Profiling vector preparations by Adeno-Associated Virus Genome Population Sequencing achieves full-genome resolution and reveals heterogenic human-vector chimeras

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization for multiple therapeutic applications. Despite this new era for gene therapy, the integrity of packaged vector genomes during rAAV production is often overlooked. Encapsidation of partial vector genomes can negatively impact therapeutic efficacy and potential safety. Using high-throughput single-molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive library preparation steps. We have exploited this methodology to profile all heterogenic populations of self-complementary AAV genomes via bioinformatics pipelines, and have coined this approach AAV-genome population sequencing (AAV-GPseq). We show that this method can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. With AAV-GPseq, we can also detect many reverse packaged genomes that encompass bacterial sequences of plasmid origin, as well as adenovirus and rep/cap sequences originating from packaging and helper plasmids. Finally, we detect the encapsidation of sequences containing host-cell genomes that are chimeric with ITR-containing vector sequences. These discoveries pose new concerns for how we define the purity and homogeneity of viral vector preparations, and how undesired packaged sequences may impact rAAV-based clinical modalities.