AAV TRANSDUCTION TRACING IN HEMATOPOIETIC CELLS BY TARGETED SCARRING OF GENOMIC SAFE HARBORS

Abstract

AAV is a potent and broadly used gene delivery platform with potential applications to treat many genetic diseases, including incurable diseases of hematopoietic cells and the immune system. As this viral vector becomes more popular for gene therapies, it is important to accurately assess the safety and specificity of its transduction. As of now, no AAV variant has been identified with selective tropism towards hematopoietic cells. Our laboratory screened a high complexity (~5 million variants) library of AAV capsids to identify top candidates showing superior efficiency of transduction into human hematopoietic progenitors in vivo. While broadly- used, the common methodology of testing the top-enriched variants by tagging with a fluorescent reporter (such as GFP) has limited sensitivity due to potential dilution of AAV genomes in proliferating cells. Therefore, this method of detection might not reveal the full transduction profile of AAV variants among blood and non-blood cells in vivo. To address this issue, my project aims to develop a more sensitive detection method that can track transduced cells longitudinally, irrespective of their proliferation history. In particular, by using CRISPR-Cas editing, we targeted genomic safe harbor (GSH) sites to introduce a genetic “scar” in transduced cells, which can be traced with high sensitivity over time as these cells divide and differentiate to almost any downstream progeny cell type. The optimized CasMini system developed through this work can be further applied to investigate activities of various AAV serotypes, including the top-enriched variants obtained from the lab’s prior screen. Ultimately, this system will be useful to examine the broad biodistribution and potential off-target activities of a variety of AAV-based gene therapeutics.