308. Inclusion of Heterologous ITRs in Dual AAV Vectors for Retinal Gene Therapy

AAV cargo capacity limited to about 5 kb prevents their applications from treatment of inherited blinding conditions which require the transfer of genes with larger coding sequences. However, dual AAV vectors, each containing one of the two halves of a large gene expression cassette, are emerging as promising tools to overcome this limitation. However, dual AAV show lower levels of transgene expression than a single AAV vector and are associated with production of proteins shorter than expected from either the 5’- or 3’-half AAV These limitations can be in part overcome by improving the productive tail-to-head AAV genome concatemerization by including heterologous inverted terminal repeats (ITRs) at both ends of each dual AAV vector genome.We have generated dual AAV hybrid vectors with heterologous ITRs from AAV2 (ITR2) and AAV5 (ITR5) which are the most divergent among the various AAV serotypes. However, we found a significant reduction in the yields of AAV vectors with heterologous ITR2 and 5 when compared to those of vectors with homologous ITR2. In addition, the genome titer performed on ITR2 sequences results lower than that on a different part of the genome suggesting that ITRs might not be intact in AAV vectors with heterologous ITRs. Finally, the amount of full length protein relative to shorter was similar between vector with homologous and heterologous ITRs. We are currently testing dual AAV hybrid vectors with ITRs derived from other serotypes divergent from AAV2 which could overcome some of the limitations we have found using ITR5.