The experiments proposed in the projects of this application involve the study ofthe immune responses directed to the viral vector capsid and the transgene product in AAV-mediated gene transfer. Thus, the Vector Core is an essential constituent of this proposal, as it will provide support to the experimental activities outlined. All the projects require several different transgenes, reporter genes, and expression cassettes to be packaged in alternate AAV serotypes 1 through 9, together with other capsid variants, including variants with mutations at surface-exposed tyrosine residues, or with insertions of immunomodulatory viral peptides into the N-terminus of VP2, a vector capsid protein. Furthermore, the projects involve in vitro (cell culture) and in vivo (small animal models) experiments. Accomplishing the goals of each individual project will need a high degree of flexibility in terms of quality, number, and scale of each individual AAV preparation. It should be noted that differences in capsid structure of newly isolated serotypes and/or capsid mutants may require fine-tuning ofthe purification and formulation protocols, therefore it is preferable to have expertise in AAV vector production within the program rather than relying on contract organizations. During the previous funding period we established a Vector Core with proven production capability (~8E15 vector genomes produced), able to achieve economies of scale, and, most importantly, providing reliable vectors with a high level of purity and potency. The Vector Core will continue to produce AAV vectors with consistent and comparable quality in the new proposal using a helper virus-free transfection system and gradient centrifugation purification techniques, to support proposed studies in vitro and in animal models. The Core will produce, purify, quantify, aliquot, and store the vectors and provide the requested quality controls, depending on users'request. Quality ofthe vector will be carefully monitored to maintain titer, purity, and potency. Additionally, as in the previous funding period, the Core will manage other services for the projects, including characterization of AAV vector preparations, DNA plasmid production, peptide library resuspension and storage, and other activities that serve the scope ofthe proposed application.

Public Health Relevance

Gene therapy represents a new method of treatment for previously untreatable genetic diseases. Immune responses to gene transfer vectors represent one of the last barriers to successful gene therapy. The Vector Core (Core B) utilizes a manufacturing process similar to that used for clinical vector production. Vector manufacturing by our vector core will facilitate translation of relevant pre-clinical findings into clinical studies.

National Institute of Health (NIH)
Research Program Projects (P01)
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Heart, Lung, and Blood Program Project Review Committee (HLBP)
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Wistar Institute
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Rogers, Geoffrey L; Martino, Ashley T; Zolotukhin, Irene et al. (2014) Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B. J Transl Med 12:25
Sarkar, Debalina; Biswas, Moanaro; Liao, Gongxian et al. (2014) Ex Vivo Expanded Autologous Polyclonal Regulatory T Cells Suppress Inhibitor Formation in Hemophilia. Mol Ther Methods Clin Dev 1:
Wang, Xiaomei; Moghimi, Babak; Zolotukhin, Irene et al. (2014) Immune tolerance induction to factor IX through B cell gene transfer: TLR9 signaling delineates between tolerogenic and immunogenic B cells. Mol Ther 22:1139-50
Liao, Gongxian; van Driel, Boaz; Magelky, Erica et al. (2014) Glucocorticoid-induced TNF receptor family-related protein ligand regulates the migration of monocytes to the inflamed intestine. FASEB J 28:474-84
Liao, Gongxian; O'Keeffe, Michael S; Wang, Guoxing et al. (2014) Glucocorticoid-Induced TNF Receptor Family-Related Protein Ligand is Requisite for Optimal Functioning of Regulatory CD4(+) T Cells. Front Immunol 5:35
Wu, Te-Lang; Li, Hua; Faust, Susan M et al. (2014) CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome. Mol Ther 22:42-51
Sherman, Alexandra; Schlachterman, Alexander; Cooper, Mario et al. (2014) Portal vein delivery of viral vectors for gene therapy for hemophilia. Methods Mol Biol 1114:413-26
Mingozzi, Federico; Anguela, Xavier M; Pavani, Giulia et al. (2013) Overcoming preexisting humoral immunity to AAV using capsid decoys. Sci Transl Med 5:194ra92
Buchlis, George; Odorizzi, Pamela; Soto, Paula C et al. (2013) Enhanced T cell function in a mouse model of human glycosylation. J Immunol 191:228-37
Mingozzi, Federico; High, Katherine A (2013) Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122:23-36

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