7. High-Throughput Sequencing of AAV Proviral Libraries from the Human Population Reveals Novel Variants with Unprecedented Intra-and Inter-Tissue Diversity (2016)

Recombinant adeno-associated viruses (rAAVs) remain the most promising gene delivery vehicle for human gene therapy. The development of novel rAAVs with high transduction efficiency and specific tropisms primarily relies on PCR cloning of natural variants from primate tissues, or the engineering of capsids via directed evolution and rational design. Faithful clinical translation of leading therapeutic AAV serotypes derived from non-human tissues remains a hot-button issue. Here, we employed high-throughput variant profiling of natural AAV proviral libraries from the human population to identify novel AAV variants with unique tissue tropisms. We screened 844 surgical specimens, encompassing a wide range of tissue types and disease states from 455 patients by robust signature PCR. We found that >80% of human tissues are AAV positive with notable serotype frequencies (AAV2/3 chimera > AAV8 > AAV2 > AAV6) distributed among different tissues. Notably, we also used qPCR primers against the conserved AAV rep sequence to quantify the abundance of pAAV proviral genomes in human tissues. Next, to ensure the highest return on identifying novel variants, we employed single-molecule, real-time (SMRT) sequencing to characterize full-length rep and cap sequences. Our preliminary findings from a single patient tissue sample revealed more than 800 unique DNA sequence variants (originating from single nucleotide polymorphisms, genetic recombination, and de novo mutations) that account for more than 50 unique amino acid sequence variants. We also detected significant inter-patient variation, as well as high inter-tissue variability within individual patients. These findings suggest that AAV exhibits the capacity for high genomic diversity and accelerated intra-host evolution. These characteristics, which are shared among many other viruses, are presumed to promote survival against the host immune response, and fitness in tissue-specific cellular environments. We also validated a selection of AAV8 variants by assessing their capacity for vector packaging and in vivo gene transfer. We found that a subset of variants has a stronger competency for transcytosis and tropism to liver and muscle than the conventional AAV8 serotype. Remarkably, one AAV8 variant can cross the blood-brain barrier and target the CNS more efficiently than AAV9 after intravenous injection of adult mice. Another noteworthy discovery is the sequence variability found in the assembly-activating protein (AAP) gene, which is encoded by an alternative open reading frame of the cap gene. The diversity within AAP may define the efficacy of AAV packaging among variants, and implicate its role in AAV evolution and fitness. By exploiting AAV-host tissues as a natural incubator for viral evolution to potentiate new AAV capsid sequences, and employing a strategy to profile intact capsid sequences on a high-throughput scale, this study highlights the unprecedented diversity and evolutionary capacity of AAV. Importantly, these new findings reshape the versatility of rAAV as the ideal biotherapy for targeting a range of normal and/or diseased tissues.