Establishment of High Titer Baboon Endogenous Retrovirus Envelope Pseudotyped Lentiviral Vector Production Protocol Focusing on Syncytium Formation and Exfoliation

Introduction

Cytokine stimulation of hematopoietic stem cells (HSCs) for the expression of virus entry transporter is inevitable to obtain high transduction efficiency for VSVg envelope pseudotyped lentiviral vectors (VSVg-LVs). However, those stimulation induce apoptosis in HSCs derived from patients affected by DNA repair disorder, the establishment of novel transduction methods has been warranted. Sodium-dependent neutral amino acid transporter type-1 and type-2, which are the entry transporter for Baboon endogenous retrovirus (BaEV-Rless) envelope pseudotyped lentiviral vectors (BaEV-Rless-LVs), are highly expressed on the surface of HSCs in absence of cytokine stimulation. Therefore, BaEV-Rless-LVs could be an effective gene therapy tool to increase transduction in HSC with DNA repair-related mutations. However, production of high titer BaEV-Rless-LVs proved to be challenging compared to VSVg-LVs, as the titer of BaEV-Rless-LVs was one thousandth of VSVg-LVs. 293T cells transfected with helper plasmids, including BaEV-Rless plasmid, showed morphological changes such as syncytium formation and exfoliation, likely impairing cell viability and virus production. Therefore, for clinical applications, improved production of BaEV-Rless-LVs is mandatory. To this end, we modified the protocol to prevent the characteristic morphological changes, syncytium formation and exfoliation, which could be the causes of low virus titer.

Methods

In the attempt to increase viral production, we modified the VSVg-LVs "basic protocol" modified to produce BaEV-Rless-LVs: co-transfection of 0.44 µg of BaEV-Rless envelope plasmid, 1.464 µg of the self-inactivating HIV-1 derived vector encoding green fluorescent protein (GFP) under the control of spleen focus foamy virus promoter (SIN-HIV-SFFV-GFP), 0.336 μg of Rev regulatory plasmid and 0.74 μg of RRE plasmid to 293T cells per well of 6-well-plate, respectively. Virus titration was performed on 293T cells using serial dilution of the viral preparations and assessing 293T GFP⁺ cells using flow cytometry analysis. To investigate in detail the time-course of syncytium formation and exfoliation of transfected 293T cells, we used by time-lapse analysis to assess critical culture conditions and to guide us on how to further modify the protocol.

Results

Time-lapse analysis revealed that syncytium formation initiated at 8-hours post-transfection of the helper plasmids and terminated at 18-hours post-transfection. (Figure 1). Exfoliation started at 20-hours post-transfection, soon after completion of syncytium formation. As we hypothesized that the syncytium formation and exfoliation might be the cause of the low viral vector production, we modified three points of the basic protocol: the amount of BaEV-Rless plasmid, harvest timing of virus supernatant and use of poly-l-lysine (PLL) coated plate to avoid syncytium formation and exfoliation of 293T cells. First, to clarify whether the amount of BaEV-Rless plasmid was proportional to syncytium formation and virus titer, we changed the amounts of BaEV-Rless plasmid and compared titers. Interestingly, increasing 1.2-fold of the amount of plasmid raised the titer 3-fold despite syncytium formation. In contrast, even though smaller amounts of BaEV-Rless-plasmid did not induce syncytium formation and exfoliation, the titer of BaEV-Rless-LVs was reduced and/or undetectable. Next, we identified the optimal harvest time for viral titer. Harvest of viral supernatant at 48-hours post-transfection was associated with the highest titer despite completion of syncytium formation and exfoliation of 293T cells. Finally, we compared the titers with or without using PLL coated plate. Using PLL coated plate prevented exfoliation of transfected 293T cells and doubled the titer. Eventually, combination of using 1.2-folds of BaEV-Rless-plasmid, harvest of virus supernatant at 48-hours post-transfection and using PLL coated plate resulted in a 10-fold titer increase compared with the basic protocol (Figure 2).

Conclusion Our novel high titer protocol for BaEV-Rless-LVs results in 10-fold titer increase compared to the basic protocol, and it could be utilized to develop clinical applications for DNA repair-related disorders.

Rivella:Vifor: Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Cambridge Healthcare Res: Consultancy; Celgene Corporation: Consultancy; Catenion: Consultancy; First Manhattan Co.: Consultancy; FORMA Therapeutics: Consultancy; Ghost Tree Capital: Consultancy; Keros Therapeutics: Consultancy; venBio Select LLC: Consultancy; Rallybio, LLC: Consultancy; BVF Partners L.P.: Consultancy; Techspert.io: Consultancy; Slingshot Insight: Consultancy; Sanofi Aventis U.S.: Consultancy; Protagonist Therapeutics: Consultancy; Noble insight: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Membership on an entity's Board of Directors or advisory committees.