EXPRESSING FULL-LENGTH DYSTROPHIN WITH AAV
Dongsheng Duan
Department/ Educational Institution Type:
Grant 5R21NS062934-02 from National Institute Of Neurological Disorders And Stroke
Abstract: Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). There is no cure for these devastating diseases. An effective therapy relies on treating all affected muscles in the body. Adeno-associated virus (AAV) can efficiently deliver a therapeutic gene to all body muscles. Therefore, replacing the defective dystrophin gene with AAV gene therapy holds great therapeutic promise. However, AAV vector has a 5 kb packaging capacity. This is insufficient to deliver the 11.06 kb full-length dystrophin coding sequence. For this reason, current AAV gene therapy is forced to use the massively truncated mini- or micro-dystrophin genes. In this application, we propose to develop a novel tri- AAV system to express the full-length protein. The combined capacity of three AAV vectors provides enough room for the full dystrophin coding sequence. However, random recombination among different AAV vectors will lead to very inefficient reconstitution of the full-length protein. We, among others, have recently developed a series of novel approaches to facilitate unidirectional recombination between different AAV vectors. In this project, we will apply these new technologies to the development of the tri-AAV full-length dystrophin vectors. Our specific aims are (1) to design and construct full-length dystrophin tri-AAV vectors, and (2) to evaluate the full-length dystrophin tri-AAV vectors in dystrophin-deficient mdx mice by local gene transfer. We believe that this high payback translational project will likely lead to a breakthrough in AAV-mediated DMD gene therapy in the future. Duchenne muscular dystrophy (DMD) is a life threatening disease affecting a fairly large population (more than one in 3,500 newborns). It is caused by dystrophin gene mutations. The best therapy is to replace the mutated gene with a normal gene. AAV vector can express truncated versions of the dystrophin gene but it cannot express the full-length protein. In this translational project, we propose to develop a novel tri-AAV system to express the full-length dystrophin protein. If successful, this project will lead to a big breakthrough in DMD gene therapy
Keywords: 0-6 weeks old; AAV vector; Abbreviations; adeno associated virus group; adeno-associated viral vector; adeno-associated virus vector; Adeno-Associated Viruses; Affect; Anterior; Antibodies; Applications Grants; base; Becker dystrophy; Becker muscular dystrophy; Becker muscular dystrophy (BMD); Becker pseudohypertrophic muscular dystrophy; Becker-Kiener muscular dystrophy; benign X-linked recessive muscular dystrophy; biological signal transduction; Blotting, Western; Carrying Capacities; Cell Communication and Signaling; Cell Signaling; Cells; Cessation of life; childhood pseudohypertrophic muscular dystrophy; classic X-linked recessive muscular dystrophy; Code; Coding System; Complex; Death; Deoxyribonucleic Acid; Dependovirus; design and construction; Development; Disease; disease/disorder; Disorder; Disorder of muscle, unspecified; DNA; DNA Alteration; DNA mutation; DNA Recombination; DNA recombination (naturally occurring); DNA Sequence; Duchene; Duchenne; Duchenne de Boulogne muscular dystrophy; Duchenne disease; Duchenne dystrophy; Duchenne muscular dystrophy; Duchenne muscular dystrophy (DMD); Duchenne myodystrophy; Duchenne pseudohypertrophic muscular dystrophy; Duchenne syndrome; Duchenne-Griesinger syndrome; Duchenne/Becker muscular dystrophy; Duchenne/Becker muscular dystrophy (DMD/BMD); Dystrophin; effective therapy; Ellis-van Creveld (EvC) syndrome; Foundations; Future; Gene Alteration; Gene Delivery; Gene Mutation; gene product; gene therapy; Gene Therapy Vectors; Gene Transduction Agent; Gene Transduction Vectors; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genes; Genetic Alteration; Genetic Change; Genetic defect; Genetic Intervention; Genetic mutation; Genetic Recombination; genetic therapy; Genome; genome mutation; Glycoproteins; Grant Proposals; Grants, Applications; Head; heavy metal lead; heavy metal Pb; homologous recombination; homology (molecular); Hybrids; Inbred mdx Mice; Infant, Newborn; Infection; Inflammation; INFLM; Injection of therapeutic agent; Injections; Injury; Intervention, Genetic; Intracellular Communication and Signaling; Inverted Terminal Repeat; Lead; Length; Life; Measures; Mediating; Mice, Inbred mdx; micro-dystrophin; microdystrophin; mild X-linked recessive muscular dystrophy; mini-dystrophin; minidystrophin; Molecular Biology, Gene Therapy; Morphology; Mouse, mdx; Muscle; Muscle Disease; Muscle disease or syndrome; Muscle Disorders; Muscle Tissue; Muscular Diseases; muscular disorder; Muscular Dystrophies; Muscular Dystrophy, Becker; Muscular Dystrophy, Duchenne; Muscular Dystrophy, Pseudohypertrophic; Mutate; Mutation; Myodystrophica; Myodystrophy; Myopathic Conditions; Myopathic disease or syndrome; Myopathic Diseases and Syndromes; Myopathy; Myopathy, unspecified; Natural regeneration; Neural Constitutive Nitric Oxide Synthase; neuronal nitric oxide synthase; new approaches; new technology; newborn human (0-6 weeks); Newborn Infant; Newborns; Nitric Oxide Synthase Type I; nNOS enzyme; NOS 1 protein; novel; novel approaches; novel strategies; novel strategy; Pathogenesis; Patients; Pb element; Phenotype; Physiologic; Physiological; Play; Polyadenylation; Population; premature; Problem Solving; progressive muscular dystrophy of childhood; progressive muscular dystrophy, Becker type; Progressive Muscular Dystrophy, Duchenne Type; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); protein blotting; Protein Region; Proteins; pseudohypertrophic adult muscular dystrophy; Pseudohypertrophic Muscular Dystrophy, Childhood; pseudohypertrophic muscular paralysis; pseudohypertrophic progressive muscular dystrophy, Duchenne type; Recombination; Recombination, Genetic; reconstitute; reconstitution; Recovery; regenerate; Regeneration; restoration; RNA Polyadenylation; Role; Sarcolemma; Sequence Alteration; Sequence Homology; Series; Serotyping; Signal Transduction; Signal Transduction Systems; Signaling; social role; System; System, LOINC Axis 4; Tail; Therapeutic; therapeutic gene; Therapy, DNA; transduction efficiency; transfer of a gene; transgene expression; vector; Viral; Viral Vector; Western Blotting; Western Blottings; Western Immunoblotting; X-linked dilated cardiomyopathy; X-linked dilated cardiomyopathy (XLCM); X-linked muscular dystrophy; X-linked recessive muscular dystrophy
Project start date: 2008-07-01
Project end date: 2010-12-30
Budget start date: 1-JUL-2009
Budget end date: 30-DEC-2010
PFA/PA: PAR-06-203
5R21NS062934-02 (2009): $196219
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Dongsheng Duan
Expressing Full-length Dystrophin With AAV
Dongsheng Duan, Associate Professor
Molecular Microbiology And Immunologyuniversity Of Missouri-columbia
310 Jesse Hall
columbia, Mo 65211
Grant 1R21NS062934-01 from National Institute Of Neurological Disorders And Stroke IRG: NSD
Abstract: Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). There is no cure for these devastating diseases. An effective therapy relies on treating all affected muscles in the body. Adeno-associated virus (AAV) can efficiently deliver a therapeutic gene to all body muscles. Therefore, replacing the defective dystrophin gene with AAV gene therapy holds great therapeutic promise. However, AAV vector has a 5 kb packaging capacity. This is insufficient to deliver the 11.06 kb full-length dystrophin coding sequence. For this reason, current AAV gene therapy is forced to use the massively truncated mini- or micro-dystrophin genes. In this application, we propose to develop a novel tri- AAV system to express the full-length protein. The combined capacity of three AAV vectors provides enough room for the full dystrophin coding sequence. However, random recombination among different AAV vectors will lead to very inefficient reconstitution of the full-length protein. We, among others, have recently developed a series of novel approaches to facilitate unidirectional recombination between different AAV vectors. In this project, we will apply these new technologies to the development of the tri-AAV full-length dystrophin vectors. Our specific aims are (1) to design and construct full-length dystrophin tri-AAV vectors, and (2) to evaluate the full-length dystrophin tri-AAV vectors in dystrophin-deficient mdx mice by local gene transfer. We believe that this high payback translational project will likely lead to a breakthrough in AAV-mediated DMD gene therapy in the future. Duchenne muscular dystrophy (DMD) is a life threatening disease affecting a fairly large population (more than one in 3,500 newborns). It is caused by dystrophin gene mutations. The best therapy is to replace the mutated gene with a normal gene. AAV vector can express truncated versions of the dystrophin gene but it cannot express the full-length protein. In this translational project, we propose to develop a novel tri-AAV system to express the full-length dystrophin protein. If successful, this project will lead to a big breakthrough in DMD gene therapy
Project start date: 2008-07-01
Project end date: 2010-06-30
DUAL AAV VECTORS FOR DUCHENNE MUSCULAR DYSTROPHY THERAPY
Dongsheng Duan
University Of Missouri-columbia, 310 Jesse Hall, Columbia, Mo 65211
Grant 5R01AR049419-08 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Abstract: Dystrophin gene mutations lead to Duchenne muscular dystrophy (DMD), a severe muscle disease that affected nearly all muscles in the body. A cure for DMD requires body-wide therapy. Adeno-associated virus (AAV) is currently the only viral vector that can efficiently transduce whole body muscle. Thus AAV is the vector-of-choice for DMD gene therapy. Despite great promise, AAV gene therapy is challenged by the small viral packaging capacity (5 kb maximal). The 11.5 kb full-length dystrophin coding sequence cannot be delivered by a single AAV vector. To overcome this obstacle, investigators have developed abbreviated micro/mini-dystrophin genes. Microgenes can fit into a single AAV but it cannot restore muscle force to the normal level. A 6 kb H2-R19 minigene fully recovers muscle force. My first and only R01 was to develop dual AAV vectors to express the 6 kb minigene in a single muscle in mdx mice, a mild mouse model for DMD. We have successfully accomplished and surpassed this goal. In this renewal, we will further advance dual AAV gene therapy for DMD. Despite the fact that the 6 kb minigene can fully restore muscle force, this minigene cannot restore neuronal nitric oxide synthase (nNOS) to the sarcolemma. The loss of sarcolemmal nNOS leads to functional ischemia in mdx mice and DMD patients. We recently developed a novel 7 kb minigene that recruits nNOS to the sarcolemma. In this renewal, we will dissect out the structure motif(s) responsible for nNOS recruiting in the dystrophin gene. Furthermore, we will establish the therapeutic advantage(s) of the 7 kb minigene in transgenic mice by measuring muscle force and blood perfusion. Most importantly, we will develop novel dual AAV vectors to express the 7 kb minigene. We have previously demonstrated an efficient mini-dystrophin gene therapy with a pair of the trans-splicing AAV (tsAAV, a dual vector approach). This proof-of-principle study is performed in mdx mice by local injection. In this renewal, we will test the hypotheses that (1) systemic whole body dual AAV vector gene transfer can be achieved in a symptomatic dystrophin/utrophin double knockout (dko) mouse DMD model; (2) furthermore, systemic minigene therapy with dual AAV vectors can ameliorate muscle pathology, restore muscle force and blood perfusion in dko mice. Our long-term goal is to develop an effective minigene therapy for DMD patients. Establishing systemic gene transfer in a large animal model is a logical next step. We have recently characterized a Corgi dog model for DMD. However canine muscle has been notoriously difficult to transduce with any vector. We have now overcome this hurdle and achieved systemic single AAV transduction in wild type newborn dogs. This is the first demonstration of a whole body gene transfer in a large animal model. In this renewal, we will test the hypotheses that (1) systemic dual AAV vector transduction can be achieved in newborn dogs; (2) systemic dual AAV minigene therapy is feasible in neonatal dystrophic dogs. In summary, our study will establish the foundation for dual AAV minigene gene therapy in DMD patients in the future. PUBLIC HEALTH RELEVANCE. Duchenne muscular dystrophy is a life threatening disease affecting a fairly large population (more than one in 3,500 newborns). It is caused by dystrophin gene mutations. Dual AAV-mediated minigene therapy hold great promise to cure the disease. Our work will advance current dual vector therapy by providing a functionally superior minigene, by targeting all body muscles in a symptomatic mouse model, and by systemic approach in a dog model. Our findings will pave the way to eventually move dual AAV gene therapy to human trials in the future
Keywords: 0-6 weeks old; 21+ years old; AAV vector; Abbreviations; Address; Adeno-Associated Viruses; Adult; Affect; Alkaline Phosphatase; Animal Model; Animal Models and Related Studies; Animals; Applications Grants; Back; Backcrossings; Biopsy; C-terminal; CD8; CD8B; CD8B1; CD8B1 gene; Canine Species; Canis familiaris; Cell-Extracellular Matrix; Cellular Matrix; Clinical; Code; Coding System; Collaborations; Contracting Opportunities; Contracts; Cytoskeletal System; Cytoskeleton; DNA Alteration; DNA Recombination; DNA mutation; DNA recombination (naturally occurring); Data; Dependovirus; Diagnosis; Disease; Disorder; Disorder of muscle, unspecified; Dogs; Dorsum; Duchene; Duchenne; Duchenne de Boulogne muscular dystrophy; Duchenne disease; Duchenne dystrophy; Duchenne muscular dystrophy; Duchenne muscular dystrophy (DMD); Duchenne myodystrophy; Duchenne pseudohypertrophic muscular dystrophy; Duchenne syndrome; Duchenne-Griesinger syndrome; Dystrophin; Dystrophin-Related Protein; Dystrophin-Related Protein 1; ECM; Ellis-van Creveld (EvC) syndrome; Evaluation; Extracellular Matrix; Face; Fibrosis; Foundations; Funding; Future; Gene Alteration; Gene Delivery; Gene Mutation; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; GeneHomolog; Genes; Genetic Intervention; Genetic Recombination; Genetic mutation; Goals; Grant Proposals; Grants, Applications; Homolog; Homologous Gene; Homologue; Human; Human, Adult; Human, General; INFLM; Immune response; Inbred mdx Mice; Infant, Newborn; Infiltration; Inflammation; Injection of therapeutic agent; Injections; Intervention, Genetic; Investigators; Ischemia; Knock-out; Knockout; LYT3; Lead; Length; Letters; Life; Light; Link; Mammals, Dogs; Mammals, Mice; Man (Taxonomy); Man, Modern; Measurement; Measures; Mechanics; Mediating; Membrane; Mice; Mice, Inbred mdx; Mind; Modeling; Molecular; Molecular Biology, Gene Therapy; Monitor; Mouse, mdx; Murine; Mus; Muscle; Muscle Disease; Muscle Disorders; Muscle Tissue; Muscle disease or syndrome; Muscle, Skeletal; Muscle, Voluntary; Muscular Diseases; Muscular Dystrophies; Muscular Dystrophy, Duchenne; Muscular Dystrophy, Pseudohypertrophic; Myodystrophica; Myodystrophy; Myopathic Conditions; Myopathic Diseases and Syndromes; Myopathic disease or syndrome; Myopathy; Myopathy, unspecified; NOS 1 protein; Natural regeneration; Necrosis; Necrotic; Neonatal; Neonatal Screening; Neural Constitutive Nitric Oxide Synthase; Newborn Infant; Newborn Infant Screening; Newborns; Nitric Oxide Synthase Type I; Orthophosphoric-monoester phosphohydrolase (alkaline optimum); Pathogenesis; Pathology; Patients; Pb element; Phase; Photoradiation; Physiologic; Physiological; Population; Problem Solving; Progressive Muscular Dystrophy, Duchenne Type; Proteins; Pseudohypertrophic Muscular Dystrophy, Childhood; Recombination; Recombination, Genetic; Recovery; Recruitment Activity; Regeneration; Research; Research Personnel; Researchers; Rod; Rod Photoreceptors; Rods (Eye); Rods (Retina); Role; Sample Size; Sarcolemma; Sequence Alteration; Signaling Molecule; Skeletal Muscle Tissue; Skeletal muscle structure; Spectrin; Staging; Structure; T-Cells; T-Lymphocyte; Testing; Therapeutic; Therapy, DNA; Thymus-Dependent Lymphocytes; Time; Trans RNA Splicing; Trans-Splicing; Transgenic Mice; Transgenic Organisms; Treatment Efficacy; UTRN Protein; Utrophin; Viral Packaging; Viral Vector; Virus Packagings; Work; X-linked dilated cardiomyopathy; X-linked dilated cardiomyopathy (XLCM); X-linked muscular dystrophy; X-linked recessive muscular dystrophy; adeno associated virus group; adeno-associated viral vector; adeno-associated virus vector; adult human (21+); alkaline phosphomonoesterase; base; benign X-linked recessive muscular dystrophy; canine; childhood pseudohypertrophic muscular dystrophy; classic X-linked recessive muscular dystrophy; design; designing; disease/disorder; domestic dog; experiment; experimental research; experimental study; facial; gene product; gene therapy; genetic therapy; glycerophosphatase; heavy metal Pb; heavy metal lead; hemodynamics; host response; immunoresponse; improved; insight; intracellular skeleton; meetings; membrane structure; mild X-linked recessive muscular dystrophy; mini-dystrophin; minidystrophin; model organism; mouse model; muscle degeneration; muscular disorder; muscular dystrophy mouse model; nNOS enzyme; neuronal nitric oxide synthase; newborn human (0-6 weeks); newborn screening; novel; perfusion (blood); progressive muscular dystrophy of childhood; pseudohypertrophic adult muscular dystrophy; pseudohypertrophic muscular paralysis; pseudohypertrophic progressive muscular dystrophy, Duchenne type; public health relevance; reconstitute; reconstitution; recruit; regenerate; research study; restoration; rod cell; social role; success; therapeutic efficacy; therapeutic gene; therapeutically effective; thymus derived lymphocyte; transduction efficiency; transfer of a gene; transgenic; vector; vector genome
Project start date: 2008-06-15
Project end date: 2013-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-07-125
5R01AR049419-08 (2010): $395419
5R01AR049419-07 (2009): $423864
3R01AR049419-07S1 (2009): $521831
Exploring Systemic AAV Gene Delivery In The Dystrophic Dog
Dongsheng Duan, Associate Professor
Molecular Microbiology And Immunologyuniversity Of Missouri-columbia
310 Jesse Hall
columbia, Mo 65211
Grant 1R21AR057209-01A1 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: NSD
Abstract: Duchenne muscular dystrophy (DMD) is the most prevalent lethal childhood genetic disease. It is caused by dystrophin gene mutation. Numerous progresses have been made in the last decade in developing DMD gene therapy. Impressive whole body rescue has been reported in DMD mouse models with adeno-associated virus (AAV)-mediated micro-dystrophin gene therapy. An important next step prior to human trial is to demonstrate the therapeutic efficacy of the micro-dystrophin gene in canine DMD models. Canine muscle has been notoriously difficult to transduce because of the strong cellular immune response. Furthermore, DMD affects nearly every muscle in the body. Only systemic gene transfer can truly ameliorate the disease. We recently developed an efficient whole body muscle transduction protocol in normal neonatal dogs. This is the first demonstration that systemic AAV delivery can reach multiple muscles in a large animal. To advance DMD gene therapy studies, we have recently established a Corgi dog model for DMD. The affected dogs share the same dystrophin gene mutation and display the same clinical phenotype as human patients. In this translational R21 project, we will apply these exciting findings to the therapeutic canine R4-23/C microgene. We will perform systemic AAV delivery in neonatal dystrophic Corgi dogs. The primary goal is to achieve wide spread muscle transduction. We will also perform preliminary efficacy evaluation using a comprehensive set of histopathology endpoints. Our study will set up a foundation to thoroughly evaluate AAV micro-dystrophin gene therapy in dog models in the future. Project Narrative Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutation. Currently, there is no cure. Adeno-associated virus-mediated gene therapy has shown a great promise to ameliorate this disease. In this study we will develop novel techniques to achieve whole body gene transfer in newborn DMD dogs. The majority of DMD patients can be diagnosed through neonatal screening. Our study will open the door for neonatal gene therapy in human patients in the future
Project start date: 2008-09-18
Project end date: 2010-08-31
Dual AAV Vectors For Duchenne Muscular Dystrophy Therapy
Dongsheng Duan, Assistant Professor
University Of Missouri-columbia 310 Jesse Hall Columbia, Mo 65211
Grant 5R01AR049419-05 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases IRG: MEDB
Abstract: Duchenne muscular dystrophy (DMD) is the most common form of inherited muscle disease. It usually leads to death from respiratory or cardiac failure by age 20. Currently, no effective treatment is available for this fatal disease. DMD is an X-linked genetic disease caused by dystrophin gene mutation. Gene therapy represents a very promising avenue to cure DMD. Recombinant adeno-associated virus (rAAV) mediates high-level persistent transgene expression in muscle. Recent clinical trials have further confirmed the efficiency and the safety of rAAV vectors in muscle. However, rAAVmediated DMD gene therapy has been significantly limited by the small viral packaging capacity. Only the highly truncated C-terminal-deleted versions of "micro-dystrophin" genes have been attempted. Both clinical and transgenic studies show that the C-terminal-inclusive larger genes (such as the 6.0-6.3kb "mini-dystrophin" genes and the approximately 4.7kb "C-terminal-inclusive micro-dystrophin" genes) are therapeutically superior. Unfortunately the strong therapeutic expression cassettes derived from these genes are too large to be packaged in a single AAV virion. We have recently developed several dual vector approaches to expand AAV packaging capacity. Among these, the concatamerization-based "trans-splicing" and "cis-activation" strategies hold great promise for delivering the C-terminal-inclusive larger dystrophin genes. However, the expression level achieved so far is not sufficient for DMD gene therapy. In this proposal, we plan to extend our previous findings and further explore the molecular mechanisms underlying these methods, in the hope of improving the transduction efficiency for DMD gene therapy. In particular, we will try to identify and overcome the rate-limiting barriers to transgene expression. These include problems associated with dual vector co-infection, concatamerization of AAV genome inside cell, and transcription, splicing, and stability of AAV concatamers. More important, we will apply this newly obtained information to generate the most effective trans-splicing and cis-activation AAV vectors for the C-terminal-inclusive larger dystrophin genes. Therapeutic potentials of these newly developed AAV vectors will be rigorously tested in the limb muscle, diaphragm, and heart of the murine DMD model (mdx mouse). A comprehensive array of assays will be used to examine the level of gene expression and the functional improvement in muscle histology and contraction. To address safety concerns, we also plan to evaluate the potential deleterious effects from putative truncated protein production in the trans-splicing method. Taken together, our findings will lead to the eventual application of these very promising dual AAV vector strategies to the human DMD gene therapy.
Keywords: adeno associated virus group, dystrophin, gene mutation, gene therapy, muscular dystrophy, nonhuman therapy evaluation, RNA, diaphragm, gene expression, genetic transcription, muscle contraction, myocardium, sex linked trait, laboratory mouse, polymerase chain reaction, transfection /expression vector
Project start date: 2003-07-14
Project end date: 2008-04-30
NOVEL HYBRID AAV VECTORS FOR CYSTIC FIBROSIS GENE THERAPY
Dongsheng Duan, Associate Professor
University Of Missouri-columbia, 310 Jesse Hall, Columbia, Mo 65211
Grant 5R21DK076552-02 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Cystic fibrosis (CF) is the most common autosomal recessive disease. This lethal disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Currently, there is no cure. Progressive loss of lung function is the leading cause of morbidity and mortality in CF. Delivering a functional CFTR gene to airway cells may provide a permanent remedy for this relentless disorder. Adeno-associated virus (AAV) has been at the forefront for CF gene therapy. Several clinical trials have been performed with AAV vector. Despite promising results in safety and DNA level gene transfer, these trials have failed to yield enough CFTR protein in patients. The lack of a strong promoter is largely responsible for the low level protein expression. AAV is one of the smallest viruses. Its 5kb packaging capacity is barely enough to carry the full-length CFTR cDNA. There is no room for a potent promoter. Novel strategies are urgently needed to expand AAV packaging capacity. Several dual vector approaches, including trans-splicing and overlapping, hold promise to deliver a strong full- length CFTR cDNA expression cassette. Unfortunately, their transduction efficiency is too low to be useful. Furthermore, the success of these traditional dual vector approaches is highly dependent on the transgene sequence and they may not work for the CFTR gene. To overcome the inherent limitation in the traditional dual vector approaches, we have now developed the hybrid vector system, a noval second generation dual vector strategy. This is a generic approach independent of the transgene sequence
Keywords: 5`-Adenylic acid, homopolymer; AAV vector; Adeno-Associated Viruses; Biochemical; CF patients; CFTR; CFTR Protein; CMV promoter; Cells; Clinical Trials; Clinical Trials, Unspecified; Code; Coding System; Complementary DNA; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Recombination; DNA recombination (naturally occurring); DNA, Complementary; Deoxyribonucleic Acid; Dependovirus; Disease; Disorder; Drugs, Nonproprietary; Electrophysiology; Electrophysiology (science); Gene Expression; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Generations; Generic Drugs; Genes; Genetic Alteration; Genetic Change; Genetic Intervention; Genetic Recombination; Genetic defect; Hybrids; In Vitro; Intervening Sequences; Intervention, Genetic; Introns; Inverted Terminal Repeat; Lead; Length; Lung; Methods; Molecular; Molecular Biology, Gene Therapy; Morbidity; Morbidity - disease rate; Mortality; Mortality Vital Statistics; Mucoviscidosis; Mutation; Neurophysiology / Electrophysiology; Patients; Pb element; Phase; Poly A; Poly(rA); Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteins; RNA Splicing; Recombination; Recombination, Genetic; Respiratory System, Lung; Respiratory physiology; Safety; Schools, Medical; Series; Serotyping; Splicing; System; System, LOINC Axis 4; Therapeutic; Therapy, DNA; Trans RNA Splicing; Trans-Splicing; Transgenes; Virion; Virus; Virus Particle; Viruses, General; Work; adeno associated virus group; adeno-associated viral vector; adeno-associated virus vector; base; cDNA; cDNA Expression; clinical investigation; cystic fibrosis patients; cystic fibrosis transmembrane regulator; disease/disorder; fusion gene; gene product; gene therapy; generic; genetic therapy; genome mutation; heavy metal Pb; heavy metal lead; in vivo; lung function; medical schools; new approaches; novel; novel approaches; novel strategies; novel strategy; patients with CF; patients with cystic fibrosis; polyadenylate; protein expression; pulmonary; reconstitute; reconstitution; respiratory function; size; success; transduction efficiency; transfer of a gene; transgene expression; vector
Project start date: 2007-07-15
Project end date: 2010-06-30
Budget start date: 1-JUL-2008
Budget end date: 30-JUN-2010
PFA/PA: PA-06-155
5R21DK076552-02 (2008): $0
Sponsored Links Excellgen http://Excellgen.com
DUCHENNE CARDIOMYOPATHY GENE THERAPY
Dongsheng Duan, Associate Professor
University Of Missouri-columbia, 310 Jesse Hall, Columbia, Mo 65211
Grant 1R01HL091883-01A2 from National Heart, Lung, And Blood Institute
Abstract: Cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy (DMD), the most common childhood lethal muscle disease. DMD is caused by dystrophin gene mutation and there is currently no cure. Adeno-associated virus (AAV)-mediated micro/mini-dystrophin gene therapy has shown great promise in ameliorating Duchenne skeletal muscle disease. However, we recently found that the abbreviated genes that were developed for treating skeletal muscle disease may not completely fulfill the needs of the heart. Here, we hypothesize that Duchenne cardiomyopathy gene therapy may require a specific dystrophin domain that is missing in the current available micro/minigenes. On reviewing Duchenne cardiomyopathy-related clinical reports over the last 17 years, we identified a putative heart protection domain in the dystrophin gene. In this proposal, we will test whether we can achieve better cardiac rescue by including the putative heart protection domain in the micro/minigenes. Specifically, novel micro/minigenes carrying the putative heart protection domain will be generated. AAV will be used to deliver these micro/minigenes to the heart in the mouse models of Duchenne cardiomyopathy. Comprehensive anatomic, cellular, biochemical, and physiological assays will be used to monitor cardiac rescue. The therapeutic efficacy of new micro/minigenes will also be compared to that of the current micro/minigenes. Our long-term goal is to develop an effective AAV gene therapy to treat patients. A critical step before initiating human trial is preclinical evaluation in the canine DMD model. We hypothesize that AAV gene therapy can ameliorate cardiomyopathy in the golden retriever muscular dystrophy (GRMD) model. The best micro/minigenes identified in the murine model will be delivered to neonatal GRMD puppy by systemic AAV gene transfer. Normal dogs and saline injected GRMD dogs will be included as controls. Progression of the heart disease as well as gene transfer efficiency will be carefully monitored using a comprehensive panel of anatomic, histological, cellular, biochemical, and physiological assays we already developed. Taken together, our study will significantly advance Duchenne cardiomyopathy gene therapy. Duchenne muscular dystrophy (DMD) is a lethal disease affecting a fairly large population of patients (~one in 3,500 newborn boys). DMD related heart disease significantly reduces the life quality and life span of patients. Here we propose to develop AAV gene therapy to treat Duchenne heart disease. Our findings will pave the way to eventually cure DMD
Keywords: 0-6 weeks old; AAV vector; Address; Adeno-Associated Viruses; Affect; Anatomic; Anatomical Sciences; Anatomy; Animal Model; Animal Models and Related Studies; Assay; Bioassay; Biochemical; Biologic Assays; Biological Assay; Canine Species; Canis familiaris; Cardiac; Cardiac Diseases; Cardiac Disorders; Cardiomyopathies; Cause of Death; Childhood; Clinical; Clinical Data; Clinical Trials; Clinical Trials, Unspecified; Code; Coding System; DNA Alteration; DNA mutation; Dependovirus; Disease; Disorder; Disorder of muscle, unspecified; Dogs; Duchene; Duchenne; Duchenne de Boulogne muscular dystrophy; Duchenne disease; Duchenne dystrophy; Duchenne muscular dystrophy; Duchenne muscular dystrophy (DMD); Duchenne myodystrophy; Duchenne pseudohypertrophic muscular dystrophy; Duchenne syndrome; Duchenne-Griesinger syndrome; Dystrophin; Ellis-van Creveld (EvC) syndrome; Exons; Foundations; Future; Gene Alteration; Gene Delivery; Gene Mutation; Gene Transfer; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genes; Genetic Alteration; Genetic Change; Genetic Intervention; Genetic defect; Genetic mutation; Goals; H2 gene; Heart; Heart Diseases; Human; Human, General; Inbred mdx Mice; Infant, Newborn; Injection of therapeutic agent; Injections; Intervention, Genetic; Jugular Veins; Lead; Length; Length of Life; Life; Longevity; Mammals, Dogs; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Mice; Mice, Inbred mdx; Modeling; Molecular Biology, Gene Therapy; Monitor; Mouse, mdx; Murine; Mus; Muscle Disease; Muscle Disorders; Muscle disease or syndrome; Muscle, Skeletal; Muscle, Voluntary; Muscular Diseases; Muscular Dystrophies; Muscular Dystrophy, Duchenne; Muscular Dystrophy, Pseudohypertrophic; Mutation; Mycocardium Disease; Myocardial Diseases; Myocardial Disorder; Myocardiopathies; Myodystrophica; Myodystrophy; Myopathic Conditions; Myopathic Diseases and Syndromes; Myopathic disease or syndrome; Myopathy; Myopathy, unspecified; Neonatal; Newborn Infant; Newborns; Patients; Pb element; Physiologic; Physiological; Progressive Muscular Dystrophy, Duchenne Type; Proteins; Pseudohypertrophic Muscular Dystrophy, Childhood; QOL; Quality of life; Reporting; Saline; Saline Solution; Science of Anatomy; Sequence Alteration; Serotyping; Skeletal Muscle Tissue; Skeletal muscle structure; Structure of jugular vein; Structure-Activity Relationship; Tail; Testing; Therapeutic Effect; Therapy, DNA; Treatment Efficacy; Veins; X-linked dilated cardiomyopathy; X-linked dilated cardiomyopathy (XLCM); X-linked muscular dystrophy; X-linked recessive muscular dystrophy; adeno associated virus group; adeno-associated viral vector; adeno-associated virus vector; anatomy; base; benign X-linked recessive muscular dystrophy; boys; canine; chemical structure function; childhood pseudohypertrophic muscular dystrophy; classic X-linked recessive muscular dystrophy; clinical investigation; disease/disorder; domestic dog; early onset; effective therapy; gene product; gene replacement therapy; gene therapy; genetic therapy; genome mutation; heart disorder; heart function; heavy metal Pb; heavy metal lead; life span; lifespan; mild X-linked recessive muscular dystrophy; mini-dystrophin; minidystrophin; model organism; mouse model; muscular disorder; myocardium disorder; newborn human (0-6 weeks); novel; patient population; pediatric; preclinical evaluation; progressive muscular dystrophy of childhood; pseudohypertrophic adult muscular dystrophy; pseudohypertrophic muscular paralysis; pseudohypertrophic progressive muscular dystrophy, Duchenne type; public health relevance; structure function relationship; therapeutic efficacy; therapeutically effective; transfer of a gene; vector
Relevance: Public Health Relevance Duchenne muscular dystrophy (DMD) is a lethal disease affecting a fairly large population of patients (~one in 3,500 newborn boys). DMD related heart disease significantly reduces the life quality and life span of patients. Here we propose to develop AAV gene therapy to treat Duchenne heart disease. Our findings will pave the way to eventually cure DMD
Project start date: 2010-07-01
Project end date: 2014-04-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
1R01HL091883-01A2 (2010): $520740