506. Production of Recombinant Adeno- Associated Virus Type 1 Vector Using a Herpes Simplex Virus Based System

Abstract

Top of pageAbstract Recombinant adeno-associated virus (rAAV) is an attractive vector for use in gene therapy treatment of various diseases. However, attaining high-level of rAAV has so far been proven problematic and the usefulness of rAAV vectors has been limited by the low production yield with current production methods. Previously we reported a highly scalable method for rAAV serotype 2 (AAV-2) production using two recombinant herpes simplex viruses (rHSV). One rHSV (rHSV-rep2cap2) expresses rep and cap of AAV-2 and provides helper function for AAV replication and packaging, and the other rHSV (rHSV-TR-GFP) contains the reporter gene green fluorescent protein (GFP) flanked by the inverted terminal repeats (ITR) of AAV-2. Both rHSV vectors are replication defective in non complementing cell lines because of the deletion of an essential gene. This rHSV-based system markedly increased the yield of rAAV production when compared to other traditional AAV production methods. It is well known that different serotypes of AAV have distinct host ranges and cell type preferences, and AAV vectors pseudo- typed with serotype one capsid have been shown to be particularly useful in transducing muscle cells. Here we report the development and initial characterization of an rHSV based production system for rAAV-1. Using a replication defective rHSV as the backbone, the AAV-2 Rep and AAV-1 Cap genes were engineered into rHSV by homologous recombination to generate the helper virus rHSV- rep2cap1. The ability of this virus to produce rAAV-1 was tested by co-infecting HEK-293 cells with rHSV-rep2cap1 and rHSV-TR- GFP. AAV genome titers determined by real time PCR showed that rAAV-1-GFP was packaged with a similar efficiency as the positive control rAAV-2-GFP. The infectivity of the produced rAAV-1-GFP was also examined and the results showed that rAAV-1-GFP was able to transduce C12 cells at an expected efficiency when compared to that of AAV-2-GFP. Studies are currently underway to evaluate these rAAV-1 vectors for gene delivery in vivo. In conclusion, this study suggests that the rHSV-based rAAV production system has a great potential for large scale rAAV production of various serotypes and this system may be able to generate sufficient quantities of rAAV for preclinical and clinical applications.