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A dual-reference study design for understanding and improving AAV genome size analysis.

Yali Sun1, Zhi-Xiang Lu1, Michael Miller1

  • 1Revvity, Inc, Waltham, Massachusetts, USA.

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Summary

We developed a fast and accessible microfluidic assay for characterizing recombinant adeno-associated virus (rAAV) genomes. This method uses a novel single-stranded DNA (ssDNA) reference standard for accurate size assessment, improving gene therapy quality control.

Keywords:
AAV genome integritydual referencemicrofluidic capillary electrophoresisssAAVssDNA 7K Ladder

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Area of Science:

  • Molecular Biology
  • Gene Therapy
  • Analytical Chemistry

Background:

  • Recombinant adeno-associated virus (rAAV) is a key gene delivery platform due to its long-lasting effects and low immunogenicity.
  • Accurate characterization of rAAV genome integrity and purity is essential for clinical efficacy and patient safety.
  • Existing methods for rAAV genome size assessment are often time-consuming, inaccessible, or lack appropriate reference standards for single-stranded DNA (ssDNA).

Purpose of the Study:

  • To develop a rapid, accessible, and reliable assay for characterizing the size and integrity of rAAV genomes.
  • To establish a suitable reference standard for analyzing long single-stranded DNA (ssDNA) fragments in rAAV preparations.

Main Methods:

  • Development of a single-stranded DNA (ssDNA) assay utilizing a microfluidic capillary electrophoresis platform.
  • Implementation of a novel ssDNA reference standard for accurate size determination of rAAV genomes.
  • Evaluation of assay sensitivity, speed, and throughput for high-volume sample analysis.

Main Results:

  • The developed ssDNA assay provides accurate size calling for ssDNA fragments.
  • Achieved a detection sensitivity of approximately 89 pg/µL (3 × 10^10 GC/mL AAV) for a 5.1 kb ssDNA fragment.
  • Demonstrated a rapid turnaround time of approximately 100 seconds per sample, enabling high-throughput analysis.
  • Observed potential for ssDNA annealing post-capsid release, forming double-stranded DNA (dsDNA) peaks dependent on sample processing.

Conclusions:

  • The microfluidic ssDNA assay offers a fast and accessible method for rAAV genome characterization.
  • The use of a specific ssDNA reference standard is crucial for accurate size analysis.
  • Recommend employing dual-reference standards and orthogonal methods to comprehensively assess rAAV genome size and integrity, mitigating risks of mischaracterization due to ssDNA annealing.