AAV yield, bioactivity, and particle heterogeneity are impacted by genome size and non-coding DNA elements

Adeno-associated virus (AAV) vectors currently represent the most attractive platform for therapeutic gene delivery. Ensuring efficient AAV production and vector integrity, defined by efficient packaging of full-size genomes, high full/empty ratios, and optimal bioactivity, therefore is of utmost importance. However, during AAV production, not only capsids that carry the full-size genome but also empty as well as capsids containing partial or oversized genomes are produced. To systematically investigate the impact of AAV genome size on packaging efficiency, genome integrity, (over)full/partial/empty ratio, and bioactivity, we designed single-stranded CMV-EGFP-poly(A) expression cassettes with successively increasing sizes (2–5 kb), by inserting non-coding stuffer sequences either upstream or downstream of the CMV-EGFP-poly(A) sequence. Our results demonstrate a systematic decrease of AAV yields and bioactivity with increasing expression cassette size. While significant overfilling was observed at expression cassette sizes <2.5 kb, an increase in partially filled capsids, due to genome truncation, was observed >4.5 kb. Surprisingly, the losses in productivity and bioactivity were not observed with a second stuffer sequence. Therefore, while the insertion of non-coding DNA is a straightforward approach to “right-size” the AAV genome and optimize CMC aspects, careful selection of the DNA sequence is required to assure AAV quality.