Executive Summary

The structural integrity of inverted terminal repeats (ITRs) in plasmid DNA is a primary determinant of recombinant AAV vector yield and quality. Due to their palindromic nature, ITRs are susceptible to recombination and deletion during plasmid amplification in E. coli, which directly compromises the production of full, functional viral genomes. We employ a quantitative PCR (qPCR) based assay to rapidly determine the percentage of plasmids with intact ITRs, providing a key quality control metric that de-risks downstream manufacturing and strengthens the CMC data package for AAV programs.

The CMC Challenge of ITR Recombination

During propagation in standard bacterial strains, ITR sequences are prone to homologous recombination, leading to deletions and rearrangements. This results in a heterogeneous plasmid population where a fraction of the DNA lacks the complete sequences necessary for replication and packaging. Using such a population for AAV production has two negative consequences:

• Reduced Functional Titer: Only plasmids with two intact ITRs can generate a genome that is efficiently packaged. A compromised plasmid lot directly reduces the output of full, transgene-expressing vectors.

• Process Inefficiency: The manufacturing process proceeds regardless of plasmid quality, consuming expensive reagents and capacity to produce a higher ratio of empty or partially filled capsids, complicating downstream purification.

qPCR as a Quantitative Solution

To mitigate this risk, a quantitative assessment of ITR integrity is a necessary quality control step. Compared to older, semi-quantitative methods like Sanger sequencing or restriction enzyme digests, a qPCR-based assay offers superior speed, sensitivity, and throughput. This method provides a clear percentage of intact molecules, enabling data-driven decisions on which plasmid lots are suitable for advancing into large-scale production.

This level of analytical rigor for starting materials is consistent with a broader strategy of controlling for factors that can influence in vivo outcomes. Research has shown that components within a vector preparation can activate innate immune pathways, such as those involving Toll-like receptor 9 (TLR9), potentially triggering adaptive immune responses against the transgene product and reducing long-term expression (PMID: 31703913). Ensuring the highest purity and integrity of the starting plasmid DNA is a fundamental step in minimizing variables that could contribute to unwanted immunogenicity. A well-characterized product is better positioned to achieve sustained efficacy, a principle demonstrated in studies showing how managing the host immune response is key to enabling robust transgene expression (PMID: 26022732).

Our process integrates this assessment as a standard release test for all research and GxP-grade plasmid preparations, ensuring that every AAV production run at our >100,000 sq ft Philadelphia facility begins with well-characterized, high-quality starting material. This analytical oversight is part of the framework that has supported our clients in achieving their IND milestones.

“We started collaborating with UPenn Vector core in 2023 and the AAV vector which they manufactured laid a foundation for development of a gene therapy candidate which will enter soon preclinical studies. The key people from UPenn Vector Core joined Franklin Biolabs and our partnership transitioned without interruption… Franklin Biolabs is an essential collaborator in our AAV-vector based gene therapy candidate development, and we hope to continue the partnership for years to come.”
– Biotech Partner

By identifying and rejecting suboptimal plasmid lots early, we protect project timelines and budgets, contributing to a more predictable path toward the clinic.