503. A Novel Herpes Simplex Virus Helper Based Production of Adeno- Associated Virus Vectors for Treatment of Retinal Angiogenesis

Abstract

AGTC's proprietary rAAV vector production method uses recombinant herpes simplex virus (HSV) as the helper agent for rAAV production. This production method has been demonstrated to be both highly scalable and productive compared to other rAAV production methods such as the transient co-transfection or adenovirus co-infection process. rAAV production by rHSV-1 methodology requires two recombinant HSV-1 vectors, rHSV-1- rep2cap2 which expresses the AAV 2 rep and cap genes, and a second rHSV-1 that contains the therapeutic gene of interest flanked by the AAV-2 inverted terminal repeats.Both rHSV vectors are deleted in an essential gene for HSV replication and hence they are propagated only in a complementing cell line. Here we report the construction and characterization of rHSV-TR-shFlt-1 that contains a novel soluble hybrid version of Flt-1, the receptor for vascular endothelial growth factor (VEGF). This novel shFlt-1 is a high-affinity binder of VEGF, a central player in pathologic neovascularization in both wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). To construct rHSV-TR-shFlt-1, a shuttle plasmid containing the expression cassette of sFlt-1 flanked with homologous HSV-1 sequences was first constructed and co-transfected with rHSVd27.1- TR-GFP viral DNA into complementing V27 cells. rHSV-TR-shFlt-1 was screened against the parental rHSV-1-GFP viruses that express fluorescent GFP, and the homologous recombination was further confirmed by Southern blot analysis. Selected rHSV-TR-shFlt-1 clones were propagated through 10 passages and their ability to produce rAAV2-TR-shFlt-1 vector was examined by real-time PCR. The expression level, size, and in-vitro biological activity of sFlt-1 expressed in rAAV2-TR-shFlt-1 transduced cells were determined by ELISA, immunoblot, and the HUVECs proliferation assay respectively. Stability studies indicated that the rHSV-TR-shFlt-1 helpers maintained their ability to produce rAAV comparable to that of the positive control rHSV-1-GFP through 10 passages. Preliminary data from the oxygen-induced retinopathy (OIR) mouse model suggests that rAAV2-shFlt-1 derived from rHSV1- TR-shFlt-1 is able to inhibit retinal neovascularization, and there is no significant difference compared to rAAV2-shFlt-1 vector made by the transfection method. To our knowledge this is the first report of the construction and characterization of a recombinant HSV helper virus to produce a rAAV vector encoding a therapeutic gene for inhibition of ocular angiogenesis. Further preclinical evaluation of the AAV2-shFlt-1 is in the process.