Peeling The Onion: A Stratified Approach To rAAV DNA Purity And Integrity Characterization

By Melissa Carver, Ph.D.,; Stephen Ollosi, MS; Austin E. Smith Ph.D.; and Michael Murphy Ph.D.

The past decade has witnessed significant advancements in gene therapies for rare diseases, with a crucial focus on the safe delivery of therapeutic DNA to patients. The recombinant Adeno-Associated Virus (AAV) has emerged as a proven and safe platform for gene therapy. As the field progresses, it is essential that the quality control tests for assessing AAV product safety also evolve.

In this context, we present a variety of molecular biology and biochemical methods designed to evaluate the purity and integrity of DNA in AAV products. Each method offers distinct insights and limitations regarding DNA quality. Assessing genomic integrity and DNA purity can be accomplished through techniques involving intact capsids, extracted DNA, and sequencing.

Comprehensive evaluation requires employing multiple methods, such as analyzing intact capsids, measuring DNA length, and using sequence-specific techniques like multiplexed ddPCR, which provides quantifiable results not obtainable through other approaches. We emphasize that a combination of these methods is essential to fully characterize the DNA purity and integrity of recombinant AAV products. Together, these tools offer rapid and effective means to evaluate the genomic integrity and purity of gene therapy products.