In recent years, there has been an increase in the number of registered clinical trials evaluating recombinant adeno-associated virus (AAV)-based gene therapies, in part due to AAV’s ability to effectively deliver genes to target cells with minimal side effects. While approved therapies have the potential to change lives, they also represent some of the costliest drugs on the market, reflecting in part the cost to produce them. Some of the high production costs are driven by inefficiencies in generating sufficient quantities of AAV particles. A therapy may require anywhere from 1011 to 1016 viral genomes per dose1; cell stacks or large bioreactors may produce only a handful of doses per run, presenting a substantial bottleneck in manufacturing.2 To reduce costs and realize the full potential of gene therapies, improvements in productivity are essential.

One area for improvement is the transfection process used in the upstream manufacturing of AAV gene therapies. The TransIT-VirusGEN® transfection solution includes both polyamine-containing polymers and lipids that help overcome barriers during transfection, enabling high transfection efficiency and low cellular toxicity. The polymer facilitates nucleic acid condensation, binding, and uptake by the cells, while the lipid promotes endosomal escape.

The RevIT™ AAV Enhancer can be used in conjunction with both the VirusGEN solution and conventional polymeric transfection reagents to produce 2–4X higher genome titers in suspension HEK 293 cells. The enhancer is simple to use, easily integrates into existing workflows, and produces high-quality titers across a range of AAV serotypes and cell growth media. When combined with the TransIT-VirusGEN solution, the enhancer enables use of lower amounts of plasmid DNA (pDNA), which represents a key cost-saving opportunity.

Improved genome titers across multiple AAV serotypes

The efficacy of RevIT AAV Enhancer was assessed across multiple serotypes (AAV2, AAV5, AAV8, and AAV9) in 293-VP 2.0 cells (Thermo Fisher) using TransIT-VirusGEN and the single-component polymeric transfection reagents (Figure 1). In all serotypes, use of the RevIT AAV Enhancer in conjunction with TransIT-VirusGEN increased genome titers 1.7- to 2.4-fold compared to the TransIT-VirusGEN control. The RevIT AAV Enhancer plus TransIT-VirusGEN condition also delivered up to 6-fold higher genome titers and 2.9-fold higher percent full capsids. RevIT AAV Enhancer increased genome titers 1.7- to 2.2-fold with other transfection reagents compared to their respective controls, demonstrating broad applicability.

Figure 1. RevIT™ AAV Enhancer increases genome titers across multiple serotypes and transfection platforms.

The enhancer was also tested in AAV2, AAV5, AAV8, and AAV9 serotypes using 293-VP 2.0 cells grown in either Viral Production Medium (Thermo Fisher) or BalanCD HEK 293 Medium (Irvine Scientific). There was little to no difference in performance across these growth media formulations (Figure 2). Together, these data demonstrate broad serotype and growth medium compatibility when using RevIT AAV Enhancer in conjunction with TransIT-VirusGEN and single-component polymeric transfection reagents.

Figure 2. RevIT™ AAV Enhancer is effective in multiple cellular growth media formulations.

Cost savings achieved with lower pDNA doses

Protocols for commonly used single-component polymeric transfection reagents recommend pDNA doses of 1 μg/106 cells (for example, 3 μg/mL at a density of 3 × 106 cells/mL) for triple-transfection-mediated AAV production in suspension cells. In contrast, the TransIT-VirusGEN protocol recommends a pDNA dose of 2 μg/mL of cell culture regardless of cell density, which represents a 33% decrease in pDNA usage. Scaling pDNA dosage by cell density does not improve viral titers, even when RevIT AAV Enhancer is employed, suggesting that maintaining a pDNA dose of 2 μg/mL cell culture with TransIT-VirusGEN can reduce the usage of valuable pDNA and save on manufacturing costs.

Lower amounts of pDNA with RevIT AAV Enhancer were tested, demonstrating that the enhancer allowed for a decrease in pDNA doses to as low as 0.75 μg/mL in some serotypes while still maintaining high genome titers (Figure 3). This allows for up to a 75% decrease in pDNA usage compared to a traditional DNA dose method of 3 μg/mL pDNA for a density of 3 × 106 cells/mL. Decreasing pDNA doses also led to a higher percentage of full capsids, demonstrating that drastic cost savings and higher quality AAV can be achieved using lower pDNA doses in conjunction with RevIT AAV Enhancer.

Figure 3. Lower pDNA doses can be used to achieve high genome titers and high percent full capsids with RevIT™ AAV Enhancer.

A proven strategy to increase titers and decrease costs

Use of the RevIT AAV Enhancer substantially increases AAV genome titers across multiple serotypes and transfection platforms, including the TransIT-VirusGEN transfection reagent and polymeric transfection reagents. Simple optimization allows for fast and easy integration of RevIT AAV Enhancer into existing AAV manufacturing workflows to increase titers by 2–4X. The enhancer also enables reductions in the amount of pDNA required during the transfection process, leading to considerable cost savings in AAV-based gene therapy manufacturing.

Download our white paper on use of the RevIT AAV Enhancer for additional studies and
details on materials and methods:(

Becky Reese, PhD, is senior scientist, Jennifer Swanson is R&D associate scientist III, Austin Storck is associate scientist III , and Laura Juckem, PhD, is vice president of research and development at Mirus Bio.


1. Au HK, Isalan M, Mielcarek M. Gene therapy advances: A Meta-Analysis of AAV Usage in Clinical Settings. Front. Med. (Lausanne) 2022; 8.

2. Clément N, Grieger JC. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol. Ther. Methods Clin. Dev. 2016; 3: 16002. 

Previous articleMicrospectral Analysis Safety
Next articleMultiomics Opens the Door: More Data, More Insight into Biology’s Complexity