Development of 2LTRZFP-expressing induced pluripotent stem cells as a potential anti-HIV-1 gene therapy against viral integration

Highly active antiretroviral drug is the standard treatment for HIV-1 infection to suppress the viral load. However, this treatment does not completely eradicate the virus; it simply decreases the viral load to undetectable levels. The development of a novel therapy to cure the disease is essential. Previously, we developed an engineered zinc finger protein (ZFP) that specifically binds to the 2-LTR-circle junction (2LTRZFP), the target site for viral integrase, preventing HIV-1 integration in human CD34+ hematopoietic stem/progenitor cells (HSPCs) and macrophages. Although the transduction efficiency of 2LTRZFP was ∼50%, purifying and expanding the 2LTRZFP-expressing HSPCs proved difficult. In addition, the batch-to-batch variability in transduction efficiency could have a major impact on the therapeutic efficacy. In this study, we introduced the 2LTRZFP into human induced pluripotent stem cells (iPSCs) followed by clonal isolation and functional validation of the 2LTRZFP. Upon the HIV-1 challenge, the 2LTRZFP protein was found to inhibit the viral integration in iPSCs, iPSC-derived HSPCs, and macrophages. The engineered iPSC clone could be differentiated into functional macrophages, as evidenced by M1 and M2 polarization, and phagocytosis. Our finding revealed that the 2LTRZFP did not perturb the macrophage differentiation process. Therefore, the 2LTRZFP-expressing iPSCs could provide an unlimited supply of HIV-1-resistant HSPCs for transplantation, potentially leading to HIV-1-resistant blood cells. The knowledge obtained from this study will provide a cornerstone for HIV-1 gene therapy using HSPC transplantation as a sustainable HIV-1 treatment in the future.