Discovery Strategies 2001
Novel Mechanisms for Regulating Gene Expression in vivo
The post-genomics era is increasing the demand for new tools and technologies that improve our understanding of how genes and proteins interact at the level of the whole organism. The mouse continues to be a platform of choice for evaluating the effects of altering gene expression in vivo. However, problems of low throughput, embryonic lethality, toxicity, immune system recognition, tissue specific gene expression and therapeutic protein delivery have yet to be completely overcome. This conference will present some of the new technologies, and newly improved methodologies for altering gene expression and delivering therapeutic gene products to the whole animal. Topics include ribozymes, adenovirus vectors, large scale, high throughput knockout/in and transgenic generation, tissue specific promoters, temporal and spatial regulation, and antisense or antigene triplet forming oligonucleotides.
Speaker List:
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Molly Bogue |
Mouse Phenome Project in Discovery Research and Database Demonstration |
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Carlo Catapano |
Targeting Oncogenic Transcription Factors with Triplex DNA Forming Oligonucleotides |
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Sheila Connelly |
Improved Adenoviral Vectors for Hemophelia Gene Therapy |
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Nick Dean |
Gene Knock-down and Functionalization in Animals Using Antisense Oligonucleotides |
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Peter Glazer |
Specific Mutations Induced by Triplex-forming Oligonucleotides in Mice |
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John Murphy |
Viral Vectors as Tools for Functional Genomics |
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Pam Pavco |
Development of Synthetic Ribozymes for Therapeutic Uses |
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Richard Pestell |
Ponasterone Regulated Transgenics to Study Breast Cancer in the Mouse |
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Victor Rivera |
Regulated Delivery of Secreted Proteins |
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John Rossi |
Strategies for Enhancing the Biological Efficacy of Intracellulary Expressed Ribozymes |
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Alexy Terskikh |
Fluorescent Timer: A New Tool For Monitoring Promoter Activity Over Time |
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Flossie Wong-Staal |
An Inverse Genomics Approach for Gene Discovery |
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Kevin Seburn |
High-throughput Phenotyping: Screening Mice for Neurological Mutations in the JAX-Neuroscience Mutagenesis Facility |
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Targeting Oncogenic Transcription Factors with Triplex DNA Forming Oligonucleotides
Carlo Catapano, MD, PhD
MUSC, Hollings Cancer Center
Triplex forming oligonucleotides that bind to homopurine:homopyrimidine sequences in DNA in a sequence-specific manner can be used as selective gene repressors. This strategy has proven to be successful in various experimental systems and may provide the means to design novel DNA sequence-specific and molecular targeted cancer therapeutics. Data relative to the targeting of oncogenic transcription factors, such as c-myc and ets2, in cancer cells will be presented. These genes are frequently over-expressed in cancer cells and have important roles in pathogenesis and progression of a variety of cancer types. Downregulation of the expression of these genes by triplex forming oligonucleotides effectively inhibits growth and induces apoptotic death of cancer cells. The strategy underlying the design and use of triplex forming oligonucleotides, potential pitfalls and possible applications of this approach to prevention and therapy of cancer will be discussed.
Improved Adenoviral Vectors for Hemophelia Gene Therapy
Sheila Connelly
Novartis Pharmaceutical
Adenoviral vectors provide a promising system for the treatment of hemophilia A, a common congenital bleeding disorder resulting from a deficiency of blood coagulation factor VIII (FVIII). We have generated potent human FVIII-encoding adenoviral vectors which, when delivered intravenously, achieve physiological levels of FVIII in mice, dogs, and monkeys. Vector administration to hemophiliac mice and dogs resulted in complete phenotypic correction, and in mice, therapeutic FVIII levels were maintained for at least one year. In hemophiliac dogs, expression was short-term due to an immune response against the FVIII protein. In normal monkeys, administration of an epitope-tagged human FVIII-encoding vector resulted in expression of physiological levels of FVIII, sustained for 14-28 days. Analysis of liver biopsy samples revealed the presence of vector DNA at all time points in the study, days 7, 28, and 56. The monkeys displayed a variable antibody response to the vector-mediated FVIII expression. One animal developed a low-level transient inhibitor, one animal displayed an inhibitor titer that continued to increase for the duration of the study, and two animals did not develop FVIII inhibitory antibodies. As a step to achieve more sustained expression in large animal models, and toward reducing vector hepatotoxicity and immunogenicity, we have developed a gutless adenoviral vector. Gutless vectors are devoid of all viral genes and must be grown in the presence of a helper virus, usually an E1-deficient early generation adenoviral vector. The helper virus supplies the structural proteins required for gutless vector replication and packaging. The FVIII gutless vector grew to high titers, and could be reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome, and helper virus contamination levels were reproducibly <0.5%. Comparison of the gutless vector to an early generation, Av3 vector, following IV administration to FVIII-deficient mice revealed expression of 10-fold higher FVIII levels in the gutless vector-treated animals which were sustained for at least 10 months. In contrast, mice that received the Av3 vector displayed FVIII levels at or below the limit of sensitivity of the assay at 3 months. A dose-response study comparing FVIII expression levels and vector-induced liver toxicity of both vectors revealed 10-20-fold higher FVIII expression levels in mice treated with the gutless vector at all doses analyzed. Furthermore, liver toxicity, measured by serum levels of liver enzymes, revealed that the gutless vector was significantly less toxic than the Av3 vector. These data suggest that the gutless FVIII vector is improved over early generation vectors with respect to level and duration of FVIII expression, and the superior liver toxicity profile. Current effort is focused on the development of vector production technology and vector evaluation in clinically relevant large animal models.
Gene Knock-down and Functionalization in Animals Using Antisense Oligonucleotides
Nicholas M. Dean, Ph.D.
Isis Pharmaceutical
The increase in both the amount, & rate of accumulation of genomic information currently available has created new & difficult challenges for the pharmaceutical industry. These include how best to rapidly & efficiently, identify genes responsible for complex diseased phenotypes and how to fully utilize this information to develop new & specific classes of drugs. Key in this process is the ability to determine gene function in animals. Genetic techniques for regulating gene expression in animals can be difficult and time consuming. Recent advances in antisense oligonucleotide chemistry & lead sequence selection have greatly facilitated the use of antisense for in vivo studies. Unlike genetic knock-outs, antisense effects can be regulated like any pharmacological agent – for example gene expression can be titrated by using different antisense doses. In addition, multiple antisense can be dosed simultaneously allowing for multiple gene knock-downs. Combined with DNA array analysis and other phenotypic assays, this approach offers a powerful technology to both identify novel cellular networks & signaling “cassettes� in animals, as well as to validate genes as attractive drug targets.
Specific Mutations Induced by Triplex-forming Oligonucleotides in Mice
Peter Glazer
Yale University
Triplex-forming oligonucleotides (TFOs) recognize and bind to specific duplex DNA sequences and have been used extensively to modify gene function in cells. Although germline mutations can be incorporated using embryonic stem cell technology, little progress has been made toward introducing mutations in somatic cells of living organisms. Here we demonstrate that TFOs can induce mutations at specific genomic sites in somatic cells of adult mice. Mutation detection was facilitated using transgenic mice bearing chromosomal copies of the supF and cII reporter genes. Mice treated with a supF-targeted TFO displayed ~5-fold greater mutation frequencies in the supF gene compared to mice treated with a scrambled sequence control oligomer. No mutagenesis was detected in the control gene (cII) with either oligonucleotide. These results demonstrate that site-specific, TFO-directed genome modification can be accomplished in intact animals.
Viral Vectors as Tools for Functional Genomics
John E. Murphy, Ph.D.
Bayer Biotechnology
Adenoviral and adeno-associated viral (AAV) vectors are being used to study the efficacy of candidate protein therapuetics in mouse disease models. One of the primary advantages of viral vectors is that the protocol for production of the vector is independent of the gene expressed by the vector. Once introduced into an animal or cell, the viral vector directs production of the desired protein for a sustained period of time. Thus, one can rapidly proceed from novel genes to testing function in an animal model without developing a customized protein purification system for each new gene product.
The choice of a vector system is dependent on the ultimate application. Adenoviral vectors are easy to produce, can efficiently infect most cell types in vivo and in vitro and lead to high level gene expression when administered to an animal by various routes. Early generation adenoviral vectors induce an inflammatory response that limits the duration of transgene expression and complicates interpretation of in vivo results. While difficult to produce, AAV vectors have been shown to lead to life-long transgene expression with little inflammatory response. Thus, first generation adenoviral vectors are most appropriate for studies of short duration (1-2 weeks), while AAV vectors are best suited for studying transgene effects in chronic or genetic disease models. Specific examples of the use of adenoviral vectors in angiogensis, cancer and fibrosis models and AAV vectors in asthma and obesity models will illustrate the strengths and limitations of each system.
Development of Synthetic Ribozymes for Therapeutic Uses
Pamela A. Pavco
Ribozyme Pharmaceutical, Inc.
The use of ribozymes to reduce gene expression represents a novel approach to the treatment of disease. Ribozymes are enzymatic RNA molecules that can be designed to cleave a specific target RNA sequence and thus may offer significant specificity advantages over other approaches. Synthetic ribozymes are chemically modified to improve their resistance to nuclease degradation while retaining catalytic cleavage activity. The screening of nuclease resistant ribozymes in the context of several therapeutic targets has resulted in lead ribozymes with significant activity in cell culture and efficacy in multiple in vivo models. These ribozymes have also demonstrated 4a safe toxicology profile in rodents and primates and a plasma pharmacokinetic profile that supports subcutaneous dosing. Four ribozymes are currently in development.
ANGIOZYME™ targets the mRNA of the VEGF receptor-1 (VEGF-R1) to act as a potential antiangiogenic agent. Preclinically, treatment with ANGIOZYME results in specific reduction of VEGF-R1 mRNA, inhibition of endothelial cell growth in cell culture and significant inhibition of primary tumor growth and metastasis in mouse models. ANGIOZYME was well tolerated in Phase I and I/II trials and is currently being evaluated in several Phase II trials. The antiviral ribozyme HEPTAZYME™ targets a site within the highly conserved 5’-untranslated region (UTR) of the Hepatitis C virus (HCV). In cell culture, treatment with HEPTAZYME inhibited replication of an HCV-poliovirus chimera where the HCV 5’ UTR is required for translation. HEPTAZYME™ was well tolerated in Phase I and I/II clinical studies and is entering a Phase II trial. HERZYME™ targets the proto-oncogene HER2/neu which is overexpressed in ~30% of breast and ovarian cancers. In cultured cells, treatment with HERZYME inhibited cancer cell proliferation and reduced levels of HER2 mRNA. Interestingly, HERZYME is a novel ribozyme motif, a Zinzyme™, developed using in vitro selection to have improved cleavage activity under physiologic conditions. HepBzyme™ targets a conserved site present in several overlapping Hepatitis B viral (HBV) mRNA transcripts. Antiviral activity of HepBzyme has been demonstrated in cell culture by a decrease in viral proteins (HBsAg, HBeAg) and HBV DNA following treatment. In an HBV transgenic mouse model, treatment with HepBzyme significantly reduced viremia as compared to control-treated animals.
Ponasterone Regulated Transgenics to Study Breast Cancer in the Mouse
Richard G. Pestell, M.B.B.S, M.D., Ph.D., F.R.A.C.P
Albert Einstein Cancer Center
The ability to regulate temporal and spatial specific expression of target genes in transgenic mice will facilitate analysis of gene function and enable the generation of murine models of human diseases. The genetic analysis of mammary gland tumorigenesis requires the development of mammary gland-specific transgenics that are tightly regulated throughout the adult mammary epithelium. Analysis of genes implicated in mammary gland tumorigenesis has been hampered by mosaic transgene expression and the findings that homozygous deletion of several candidate genes (cyclin D1, Stat5A, prolactin receptor) abrogates normal mammary gland development. We have developed transgenic mouse lines in which sustained transgene expression is inducibly regulated, both specifically and homogeneously in the adult mammary gland epithelium. Transgenes encoding RXRa and a chimeric ecdysone receptor, were placed under control of a modified MMTV-LTR, to target mammary gland expression. These transgenic "receptor" lines were crossed with transgenic "enhancer" lines in which the ecdysone/RXR binding site induced ligand-dependent expression of transgenic b-galactosidase. Pharmacokinetic analysis of a highly bioactive ligand (Ponasterone A), identified through screening ecdysteroids from local plants, demonstrated sustained release and transgene expression in vivo. We have subsequently generated receptor lines that are also transgenic for mammary targeted oncogenes. We have constructed enhancer lines expressing oncogenes or dominant negative mutants of signaling pathways implicated in the induction and/or maintenance of mammary tumorigenesis. Transgenic models with both tightly regulated and homogeneous transgene expression and sustained in vivo using ligands readily extracted from local flora, have broad practical applicability for genetic analysis of mammary gland disease.
Regulated Delivery of Secreted Proteins
Victor Rivera, Ph.D.
ARIAD Pharmaceuticals
Safe and effective delivery of secreted therapeutic proteins by gene therapy will require regulatory systems that permit the natural level and kinetics of protein expression to be reproduced. Delivery of proteins such as erythropoietin (Epo), for the treatment of anemia, should occur on a timescale of days or weeks. To control expression of proteins with these kinetics we have developed a system for pharmacologic control at the level of transcription. Gene expression is made dependent upon a transcription factor whose activity is regulated by the orally bioavailable drug rapamycin, or non-immunosuppressive derivatives (rapalogs). Using this system, long-term, tightly regulated expression of Epo has been demonstrated in mice upon intramuscular delivery using AAV vectors. Regulated expression of Epo for over 2 years has also been achieved in non-human primates. Recently we developed transcription factors with greatly enhanced activation potency, to improve the persistence and robustness of protein expression in vivo. The new factors also allow delivery of the entire regulatory system using a single retroviral vector. Cells transduced with this single virus system display negligible basal expression, permitting delivery of highly toxic proteins, and give induction ratios of at least 3 orders of magnitude in response to a rapalog.
Delivery of proteins such as insulin, for the treatment of diabetes, or parathyroid hormone, for the treatment of osteoporosis, must occur rapidly and transiently, on a time scale of hours. To control expression of proteins with these kinetics we have developed a system that allows direct pharmacologic control of protein secretion. By engineering the protein so that it accumulates as aggregates in the endoplasmic reticulum, secretion can be stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid, transient production of insulin has been achieved in vitro and in vivo, resulting in a transient correction of glucose levels in hyperglycemic mice. The latest data from preclinical evaluation of these systems in rodent and primate models will be presented.
Strategies for Enhancing the Biological Efficacy of Intracellulary Expressed Ribozymes
John Rossi, Ph.D.
Beckman Research Institute
Ribozyme testing in vitro depends upon the free diffusion of ribozymes and target RNA to achieve functional interactions. The potential for ribozyme- substrate interaction inside of cells is complicated by the fact that protein-RNA interactions govern all RNA movement. Proteins interact with all classes of RNA from the moment they are synthesized until they are degraded. For many RNAs it is known that movement from the nucleus to their final destination follows specific trafficking networks or patterns. In order to make trans-acting ribozymes more effective in cells, we have developed two strategies for enhancing ribozyme-target interactions. The first strategy is to identify accessible sites for ribozyme pairing on native RNAs. To do this we have created a novel scheme for identifying ribozyme-pairing targets on RNAs in cell extracts. This approach takes advantage of protein-RNA interactions that either block or enhance ribozyme accessibility. The sites that are most accessible in extracts are also the sites in cell culture, and in vivo. Using this procedure we have identified accessible sites six different RNAs ranging from HIV Tat through MDM2 and the DMPK mRNA that is associated with myotonic dystrophy. Results from several of these studies will be reviewed. Equally important to site accessibility is the co-localization of ribozyme and target RNAs. Several examples of ribozyme-target co-localization will be presented. The strategies for co-localization rely upon cis-appended RNA sequences that direct the ribozyme transcript to the same sub-cellular compartment as the target RNA. Examples will be presented demonstrating the use of 3’ untranslated regions that have “zipcode� sequences that traffic and localize RNAs to specific sub-cytoplasmic localization. In transgenic fruitflies expressing a target RNA with either the Drosophila oskar or bicoid 3’ UTRs, we observed strong co-localization of the ribozyme and target in oocytes that was accompanied by a two to three fold enhancement of ribozyme function. Another co-localization strategy utilizes a small nucleolar RNA to direct a ribozyme to the nucleolus through which HIV RNAs traffic.
Overall, we have developed useful strategies for optimizing ribozyme efficacy in cells. These strategies should make it possible to develop effective ribozymes for target validation and therapeutic applications.
Fluorescent Timer: A New Tool For Monitoring Promoter Activity Over Time
Alexey Terskikh, Ph.D.
Stanford University
We discovered a mutant of the red fluorescent protein drFP583 (called Fluorescent Timer) that changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression, e.g. for in vivo labeling to measure expression from the heat-shock-dependent promoter in C. elegans to the Otx-2 promoter in developing Xenopus embryos. Importantly, the fluorescent timer can be used to monitor both activation and downregulation of target promoters on the whole-organism scale.
An Inverse Genomics Approach for Gene Discovery
Dr. Flossie Wong-Staal
UC San Diego & Immusol
We have developed a novel inverse genomics approach based on a random hairpin ribozyme library. Expression of this library in cells and subsequent selection for a given phenotype yield single ribozymes responsible for induction of that phenotype. The target recognition sequences of these ribozymes are then used to identify the corresponding target gene(s). We have used this strategy to identify cellular genes of interest in several systems relevant to cancer and infectious diseases, including: tumor suppressor genes, regulatory genes for BRCA1, and genes involved in HCV and HIV replication. All candidate genes were validated by demonstrating that ribozymes designed against additional sites in the mRNA conferred the same phenotypic changes.
