DNA contamination within recombinant adeno-associated virus preparations correlates with decreased CD34 cell clonogenic potential.

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPCs), but they have many outstanding concerns. Specifically, their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging, a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally, this process results in the packaging of complete viral particles only containing rAAV genomes; however, this is not the case. Through this study, we were able to leverage single-stranded virus (SSV) sequencing, a next-generation sequencing-based method to quantify all DNA species present within rAAV preps. From this, it was determined that much of the DNA within some rAAV preps is not vector-genome derived, and there is wide variability in the contamination by DNA across various preps. Furthermore, we demonstrate that transducing CD34 HSPCs with preps with higher contaminating DNA resulted in decreased clonogenic potential, altered transcriptomic profiles, and decreased genomic editing. Collectively, this study characterized the effects of DNA contamination within rAAV preps on CD34 HSPC cellular potential.