Gene Delivery - non-viral Systems and in vivo Applications

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Overview

Using gene therapy for the treatment and prevention of disease on a genetic level has not yet reached its full potential.

One of the major challenges in using in vivo gene therapy is finding efficient and stable ways to introduce genes into target cells. As more efforts are focused on developing the non-viral gene transfer system, signifcant progress has been made in the area of targeted gene delivery. For example, Nanometric particles are being developed to transfer genes specifically to blood vessels and to target cells in vivo. Physical stimuli such as electroporation or using magnetic force are being optimized to enhance the gene transfer.

This 2nd annual conference addresses a variety of approaches to overcome technical hurdles to optimize and adapt gene delivery systems for specific purposes. The state-of-the-art knowledge will be presented on such topics such as:

DNA Delivery
Electroporation
Magnetofection
Synthetic Gene Transfer
Targeted Gene Delivery
In Vivo Applications
Stability of DNA Particles
Improving Gene Potency
Lipid Based Gene Delivery Platforms
RNAi

This is a great opportunity to find out what the major players in the area of non - viral gene delivery are working on. Meet this international group of experts and take advantage of their insights on how to work towards the full potential of human gene therapy.
Register early - space is limited.

Agenda

Monday, December 9, 2002

8:55 Chairperson's Opening Remarks
James Hagstrom, Vice President, Mirus Corporation

Mechanical / Physical Gene Delivery

Hydrodynamic delivery of pDNA has shown great promise as a method whereby the mammalian liver can be used as a depot to produce therapeutic amounts of a secreted transgene product. However, it is not clear how or if this approach can be made practical for gene therapy in humans. We have been using rabbits to evaluate alternative catheter-based approaches for delivering pDNA to the liver. We are also evaluating variables such as delivery volumes and rates for their effects on both expression and toxicity, with the ultimate aim of arriving at an optimal catheter-based protocol.

9:35 Naked Plasmid DNA Delivery via the Coronary Venous System for the Treatment of Ischemic Heart Disease
Hans Herweijer, Director of Pre-Clinical research, Mirus Corporation

Myocardial uptake of plasmid DNA has several advantages over virally-mediated gene transfer in the heart. Yet, this method has been limited to direct intramyocardial injections necessitating a thoracotomy or the use of complex endomyocardial mapping catheters. We report a percutaneous method for achieving high levels of gene transfer using elevated pressure retrograde coronary venous injections into specific myocardial beds.

10:10 Magnetofection: Enhancing and Targeting Gene Delivery by Magnetic Force
Christian Plank, Institute of Experimental Oncology, Technische Universitaet Muenchen, Germany*

In order to overcome the limitations to gene delivery and therapy associated with insufficient vector accumulation at target sites, we associated gene vectors with superparamagnetic nanoparticles and performed gene delivery under the influence of magnetic fields. In this manner, the vector dose was accumulated on target cells, reducing the duration of gene delivery to minutes and enhancing vector efficacy up to several thousand fold. Magnetofection is universally applicable to viral and nonviral gene vectors, to plasmid DNA and antisense oligonucleotides. Efficient transfection of otherwise resistant primary cells was achieved as well as localized gene delivery in the gastro-intestinal tract and in the vascular compartment in vivo. Magnetofection provides a novel tool for automated high throughput gene screening in vitro and can help overcome fundamental limitations to ex vivo and in vivo gene therapy. *In collaboration with Ulrike Schillinger, Franz Scherer, Martina Anton and Bernd Gaensbacher.

10:45 Refreshment Break and Poster/Exhibit Viewing

11:15 Electroporation Assisted Gene Delivery for in vivo DNA Therapy
Lei Zhang, Ph.D., Senior Scientist, Project Leader, Genetronics, Inc.

Electroporation is evolving rapidly and gaining significant momentum as a therapeutic strategy to treat inherited and acquired diseases. Among several non-viral methods, Electroporation, as a physical means, has demonstrated its superior efficiency, simplicity, and reproducibility. This talk will highlight the recent progress we have made in the development of plasmid-based gene therapy via targeting multiple accessible tissues. The potential of using a clinical grade electroporation system will also be discussed.

11:50 Plasmid Delivery to Skeletal Muscles Using in vivo Electroporation
Iacob Mathiesen, CEO of Inovio AS, Norway

Skeletal muscle is often the target of choice for genetic immunization or for production and secretion of therapeutic protein into the circulation. Efficiency of plasmid delivery to skeletal muscle is enhanced more than 100 fold if brief electrical pulses are applied at the site of DNA injection. Recent developments and experience from large and small animal studies will be discussed.

12:25 A Novel Approach to Gene-Based Therapies: Gene Delivery to the Salivary Glands and Intestines
Roland Scollay, Ph.D., President and CEO, Genteric, Inc.

Genteric uses local gene delivery to the gastro intestinal organs to deliver both systemic protein therapeutics and DNA vaccines. DNA is delivered orally to the intestines or via catheter to the salivary glands, using proprietary formulations. Data will be presented showing gene delivery with non-viral vectors resulting in therapeutic levels of proteins in the systemic circulation. In addition we will show the robust systemic and mucosal immune responses which can be generated when this approach is used for DNA vaccination.

1:00 Luncheon Sponsored by The Knowledge Foundation, Inc.

2:25 Chairperson's Remarks
Lei Zhang, Ph.D., Senior Scientist, Project Leader, Genetronics, Inc.

Targeted Gene Delivery

Tosk has expanded its gene delivery technologies to include the currently named OMG technology. This technology brings targeted gene delivery out of the cell culture environment and into the animal. Using the OMG technology to constructing knock-outs and knock-ins, as well as gene therapy applications in mice and rats will be presented.

3:05 Development and Characterization of a Lipid-Polycation-DNA (LPD) Lipopolyplex for Systemic Gene Delivery
Ralph W. Paul, Ph.D., Director, Technology Discovery, Targeted Genetics Corporation

Our goal has been the development of a gene delivery platform that is lipid based, systemically deliverable and targeted to specific cellular sites. To permit specific gene delivery and realize optimal transgene effectiveness the system must have minimal non- specific transfection and immune effects. Such a platform technology was generated by combining cationic and polyethylene glycol bearing lipids with polycation compacted DNA and specific cellular targeting ligands. The characterization and use of these formulations in xeno- and syngeneic tumor efficacy models will be illustrated.

3:40 Refreshment Break and Poster/Exhibit Viewing

4:10 Tumor Regression by Targeted Gene Delivery to the Neovasculature
David A. Cheresh, Department of Immunology, The Scripps Research Institute

Efforts to influence the biology of blood vessels by gene delivery have been hampered by a lack of targeting vectors specific for endothelial cells in diseased tissues. Here we show that a cationic nanoparticle (NP) coupled to an integrin alphavbeta3-targeting ligand can deliver genes selectively to angiogenic blood vessels in tumor-bearing mice. The therapeutic efficacy of this approach was tested by generating NPs conjugated to a mutant Raf gene, ATPmu-Raf, which blocks endothelial signaling and angiogenesis in response to multiple growth factors. Systemic injection of the NP into mice resulted in apoptosis of the tumor-associated endothelium, ultimately leading to tumor cell apoptosis and sustained regression of established primary and metastatic tumors.

4:45 Targeting of Cancer Cells with Folate-Conjugated Nanometric Particles
Jean-Paul Behr, Laboratoire de Chimie Genetique associe CNRS/Universite Louis Pasteur de Strasbourg, Faculte de Pharmacie, France

The size of condensed DNA particles is a key determinant for diffusion towards target cells in vivo as well as for subsequent intracellular trafficking. The smallest complexes are obtained when each plasmid molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C14COrn). The resulting particles were stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles remain anionic (zeta potential = -45 mV) and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended form. Moreover, the dimerized (C14COrn)2 lipid was found to be an efficient transfection reagent for various cell lines.
In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-Folate residues. DNA was condensed into monomolecular particles as described above and was coated by mixing with DPPE-PEG-Folate. Physicochemical data showed particles coated with 2% of DPPE-PEG3400-Folate to remain monomolecular and to be stable in the cell-culture medium. Caveolae-mediated cell entry into folate receptor-expressing cells was observed by confocal microscopy.

5:20 End of Day One

Tuesday, December 10, 2002

8:55 Chairperson's Remarks
Suzie Hwang Pun, Senior Scientist, Insert Therapeutics, Inc.

Optimizing Gene Delivery

Most plasmids contain a cDNA as the gene for a given therapeutic protein. cDNA's are expressed, however, at lower levels than their genomic counterparts, and may add an inefficiency factor to non-viral gene delivery. Therefore cDNA's must be optimized to produce high levels of a single transcript that is optimally accessed and translated by the cellular machinery. To this end we have focussed on ways to improve cDNA expression by engineering sequence elements in both coding and non-coding regions of a cDNA.

9:35 What Viral Vectors have to Learn from Synthetics: Parsing Parameter Space to Optimize Function
David Schaffer, Ph.D., Assistant Professor, Department of Chemical Engineering & The Helen Wills Neuroscience Institute, University of California at Berkeley

Both viral and synthetic vectors have made significant progress over the past decade. Viral vectors harness the evolved abilities of their parent viruses to deliver genes, but they have a number of shortcomings that stem from the fact that never never intended them to serve as therapeutics. We are implementing a chemical and genetic engineering to enhance their functions, in essence using a synthetic approach to optimize viral vectors.

In vivo application

Intradigm has optimized nucleic acid delivery into animal disease tissues for gene expression or inhibition with very low vector background. The unique technology combines disease perturbation with pathway analysis to discover disease-controlling protein that are drug targets and drug candidates, and operates directly in animal disease models. The technology yields targets controlling complex, multi-cellular pathways missed by high throughput cell-based methods.

10:45 Refreshment Break and Poster / Exhibit Viewing

11:15 Development of Gene-Matrix Combinations for Tissue Regeneration
Barbara Sosnowski, Ph.D., Vice President Technology Development, Selective Genetics, Inc.

We have utilized a delivery system to injured tissue, in which a relevant gene is placed within a biodegradable matrix and is taken up by the tissue repair and progenitor cells migrating into the matrix. These gene activated matrices have the potential to regenerate tissue defects which occur in skin, develop collateral arteries in ischemic tissue and build new bone.

11:50 In Vivo Properties of a Liposomal Delivery System for Antisense and Immunostimulatory Oligonucleotides
Sean Semple, Senior Scientist, Inex Pharmaceuticals Corp.

We have developed an intravenous delivery system for polynucleotides that uses an ionizable aminolipid to facilitate encapsulation, thereby minimizing positive charge and toxicity in final formulation. Studies in rodents and non-human primates have shown this formulation to be well tolerated. The in vivo properties of this delivery system will be discussed, using oligonucleotides as examples. Particular emphasis will be given to biological activity and mechanism of action in vivo.

12:25 Using Microorganism Proteomes for DNA Vaccine Antigen Discovery
Dr. Philip L. Felgner, Chief Scientific Officer, Gene Therapy Systems, Inc.

Protein subunit vaccines and DNA vaccines are safer vaccine alternatives compared to live attenuated or killed microorganism vaccines, but technology for identifying the affective antigens for use in subunit vaccines is lacking. Gene Therapy Systems, Inc. has developed high-throughput approaches for making PCR fragments transcriptionally active so that they can be rapidly expressed in a variety of different expression systems. These methods can be used to generate complete proteomes consisting of 4-6,000 individual purified proteins from different microorganisms within two to three weeks. The proteomes can be used to scan the immune responses from infected individuals, and bioinformatic approaches can be applied to identifying the antigens that are necessary for protection. This technology is being applied to the development of subunit and DNA vaccines against malaria, tuberculosis, anthrax, and smallpox. A "Peptide Nucleic Acid Dependent Gene Chemistry" approach also developed at Gene Therapy Systems may improve the delivery of DNA vaccines to make them more effective.

1:00 Lunch on Your Own

2:25 Chairperson's Remarks
Jean-Paul Behr, Laboratoire de Chimie Genetique associe CNRS/Universite Louis Pasteur de Strasbourg, Faculte de Pharmacie, France

RNAi

Small interfering RNAs (siRNA) are short double stranded RNA fragments which when delivered into mammalian cells, can facilitate highly efficient, gene-specific inhibition of expression. This inhibitory effect, termed RNA interference (RNAi) is very robust, reaching 90% for the targeted gene. However, the ability to achieve efficient RNAi activity is dependent on effective delivery. This talk will highlight our recent work in developing technologies for effectively delivering siRNAs into both mammalian cells in culture as well as tissues in adult mice.
Polymers

3:05 Polymeric Delivery System for Intracellular Delivery of Biomolecular Therapeutics
Patrick S. Stayton, Dept. of Biochemistry, University of Washington*

The biotechnology and pharmaceutical industries have developed a wide variety of potential therapeutics based on the molecules of biology: DNA, RNA and proteins. While these therapeutics have tremendous potential, effectively formulating and delivering them has also been a widely recognized challenge. We have developed new ŇsmartÓ pH-responsive polymeric carrier systems that incorporate biomolecular drugs such as plasmid and antisense DNA and enhance their intracellular delivery through a biomimetic strategy. *In collaboration with Allan S, Hoffman, University of Washington.

3:40 Refreshment Break and Poster / Exhibit Viewing

4:00 Tumor Targeted, Cyclodextrin Polycation-Based Gene Vector for Systemic Delivery
Suzie Hwang Pun, Senior Scientist, Insert Therapeutics, Inc.

Abstract not available at time of print. Please go to www.knowledgefoundation.com for the latest program updates.

4:35 Biopolymers as Tools to Facilitate Delivery of Nucleic Acids
Robert G. Urban, Ph.D., VP Technology,ZYCOS, Inc.

Biopolymers have proven to be useful in the delivery of pharmaceutical compounds in a number of clinical settings. More recently the protective and time-releasing properties of these polymers have been explored as a formulations chemistries for gene delivery. A collection of experiences in developing these systems for in vivo target validation and then into human clinical testing will be reviewed.

5:10 Receptor-Mediated Gene Delivery
Revati Tatake, Ph.D., Principal Scientist, Boehringer Ingelheim Pharmaceuticals, Inc.

Cell surface receptors can be exploited to enhance gene delivery into cells. This receptor-mediated endocytosis has been used to markedly increase the efficiency of transfection in mammalian cells.

5:45 End of Conference

Call for Posters

Call for Posters
Industry 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 November 18, 2002 for inclusion in conference documentation. Additional poster submissions will be accepted until December 4, 2002 but may not be included in conference documentation.

Size of Posterboard: 3x4 feet (90 x 120 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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