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

Area of Science:

• Molecular Biology
• Gene Therapy
• Biotechnology

Background:

• Adeno-associated virus (AAV) vectors are a leading platform for gene therapy.
• Efficient AAV production requires optimal vector integrity, including full-size genome packaging and high full/empty ratios.
• Current AAV production methods yield a mix of full, empty, and partially filled capsids.

Purpose of the Study:

• To investigate how AAV genome size impacts packaging efficiency, genome integrity, capsid ratios, and bioactivity.
• To evaluate the effect of inserting non-coding stuffer sequences on AAV vector characteristics.
• To determine the optimal strategy for 'right-sizing' AAV genomes for therapeutic applications.

Main Methods:

• Designed single-stranded CMV-EGFP-poly(A) expression cassettes with sizes ranging from 2-5 kb.
• Inserted non-coding stuffer sequences upstream or downstream of the expression cassette.
• Analyzed AAV yields, bioactivity, full/empty ratios, and genome integrity using varying cassette sizes.

Main Results:

• AAV yields and bioactivity decreased systematically as expression cassette size increased.
• Overfilling was observed with cassettes <2.5 kb, while genome truncation and partial filling increased >4.5 kb.
• A second stuffer sequence unexpectedly did not cause similar losses in productivity and bioactivity.

Conclusions:

• Inserting non-coding DNA is a viable method to optimize AAV genome size for manufacturing (CMC) aspects.
• The specific DNA sequence used for genome 'right-sizing' is critical for maintaining AAV quality and bioactivity.
• Careful sequence selection is necessary to ensure consistent AAV vector performance in gene delivery.