Quantifying DNA point mutations in commercially available AAV reporter vectors and plasmids

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Adeno-associated virus (AAV) vectors are crucial for gene therapy.
• Accurate quantification of mutations in AAV products is essential for safety and efficacy.
• Existing sequencing methods may vary in their precision for detecting low-frequency mutations.

Purpose of the Study:

• To compare the accuracy of three sequencing methods for quantifying point mutations in AAV products.
• To evaluate single-template amplification with Sanger sequencing (STA-Sanger), Illumina sequencing, and Oxford Nanopore Technologies (ONT) sequencing.
• To assess the consistency and reliability of mutation detection across different platforms.

Main Methods:

• Sequencing of the green fluorescent protein (GFP) transgene from AAV9 reporter vectors and their plasmids.
• Utilizing STA-Sanger, Illumina NGS, and ONT NGS platforms.
• Analysis of per-base mutation rates and non-reference majority variant frequency (MVF) correlations.

Main Results:

• STA-Sanger and Illumina showed similar low vector per-base mutation rates (0.016% and 0.013%, respectively).
• ONT sequencing exhibited significantly higher per-base mutation rates (vector: 2.2%, plasmid: 1.3%).
• ONT demonstrated stronger correlation among vector lots for MVF (R² = 0.91) compared to Illumina (R² = 0.46).

Conclusions:

• Evidence of single-nucleotide mutations in AAV products was observed.
• A lack of consistency among sequencing platforms highlights potential challenges in AAV quality control.
• The AAV industry needs to develop more accurate sequencing methodologies for standardized QC protocols.