One Stone, Two Birds: Engineering Rep-Cap Plasmids To Enhance AAV Manufacturability And Potency

By Xiaojun Liu, Yeonji Kim, Chris Brown, and Jing Zhu

Recombinant adeno-associated virus (rAAV) vectors are pivotal in gene therapy due to their proven safety and efficacy for therapeutic gene delivery. However, low production yields and vector heterogeneity remain significant barriers to scalable, high-quality manufacturing. In this study, we introduce a novel strategy to address these challenges through the engineering of Rep-Cap plasmids, a critical component of the triple-plasmid transfection system.

Using rational design and synthetic biology approaches, we optimized Rep-Cap plasmid sequences by incorporating regulatory elements for synchronized gene expression, fine-tuning the VP1/VP2/VP3 protein ratio, optimizing codon usage, and eliminating unstable or recombinogenic regions. Engineered plasmid variants were assessed in proprietary and commercial suspension HEK293 cell platforms across multiple AAV serotypes, including AAV2, AAV6, and AAV8.

Our optimized Rep-Cap plasmids yielded substantial improvements in AAV production and quality. Specifically, vector yields increased by 2- to 10-fold, exceeding 2E15 VG/L, while the ratio of full-to-empty capsids improved by 50%, indicating enhanced vector integrity. Transcriptomic analyses revealed balanced and sustained expression of Rep and Cap proteins, reduced cellular stress, and improved packaging efficiency. Comprehensive characterization, including TEM, AUC, CE, and NGS assays, confirmed the quality of the AAV vectors produced using these plasmids.

Beyond improving production, the engineered Rep-Cap plasmids demonstrated scalability across serotypes in suspension-based systems and retained robust functional performance. In vitro and in vivo functional assays validated the enhanced bioactivity and safety of the resulting vectors, highlighting the translational potential of this approach.

Our findings underscore the dual benefits of engineering Rep-Cap plasmids: enhancing AAV manufacturability and potency. This strategy offers a scalable, versatile solution to current manufacturing challenges, meeting the growing demand for high-quality AAV vectors in clinical and commercial applications.