IL-15/IL-15Rα-targeted CRISPRa boosts NK cell effector function against HIV-infected cells

Like many other immune cell types, natural killer (NK) cells exhibit impaired functionality in people with HIV (PWH), limiting their ability to efficiently target and eliminate HIV-infected cells and contributing to the persistence of the HIV reservoir. Recently, adoptive NK cell therapy using activated or engineered NK cells has emerged as a promising immunotherapeutic strategy for cancer; however, its application to HIV remains in the early stages of development. NK cells primarily rely on interleukin-15 (IL-15) signaling for their development, survival, and maintenance, and dysregulated IL-15 signaling has been reported in PWH. We hypothesized that simultaneous enhancement of IL-15 and IL-15 receptor alpha (IL-15Rα) expression would improve NK cell effector function and promote clearance of HIV reservoir. Utilizing dCas9-VPR-mediated multiplex CRISPR activation (CRISPRa), we successfully upregulated both IL-15 and IL-15Rα expression in the same primary NK cells without altering the genomic sequence. CRISPRa-treated NK cells displayed increased IL-15 and IL-15Rα transcript levels, as measured by RT-qPCR and significantly elevated surface expression of IL-15Rα, as determined by flow cytometry. Enhanced IL-15/IL-15Rα expression increased NK cell metabolic activity and cytotoxicity against target K562 cells, a chronic myelogenous leukemia cell line, indicating improved effector function. Furthermore, in co-culture experiments using an HIV-positive T-cell line, IL-15/IL-15Rα-CRISPRa-treated NK cells demonstrated enhanced target cell elimination and increased secretion of IFN-γ. Together, these findings provide a proof of concept that multiplex CRISPRa can be used to enhance NK cell function, representing a novel strategy to improve persistence and therapeutic efficacy in HIV and other disease settings.