87. Towards Renal-Targeted Gene Delivery: Identification of Clades A, B, E and F Adeno-Associated Virus Sequences from Human Urine Samples

Abstract

Adeno-associated virus (AAV) vectors have been successful in gene therapy for a range of inherited and acquired diseases. However, the development of efficient AAV-based gene delivery systems to treat renal diseases is lagging behind, mainly due to the unavailability of highly kidney-tropic AAV serotypes. To identify novel AAV capsid mutants with a natural renal tropism, we tested human urine samples for the presence of AAV sequences. We treated a combination of 293T, HeLa and Vero cells with 93 individual clinical urine samples, followed by one day of infection with high titer adenovirus 5 for AAV expansion. The majority of samples, 69%, were positive for AAV DNA by capsid specific, N-terminus nested PCR. Out of these samples 63% were AAV2-like (Clade B), 27% AAV8-like (Clade E), 1.6% AAV1-like (Clade A) and 8.7% had chimeric cap sequences (Clades B-E, B-F, F-B-E, F-E and E-B hybrids). In characterizing the AAV rep-cap junction region from DNA samples that were not uniquely AAV2 cap positive, the sequences were most closely related to the following: AAV1 rep-AAV1 cap, AAV2 rep-AAV2cap, AAV9 rep-AAV9 cap and AAV2 rep-AAV8 cap. Analysis of full length capsid sequences from overlapping PCRs revealed AAV8-like sequences, AAV2-like sequences and chimeric sequences of AAV8, AAV9 and rhesus isolates. We have cloned these AAV variants into the pXR plasmid and are currently testing their tissue tropisms in vivo. This study is the first to report frequent detection of AAV sequences in human urine. AAV vectors with novel AAV capsid variants may accelerate renal-targeted AAV gene therapy applications.