UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

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). Green open access

[thumbnail of 1-s2.0-S2329050122000092-main.pdf]
Preview
Text
1-s2.0-S2329050122000092-main.pdf - Published Version

Download (2MB) | Preview

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.ucl.ac.uk/id/eprint/10143775
Downloads since deposit
15Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item