Enzymatically amplified linear dbDNA TM as a rapid and scalable solution to industrial lentiviral vector manufacturing.

Autor: Barreira M; Touchlight Genetics Ltd, Hampton, TW12 2ER, United Kingdom.; Cell and Gene Therapy Catapult, Guy's Hospital, London, SE1 9RT, United Kingdom., Kerridge C; Touchlight Genetics Ltd, Hampton, TW12 2ER, United Kingdom., Jorda S; Touchlight Genetics Ltd, Hampton, TW12 2ER, United Kingdom.; Medical Research Institute La Fe, 46026, Valencia, Spain., Olofsson D; Omiqa Bioinformatics GmbH, Altensteinstraße 40, 14195, Berlin, Germany., Neumann A; Omiqa Bioinformatics GmbH, Altensteinstraße 40, 14195, Berlin, Germany., Horton H; Touchlight Genetics Ltd, Hampton, TW12 2ER, United Kingdom., Smith-Moore S; Touchlight Genetics Ltd, Hampton, TW12 2ER, United Kingdom. sarah.moore@touchlight.com.
Jazyk: angličtina
Zdroj: Gene therapy [Gene Ther] 2023 Feb; Vol. 30 (1-2), pp. 122-131. Date of Electronic Publication: 2022 May 24.
DOI: 10.1038/s41434-022-00343-4
Abstrakt: Traditional bacterial fermentation techniques used to manufacture plasmid are time-consuming, expensive, and inherently unstable. The production of sufficient GMP grade material thus imposes a major bottleneck on industrial-scale manufacturing of lentiviral vectors (LVV). Touchlight's linear doggybone DNA (dbDNA TM ) is an enzymatically amplified DNA vector produced with exceptional speed through an in vitro dual enzyme process, enabling industrial-scale manufacturing of GMP material in a fraction of the time required for plasmid. We have previously shown that dbDNA TM can be used to produce functional LVV; however, obtaining high LVV titres remained a challenge. Here, we aimed to demonstrate that dbDNA TM could be optimised for the manufacture of high titre LVV. We found that dbDNA TM displayed a unique transfection and expression profile in the context of LVV production, which necessitated the optimisation of DNA input and construct ratios. Furthermore, we demonstrate that efficient 3' end processing of viral genomic RNA (vgRNA) derived from linear dbDNA TM transfer vectors required the addition of a strong 3' termination signal and downstream spacer sequence to enable efficient vgRNA packaging. Using these improved vector architectures along with optimised transfection conditions, we were able to produce a CAR19h28z LVV with equivalent infectious titres as achieved using plasmid, demonstrating that dbDNA TM technology can provide a highly effective solution to the plasmid bottleneck.
(© 2022. The Author(s).)
Databáze: MEDLINE
Externí odkaz: