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It’s been an explosive few years for gene therapies. The ability to correct genetic mutations at the molecular level offers hope for curing previously untreatable conditions. However, despite the significant progress and the approval of new gene therapies, there are still challenges across the preclinical and clinical landscape.
Chief among them are the delivery and manufacturing of these therapies. Effective and efficient delivery mechanisms are crucial for ensuring that therapeutic genes reach their target cells. Simultaneously, scalable and cost-effective manufacturing processes are essential to make these treatments accessible and affordable for a broader range of patients.
Adeno-associated virus (AAV) has emerged as the preferred vector for delivering therapeutic transgenes and gene editing tools into cells. AAV-based therapies have shown considerable promise in treating rare genetic disorders, yet to realize their full potential; we must develop more manufacturable drugs that can be produced at scale and reasonable cost.
In the following blog, we’ll discuss how AAV-based gene therapies are manufactured and some of the problems that can arise during the process.
Understanding the AAV Manufacturing Process
The AAV therapeutic manufacturing process is intensive, with each dataset for a single construct taking about three weeks and costing approximately $50,000.1 Given the need to test multiple designs, the costs and time required for therapeutic development can escalate. The process involves several critical steps: Designing the viral construct with the right components, producing and purifying plasmids to cGMP standards, and using various systems for AAV production, each with its own complexities.2
Plasmid Construction, Development, and Preparation
The process begins with the production and purification of plasmid DNA, incorporating essential genetic elements for AAV vector production. This step is followed by rigorous quality control tests to ensure the plasmid is contaminant-free and meets the required genetic integrity standards. Finally, production is scaled up to produce sufficient quantities of plasmid DNA for large-scale viral vector manufacturing.
Cell Expansion
Following plasmid QC and scale-up, AAV manufacturers must select an appropriate cell line that efficiently supports AAV production and propagation. The culture conditions can be optimized to maximize cell growth and viability. Finally, the cell cultures are scaled up to the volume required for commercial production while maintaining cell health and productivity.
Plasmid Transfection
Plasmid DNA is then introduced into host cells using lipofection or another similar method as part of the delivery process.3 Transfection efficiency is closely monitored to achieve high rates of DNA uptake and expression rates. Continuous optimization involves adjusting parameters to improve the transfection efficiency, ensuring cells are primed for optimal viral vector production.
AAV Production
The process begins by producing AAV vectors using cells transfected with plasmid DNA. Viral particles are then harvested from the cell culture at the optimal time post-transfection to ensure high yield and activity. This stage is critical for quality assurance, involving tests to confirm the vectors' integrity, potency, and purity before proceeding further in the production process.
Purification
The purification process involves removing cellular debris and impurities using ultracentrifugation, chromatography, filtration, etc. The viral vector preparations are then concentrated to achieve the desired potency levels. Finally, the purification process is validated to ensure consistent quality and functionality of the viral vectors.
Fill & Finish
Following purification, the viral vector undergoes formulation to stabilize its integrity and efficacy in suitable conditions. Subsequently, the final product is filled into sterile vials or syringes under aseptic conditions to prevent contamination. Finally, the product is meticulously packaged and labeled to meet regulatory standards before distribution.
Persistent Manufacturing Challenges Distinguishing AAV Empty/Full Capsid Problem
During AAV-based gene therapy manufacturing, various impurities arise, such as empty AAV capsids lacking genomic DNA.4 Additionally, sequencing and molecular methods revealed that partially-filled capsids containing truncated genomes are another impurity generated during manufacturing. Collectively, these non-therapeutic capsids complicate dose calculations and increase immunogenic load, raising safety concerns.5 Accurate quantification of capsids with complete genomes is crucial to ensure the effective payload is delivered without exceeding safety thresholds.
Downstream techniques like size exclusion and anion-exchange chromatography can remove some empty capsid impurities but may not eliminate partially filled capsids. Pre-manufacturing methods such as analytical ultracentrifugation and scanning electron microscopy can identify these capsids but are not high-throughput.4 Full/empty capsid ratios and partially filled capsid analysis are critical quality attributes (CQAs) for gene therapy candidates. These ratios can be assessed by comparing viral genomes quantified by qPCR or ddPCR to viral particles measured by ELISA. However, the lack of ELISA antibodies for all AAV serotypes hinders standardization.
Optimizing AAV Payloads to Enhance Potency, Boost Specificity, and Minimize Immunogenic Response
Reducing AAV manufacturing costs depends on minimizing impurities and standardizing detection methods6. For example, Hemgenix production using a baculoviral expression system can achieve full-to-empty capsid ratios between 50% and 80%.7,8 Improving these ratios or reducing downstream purification needs could significantly lower gene therapy costs.
Form Bio’s AI platform provides a standardized, computational method for designing and producing better AAV gene therapies faster. Our recently launched In Silico Assays™ allow AAV gene therapy developers to access design risk assessments in under 24 hours before running any in vitro assays.
Our team has also published standard methods for analyzing and reporting rAAV data generated from PacBio’s HiFi sequencing method.9 These methods help determine how pure AAV vectors are and identify any impurities and improves rAAV design and quality control.
FAQs
How are AAVs manufactured?
Adeno-associated viruses (AAVs) are manufactured by transfecting host cells, typically HEK293 cells, with plasmids containing the AAV genome, replication genes, and helper virus genes. These cells then produce the viral particles, which are harvested from the cell culture. The AAV particles are subsequently purified through techniques such as ultracentrifugation or chromatography to remove impurities and concentrate the virus.
How long does it take to make AAV?
Producing AAV typically takes several weeks, including cell culture growth, transfection, and viral particle production, which usually spans around 7-10 days. Post-production, purification and quality control testing can take an additional 1-2 weeks. Overall, the entire process from start to finish generally takes about 3-4 weeks.
How long is the typical AAV genome?
The single-stranded DNA genome of AAV is 4.8 kb
How long does an AAV last?
Adeno-associated viruses (AAVs) are stable for several months to years when stored properly at -80°C. Once administered, the AAVs can persist in host tissues for months to years, with gene expression typically lasting several months. The duration of gene expression depends on factors such as the target tissue and the specific AAV serotype used.
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- Integrating AAV & AI For Gene Therapy Innovation | Form Bio. Accessed June 20, 2024.
- The Role of AAV in Effective Gene Therapy | Form Bio. Accessed February 16,
- Kimura T, Ferran B, Tsukahara Y, et al. Production of adeno-associated virus vectors for in vitro and in vivo applications. Sci Rep. 2019;9(1):13601.
- Penaud-Budloo M, François A, Clément N, Ayuso E. Pharmacology of Recombinant Adeno-associated Virus Production. Mol Ther - Methods Clin Dev. 2018;8:166-180.
- FDA. Cellular, Tissue, and Gene Therapies Advisory Committee. Meeting #70. Toxicity Risks of Adeno-associated Virus Vectors for Gene Therapy. September 2-3, 2021. Accessed March 8, 2023.
- FDA Clinical Trial Guidance For AAV Gene Therapy | Form Bio. Accessed September 13, 2023.
- Destro F, Joseph J, Srinivasan P, et al. Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system. Mol Ther - Methods Clin Dev. 2023;30:122-146.
- Destro F and John J, MIT. Mechanistic Modeling To Optimize rAAV Production For Gene Therapy. Cell and Gene. Published August 11, 2023. Accessed June 3, 2024.
- Talevich E, Tseng E, Diallo A, et al. Standardized Nomenclature and Reporting for PacBio HiFi Sequencing and Analysis of rAAV Gene Therapy Vectors. Published online May 12, 2024. Accessed June 4, 2024.
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