Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids.
Adeno-associated virus (AAV) vectors are highly efficient tools for use in gene therapy. Current production methods rely on plasmid transfection and are not generally considered amenable to scale-up. To improve recombinant AAV (rAAV) vector production in terms of both final titre and simplicity, we constructed recombinant herpes simplex virus (HSV) vectors, either disabled (ICP27 deleted) or nondisabled, encoding the AAV rep and cap genes. We also integrated an rAAVGFP construct into the nondisabled vector and also into a second pair of HSV vectors (disabled and nondisabled) not expressing rep and cap. Transgene incorporation and expression was confirmed by Southern and Western blot, respectively. Optimal double-infection ratios were established for disabled and nondisabled pairs of rep/cap-expressing and rAAVGFP-containing vectors, resulting in up to 1.55 x 10(12) rAAV capsids and 4 x 10(8) expression units from approximately 1 x 10(7) BHK producer cells. Functionality of the prepared vector was confirmed by the detection of abundant green fluorescent protein (GFP) expression following injections of rAAV preparations into the rat brain. This paper therefore describes a simple, efficient, and transfection-free rAAV production process based on the use of HSV and not relying on a proviral cell line that, with appropriate scale-up, could yield quantities of rAAV sufficient for routine clinical use.
Department of Immunology and Molecular Pathology, The Windeyer Institute, University College London, London W1T 4JF, UK.
M J
MJ
Booth
A
A
Mistry
X
X
Li
A
A
Thrasher
R S
RS
Coffin
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000096:biosynthesis*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D019076:Transgenes; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D049452:Green Fluorescent Proteins