Detailed Program Schedule
Wednesday, May 28
12:30 pm - 2:30 pm
Room 311
Chair
Sonia Skarlatos, PhD
Speakers
Sonia Skarlatos, PhD
Introduction
James W. Wilson, MD, PhD
PennVector: A Resource for Research Grade Vectors
Janet Benson, PhD
Preclinical Toxicology and Biodistribution Assessments Supporting Development of Gene Therapy Products
J. Fraser Wright, PhD
Adeno-associated Virus Vectors to Support Clinical Studies
Lakshmi Sastry, PhD
Lentiviral Vector Production
Susan B. Sepelak, MS, MSM
Program Management and Investigator Access to Resources
12:30 pm - 3:30 pm
Room 312
Co-Chairs
Samuel C. Wadsworth, PhD
Geoff Symonds, PhD
Speakers
Samuel C. Wadsworth, PhD
Challenges in Vector Production
A central issue for gene therapy since its inception has been the ability to manufacture vectors in a consistent and reliable manner. This issue pertains to the earliest stages of research where confirmation of basic findings by others is essential. This issue increases in importance as projects move from the discovery stage, frequently conducted in academic laboratories, on to development and e0ventually into the clinic, with the latter stages conducted in a more regulated environment whether in academia or industry. For exciting basic research findings to be brought to fruition, ever more reliable manufacturing processes are needed with vector comparability being key. This session will explore manufacturing in a subset of vector systems focusing on the transitions from discovery to the clinic.
Larry A. Couture, PhD
Vector Production in Support of Early Phase Clinical Trials
City of Hope’s Center for Biomedicine and Genetics is a multi-product manufacturing facility producing clinical-grade lentivirus, adenovirus and oncolytic vectors, plasmid DNA, recombinant proteins, bacterial products, and cell-based products. As a recent NGVL designated production center, the CBG established 500mg-1g scale plasmid DNA manufacturing capability using Wave Bioreactor technology. This process has proven superior to stir tank bioreactor methods in our hands and resolved very low level prior product contaminations detectable using a sensitive PCR release test in fermentor based processes. In addition, an up to150 liter lentivirus production process has been developed and used to produce material currently in clinical trials. Methodologies for Wave reactor plasmids DNA production and lentivirus production and clarification/concentration processes will be described.
Philip Ng, PhD
Production of Helper-Dependent Adenoviral Vectors
Helper-dependent adenoviral vectors (HDAds) are devoid of all viral coding sequences, retaining only minimal non-coding Ad cis-acting sequences required for replication and encapsidation. Consequently, HDAds can transduce target cells with high efficiency to mediate long-term transgene expression without chronic toxicity thus having tremendous potential for gene therapy. HDAd are produced by co-infecting Cre-expressing 293 cells with an E1-deleted helper Ad bearing a packaging signal flanked by loxP sites. Cre-mediated site-specific recombination between the loxP sites results in excision of the packaging signal from the helper virus genome, rendering it unpackageable but still able to replicate and trans-complement propagation of the HDAd. Methods for the rescue, amplification, large-scale production and purification of HDAd will be presented. Characterization of HDAd in terms of helper virus contamination and vector infectivity will also be discussed.
Wim Hermens, PhD
A Modular and Disposable Manufacturing Platform for Adeno-Associated Viral (AAV) Vectors
A major aim of AMT is the development of gene-based therapies for “orphan” diseases. To take advantage of an accelerated regulatory pathway for such rare diseases, a modular and easily scalable production system is preferred in order to anticipate on the manufacturing demands for pre-clinical and clinical studies as well as the studies required to show regulatory compliance.
AMT has further developed the baculovirus-expression vector system for AAV vectors as described by Urabe et al., 2002. The serum free insect cell culture line of Spodoptera frudiperda (SF+) and the baculovirus backbone was obtained from Protein Sciences Corporation. AMT has made significant improvements with regard to the stability of the baculoviruses and the infectivity of the AAV vector produced in insect cells. AMT has fully GMP qualified banks of the insect cells and the baculoviruses (Master and Working Seed Virus). The use of three different baculoviruses encoding the Rep genes the Cap genes and the vector sequence for AAV production makes the manufacturing platform highly modular.
AMT is currently using the baculovirus expression system on 50 L scale. The manufacturing platform is fully disposable apart from the equipment for column chromatography. The disposable approach makes the platform very flexible since cleaning validation is reduced to a minimum upon a product change. The major disposable parts in the process are Wave bioreactors, disposable bags for holding of process intermediates and a fully disposable UF/DF set-up. Besides these disposables AMT has decided to perform the high capacity affinity chromatography step in a single use mode in order to prevent lengthy cleaning and re-use studies for the regulatory package.
In this presentation the manufacturing platform will be presented for the lead product of AMT, which is an AAV1 vector expressing the Lipoprotein Lipase (LPL) gene (AMT-011). Currently, the GMP-compliant process has successfully been subject to process validation studies and viral clearance studies in order to ensure regulatory compliance required for commercial manufacturing of AMT-011.
J. Fraser Wright, PhD
Towards Fully Scalable Purification of AAV Vectors
A division of The Center for Cellular and Molecular Therapeutics at The Children’s Hospital of Philadelphia, the AAV Clinical Vector Core is a multi-product facility that manufactures and certifies adeno-associated virus-based gene transfer vectors for use in clinical studies. The facility supports currently open clinical trials for hemophilia B and Leber Congenital Amaurosis (LCA), and is the designated NHBLI Gene Therapy Resource Program Clinical AAV Core Laboratory. Manufacturing capacity is ~2x1015 vg per clinical lot, providing highly purified vector of several AAV serotypes, adequate to support most early phase clinical applications. The serotype-specific manufacturing processes used emphasize clinical product purity (including substantial removal of non-vector, virus-related subpopulations) as well as process scalability. Major objectives for ongoing process development and optimization are: 1) maintaining high clinical vector quality thereby ensuring optimal product efficacy and safety; and 2) enhancement of process scalability to successfully support promising AAV-based products through late-stage clinical studies. This presentation will describe our experience in the development and implementation of scalable purification strategies, focusing on ion-exchange chromatography methods.
Geoff Symonds, PhD
Progress in Vector Production
A gene therapy product must be “manufacturable” under GMP conditions so that the product is of known quality, quantity and potency. The presentations today address the basic issues of production: application to early clinical trials, consistency of product, modular platform, disposable components and scalability. By necessity, examples have been chosen but the lessons are widely applicable. At the end of this session an answer to the central question: “what does it take to make a gene therapy product” will have been given. By way of conclusion this presentation will summarize what answer we have thus far.
12:30 pm - 3:30 pm
Room 304/306
Chair
Gloria Gonzalez-Aseguinolaza, PhD
Speakers
Ding Ma, MD, PhD
Adjuvant ADV-TK Administration Improves Outcome of Liver Transplantation in Patients with Advanced Hepatocellular Carcinoma
Most patients with hepatocellular carcinoma (HCC) are not candidates for liver transplantation because of advanced disease. ADV-TK therapy is an established adjuvant treatment in cancer, especially because of its chemotherapeutic efficacy-promoting ability and its hepatotropic characteristics. Forty-five HCC patients with tumors larger than 5 cm in diameter without metastases in lung or bones as detected by CT or MRI scans, participated in the study over a follow-up period of 50 months. Twenty-two patients received liver transplant only and 23 received liver transplant combined with ADV-TK therapy. The recurrence-free survival and the overall survival in the liver transplantation plus ADV-TK therapy group were 43.5% and 69.6% at three years; both values were significantly higher than those in the liver transplantation only group (9.1% and 19.9%, respectively). The results of multivariant and subgroup analysis will be presented which indicate that lack of vascular invasion is an important criteria for selection for liver transplant and ADV-TK therapy.
Dominic Wells, PhD
Gene Therapy Trials for Duchenne Muscular Dystrophy
Recent promising pre-clinical studies showing the potential to treat multiple muscles by regional or systemic delivery of a variety of gene therapy reagents have stimulated the initiation of a range of clinical trials aimed at restoring the expression of dystrophin in Duchenne muscular dystrophy (DMD). Ongoing trials include the use of adeno-associated virus for the transfer of microdystrophin, antisense molecules to induce exon-skipping to restore the reading frame and drugs to promote read through of premature stop mutations. The status of these trials will be reviewed and, in the case of AAV and antisense based therapies, issues associated with regional or systemic delivery will be considered.
Keiya Ozawa, MD, PhD
AAV Vectors for Gene Therapy of Parkinson’s Disease
AAV vectors are considered to be promising gene-delivery vehicles for gene therapy, because they are derived from non-pathogenic virus, efficiently transduce non-dividing cells, and cause long-term gene expression. Parkinson’s disease is one of the most promising candidates of gene therapy. As for the initial step of clinical application, AAV vector-mediated AADC (aromatic L-amino acid decarboxylase; the enzyme converting L-DOPA to dopamine) gene transfer in combination with oral administration of L-DOPA is appropriate, since dopamine production can be regulated by adjusting the dose of L-DOPA. The other applications of AAV vectors will also be discussed.
Robin R. Ali, PhD
Gene Therapy for Early Onset Severe Retinal Degeneration Due to Defects in RPE65
Gene therapy can improve retinal function and visual behaviour in animal models with defects in RPE65. The purpose of this study was to determine whether gene therapy can be safe and effective in humans. In a phase I/II clinical trial, we have delivered subretinally recombinant adeno-associated virus (rAAV) vector expressing human RPE65 under the control of a human RPE65 promoter in 3 adult human subjects with early onset severe retinal degeneration associated with defects in RPE65. We have examined systemic vector dissemination and immune responses following vector delivery and have assessed visual function using a range of electrophysiological and psychophysical techniques.
Elizabeth Rakoczy, PhD
Preparations for a Phase I/II Secretion Gene Therapy Trial of Age Related Macular Degeneration
A pre clinical trial for the wet form of AMD was conducted by subretinal injection of AAV.sFlt-1 (a recombinant adenoassociated virus carrying the soluble vascular endothelial growth factor receptor 1 gene) into the eyes of non-human primates (macaca fascularis). Functional analysis by electroretinography did not show any changes following the injection and ophthalmic examination using fundus photography and fluorescein angiography did not detect any abnormalities, except for minor conjunctivitis over the first 1-3 days post surgical procedure, but this resolved through the use of tobramycin eye drops. Biodistribution studies showed the absence of the AAV2 capsid in the plasma by AAV2 ELISA, the presence of the AAV.sFlt construct in the retina but not in other tissues sampled (optic nerve, liver, spleen, heart, lungs, cornea, lymph nodes) by qPCR, statistically significant increase in sFlt-1 protein levels in the 3/5 AAV.sFlt-1 injected eyes at 12 months post injection (p < 0.05) but not in the plasma. Flow cytometry conducted to assess changes in immune cell subset population did not detect any significant changes in T cell, B cell, natural killer cell, dendritic cell and macrophage populations at 2 weeks 1, 3, 9 and 12 months post injection in 4/5 monkeys injected with AAV.sFlt-1 and 2/2 monkeys injected with AAV.gfp. One monkey, showing a pre-activated phenotype at day zero subsequently developed a transient increase in CD4 and CD8 T cells at 14 days post injection suggesting that patients with an activated immune system may have to be rescheduled or excluded from the trial.
Christoph Klein, MD
Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich-Syndrome
Wiskott Aldrich Syndrome is a life-threatening immune-disorder characterized by bleeding secondary to microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to lymphoma. Based on extensive preclinical studies, a clinical gene therapy protocol was developed at Hannover Medical School. We here present a preliminary analysis of the first two patients treated in 2006. Both patients showed evidence of long-term gene marking in myeloid and lymphoid cells. Furthermore, the majority of peripheral thrombocytes showed evidence of WASP expression. Functional reconstitution was documented in dendritic cells (podosome formation), T-cells (proliferation in response to CD3-signaling), and NK-cells (formation of immunological synapse). Clinically, patients are in excellent condition more than one year after gene therapy. Of note, they did not develop any severe infections or bleeding. Eczema and autoimmunity resolved completely. Molecular insertion site analysis yielded a polyclonal pattern of hematopoiesis. In sum, hematopoietic stem cell gene therapy may be of benefit for patients with WASP deficiency – more extensive studies and longer follow-up are needed to determine the safety profile of this experimental approach.
Nathalie Cartier-Lacave, MD
Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector Hemonstrates Expression of the Therapeutic Protein in High Percentage of Lymphocytes and Monocytes in Two Patients with X-Linked Adrenoleukodystrophy
We report preliminary results in two children with cerebral X-linked adrenoleukodystrophy (ALD) who received hematopoietic stem cell (HSC) gene therapy using a HIV1-derived lentiviral vector. Mobilized peripheral blood CD34+ cells were transduced ex vivo with a non-replicative HIV1-derived lentiviral vector expressing the ALD cDNA. Transduced CD34+ cells were infused to ALD patients after full myeloablation with cyclophosphamide and busulfan. The percentage of corrected lymphocytes and monocytes in the peripheral blood of treated patients remained stable from day 60 to the last follow-ups (15-20%). Tests assessing vector-derived RCL and vector mobilization were negative up to the last follow-ups.
These early results support that ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization, and that a high percentage of hematopoietic progenitors were transduced expressing ALD protein in the short term . HSC gene therapy appears to have short term neurological effects comparable with allogeneic HSC transplantation.
The goal of the ASGT Education Program is to provide participants with a broad background and introduction to topics relevant to the field of gene therapy. Education Sessions will be subdivided into two headings. “Topical Review” sessions will provide a broad perspective of an area of importance to the gene therapy community such as major vector systems, specific target organs or diseases, and funding/regulatory issues. “Emerging Field Review” sessions will provide a broad perspective of a technological or scientific discipline that has recently become of great significance to our members.
4:00 pm - 5:30 pm
Room 304-306
Regulatory RNAs are small RNAs that control RNA expression by targeting transcripts for cleavage or translational repression. Found across plants, animals and viruses, these genetic rheostats play critical roles in a wide spectrum of activities ranging from normal development to diseases states such as cancer and heart disease. In this session, we will define and discuss the various classes of regulatory RNAs, outline their potential application as drug discovery and validation tools, and conclude by discussing their potential application as therapeutics for various human maladies.
Chair
Terry Hermiston, PhD
Speakers
Phillip Zamore, PhD
Overview on Regulatory RNAs: sh, i, si, micro and ribozymes
In animals, small silencing RNAs regulate gene expression, defend against viral infection, and protect the genome from transposon mobilization. Three classes of small RNAs have been described in flies and humans: small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-associated RNAs (piRNAs). Each of these small RNA classes is produced by a distinct biogenesis pathway and functions through a specific subtype of Argonaute effector protein. Although the siRNA, miRNA, and piRNA pathways are separate, there is considerable functional and genetic overlap among them. How are these three small RNA classes made? How do they function? And what small RNA features and cellular pathways act to distinguish these three types of small silencing RNAs, despite their fundamental chemical similarity?
William C. Hahn, MD, PhD
Functional Genomics, Experimental Models and Cancer
The development of cancer is a multi-step process in which normal cells sustain a series of genetic alterations that together program the malignant phenotype. Recent advances in genomics now make it possible to consider enumerating all of the genetic lesions in specific cancers. While these approaches will yield critical information regarding the identify, number, and types of alterations found in human tumors, further progress in understanding the molecular basis of malignant transformation depends upon the application of genome scale tools to functionally annotate the cancer genome. Over the past several years, we have developed genome scale RNAi libraries and open reading frame expression libraries that permit a systematic evaluation of genes involved in cancer initiation and mainteance. Using these libraries, we have now performed screens in a panel of human cancer cell lines to systematically identify cancer vulnerabilities. By combining these functional approaches with information derived from mapping the structural abnormalities present in cancer genomes, we have identified several genes that contribute to cancer development. Taken together, these studies suggest that combining forward and reverse genetic approaches with information derived from the cancer genome anatomy mapping projects will yield a comprehensive list of cancer vulnerabilities.
Phillip A. Sharp, PhD
Therapeutic Application of Regulatory RNAs
RNA interference has provided new methods for silencing specific genes in vertebrate cells. siRNAs with their 5’ and 3’ structures are actively recognized during assembly into protein complexes that efficiently target mRNAs. These complexes normally function in microRNA control of cellular genes and thus are present in all cell types. Understanding the potential of siRNAs as a new type of therapeutic agents and the roles of microRNAs in disease processes is rapidly advancing.
4:00 pm - 5:30 pm
Room 100
Gene modification of stem cells may play important roles for therapy of genetic diseases. Each clinical target requires specific types and designs of vectors. Primary immune deficiency diseases have been successfully treated using retroviral vectors for gene transfer to hematopoietic stem cells (HSC), and lentiviral and foamy viral vectors hold prospects for greater efficacy and safety. Hemoglobin disorders (sickle cell disease and thalassemia) are very common and allogeneic HSC transplants may be therapeutic but are limited by donor availability and immunological reactions. Gene therapy for hemoglobin disorders requires efficient transfer of globin genes to autologous HSC with suitably high expression in erythrocytes. Pluripotent stem cells, from embryonic cells or induced from mature cells, may have wide-spread applications for cell and gene therapy, but present unique challenges for effective gene modification.
Chair
Donald B. Kohn, MD
Speakers
Fabio Candotti, MD
Gene Transfer Vectors for Primary Immunodeficiency Diseases
Punam Malik, MD
Thalassemias
Hemoglobinopathies - beta thalassemia and sickle cell disease are the most common single gene disorders that amenable to gene therapy. The advent of lentiviral vectors have revolutionized this field, and correction of animal and human models has been shown. This talk will focus on the current status of gene therapy for beta-thalassemia and sickle cell disease and the progress towards clinical trials for these diseases.
Robert G. Hawley, PhD
Vectors for Human Pluripotent Stem Cell Transduction
Human pluripotent stem cells, whether isolated from blastocysts or induced from somatic cells, provide a powerful in vitro system to investigate the early stages of human development. Irrespective of the cell source, much work will be required to translate basic findings in stem cell biology into safe and effective clinical treatments in the emerging field of regenerative medicine. This presentation discusses the state of the art and the continuing challenges in the development of efficient gene delivery approaches involving human pluripotent stem cells, with particular regard to the biosafety aspects of various integrating vector systems and the strategies being explored to protect against transgene silencing while reducing the risk of insertional mutagenesis: the primary focus will be on recent advances made in gammaretroviral, lentiviral and foamy virus vector designs toward these goals.
4:00 pm - 5:30 pm
Room 302
Much of recent gene therapy work has been devoted to improving vector function in vitro and in vivo. Now that improved vectors are available for use, the applications in vivo are now confronting significant pharmacologic and immunologic barriers that prevent vectors from reaching target cells, mistarget them to non-target tissues, and that drastically reduce the persistence of genetic modification. This review session will discuss the basic biology of these barriers, how they impede effective gene therapy, and approaches to circumvent these barriers.
Co-Chairs
Joseph Zabner, MD
Michael A. Barry, PhD
Speakers
Hildegund CJ Ertl, MD
Immune System Barriers
In hepatic human gene replacement trials with an AAV-2 vector expressing F.IX the observed lack of sustained F.IX expression is assumed to reflect removal of transduced cells by AAV-specific CD8+ T cells. Attempts to establish mouse models to recapitulate this finding initially failed. Mice immunized with adenoviral vectors expressing AAV capsid or with AAV vectors developed CD8+ T cells in blood, lymphatic tissues, and liver to epitopes shared between AAV2 and AAV8, and serotype-specific neutralizing antibodies. When mice are infused at the height of the T cells’ effector phase with a heterologous AAV vector expressing human factor IX under a hepatocyte-specific promoter sustained hepatic factor IX expression can be achieved despite the presence of AAV capsid-specific CD8+ T cells in the liver. In contrast transfer of AAV vectors at late times after the initial immunization fails to result in F.IX expression. These results will be discussed.
John H. Wolfe, VMD, PhD
Barriers to CNS Gene Delivery
Gene delivery to the central nervous system (CNS) is restricted by the blood-brain barrier, by the complexities of surgery including the potential for damage from the procedures, and by the specialized functions in different sub-regions of the brain. A number of methods and vectors have been explored to deliver genes and/or proteins into the CNS. Their utility depends on whether focal vs global delivery is needed and whether the gene itself needs to be delivered into the diseased cells or the protein produced by a genetically corrected cell can be used by other diseased cells. Strategies that have been attempted to circumvent these restrictions will be discussed.
Michael A. Barry, PhD
Targeting and Detargeting Gene Therapy Vectors
One approach to theoretically improve gene therapy and oncolytic virus efficacy and safety is to physically target the viruses to cells by modifying their ligand-receptor interactions. Virus targeting will likely require at least two steps: 1) “retarget” virus by adding new ligands to the vector to mediate binding to receptors expressed on target cells and 2) “detarget” by blocking promiscuous binding to non-target cells. Approaches to apply these targeting and detargeting strategies in the context of the pharmacology living animals will be discussed.
4:00 pm - 5:30 pm
Room 112
Cancer gene therapy has made significant progress over the recent years with numerous vectors applications in clinical trials. This topical review will cover a variety of subjects dealing with (i) the recent progress made identifying a side population of cancer stem cells in patients with leukemias and some solid tumors examining their role and new potential therapies based on their drug resistance properties; (ii) the use of novel viral vectors such as Measles, Sindbis virus and others as cancer therapeutics: and (iii) the use of vectors to deliver targeted therapeutics for cancer gene therapy such as use of specific single-chain antibodies.
Chair
William F. Goins, PhD
Speakers
Andre Lieber, MD, PhD
Epithelial to Mesenchymal Transition and Cancer Stem Cells
I will attempt to summarize published reports in support of the existence of leukemia and solid tumor stem cells and will discuss progress and problems in therapeutic targeting of cancer stem cells.
Furthermore, I will present studies from our lab on ovarian cancer. We found that a subpopulation of ovarian cancer cells expresses both epithelial and mesenchymal markers. These epithelial-mesenchymal hybrid cells are enriched for pluripotent, tumor-initiating, CD133high, and CD44high cells. In situ, these putative ovarian cancer stem cells are localized within nests of epithelial cancer cells that are surrounded by tumor stroma.
Stephen J. Russell, MD, PhD
Novel Viral Vectors for Cancer
Of the more than 2000 known viral species, most should be amenable to genetic modification, providing a basis for an enormous diversity of replication-competent or replication-defective viral vectors. “Novel” is a nebulous term that is frequently used when describing not just new viral platforms, but also established viral platforms into which new genes have been inserted, or for which novel targeting strategies have been developed. This presentation will briefly overview some recent developments in this area, focusing primarily on replication-competent oncolytic viruses and strategies that can be used to control their host-range properties.
Paul B. Fisher, MPH, PhD
Targeted Therapeutic Vectors for Cancer
In principle, conditionally replication competent adenoviruses (CRAds) should provide effective reagents for cancer therapy. However, limitations of cancer-specific targeting, inadequate gene delivery and immune clearance provide significant barriers restricting therapeutic effectiveness, particularly in the context of metastatic disease. Recent studies demonstrate that combining cancer-specific targeting of viral replication with immune modulation or a cancer-selective apoptosis-inducing gene may address these limitations resulting in effective therapies for both primary and metastatic cancers. This session will overview the development of prototype bipartite CRAds, called Cancer Terminator Viruses, with profound anticancer activity resulting from interferon gamma or MDa-7/IL-24 production when replication occurs uniquely in tumor cells.
4:00 pm - 5:30 pm
Room 309
The launch of several gene therapy trials internationally is the result of decades of careful study regarding the causes of ocular degeneration, both genetic and acquired. This session will review recent advances in delivering therapeutics to the eye and also explore unique aspect to treating diseases of the eye.
Chair
John A. Chiorini, PhD
Speakers
Bill Hauswirth, PhD
AAV Vectors for Retinal Gene Therapy
The behavior of AAV vectors will be discussed in terms of their ability to transduce specific subsets of cells in the retina of various mammalian species. The critical variables are the intraocular site of vector administration, the AAV serotype and the promoter used to express the potentially therapeutic cDNA. In addition, examples of gene therapy for retinal diseases in animal models based on these principles will be presented. This will include AAV vectors targeting rods for X-linked Retinoschisis, targeting cones for Achromatopsia or targeting retinal pigmented epithelial (RPE) cells for the RPE65 form of Leber Congenital Amaurosis.
Peter A. Campochiaro, MD
Non-Viral Ocular Gene Transfer
In vivo nonviral gene transfer is inefficient compared to viral vector-mediated gene transfer, but since there are no viral proteins, incitement of an immune response is generally not a problem and repeated administration is feasible. It is possible to overcome the inefficiency of nonviral gene transfer in the eye, because the eye is a closed compartment increasing duration and concentration of exposure to transducing agents. Retinal pigmented epithelial cells are particularly good target cells because they take up DNA to a much greater extent than other cells types in the eye, possibly because they are phagocytic. Nonviral gene transfer is particularly useful as a research tool for promoter analysis and examples of this application will be provided. Similar principles apply to oligonucleotide-based therapies which will also be discussed.
Weng Tao, MD, PhD
Application of Enacapsulated Cell Technology for Ophthalmic Diseases
Ophthalmic disorders represent a rapidly growing disease area that is associated with the aging population. Their sight is threatened by age related macular degeneration (AMD), diabetic retinopathy, glaucoma, or retinitis pigmentosa (RP). Few effective treatments for these disorders are available to date, in part due to lack of effective delivery of therapeutic molecules to the retina. The encapsulated cell technology (ECT) allows the controlled, continuous, and long-term administration of protein drugs in the eye, where therapeutic agents are needed, and not to subject the host to systemic exposure. Furthermore, the implants can be retrieved, providing an added level of safety. CNTF has been shown to protect the retina from degeneration in animal models and ECT based delivery of CNTF protected photoreceptors in the rcd1 dog model of retinitis pigmentosa. Currently, ECT-CNTF is in clinical development for RP and dry AMD
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5:30 pm – 5:45 pm
5:45 pm - 7:15 pm
Room 304 - 306
The education sessions provided by the non-viral vector committee will focus on experimental approaches that can be applied to the improvement of non-viral vectors.
Combinatorial Approaches to Improving Polymers of Polyplex Formation:
This use of combinatorial and high throughput methods to identify new materials for the production of non-viral polyplexes. The key factors including appropriate experimental designs that should be considered when undertaking combinatory approaches will be emphasized in the presentation.
Understanding, Managing and Abrogating the Toxicity of Non-Viral Vectors in Nucleic Acid Delivery:
The session will review adverse events associated with non-viral vectors including immune stimulation and complement activation. Strategies to ameliorate them will be discussed.
Application of Synthetic Peptide Bioconjugates in Targeted Delivery of DNA In Vivo:
The presentation will identify current peptide-based non-viral gene vectors and discuss approaches for their assembly, characterization and identification of the mechanism of gene delivery.
Chair
Francis C. Szoka, Jr., PhD
Speakers
David A. Putnam, PhD
Combinatorial Approaches to Improving Polymers of Polyplex Formation
The structure/function relationships that govern the delivery of DNA and siRNA by synthetic vectors are largely undefined. To better understand the fundamental basis of transfection by polycationic vectors, some investigators have evoked a combinatorial approach. Using a combinatorial paradigm, these investigators are able to more completely map the structure/function parameter space for transfection, and begin to understand how polymer architecture and composition influences activity. This session will outline the combinatorial approach toward new vector development.
Ian MacLachlan, PhD
Understanding, Managing and Abrogating the Toxicity of Non-Viral Vectors in Nucleic Acid Delivery
Kevin G. Rice, PhD
Application of Synthetic Peptide Bioconjugates in Targeted Delivery of DNA In Vivo
Synthetic peptides have been functionalized into bioconjugates to improve the delivery of DNA to the nucleus of target cells in vivo. In the circulation, PEGylated-peptides and glycopeptides protect DNA polyplexes from degradation and target DNA across the cell membrane. Inside the cell, fusogenic peptides facilitate the endosomal escape of DNA into the cytosol. Nuclear targeting peptides are designed to facilitate the transport of DNA across the nuclear membrane. The architecture of assembling peptide bioconjugates to form DNA polyplexes is fundamental to improving the efficiency of gene transfer in vivo.
5:45 pm - 7:15 pm
Room 100
Gene transfer vectors integrate into the human genome in a non-random fashion, behaving in most cases like the viruses they originate from. Recent advances in genome-wide sequencing technology, bioinformatics and molecular genetics are providing an unprecedented wealth of information about viral integration “preferences” and the complex interactions between viral pre-integration complexes and chromatin components. Uncontrolled insertion of foreign DNA into the genome may have deleterious consequences, and is a serious safety concern for gene therapy applications. New molecular tools are being developed to target transgene integration and increase the frequency of homologous recombination. These advances will be discussed in the context of developing better and safer gene transfer technology.
Chair
Fulvio Mavilio, PhD
Speakers
Frederic D. Bushman, PhD
Molecular Basis of Retroviral Integration
To correct genetic diseases, it is generally necessary to integrate a therapeutic vector into a chromosome of the host cell. Retroviral vectors have been widely used, but integration has resulted in insertional activation of oncogenes and adverse events in patients. The lecture will cover the mechanism and consequences of integration, with an emphasis on use of new sequencing and bioinformatics methods.
Alan Engelman, PhD
Nucleoprotein Complex Interactions during HIV DNA Integration
Integration is essential for HIV replication and stable lentiviral vector transduction. The integrase protein, which works in the context of the viral preintegration complex, interacts with cellular chromatin in a nonrandom fashion, as HIV and other lentiviruses preferentially target active genes. Recent results have revealed that interactions between integrase and the cellular chromatin factor LEDGF/p75 determine the frequency and distribution of HIV integration. The LEDGF/p75-integrase interface therefore creates opportunities for novel drug design as well as to modulate the distribution of HIV integration along human chromosomes.
Matthew H. Porteus, MD, PhD
Developing Homologous Recombination for Gene Therapy
In the last several years the use of integrating viruses have begun to show promise in the treatment of genetic diseases of the hematopoietic system. Unfortuantely, those trials have also shown that the uncontrolled integration of such viruses poses an oncogenic risk. A potential solution to insertional oncogenesis caused by uncontrolled integration is to use homologous recombination to either directly “correct” disease causing mutations or to control the target site that the transgene integrates into. The spontaneous rate of homologous recombination is too low to be of clinical use, but if a site-specific DNA double-strand break is created, homologous recombination can be increased at the site of the break to levels that would be therapeutically useful. There are several different ways that site-specific double-strand breaks can be created and in this session I will discuss some of these methods with an emphasis on the efficacy and safety of using zinc finger nucleases to stimulate homologous recombination.
5:45 pm - 7:15 pm
Room 302
Immune responses to vectors and transgene products are a major hurdle for gene therapy. T cell responses are a central component of adaptive, antigen-specific immunity. This session will review basic concepts of T cell activation with particular emphasis on co-stimulatory signaling pathways. Strategies for blocking co-stimulation in gene transfer to achieve immune tolerance will be explained. Finally, advantages and potential problems of immune suppression protocols are being discussed.
Chair
Roland W. Herzog, PhD
Speakers
Cox Terhorst, PhD
Signaling between Antigen Presenting Cells and Effector/Regulatory T Cells
Carol H. Miao, PhD
Recent Advances in Immune Modulation
Successful gene therapy protocols rely on the hypo-responsiveness of the immune system to transgene products generated from gene transfer vectors. In order to prevent cytotoxic lymphocyte or antibody formation induced by transgene expression, various strategies derived from recent advances in immune tolerance induction protocols have been tested in gene therapy model systems. Current immunosuppressive drugs were used to nonspecifically target T-cell activation, clonal expansion, and differentiation into effector cells. Aside from induction of central tolerance, peripheral tolerance to transgenes may be achieved by several different pathways including deletion of activated/effector T cells by depleting antibodies, generation of T cell apoptosis or anergy by costimulation blockade, and active suppression by T regulatory cells. This presentation will review the development of these strategies using various immune modulation protocols to induce long-term immune tolerance specific to the transgene product.
Terry Strom, MD
Immune Suppression Therapy: Opportunities and Risks
Creation of T cell tolerance (T cell unresponsiveness to a specific antigen) as means to produce ideal outcomes in transplant recipients or patients with an autoimmune disease has proven most difficult. Yet it is easy to tolerize T cells in a test tube with a variety of T cell directed therapeutic agents. We are pursuing the hypothesis that adverse forms of innate immunity, e.g., TLRs, pro-inflammatory cytokines or certain dendritic cells, can negate the ability of T cell directed strategies to produce T cell tolerance. To this end we have learned that following antigen activation the presence of select cytokines can preclude recruitment of naïve T cells into the pool of Foxp3+ regulatory T cell. Similarly certain aspects of innate immunity can deprogram regulatory T cells. Regulatory cell change molecular phenotype and loss regulatory capacity following interaction with certain elements of the innate immune system. In accordance we have tested new short term therapeutic protocols that 1- mix both T cell and select inflammation altering components (power mix) and/or 2- use an acute phase reactant protein that acts directly upon adverse inflammation to sharply tip the balance of inflammatory cytokines toward an anti-inflammatory mode. Both approaches have been used with surprising success in creating immune tolerance in daunting preclinical models of autoimmune diabetes and transplantation. As result, therapeutic trials have been provisionally funded in which patients with new onset type 1 diabetes will receive short courses of therapy with either power mix or alpha1 anti-trypsin (an acute phase reactant).
5:45 pm - 7:15 pm
Room 112
Combination of multiple targeting levels is key for developing viruses into effective cancer therapeutics. Different targeting principles will be presented, and their validation discussed based on efficacy in pre-clinical models and cancer clinical trials. Reprogramming of virus particle activation and of receptor interactions are examples of cell entry targeting. Tissue-specific transcription and replication illustrate post-entry targeting. Strategies resulting in preferential spread of replicating vectors in tumors with innate immunity defects will be discussed in the context of temporary host immunosuppression.
Chair
Roberto Cattaneo, PhD
Speakers
Roberto Cattaneo, PhD
Entry Targeting: Proteolytic Activation and Receptor Interactions
Cell entry of virus particles can be reprogrammed at two different levels: proteolytic activation, which can be made dependent of cancer-specific proteases, and receptor binding, which can be retargeted to proteins expressed on the surface of cancer cells. We will review how the specificity of cleavage of the fusion protein of enveloped viruses was made dependent on metalloproteinases expressed in the tumor matrix. We will also review how single-chain variable fragment (scFv) antibodies are displayed on the envelope of oncolytic viruses, and entry of these replication-competent viruses is restricted to cells expressing the designated receptor. Finally, we will present recent data documenting that scFv-based receptor targeting technology can be applied to restrict transduction of lentiviral vectors to relevant cell types, improving safety.
Dirk M. Nettelbeck, PhD
Transcriptional Targeting
The principle of cancer-specific transcription and replication of oncolytic viruses will be presented. This principle has been established and optimized for transcriptional targeting of DNA virus replication. It is based on the insertion of cellular transcription control elements into viral genomes in order to direct viral gene expression in a cell-type selective manner. It will be discussed how different promoter elements have been combined and tested, and how the fidelity of these constructs can be ensured after transfer in the virus genome. It will also be shown which viral genes have been regulated, ultimately resulting in oncolytic viruses with a high degree of selectivity.
E. Antonio Chiocca, MD, PhD
The Host Response to Viral Oncolysis
Clinical trials of oncolytic viruses (OVs) for cancers have revealed that these agents are well tolerated. However, clear-cut evidence of efficacy has been elusive. A fundamental issue continues to limit the efficacy of cancer virotherapy: the response of the host to the ongoing viral infection. This response occurs at the level of intracellular tumor defenses to the viral infection, extracellular stromal reactions to the propagating virus, and, importantly, active host defenses consisting of innate immune as well as inflammatory angiogenic responses. The combination of these host responses severely limit and curtail tumor cell infection and viral replication, thus limiting anticancer efficacy. We thus believe that such host responses will have to be circumvented, even temporarily and/or partially, in order to fulfill the promising anticancer effects of these agents.
5:45 pm - 7:15 pm
Room 309
The dystrophin gene is one of the first few disease genes identified. Mutations in the dystrophin gene lead to Duchenne muscular dystrophy (DMD). The lessons learned from DMD gene therapy development illustrate an excellent example on how to rationally design and implement gene therapy studies. In this session, we will discuss issues related to the use of appropriate animal models, the construction of an effective therapeutic gene and the gene delivery strategies. This will be an excellent learning opportunity for investigators who just start in this field, especially these who are interested in muscle-related gene therapy.
Chair
Dongsheng Duan, PhD
Speakers
Xiao Xiao, PhD
From the Disease to the Gene: A Historic Perspective of DMD Gene and Cell Therapy
Bruce F. Smith, VMD, PhD
Intermediate Animal Models for Muscle Gene Therapy
The translation of Gene Therapy, in vivo, from murine models to human patients, requires that questions of mass (dose) and immune response be addressed. A number of spontaneously occurring models of inherited muscle disease have been identified in the dog and cat over the past several decades. These models are termed “intermediate” models because they represent both a stepping-stone between murine and human studies and because the animals are intermediate in size between mice and humans. These models have many of the characteristics of their human disease counterparts. Importantly, they have intact, out-bred, immune systems, mimicking the more complicated scenario of human patients. In addition, due to the underlying mutations, many of these models show similar subtle variation in phenotype to those seen in humans. Thus, the selection of the correct intermediate model may be a critical step in the progression towards clinical success. This presentation will review currently available canine and feline models, focusing on Duchenne-like muscular dystrophy, but addressing other muscle disease models as well. In addition to information on the disease phenotypes, practical considerations for working with these models such as breeding intervals, care of affected offspring and other common issues encountered will be presented in order to provide a perspective on how to best incorporate these intermediate models into a translational plan for muscle gene therapy.
Dongsheng Duan, PhD
Design Dystrophin for Duchenne Muscular Dystrophy Gene Therapy
The dystrophin gene is the first human disease gene cloned by positional cloning. It is also one of the largest genes with a ~12 kb coding sequence. Mutations in the dystrophin gene lead to Duchenne and Becker muscular dystrophy as well as X-linked dilated cardiomyopathy. A promising approach to treat these diseases is to introduce a functional gene by gene replacement therapy. However the large size of the dystrophin coding sequence presents a challenge. It cannot fit into many gene therapy vectors. A strategy to overcome this hurdle is to design synthetic genes that are substantially abbreviated but still functional. The full-length dystrophin protein contains four major domains including the N-terminal, rod, cysteine-rich and C-terminal domains. The rod domain can be further divided into 24 spectrin-like repeats and four hinges. In this discussion, we will review biological implications from retaining and/or deleting different regions in the synthetic dystrophin gene. We will also discuss the pros and cons of the current mini- and micro-genes and strategies to further improve them to meet the clinical need.
7:00 pm – 9:00 pm
Room Exhibit Hall A, Plaza Level
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