The adeno-associated virus (AAV) is a dependent parvovirus whose unique biology recommends it as a safe and efficient vector for gene therapy. In the absence of co-infection with a helper virus (adenovirus), the wtAAV integrates and persists in the host cell genome in a latent state, a property that would be attractive if reproduced by recombinant AAV (rAAV) vectors. We recently demonstrated the ability of muscle to serve as a platform for rAAV gene therapy in vivo, both in mouse and in a large animal model (the Chapel Hill strain of hemophilia B dogs). The overall goals of the proposed research are to improve AAV muscle gene therapy for hemophilia by investigating the molecular steps involved in rAAV transduction in primary muscle cells and by optimizing rAAV/F.IX vectors.
Specific Aim I. Analysis of conversion of ssDNA to HMW DNA in vivo: A: Determine the molecular fate of rAAV vectors of skeletal muscle in vivo.
This aim will require examining total genomic DNA from rAAV-infected muscle to determine the time course for conversion of input ssDNA to a form capable of persistence (HMW DNA). Levels of transgene expression and of DNA replication activity will be investigated in parallel to define a mechanism for the apparent amplification of transgene expression over time in non-dividing cells (mature muscle) following rAAV gene delivery. B: Determine the capacity of skeletal muscle to integrate rAAV in vivo. The ability of wild-type AAV to integrate into the host cell genome has led to the unproven assumption that sustained expression from rAAV vectors occurs via transcription form integrated rAAV sequences. Using a mouse model developed in our laboratory, which has the human integration site for wtAAV, we will seek to demonstrate stable vector (rAAV/F.IX) integrated into the skeletal myocyte genome.
Specific Aim II. To test whether low levels of F.IX expression following rAAV gene therapy can be improved by higher specific activity F.IX variants and by repeat administration of rAAV/F.IX with alternative capsid structures. A: Factor IX variants have been constructed to study interactions of the protein with neighboring clotting cascade proteins. Constructs, including a chimeric protein with the EGF-1 domain of F.IX replaced by that of factor VII, and F.IX including a single point mutations in the catalytic domain will be investigated after in vivo delivery using rAAV vectors. B: Readministration of transgene using alternative serotype rAAV. While cellular immune response does not limit rAAV transgene expression, neutralizing antibodies predictably develop. By using alternative serotypes of capsid virus for packaging of transgene (AAV2, AAV3, AAV4), sequential administrations may elude the development of neutralizing antibodies to AAV, and allow augmented transgene expression after re-administration of the therapeutic vector.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Clinical Investigator Award (CIA) (K08)
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Special Emphasis Panel (ZHL1-CSR-Y (O1))
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Tenenbaum, L; Lehtonen, E; Monahan, P E (2003) Evaluation of risks related to the use of adeno-associated virus-based vectors. Curr Gene Ther 3:545-65
Monahan, Paul E; Jooss, Karin; Sands, Mark S (2002) Safety of adeno-associated virus gene therapy vectors: a current evaluation. Expert Opin Drug Saf 1:79-91