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

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 (dbDNATM) 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 dbDNATM can be used to produce functional LVV; however, obtaining high LVV titres remained a challenge. Here, we aimed to demonstrate that dbDNATM could be optimised for the manufacture of high titre LVV. We found that dbDNATM 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 dbDNATM 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 dbDNATM technology can provide a highly effective solution to the plasmid bottleneck.

Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome.

Adeno-associated virus (AAV) is a small human Dependovirus whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at the rep ORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)-PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity.