Structural analysis of recombinant AAV vector genomes at single-molecule resolution

Area of Science:

• Molecular Biology
• Gene Therapy
• Bioinformatics

Background:

• Recombinant adeno-associated virus (AAV) vectors are crucial for in vivo gene therapy.
• Genome heterogeneity in AAV vectors poses a significant safety concern.
• Accurate characterization of AAV genome structure is essential for therapeutic development.

Purpose of the Study:

• To develop and validate a novel computational pipeline for analyzing AAV genome heterogeneity.
• To enable precise classification of individual AAV genomes and their structural variants.
• To provide insights into sequence and structural features influencing AAV genome diversity.

Main Methods:

• Development of a long-read, read-level analysis pipeline utilizing PacBio sequencing data.
• The pipeline integrates a tiling step for read alignment and a parsing step using formal grammar for classification.
• Classification into five structural categories: expected, truncated, snapback, truncated snapback, and others, with detailed annotation.

Main Results:

• The pipeline achieved high accuracy in classifying AAV genome structures from both single-stranded and self-complementary vectors.
• Analysis revealed distinct genome structure patterns, with the majority classified as expected full-length species.
• Snapback genome breakpoints often clustered at specific sites, suggesting roles for secondary structures and other mechanisms.

Conclusions:

• The developed pipeline offers a comprehensive framework for evaluating AAV genome diversity at single-vector resolution.
• Insights into AAV genome heterogeneity can guide vector design and production for improved safety and efficacy.
• The method demonstrates robustness against sequencing errors, enhancing its utility for AAV therapeutic development.