Partial genome content within rAAVs impacts performance in a cell assay-dependent manner

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• Recombinant adeno-associated viruses (rAAVs) are key gene delivery vectors.
• Packaging of incomplete viral genomes into rAAV capsids is a significant concern.
• Limited data exists on the impact of partial genomes on rAAV performance.

Purpose of the Study:

• To investigate the effect of partial self-complementary EGFP genomes on rAAV vector performance.
• To characterize different rAAV populations based on genome content and density.
• To correlate genome integrity with gene expression efficiency.

Main Methods:

• Isolation of rAAV populations using cesium chloride density gradient ultracentrifugation.
• Characterization of viral genomes via alkaline gels, Illumina Mi-Seq, SEC-MALS, and CD-MS.
• Quantification of DNA and assessment of EGFP expression using ddPCR, live-cell imaging, and ELISA.

Main Results:

• Three distinct rAAV populations (light, moderate, heavy) were isolated.
• The light fraction, containing partial genomes, showed reduced EGFP expression.
• DNA delivery efficiency was similar across fractions, but the light fraction lacked promoter regions, impairing expression.

Conclusions:

• rAAVs with partial genomes can deliver DNA but exhibit diminished vector performance.
• Incomplete genomes, particularly lacking regulatory elements, compromise gene expression.
• Vector integrity is critical for effective gene therapy applications using rAAVs.