Most experiments proposed in this grant require production of AAV vector from plasmids that the individual researches design using standard recombinant DNA techniques. The goal of this (research grade) Vector Core facility is therefore the production of research grade AAV vector for pre-clinical studies in tissue culture, mice, hemophilic dogs, and other experimental systems. This function will ensure the availability. of recombinant AAV for the different projects that involve experiments with AAV vectors for expression of therapeutic transgenes. AAV vector is produced in a helper virus-free system based on large-scale plasmid transfection of HEK-293 cells using two helper plasmids that supply AAV rep/cap and adenoviral helper functions and a third plasmid encoding the recombinant vector. This will allow production of a variety of different vectors for the investigators. The use of a core facility will provide reproducible yield and purity of vector, and will be more cost- effective than vector production in individual laboratories, in particular for experiments that involve large animal models. The service of the Core will include large-scale preparation of helper and vector plasmids, large- scale transfection of HEK-2934 cells using calcium phosphate participation, recovery and purification of recombinant AAV by cell lysis followed by column chromatography of gradient centrifugation, dialysis, sterile filtration, and storage of purified vector, and quantitative slot blot hybridization. The Core will perform additional tests on purity and sterility of vector, if required for the intended experiment, and assist the researchers that requested the vectors with set up of functional assays. Standard vector preparations are expected to yield approximately vector genomes, while scale up will result in production of approximately 10/14 vector genomes per preparation. This will primarily by achieved by use of affinity column chromatography for vector purification.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project--Cooperative Agreements (U01)
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Special Emphasis Panel (ZHL1-CSR-C (S2))
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Stanford University
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Margaritis, Paris (2010) Long-term expression of canine FVIIa in hemophilic dogs. Thromb Res 125 Suppl 1:S60-2
Margaritis, Paris; Roy, Elise; Aljamali, Majed N et al. (2009) Successful treatment of canine hemophilia by continuous expression of canine FVIIa. Blood 113:3682-9
Bedi, Maninder S; Alvarez Jr, Rene J; Kubota, Toru et al. (2008) Myocardial Fas and cytokine expression in end-stage heart failure: impact of LVAD support. Clin Transl Sci 1:245-8
Aljamali, Majed N; Margaritis, Paris; Schlachterman, Alexander et al. (2008) Long-term expression of murine activated factor VII is safe, but elevated levels cause premature mortality. J Clin Invest 118:1825-34
Akache, Bassel; Grimm, Dirk; Shen, Xuan et al. (2007) A two-hybrid screen identifies cathepsins B and L as uncoating factors for adeno-associated virus 2 and 8. Mol Ther 15:330-9
Chen, Jian; Wu, Qi; Yang, Pingar et al. (2006) Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy. Mol Ther 13:260-9
Bedi, Maninder; McNamara, Dennis; London, Barry et al. (2006) Genetic susceptibility to atrial fibrillation in patients with congestive heart failure. Heart Rhythm 3:808-12
Grimm, Dirk; Pandey, Kusum; Nakai, Hiroyuki et al. (2006) Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype. J Virol 80:426-39
Inagaki, Katsuya; Fuess, Sally; Storm, Theresa A et al. (2006) Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther 14:45-53
Manno, Catherine S; Arruda, Valder R; Pierce, Glenn F et al. (2006) Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 12:342-7

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