3rd Annual Conference: GENE DELIVERY - Non-viral Systems & In Vivo ApplicationsNovember 11 - 13, 2003 • Shelter Pointe Hotel • Boston, MA USA [ Overview | Agenda | Call for Posters | Register ] View Electronic Brochure (PDF)OverviewDelivering genetic materials to target cells with the utmost efficiency is the key hurdle in using gene therapy for the development of new therapeutics. In recent years, scientist have focused increasingly on using non-viral systems to overcome this challenge. This 3rd conference on gene delivery systems presents various ways to improve polymeric delivery systems. In addition, other novel approaches such as using bacteria as DNA carriers or using artificial chromosomes will be presented. Various method to faciliate the delivery by using adjuvants or photochemical internalization as well as results of non-viral gene delivery in clinical trials will be explored. Don’t miss the opportunity to learn from this international assembly of experts as they highlight the major achievements and challenges on topics such as: Intracellular targeting — all new this year! — RNA has demonstrated great potential as a therapetic tool. However, efficient delivery of RNA to target remains a major challenge. This workshop focuses on AgendaTuesday, November 11, 2003RNA Delivery Workshop1:15 Registration, Poster/Exhibit Set-Up, Coffee & Pastries 1:55 Chairperson’s Remarks 2:00 Lentivirus-Delivered Stable Gene Silencing by RNAi in Primary Cells Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as S. cerevisiae, D. melanogastor, and C. elegans but similar techniques have proven difficult to apply to mammalian systems. While manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP)using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells. 2:40 RNA Interference in the Mammalian Retina The eye is the ideal organ for the effective delivery of siRNA leading to sustained RNAi in the ocular target cells. The enclosed nature of the eye and its separation from the systemic circulation provide an ideal opportunity for local delivery. We have developed technology to achieve efficient delivery of siRNA to the cells of the mammalian retina and have shown that prolonged RNAi is mediated in these cells. 3:20 Functional Antisense Delivery using NeuGene Technology No abstract available at time of print. 4:00 Refreshment Break 4:30 Delivery of siRNA and siRNA Expression Vectors in vivo Data will be presented demonstrating the efficient delivery of siRNA and siRNA expression cassettes in vivo. Effective downregulation of specific gene expression has been obtained in small (mice) and large research animals. These delivery methods enable in vivo target validation studies and the evaluation of therapeutic RNA interference approaches. 5:10 Novel Intracellular Single-Stranded DNA Expression System and its Applications A novel expression system has been developed that can generate intracellularly any single stranded DNA (ssDNA) molecule, such as a triplex-forming oligonucleotide (TFO), antisense oligodeoxynucleotide (ODN), DNA enzyme and aptamer. 5:50 End of Workshop Wednesday, November 12, 20038:00 Registration, Poster / Exhibit Set-Up, Coffee and Pastries 8:55 Chairperson’s Opening Remarks Mechanical / Physical Delivery9:00 Non-Viral Gene Delivery for Therapeutics Gene delivery can be accomplished using an electric field to induce transient cell membrane permeability (electroporation). This approach has the distinct advantage of not requiring the inclusion of a secondary agent, e.g. a lipid, viral package or carrier protein) any of which can be immunogenic or toxic. 9:35 Pulmonary Delivery of Non-Viral Gene Therapies Using the AERx System Abstract not available at time of print. 10:10 Light-Directed Gene Delivery by Photochemical Internalisation (PCI) Photochemical Internalisation (PCI) is a technology where gene delivery can be induced by illumination, constituting a possible method for efficient, site-specific in vivo gene delivery. The technology is based on light-induced rupture of endocytic vesicles, and can enhance gene delivery with different non-viral vectors, as well as delivery of oligonucleotides and some viral vectors. 10:45 Refreshment Break and Poster / Exhibit Viewing Liposomes / Polymers11:15 Issues in the Systemic, Non-Viral Delivery of Nucleic Acids Mark E. Davis, Schlinger Professor of Chemical Engineering and Executive Officer for Chemical Engineering, California Institute of Technology Non-viral nucleic acids delivery systems have the potential to yield therapeutics that can be administered systemically. In order to do so, numerous obstacles must be overcome. Barriers in extracellular and intracellular transport will be discussed, and emphasis placed on issues that need to be addressed in creating a functioning system. Example will involve all types of nucleic acids including siRNA and plasmids. 11:50 Highly Effective DNA Nanoparticles for Intrapulmonary Gene Delivery Copernicus Therapeutics has developed DNA nanoparticles in which single molecules of DNA are condensed with PEG-substituted lysine polymers. These nanoparticles efficiently transfect post-mitotic airway cells in animals without induction of significant inflammation or an immune response. DNA nanoparticles encoding hCFTR have been administered to cystic fibrosis patients in a recent phase I/II clinical trial. No significant adverse events were observed and quite favorable gene transfer and expression endpoints were noted. Copernicus is developing DNA nanoparticles as therapeutics for cystic fibrosis and other genetic diseases. 12:25 Luncheon, Sponsored by The Knowledge Foundation, Inc. 2:00 Chairperson’s Remarks 2:05 Active Nucleic Acid Delivery to Cell Nuclei Live-cell multiple nanoparticle tracking was used to show that certain polycation/nucleic acid delivery systems are actively transported within cells to the perinuclear region within minutes (Suh et al, PNAS 2003). However, DNA unpacking from non-degradable polymeric carriers is slow, which may limit transfection efficiency in non-dividing cells. A new degradable family of polycationic nucleic acid carriers was developed that: (i) condenses DNA into sub-100 nm complexes; (ii) releases intact DNA steadily over many days in buffer; (iii) can be readily modified with targeting moities, endosomolytic peptides, NLS sequences, etc.; and (iv) exhibits reduced toxicity compared to PEI. The transport of these and other polymeric carriers through relevant biological barriers will be discussed. 2:40 Cationic Lipid Systems For Intramuscular Delivery of This presentation will describe the optimization of DNA plasmids expressing detoxified Bacillus anthracis antigens and their formulation with cationic lipid systems for intramuscular administration. The preclinical testing of the plasmid-based Anthrax vaccine in mice and rabbits will be exemplified. Product safety and preclinical pharmacology, including immunogenicity and protection against aerosolized B. anthracis spore inhalation challenge, will be reported. 3:15 Refreshment Break and Poster / Exhibit Viewing NLS Sequences3:45 Nuclear Localization Sequence (NLS) - Templated Non-Viral Gene Delivery Vectors: Investigation of Intracellular Trafficking Events of LMD and LD Vector Systems Michael Keller, Ph.D., Senior Scientist, IC-Vec Ltd, UK The impact of a nuclear localization sequence (NLS) containing peptide on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and a SV40 NLS peptide to condense pDNA prior to formulation with DC-Chol/DOPE liposomes to give LMD and LND vectors, respectively. Fluorescent labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. 4:20 Plasmid DNA Extracellular and Intracellular Targeting NLS bioconjugates and their possibility for gene intracellular targeting • Strategies for site targeted covalent binding to plasmid DNA via triple helix 4:55 Selected Oral Poster Presentations 5:15 End of Day One Thursday, November 13, 20038:15 Coffee and Pastries, Poster / Exhibit Viewing 8:55 Chairperson’s Remarks Novel Delivery Systems9:00 Bactofection - Delivery of Plasmid DNA by Bacterial Carriers Bactofection is a novel approach which exploits intracellular bacteria as delivery system for plasmid DNA. Attenuated strains of intracellular bacteria like Salmonella, Shigella and Listeria spp. can deliver eukaryotic expression vectors to human host cells. After infection of the cell, the bacterial carriers release the expression vectors, which can enter the nucleus, ultimately resulting in expression of plasmid-encoded proteins by the host cell. In vivo, bactofection with antigen expression vectors elicits humoral and cellular immune responses against plasmid-encoded antigens and protection in several animal models of infectious diseases and tumors. 9:35 Safety Features of the Sleeping Beauty Transposon System For Non-Viral Gene Therapy The Sleeping Beauty (SB) Transposon System (SB) is a highly efficacious method for inserting precise sequences of DNA into human chromosomes. SB transposons integrate semi-randomly, raising the same safety questions of insertional mutagenesis that pertain to retrovirus and lentivirus vectors. We have modified the transposons to reduce potential effects of gene activation. We have also examined the patterns of SB transposon integration and found that transposons integrate less frequently in genes and their transcriptional regulatory elements than retroviruses and lentiviruses. Together, these features suggest that SB transposons will be safer vectors than viruses for long-term expression of therapeutic genes. 10:10 The ACE System: A Chromosome-Based Platform for Adult Stem Cell Mediated Gene Therapy Chromos’ ACE System is a unique and proprietary gene expression technology that functions as a vehicle for transporting genes into the nucleus of target cells where they can be expressed to produce one or more proteins in a controlled, predictable and stable manner. In contrast to viral vectors, ACEs are maintained as stable, autonomous, and non-integrating vectors that provide high, long-term transgene expression. We are currently developing ACEs for ex vivo gene therapy applications for a variety of clinical indications. The highly efficient loading of genes, ACE isolation by flow cytometry, subsequent transfer into human mesenchymal stem cells, and transgene expression in vitro and in vivo will be described. 10:45 Refreshment Break, Poster / Exhibit Viewing Optimizing Gene Delivery11:15 DNA-Lipid Complexes for Gene Therapy: Towards a Molecular Level Understanding of Transfection Cyrus R. Safinya, Professor, Materials & Physics Departments, Biomolecular Science and Engineering Program, University of California, Santa Barbara Cationic liposomes (CLs) are used as non-viral gene vectors in worldwide human clinical protocols of gene therapy. Because our understanding of the mechanisms of action of CL-DNA complexes remains poor, transfection efficiencies remain low compared to gene delivery with viral vectors. Recent studies are described including, 3D laser-scanning confocal microscopy imaging of CL-DNA pathways and interactions with cells, and synchrotorn x-ray diffraction for structure determination. The use of these modern techniques, when combined with gene expression assays to measure transfection efficiencies, is beginning to unravel structure-function relations with new insights for enhancing transfection efficiency. 11:50 RNA- and Codon-Optimized Genes to Increase Vaccine Efficacy and Safety In addition to favorable gene delivery, the potency and safety of innovative e.g. DNA and RNA-based vaccines is in most cases largely determined by the production efficiency and intrinsic adjuvant properties of the delivered gene. By employing a rational, software assisted gene design, it is now possible to increase nuclear and cytoplasmic RNA stability, to support nuclear translocation of RNAs, to enhance translation and, at the very same time, to increase immunogenicity and safety of nucleic acid based vaccines. Supportive data on the development, preclinical evaluation and potency of HIV-1 candidate vaccines relying on RNA- and codon-optimized genes in non-human primates will be provided. 12:25 Lunch on Your Own 2:00 Chairperson’s Remarks 2:05 Enhancement of Vaccine Potency Through Improved Delivery The essence of the talk will be • facilitated delivery of DNA vaccines • formulations • physical delivery • delivery of adjuvants. 2:40 Induction of Angiogenesis by Engineered Zinc Finger Sangamo BioSciences is focused on the research and development of novel transcription factors for the regulation of gene expression. Sangamo’s proprietary technology enables the engineering of a class of DNA-binding proteins known as zinc finger DNA-binding proteins, or ZFPs. By engineering ZFPs so that they can recognize a specific gene and fusing them to a suitable functional domain, Sangamo has created ZFP transcription factors (ZFP TFs) that can control gene expression and, consequently, cell function. Our most advanced therapeutic development program involves the use of transcription factors for the treatment of cardiovascular disease. We have demonstrated that specifically designed transcription factors for ischemic cardiovascular and peripheral vascular disease delivered by plasmid DNA can regulate the endogenous VEGFA gene in vivo and evoke a therapeutic effect. 3:15 Refreshment Break, Poster / Exhibit Viewing 3:45 Genetic Adjuvants for Particle-Mediated DNA Vaccines Plasmid DNA vectors encoding the heat-labile enterotoxin from E. coli (LT) are potent adjuvants for particle-mediated DNA vaccines. Typical effects observed include a 10 to 100-fold reduction in the antigen-specific IgG1-to-IgG2a ratio and a 10-fold or greater enhancement in CD4+ and CD8+ IFN-g ELISPOT responses as well as lymphoproliferative responses. The use of particle-mediated DNA vaccine delivery to the skin allows for realization of the full adjuvant potential of LT without local or systemic toxicity. 4:20 Chemical Enhancers of Non-Viral Salivary Gland Gene Transfer Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, non-viral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable enhanced non-viral gene transfer in salivary glands and other tissues. Our efforts to identify and transcend these barriers have resulted in the discovery of a group of compounds that enhance non-viral, salivary gland gene transfer by four orders of magnitude relative to unformulated DNA. 5:00 End of Conference Call for PostersIndustry and academic scientists are encouraged to submit poster titles for this event. One-page abstracts (8 1/2” x 11” with 1-inch margins) must be submitted no later than October 20, 2003 for inclusion in conference documentation. Additional poster submissions will be accepted until November 3rd, 2003 but may not be included in conference documentation. Size of Posterboard: 3x4 feet / 90 (h)x 120 (w) cm Note: If you are submitting a poster, you MUST be registered and paid in advance to ensure that a posterboard is reserved for you.
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