AAV's Little Big Problem...
Gene therapy holds the promise to transform the treatment of genetic diseases by delivering therapeutic genes directly into patients' cells. Among the available delivery platforms, adeno-associated virus (AAV) vectors have emerged as a leading choice due to their favorable safety profile and ability to mediate long-term gene expression. However, efficient production of high-quality AAV vectors remains a challenge, as vector preparations often contain a mixture of full, empty, and partially filled capsids. Genome size is a critical factor influencing AAV packaging efficiency, vector integrity, and bioactivity. To systematically assess the effects of genome length on AAV performance, the Theory of Complex Systems and Neurophysics Group (Benjamin Lindner) designed a series of single-stranded AAV vectors with increasing expression cassette sizes by inserting non-coding stuffer sequences. If you want to learn more about how these size variations affect AAV yield, genome packaging, and functional output and receive insight into the design considerations needed to optimize vector quality for clinical applications, check out their Molecular Therapy Methods & Clinical Development Article!
Abstract
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.