Combined Plasmid Redesign and Transfection Optimization Significantly Increases Upstream AAV Titers While Maintaining Vector Quality and In Vivo Potency
Aug 2026· Microorganisms· Vol 14· 0 citations· 43 references
Medicine
TL;DR
Across multiple AAV capsids and independent production runs, the optimized process reproducibly increased crude harvest titers by approximately 10- to 33-fold relative to the standard process, while maintaining key vector quality attributes.
Abstract
A high manufacturing cost of goods (CoG) remains a critical barrier to the broad clinical adoption of gene therapies and is driven in part by limited productivity in adeno-associated virus (AAV) manufacturing. Here, we report an optimized AAV production process developed to markedly increase upstream titers while preserving vector quality and potency. The process combines a redesigned plasmid system, an optimized plasmid ratio, and a novel synthetic transfection reagent and was benchmarked against a conventional triple-plasmid/PEI MAX workflow. Across multiple AAV capsids and independent production runs, the optimized process reproducibly increased crude harvest titers by approximately 10- to 33-fold relative to the standard process, while maintaining key vector quality attributes. Notably, within the detection limits of the assay, rcAAV was undetectable at 1 × 1010 vg input with the optimized process, whereas the conventional triple-plasmid (with native Rep-Cap sequence)/PEI MAX workflow remained rcAAV-positive under identical conditions. Importantly, the in vivo potency was comparable to that of vectors produced by the conventional process. These results position our optimized AAV production process as a promising strategy to materially reduce AAV manufacturing CoG per patient.
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
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
Viral vectors for gene and cell therapy are typically produced by transfecting plasmids into mammalian cells. Because plasmid DNA is not replicated in these cells, large quantities are required to express sufficient viral proteins for vector packaging, contributing significantly to manufacturing costs and plasmid-derived contaminants. Here, we harness the DNA replication machinery of human adenovirus to efficiently amplify plasmid DNA within HEK293 cells for adeno-associated virus (AAV) vector production. This approach enables vector manufacturing with 10-20-fold less plasmid encoding AAV viral genes, while replication in cells achieves up to 400-fold amplification to support packaging. Coordinated amplification and expression of viral genes recapitulate wildtype AAV biology, conferring gene therapy vectors with beneficial attributes such as high packaging efficiency and infectivity. Implementing this process during AAV vector production increases yield, full capsid ratio, reduces plasmid backbone contaminants, and notably, enhances vector potency up to threefold both in vitro and in vivo. AAV vector production with robust plasmid DNA in cellulo replication (AAVPCR) proves effective across serotypes and transgenes, offering broad potential to improve the safety, efficacy, and affordability of AAV gene therapies. Moreover, plasmid DNA replication in mammalian cells may enable diverse applications in the biomanufacturing of gene and cell therapy vectors.
Hao Liu, Ailing Du, Nan Liu et al.· Molecular Therapy· 0 citations
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.
Sirimar Laosinwattana, Yiwen Li, Braulio Carrillo Sanchez et al.· Biotechnology and Bioenginee...· 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
Recovery of adeno-associated virus (AAV) vectors from culture supernatants has attracted increasing interest as a strategy to simplify downstream processing and improve product purity. However, several AAV serotypes can be re-internalized by producer cells, raising the possibility that newly released particles are recaptured during production and thereby limiting extracellular yield. Because AAV entry is mediated by the universal receptor KIAA0319L (AAVR), receptor-dependent re-uptake may contribute to this limitation, yet its role in vector manufacturing remains unexplored. Here, we investigated whether disruption of AAVR enhances supernatant-based AAV recovery. Using CRISPR-Cas9-mediated genome editing, we generated AAVR-knockout (AAVR-KO) HEK293-EB producer cells by deleting exon 2 containing the translational start codon. AAVR ablation abolished susceptibility to multiple AAV serotypes and markedly reduced cellular uptake of extracellular particles. In supernatant AAV vector recovery assays, AAVR knockout increased supernatant vector genome recovery for selected serotypes in a representative clone, although this effect varied among independently isolated clones. Complementary AAVR overexpression reduced accumulation of extracellular AAV1 vector genomes, supporting a role for AAVR expression levels in supernatant recovery while also suggesting additional effects on production-related cellular processes. These findings identify AAVR-dependent cellular uptake or retention as a modifiable post-release process that can influence supernatant-based AAV vector recovery.