Thesis

hESC-derived NK cells expressing CD4/ζ CAR for enhanced anti-HIV-1 cytotoxic activity in vitro

Cell-based approaches utilizing hematopoietic stem cells (HSC) and T cells with naturally occurring, or experimentally introduced, genetic modifications that confer either HIV resistance or enhanced anti-HIV properties have emerged recently as viable approaches to treating HIV infection. However, a number of technical issues, such as a lack of protocols to maintain and expand HSC in vitro, inefficient gene transfer technologies, and the induction of T cell exhaustion (due to extensive in vitro expansion prior to adoptive transfer) have impeded the progress in this area. Human embryonic stem cells (hESC), which can be used to derive any type of immune cells including natural killer (NK) cells, may be a superior source of genetically modified cells compared with primary NK cells or HSC. hESC can be maintained in culture in an undifferentiated state indefinitely, and as such they can be extensively genetically manipulated, characterized for potential genotoxic events at the nucleotide level, and expanded to clinically relevant quantities. Given that these cells can be clonally expanded from a single cell, virtually all of the progeny of the genetically engineered hESC will carry the same modification at the same genomic position. Furthermore, as these cells can be effectively cryopreserved and differentiated into the desired lineage at different time points, the same cell populations can be used for preclinical studies and patient treatment. Importantly, in the case of lymphoid lineages, due to the extended length of their telomere ends, hESC-derived NK cells are less likely than their normal counterparts to undergo cellular senescence or immunological exhaustion upon expansion. We generated hESC lines that express an anti-HIV chimeric antigen receptor (CAR), which is a fusion molecule consisting of human CD4 with the signaling domain of the CD3 complex zeta-chain. CD4 binding to HIV-1 gp120 envelope on the surface of infected cells will trigger NK-cell recognition of infected cells and activation of their effector functions through zeta-chain signaling. Importantly, this vector also carries two anti-HIV shRNA sequences that confer protection from HIV-1 infection to the transduced cells. Here we present the phenotypic and in vitro functional characterization of the hESC-derived, anti-HIV CAR-expressing, NK cells.

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