Brimble, MA;
Cheng, PH;
Winston, SM;
Reeves, IL;
Souquette, A;
Spence, Y;
Zhou, J;
... Davidoff, AM; + view all
(2022)
Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps.
Molecular Therapy - Methods and Clinical Development
, 24
pp. 280-291.
10.1016/j.omtm.2022.01.008.
(In press).
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Abstract
Recombinant adeno-associated virus (rAAV) vectors are increasingly being used for clinical gene transfer and have shown great potential for the treatment of several monogenic disorders. However, contaminant DNA from producer plasmids can be packaged into rAAV alongside the intended expression cassette-containing vector genome. The consequences of this are unknown. Our analysis of rAAV preps revealed abundant contaminant sequences upstream of the AAV replication (Rep) protein driving promoter, P5, on the Rep-Cap producer plasmid. Characterization of P5-associated contaminants after infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes, in addition to in vitro evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy.
| Type: | Article |
|---|---|
| Title: | Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.omtm.2022.01.008 |
| Publisher version: | https://doi.org/10.1016/j.omtm.2022.01.008 |
| Language: | English |
| Additional information: | This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
| Keywords: | Adeno-associated virus, gene therapy, rAAVP5, vectorology, contamination, viral manufacturing |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Haematology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10143775 |
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