Aug 2026· Biotechnology and Bioengineering· 0 citations· 45 references
Medicine
TL;DR
This study highlights the potential of using evolutionary coupling analysis to guide AAV capsid engineering, suggesting that conservative mutations informed by evolutionary data can improve AAV stability and efficacy.
Abstract
Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. However, to unlock the full potential, their manufacturing yields, stability, and efficacy must all be improved. Rational design is limited by poor predictability and the potential impact of mutations on multiple important vector properties. Directed evolution requires little knowledge or predictability, but requires large libraries, and lacks appropriate selection methods to ensure that all properties needed for successful translation are achieved. Here we bring semi-rational design to AAV capsid engineering, combining co-evolutionary coupling and consensus-based design. We designed 110 mutants at sites with different degrees of coupling and mutated them either back to consensus or with various residue similarities according to BLOSUM62. Seven were selected based on their yields and transduction efficiencies before and after heat treatment. Our results demonstrate that mutations at sites within coupled networks exhibited the highest success rates. The BLOSUM62 selection strategy and consensus design approaches had similar success rates. K-means clustering of mutants further revealed that five of the successful mutants clustered in regions with moderate coupling scores and positive BLOSUM62 values. This study highlights the potential of using evolutionary coupling analysis to guide AAV capsid engineering, suggesting that conservative mutations informed by evolutionary data can improve AAV stability and efficacy.
This platform provides a powerful approach for engineering next-generation AAV vectors with customizable targeting profiles for diverse therapeutic applications and preserves AAV integrity and infectivity, and enables programmable retargeting of AAV tropism toward disease-relevant receptors.
Quan Pham, Jake Glicksman, Abhishek Chatterjee· Methods in molecular biology· 0 citations
Recombinant adeno-associated virus (rAAV) is a preferred vector in gene therapy, although high production costs inhibit widespread adoption. The most common approach for rAAV production involves transfection of HEK293 cells with three plasmids: pTransgene, pRep/Cap and pHelper. Producing sufficient amounts of these plasmids accounts for up to 40% of total batch costs. Initially, this work aimed to increase plasmid yields by replacing the backbones. While this approach increased pHelper yields, pRep/Cap and pTransgene yields were unaffected. A possible reason was identified: pTransgene contains inverted terminal repeat (ITR) sequences that are essential for rAAV production. ITRs have strong secondary structures (including hairpin loops termed B and C arms) that likely interfere with plasmid production. Therefore, targeted deletions were performed within the ITRs. Partial deletions in both the B and C arms of the ITR were most beneficial, as both plasmid yield and transgene expression increased. Importantly, partial deletions did not reduce rAAV yield, as had been previously observed when the B and C arms were fully deleted. In summary, we report a 140% increase in pHelper plasmid production, while the most successful ITR variant increased pTransgene plasmid yields by 57% and transgene expression by 28%, without reducing rAAV yields or transduction efficiency.
Nicholas Donohue, Alexandra Bogdanovic, James Conheady et al.· International Journal of Mol...· 0 citations
This article aims to provide a working framework for verifying the potency, genomic integrity, and clinical safety of vector-based gene therapies—one intended to be useful both to laboratories developing these products and to those responsible for regulating them.
Yusra A. Radeef, Z. Abdullah, Eman Fadhel Abbas Awadh· International Journal of Mul...· 0 citations
This study generated a novel recombinant AAV vector rAAV.hu.hu.S17, derived from the human spleen isolate AAV.hu.S17, and systematically evaluated its capsid features, in vitro transduction, and in vivo tissue tropism.
Wenyan Guo, Jiawen Sun, Fei Wang et al.· Journal of Genetic Engineeri...· 0 citations
The data support the genomic safety of rAAV6 and its applicability to hematological gene therapy and functional enrichment analysis indicated associations with general cellular and structural processes, without enrichment in oncogenic pathways.
H. Lee, Nayoung Park, In-Byung Park et al.· International Journal of Ste...· 0 citations
Establishing robust, serotype-specific AAV RSMs and harmonised standard operating protocols (SOPs) are essential for advancing AAV gene therapy and ensuring accuracy, reproducibility, and safety across research, development, and clinical manufacturing.
Saleheen Khan, Nathalie Van den Berghe, Els Henckaerts· Journal of Visualized Experi...· 0 citations