Efficient and Robust NK-Cell Transduction With Baboon Envelope Pseudotyped Lentivector

Abstract

NK-cell resistance to transduction is a major technical hurdle for developing NK-cell immunotherapy. By using Baboon envelope pseudotyped lentiviral vectors (BaEV-LVs) encoding eGFP, we obtained a transduction rate of 23.0 ± 6.6% (mean ± SD) in freshly-isolated human NK-cells (FI-NK) and 83.4 ± 10.1% (mean ± SD) in NK-cells obtained from the NK-cell Activation and Expansion System (NKAES), with a sustained transgene expression for at least 21 days. BaEV-LVs outperformed Vesicular Stomatitis Virus type-G (VSV-G)-, RD114-and Measles Virus (MV)-pseudotyped LVs (p < 0.0001). mRNA expression of both BaEV receptors, ASCT1 and ASCT2, was detected in FI-NK and NKAES, with higher expression in NKAES. Transduction with BaEV-LVs encoding for CAR-CD22 resulted in robust CAR-expression on 38.3 ± 23.8% (mean ± SD) of NKAES cells, leading to specific killing of NK-resistant pre-BALL -RS4;11 cell line. Using a larger vector encoding a dual CD19/CD22-CAR, we were able to transduce and re-expand dual-CAR-expressing NKAES, even with lower viral titer. These dual-CAR-NK efficiently killed both CD19 KO-and CD22 KO-RS4;11 cells. Our results suggest that BaEV-LVs may efficiently enable NK-cell biological studies and translation of NK-cell-based immunotherapy to the clinic.

Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy

“…Surprisingly, the use of BaEV pseudotyped LVs has overcome this obstacle and showed a 20-fold increase in the transduction efficiency when compared to the VSV-G pseudotyped LVs. What is more important, CAR expressing NK cells showed improvement in functionality and a higher ability to kill cancer cells [40,41]. This is proof of concept for the generation of CAR-NK cells in vitro, which may become powerful immunotherapeutic products in the future.…”

“…Additionally, BaEV-LVs were able to transduce resting naïve B cells and memory B cells (20-40% efficacy) which had not been reported before [42]. In the case of T cells, recent data demonstrated that BaEV pseudotyped LVs are capable of transducing not only naïve T cells but also early thymocytes and natural killer cells with high transduction rates (up to 80%; Table 1) [39][40][41].…”

Highly Efficient Generation of Transgenically Augmented CAR NK Cells Overexpressing CXCR4

“…Similar high gene transfer rates to primary NK cells were reached using Vecotfusin-1 as an enhancer, which might even be increased when using a different viral envelope. For Vectofusin-1, it was recently demonstrated that transgene delivery to primary NK cells can be improved by pseudotyping LVs with the envelope protein from endogenous feline virus (RD114) or baboon endogenous retrovirus (BaEV) instead of VSV-G ( 5 , 6 ). In the field of CAR T-cells gamma-retroviral vectors or VSV-G pseudotyped LVs are most commonly used for their generation.…”

“…Transduction efficiencies of NK cells vary tremendously depending on the cell source, the viral vector system, and the transduction enhancer used ( 4 ). Lentiviral vectors (LVs) pseudotyped with the glycoprotein of the vesicular stomatitis virus (VSV-G) are classically used to generate CAR T-cells but are less efficient for NK cells with transduction efficiencies of 20–40% ( 5 , 6 ). Therefore, optimization of gene transfer protocols for VSV-G pseudotyped LV for the generation of CAR NK cells is urgently required.…”

You Have Got a Fast CAR: Chimeric Antigen Receptor NK Cells in Cancer Therapy

“…Optimization of the virus packaging identified a higher transduction efficiency for primary NK cells when a Baboon envelope pseudotyped lentivirus (BaEV-LV) was used [105]. The previous standard was vesicular stomatitis virus GP (VSV-G) [106].…”