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-Structure paper
Title | Cellular and Structural Characterization of VP1 and VP2 Knockout Mutants of AAV3B Serotype and Implications for AAV Manufacturing. |
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Journal, issue, pages | Hum Gene Ther, Vol. 33, Issue 21-22, Page 1142-1156, Year 2022 |
Publish date | Sep 9, 2022 |
Authors | Iker Arriaga / Aitor Navarro / Amaia Etxabe / César Trigueros / R Jude Samulski / Philippe Moullier / Achille François / Nicola G A Abrescia / |
PubMed Abstract | AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for ...AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WT > VP2 > VP1 > VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing. |
External links | Hum Gene Ther / PubMed:36082996 |
Methods | EM (single particle) |
Resolution | 3.1 - 25.0 Å |
Structure data | EMDB-15286, PDB-8a9u: EMDB-15287: Broken-(Dis)assembled AAV3B-VP2KO particle |
Source |
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Keywords | VIRUS / Adeno-Associated Virus / AAV / AAV3B / AAV serotype 3B / Full / VP1KO |