A Highly Precise Method for the Quantitation of rAAV Cellular Uptake by ddPCR

Area of Science:

• Gene Therapy Analytics
• Molecular Biology
• Biotechnology

Background:

• Recombinant adeno-associated virus (rAAV) is a key vector in human gene therapy.
• Accurate infectious titer assays are crucial for rAAV quality, potency, and stability assessment.
• The current standard, TCID50, is laborious and exhibits high variability.

Purpose of the Study:

• To develop a novel and precise method for quantifying rAAV cellular uptake *in vitro*.
• To leverage droplet digital PCR (ddPCR) technology for enhanced gene therapy analytics.
• To establish a platform assay for various rAAV serotypes and target genes.

Main Methods:

• Utilized droplet digital PCR (ddPCR) technology for absolute DNA copy quantitation.
• Developed a method to measure rAAV cellular uptake *in vitro* using a stable AAV receptor (AAVR) cell line.
• Validated the assay's consistency with TCID50 and assessed its precision and stability-indicating properties.

Main Results:

• The developed ddPCR-based assay demonstrated high precision, surpassing the traditional TCID50 method.
• The assay showed consistency with TCID50 results, confirming its validity.
• The method is stability-indicating and suitable for various AAV serotypes and target genes.

Conclusions:

• A novel, accurate, and precise rAAV cellular uptake assay was successfully developed using ddPCR.
• This method overcomes limitations of the TCID50 assay, offering improved sensitivity and reliability for rAAV product quality assessment.
• The assay serves as a valuable tool for initial rAAV product evaluation and can complement other potency assays in late-stage gene therapy development.