ArticleMolecular therapy. Methods & clinical development2024
Producing high-quantity and high-quality recombinant adeno-associated virus by low-cis triple transfection.
Article in Molecular therapy. Methods & clinical development, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 12 citations in OpenAlex.
- Omics-guided engineering of CHO cells reveals host targets to support AAV production.Molecular therapy. Advances · 2026Article
- Anion-Exchange Membrane Chromatography for the Separation of Empty and Full Adeno-Associated Viral Capsids.Biotechnology journal · 2026Article
- An engineered UGA suppressor tRNA gene for disease-agnostic AAV delivery.Nature biotechnology · 2026Article
- Characterization and quantitation of baculoviral DNA in rAAV vectors produced in Sf9 cells.Molecular therapy. Methods & clinical development · 2025Article
- Reduced rAAV interference enhances rcAAV detection sensitivity.Molecular therapy. Methods & clinical development · 2025Article
- Development of an HEK293 Suspension Cell Culture Medium, Transient Transfection Optimization Workflow, and Analytics for Batch rAAV Manufacturing.Biotechnology and bioengineering · 2025Article
- Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors.Molecular therapy. Methods & clinical development · 2025Article
- Advancing AAV technology: From capsid design to scalable manufacturing.Molecular therapy. Methods & clinical development · 2025Article
- AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities.Molecular therapy. Nucleic acids · 2025Article
- Tracking Particle-Encapsulated DNA Across the Anion-Exchange Chromatography Fractions of Recombinant Adeno-Associated Virus Using Droplet Digital PCR and High-Throughput Sequencing.Biotechnology journal · 2025Article
- Developing AAV-delivered nonsense suppressor tRNAs for neurological disorders.Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics · 2024Review
Corrections and comments
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Authors and funding
20 authors at 1 institution in 1 country.
Funding
Abstract
Recombinant adeno-associated virus (rAAV)-based gene therapy is entering clinical and commercial stages at an unprecedented pace. Triple transfection of HEK293 cells is currently the most widely used platform for rAAV manufacturing. Here, we develop low-cis triple transfection that decreases transgene plasmid use by 10- to 100-fold and overcomes several major limitations associated with standard triple transfection. This new method improves packaging of yield-inhibiting transgenes by up to 10-fold, and generates rAAV batches with reduced plasmid backbone contamination that otherwise cannot be eliminated in downstream processing. When tested in mice and compared with rAAV produced by standard triple transfection, low-cis rAAV shows comparable or superior potency and results in diminished plasmid backbone DNA and RNA persistence in tissue. Mechanistically, low-cis triple transfection relies on the extensive replication of transgene cassette (i.e., inverted terminal repeat-flanked vector DNA) in HEK293 cells during production phase. This cost-effective method can be easily implemented and is widely applicable to producing rAAV of high quantity, purity, and potency.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.