Molecular characterization of novel Adeno-associated virus variants infecting human tissues

Abstract

Despite the many advantages with Adeno-associated virus (AAV) based vectors for gene therapy, certain barriers related to host permissivity and immune response precludes their widespread application in humans. A comprehensive study of the distribution and complexity of naturally occurring AAV in human tissues should facilitate their optimal utilization for gene therapy and tissue targeting in humans. A total of 205 samples, comprising 198 tissue samples from individuals of Indian origin and 7 different cell lines were investigated. A panel of 8 primate samples was used as controls. DNA from these samples was screened for the AAV capsid specific signature regions by a modified PCR and DNA sequencing approach. Further, we generated a single point mutation (S224A) in AAV3 vector, analogous to the mutation identified in a novel AAV3 sequence variant isolated from a peripheral blood stem cell (PBSC) sample. We further studied the infectivity of these vectors in HeLa and HS5 cells in vitro. Of the 205 samples analyzed, an AAV specific signature DNA sequence was detected in 92 samples (45%), including 85 out of 198 human tissues and in all the 7 human cell lines investigated. DNA sequencing analysis showed that AAV6(34%) was the most common serotype and identified predominantly in PBSCs. Interestingly, a comparative genotypic analysis in primate samples identified AAV3 specific DNA in most of the bone marrow or liver tissue analyzed (n = 7/8) suggesting species-specific differences in AAV infectivity. Further characterization of an AAV3 serotype variant isolated from the PBSCs was non-infectious in vitro, possibly due to altered receptor affinity. Our data outlines the genetic diversity and the distribution of AAV serotypes infecting humans and provides a basis for their further characterization to generate efficient gene delivery vectors.