Related Experiment Video
Updated: Jun 14, 2025
Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System
Published on: January 13, 2022
Characterization of residual microRNAs in AAV vector batches produced in HEK293 mammalian cells and Sf9 insect cells
Magalie Penaud-Budloo1, Emilie Lecomte1, Quentin Lecomte1
1Nantes Université, CHU Nantes, INSERM, TARGET, 44000 Nantes, France.
Abstract:
With more than 130 clinical trials and 8 approved gene therapy products, adeno-associated virus (AAV) stands as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as microRNAs (miRNAs), has not been investigated previously. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the recombinant AAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.
Insights
MicroRNAs (miRNAs) are present in adeno-associated virus (AAV) gene therapy products. These residual miRNAs, found in all tested batches, may impact the safety and efficacy of AAV therapies.
Area of Science:
- Molecular Biology
- Gene Therapy
- Biotechnology
Background:
- Adeno-associated virus (AAV) is a widely used vector for in vivo gene therapy, with numerous clinical trials and approved products.
- Residual DNA is a critical quality attribute in AAV batches due to potential genotoxic and immunogenic risks.
- The presence and impact of small cell-derived RNAs, specifically microRNAs (miRNAs), in AAV preparations have not been previously investigated.
Purpose of the Study:
- To investigate the presence of microRNAs (miRNAs) in purified adeno-associated virus (AAV) batches.
- To determine if miRNA presence is dependent on the production cell line or AAV capsid serotype.
- To explore methods for reducing residual miRNA levels in AAV preparations.
Main Methods:
- Purification of adeno-associated virus (AAV) batches produced in mammalian and insect cell lines.
- Detection and quantification of microRNAs (miRNAs) in purified AAV preparations.
- Assessment of miRNA levels following immunoaffinity chromatography with tangential flow filtration and RNase treatment.
Main Results:
- MicroRNAs (miRNAs) were detected in all tested adeno-associated virus (AAV) batches, irrespective of the production cell line or capsid serotype (serotypes 2 and 8).
- The quantity of co-purified miRNAs correlated with their abundance in the respective production cells.
- Immunoaffinity chromatography and RNase treatment significantly reduced residual miRNA levels, suggesting they are largely non-encapsidated.
Conclusions:
- This study provides the first evidence that microRNAs (miRNAs) are consistently co-purified with adeno-associated virus (AAV) particles.
- Residual miRNAs represent a previously unrecognized quality attribute in AAV preparations.
- Further research is necessary to elucidate the potential impact of these co-purified miRNAs on the safety and efficacy of AAV-mediated gene therapies.
