Method helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6.

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Author	Grimm, Dirk Kay, Mark A. Kleinschmidt, Juergen A.
Abstract	We present a simple and safe strategy for producing high-titer adeno-associated virus (AAV) vectors derived from six different AAV serotypes (AAV-1 to AAV-6). The method, referred to as “HOT, ” is helper virus free, optically controllable, and based on transfection of only two plasmids, i.e., an AAV vector construct and one of six novel AAV helper plasmids. The latter were engineered to carry AAV serotype rep and cap genes together with adenoviral helper functions, as well as unique fluorescent protein expression cassettes, allowing confirmation of successful transfection and identification of the transfected plasmid. Cross-packaging of vector DNA derived from AAV-2,-3, or-6 was up to 10-fold more efficient using our novel plasmids, compared to a conservative adenovirus-dependent method. We also identified a variety of useful antibodies, allowing detection of Rep or VP proteins, or assembled capsids, of all six AAV serotypes. Finally, we describe unique cell tropisms and kinetics of transgene expression for AAV serotype vectors in primary or transformed cells from four different species. In sum, the HOT strategy and the antibodies presented here, together with the reported findings, should facilitate and support the further development of AAV serotype vectors as powerful new tools for human gene therapy. Key Words: adeno-associated virus, gene therapy, serotypes, vector production
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Subject Keyword	Optically Controllable Adeno-associated Virus Vector Method Helper Virus-free Two-plasmid-based Production Aav Serotype Vector Adenoviral Helper Function Hot Strategy Different Aav Serotypes High-titer Adeno-associated Virus Cap Gene Assembled Capsid Powerful New Tool Human Gene Therapy Unique Cell Tropism Vector Production Successful Transfection Vp Protein Aav Serotype Rep Adeno-associated Virus Aav Vector Construct Different Specie Key Word Novel Plasmid Unique Fluorescent Protein Expression Cassette Transformed Cell Useful Antibody Helper Virus Transgene Expression Novel Aav Helper Plasmid Safe Strategy Vector Dna Aav Serotypes Gene Therapy Conservative Adenovirus-dependent Method Transfected Plasmid
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