Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids

Abstract

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 rAAVGFPcontaining vectors, resulting in up to 1.55 Â 10 12 rAAV capsids and 4 Â 10 8 expression units from approximately 1 Â 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.

Induction of Robust Immune Responses against Human Immunodeficiency Virus Is Supported by the Inherent Tropism of Adeno-Associated Virus Type 5 forDendritic Cells

“…Difficulty in producing hightitered AAV stocks has limited the clinical utility of this class of vectors. To resolve the problem, AAV vectors have been produced utilizing baculovirus in insect cells (42,56,58) and herpes simplex virus (8,14). In this study, HEK293 cells were infected with 10 5 vp/cell of AAV-HIV vectors and the expression of AAV-HIV vectors was confirmed by Western blotting (Fig.…”

Viral vectors for in vivo gene transfer in Parkinson's disease: Properties and clinical grade production

“…2), scale-up of production to the levels that might be needed for wide distribution of an approved vector for PD treatment has been an issue. Several potential scale-up procedures have been published (Booth et al, 2004;Conway et al, 1997;Kohlbrenner et al, 2005) and all are amenable to cGMP procedures (see below) (Farson et al, 2004;Snyder and Francis, 2005).…”

Herpes simplex virus type 1/adeno-associated virus hybrid vectors

“…ICP4, ICP27), which have been developed in order to reduce pathogenicity and cytotoxic effects in vector infected cells, can also be used as helper viruses for the production of rAAV vectors. Specifically, the ability of rHSV-1 that lack the ICP27 gene to efficiently act as a helper virus for rAAV production has been demonstrated [210] ; rAAV production in the absence of ICP27 appeared to be even enhanced. This may be due to the role of ICP27 in regulating transcription and translation of viral and cellular genes, for instance in the inhibition of splicing of host and AAV transcripts, which reduces synthesis of Rep and Cap proteins.…”

“…Moreover, when allied to infection of a cell line that provides the rAAV template to be packaged, transfection steps can be avoided entirely for the production of rAAV [161,211]. A protocol with a single infection step can also be accomplished by inserting an AAV ITR-flanked transgene (rAAV genome) cassette into the genome of the rHSV-1 helpervirus [210].…”