receptor of choice. Such re-targeted lentiviral vectors allow the restriction of gene transfer to basically any cell type of interest including quiescent lymphocytes with an unprecedented degree of efficiency, thus opening new options for in vivo gene therapy applications.
PâquesFCellectis SA and Cellectis Genome Surgery, 102 avenue Gaston Roussel, 93235 Romainville Cedex, France, E-mail: paques@cellectis.com
Targeted approaches for gene therapy and the use of meganucleases
Session: Educational Session 3a
In spite of significant advances in gene transfer strategies for gene therapy, there is a strong emphasis on the development of alternative methods that provide a better control of transgene expression and insertion patterns. Different types of molecular tools can target a desired transgene or gene modification in a well defined locus. The use of engineered endonucleases with tailored specificities appears as one of the most promising approaches. Three different types of such endonucleases have been used so far: Zinc Finger Nucleases, Chemical Endonucleases, and Meganucleases. We will survey the different types of targeted strategies with a special emphasis on Meganucleases. Meganucleases are the most specific natural endonucleases, and their function is to induce genome engineering events. These proteins have long proved difficult to engineer. However, recent advances in protein engineering have opened the door to a wider use of these endonucleases. We will describe the meganuclease properties, the early experiments that identified meganucleases as ideal tools for genome editing, the emergence of meganuclease engineering strategies in different laboratories, and the most recent developments illustrating the potential of these molecular scissors in gene therapy.
CathomenToniExperimental Hematology, Hannover Medical School, Germany, E-mail: cathomen.toni@mh-hannover.de
Zinc-finger nucleases: finding the balance between activity and toxicity
Session: Educational Session 3a
Recent advances in generating customized zinc finger nucleases (ZFNs) hold great promise to pave the way for therapeutic genome engineering strategies. ZFNs consist of a highly specific artificial DNA-binding domain fused to a non-specific nuclease domain. Upon binding to the target site ZFNs introduce a DNA double strand break (DSB) at the pre-selected site in the human genome. The activated cellular DNA repair pathways can subsequently either be harnessed to disrupt a gene or to correct an inborn mutation in the genome. Gene editing frequencies of up to 50% of treated cells in the absence of selection demonstrate the power of this emerging technology. Although this frequency is high enough to contemplate clinical applications some hurdles remain, including the potential genotoxicity associated with both ZFN expression and vector integration. In my talk I will show how structural changes in the ZFN architecture affect the balance between ZFN activity and ZFN-associated cytotoxicity. Furthermore, I will present a ZFN-mediated gene targeting system based on vectors derived from adeno-associated virus (AAV) and show how transient cell cycle arrests affect the balance between gene targeting and AAV vector integration.
IzsvakSuzanna
Non-viral gene transfer using integrating vector systems
Session: Educational Session 3a
Non-viral integrating vector systems represent a novel technology that opens up new possibilities for gene therapy. Similarly to retroviruses, these elements integrate into the chromosomes of host cells, but their life-cycle does not involve reverse transcription, and they are not infectious. Due to stable chromosomal insertion, these systems can result in robust, long-term expression of the integrated transgene. The recombinase toolkit includes the bacteriophage phiC31, Sleeping Beauty (SB), piggyBac and Tol2. Some of these vectors are being tested in a clinical setting, and show clear advantages over other gene transfer methods. The hyperactive SB100X is the first plasmid-based vector system that is able to overcome the efficacy problem of non-viral vectors. Compared to viral vectors, DNA-based transposon vectors are less expensive to manufacture, and have favorable safety profile, including reduced immune complications. They have no strict limitation of the size of expression cassettes, and are less prone to reverse transcriptase-induced mutations/rearrangements than retroviral vectors. The SB vector has inert transcriptional activities, fairly random genomic insertion pattern, and particularly favorable attributes for stable, long-term expression in various primary and stem cells. These advantages would facilitate the implementation of clinical trials based on recombinase vectors. In agreement with this optimism, the first human trial utilizing the Sleeping Beauty transposon system has been initiated.
My presentation addresses several issues of developing non-viral, safe, and effective therapeutic vectors. I conclude that gene therapy strategies using non-viral integrating vector systems could reduce the risk of insertional mutagenesis and immunogenicity problems imposed by viral vectors.
GalyAnneBoisgeraultFlorenceSudresMurielRyffelBernhardUMR951, Généthon, Evry, FranceIEM UMR6218, Orléans, France
Adaptive immune responses to gene transfer with viral vectors: Focus on the use of rAAV for the treatment of hereditary diseases
Session: Educational Session 3b
Recent gene therapy clinical studies have highlighted the importance of immunity against viral vector components, for the safety and therapeutic benefit of the approach. Furthermore, the induction of immune responses against gene-modified cells remains a major concern for the efficacy of gene transfer in null individuals. Recombinant adeno-associated virus (AAV) vectors are widely used for the treatment of hereditary diseases because of their broad tropism and low inflammatory properties. We will focus on this vector to present key mechanisms underlying the complex immune reactions following viral vector-mediated gene transfer and to present novel modalities that ensure the persistence of gene-corrected cells in this context.
Recent advances have shown a crucial role of innate immunity in shaping humoral and cellular immune responses to rAAV. While the induction of an adaptive immune response to rAAV2 depends on the TLR9-MyD88-type I IFN pathway, requirements appear to be partially distinct for rAAV1. Specific strategies may therefore be conceived to interfere with the recognition of the viral proteins and genome components of rAAV vectors.
The persistence of gene transfer into solid tissues is compromised by effector immune responses that develop following antigenic presentation of the transgene. Novel tissue-specific expression strategies exploiting the mir142.3p constitute an efficient and practical approach to curtail the induction of transgene-specific T and B cell reactivity in the context of rAAV-mediated gene transfer.
The interplay between anti-viral and anti-transgene immune responses remains unclear, but several approaches exist and may be combined to improve the outcome of rAAV-mediated gene therapy in humans.
Acute myocardial infarction remains a leading cause of mortality and morbidity worldwide. The infarcted human heart heals by scar formation, and large myocardial infarctions typically result in heart failure. While endogenous mechanisms leading to a replacement of lost cardiomyocytes do exist in the adult mammalian heart, these mechanisms are not sufficient for meaningful tissue regeneration. Randomized clinical trials indicate that intracoronary delivery of bone marrow (stem) cells leads to an improvement in systolic function in patients after myocardial infarction. This improvement in contractile function, however, does not appear to be related to tissue regeneration and myocyte formation. Instead, paracrine effects on infarct healing, inflammation, and angiogenesis in the infarct border zone appear to play a more important role. Identification of these paracrine mediators may enable the development of specific therapies promoting post-infarct repair and adaptation.
VandenDriesscheThierryUniversity of Leuven, Flanders Institute of Biotechnology, Belgium, E-mail: thierry.vandendriessche@med.kuleuven.be
Gene therapy for hemophilia
Session: Educational Session 4b
Protein substitution therapy (PST) with plasma-derived or recombinant clotting factors has greatly improved the quality of life and prolonged the life expectancy of patients suffering from hemophilia. Despite this significant progress, there are several challenges that would still need to be addressed. In particular, there is a need to develop improved clotting factors with prolonged half-lives that require less frequent infusions. Moreover, continuous production of clotting factors following gene therapy (GT) would be ideal since it would obviate the need for repeated clotting factor administration. Finally, it is essential to develop approaches that minimize the risk of neutralizing antibodies (clinically referred to as inhibitors) following GT. To assess the efficacy and safety of novel GT approaches for hemophilia it is necessary to conduct preclinical studies in animal models. The availability of small animal models and in particular hemophilia A and B mice with targeted disruption of the factor VIII and IX genes contributed to a better understanding of the efficacy and safety of GT approaches and paved the way towards the validation of new concepts and mechanisms. Moreover, hemophilia A and B dogs are available that have greatly facilitated translational research in hemophilia. These preclinical models are well suited to compare the relative immunogenicity of different GT approaches and to develop improved immunomodulatory strategies that reduce the risk of inhibitor development. Preclinical studies suggest that AAV and lentiviral vectors are among the most promising vectors for GT of hemophilia. It is particularly encouraging that favorable therapeutic outcomes in these large animal models correlated often with promising results in clinical trials that strenghtens their intrinsic predictive value. Though these animal models help to bridge the gap from the bench to the bedside, there are some patient-specific issues that can only be addressed in clinical studies.
UckertWolfgangMax-Delbrück-Center of Molecular Medicine, Humboldt University Berlin, Germany, E-mail: wuckert@mdc-berlin.de
Gene-modified T cells to treat cancer
Session: Educational Session 4b
Over the past decade, the genetic introduction of T cell receptor (TCR) genes into T cells has been developed as a strategy to induce defined antigen-specific T cell immunity. The potential value of TCR gene therapy was established in mouse models and the feasibility of infusion of TCR-modified autologous T cells was shown in phase I clinical trials in cancer patients. One of the next important steps will be to transform TCR gene transfer from an experimental technique into a robust clinical platform. This requires further optimization/modification of TCR genes to yield efficient TCR expression and subsequently T cells with high functional avidity against the new defined antigen specificity as well as the introduction of safety modules in TCR gene-modified cells to terminate TCR gene therapy in case of severe side effects (e.g. autoreactivity). In this presentation, different aspects to improve TCR gene therapy by optimizing the transfer vector, the TCR genes, the TCR transgene cassette and the development of a new safety module, based on a TCR-intrinsic mechanism, to eliminate TCR gene-modified T cells. after adoptive transfer will be discussed.
StripeckeRenataLymphatic Cell Therapy Laboratory, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Germany, E-mail: stripecke.renata@mh-hannover.de
Gene Modified Dendritic Cells for Cancer Therapy
Session: Educational Session 4b
Dendritic cells (DCs) are key players in the innate and adoptive immune responses. DCs can be obtained ex vivo upon culture of hematopoietic precursors (CD34 + , monocytes) in the presence of cytokines leading to their differentiation and maturation. Ex vivo grown DCs have been explored during the last decade as a cellular vaccine modality to enhance immunity against weakly stimulatory cancer (self ) antigens. Genetic manipulation of DCs and their precursors are underway to improve potent, specific and enduring therapeutic anticancer responses. Several strategies have been devised in order to enhance DC longevity, bio-distribution, inflammatory potential and antigen presentation. The pioneering work reporting efficient viral gene delivery into DCs was performed with adenoviral vectors (AdV), which are highly immunogenic in humans, hampering immune responses to weaker “self” tumor antigens. In contrast, lentiviral vectors (LVs) offer persistent, non-toxic, and non-immunogenic gene delivery into dendritic cells. Therefore, LVs have more recently emerged as a robust and practical experimental platform for gene delivery and rational genetic re-programming of DC. Here, we present the status quo of the LVs system evaluated for ex vivo and in vivo gene delivery into DCs for cancer immunotherapy. Improvements of the LV design in order to further grant higher versatility, efficacy and bio-safety for DC vaccine development are presented. Finally, LVs have recently reached the clinical gene therapy arena, prompting their use as clinical cancer vaccines in the near future.
NettelbeckDirk MHelmholtz-University Group Oncolytic Adenoviruses, German Cancer Research Center and Dept. of Dermatology, Heidelberg University Hospital, E-mail: d.nettelbeck@dkfz-heidelberg.de
Oncolytic Viruses
Viral oncolysis aims at the destruction of tumors by productive virus infection. Research towards this aim has gained momentum in recent decades based on the advent of opportunities for genetic engineering of viruses with desired properties. These efforts overlap with viral vector development in gene therapy research, especially with respect to virus tropism modification, virus production and toxicity. Furthermore, for several oncolytic viruses it is feasible to insert transgenes without loss of their replication potential, thus facilitating the combination of oncolysis with gene therapy strategies (i.e. by replication-competent gene transfer vectors alias “armed” oncolytic viruses).
Tumor-specificity of virus replication is the key feature of oncolytic viruses, which have been derived from several different virus families. Some viruses possess intrinsic oncotropism, often based on differences in innate anti-viral functions of tumor cells versus normal cells. For other viruses, replication can be targeted to tumor cells by genetic engineering. One strategy is to restrict viral cell entry to specific cells types by engineering of viral envelope or capsid proteins. Alternatively, post-entry steps of virus replication have been modulated. Transcriptional targeting of the expression of essential viral genes and regulating the stability of viral mRNAs by tissue-specific microRNAs are two examples for this strategy. Several clinical studies have demonstrated a favorable safety profile for oncolytic viruses applied locally or systemically. However, these studies have also underlined that oncolytic viruses with increased potency are needed for effective viral oncolysis in patients. Strategies that are being pursued towards this end are the mutagenesis of viruses for improving their lytic activity, the combination of viral oncolysis with established therapeutic regimens, or the engineering of “armed” oncolytic viruses.
BoschFatimaCenter of Animal Biotechnology and Gene Therapy, Universitat Autonoma Barcelona, Spain, E-mail: fatima.bosch@uab.es
Gene therapy for diabetes: moving towards the clinic?
Session: Adenoviral and AAV vectors
Diabetes mellitus is a chronic disease with severe secondary complications driven by poor glycemic control for which there is no cure. Although insulin therapy allows type 1 diabetic patients to lead normal, active lives, it has associated risks, mainly acute hypoglycaemia and development of retinopathy, nephropathy and neuropathy. Gene therapy is a new approach to treat diabetes where the main aim is to develop a way to engineer tissues to maintain normoglycaemia. We previously demonstrated the feasibility of a potential gene therapy strategy based upon engineering skeletal muscle to take up glucose by co-expressing insulin and glucokinase (GK). Both genes act synergistically such that local production of human insulin (hIns) improves glucose uptake, whereas GK acts as a glucose sensor in a concentration-dependent manner to facilitate glucose disposal. We showed reversal of diabetic hyperglycemia in AAV1-treated diabetic mice. Our objective was to undertake a pre-clinical assessment of this combined therapy in diabetic beagle dogs as a step towards treatment of human diabetes. We wished to determine (1) the feasibility and relative efficiency of AAV1-CMV-hIns treatment in large animals; (2) the minimal effective dose of AAV1-CMV-hIns; (3) the feasibility of co-injecting AAV1-CMV-hIns and AAV1-CMV-GK; and finally (4) the duration of effects, as well as any biochemical and safety issues. Our results indicate that a single viral injection can mediate long term survival for greater than two years with sustained benefit to glycemic control, body weight and with no detriment to physical performance. In this presentation, this new gene therapy approach for type 1 diabetes from our group will be discussed.
BüningHildegardDepartment I of Internal Medicine and ZMMK, University of Cologne, Germany, E-mail: hildegard.buening@uk-koeln.de
AAV targeting and re-targeting - what makes the difference?
Session: Adenoviral and AAV vectors
The adeno-associated viral vector (rAAV) platform is one of the most commonly utilized gene transfer system. Although various serotypes and variants have been developed as vectors, rAAV2 is still the dominant serotype used in clinical trials. A weakness of rAAV2 (and other serotypes) is their propensity to accumulate in liver tissue following i.v. injection, and to transduce non-target cells if applied locally. In order to develop cell-type specific vectors, targeting techniques have been developed to alter the natural tropism of AAV. By insertion of ligands at exposed surface positions, we generated targeting vectors able to transduce wild-type non-permissive cells. Furthermore, vectors with improved transduction efficiency for wild-type permissive cells and/or with improved specificity were developed. The introduction of amino acid substitutions (R585A/R588A) significantly improved receptor binding of ligands inserted at position 587 or 453 revealing the impact of capsid residues on the efficiency of targeting vectors. The same residues are critical for the binding of AAV2 to heparan sulphate proteoglycan (HSPG). Due to destruction of the HSPG binding motif, AAV targeting vectors with insertions at position 587 were expected to transduce cells HSPG independently. However, the peptide itself can convey AAV targeting vectors with the ability to interact with HSPG. The respective vectors enter cells very efficiently, but lacked cell type-specificity and failed to de-target from liver tissue. In contrast, rAAV targeting vectors displaying non-HSPG binding peptides selected by AAV peptide display possessed a restricted tropism. Although these vectors differed from AAV2 in their cell entry mechanism, they were efficiently processed intracellularly.
Mechanisms underlying increased potency of postnatal cells and uses of such cells
Session: Bone marrow and tissue resident stem cells
During the last 5–7 years we and others demonstrated that cells can be cultured from bone marrow, cord blood, testis and perhaps other postnatal tissues that have the ability to differentiate into multiple cell types, including mesoderm, endoderm and ectoderm. We termed these cells multipotent adult progenitor cells (MAPC). However, the greater potency of MAPC and other cells from somatic tissues is still less than that of Embryonic Stem Cells (ESC). Transcriptome analysis demonstrated that rodent MAPC differ significantly from other mesenchymal stem cells (MSC), but that rodent MAPC express a number, but not all, genes specifically expressed in ESC, which are known to be important for their pluripotency (MAPC express e.g. Oct4; not Nanog and Sox2), even though rodent MAPC also express genes known to be associated with primitive endoderm. Interestingly, rodent MAPC express four of the six factors recently identified to be capable of reprogramming mouse and human fibroblasts to induced pluripotent stem cells (iPSC). Studies aimed at further delineating the mechanism underlying the greater potency of MAPC, whether pre-existing in vivo or induced in culture, will be discussed, as well as studies aimed at identifying the developmental stage with which MAPC can be compared, and studies aimed at inducing lineage specification of (near) pluripotent stem cells. In addition, the possible uses in medicine of de-differentiated cells, whether MAPC or iPSC, will be discussed.
Klinikum rechts der Isar und Deutsches Herzzentrum – Technische Universität München, I. Medizinische Klinik - Molekulare Kardiologie, Ismaninger Strasse 22, 81675 München, Germany
Wellcome Trust Centre for Stem Cell Research -, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, United KingdomInstitut für Medizinische Mikrobiologie, Immunologie und Hygiene – Technische Universität München, Trogerstraße 30, 81675 München
Institut für Pharmakologie – Technische Universität München, Biedersteiner Straße 29, 80802 München
Induced pluripotent stem cells as a source for multipotent ISL1+ cardiovascular progenitors
Session: Bone marrow and tissue resident stem cells
Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modelling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis the major lineages of the mature heart, cardiomyocytes, smooth muscle and endothelial cells, arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2-5. However, it remains unclear whether a comparable precursor population is present during human cardiogenesis. Here we show, using genetic fate-mapping, that Isl1+ multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all three cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1+ progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.
NewsomePhilipCentre for Liver Research, University of Birmingham, UnitedKingdom, E-mail: p.n.newsome@bham.ac.uk
Stem cell therapy in liver disease
Session: Bone marrow and tissue resident stem cells
Stem Cell Therapy in Liver DiseaseLiver disease is the fifth highest cause of death in the UK and is the only one of the top five that continues to rise. Currently the only curative treatment for patients with end-stage liver disease is liver transplantation, but this is no longer sufficient due to the rising demand. While new strategies are being implemented to increase the supply of donors, other treatments, such as those involving stem cells are being considered. Animal studies have demonstrated that mobilisation of bone marrow stem cells with GCSF leads to replenishment of the hepatocyte population, an effect which is mediated by increased proliferation of endogenous hepatocytes. This action was biologically significant as it led to the survival of mice which would have otherwise died of liver injury. Further work indicated that infusions of HSCs aspirated from bone marrow can reduce the amount of scarring in chronically injured livers. Some of these effects have also been seen with MSCs, albeit to a lesser extent.On the basis of these encouraging findings, a number of clinical studies, across many different countries have been undertaken. Most of the clinical studies have been small, with no control group, but the majority have suggested that infusions of adult bone marrow stem cells are safe, and also exert a positive effect on liver function. The mechanism of action of the infused stem cells in the human setting is unresolved, although there is a suggestion that part of the effect may be mediated through stimulation of endogenous hepatocytes. None of the studies have been adequately powered, and thus larger randomised studies are needed before the beneficial effects can be proven.
AbkenHinrichCenter for Molecular Medicine Cologne, University of Cologne, Germany, E-mail: hinrich.abken@uk-koeln.de
Engineered T cells redirected by 2nd and 3rd generation immunoreceptors - The weal and woe of costimulation
Session: Cancer: immunity
For use in adoptive immunotherapy, strategies have been developed to equip effector T cells with defined specificity by expression of recombinant antigen receptors (CARs, immunoreceptors). The immunoreceptors consist of an extracellular antibody derived binding domain and an intracellular signalling domain, mostly the CD3z domain. Second generation immunoreceptors in addition incorporated the intracellular CD28 signalling domain in order to provide appropriate costimulation, increase IFN-g secretion and prolong T cell survival. CD28 costimulation overcomes TGF-b mediated repression in T cell proliferation but does not alter the affinity dependent threshold in T cell activation. On the other hand, CD28 costimulation makes redirected T cells more sensitive to repression by regulatory T cells than CD3z signalling only. The other members of the CD28 costimulatory family, including 4-1BB and OX40, have different impact on redirected effector functions of CD4+ and CD8+ T cells. Combined costimulation by CD28 and OX40 in 3rd generation immunoreceptors, however, is required to redirect CCR7− T cells, in contrast to CCR7+ T cells, in order to provide an effective anti-tumor response in vivo. Taken together, 2nd and 3rd generation immunoreceptors have differential impact on redirected T cell activation which has fundamental consequences for the immunotherapy of malignant diseases.
JuneCarlUniversity of Pennsylvania Philadelphia, PA, USA
Promises and challenges of engineered T cell therapies
Session: Cancer: immunity
While there are exciting examples of successful clinical strategies to mobilize the immune system to attack cancer cells, overall the results have been disappointing in randomized clinical trials. We are exploring the use of engineered T cells bearing chimeric receptors and strategies to augment their antitumor efficacy in adoptive transfer settings. The surface membrane glycoprotein mesothelin is a promising target for the immunotherapy of mesothelioma, ovarian, and pancreatic tumors due to the uniform overexpression of mesothelin and the benign phenotype of mesothelin null mice. We hypothesize that previous trials of adoptive immunotherapy for cancer that have used CTL have failed due to poor trafficking to sites of tumor, and insufficient effector functions to self antigens. Our preclinical data indicates that use of lentiviral engineered T cells with chimeric receptors that incorporate a ‘tumor resistance genotype’ should have improved function for cancer immunotherapy. We have tested mesothelin redirected T cells in humanized mouse models bearing tumor xenografts. The T cells are able to eradicate large, well established tumors at an in vivo E:T ratio of at least 1:70. As a complementary strategy, we have engineered artificial antigen presenting cells (aAPC) to express ligands for either CD28 or ICOS. These aAPC appear to be useful to reprogram T cells, and increase the antitumor efficacy of adoptively transferred T cells. In ongoing clinical trials testing adoptive transfer of T cells after retroviral or lentiviral gene transfer we find that 1) the T cells engraft and persist at high levels for 10 years or more, indicating that central memory T cells with “stem cell like qualities” can be transduced, and 2) rectal mucosal biopsy studies taken from patients after adoptive transfer indicate that the T cells traffic with high efficiency to IEL. Finally, our preclinical studies with B. Jakobsen testing TCRs engineered for high affinity indicate the ability to “convert” polyclonal T cells to monoclonal T cells with potent redirected specificity for surrogate antigens, suggesting that tumor antigens for which substantial repertoire limitations in the natural pool of available T cells can be targeted with the adoptive transfer of engineered T cells.
References
CarpenitoCet al.2009. Control of large established tumor xenografts with genetically re-targeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci U S A, 106:3360–3365.
MiloneMCet al.2009. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased anti-leukemic efficacy in vivo. Mol Ther. in press.WangGPet al.2009. Analysis of Lentiviral Vector Integration in HIV+ Study Subjects Receiving Autologous Infusions of Gene Modified CD4+ T Cells. Mol. Ther, 17:844–50.PerezEEet al.2008. Establishment of HIV-1 Resistance in CD4(+) T cells by Genome Editing using Zinc-Finger Nucleases. Nature Biotechnology, 26:808–816.Varela-RohenaA. et al.2008. Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor. Nature Med, 14:1390–1395.JuneCH. 2007. Principles of adoptive T cell cancer therapy. J Clin Invest, 117:1204–1212.JuneCHet al.2009. Lymphocyte Subset Engineering: tools, trials and tribulations. Nature Reviews Immunology, 9:704–716van HeijstJeroen1GerlachCarmen1SwartErwin1SieDaoud2Nunes-AlvesClaudio3KerkhovenRon2ArensRamon1Correia-NevesMargarida3SchepersKoen1SchumacherTon1
Division of Immunology, The Netherlands Cancer Institute, Netherlands
Central Microarray Facility, The Netherlands Cancer Institute, Netherlands
Life and Health Sciences Research Institute, University of Minho, Portugal, E-mail: t.schumacher@nki.nl
Fate analysis in the T cell lineage
Session: Cancer: immunity
T cells, as well as many other cell types, are composed of phenotypically and functionally distinct subsets. However, for most of these populations it is unclear whether they develop from common or from separate progenitors. To address such issues, we developed a novel approach-termed cellular barcoding- that allows the dissection of lineage relationships. We previously demonstrated that the labeling of cells with unique genetic tags coupled to a microarray-based detection system can be used to analyze family relationships between the progeny of such cells (1). Here we have used this technology to address two fundamental questions:- Do effector and memory T cells share common progenitors within the naive T cell pool (Gerlach et al., unpublished)?- Is the magnitude of T cell responses regulated by T cell recruitment or by clonal burst size (2)?
Schepers et al. J Exp Med205 2309 (2008)
van Heijst et al. Science in press0 0 (2009)
BreitbachCaroline1BellJohn2KirnDavid1
Clinical and Translational Research, JENNEREX Biotherapeutics, United States of America
Cancer Therapeutics, Ottawa Hospital Research Institute, Canada, E-mail: dkirn@jennerex.com
Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer
Session: Cancer: oncolytic viruses
Engineered viruses have been developed for cancer therapy both as non-replicating gene therapy agents and as cancer vaccines. Oncolytic viruses, in contrast, were developed to replicate within and subsequently lyse cancer cells. Clinical efficacy to date with each of these approaches has been limited by multiple factors including the inability to infect enough tumor cells in vivo locally within a tumor or systemically, and resistance of complex advanced tumors to a single mechanism-of-action (MOA). Over the last several years, however, a novel therapeutic class has emerged that combines the best features of all three approaches: targeted and armed oncolytic poxviruses. Recent preclinical and clinical results demonstrate convincingly that products from this therapeutic class can achieve highly selective and potent cancer destruction systemically through a multi-pronged MOA. Given recent clinical validation, we expect this therapeutic class to expand rapidly.
SeymourLeonardDept Clinical Pharmacology, University of Oxford, Great Britain, E-mail: len.seymour@clinpharm.ox.ac.uk
Systemic delivery of virotherapy for cancer
Session: Cancer: Oncolytic viruses
Therapeutic microbes delivered via the bloodstream encounter many host defences and anatomical barriers that must be surmounted to enable their access to disseminated cancers. This is particularly true for adenovirus type 5 in humans, where most recipients have powerful pre-existing adenovirus-neutralising activity. We have recently shown that human (but not murine) erythrocytes provide an additional barrier by sequestering adenovirus onto the Coxsackie and Adenovirus Receptor and (via antibodies) complement receptor 1. Coating adenovirus with a layer of hydrophilic polymer can prevent this interaction and allow virus to circulate free in the plasma, showing passive targeting to disseminated tumours and mediating good anticancer efficacy. Entry of polymer-coated virus particles into the tumour mass is a product of fluid transfer, and is directly proportional to the area under the plasma concentration-time curve. Increasing extravasation of fluid through tumour-associated endothelium using permeability-enhancers such as Tumour Necrosis Factor alpha can improve virus particle entry into tumours over 100-fold, reaching as high as 10% injected dose (virus particles) per tumour. This provides the possibility for highly efficient targeting to tumours and good anticancer efficacy.
Kirn, DH Nature Reviews Cancer9 64-71 (2009)
Park, B Lancet Oncology9 533-42 (2008)
GiaccaMauroMolecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
Searching for genes inducing myocardial protection, myocardial repair or neoangiogenesis by in vivo gene transfer using AAV vectors
Session: Cardiac gene therapy
The identification of novel genes and pathways promoting new blood vessel formation in ischemic conditions, controlling prenatal cardiomyocyte renewal or regulating cardiomyocyte survival during adult life holds paramount interest for the development of new therapeutic approaches for patients with ischemic cardiomyopathy and heart failure. Over the last several years, we have exploited the properties of AAV vectors to explore the biological effects and the therapeutic potential of over 50 different genes potentially useful to promote therapeutic angiogenesis and sustain myocardial function in small and large animal models of cardiovascular disorders. The genes investigated to date include various VEGF isoforms, angiopoietin1, IGF-1, Notch ICD, PDGF-B, TRAIL, Netrin 1, Islet-1 and genes involved in iPS formation.
These studies have led to the identification of a few novel regulators of cardiomyocyte function (e.g. VEGF-B) and cardiomyocyte proliferation (e.g. Notch1/Jagged1), and have disclosed the role of defined mononuclear cell populations in the regulation of blood vessel formation (e.g. NEMs, Neuropilin-1-Expressing Mononuclear cells, which are specifically recruited close to newly formed vessels by the prolonged expression of VEGF-A165). Together, these studies have indicated that the role of VEGF in the heart extends beyond angiogenic properties and point toward VEGFR-1 signaling as an important mediator of cardiomyocyte protection, with clear therapeutic implications.
HinkelRabeaBock-MarquetteIldikoKupattChristianMedizinische Klinik I, Klinikum Grosshadern, LMU, Germany
Thymosin ß4 induced neovascularization: how gene therapy may complement cell therapy
Session: Cardiac gene therapy
We have used murine embryonic endothelial progenitor cells (eEPCs, Tie-2, c-Kit+, Sca-1+, Flk-1 low, MHC-1-) in a pig model of acute ischemia (I, 1 h LAD occlusion)/reperfusion (R, 24 h). When 5x106 eEPCs were infused retrogradely at the end of ischemia (at 55 min), acute I/R injury was significantly attenuated, unless Wortmannin was co-applied (Kupatt et al., Circ 2005). Screening the eEPC transcriptome for paracrine factors, which are capable of activating PI3K-AKT, Thymosin ß4 (TB4) was identified. Indeed, retrograde application of TB4 peptide was diminishing infarct size to a similar extent as the eEPC retroinfusion (Hinkel et al., Circ 2008).
In a different pig model of chronic hibernating myocardium (reduction stent implantation), regional myocardial dysfunction depends solely on malperfusion, not infarcted myocardium. Since eEPC retroinfusion improved hibernating myocardium significantly, we tested the paracrine potential of forced TB4 overexpression to induce therapeutic neovascularization, utilizing a current cardiomyotropic AAV 2/9 vector for longterm expression. The AAV 2/9 TB4 vector or a LacZ vector was retroinfused at the 4 week timepoint. At 8 weeks, collateral count and perfusion were significantly improved in the AAV 2/9 TB4, sufficing to improve myocardial function of the hibernating region.
We conclude that an AAV 2/9 based gene therapeutic approach utilizing paracrine effectors of progenitor cells, e.g. TB4, may complement cell therapy in cases where viability, homing or survival of progenitor cells from patients with manifest atherosclerosis are limited.
ThumThomasMolecular and Translational Therapeutic Strategies, Medical School Hannover, Germany, E-mail: thum.thomas@mh-hannover.de
MicroRNAs regulate angiogenesis in the infarcted heart
Session: Cardiac gene therapy
Myocardial infarction (MI) results in the irreversible loss of cardiac muscle, triggering remodelling processes and the development of heart failure. Insufficient myocardial capillary density within the surviving myocardium after MI has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. MicroRNAs are small non-coding RNAs that regulate gene expression by binding to a group of messenger RNAs and thus represent promising therapeutic targets. We identified several microRNA to be primarily expressed in endothelial cells and endothelial progenitor cells which are upregulated following MI and/or hypoxic conditions. These miRNAs induce endothelial cell apoptosis and abolished endothelial capillary network formation. Specifically in endothelial progenitor cells, a miRNA reduced nitric oxide bioavailability resulting in impaired functional capacity. Several of the effects are mediated through targeting of the endothelial-enriched transcription factors and the respective downstream signalling cascades. In a mouse model of MI, blocking of endothelial miRNAs by systemic administration of a specific antagomir enhances angiogenesis in the infarcted heart and preserves cardiac function. Our findings indicate specific microRNAs to act as a critical regulator of endothelial cell apoptosis and angiogenesis, suitable for therapeutic intervention in the setting of ischemic heart disease.
MummeryChristineDepartment of Anatomy and Embryology, Leiden University Medical Centre, The Netherlands
Cardiovascular derivatives of embryonic stem cells in cardiac repair and drug discovery
Session: Cardiovascular System
Derivation of heart and vascular endthelial cells from human pluripotent stem cells (embryonic stem cells or HESCs and induced pluripotency stem cells or hiPS cells) is an area of growing interest both as a route to cell therapy for the heart and as a platform for drug discovery and toxicity. Understanding the underlying developmental mechanisms that control differentiation of pluripotent cells to cardiac progenitors and their derivatives and mimicking these in defined culture conditions in vitro is now essential for moving the field forward (1,2). Culture conditions have now been sufficiently refined that cardiomyocyte and vascular differentiation is a fairly efficient and reproducible process. Microarray analysis of modulations in gene expression during differentiation for example has shown that the major known cardiac genes are upregulated but that novel genes are also expressed (3). Genetically marked HESCs have been produced in which expression of the green fluorescent protein marker is retained after differentiation (4). This has permitted unambiguous tracing of cardiomyocytes following transplantation into a mouse heart (5). Long term survival of the cells and integration into the host heart has been observed and the ability of these cells to restore cardiac function in mice that have undergone myocardial infarction has been investigated. Whilst there is early improvement in hearts receiving hESC-derived cardiomyocytes compared with hearts receiving non-cardiomyocyte derivatives, this is not sustained. Cardiac repair using stem cell derived cardiomyocytes will likely require more than efficient cardiomyocytes production. More immediate applications of HESC-derived cardiomyocytes and vascular endothelial cells in drug discovery and disease are now close to implementation. Results of these studies, in particular drug responses of HESC-derived cardiomyocytes will be shown.
Itskovitz-EldorJosephMD DScDepartment of Obstetrics and Gynecology, Rambam Health Care Campus, Stem Cell Center, Faculty of Medicine, Technion POB 9602, Haifa 31096, Israel, E-mail: Itskovitz@rambam.health.gov.il
Culture and scale-up of human pluripotent stem cells in defined conditions
Session: Cell manufacturing
Human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are pluripotent cells isolated from blastocysts. Traditionally these cells have been cultured with a supportive layer in 2D culture, which allows their continuous growth in the undifferentiated state. However, any future use of these cells in the clinic or industry will require a scalable, reproducible and controlled culture system.
A suspension culture for undifferentiated pluripotent stem cells will be presented, which is either static or dynamic and includes medium consisting of serum replacement, bFGF and interleukins, mainly the IL6-Il-6 receptor chimera. Four hESC lines cultured as small spheroids maintained all typical ESC features following prolonged culture of over 50 passages (160 doublings). In addition, when applied onto a dynamic system for 10 days, the number of cell clumps increased 26-fold and cell number increased 100-fold, all the while maintaining ESC characteristics, including stable karyotype and pluripotency. The proposed suspension system is suitable for both the routine culture of hESC and iPSC in 3D and for mass production of pluripotent cells for therapeutic and industrial ends.
Institute of Medical Biology - IMB, AStar, Singapore
Department of Cardiac, Thoracic, Transplantation and Vascular Surgery - HTTG, Hannover Medical School - MHH, Germany, E-mail: zweigerdt.robert@mh-hannover.de
Up scaling generation and enrichment of pluripotent stem cell-derived cardiomyocytes
Session: Cell manufacturing
Myocardial infarction patients can survive ischemia-reperfusion injury that affects up to 30% of the left ventricle, which can translate into the irreversible loss of about 1–2 billion functional heart muscle cells. Cardiomyocytes from pluripotent human stem cell sources such as embryonic stem cells (hESC) or induced pluripotent stem cells (hiPS) hold great promise for the development of cell therapeutics but significant technical hurdles exist, particularly the efficient production of large cardiomyocyte numbers in high purity. We have progressed towards the scalable generation of cardiomyocytes in suspension culture in bioreactors. The initial protocol relied on inducing stem cell differentiation in clumps, which is a difficult-to-control method limiting reproducibility. Our current aim is to develop single cell inoculation of hESC/hiPS expansion and differentiation and translating such processes to stirred tank reactors. Prior work on mouse ESC-derived cardiomyocytes suggests that the controlled environment and optimized culture strategies in instrumented bioreactors can highly improve cell yields and reproducibility.
SensebéLuc12TarteKarin3
Etablissement Français du Sang (EFS) & Groupe d'Etude des Cellules Souches Mésenchymateuses, Tours, France
University of Tours EA3855, Tours, France
Inserm U917, Rennes, France
Release criteria and safety aspects of clinical scale mesenchymal stromal/stem cells - what is relevant?
Session: Cell manufacturing
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, simple and efficient processes are used and allow producing hundred millions of MSC in a short period of time using simple medium supplemented with either FCS+ FGF2 or platelet lysate. Due to conflicting data on the risk of transformation, the regulatory authorities required a survey of karyotype for cultivated MSC. Five out 20 different productions showed the presence of aneuploidy at the end of passage 1 (P1), targeting chromosome 5 and less consistently chromosomes 8 and 20, with no associated chromosome structural abnormalities. On these five productions with aneuploidy, 3 were from the same donor, among which 2 were obtained with FCS/FGF2 and one with platelet lysate. Abnormalities did not persist in culture along time. Presenting or not aneuploidy, all 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 human MSC whatever the process used, and suggest there is no occurrence of transformation of cultivated MSC. Finally, karyotype could not be a relevant control and a new approach of controls is needed.
BoniniMaria ChiaraExperimental Emathology, San Raffaele Scientific Institute, Italy, E-mail: bonini.chiara@hsr.it
Gene therapy for leukemia: alloreactivity and beyond
Session: Cell therapy and the role of genetic modification
Most of the antileukemic potential of allogeneic hematopoietic stem cell transplantation (HSCT) resides in alloreactivity towards patient-specific antigens, such as minor and major histocompatibility antigens by donor lymphocytes. Unfortunately alloreactive cells may result in detrimental effects such as graft-versus-host-disease (GvHD), or may be ineffective in controlling leukemias. We recently showed that, upon in vivo selective pressure by donor T cells, acute myeloid leukemia can undergo genomic rearrangements which result in loss of the patient-specific HLA haplotype and leukemia relapse after haploidentical HSCT (Vago et al. NEJM, 2009). Gene transfer technologies may allow to overcome these hurdles. In a phase I-II clinical trial enrolling 54 pts undergoing haploidentical HSCT, we showed that the transfer of a suicide gene into donor lymphocytes, allows to provide rapid and effective immune reconstitution, and control of GvHD (Ciceri, Bonini et al., Lancet Oncology 2009). By taming the toxicity of allo-HSCT the suicide gene approach renders haploidentical HSCT a feasible, safe and more effective option for cancer patients of all ages. To further increase the potency of donor lymphocytes against leukemia, we developed protocols for gene manipulation of central memory (TCM) T lymphocytes, cells that share many chracteristics with stem/progenitor cells, namely the ability to self-renew and to differentiate into a progeny of effectors, thus representing the ideal source of long-term anti-tumor immunity. We are currently exploiting this concept for a genetic editing of T cell specificities, to generate TCM lymphocytes specific for leukemic antigens, involved in oncogenic transformation, for a safe, effective and persistent treatment of leukemia.
FibbeWillem EDepartment of Immunohematology and Blood Transfusion, Leiden University Medical Center PO Box 9600, 2300 RC Leiden, The Netherlands
Mesenchymal stem cell therapy, a cellular form of immune suppression?
Session: Cell therapy and the role of genetic modification
Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have emerged as a promising therapeutic modality for tissue regeneration and repair. The interest in MSC therapy has been further raised by the observation that MSCs are able to modulate immune responses in vitro and in vivo. These properties may be used in clinical therapy in the context of allogeneic stem cell transplantation or treatment of auto-immune disorders.
MSCs are known to secrete a number of cytokines and regulatory molecules implicated in regulation of hematopoiesis. These characteristics have generated clinical interest to use MSCs to enhance hematopoietic stem cell engraftment. Indeed animal models provide experimental evidence that MSCs facilitate engraftment. In addition to providing critical growth factors, MSCs display immunosuppressive properties that might facilitate engraftment. In vitro studies with human, baboon, and murine MSCs demonstrated that MSCs suppress the proliferation of T cells induced by alloantigens or mitogens. Furthermore, MSCs have been reported to induce T cell division arrest, to inhibit the differentiation and maturation of dendritic cells, and to decrease the production of inflammatory cytokines by various immune cell populations. In line with their immuno-suppressive capacities in vitro, animal studies indicate that MSCs also display immunosuppressive capacities in vivo. Allogeneic MSCs may prolong skin allograft survival in immunocompetent baboons and may prevent the rejection of allogeneic tumor cells in immunocompetent mice. The mechanisms underlying these effects of MSCs have not been clearly identified. Although conflicting results have been reported, most studies agree that soluble factors are involved. The therapeutic application of the immunosuppressive properties of MSCs has already been exploited in the clinical setting for the treatment of acute graft-versus-host disease after allogeneic stem cell transplantation and for prevention of graft rejection after haplo-identical transplantation. Recently, studies have been initiated in auto-immune disorders, including Crohn's disease and in modulating chronic kidney rejection after kidney transplantation.
LamersCorTranslational Tumor Immunology, Department of Medical Oncology, Erasmus MC – Daniel den Hoed, Rotterdam, The Netherlands, E-mail: c.lamers@erasmusmc.nl
Retargeted effector cells for adoptive cancer immunotherapy
Session: Cell therapy and the role of genetic modification
Adoptive transfer of antigen-specific T cells has shown therapeutic success in the treatment of viral infections and metastatic cancer, in particular melanoma. T cells specific for the antigen of interest can be generated in vitro and adoptively transferred back to provide patients with large numbers of immune-competent T cells. Adoptive T cell therapy, however, is a patient-tailored treatment that is not universally applicable. Transfer of genes encoding antigen-specific receptors into T cells (i.e., genetic T cell retargeting) represents an attractive alternative to introduce tumor-specific immunity. The available tumor antigen-specific receptors comprise (i) antibody-based receptors (i.e., conventional antibodies or T cell receptor (TCR)-like antibodies that are non-MHC restricted), and (ii) natural TCRs or optimized TCRs that are MHC restricted. To date, tumor antigen-specific gene-modified T lymphocytes are tested in several preclinical tumor models and in a restricted number of clinical studies, for the treatment of a.o., renal cell cancer, melanoma and prostate cancer. The presentation will review our experience with the clinical use of autologous gene-modified T lymphocytes in cancer immunotherapy, and present both effectiveness and challenges of this experimental immunogene therapy and how challenges are currently addressed in our preclinical work.
KlatzmannDavidMaurySébastienDarrasse-JèzeGuillaumeBergotAnne-SophiePodsypaninaKatrinaLemoineFrançoisCohenJosé L.Immunology-Immunopathology-Immunotherapy; UPMC-CNRS-INSERM-AP-HP, Paris, France; *Memorial Sloan-Kettering Cancer Center, New York, USA, E-mail: david.klatzmann@chups.jussieu.fr
Regulatory T cells and cancer immunotherapy: revision of the immunosurveillance of cancer paradigm and therapeutic consequences
Session: Cell therapy and the role of genetic modification
The immunosurveillance of cancer paradigm, originating from the early 20th century - Paul Ehrlich, 1909 - postulates that cancer cells are constantly produced by our organism, and immediately rejected by our immune system; it is only when cancer cells evade this surveillance that tumors develop. This paradigm is widely accepted and propagated despite the facts that (i) the numerous additive molecular events required for transformation makes it unlikely that we are constantly producing cancer cells, and (ii) long term follow-up of immuno-compromised patients revealed an increased occurrence of only virus-induced tumors.
We recently observed1 that tumor emergence is indeed sensed by the immune system, but rather by regulatory/suppressor T cells (Tregs) than by effector T cells. Immunosurveillance exists, but is one that works to tolerate rather that reject tumors. This, and other observations showing the involvement of Tregs to imprint a tolerant tumor environment, has important therapeutic implications. This led us to implement two clinical trials2 in which we manipulate Tregs for cancer immunotherapy, and which results are very encouraging.
BaderP.1SoerensenJ.1KoehlU.1KreyenbergH.1JarischA.1WeberG.1WillaschA.1KuciS.1PonstinglE.1KrennT.2MoskovitsJ.1EsserR.1TonnT.3KlingebielT.1University Children's Hospital Frankfurt a. Main, Germany;University Children's Hospital Homburg Saar, Germany;
Institute for Transfusion Medicine, University Hospital Frankfurt a. Main
Allogeneic transplantation from alternative donors in children and young adults
Session: Cell therapy clinical trials
Introduction: We have started a prospective trial in children and adolescents with malignant and non-malignant diseases to study the engraftment rate after transplantation of CD3/CD19 depleted peripheral stem cells of haploidentical related or unrelated donors. So far 53 children and adolescents were enrolled. Patients were transplanted for ALL (n = 16), AML (n = 12), MDS (n = 3), rhabdomyosarcoma (RMA, n = 12), other solid tumors (n = 3), and non malignant diseases (n = 7). The patients received a reduced intensity regimen consisting of fludarabine, melphalan, thiotepa and OKT3 (n = 53).
Results: 53/53 patients showed primary engraftment (100%) with a median time to reach more than 1000/μL ANC and more than 20,000/μL platelets of 14 (range 14–16) and 15.5 days (range 0–22), respectively. For patients with acute leukemia (n = 28) the cumulative incidence (CI) of GVHD grade II was 0.19 and for grade III 0.04 respectively. Chronic GVHD did not occur. Event-free survival for patients with acute leukemia (n = 28) was 0.46 and for patients with acute leukemia who were in remission (n = 21) at the time of transplant was 0.60. Cumulative incidence of non relapse mortality in these patients was 0.12. When analysing for disease entities, patients with ALL (n = 12) and AML (n = 9) in remission had a pEFS of 0.5 and 0.78, respectively. All patients with ALL (n = 4) and AML (n = 3) who were not in remission at the time of transplant developed a subsequent relapse and died. All patients with non-malignant diseases (n = 7) survived.
Conclusion: The preliminary results show that rapid and sustained engraftment could be achieved using CD3/CD19-depleted peripheral allografts.
HoAnthonyMedizinische Klinik V, University of Heidelberg, Germany, E-mail: anthony_dick.ho@urz.uni-heidelberg.de
Mechanisms of stem cell mobilization for clinical transplantations
Session: Cell therapy clinical trials
In 1985 the Heidelberg group performed one of the first successful transplantations using stem cells collected from the circulation blood instead of from the marrow. Nowadays, peripheral blood stem cells (PBSC) have largely replaced bone marrow-derived cells in autologous and allogeneic transplants. The main disadvantage of PBSC is that they have to be “mobilized” from the marrow. Stem cells adhere to their bone marrow niche by interactions between SDF1α, which is expressed by bone marrow stromal cells, and CXCR4, which is expressed on CD34+ cells. G-CSF mobilizes stem cells from the marrow niche by secretion of neutrophil-associated extracellular proteases which subsequently release HSC from their niche, whereas Plerixafor, the first of a novel class of agents that directly inhibit the CXCR4-SDF1α cell-cell interaction and release stem cells into the circulating blood. In a retrospective analysis of 890 patients scheduled to receive autologous transplants in Heidelberg from 2003–2008, 108 pts (12.1%) were found to be “Poor Mobilizers” (PM). Secondary strategies to mobilize HSC from the PM who failed to achieve an adequate collection included: another cycle of induction chemotherapy+G-CSF, Plerixafor, or bone marrow harvest. In patients who received Plerixafor as a secondary attempt within this series, the goal of collecting 2.0 × 106 CD34+ cells per Kg body weight could be accomplished and all patients were transplanted successfully. Within an international trial for patients with NHL and MM, we have enrolled 31 patients from Heidelberg and have demonstrated that the combination of G-CSF and Plerixafor (G+P) resulted in a significant increase in CD34+ cell yield after apheresis and an absolute increase in the primitive subset of CD34+/CD38- cells collected. Moreover, G+P administration resulted in a rapid and sustained neutrophil and platelet engraftment of the mobilized HSC. Parallel to these clinical trials, Plerixafor has been demonstrated to mobilize leukemia stem cells from their niche in preclinical experiments.An understanding of the molecular mechanisms controlling the interactions between stem cells and their marrow niche has led to the development of a new class of therapeutics, i.e. inhibitors of CXCR4-SDF1α, that specifically mobilize more potent stem cells from the marrow into the circulating blood. Our preclinical data also indicated we could “mobilize” leukemia stem cells from their niche and render them vulnerable to chemotherapy.
Departments of Surgery, Hepatology, Haematology, and Radiology at Hammersmith Campus, Imperial College London, UK, E-mail: nagy.habib@imperial.ac.uk
Clinical application of adult bone marrow-derived stem cell progenitors
Session: Cell therapy clinical trials
Adult stem therapy could solve the problem of diseases of the liver and heart, diabetes, and stroke. The view is predicated upon the evidence that stem cells, particularly those in haematopoietic tissue, have the ability to develop into endodermal, mesodermal and ectodermal cell types.
Several sources of stem cells have been proposed as sources for cell therapy. Embryonic stem cells are the most potent, but may be tumorgenic when transplanted in vivo and their use is beset by ethical issues. Adult stem cells may be found in any tissues, but haematopoietic tissue is most accessible. From laboratory studies we identified a CD34+ subpopulation with a morphology associated to that of primitive stem cells. We used this population for a phase I safety and toxicity study of cell therapy in 5 patients with liver disease. Following granulocyte-colony stimulating factor (G-CSF) administration and a leukapheresis procedure CD34+ cells were selected and re-infused into the patient via either the portal vein or hepatic artery. No complications or specific side effects related to the procedure were observed. Three of the five patients showed an improvement in serum bilirubin and four of five in serum albumin.
A phase II study was commenced in 2006. Nine patients with alcoholic liver cirrhosis were given G-CSF followed by a leukapheresis procedure. The bone marrow-derived stem cell progenitors were expanded in vitro and reinfused into the hepatic artery. On average a 5 fold expansion in cell numbers was achieved. No treatment related side effects or toxicities were observed. There were significant decreases in serum bilirubin (p < 0.05) 4, 8 and 12 weeks post infusion. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) showed improvement through the study period and were significant (p < 0.05) one week post infusion. The Child-Pugh score improved in 7 out of 9 patients while 5 patients had improvement of ascites on imaging. The clinical and biochemical improvement in the study group is encouraging.
Currently we are investigating the therapeutic potential of this source of stem cells in patients with diabetes type 1 and a successful renal transplant, patients who have suffered acute total anterior and patients with cerebral ischaemia.
KlatzmannDavidImmunology-Immunopathology-Immunotherapy; UPMC-CNRS-INSERM-AP-HP, Paris, France, E-mail: david.klatzmann@chups.jussieu.fr
The early days of AIDS/HIV research: extraordinary times, (extra)ordinary people
Session: ESGCT presidential symposium
The identification of AIDS in the early 80ies triggered extraordinary research. Extraordinary because it immediately mixed the most intriguing, exiting, stimulating clinical and fundamental science with the most acute societal, media, money, and sex issues.
Beside the big scientific fights that ensued, and occupied the scene, this was also the occasion of formidable collaborative efforts between clinicians, researchers and patients that were the real drivers of the soon to come successes.
Lessons should be taken from these times that are pertinent to gene therapy development.
VandenDriesscheThierryVesalius Research Center, Flanders Institute for Biotechnology, VIB, University of Leuven, Belgium, E-mail: thierry.vandendriessche@med.kuleuven.be
Darwinian evolution and selection in gene therapy
Session: ESGCT presidential symposium
The principles of evolution and selection can be applied to the field of gene therapy to improve vector performance at the level of (i) vector entry (ii) transgene persistence and (iii) expression levels. (i) The limited efficiency of viral vector entry into the desired target cells can be overcome by selecting capsid variants from a library of mutants that exhibit the highest propensity for binding and entry into the desired target cells. This approach has been used successfully for the selection of AAV vectors with altered tropism, increased target cell specificity and reduced immunoreactivity. Moreover, the naturally occuring diversity of AAV serotypes also presents with unprecedented opportunities to alter vector tropism by pseudotyping. (ii). Hence, there is a need to develop robust integrating non-viral vectors for gene therapy. This has been accomplished by generating a library of Sleeping Beauty (SB) transposase mutants and subsequent selection of hyperactive SB mutants resulting in higher transposition efficiencies in clinically relevant primary target cells, including CD34+ HSC and hepatocytes. (iii) The combination of transcription factor binding sites (TFBS) is key in dictating high-level tissue-specific expression. A novel data mining algorithm was used to identify highly expressed tissue-specific promoters that were relatively enriched in these TFBS motifs and that were evolutionary conserved among divergent species. The incorporation of these conserved motifs in gene delivery vectors resulted in robust hepatocyte-specific gene expression. However, paradoxically, the principles of evolution by natural selection also account for some of the adverse events in the field. In conclusion, the concepts laid down by Darwin 150 years ago offer unique opportunities to combat disease and alleviate human suffering by gene therapy.
BainbridgeJames1RobbieScott1SmithAlexander1BarkerSusan1FitzkeFred1MooreAnthony1YzerSuzane2van den BornIngeborgh2RubinGary1AliRobin1
UCL Institute of Ophthalmology and Moorfields Eye Hospital Biomedical Research Centre for Ophthalmology. London, UK
Rotterdam Eye Hospital, The Netherlands, E-mail: r.ali@ucl.ac.uk
Clinical trial of gene therapy for inherited retinal dystrophy caused by RPE65 deficiency
Session: Eye and brain
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 young adults and children. We administered subretinally a rAAV-2/2 vector expressing RPE65 cDNA under the control of a human RPE65 promoter. Recently we have increased the vector dose from 1e11vg to 1e12vg. Examination of systemic vector dissemination, immune responses, electrophysiology, retinal imaging and detailed psychophysical assessments of visual function suggest that subretinal administration 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.
AuricchioAlbertoTelethon Institute of Genetics and Medicine (TIGEM), Dept. of Pediatrics, Federico II University, Italy, E-mail: giovannini@tigem.it
Advancements in AAV-mediated retinal gene transfer for inherited retinal diseases
Session: Eye and brain
Gene delivery represents a promising strategy for treatment of inherited blinding retinal diseases. The eye is enclosed, immune-privileged and small, thus requiring low amount of vectors for efficient transduction. The most promising gene transfer system for the retina derives from the small adeno-associated virus (AAV). More than 100 AAV serotypes with different capsids have been isolated and many have been converted in gene transfer vectors. We observed that capsids are the main determinants of AAV vector transduction and we described the retinal transduction characteristics of several AAV serotypes, including novel ones as they become available. The combination of the appropriate AAV serotype, intraocular administration route and regulatory elements allows to efficiently target gene expression to specific cells in the retina of small and large animals of retinal diseases, including retinitis pigmentosa, albinism, Stargardt's disease and age-related macular degeneration. After a decade of successful proof-of-principle of AAV-mediated gene transfer in animal models of retinal diseases, the first clinical trials testing the safety and efficacy of AAV-mediated gene transfer to the retina of patients with Leber Congenital Amaurosis, a rare form of childhood blindness, have recently started. Results one and half year after vector administration look extremely promising.
MBB Molecular Neurobiology, Karolinska Institute Stockholm, Sweden
Howard Florey Institute, Carlton South Victoria, AustraliaUniversidad Autonoma, Madrid, SpainUniv de Castilla La Mancha, Albacete, Spain, E-mail: ernest.arenas@ki.se
Enhanced dopaminergic differentiation of neural and embryonic stem cells by Wnt5a
Session: Eye and brain
Parkinson's disease has been identified as a priority target for cell replacement therapy because: (a) Neural degeneration is mainly circumscribed to midbrain dopaminergic neurons, and (b) grafting of human ventral midbrain fetal tissue, under specific conditions, improve motor symptoms and reduce the need of L-DOPA in parkinsonian patients. Embryonic and neural stem cells have been suggested as adequate cellular sources for dopamine cell replacement therapy because of their capacity to generate large numbers of midbrain dopaminergic (DA) neurons. However, several challenges need to be overcomed in order to develop stem cells as a clinical tool for cell replacement therapy. These challenges include the need to achieve homogenous, correctly specified, functional midbrain DA neurons; and to improve safety, by eliminating the risk of tumor formation. Efforts to elucidate the cellular mechanisms and molecular programs that regulate both processes have lead to the identification of factors such as Wnt5a, which reduce excessive proliferation and enhance DA differentiation in vitro and in vivo (1,2). Moreover, transplantation of Wnt5a-treated rodent midbrain neural stem/progenitor cells enhanced anatomical and behavioral recovery of parkinsonian mice, in the absence of tumors (1). We now examine whether Wnt5a can exert similar functions on the DA differentiation of embryonic stem cells. Our results indicate that Wnt5a improves the efficiency of protocols for the in vitro DA differentiation of both mouse and human embryonic stem cells. Our data suggest that Wnt5a may contribute to facilitate the development of embryonic stem cell replacement therapies for Parkinson's disease.
Parish Journal of Clinical Investigation118 149–160 (2008)
Andersson PLoS ONE3 3517 (2008)
FerrariGiulianaSan Raffaele Telethon Institute for Gene Therapy, Fondazione Centro San Raffaele del Monte Tabor, Italy, E-mail: giuliana.ferrari@hsr.it
Preclinical assessment for beta-thalassemia major demonstrates efficacy and safety of lentiviral mediated gene transfer
Session: Focus: gene therapy of globinopathies
Beta-thalassemia is a common monogenic disorder due to mutations in the beta-globin gene and gene therapy, based on autologous transplantation of genetically corrected hematopoietic stem cells (HSCs), holds the promise to treat all the patients lacking a compatible allogeneic bone marrow (BM) donor. We recently showed long-term correction of murine beta-thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector, expressing a transcriptionally regulated human beta-globin gene (Miccio et al., PNAS, 2008). Here, we report successful correction of thalassemia major in human cells, by studying a large cohort (n = 43) of pediatric patients of diverse ethnic origin, carriers of different beta0 and beta+ mutations and all candidates to BM transplantation. Extensive characterization of BM-derived CD34+ cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of HbA synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of hematopoietic progenitors. Gene expression program of treated CD34+ cells is not specifically altered in thalassemic samples with respect to normal ones. Analysis of vector integration sites shows preferential targeting of transcriptionally active regions, without bias for cancer-related genes. Overall, these results provide a solid rationale for the translation to clinical application of GLOBE lentiviral vector.
AntoniouMichaelKaoVincentFerreiraSoniaNuclear Biology Group, King's College London School of Medicine, Department of Medical and Molecular Genetics, Guy's Hospital, London, UK, E-mail: michael.antoniou@genetics.kcl.ac.uk
Novel designs for improving reproducibility and levels of β-globin production from lentiviral vectors for targeting the haemoglobinopathies
Session: Focus: gene therapy of globinopathies
The high level and erythroid specific expression of the human β-globin gene is mediated by the locus control region (βLCR) working cooperatively with local gene promoter and enhancer elements. The βLCR consists of 5 elements (HS1/HS2/HS3/HS4/HS5) distributed over a 16kb interval, possesses a dominant chromatin remodelling and transcriptional activating capability and thus able to confer on integrated transgenes full physiological levels of expression that are proportional to copy number. Given its large size, by necessity all LVs constructed to date incorporating the βLCR contain only “microlocus” versions of this element (μβLCR) combining in most cases small fragments encompassing the most transcriptionally active HS2, HS3 and HS4 sites with a total size of approximately 2.5-3.5kb. Although a number of μβLCR + mini-β-globin gene LVs has been shown to successfully rescue the disease phenotype in mouse models of β-thalassaemia and sickle cell anaemia, it is evident that the μβLCR is, not surprisingly, far less potent a transcriptional activator than the complete element. All versions of LVs with μβLCR cassettes even when flanked by the cHS4 insulator element, show a high variability in expression between different integration sites with data suggesting that at least 50% of vector integration events will fail to express at a therapeutic level. As a result, current generation μβLCR LVs can only achieve reproducible therapeutic levels of β-globin production when present at an average of two vector copies per cell and in some cases higher depending on vector design. This level of μβLCR LV performance clearly compromises both efficacy and safety. This presentation will introduce our efforts to address these problems at (i) a transcriptional level by augmenting μβLCR function with the ubiquitous chromatin opening element (UCOE) from the human HNRPA2B1/CBX3 locus and (ii) at a post-transcriptional level by promoting more efficient pre-mRNA 3′end processing with the β-globin transcriptional termination region.
LeboulchPhilippeM.D.123the LentiGlobin clinical trial study group
CEA, Institute of Emerging Diseases and Innovative Therapies (iMETI), Fontenay-aux-Roses 92265, France
Inserm U962 and University Paris XI, CEA-iMETI, Fontenay-aux-Roses 92265, France
Genetics Division, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02115, USA, E-mail: pleboulch@rics.bwh.harvard.edu
Conversion to Transfusion Independence with Partial Clonal Dominance after Lentiviral Gene Therapy for Severe Human Beta-Thalassemia
Session: Focus: gene therapy of globinopathies
An 18 year old male with severe βE/β0-thalassemia and no HLA-matched sibling donor was transplanted after Busulfex-mediated myeloablation with autologous bone narrow CD34+ cells transduced ex vivo with a lentiviral vector expressing a marked βA-T87Q-globin gene. Before transplantation, the patient was dependent on monthly transfusions since age 3 (2 to 3 RBC packs each time; 157 ml RBCs/kg the year before transplant) with growth retardation and spontaneous hemoglobin (Hb) levels between 4 and 6 g/dL, splenectomized since age 6, and under iron chelation therapy since age 8. Hydroxyurea therapy was ineffective. Twenty five months after gene therapy, Hb levels are between 9.3 and 9.9 g/dL, of which up to 3.7 g/dL contains βA-T87Q-globin, the remainder being HbE and HbF. No transfusion has been provided for the last 13 months, and there is near physiological levels of βA-T87Q-globin expression on a per gene basis. Half of the therapeutic effect derives from a partially dominant cell clone with vector insertion within the HMGA2 gene, although βA-T87Q-globin expression output shows little position dependency. While hematopoietic homeostasis is currently maintained, this observation together with recent results of deep sequencing analysis of other gene therapy clinical trials question whether therapeutic potency can be entirely dissociated from vector-mediated genomic effects.
DropulicBoroKeefeRobertBaranyiLajosFisherMichaelBio Therapeutics, Lentigen Corporation, United States of America, E-mail: nina.weddle@lentigen.com
Virus like particle production for H5N1 influenza using lentiviral technology
Session: Genetic vaccines
Traditional egg-based Influenza Live Virus, inactivated vaccines have major limitations and drawbacks, particularly for use in a Pandemic setting. High Pathogenicity Avian Influenza (HPAI) viruses, such as the H5N1 strains currently circulating in Asia, are too virulent to grow in eggs, and require a biohazard containment facility to handle. Lentigen has developed a fast, recombinant and highly immunogenic Virus-Like Particle (VLP) vaccine for H5N1 Influenza, based on the use of lentiviral technology to permanently transduce 293 cells to produce wholly Influenza VLPs. One of the advantages of the system is the continuous production of VLPs into the supernatant of medium, obviating batch production methods and reducing the scale and therefore expense for vaccine production. Influenza A H5N1 Vietnam 1203 Matrix 1, Hemagglutinin 5 (HA5), and Neuraminidase (NA1) genes were engineered into a lentiviral vector as a single genetic element, and the vector used to transduce HEK 293 cells. A cell line containing 25 copies per cell was selected and grown at high density, producing VLPs that budded into the supernatant. Concentrated supernatants were harvested and tested for the presence of the M1, HA, and NA proteins. M1 and HA were detected by Western Blot. HA was detected by ELISA and RBC agglutination, while NA was detected by ELISA and the Neuraminidase Umberifyll assay.
EPIXIS S.A., F-69007 Lyon and F-75013 Paris, France
Université de Lyon, UCB-Lyon1, IFR128; INSERM, U758; ENS de Lyon, F-69007 Lyon, France.CEA, F-92265 Fontenay aux Roses, FranceUPMC Univ Paris 06, UMR 7211, Paris, F-75013 Paris, France.Institut Pasteur, CNRS, URA3015, LVGV, F-75015 Paris, France, E-mail: cd@epixis.com
RetroVLP-based vaccine candidates against Hepatitis C induce both cellular and broadly neutralizing humoral immune responses
Session: Genetic vaccines
Background: The disease burden and public health impact of chronic HCV infection continues to be a major problem; more than 170 million people worldwide are chronically infected by HCV, which is the causative agent of chronic hepatitis C, cirrhosis, and finally liver cancer. Current treatment is not effective in all patients and is frequently associated with unacceptable side effects. Clearly, a need exists for improved treatments and one such strategy is the use of therapeutic vaccines.
Technology: The expression of Moloney murine leukemia virus (Mo-MLV) gag proteins is sufficient to generate retrovirus-like particles (retroVLPs). These can be used as antigen-display platforms by pseudotyping with heterologous envelope proteins, or by insertion of epitopes in structural constituents. In the present study, we investigated the use of such retroVLPs, pseudotyped with the HCV envelope proteins E1 or E1E2, as hepatitis C immunogens. Aiming to improve the E1 responses, wild type E1 and modified E1 (E1G) were compared. Furthermore, aiming to circumvent in vitro production of the retroVLPs, their immunogenicity was compared with DNA plasmid vectors expressing the retroVLPs in vivo (plasmo-retroVLPs). Experiments were conducted in mouse and macaque.
Results: Data obtained in mouse and macaque were coherent and consistent. RetroVLPs induced both cellular and humoral immune responses, including broadly cross-neutralizing responses, as demonstrated in both pseudotyped particles or cell cultured virus assays. Furthermore, through the use of retroVLP-E1G or retroVLP-E1E2, induction of E1 and/or E2 specific antibody responses could be steered and unprecedented E1 responses could be induced. Importantly, in vitro-produced retro-VLPs and plasmo-retroVLPs induced responses of similar magnitude.
Conclusion: RetroVLPs pseudotyped with HCV envelope proteins represent a promising Hepatitis C vaccine candidate. Our plasmo-retroVLP-E1G represents a unique, easy to produce, vaccine candidate that is now entering preclinical development.
SchroederTimmInstitute of Stem Cell Research, Helmholtz Zentrum München, Germany, E-mail: timm.schroeder@helmholtz-muenchen.de
Tracking stem cells at the single cell level: New tools for old questions
Session: Hematopoiesis
The hematopoietic system is the classical mammalian stem cell system. It constantly maintains the right number and types of blood cells in our body. Throughout lifetime, enormous numbers of cells of the more than 10 different blood lineages are being produced from hematopoietic stem cells (HSC). Despite intensive research, many long-standing questions in hematopoiesis research remain unsolved. One major reason is the fact that hematopoiesis usually is followed by analyzing the fate of populations of cells - rather than individual cells - at very few time points of an experiment, and without knowing their individual identities. Real-time tracking of individual cells in culture, tissues or whole organisms would be an extremely powerful approach to fully understand the developmental complexity of hematopoiesis. However, many required tools are still under development and their application remains difficult. We have developed a computer aided culture and imaging system to follow the fate of individual cells over long periods of time. New software was programmed, helping to record and display the divisional history, position, properties etc. of all individual cells in a culture over many generations. I will discuss how we used this system to answer long standing questions in hematopoiesis research.
BoztugKSchmidtMSchwarzerABanerjeePPAvedillo DíezADeweyDBöhmMNaundorfSKühlckeKBlasczykRKondratenkoIMaródiLOrangeJvon KalleCKleinCDepartment of Pediatric Hematology/Oncology, Hannover Medical School, Germany, E-mail: klein.christoph@mh-hannover.de
HSC Gene Therapy in Two WAS Patients
Session: Hematopoiesis
Wiskott-Aldrich syndrome (WAS) is a complex primary immunodeficiency disorder associated with thrombocytopenia, eczema, autoimmunity and increased risk of malignancies. We here report our experience in two WAS patients treated by transfusion of autologous, genetically modified hematopoietic stem cells (HSCs). We demonstrate sustained WASP expression in HSCs, lymphoid and myeloid cells, and thrombocytes up to 2.5 years after gene therapy. T lymphocytes, natural killer (NK) cells, and monocytes were functionally corrected, as shown by in vitro assays. B cells produced specific antibodies after vaccination in vivo. The patients' clinical condition markedly improved with respect to hemorrhagic diathesis, eczema, autoimmunity, and predisposition to severe infections. Genome-wide molecular insertion site analysis demonstrated targeting of multiple genes controlling growth and differentiation and sustained polyclonal hematopoiesis. Preliminary results in 6 additional patients will also be presented. Our data provide proof-of-principle that HSC gene therapy for WAS is feasible and effective at correcting platelet dysfunction, autoimmunity, and susceptibility to infection.
AdjaliOumeyaUMR649, INSERM, France, E-mail: oumeya.adjali@univ-nantes.fr
Induction of tolerance to the transgene after AAV-based gene transfer to the skeletal muscle of nonhuman primates
Session: Immunity and tolerance
Recombinant adeno-associated virus (rAAV) provides a clinically relevant platform for efficient and sustained gene therapy. However, recent gene transfer studies into animal models and humans indicate that the risk of transgene and/or capsid-specific immune responses is not negligible and depends on multiple factors. Among them, the route of vector delivery is an important one although poorly addressed in large animal models. The intramuscular (IM) route is often associated to immunotoxicity. We will discuss this issue in the model of nonhuman primate (NHP). As an alternative to the classical IM route, we developed a regional intravenous (RI) delivery protocol in the macaque and monitored the host immune response towards the transgene. In contrast to the IM route, the RI delivery of the rAAV allowed stable expression of the transgene. Cell-based immunomodulation strategies to prevent anti-transgene immune responses will be also addressed. Tolerogenic dendritic cell (DC)-based protocols have not been yet explored in NHP gene transfer studies. We have generated immature macaque bone marrow derived DC (iBMDC) and characterized their in vitro immunomodulatory effects. Preliminary results on the ability of these cells to induce in vivo transgene tolerance in the macaque following IM delivery of rAAV will be presented. Altogether, these strategies may contribute to the development of optimal and safe rAAV-based gene transfer protocols to patient's skeletal muscle.
BarkerNickClevers Group, Hubrecht Institute, Netherlands, E-mail: n.barker@niob.knaw.nl
Lgr5 stem cells in self renewal and disease
Session: Liver, intestine, pancreas
We recently showed the Wnt target gene, Lgr5, to be specifically expressed on crypt-base columnar cells located at the base of the intestinal crypts. Using in-vivo lineage tracing we have proven these cells to be the stem cells of the small intestine and colon (Barker et al. (2007) Nature 449:1003). Using a similar strategy we also demonstrated that Lgr5 marks cycling stem cells in the hair-follicle (Barker et al. (2008) Nature Genetics 40:1291). Ongoing lineage tracing experiments in the stomach and mammary gland strongly indicate that Lgr5 is also a bona-fide marker for adult stem cell populations in these tissues. Using Lgr5-EGFP-ires-CreERT2 mice to selectively induce deletion of the APC tumor suppressor gene in the intestinal stem cells we recently proved that these Lgr5+ve stem cells are the cells-of-origin of colon cancer (Barker et al. (2009) Nature 457: 608. Epub 2008 Dec 17). This work revealed the presence of a minor population of Lgr5+ve cells within intestinal tumors, which are likely to be stem cells fuelling the growth of the cancer (the cancer stem cells). Inducible deletion of both Lgr5 and the closely related gene Lgr4 in the intestine causes rapid stem cell loss and subsequent crypt death, demonstrating a partially redundant role for these orphan GPCR's in maintaining intestinal stem cell viability.
Pulmonary, Allergy and Critical Care Medicine, Duke University, United States of America
Human Vaccine Institute, Duke University, United States of America, E-mail: roxana.teisanu@duke.edu
Immunophenotypic subsetting of functionally distinct lung epithelial progenitor cells
Session: Lung replacement and regeneration 2
Epithelial injury followed by defective repair is an important component of several lung diseases. The repair process largely depends on proliferation and differentiation of endogenous epithelial stem cells. Mouse bronchiolar stem cells have been identified in vivo based on functional characteristics including naphthalene resistance, long-term retention of labeled DNA precursors, and dual expression of markers for airway (CCSP) and alveolar (pro-SPC) epithelium. We determined that Epithelial Cell Adhesion Molecule (EpCAM) and Integrin α6 are expressed on the cell surface of alveolar and bronchiolar epithelial cells, and that low levels of Sca-1 expression characterize the bronchiolar epithelium. Within the Sca-1low EpCAMpos Integrinα6pos population of bronchiolar epithelial cells, autofluorescence (AF) levels distinguish the facultative transit-amplifying population (AFhi) from bronchiolar stem cells (AFlow). Use of transgenic animal models allowing expansion or depletion of the stem cell pool and lineage tracing allowed us to determine the identity of cells isolated based on their cell surface phenotype and autofluorescence characteristics. Injury models were used to validate the functional characteristics of the two fractions of bronchiolar progenitors. In conclusion, we have developed and validated a fractionation approach for the generation of enriched preparations of bronchiolar stem and Clara cells from the mouse lung. These data enable establishment of robust in vitro and transplantation assays to further validate the functional behavior of stem and facultative TA (Clara) cells and allows analysis of gene expression profile of the two populations towards a better understanding of unique features of the bronchiolar stem cell compartment.
Department of Cardiac-, Thoracic-, Transplantation and Vascular Surgery, LEBAO, Hannover Medical School, Germany
Endothelialization of poly 4-methyl-1-pentene (PMP) gas exchange membranes towards bioartificial lung development
Session: Lung replacement and regeneration 2
Polymeric materials are widely used in biomedical devices and bioartificial organs with blood contacting surfaces. To maintain their functionality these devices should completely prevent the activation of the coagulation system and subsequent clot formation, thus the need for materials with improved blood compatibility is high. Surface endothelialization is considered an important tool to optimize the blood compatibility of biomaterials because a functional endothelial cell layer on an artificial material may be able to control hemostasis and therefore provide a solution to improve the biocompatibility of these materials. Here we report on endothelialization of polymethylpenthene (PMP) gas exchange membranes used in membrane oxygenators and artificial lung systems using human cord blood derived endothelial cells (hCBEC). We achieved complete endothelialization of PMP membranes with the hCBEC wich could be maintained for several weeks. Apart from the generation of a complete endothelial monolayer onto the surface of a biomaterial, biological functionality of seeded cells is a prerequisite for the desired contribution to the performance of the endothelialized surface. We found that the cells maintained their endothelial characteristics and functionality while cultivated on the PMP membranes. Endothelialization resulted in significant lower platelet adhesion and activation compared to unseeded membranes. Of importance, the endothelial layer had no mayor impact on gas permeability of PMP membranes. This study is a first promising step towards the development of a biofunctionalized surface for the use in blood contacting medical devices, particularly in artificial lungs.
CossuGiulioDivision of Regenerative Medicine, San Raffaele Scientific Institute, Italy, E-mail: cossu.giulio@hsr.it
Towards a cell therapy for Duchenne Muscular Dystrophy
Session: Muskulo skeletal system
Mesoangioblasts are recently characterized progenitor cells, associated with the vasculature and able to differentiate into different types of solid mesoderm, including skeletal muscle (1). When both wild type or dystrophic, genetically corrected, mesoangioblasts were delivered intra-arterially to dystrophic muscle of α-sarcoglycan KO mice (a model for limb girdle muscular dystrophy), they resulted in a significant functional amelioration of the dystrophic phenotype (2). Intra-arterial delivery of wt mesoangioblasts, non DLA matched to GRMD dystrophic dogs resulted in a partial recovery of muscle morphology and function, dystrophin expression and clinical amelioration. Delivery of autologous mesoangioblasts expressing human micro-dystrophin did not cause a comparable amelioration, despite widespread micro-dystrophin expression (3). Human adult mesoangioblasts were isolated and expanded in vitro from muscle biopsies: they were shown to correspond to a subset of pericytes (4). Based on these results, a monocenter, prospective, non-randomised, clinical phase I study of cell therapy with HLA-matched donor human mesoangioblasts in DMD patients started in June 2009, with a one year preliminary study (involving 30 DMD patients, aged 5–10), required to validate outocome measures. Three out of these patients will undergo successive intra-arterial transplantations at escalating doses of cells under a continuous regime of immune suppression. Safety will be the primary objective of the study. However it is expected that transplantation of mesoangioblasts will result in a detectable increase in muscle strenght and consequent clinical amelioration or stabilization.
FryeMichaelaWellcome Trust Centre for Stem Cell Research, University of Cambridge, UnitedKingdom, E-mail: michaela.frye@cancer.org.uk
The RNA methyltransferase Misu (NSun2) mediates functions of Myc in normal skin and cancer
Session: Neural and ectodermal stem cells
The proto-oncogene c-Myc functions as an important regulator for stem cell maintenance and differentiation in normal tissues. Activation of Myc in skin induces stem cells to exit the stem cell compartment, increases proliferation of progenitor cells and subsequently leads to lineage-specific differentiation into sebaceous glands and interfollicular epidermis. One important down-stream target gene of Myc is the RNA methyltransferase Misu (Nsun2). Misu mediates Myc-induced proliferation in primary human keratinocytes and squamous cell carcinomas. Here, we demonstrate that Misu exerts its effect on cell growth in cancer cells by stabilizing the mitotic spindle. Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA-protein complex and the presence of both RNA and Misu is required for correct spindle assembly. Misu mediates its function at the spindle by recruiting Nucleolar and Spindle-Associated Protein (NuSAP), an essential microtubule-stabilizing and bundling protein. To determine the function of Misu in healthy skin, we analysed a loss-of-function mouse model and catalytic dead mutants of Misu and demonstrated that Misu stimulates terminal differentiation of epidermal cells. In conclusion, Misu represents an important down-stream effector of Myc in controlling epidermal stem cell differentiation and cancer.
Minasi Development129 2773 (2002)
Sampaolesi Science301 487 (2003)
Sampaolesi Nature444 574 (2006)
Dellavalle Nature Cell Biology9 255 (2007)
KempermannGerdGenomics of Regeneration, CRTD - Center for Regenerative Therapies Dresden, Germany, E-mail: gerd.kempermann@crt-dresden.de
Activity-dependent regulation of adult neurogenesis in vivo and neural stem cells in vitro
Session: Neural and ectodermal stem cells
In the two neurogenic regions of the adult brain, the hippocampus and the olfactory bulb, neural precursor cells lifelong generate new neurons. This adult neurogenesis is regulated by behavioral activity, most notably voluntary physical exercise and the exposure to complex environments. One of the key questions in the field is, how this macroscopic activity is translated to the precursor cells themselves. Neural precursor cells can respond to excitation in vitro and translate this stimulus into a developmental program. More specifically, as demonstrated in co-culture models, the precursor cells can sense synchronous network activity. The effects of the neuronal network onto the precursor cells are partly mediated by BDNF. Presumably, as recent results about the role of the immune system in the control of adult neurogenesis suggest, additional pathways involve immune cells and secreted cytokines.
CathomenToniExperimental Hematology, Hannover Medical School, Germany, E-mail: toni.cathomen@gmail.com
Targeted genome engineering by zinc-finger nucleases
Session: Persisting transgenes
Recent advances in generating customized zinc-finger nucleases (ZFNs) hold great promise to pave the way for genome engineering strategies in human gene therapy. ZFNs consist of an artificial DNA-binding domain fused to a non-specific nuclease domain. Upon binding to the target site, ZFNs introduce a DNA double strand break at a pre-selected site in the human genome. The activated cellular DNA repair pathways can be harnessed subsequently to disrupt a gene (gene knockout) or to correct an inborn mutation in the genome (gene targeting). Gene editing in up to 50% of treated cells in the absence of selection demonstrate the power of this emerging technology and is high enough to contemplate clinical applications. In this educational talk the basics of the ZFN technology will be explained, including engineering platforms and assays to evaluate the activity and toxicity of ZFNs. Furthermore, some of the remaining hurdles, such as the low frequency of ZFN-mediated gene targeting in human somatic stem cells and iPS cells as well as the assessment of ZFN specificity will be discussed.
Max Delbrück Center for Molecular Medicine, Berlin, Germany
Department of Human Genetics, University of Aarhus, Aarhus C, Denmark
Flanders Institute for Biotechnology (VIB), Vesalius Research Center, University of Leuven, Leuven, Belgium
Transposons as nonviral, integrating gene vector systems
Session: Persisting transgenes
DNA-based transposons are natural gene delivery vehicles that integrate into the chromosomes of host cells. We compared the Sleeping Beauty (SB), piggyBac and Tol2 transposons with respect to overall activity, target site selection and transgene copy number as well as long-term expression in human cells. SB was the most efficient system under conditions where the availability of the transposon DNA is limiting the transposition reaction, including hard-to-transfect hematopoietic stem/progenitor cells. All three systems provided long-term transgene expression in human HeLa cells with minimal signs of silencing. We have generated large numbers of SB insertions in K562 cells by transduction with an integrase-defective lentivirus/SB hybrid vector. Recovery of the transposon insertions by LAM-PCR and high-throughput sequencing on the Solexa platform allowed us to unambiguously map >8000 unique SB insertions. The data show random genomic insertion of SB with respect to RefSeq genes, which represents a significant advantage over commonly used integrating viral vectors. Nevertheless, random genomic insertion can lead to genotoxic effects due to mutagenesis of cellular genes. We manipulated SB's target site selection in order to achieve target-selected transposition into predetermined loci or chromosomal regions. To that end, we designed chimeric targeting proteins, in which the module responsible for target DNA binding can be a natural DNA-binding protein or domain (such as the tetracyclin repressor or the DNA-binding domain of the Adeno Associated Virus Rep protein), or an artificial protein such as a designer zinc finger. We provide proof of principle for target-selected transposition of SB.
Scientific Direction, Cellectis Genome Surgery, France
Development, Cellectis Genome Surgery, France
Scientific Direction, Cellectis SA, France, E-mail: paques@cellectis.com
Meganucleases for gene therapy
Session: Persisting transgenes
Most current gene therapy approaches for monogenic inherited diseases rely on gene transfer, by random integration into the genome of patient's cells, of a functional copy of the mutated gene. However, a growing interest for targeted approaches, ranging from the targeted insertion of the functional gene into a chosen locus (“safe harbour” strategy) to the precise editing of the deleterious mutation (gene correction), is manifest. Such targeted approaches include the use of very specific endonucleases that can induce high frequencies of homologous gene targeting in the vicinity of their cleavage site. Meganucleases, the most specific natural endonucleases represent ideal tools for targeted approaches, and we engineered tailored meganucleases recognizing human genes, such as XPC and Rag1, or viruses. Highly efficient gene targeting could be induced in human cells. However, meganucleases could also represent a new class of antiviral agents. The majority of current antiviral treatments are based on the hindrance of productive viral replication by agents that inhibit virally encoded proteins. Many chronic viral infections are due to double-stranded DNA viruses or viruses that involve a double-stranded DNA intermediate during their replicative cycle. Meganucleases could cleave and either partially excise or eliminate viral DNA from infected cells. We have shown that the expression of the I-SceI meganuclease can prevent the infection with a recombinant Herpes Simplex Virus (HSV) containing the endonuclease recognition site. In addition, we have explored the antiviral potential of redesigned meganucleases. We are currently conducting a survey of the properties of these proteins in vivo.
von KalleChristofNational Center for Tumor Diseases Heidelberg (NCT), Germany
Efficient pharmakodynamics studies in clinical gene therapy
Session: Persisting transgenes
The therapeutic benefit for patients undergoing gene therapy with integrating retroviral vectors could impressively demonstrated in a variety of monogentic inherited diseases. The occurrence of unwanted clonal proliferation and oncogenesis in various patients elicited intense and indispensable efforts to determine the integration profiles of the vectors used, to assess vector biosafety and to follow the clonal fate of gene-modified cells in vivo. To ensure efficient pharmacodynamics studies in clinical gene therapy trials, we have developed novel bioinformatical and practical tools for comprehensive (LAM-PCR mediated) integration site analyses and next generation sequencing technologies (GSFlx and GSFlx Titanium; 454/Roche). We have created a precise model for determining the genomic accessibility of viral integrations by annotating the human genome with recognition motifs of each frequently cutting restriction enzyme, including terms in which it is technically feasible to amplify, sequence and map viral integration sites. Most importantly, our modelling defines a priori optimal enzyme combinations for distinct vector - cell gene transfer settings on the genome and subgenome level. A newly developed non-restrictive (nr) variant of the LAM-PCR is able to identify all existing integration sites present in a transduced sample. Both, LAM-PCR and nrLAM-PCR have been efficiently combined with the GSFlx sequencing system and downstream automated data mining, aiming to reach highly efficient and first ever comprehensive and quantitative IS assays. Thus, genom-wide comprehensive integration site sequencing allow efficient pharmacodynamics studies in preclinical and clinical gene therapy trials where integrating vectors systems have been used.
RudolphCarstenPediatrics, Ludwig Maximilians University, Germany, E-mail: carsten.rudolph@med.uni-muenchen.de
Transcript replacement therapy for the treatment of inherited lung diseases
Session: Physicochemical platforms
Several clinical studies have shown proof-of-principle for gene replacement therapy of the lung. However, neither treatment with viral nor nonviral vectors resulted in the ultimate cure of patients. Major obstacles are safety concerns associated with the use of viral vectors including either immunogenicity risks which may exclude their re-administration or genotoxicity risks which may lead to tumor formation. The use of nonviral vectors is predominantly limited by their low gene transfer efficiency. Here, we investigated whether transcript replacement therapy by messenger RNA (mRNA) delivery may be used as an alternative to achieve therapeutic expression levels in the lung in vivo. We demonstrate for the first time that transcript replacement therapy by mRNA delivery leads to therapeutic expression levels in the lungs. Using aerosolized mRNA coding for human Surfactant Protein B (SP-B), we achieved protection of SP-B deficient mice from respiratory failure and death, together with maintenance of normal lung function in the absence of lung inflammation. Therefore, we believe that transcript replacement therapy by mRNA delivery bears great potential as an alternative to commonly used gene vectors and should be considered as new option for treating inherited lung diseases.
WagnerErnstPharmaceutical Biotechnology, University of Munich, Germany, E-mail: erwph@cup.uni-muenchen.de
‘Synthetic viruses’ - Dynamic polymer nanosystems for therapeutic pDNA and RNA delivery
Session: Physicochemical platforms
In gene therapy and related therapeutic nucleic acid delivery, the different tasks at the different extra- and intra-cellular locations ask for dynamic carriers. Like natural viruses, carriers have to be bioresponsive, sensing their current environment and reacting by facilitating the next delivery step. For this purpose polymers can be equipped with molecular sensors (such as sensitive chemical bonds or conformations) able to respond to applied physical stimuli (e.g. hyperthermia, light) or endogenous biological triggers (e.g. in the endosome or cytosol) [1]. PEG shielding together with receptor targeting may enhance specificity and efficacy of the initial delivery step but hamper subsequent steps. pH-labile bonds can be incorporated to trigger cleavage of PEG in endosomes [2,3]. EGFR targeted polyplexes reversibly shielded with endosomal-labile PEG mediated enhanced tumor specific gene transfer in tumor-bearing mice as compared with stably shielded polyplexes [3]. Dual-responsive siRNA polymer conjugates were designed for enhanced extracellular stability and intracellular release [4].
SchölerHans R.Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, 48149, Germany
Induction of pluripotency in adult stem cells: iPS, piPS and gPS
Session: Pluripotent cells
Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, was first described for fibroblasts and initially required the introduction of the virally expressed transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Recently, pluripotent stem cells could be induced using recombinant proteins (1). We previously reported that Oct4 alone is sufficient to directly reprogram adult mouse neural stem cells (NSCs) to iPS cells (2). In my talk I will present the generation of one-factor (1F) human iPS from human fetal NSCs (1F hNiPS) by ectopic expression of OCT4 alone (3). 1F hNiPS cells resemble human embryonic stem cells (ESCs) in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is also sufficient to reprogram human NSCs to pluripotency. In addition, I will show that mouse pluripotent stem cells can be derived from established adult unipotent stem cell lines without virally expressed transcription factors. Germline stem cells (GSCs) are unipotent cells of the testis capable of self-renewing and of giving rise to sperm. GSCs express endogenous levels of Oct4 and Klf4 like ESCs, while they express lower levels of Sox2 and c-Myc. Genome-wide gene expression profiling demonstrates that GSCs are more closely related to ESCs than other cell types that had been previously reprogrammed, which puts germline pluripotent stem (gPS) cells converted from GSCs even closer to ESCs than iPS cells. gPS cells were also derived after clonal expansion from single GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation analyses, including germ cell contribution and germ cell transmission. We also showed that functional cardiomyocytes and neural cells could be derived by in vitro differentiation of gPS cells. Various systems to derive pluripotent stem cells will advance the field further towards understanding reprogramming and generating patient-specific cells.
Wagner E. Expert Op. Biol. Ther.7 587–593 (2007)
Knorr V. et al.Pharm. Res.25 2937–2945 (2008)
Fella C. et al.Eur. J. Pharm. Sc.34 309–320 (2008)
Meyer M. et al.Mol. Pharm.6 752–762 (2009)
BuerenJuanHematopoiesis and Gene Therapy, CIEMAT, Spain, E-mail: juan.bueren@ciemat.es
Advances of gene therapy and cell reprogramming in fanconi anemia
Session: Pluripotent cells
Fanconi anemia (FA) is a rare inherited syndrome, mainly characterized by bone marrow failure (BMF) and cancer predisposition. The low number and poor quality of the hematopoietic stem cells (HSCs) present in the BM of FA patients accounts for their BMF and has limited the efficacy of gene therapy in FA. Aiming to generate disease-free HSCs from non hematopoietic somatic tissues, combined procedures of gene therapy and cell reprogramming were applied to skin fibroblasts and keratinocytes from FA-A patients. Because FA cells are genetically instable, FA skin cells were first transduced with lentiviral vectors carrying the FANCA gene, and then reprogrammed with OCT4, SOX2, KLF4 and c-MYC retroviral vectors. iPS-like colonies from three out of six FA patients were obtained. Reprogrammed colonies fulfilled all the criteria of colony morphology, growth properties, expression of pluripotency-associated markers and differentiation potential, required for iPS cells. All FA-iPS clones, but not the original patient fibroblasts, expressed FANCA. After in vitro differentiation in OP9 cells, FA iPS cells generated CD34+ and CD45+ cells. Moreover, iPS-derived CD34+ cells produced healthy erythroid and myeloid colonies in methylcellulose cultures. Our results show for the first time the feasibility of generating genetically-corrected hematopoietic progenitors from skin cells of FA patients, opening new perspectives for the therapy of BMF syndromes of genetic etiology.
ChenJiekaiPeiDuanqingStem Cell and Cancer Biology Group, Guangzhou Institute of Biomedicine and Health CAS, China, E-mail: duanqing.pei@gmail.com
Optimized culture condition for reprogramming mouse fibroblasts by defined factors
Session: Pluripotent cells
Reprogramming somatic cells to pluripotent states by combinations of defined factors remains inefficient and ariable across experimental settings. We hypothesize that while the reprogramming factors are essential to start the eprogramming engine in somatic cells, the cell culture conditions determine the efficiency of the reprogramming rocess. We derived an optimized culture medium, iPS-SF1, that facilitates the efficient and reproducible generation of mouse induced pluripotent stem cell (iPS) lines. We found that serum is a potent inhibitor and bFGF an enhancer of reprogramming. iPS-SF1 may become a standard basal media for screening small chemicals and mechanistic studies.
MingerStephenStem Cell Biology Laboratory, Kings College London, United Kingdom, E-mail: stephen.minger@kcl.ac.uk
Therapeutic and research potential of human stem cells
Session: Pluripotent cells 2
There has been significant interest in the therapeutic and scientific potential of stem cells since reconstitution of the haematopoietic system was first realized by bone marrow transplantation in the 1960s. The isolation of tissue-specific, multipotent stem cells from adult organs and the derivation of pluripotent human embryonic stem cells offer the potential for regeneration of a number of different tissues and organs susceptible to age-related degenerative conditions and traumatic injury. In the not-too-distant future, it will be possible to repair heart tissue damaged by myocardial infarction, to replace neuronal cells lost in Parkinson's and Alzheimer's diseases, to transplant new insulin producing cells for diabetics and myelinating cells for individuals afflicted with multiple sclerosis, and to replace bone and cartilage lost through aging and inflammatory disease. In addition, the generation of specific populations of defined subtypes of human cells has tremendous potential to revolutionize the fields of drug discovery and investigation into the cellular bases of human disease. The newly emerging field of Regenerative Medicine will fundamentally alter clinical medicine and significantly influence our perceptions of aging, health and disease, with a myriad of consequences for society at large.
Department of Pediatric Endocrinology Neurology, Hopital Saint-Vincent de Paul, Germany
Translational Oncology, German Cancer Research Center, Germany
Department of Biotherapy, Hopital Necker-Enfants Malades, France
Department of Pediatric Immuno-Hematology, Hopital Necker-Enfants Malades, France, de Paul, France
Hematopoietic stem cell gene therapy for X-linked adrenoleukodystrophy: polyclonal distribution of lentiviral vector
Session: Progress in clinical gene therapy
We have initiated a gene therapy trial in 3 patients with cerebral adrenoleukodystrophy (X-ALD), a devastating demyelinating disease of the CNS. Autologous transplantation of CD34+ cells transduced with a HIV-1 lentiviral SIN-vector allowed stable and sustained restoration of the genetic defect in hematopoietic stem cells up to 30 months after transplantation. Based on: 1) LAM-PCR and extensive pyrosequencing; 2) analysis of retroviral frequency of LAM-PCR amplicons; 3) analysis of sample purity and elimination of collision, hematopoiesis remained polyclonal with no signs of clonal dominance or even premalignant disproportional distribution of cellular contributions. Integration site (IS) distribution was analyzed in ex vivo transduced cells prior to reinfusion and on engrafted cells, revealing gene coding regions as preferred targets for lentiviral vector integration. Identical IS were identified in myeloid and lymphoid lineages, indicating successful ex vivo transduction of hematopoietic stem cells. Our molecular follow-up presents the first molecular data on lentiviral clinical gene therapy in humans. Although a longer follow-up will be necessary, this high throughput distribution analysis of the IS repertoire suggests that the potential for genotoxicity of lentiviral vectors in this application is low. Lentivirus vectors thus offer great promise for safe and effective correction of human stem cells.
KiemHans-Peter12
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109
Department of Medicine, University of Washington, Seattle WA 98195, USA, E-mail: hkiem@fhcrc.org
MGMTP140K-mediated in vivo selection and chemoprotection of long-term repopulating cells in nonhuman primates
Session: Progress in clinical gene therapy
In vivo selection of genetically modified hematopoietic repopulating cells has many potential therapeutic applications. For some applications which will likely require relatively high levels of gene marking, such as hemoglobinopathies and anti-HIV strategies, in vivo selection may be required to increase initially low levels of gene-modified cells. We have shown efficient post-transplantation selection of macaque (M. nemestrina) long-term hematopoietic repopulating cells using methylguanine methyltransferase (MGMTP140K) in a primate model following ablative conditioning. We were able to increase pre-chemotherapy gene marking levels of 11.3% in granulocytes and 15.3% in lymphocytes to a post-chemotherapy gene marking level of 76.9% in granulocytes and 49.0% in lymphocytes. More recently we explored in vivo selection after nonmyeloablative conditioning. Using the macaque we have tested targeted busulfan for pretransplant conditioning to provide sufficient myelosuppression and facilitate engraftment of chemoprotected hematopoietic stem cells while minimizing extra-hematopoietic toxicity. Following reduced-intensity conditioning with busulfan (4 mg/kg/day for 2 days) and infusion of gene-modified cells, there was moderate myelosuppression. Following stable hematopoietic recovery, we observed about 4% gene marking in total white blood cells that, following a single cycle of O6BG (x2) and BCNU, rose to about 16% gene marking. Gene marking has been stable for more than 9 months following chemotherapy. Clonality studies before and after in vivo selection are underway. In summary, we were able to show efficient engraftment and selection of MGMT-P140K gene-modified cells after myeloablative and reduced-intensity conditioning.
RussellStephen JDepartment of Molecular Medicine, Mayo Clinic, Rochester MN 55905, USA, E-mail: sjr@mayo.edu
Trackable, NIS-expressing oncolytic viruses
Session: Progress in clinical gene therapy
Animal studies have shown that oncoytic viruses spread preferentially in neoplastic tissues, directly killing infected tumor cells and provoking an immune/inflammatory response that further amplifies tumor cell killing, while causing minimal damage to normal tissues. However, the results from human oncolytic virotherapy trials have to date fallen far short of expectations based on successes in animal cancer models. Inferior therapy outcomes in humans could be a consequence of inefficient, inaccurate delivery of the virus to sites of tumor growth, failure/premature termination of intratumoral virus propagation, or long-term survival of chronically virus-infected tumor cells. Intensive monitoring of these parameters is easily achieved in rodent therapy models through detailed analysis of tissues explanted from experimental animals following euthanasia at various timepoints after initiation of therapy. Clearly this approach is not applicable to human studies. We are therefore exploiting a NIS-based monitoring system to facilitate repeated (as often as daily) noninvasive radioisotopic imaging to determine the number and location of virally infected cells in mammals treated with an oncolytic virus. This approach is expected to be equally applicable in mice and humans. NIS is the thyroid al sodium iodide symporter, a protein expressed on the surface of thyroid follicular cells that mediates uptake and concentration of radioiodine. NIS expression can be monitored noninvasively, quantitatively and repeatedly by imaging the biodistribution of radioactive isotopes (eg. I-123, I-124, I-125 or Tc99M) using gamma camera, SPECT or PET imaging modalities. These isotopes are quantitatively detected even when localized deep in the body because gamma rays are only minimally attenuated by the tissues through which they pass. Based on these considerations we generated oncolytic measles viruses and vesicular stomatitis viruses coding for NIS. Preclinical studies were conducted to demonstrate how radioisotopic imaging of NIS gene expression can be used to noninvasively monitor the intratumoral propagation of these oncolytic viruses in living animals. Clinical trials evaluating the approach in human cancer patients are currently underway.
D'ApoteLuciaRegulatory Affairs and Organisational support, EMEA, United Kingdom, E-mail: lucia.dapote@emea.europa.eu
The EMEA-CAT: Tools to overcome regulatory barriers to commercialisation of Advanced Therapies
Session: Safety and regulatory affairs
In January 2009 the Committee for Advanced Therapies (CAT) started operating at the EMEA. The CAT is one of the most prominent feature of the new legislation on Advanced Therapy Medicinal Products (ATMPs) which are based on gene therapy, somatic cell therapy or tissue engineering. The new legislation has established a dedicated path for the marketing authorisation of ATMPs, specific tools and incentives to foster research and development in this field and to facilitate their access to the EU market.The evaluation of ATMPs often requires very specific expertise, which goes beyond the traditional pharmaceutical field and covers borderline areas such as biotechnology, medical devices, mechanical sciences, transplantation and surgery. The CAT is a multidisciplinary scientific committee that possesses all the expertise to evaluate ATMPs.A recent survey has highlighted that regulatory uncertainties are perceived as one of the principal barrier to the development of new ATMPs. The new regulatory framework and the operation of the CAT is expected to overcame this barrier by establishing a central point of reference for companies, research groups and academia operating in the field, providing specialised expertise for scientific advice, offering dedicated regulatory tools and financial incentives. Early interactions between developers, manufacturers and regulators on scientific and regulatory challenges are also expected to improve the chance to successfully develop and market ATMPs.
De BeckkerGertnvTiGenix
The ChondrOcelect® example: Managing the regulatory expectations under the new ATMP Framework for tissue engineered products
Session: Safety and regulatory affairs
On 26 June 2009, the European Medicines Agency recommended the first marketing authorisation for an advanced medicinal product, following a positive opinion from the Agency's Committee for Advanced Therapies (CAT) and the Committee for Medicinal Products for Human Use (CHMP).
ChondroCelect®, from TiGenix nv, is a cell-based medicine that is used to repair defects in the cartilage of the femoral condyle (the end of the thighbone) in the knee. It consists of chondrocytes (cartilage-forming cells) that are taken from a healthy region of the patient's cartilage, grown outside the body, and then re-implanted during surgery.
This presentation will focus on the regulatory challenges experienced during the regulatory approval process and the key learning's for the successful development of a tissue engineered product.
KingNancy M. P.JDDepartment of Social Sciences and Health Policy, Center for Bioethics, Health, and Society, Wake Forest University, Winston-Salem, North Carolina, USA, E-mail: nmpking@wfubmc.edu
Genetic Manipulation in Regenerative Medicine: Research Ethics Reminders and Recommendations
Session: Safety and regulatory issues
Genetic manipulation technologies are important in regenerative medicine research. The lessons learned from gene transfer research are therefore important for regenerative medicine. Two examples of the intersection between genetics and regenerative medicine highlight the principal research ethics issues to consider:
Creating induced pluripotent stem cells (iPS cells) almost always requires the addition of new genetic material. In the future, when iPS cells are introduced into humans, the risks of harm must neither be exaggerated nor disregarded, and long-term follow-up will be essential. Creating iPS cell lines also represents the “personalization” of experimental medicine through study of particular diseases. Patients with conditions thought to have genetic associations provide somatic cells to investigators, who grow iPS cell lines and insert them into animals to create disease models, identify associated genes, and begin developing potential treatments. This research raises intellectual property questions, requires scrutiny of relationships between disease advocacy groups and investigators, and implicates the many ethical considerations associated with biobanking and with creation of “humanized” chimeric animals.
Many experimental regenerative medicine technologies use transgenes to increase effectiveness. Endogenous regeneration may “seed” a failing organ with genetically altered stem cells, to replace or reprogram defective cells. Regrown solid organs may be infused with growth factor genes to promote development of vasculature for oxygenation. These and similar examples raise research ethics issues familiar to gene transfer: balancing harms and benefits, the therapeutic misconception, and the choice between standard therapies and experimental interventions.
Ethical recommendations for regenerative medicine research will be provided.
Lasers for cell imaging and manipulation – biophotonics in regenerative science
Session: Stem cells and genetic modification
In regenerative biology, several aspects of laser radiation can be employed. On one hand, highly precise imaging using laser microscopy based on linear and nonlinear microscopy allows the imaging and tracking of specific cells or tissues, based on intrinsic fluorophores as well as fluorescent markers. Thereby, structures like tissue matrices can be imaged in combination with adherent cells attaching on these structures at the same time. The use of ultrashort laser pulses (approximately 140 femtoseconds) for imaging allows the use of near infrared laser light, leading to penetration depths of up to a millimeter. If higher laser powers are applied, the very same laser can be used to manipulate cells or cell cultures with subcellular precision. The nonlinear absorption due to the high peak intensities at the submicrometer focus can be used to ablate cellular structures or to perforate the cell membrane, enabling the delivery of foreign molecules into the cell. Possible applications would be laser transfection or nanoparticle transfer into cells.
Studies with different cell lines will be presented, showing the precise and minimal invasive cell handling of valuable cells as for example stem cells or primary cells. Moreover, further applications of different imaging modalities of laser microscopy in tissue engineering will be shown and discussed.
BakerAndyCardiovascular and Medical Sciences, University of Glasgow, United Kingdom, E-mail: ab11f@clinmed.gla.ac.uk
Adenovirus: coagulation factor interactions and their impact on virus tropism
Session: Vector targeting 1
The development of adenoviral serotype 5 (Ad5) based vectors for systemic applications is limited by their inherent tropism for liver and spleen, precluding the ability to retarget to sites of disease. Recent studies have elucidated the mechanism underlying much of this tropism and the consequence of this infectivity profile1–4. Using a range of techniques we have shown the precise role of coagulation factor X in mediating liver uptake of adenovirus in rodent models. Recently, we modeled our cryoelectron microscopy data from the Ad5:FX interaction. We observed contact points within the hexon in hypervariable regions 5 and 7. We created vectors that had defined mutagenesis of key amino acids in each region and additional vectors with entire hypervariable region swaps. In both cases amino acids from Ad26 were used. The impact of these modifications in the Ad5 hexon on in vivo tropism of the virus and on pre-existing immunity in human subjects will be discussed.These findings highlight the fundamental importance of the hexon:FX interaction dictating in vivo tropism as well as novel avenues for vector retargeting to alternate sites in vivo.
Parker AL et al,Blood (2006)
Waddington SN et al,Cell (2008)
Kalyuzhniy, O et al,PNAS (2008)
Di Paola, N et al,Immunity (2009)
KreppelF.PrillJ.-M.EspenlaubS.EnglerT.KochanekS.
Modulation of the hepatocyte tropism of Ad5-based gene transfer vectors by specific geneti-chemical modification of the hexon capsomere
Session: Vector targeting 1
The application of Ad5-based gene transfer vectors by systemic delivery is significantly hampered by interactions of the vector particles with cellular and non-cellular blood components. For example blood coagulation factor X binds to the hexon capsomere and this interaction mediates the hepatocyte tropism of Ad5-based vector particles. Furthermore, a large majority of vector particles is scavenged by Kupffer cells and thereby removed from circulation. Therefore, all attempts to systemically deliver Ad5-based vectors for gene transfer to diverse tissues such as disseminated tumors or even to hepatocytes have to control these interactions. Here we present evidence that, depending on the PEG size used, specific PEGylation of only the hexon capsomere allows for both significantly de- or increased transduction of hepatocytes. We demonstrate that this technology can reduce the interaction of vector particles with blood coagulation factors and, importantly, with Kupffer cells.
Based on the geneti-chemical capsid modification technology we specifically PEGylated the HVR5 of hexon with differently sized PEG molecules (750 Da, 2 kDa, 5 kDa). We injected 2E10 specifically PEGylated particles of an EGFP-expressing Ad vector into Balb/c mice and analyzed transgene expression and vector copy numbers in the liver 72 h later. Small PEGs (750 Da, 2 kDa) specifically coupled to hexon led to 60–77% decreased transgene expression in hepatocytes, which could be corroborated by histological analysis and quantitative PCR. Direct comparison of transduction efficiencies in mice pretreated with warfarin and therefore depleted of vitamin K-dependent blood coagulation factors suggested that the PEGylated vector particles showed decreased interaction with blood coagulation factor X.
Surprisingly the largest 5 kDa PEG molecule specifically coupled to hexon led to a significant 11-fold increase in hepatocyte transduction and quantitative PCR revealed an up to 6-fold increase in vector copy genomes in the liver. Additional experiments with warfarin-pretreated mice revealed that the hepatocyte transduction mediated by the vectors PEGylated at hexon with 5 kDa PEG was independent of the presence of factor X. Furthermore, the increase in transgene expression and vector copy genome numbers closely matched the increase observed in mice depleted from Kupffer cells by clodronate liposome injection and treated with unmodified vector particles. Thus, we assumed that mechanisms, that counteract factor X-dominated hepatocyte tropism can be blocked by hexon-specific PEGylation with the 5 kDa mal-PEG, but not with smaller PEG moieties. In fact, depletion of Kupffer cells by clodronate liposomes prior 5kDa-PEG-vector injection did not further improve transduction of hepatocytes or vector genome copy numbers in the liver. These data suggest that a significant amount of the PEGylated vector particles evaded from Kupffer cells.
In summary, we demonstrate that the specific chemical modification of the hexon capsomere is a very powerful tool to modulate the liver tropism of Ad5-based gene transfer vectors.
CattaneoRobertoMolecular Medicine, Mayo Clinic, United States of America, E-mail: cattaneo.roberto@mayo.edu
Targeted paramyxovirus envelopes for cancer therapy and gene delivery
Session: Vector targeting 2
We study the envelope proteins of measles virus (MV) and the related paramyxoviruses, and use them to target vectors to designated receptors (1). MV entry is mediated by two glycoproteins, the H-protein that is responsible for receptor binding and for fusion (F) protein activation. We have mapped the binding sites for three natural receptors to distinct locations on the H-protein crystal structure (2, 3), and have shown that targeting ligands fused to the carboxyl-terminus can trigger fusion. We are probing the mechanisms of fusion activation by constraining the H-protein conformation through additional disulphide bonds, and by engineering artificial ligands or 'cantilevers' at specific locations to trigger fusion. Preliminary results support the hypothesis that there are only a few pathways of fusion activation. In collaboration with the Buchholz group in Langen, Germany, we have shown that HIV-1 vectors can be pseudotyped with cytoplasmic tail-truncated MV glycoproteins, and targeted to cell types of interest (4). We have also retargeted the H-proteins of canine distemper virus (CDV) and the Tupaia paramyxovirus. Finally, we have generated a chimeric MV shielded by the retargeted CDV envelope proteins. This virus is minimally reactive with MV-neutralizing antibodies but retains oncolytic activity. Thus, paramyxovirus envelopes have increasingly broad applications in targeting and shielding, improving efficiency and safety of gene transfer and oncolysis protocols.
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WilsonJames M.University of Pennsylvania, USA, E-mail: wilsonjm@mail.med.upenn.edu
AAV immunity: Antibodies and Inflammation
Session: Vector targeting (1)
AAV mediated gene transfer in vivo is remarkable for a relative lack of T cell responses to the transgene and capsid which distinguishes it from most other viral and non viral gene transfer vehicles. A consequence of this immunobiology is stable expression of the transgene even if it is antigenic. We have undertaken a thorough evaluation of the responses of the host to AAV vectors and have found them to be quite complex. The apparent tolerance of the host to AAV encoded antigens is not present in larger animals including dogs and monkeys. We also find that pre-existing humoral responses to AAV by natural infections have a dramatic impact on vector performance impacting on both safety and efficacy. Finally, we show that innate immunity present in the host or elicited by the capsid has a major influence on the outcome of AAV gene transfer.
