Nanopore sequencing and functional screening of AAV genomes for optimal production and function

Abstract Adeno-associated virus (AAV) has become a popular tool for gene therapy due to its ability to safely and efficiently deliver genetic elements to multiple tissue types in vivo. However, production of AAV particles can be challenging and result in a heterogeneous mixture of AAV particles with incomplete genomes.  Here, we developed a system to 1) identify truncated AAV genomes and the underlying sequence elements responsible for inefficient AAV genome packaging, and 2) evaluate how these sequence elements impact AAV manufacturing and transduction. To this end, we have established protocols using the Oxford Nanopore Technologies platform to characterize sequence elements within pooled libraries of AAV particles to inform their transduction potential in vivo.  The approach presented herein identified problematic promoter sequence elements and can be used to select AAV constructs with properties necessary for safe and effective gene therapies destined for the clinic.  Biography Keith Connolly has been with Modalis Therapeutics for the past seven years, now serving as Director — Molecular Biology and Genomics. Keith’s primary role at Modalis is applying state-of-the-art techniques to address biological problems associated with gene therapy. Recently, he has been interested in applying sequencing technologies to optimize adeno-associated virus manufacturing and delivery. Keith also oversees development of a neuromuscular research program and advises on laboratory activities ranging from molecular biology to in vivo characterization of gene therapies.