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Updated: Sep 30, 2025
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Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors
Published on: April 5, 2024
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Human and Insect Cell-Produced Recombinant Adeno-Associated Viruses Show Differences in Genome Heterogeneity
Ngoc Tam Tran1,2, Emilie Lecomte3, Sylvie Saleun3
1Horae Gene Therapy Center, UMass Chan Medical School, Worcester, Massachusetts, USA.
Human Gene Therapy
|March 16, 2022
Summary
Adeno-associated virus (AAV) vector production pipelines impact genome accuracy. Even "empty" capsids contain truncated genomes, suggesting a need for improved AAV vector quantification and evaluation methods.
Area of Science:
- Gene Therapy
- Molecular Biology
- Biotechnology
Background:
- Adeno-associated virus (AAV) vectors are crucial for gene therapy, but manufacturing challenges persist.
- Ensuring the safety and efficacy of AAV vectors requires understanding the composition of vector preparations, particularly concerning empty or partially filled capsids.
- Current methods cannot fully eliminate empty or partial capsids, and their exact genome content remains largely unknown.
Purpose of the Study:
- To investigate the genome composition of DNase-resistant, encapsidated AAV vector genomes produced by two common manufacturing pipelines.
- To compare the accuracy of vector genomes generated by plasmid transfection in HEK293 cells (pTx/HEK293) versus baculovirus expression in Sf9 cells (rBV/Sf9).
- To characterize the contents of purified "empty" AAV capsids.
Main Methods:
- AAV-genome population sequencing was employed to analyze vector genome compositions.
- Two production systems were compared: pTx/HEK293 and rBV/Sf9.
- Cesium chloride gradient ultracentrifugation was used for purification of empty capsids.
Main Results:
- The rBV/Sf9 production system yielded a higher proportion of truncated and unresolved vector genomes compared to the pTx/HEK293 system.
- "Empty" capsids purified via ultracentrifugation were found to contain single truncated or unresolved inverted terminal repeat (ITR) sequences.
- The frequency of these "mutated" ITRs correlated with the abundance of inaccurate genomes across all analyzed fractions.
Conclusions:
- Manufacturing pipeline choice significantly influences AAV vector genome integrity.
- Even purified "empty" AAV capsids contain packaged genetic material, challenging current purification and quantification standards.
- These findings necessitate a re-evaluation of AAV vector evaluation and quantification methods for improved clinical safety and efficacy.
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