Richard J. Samulski - US grants

Adeno-associated virus is a dependent parvovirus requiring coinfection with another virus in order to undergo a productive infection in cultured cells. In the absence of coinfection with helper virus, the AAV genome integrates via its ends into the host genome and can later be "rescued" upon infection of the latent cell with adenovirus. The overall objective of the proposed work is to utilize the infectious AAV recombinant clone to understand the mechanism(s) of integration and rescue in human cells. Three goals are proposed: (1) To determine whether AAV-encoded activities are required for specific integration a AAV DNA (in a way that permits subsequent rescue). Preliminary experiments suggest that this may be the case. Two approaches will be taken to address this problem. First, the latently infected Detroit 6 cell line will be infected with a selectable AAV virus, AAV/NEO. The resulting neo cell lines will then be assayed for rescue upon superinfection with adenovirus. Second, cell lines expressing specific AAV genes will be generated and assayed as described for the detroit 6 line. (2) To develop an efficient system for retrieving and cloning integrated AAV sequences, and to then determine the sequences at the viral-host junctions. The lambda repressor-binding sequence will be introduced into an AAV recombinant clone, and the feasibility of retrieving a single copy of this DNA from genomic DNA by binding to a filter containing the repressor will be tested. If successful, the procedure will be utilized to clone a number of integration sites and they will be analyzed. And (3) To utilize bacterial cells for the recovery of products of the rescue process from HeLa cells transfected with the infectios clone. The products will then be analyzed to obtain clues about possible rescue mechanisms. If the products of the rescue process can be routinely isolated and identified the assay will be utilized to study the requirements for AAV rescue.

Adeno-associated virus (AAV) is a dependent parvovirus. That is, it requires coinfection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In the absence of coinfection with helper virus, the AAV genome integrates via its ends into the host genome in a site specific manner and resides there in a latent state until the cell is infected with helper virus. Then the AAV DNA is "rescued", replicates and establishes a normal productive infection. AAV has a broad host range for infectivity, it is ubiquitous in humans, it can be concentrated to titers exceeding 109 infectious units per milliliter, and is a completely nonpathogenic integrating viruses. The availability of a infectious recombinant AAV clone, helper-free packaging system, and site-specific integration have provided us with a manipulable system which has tremendous potential as a eucaryotic viral vector. The overall objective of the proposed work is to fully test the feasibility of AAV as a cell type transducing viral vector. The immediate specific aims of the proposed research are as follows: 1. To characterize AAV as a vector in some key specific cell types in culture. 2. To test the feasibility of using AAV hybrid virions for the introduction and expression of genes in the animal. 3. To test animal model systems for specific delivery and correction of well characterized genetic defects.

One of the great aspirations of gene therapy is to eventually develop technology which will provide a feasible approach to correct genetic defects and combat infectious diseases. My laboratory is engaged in studying the molecular biology of the defective human parvovirus adeno- addressed virus (AAV) in hopes of developing a safe efficient viral vector for human gene therapy. AAV is a dependent parvovirus. That is, it requires coinfection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In a lytic infection, AAV DNA replicates as a 4.7 kilobase double-stranded molecule and is packaged into virion as linear single strands of both polarities. In the absence of coinfection with helper virus, the AAV genome integrates via its ends into the host genome in a site specific manner and resides there in a latent state until the cell is infected with helper virus. Then the AAV DNA is "rescued", replicates and establishes a normal productive infection. AAV has a broad host range for infectivity (human, monkey, mouse, etc.) when coinfected with the appropriate helper. In fact, compared to the current retroviral vectors these features of AAV are of considerable interest in utilizing AAV as a viral vector. Human AAV has a number of advantages. Some of them are: (1) it is ubiquitous in humans, (2) AAV can be concentrated to titers exceeding 10(9) infectious units per milliliter, and (3) it is completely nonpathogenic integrating virus. Ongoing research is revealing that this nonpathogenic human virus is now accessible for utilization as a vector. We have developed a packaging system which allows for efficient encapsidation of foreign genes into AAV virions. We have also identified the essential cis-acting sequences required for efficient integration into host cell DNA. Finally, we have characterized wild type integration and uncovered the exciting result of site-specific integration. This last observation clearly sets apart AAV as a eucaryotic viral vector and it's potential for gene therapy in humans. The overall objective of the proposed work is to fully test the feasibility of AAV as a specific transducing viral vector for globin gene therapy.

One of the great aspirations of gene therapy is to eventually develop technology which will provide a feasible approach to correct genetic defects and combat infectious diseases. My laboratory is engaged in studying the molecular biology of the defective human parvovirus adeno- addressed virus (AAV) in hopes of developing a safe efficient viral vector for human gene therapy. AAV is a dependent parvovirus. That is, it requires coinfection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In a lytic infection, AAV DNA replicates as a 4.7 kilobase double-stranded molecule and is packaged into virion as linear single strands of both polarities. In the absence of coinfection with helper virus, the AAV genome integrates via its ends into the host genome in a site specific manner and resides there in a latent state until the cell is infected with helper virus. Then the AAV DNA is "rescued", replicates and establishes a normal productive infection. AAV has a broad host range for infectivity (human, monkey, mouse, etc.) when coinfected with the appropriate helper. In fact, compared to the current retroviral vectors these features of AAV are of considerable interest in utilizing AAV as a viral vector. Human AAV has a number of advantages. Some of them are: (1) it is ubiquitous in humans, (2) AAV can be concentrated to titers exceeding 10(9) infectious units per milliliter, and (3) it is completely nonpathogenic integrating virus. Ongoing research is revealing that this nonpathogenic human virus is now accessible for utilization as a vector. We have developed a packaging system which allows for efficient encapsidation of foreign genes into AAV virions. We have also identified the essential cis-acting sequences required for efficient integration into host cell DNA. Finally, we have characterized wild type integration and uncovered the exciting result of site-specific integration. This last observation clearly sets apart AAV as a eucaryotic viral vector and it's potential for gene therapy in humans. The overall objective of the proposed work is to fully test the feasibility of AAV as a specific transducing viral vector for globin gene therapy.

Adeno-associated virus (AAV) is a dependent parvovirus. That is, it requires coinfection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In the absence of coinfection with helper virus, the AAV genome integrates via its ends into the host genome in a site specific manner and resides there in a latent state until the cell is infected with helper virus. Then the AAV DNA is "rescued", replicates and establishes a normal productive infection. AAV has a broad host range for infectivity, it is ubiquitous in humans, it can be concentrated to titers exceeding 109 infectious units per milliliter, and is a completely nonpathogenic integrating viruses. The availability of a infectious recombinant AAV clone, helper-free packaging system, and site-specific integration have provided us with a manipulable system which has tremendous potential as a eucaryotic viral vector. The overall objective of the proposed work is to fully test the feasibility of AAV as a cell type transducing viral vector. The immediate specific aims of the proposed research are as follows: 1. To characterize AAV as a vector in some key specific cell types in culture. 2. To test the feasibility of using AAV hybrid virions for the introduction and expression of genes in the animal. 3. To test animal model systems for specific delivery and correction of well characterized genetic defects.

virus; microscopy; genome; biomedical resource; bioengineering /biomedical engineering; biomaterials;

DESCRIPTION (Taken directly from the application) Long term treatment of cystic fibrosis (CF) through gene therapy will require that the transgene be stably maintained within the transduced cell population. Further, the vector should not express viral antigens which will elicit an immune response leading to the destruction of the transduced cells. One of the most promising viral vector systems currently being developed for the treatment of CF is based on recombinant adeno-associated virus (rAAV). The AAV genome contains terminal repeat (TR) sequences at each end which promote the integration of the genome into the host chromosome. Recombinant AAV vectors offer two advantages over other viral vector systems: they can be maintained in the integrated state for the lifetime of the transduced cell and its progeny; and, the vectors can be constructed such that no viral genes remain to be expressed and to elicit an immune response. However, rAAV vectors which retain the viral rep gene are specifically integrated into a site on human chromosome 19. This represents an important safety feature in vectors with this property. Disadvantages of rAAV vectors are a stringent size limitation for packaging and difficulty in growing high titer virus stocks of suitable purity. We have created a new DNA vector, double D, which utilizes modified AAV TRs to promote integration but can be propagated as a plasmid in bacteria. This hybrid vector thus solves the problems of transgene coding capacity and production of large quantities of purified vector. We propose to develop methods for the efficient delivery of this vector into cultured cells, including airway epithelial cells, using liposome mediated transfection. This work will also entail the transient co-delivery of the AAV Rep protein in bans such that integration of the vector will be directed to the chromosome 19 site. Finally, we will utilize the double D vector to deliver the CFTR gene into cells and determine the magnitude and duration of its Expression.

DESCRIPTION (Abstract of the application) Currently, a rate limiting step to-the emerging field of gene therapy has been inefficient vector transduction. While many advances have been made in the development of various delivery systems, including retrovirus, adenovirus, herpes virus, adeno-associated virus (AAV), lenti virus, and non-viral vectors, these systems still lack the efficiency required for successful delivery in vivo. Furthermore, it will be critical to target viral vectors to specific cell types, and to dividing as well as non-dividing cells, for successful gene therapy. Along with modifying vectors for specific cell targeting, it will be essential to maintain proper endocytosis along with improved efficiency. In the proposed work we will explore the use of the small parvovirus, AAV, as a cell-specific targeting vector by manipulating the viral capsid proteins. Since the AAV virion is composed of only 3 capsid proteins encoded by overlapping reading frames, it should provide a simple system for genetic manipulation. The goal of this research is to identify and alter the region(s) of AAV capsid(s) that govern(s) its normal host range and to identify a region(s) that can be manipulated to display foreign epitopes on the surface of the capsid. Ultimately, these genetically modified capsid vectors will be tested for directed gene delivery in cultured cells with the long range goal of efficient targeted delivery in vivo.

This project is studying the molecular basis for virus adhesion, translation and disruption at surfaces. The atomic force microscope is being used to image surface bound viruses, to measure the lateral forces needed to release and translate the particles, and to apply quantitative force loads to study the elasticity of the virus capsid. The molecular level details of these properties will be informed by the molecular modification of the virus capsid by the group in Gene Therapy, while substrates will be modified by techniques developed in the Superfine Group. Over the past year we have made substantial progress in the imaging of biological systems under liquid. We have developed to techniques for AFM solution imaging: magnetic resonance and photothermal modulation. For the latter, we have a publication in press that describes our technique for oscillating the cantilever using a modulated diode laser. This is a compact, readily available technique that should find applicability in many laboratories. We have established sample preparation techniques that are being combined with adsorption and desorption experiments to get solution diffusion constants and binding energies. We have manipulated surface bound adenoviruses successfully without apparent damage to the viruses and without adhesion of the viruses to the AFM tip. Our next steps in the project involve the quantitative manipulation to get lateral binding forces, quantification of the adsorption/desorption and the molecular modification of substrates.

One of the great aspirations of gene therapy is to eventually develop technology which will provide a feasible approach to correct genetic defects and combat infectious diseases. We are engaged in studying the molecular biology of the defective human parvovirus adeno-associated virus (AAV) in hopes of developing a safe and efficient viral vector for human gene therapy. AAV is a dependent parvovirus which requires co- infection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In the absence of co-infection with helper virus, wild type (wt) AAV genome integrates via its ends into the host chromosome in a site-specific manner and resides there in a latent state until the cell is infected with helper virus. The interest in AAV as a eukaryotic vector has centered around the biology of this virus. In addition to this unique life-cycle, AAV has a broad host range for infectivity (human, mouse, monkey, etc.), it is ubiquitous in humans, and is completely nonpathogenic integrating virus. Our research pioneered the use of recombinant AAV (rAAV) as a gene delivery system for hemopoietic cells. We initiated studies testing both human globin genes as well as the Fanconi anemia C gene (FAC). Previous efforts utilizing the globin locus control region (LCR) sequence has met with difficulty using other vector systems. We have demonstrated high level, tissue specific expression of human globin genes in human erythroid cells, where expression and globin protein was detected. Although globin expression was detected after rAAV infection, transduction was low in vitro. In continued analysis of this system, we demonstrated expression and correction of lymphoblast cells from a Fanconi patient using an AAV vector carrying the Fanconi anemia gene (FAC). Utilization of this vector in patient CD34 enriched bone marrow cells demonstrated gene transduction and selective growth advantage in colony forming assay. These studies were extended to non-human primates bone marrow cells with preliminary results suggesting gene marking out to 3 months. This last observation and data from in vitro systems suggests a rate limiting step in efficient transduction of rAVV in hematopoietic progenitor cells is the lack of stable integration. The overall objective of the proposed work is to study integration in bone marrow stem cells using the well characterized wt AAv site-specific integration system and a new animal model we have developed. The long range goal is to better understand these molecular step in primary bone marrow stem cells with the ultimate goal of developing specific viral vectors with efficient transducing capability as a result of targeted integration.

A variety of virus vectors have established that effective gene delivery can be attained in non-dividing mammalian cells. For example, adeno- associated virus (AAV) vectors have been shown to stably transfer and express foreign genes in brain and muscle with little or no accompanying toxicity (McCown et al., 1996; Xiac et al., 1996). Based upon these advances, the present center grant will focus upon the use of AAV and adenovirus (AD) vectors to deliver and express genes proposed to ameliorate the adverse effects of chronic ethanol exposure, both in brain and liver. Therefore, the role of the vector core will be able to construct the cDNA cassettes, insert these cassettes into AAV of Ad vector plasmids, replicate and package the virus vectors, and finally test the function of the vectors, both in vitro and in vivo. Specifically, in Dr. Crews' section, AAV-tyrosine hydroxylase or tryptophan hydroxylase vectors will be prepared to test the involvement of dopaminergic and/or serotonergic function in rodent models of ethanol preference. In Dr. Thurman's section, AAV vectors will be prepared for the delivery of superoxide dismutase (SOD)/catalase, in order to evaluate the origin of ethanol induced oxidative damage to the liver, while Dr. Sulik will use the same AAV vectors to probe the mechanisms of ethanol teratogenicity. For Dr. Brenner, AD vectors will be prepared in order to identify the role of the immediate early genes, APl and NFkappaB, in ethanol-induced ethanol liver pathology. For Dr. Murrow, both sense and antisense AAV vectors will be prepared to delineate the role of GABAa receptor subunits in ethanol self administration, and finally, AAV vectors with antisense constructs to diazepam-binding inhibitor (DBI) and corticotrophan-releasing hormone (CRF) will be prepared for Dr. Breese to probe the role of these factors in the evolution of ethanol withdrawal anxiety. Thus, the vector core will provide the means to investigate molecular mechanisms directly involved in ethanol-induced pathologies, both in the brain and in the liver.

Current approaches to transfer genes in vivo employ neither recombinant viral vectors or non-viral delivery systems. Adeno-associated viral (AAV) vectors are non-pathogenic integrating vectors that infect both dividing and non-dividing cells. Since all viral genes are removed (96 percent of the viral genome), packaging of foreign DNA up to 5 kb can be incorporated into these vectors. Recently Dr. Samulski and his colleagues demonstrated transduction of rAAV vector expressing B- galactosidase after direct injection in rodent muscle. Vector DNA and transduced gene expression was detected for over one year without significant immune response. There was no evidence of vector toxicity in any animal treated suggesting the ability to safely and stably transfer sequences into muscle as an attractive platform for gene therapy. These studies have been extended into a large animal model (hemophilic FIX dog) using FIX sequences in place of B-galactosidase. Evidence for stable gene expression now out to 16 weeks suggests successful long term vector delivery. Analysis of vector spread was localized to site of injection and draining lymph node with infiltrating immune cells at the site of injection. Ironically this infiltration correlated with residual inactivated A helper virus and not AAV transduced cells, suggesting further improvement in vector production is still required, a focus of this grant. Analysis of gene expression follows an atypical expression curve with onset of expression 10 to 21 days after vector delivery. In addition, gene expression appears to climb with time suggesting that molecular events that may be related to viral replication are taking place. These observations of stable long term gene expression, assumed to be obtained by vector integration, is in contrast to low level replication over four months. While these results demonstrate the first example of AVV productive gene therapy in a large animal, better elucidation of the molecular fate of the vector genome is required in order for safe translation into a clinical setting. Finally, these results strongly suggest that muscle is an attractive site for AAV transduction, and that further analysis of vector preps carrying various modified FIX genes devoid of helper Ad proteins will provide meaningful preclinical information that should facilitate AAV gene therapy for this genetic disorder.

Vector Core Facility (R. Judge Samulski, Ph.D., Faculty Director; Gabriele Kroner-Lox, Ph.D., Facility Director) Started with institutional funds, the Vector Core was initiated in 1993 as part of the new UNC Gene Therapy Center with the intent of establishing a state-of-the-art facility capable of generating novel viral vectors for efficient gene delivery. The Core's specific purposes are to: maintain and distributed a library of recombinant adenovirus (rAd), recombinant adeno-associated virus (rAAV) vectors and retroviral vectors; generate new rAd, rAAV and retroviral vectors from plasmid substrates containing genes cloned by individual investigators; produce common vector stocks from viral plaques (rAd), recombinant plasmids (rAAV) or established producer cell lines (retroviruses); provide consultation on the use, design, and production of different gene delivery vectors; and supply investigators with high quality vector preparations. To date, the Vector Core has not received any Cancer Center Support Grant (CCSG) funding, although peer-reviewed LCCC members currently represent over 40% of the Vector Core's users. CCSG funding of $72, 308 (17% of total budget) will primarily support the Facility Director (50%), who is critical to making the vectors used in clinical trials, and the Core's skilled Research Technician (75%). This support will ensure that prices remain low for recombinant adenoviral vectors (one-third the cost of commercial sources) and that the facility continues to provide viral vector reagents (AAV, Lentiviral, targeting vectors) for cancer-specific research that are otherwise unavailable commercially.

One of the great aspirations of gene therapy is to develop technology that will provide a feasible approach to correct genetic defects and combat infectious diseases. We are engaged in studying the molecular biology of the human parvovirus adeno-associated virus (AAV) with the intent to develop a safe and efficient viral vector for human gene therapy. AAV is a dependent parvovirus which requires co-infection with another virus (either adenovirus or certain members of the herpes virus group) to undergo a productive infection in cultured cells. In the absence of co-infection with helper virus, the wild type (wt) AAV genome integrates via its ends into a specific host chromosomal site and remains latent until helper virus infection. The interest in AAV as a vector has centered around the biology of this virus. In addition to its unique life-cycle, AAV has a broad host range for infectivity (human, mouse, monkey, dog, etc.), it is ubiquitous in humans, and is completely non-pathogenic. Our research pioneered the use of recombinant AAV (rAAV) as a gene delivery system for central nervous system, and muscle cells, with vector expression for over 1.5 years without immune consequences or vector toxicity. With respect to the airway system, the subject of this proposal, we initiated studies that uncovered a rate limiting steps involved in vector transduction. This rate limiting step, which involved second strand synthesis of the single-stranded viral genome, could be augmented by genetic (Ad open reading frame (ORF) 6) Physical (heat shock, UV, X-ray irradiation) and chemical steps (hydroxyl urea, butyrate, etc.). This has lead to a better understanding for the initial event involved in AAV infection and more practically, has resulted in a new approach for generating Ad free rAAV preps. Our continued efforts to dissect the primary steps involved in AAV infection has recently led to the identification of the AAV receptor. We are now poised to take advantage of this information in determining in vivo which cells are true targets for AAV infection, mapping the epitopes on the virus capsid involved in binding, and utilizing this information for purifying AAV vectors. Identification of the AAV receptor, rate limiting for vector transduction and ability to efficiently transduce primary brain and muscle cells, but not airway cells in vivo, has provided a unique paradigm for studying the molecular steps involved in efficient vector transduction. The overall objective of the proposed work is to study the primary steps involved in rAAV transduction and persistence in airway cells using highly defined rAAV preps, a unique in vitro airway model, and hybrid targeting vectors capable of carrying genes larger than 5.0 kb. The long range objective is to better understand these molecular steps in primary airway cells with the ultimate goal of developing specific viral vectors with efficient targeted transducing capability of CFTR.

ABSTRACT (provided by the applicant) The goal of this PPG, Gene Therapy for Pulmonary and Hematologic Disorders, is to facilitate translation of basic knowledge of gene delivery to safe and rigorous human clinical trials. The long-range goal is to provide novel therapeutic modalities for treating monogenetic diseases such as Hemophilia and Cystic Fibrosis. The major objectives in the UNC PPG are: (1) the development of highly efficient and high titer viral vectors, optimum expression, and safe persisting delivery systems, and (2) development of novel animal models for better defining rate limiting steps involved in target cell transduction, analysis of long term high level vector gene expression, and expression of normal and mutant human genes (e.g. FIX, CFTR). Four basic science projects and four cores are proposed. The basic research projects are proposed to focus on understanding rate-limiting steps in effective gene transfer. Projects 1 and 2 focus on AAV (Samulski) and Lentivirus (Olsen and Kafri), respectively. These projects are aimed at understanding and overcoming inefficient gene delivery related to virus entry and persistent transgene expression. The goal of the proposed studies is to generate new knowledge about the safety and biological efficacy of gene delivery, which will provide information important to the design of future clinical trials. Projects 3 and 4 involve animal models for airway (Boucher) and hemophilia (Stafford) disorders, respectively. These studies aim to define the host-associated rate limiting step(s) for understanding of the cell biology of the target tissue for efficient in vivo gene delivery and translating improvements in vector development. Specifically, goals include increasing access to airway epithelia (Project 3), development of novel models (e.g. a humanized hemophilia mouse, Project 4). This work will be supported by four cores, Administrative (Core A), Vector (Core B), Animal (Core C), and Morphology (Core D). The PPG is a highly interactive program comprised of clinical investigators and expert basic laboratories designed to optimize vectors and to test their interactions with target cells in vitro and in vivo. The ultimate goal of the UNC PPG is to achieve safe, effective, gene delivery in humans.

DESCRIPTION (provided by applicant) Current approaches to transfer genes in vivo employ either recombinant viral vectors or non-viral delivery systems. We are engaged in studying the molecular biology of the human parvovirus adeno-associated virus (AAV) with the intent to using this virus as a platform for developing a novel, safe, and efficient delivery system for human gene therapy. Our research pioneered the use of? recombinant AAV (rAAV) as a gene delivery system for central nervous system, and muscle cells demonstrating vector expression for over 1.5 years without immune consequences or vector toxicity. While promising, these studies uncovered rate limiting steps involved in AAV vector transduction; namely receptor mediated entry, and conversion of singled-stranded viral genomes to double-stranded expressing templates (i.e. second-strand synthesis). In addition to this rate-limiting step, the finite packaging capacity of this virus (4.5kb) has restricted the use of this vector to small genes or cDNAs. To advance the prospects of efficient AAV gene delivery, vectors sufficient to carry larger genes must be developed. In addition, virions that specifically and efficiently target defined cell types will be required for clinical application. The focus of this grant will be to address these issues. Three approaches will be analyzed in this proposal to overcome AAV?s problem of viral entry, inefficient second-strand synthesis, and packaging constraints. We have generated exciting new mouse data supporting differential infectivity in type I vs. type II muscle using traditional AAV type II vectors, as well as increased transduction when using other AAV serotype specific vectors. These results emphasize the importance of receptor mediated AAV entry. Mapping and characterization of other AAV serotype capsid domains required for entry will be explored to facilitate more infectious vectors. AAV type II as well as serotype specific vectors 1-5 will be tested in the Chapel Hill canine model for efficient FIX gene delivery in an effort to extend current mouse studies in a large animal model. We have now developed a method for generating AAV vectors carrying duplex viral genomes. These reagents will be characterized for efficient transgene expression after vector delivery in an effort to study rate-limiting steps involved in second-strand synthesis and vector gene expression in vivo. Finally, efforts to engineer vectors that carry twice the packaging capacity of wild type AAV will be explored by using split gene vectors that rely on hetero-dimer concatemers after vector infection. The long term objective is to develop novel delivery systems that exploit the advantages of AAV viral infectivity without the disadvantages of inefficient viral entry, rate limiting steps involved in second-strand synthesis, or packaging constraints.

DESCRIPTION (provided by applicant): The development of transgenic and knockout mice and the use of viral gene delivery have allowed major advances in alcohol research and in basic research in general. Transgenic and knockout mice have provided extremely useful molecular tools to investigate the genetic basis of pathological conditions and will be used in this center grant to address critical mechanistic questions related to alcohol-induced pathogenesis in the brain and liver. Recent developments with viral vectors have allowed investigators to use these for gene delivery in vivo and in vitro as research tools aimed at elucidating the molecular and genetic basis of pathological conditions. The Viral Vector and Genotyping Core will provide center members a unique and novel opportunity to utilize recombinant viral vectors to analyze mechanisms involved in ethanol-induced psychological behavior and ethanol-induced brain and liver pathology. This core will assist center members who do not have a solid foundation in viral mediated gene transfer techniques with the necessary expertise from experienced colleagues, facilities, and experimental reagents. The Viral Vector and Genotyping Core will supply center members with expert assistance in the construction of adeno-associated virus (AAV) and adenovirus (AD) vectors and will provide high titer viral preparations necessary to investigate aspects or neural and hepatic ethanol induced pathologies not easily accomplished by inexperienced investigators. The Core will also provide critical genotyping assistance for monitoring genotype integrity of the experimental animals. The specific aims for this Core will provide each investigator with the necessary expertise, facilities, and procedures needed to produce AAV and AD vectors and will meet the demands for validating the genetics of the research animals to accomplish the goals of the proposed investigations.

One of the great aspirations of gene therapy is to develop technology that will provide a feasible approach to correct genetic defects and combat infectious diseases. We are engaged in studying the molecular biology of the human parvovirus adeno-associated virus (AAV) with the intent to develop a safe and efficient viral vector for human gene therapy. AAV is a dependent parvovirus which requires co-infection with another virus [either adenovirus (Ad) or certain members of the herpes virus group] to undergo a productive infection in cultured cells. In addition to its unique life-cycle, AAV has a broad host range for infectivity (human, mouse, monkey, dog, etc.), it is ubiquitous in humans, and is completely nonpathogenic. Our research pioneered the use of recombinant AAV (rAAV) as a gene delivery system for central nervous system, and muscle cells, with vector expression for over 1.5 years without immune consequences or vector toxicity. We initiated studies that uncovered rate limiting steps involved in vector transduction. This has resulted in a new approach for generating Ad free rAAV preps. Our continued efforts to dissect the primary steps involved in AAV infection has recently led to the identification of the AAV 2 receptor. These advances and the development of novel AAV serotypes have provided a tool box like approach to engineer airway specific AAV vectors. Identification of the AAV receptor, rate limiting steps for vector transduction and ability to efficiently transduce primary brain and muscle cells, but not airway cells in vivo, has provided a unique paradigm for studying the molecular steps involved in efficient vector transduction. The overall objective of the proposed work is to study the primary steps involved in rAAV transduction and persistence in airway cells using chimeric AAV capsid evolved to specifically infect human airway epithelial cells. The long range objective is to better understand these molecular steps in primary airway cells with the ultimate goal of developing specific viral vectors with efficient targeted transducing capability of CFTR.

DESCRIPTION (provided by applicant): The goal of the PPG (Gene Therapy for Cystic Fibrosis) is to create gene transfer vectors that will efficiently transduce cells of the lung. Major but not exclusive therapeutic targets are the epithelia of the large and small airways, which are the sites of cystic fibrosis lung disease. The major hypothesis tested in the PPG are (1) new vectors are needed, including higher capacity, better expressing AAV vectors; high titer, safe lentiviral vectors; and adenoviral vectors specifically targeted to airway epithelial receptors; and (2) that a rate-limiting variable for gene transfer efficiency in the lung is at the site of initial vector-cell interaction, including both binding and entry across the plasma membrane. Three projects and four Cores are proposed. Project 1 (Parvovirus Vectors for Airway Delivery, R.J. Samulski, P.I) proposes to design and produce new AAV vectors that increase the vector packaging size, augment the efficiency of vector entry, and increase the efficiency of expression (conversion from single strand to double strand DNA templates) using chimeric virion capsids, targeting ligands and modified viral terminal repeats. Project II (Equine Lentiviral Vector for Gene Delivery, J.C. Olsen, P.I.) proposes to develop high titer, efficiently expressing, and safe equine lentiviral vectors. Project III (Cell Biology of Airway Epithelial Gene Transfer, Raymond Pickles/R.C. Boucher and M. Peeples, PI) proposes to define the barriers and targets in the apical domain of airway epithelia, modify the barriers using either oxidant injury or more specific modulators of the tight junctions, and finally, modify vectors to target a class of receptors on the apical membrane that exhibit cellular internalization in response to agonist addition. The projects are supported by an Administrative Core (core A), a Cell Culture Core (Core B), a Vector Core (Core C) and an Imaging Core (Core D). The PPG is a highly interactive program designed to modify vectors and test their interactions with target cells in vitro and in murine models in vivo.

[unreadable] DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD), which affects one in 3500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death due to a lack of expression of the protein dystrophin in muscle tissues. Genetic replacement of defective protein with a functional, miniaturized form of dystrophin represents a promising approach for the treatment of DMD. The aforementioned minidystrophin transgene is currently being evaluated in human clinical trials using AAV vectors as agents for intramuscular delivery. The current exploratory/developmental research proposal is focused on the design and development of novel AAV vectors with enhanced gene delivery efficiency in dystrophic skeletal muscle using the mdx mouse model. Rationale for the exploratory phase of the translational research hinges on the overexpression of integrin a7[unreadable]1 in dystrophic muscle and our seminal finding that modulation of a putative integrin-binding domain on AAV2 capsids affects their ability to transduce normal skeletal muscle. We have formulated specific design strategies based on the aforementioned finding that include: (a) rational engineering of the integrin-binding domain to develop integrin a7[unreadable]1-targeted AAV vectors that transduce dystrophic muscle with high efficiency and (b) combinatorial engineering of AAV vectors that efficiently transduce dystrophic muscle in mdx mice. During the developmental phase, novel AAV vectors selected through this process will be utilized to deliver an enhanced minidystrophin transgene for therapeutic application in the mdx mouse model. Research design involves rational insertion peptide ligands to generate integrin a7[unreadable]1-targeted AAV vectors, directed evolution of dystrophic muscle-specific vectors obtained from an AAV mutant library administered into mdx mice through different routes of administration, and therapeutic application of an enhanced mini-dystrophin transgene construct in the mdx mouse model. The novel reagents developed and optimized in this proposal will be advanced for further therapeutic testing in larger animal models of muscular dystrophy and eventually in human muscular dystrophy clinical trials in the near future. Duchenne Muscular Dystrophy (DMD), which affects one in 3500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death due to a lack of expression of the protein dystrophin in muscle tissues. The current exploratory/developmental research proposal is focused on the design and development of novel AAV vectors with enhanced gene delivery efficiency in dystrophic skeletal muscle using the mdx mouse model. The novel reagents developed and optimized in this proposal will be advanced for further therapeutic testing in larger animal models of muscular dystrophy and eventually in human clinical trials in the near future. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The NIH Senator Paul B. Wellstone Centers are the centerpiece of the nation's effort to reduce morbidity and mortality from muscular disorders and are a major source of discovery and development of more effective approaches to prevention, diagnosis, and therapy. The University of North Carolina (UNC) "goal" of establishing a premier Wellstone Center in the state of North Carolina has assembled a highly interactive research group creating a dynamic program comprised of experienced clinical investigators (Drs. Powers &Wolff), expert basic laboratories (Drs. Samulski, Xiao, and Beecham), and large animal models (Dr. Kornegay) focused on developing, testing, and establishing therapeutic treatments for Duchenne Muscular Dystrophy (DMD) and other genetic muscle disorders. A major theme of the Center is to advance novel gene based therapies into the clinic for muscle disorders. The impetus for this effort stems from the fact that UNC Gene Therapy Center (GTC) has made significant strides in recent years to build the foundation that will enable development of a full-scale bench-to-bedside effort bringing gene therapy for DMD closer to a reality (e.g. GMP vector production facility, DMD dog model core). In the spirit of this mission, UNC with support from MDA has independently initiated the first gene-based therapy for DMD using a newly engineered AAV vector (Dr. Samulski) and a mini-dystrophin (dys) gene (Dr. Xiao). A common theme emerging from our Phase I studies and a primary focus of the proposal, relates to establishing a clear understanding of safety and efficacy after treatment with gene based therapeutics and advancing these efforts into selected human clinical trials that will show potential for treatment. Continued discussion with FDA for most efficient and prudent manner to advance our gene therapy based efforts to DMD patients with minimum risk and maximum benefit has lead to the proposed MDCRC Wellstone Center application. The MDCRC will consist of 3 projects (Project 1 PI- Dr. Powers coPI Dr. Wolff -, Project 2 PI-Dr. Xiao, Project 3 PI- Dr. Samulski, (Projects 1-3) and four cores: an Administrative core (PI-Dr. Jude Samulski, Co-PI-Dr. Powers) Training Core, Vector Core (PI Dr. Jeffrey Beecham) and Large animal core (PI- Dr. Kornegay).

DESCRIPTION (provided by applicant): The available data of AAV vectors in the clinic emphasize the importance of continued optimization efforts at the levels of the AAV capsid, genome and transgenic cassette. A focus of this proposal is to derive clinical AAV vector best suited for systemic disorders (MPS, Hurlers, etc.). At the capsid level, it is apparent that animal models do not always predict the human outcome and that more efficient human specific capsids are required to achieve a lower administered dose. In Aim 1, we seek to create a new paradigm of AAV vector selection for human transduction by generating the first AAV receptor expression map on tissues of mouse, primate and human origin. This tissue specific AAV receptor Atlas will be overlaid with AAV binding and transduction data in an effort to tease out regions of the capsid important for tissue specific interactions in varied backgrounds. In addition novel chimeric capsids isolated from a directed evolution strategy on primate and human livers established in a mouse model will be triaged against our receptor/binding atlas to determine if in vitro binding correlates to in vivo results. Then, capsid isolates from a primatized-liver mouse model will be investigated for primate liver transduction in vivo to determine if this strategy represents a valid method to derive primate (human & non human) liver specific AAV capsids. At the level of the AAV genome, we have assembled a panel of DNA repair dependent AAV substrates that report critical aspects of genome persistence including circularization, concatemerization and homology directed annealing. Investigations of these reagents in mutant backgrounds defective in different DNA repair pathways will offer insights into the preferred reliance on homologous recombination and non-homologous end joining mechanisms in vitro and in vivo providing a better prediction of vector performance in diseased settings (Aim 2). At the level of the vector transgene, we demonstrate in mouse liver, heart and eye a novel method to induce transgene synthesis using the IVS2- 654 intron and an anti-sense oligonucleotide. The work herein seeks to generate smaller synthetic variants that exhibit tighter control as well as altered transgene expression levels, thus providing a panel of regulatory switches which can be tailored for specific applications. Finally, a strategy is proposed to engineer an off switch for the induced transgene synthesis from IVS2-654, which may also allow the precise tuning of transgene synthesis at a fixed vector dose. Collectively, the results of the proposed experiments seek to address the observed clinical deficiencies in AAV gene therapy applications for diseases of systemic nature by our continued optimization efforts at the levels of the capsid and genome as well as the transgenic DNA cassette.

Adeno-associated Virus Vectors for Targeted and Repeat Delivery Project 1 "Adeno-associated Virus Vectors for Targeted and Repeat Delivery" will capitalize on AAV vector results derived from the on-going AAV Phase I DMD clinical trial. The advent of novel AAV vectors (serotype 1-11) and the solving of the capsid crystal structure, has allowed us and others to carry out rational design of AAV capsids to generate laboratory strains of gene delivery vehicles. Using such an approach, we have been successful in engineering AAV type 2 liver tropic vectors into a muscle tropic virus using only 5 amino acids derived from AAV type 1 capsid backbone. This novel chimeric reagent (AAV 2.5) has a distinct immune profile when compared to capsid backbone of parent serotypes and has high efficiency for muscle transduction. Due to the advantages of this chimeric AAV particle we recently initiated a Phase I Gene Therapy clinical trial for DMD with Dr. Xiao. Early information derived from these efforts has provided hypothesis driven approaches to better understand basic steps such as immune response to viral capsid, viral /receptor interaction, intra-cellular trafficking, and transgene expression. In the proposed studies, we hope to capitalize on these observations by deriving a collection of disease tropic vectors with novel immune profile that will allow for repeat administration. These objectives will be carried out by availability to AAV DNA capsid library technology, selection against patient neutralizing serum (including on-going AAV clinical trial), and use of large animal models carrying muscle specific disease traits (Project 3). Our objective is to better understand the molecular mechanism of efficient vector muscle transduction that will allow these and other novel delivery reagents for skeletal and heart specific muscle gene therapy. Success of these studies will facilitate the immediate goals of Project 2 and the long-term objective of initiating clinical trial for muscle disorders.

DESCRIPTION (provided by applicant): Proper transgene expression is essential to achieve therapeutic effect and ensure safety in many gene therapy strategies. Current regulation systems for controlling transgene expression typically require use of a special promoter and co-delivery of a cassette expressing a trans-activator protein. While these systems have been proven effective, the requirements prohibit differential regulation of multiple transgenes. Additionally, the requirement of co-delivering a trans-activator adds significantly to the payload required of the chosen gene transfer vector. To develop a new type of regulation system without such drawbacks, we are exploring a novel regulation system based on alternative splicing. To date, we have developed minimal alternative splicing introns capable of regulating transgene expression. Furthermore, we have demonstrated the feasibility of using two different ASO to independently control the expression of two different transgenes. Using AAV vector for delivering our novel regulation system, we have successfully expressed a marker gene in mouse eye in a controlled manner. Therefore, we are proposing to further develop our novel regulation system and apply the resulting technology for gene therapy of ocular diseases. Our hypothesis is that our novel regulation system could be developed to independently regulate the expression of multiple transgenes, and that differential expression of multiple potent factors inhibiting angiogenesis via different pathways would have a synergistic effect in treating ocular neovascularization. Our long-term goal is to develop novel delivery systems that exploit the advantages of AAV mediated persistent gene transfer with the ability to control the expression of the therapeutic genes being delivered. The studies described in this proposal would allow us to further optimize our novel regulation system suitable for gene therapy of clinically relevant diseases. The Specific Aims of the proposal are as follows: 1. Study the kinetics of transgene expression for our inducible system in an eye model. We have already successfully demonstrated our inducible system both in vitro and in vivo. We will focus on using marker genes in an eye model to study the kinetics including the onset, duration and level of transgene expression under various conditions of induction. 2. Develop our inducible system to differentially regulate the expression of multiple transgenes. In our regulation system, transgene expression is controlled by using antisense oligonucleotides targeting alternative splice site to modulate the alternative splicing of transgene message. Since the alternative splice site and its flanking sequences can be varied, we will develop introns with different sequences to allow differential regulation of multiple transgenes. 3. Regulate the expression of multiple transgenes in eye models. We will first validate our proposed multiple-transgene regulation system using marker genes in normal eyes and then differentially regulate the expression of multiple potent blockers for survival factors supporting neovascularization in an eye disease model in an effort to better validate this novel regulation system. PUBLIC HEALTH RELEVANCE: Regulating transgene expression is essential to achieve therapeutic effect and ensure safety in many gene therapy strategies. We are developing a novel regulation system to independently regulate the expression of multiple transgenes, such as those encoding potent factors inhibiting angiogenesis via different pathways and potentially having a synergistic effect in treating ocular neovascularization.

DESCRIPTION (provided by applicant): Alpha-1 antitrypsin (AAT) deficiency, due to the piZZ mutation, results in life-threatening lung and liver diseases in children and adults. Currently, gene addition therapy is being tested to prevent lung disease. However, this strategy does not halt liver disease progression due to the accumulation of mutant PiZZ protein in the endoplasmic reticulum of liver cells rather than normal secretion into the blood and body fluids. From our previous two year R01 application funded from the ARRA (R01DK084033), we have generated a substantial dataset, demonstrating that : 1) AAV vectors can efficiently deliver self-complimentary shRNA to knock down piZZ AAT expression at both mRNA and protein levels, resulting in improved liver pathology of piZZ mice. 2) Alternative combinatorial approaches can be used to both knockdown piZZ expression and restore functional, circulating AAT; These approaches include liver AAV-shRNA delivery followed by muscular injection of an AAV-wtAAT cassette, dual vector administration into the liver with independent vectors for shRNA and an optimized AAT cassette that avoids shRNA-based degradation, and finally delivery of a single vector that simultaneously delivers shRNA and optimized AAT into the liver. The dual-targeting strategy applied in these experiments simultaneously prevents both liver and lung disease development in AAT deficiencies. However, AAV delivery of shRNA faces three obstacles: capsid specific CTL-mediated elimination of AAV-transduced liver cells, potential off-target effects of shRNA in non-targeted, transduced tissue, and the high prevalence of neutralizing antibodies (Nab) to AAV in the human population. In response to these obstacles, the primary objectives of this renewal proposal are to: (i) investigate the effect of AAV capsid specific CTL-mediated killing on AAV-transduced liver cells in piZZ mice after treatment with scAAV/shRNA vector (ii) develop novel approaches to exclusively control shRNA expression in the liver of piZZ mice (iii) generate AAV mutants capable of liver-specific transduction and evasion of neutralizing antibody activity. The long-term goal of the proposal is to design safer and more effective AAV vectors for gene therapy in patients with AAT deficiency.

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is a very promising gene therapy vector in pre-clinical and clinical trials. However, recent studies have demonstrated that the AAV2 capsid can induce a cytotoxic T lymphocyte (CTL) response via both classical antigen presentation and cross-presentation pathways, thereby raising concerns associated with immune response to AAV vectors. In particular, it has been suggested that capsid specific CTLs eliminated AAV2 transduced liver cells and resulted in therapeutic failure in a hemophilia B clinical trial. The goal of this proposal is to understand the mechanisms of presentation of AAV capsid antigens in vitro and in vivo and more importantly, to devise strategies to evade the immune response. Our long term goals are to enhance the safety and efficacy of AAV vectors through formulation of novel immune evasion strategies. Since data from animal models have contradicted clinical observations outlined above (possibly due to poor immunogenicity of the AAV capsid in mice), we have integrated a strong immune domain OVA epitope SIINFEKL into the VP3 protein of the AAV2 capsid (AAV2-OVA). Decreased transgene expression was seen in mice with memory OVA CTLs following liver transduction with AAV2-OVA vector. In the current proposal, we will use AAV2-OVA vector to investigate the kinetics and mechanisms of AAV capsid cross-presentation in transduced cells in vitro and in vivo (Aim 1 and 2). Through these studies, we expect to thoroughly characterize the CTL response to AAV capsid proteins in mice that more accurately represents data obtained in humans. After AAV vector binds on cell surface, via endosomal uptake, AAV2 capsids must uncoat enroute to the nucleus prior to vector genome transcription. This trafficking route suggests that antigen presentation of capsids after transduction may follow a classical MHC-class I pathways. Many viruses (For example herpes, EB) evade host immune response by synthesizing small peptides called viral proteins interfering with antigen presentation (VIPR), we propose that integration of VIPR into AAV capsid evade host CTL mediated elimination of AAV transduced target cells (Aim 3). By engineering VIPRs into AAV2 capsid proteins, we will ensure that antigen presentation will be attenuated only in AAV2 transduced cells without systemic side effects on the immune system (as would be the case with immunosuppressive drugs or application of regulator T cells). These experiments rely on predetermined domains in AAV capsid proteins for incorporation of VIPR domains. A timely understanding is critical for the continued use of AAV therapy under the current protocols (i.e. without immunosuppression addendums).

DESCRIPTION (provided by applicant): The American Society of Gene & Cell Therapy (ASGCT) was founded in 1996. ASGCT's mission is to advance knowledge, awareness, and education leading to the discovery and clinical application of genetic and cellular therapies to alleviate human disease. Our Annual Meeting represents the major educational initiative of the Society and has been CME-accredited for all of our prior 14 meetings. This R13 proposal requests support for travel grants for trainees. Over 1/4 of our members are scientists-in-training working to develop clinically applicable gene- based technologies. Trainee participation in the Annual Meeting is fostered by an outstanding educational program, trainee travel awards, and recognition of outstanding scientific accomplishments through peer-reviewed trainee Excellence in Research Awards. Over the past decade, the ASGCT has offered 470 travel grants and 63 Research Awards, with almost 190 travel grants made possible by R13 grant awards from the NIH during that same time. Educational opportunities for travel awardees include exceptional plenary speakers, state-of-the-art scientific symposia, and educational sessions that review current thinking on a variety of topics. In addition to leading scientists and clinicians, the program includes ethicists and representatives from the FDA, OBA, and NIH so young scientists may gain insight into the compliance and ethical issues related to human gene therapy and cell therapy. Trainees are active presenters in oral abstract and poster presentations, and the top trainee abstracts are recognized at the Presidential Symposium. The size of the ASGCT meeting (approximately 1,700 participants) is ideally suited to expose young scientists to leaders in the field yet provide opportunities for trainees to present their work at the premier meeting in the field of gene and cell therapy. NIH support for the ASGCT Annual Meeting will allow continued educational and professional advancement of trainees in the field of cell and gene therapy and the opportunity to network with leaders in the field. PUBLIC HEALTH RELEVANCE: The American Society of Gene & Cell Therapy (ASGCT) is seeking funding for 20 travel awards for post-doctoral fellows and students who submit scientific abstracts for its 15th Annual Meeting in May 2012. Travel awards are given to students and post-doctoral fellows who apply for the awards and who receive the highest scores during the peer review process.

Adeno-associated virus (AAV) vectors have been explored extensively in numerous pre-clinical studies with varying degrees of success. A numberof these pre-clinical studies are now translating into encouraging results in phase l/ll clinical trials. In fact most recently, self-complementary (sc)AAV vectors have demonstrated sustained and potentially therapeutic levels of Factor IX in Phase I study of hemophilic B patients negative for pre-existing /\AV Ab (ASH Nov 2010 &ASCGT 2011). With 90% ofthe human population naturally infected by AAV2, and 30-50% expressing NAb, contiuned success will require resolution of this problem. For these reasons Project 1 has focused on role of N/\b in AAVFIX gene transfer and describes a proposal to better understand the biology of Ab response to AAV capsids and to engineer novel molecular approaches to overcome these limitations. HLA class II phenotypes play a major role in antigen presentation to induce antibody production. Although almost every individual produces AAV specific immunoglobulin, NAb is only detectable in half of the population, indicating that not all /\AV-specific Abs produced possess neutralizing activity. To determine which HLA class II phenotypes are associated with AAV NAb production, we will screen the HLA-class II phenotypes, AAV specific immunoglobulins and AAV NAb profile in large number of human subjects (Aim 1). HLA-class II molecules bind to peptides of 12 to 18 aa and present the complex on antigen presenting cells to induce a humoral immune response. To further elucidate w/hich epitopes from AAV capsid are bound by NAb, we will use sera from St. Jude FIX trial (see let of support, Nathawni), AAV immunized HLA-class II transgenic mice and humans with NAb positive serum for this study (Aim 2). There are several approaches being considered to evade /\AV NAb including chemical and genetic modification of AAV virion in the presence of neutralizing antibodies or randomly mutating AAV capsid. Based on the information from eiptope mapping, a novel aptamer selection against Ab will be explored (Aim 3). The broad objective of this proposal is to advance our understanding of capsid antigen presentation and examine therapies to circumvent major limitations imposed by systemic humoral immunity. RELEVANCE (See instructions): Program Project 1 objective is to better understand /V^V capsid antigen biology in novel animal models of bleeding disorders and explore molecular approaches in hope of extending the clinical success seen with scAAV FIX.

DESCRIPTION (provided by applicant): The program project grant Neutralizing Antibody (NAb) and AAV FIX Gene Therapy is a timely focused application centered around advancing exciting new clinical data that self complementary (sc)AAV Factor IX (FIX) vectors are showing promising success in clinical studies (5-8% >1yr). To further advance these successes, we have assembled a multidisciplinary approach to understand, address, and resolve the role of pre-existing NAb to AAV capsid (over 80% of general population). A primary focus of PPG relates to molecular interactions between viral capsid in antibody response, engineering next generation vectors, and development and testing in novel NAb positive animal models. This PPG consists of 4 projects and 3 cores. Project 1 FIX Gene Therapy and Role of AAV NAb (PI - Dr. R. Jude Samulski) will capitalize on the availability of St. Jude FIX trial serum samples, humanized mouse models and peptide library to understand relationship between capsid antigen presentation and Ab response. Project 2 Humanizing AAV Vectors for Gene Therapy (PI- Dr. Aravind Asokan) will focus on engineering and utilizing novel AAV vectors in an effort to avoid NAb. Project 3 Intra-articular AAV to Circumvent Systemic Neutralization (PI - Dr. Paul Monahan) will utilize PPG gene transfer reagents to understand and prevent joint complications of hemophilia in animal models with pre-existing NAb. Project 4 Generation of a Model of Hemophilia B and AAV Resistant Primates (PIs Bruce Sullenger/Dougald Monroe): will use aptamer technology to generate and validate hemophilia in Ab+ non-human primate model for gene correction with PPG vectors, along with Administrative core (Pl-Dr. Samulski, Co-Dr. Monahan), vector and animal cores (Pls-Drs. Beecham & Li). This PPG is composed of unique group of researchers with expertise in biochemistry (Dr. Monroe), molecular biology (Dr. Sullenger), virology (Drs. Asokan & Samulski), and clinical hematology (Dr. Monahan) working synergistically to bring direct benefit of vector development (AAV), molecular therapeutics (sc-opt FIX and opt FVlll), and novel NAb non-human primate hemophilic model (aptamer) to bleeding disorders. The long-term objective of this PPG is to advance basic understanding of vector-cell-animal model interactions for safe gene delivery.

DESCRIPTION (provided by applicant): Our recent clinical trial using adeno-associated viral vectors (AAV) to deliver Mini-Dystrophin to the muscle of patients with Duchenne muscular dystrophy (DMD) was met with an unexpected result; after treatment, a Dystrophin-specific T-cell response was found in two patients, which was related to revertant fiber development prior to therapy. These Dystrophin-specific T cells have the potential to eradicate all genetically modified muscle resulting in an ineffective therapy, as well as presenting a general concern for gene therapy communities in general. Currently, no strategy exists to avoid transgene-specific CTLs, whether they are pre-existing or therapy- induced, and systemic long-term immunosuppression is considered a non-viable option. However, particular viruses found in nature have evolved a potential solution to this dilemma by synthesizing small peptides that inhibit antigen presentation only in transduced cells. In preliminary experiments, we demonstrate that the cellular synthesis of these viral inhibitory peptides (termed VIPRs) prevents the surface presentation of a well-defined antigen, thus protecting transduced cells from the host's immune response. In the current proposal, we will use a canine DMD model (GRMD) to evaluate the evasion ability of VIPRs from Dystrophin- specific CTL-mediated elimination of AAV transduced muscles. First, we will test whether the utilization of VIPRs can block the induction of a Dystrophin-specific CTL response after AAV muscle injection (Aim 1). Next, we will study whether the application of VIPRs will help AAV transduced muscle fibers escape pre-existing Dystrophin-specific CTL-mediated elimination (Aim 2). To decrease the vector cassette size for efficient virion package and/or to enhance the evasion ability of VIPRs, we will optimize the VIPR domains and test the immune evasion capacity of mutant variants (Aim 3). By delivering VIPRs and mini-dystrophin in the same vector, this approach ensures that antigen presentation will be attenuated only in AAV- transduced cells without systemic side effects on the immune system (as would be the case using immunosuppressive drugs or by the application of regulatory T-cells). Collectively, this proposal outlines a promising strategy to overcome our clinical DMD observations and concerns for gene therapy studies in general, by creating an AAV vector capable of avoiding the host's immune response to a foreign protein.

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is characterized by the autoimmune-mediated destruction of the insulin producing ¿ cells of the islets of Langerhans. Our work and that of others in murine models has shown that transferring genes for immunoregulatory molecules to ¿ cells in vivo in the endogenous pancreas or in vitro in islet grafts, effectively suppresses autoimmunity and promotes islet transplantation tolerance, respectively. We have been using adeno-associated virus (AAV) vector gene transfer to manipulate the immunogenicity of murine ¿ cells in vivo and in vitro. AAV vector technology has rapidly advanced and clinical trials have demonstrated successful application of this gene delivery strategy. With this in mind, the goal of our R21 proposal is to develop AAV vectors with increased tropism for human ¿ cells in vivo and in vitro. A two-pronged approach will be taken. First, we will employ a panel of wild-type AAV capsid proteins to identify those capsids that efficiently transduce human ¿ cells in vitro, and in an in vivo model. Secondly, an effort will be made to engineer novel AAV capsid variants that selectively transduce human ¿ cells in vivo and in vitro, and which evade human AAV neutralizing antibodies. Here DNA shuffling of AAV capsid genes combined with directed evolution will be used to develop novel ¿ cell-specific AAV capsids. Experiments will also include testing the efficacy of AAV vector treatment to block immune-mediated destruction of human ¿ cells in vivo. Together, this work will provide a better understanding of the transduction properties of AAV capsids for human ¿ cells, and will identify/generate capsids that can be directly tested in the clinic. In this way, initial steps wil be taken towards our long-term goal of using AAV vectors to establish ¿ cell-specific and/or islet graft tolerance for human T1D.

Instmctions): The Administrative Core will provide (1) professional and (2) administrative functions to the PPG. The professional component led by Drs. Samulski (PI) and Monahan (Co-Pl) is designed to monitor the overall progress ofthe PPG, solve problems as they happen and provide direction for future research. Weekly meetings with the Projects and Core leaders will be held to ensure smooth, coordinated functioning ofthe projects and to develop new research directions. The administrative component will provide support services required, assuring the smooth functioning of each program element. These services will include management of budget, personnel and purchasing, and overseeing of communications, publication and copy facilities. The goal of this part of the Core will be to provide efficient administrative resources to all PPG project investigators and Core leaders so that the individual investigator's time for administrative matters will be minimized, permitting maximum time for research endeavors. This core will also be the interface with administrators of different departments and divisions within UNC as well as the University's Office of Sponsored Research and administrative interactions required by the NIH. RELEVANCE (See instructions): The Administrative Core will provide (1) professional and (2) administrative components to the Program Project Grant (PPG).The Administrative Core (Core A) is dedicated to making center-based research as cost effective and as widely available as possible, fostering successful synergistic interactions between the program investigators and collaborators.

? DESCRIPTION: Adeno-associated virus (AAV) vector has been successfully applied in phase I clinical trials in hemophilia B patients with liver targeting. However, these studies have suggested that AAV capsid specific cytotoxic T lymphocytes (CTL) have the potential to eliminate AAV transduced hepatocytes and result in the therapeutic failure. Our prior studies have demonstrated that AAV capsid antigen presentation is dose-dependent and requires capsid ubiquitination for proteasome mediated degradation. The contamination of empty virions in AAV preparation inhibits transduction from full particles of AAV vectors and potentially increases the risk of virus capsid antigen load. In this proposal we will investigate capsid antige presentation from AAV empty virions and the effect of empty particles on antigen presentation from full virus transduction (Aim 1). To decrease antigen presentation on AAV transduced cells for avoiding capsid specific CTL-mediated elimination, it has been proposed to modify the AAV capsid surface or apply proteasome inhibitors to enhance AAV transduction while lowering the effective dose or to escape capsid ubiquitination. We will study the effect of AAV mutants and proteasome inhibitors on AAV capsid antigen presentation (Aim 2). It is well-known that the transduction of AAV vectors in mouse models does not always translate into the human. Finally, we will explore the directed evolution approach combined with a rational design strategy to isolate AAV vectors with human hepatocyte specific tropism and the ability to evade a capsid specific CTL response in humanized mice (Aim 3). Elucidation of AAV empty capsid antigen presentation in vivo and the development of an AAV vector with enhanced human liver transduction and CTL immune-evasion will allow us to design safer and more effective strategies that address the current clinical complications for human liver gene therapy using AAV.

Abstract Cystic fibrosis (CF) is caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. Airway disease is the leading cause of morbidity and mortality. Gene therapy offers a potential cure for CF. Among gene delivery vehicles, adeno-associated virus (AAV) vectors have demonstrated success in clinical trials. AAV vectors have also been tested in CF patients via airway delivery for gene addition but without measurable success perhaps due to physical airway barriers and inefficient transduction. Studies have shown that higher AAV transduction of airway epithelial cells occur via the basolateral membrane in vitro and our preliminary study demonstrated that systemic administration of AAV vectors induced much more efficient lung transduction than airway delivery in mice. These findings strongly suggest that AAV can transduce airway epithelial cells via systemic administration. The efficiency of AAV transduction in airway epithelial cells after systemic delivery of AAV vectors is usually restricted by the blood vascular barrier and a high prevalence of AAV neutralizing antibodies (Nabs). In this proposal, based on our previous studies, we will explore different strategies to develop novel AAV vectors using directed evolution and AAV virion specific binding peptides via phage display technology to increase AAV vector vascular permeability for enhanced transduction in airway epithelial cells and to evade Nabs after systemic administration. Finally, the novel approaches developed in this proposal will be used to deliver optimized CRISPR/Cas9 specific for CFTR del508 to CF mice and study the long-term phenotypic correction. The results generated from these studies will allow us to develop an effective approach to treat CF patients using AAV vector mediated gene delivery. .