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Expression of the minimally polymorphic HLA-E molecule prevents NK-cell-mediated rejection of cells lacking expression of HLA-A, B and C. Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin ( B2M ) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8 + T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

Malignant melanoma is a highly aggressive and drug-resistant cancer. Virotherapy is a novel therapeutic strategy based on cancer cell lysis through selective virus replication. However, its clinical efficacy is modest, apparently related to poor virus replication within the tumors. We report that the growth compromised HSV-2 mutant ΔPK has strong oncolytic activity for melanoma largely caused by a mechanism other than replication-induced cell lysis. The ratio of dead cells (determined by trypan blue or ethidium homodimer staining) to cells that stain with antibody to the major capsid protein VP5 (indicative of productive infection) was 1.8-4.1 for different melanoma cultures at 24-72hrs p.i. Cell death was due to activation of calpain as well as caspases-7 and -3 and it was abolished by the combination of calpain (PD150606) and pancaspase (zVAD-fmk) inhibitors. Upregulation of the autopahgy protein Beclin-1 and the pro-apoptotic protein H11/HspB8 accompanied ΔPK-induced melanoma oncolysis. Intratumoral ΔPK injection (106-107 pfu) significantly reduced melanoma tumor burden associated with calpain and caspases-7 and -3 activation, Beclin-1 and H11/HspB8 upregulation and activation of caspase-1 related inflammation. Complete remission was seen for 87.5% of the LM melanoma xenografts at 5 months after treatment termination. The data indicate that ΔPK is a promising virotherapy for melanoma that functions through virus-induced programmed cell death (PCD) pathways.

Cancer is a largely incurable and fatal disease that is resistant to standard therapeutics. This resistance is mediated by enhanced proliferative and prosurvival signals caused by activating mutations in the Ras/Raf/MEK/ERK and PI3K/AKT pathways, and the contribution of highly resilient cancer stem cells (CSC). Oncolytic virotherapy is based upon cancer specific virus replication and cell lysis. The specificity of engineered oncolytic viruses (OV) is accomplished through the deletion of one or more genes that are critical to virus replication. The mutations responsible for uncontrolled proliferation of cancer cells compensate for these deletions, thereby allowing OV to target cancer cells. HSV-2 encodes a unique serine/threonine protein kinase (PK)—known as ICP10PK—that activates the Ras and PI3K survival pathways. The mutant virus ΔPK, which lacks this PK domain, exhibits severely reduced levels of virus replication and latency reactivation while simultaneously inducing programmed cell death (PCD) in neurons. This dissertation work investigated the ability of ΔPK to selectively replicate and induce PCD in cancer cells and to define the molecular mechanisms involved therein. The data demonstrate that ΔPK eradicates cancer cultures and melanoma xenografts while sparing normal cells, and is well tolerated in treated mice. ΔPK-induced lysis of melanoma monolayer cultures is dominated by caspase- and calpain-mediated PCD, but also involves autophagy induction and JNK/cJun activation. Importantly, ΔPK eradication of breast and melanoma CSC requires autophagy and/or calpain but not caspase activation, and the contribution of calpain-mediated facilitation of autophagic flux represents a novel node of cross-talk between these two death pathways. In addition, ΔPK infection of melanoma cultures concomitantly inhibits anti-inflammatory IL-10 secretion while inducing the expression and secretion of numerous pro-inflammatory cytokines. Melanoma xenografts treated with ΔPK also exhibit a marked innate immune response. Significantly, ΔPK induces its own cancer specific replication through the activation of JNK, whereas the induction of PCD has no effect on virus growth. Collectively, the data indicate that ΔPK differentially induces a spectrum of PCD and pro-inflammatory programs that preclude the selection of resistant subpopulations in both quiescent CSC and proliferative tumor cells. These mechanisms uniquely potentiate its oncolytic activity and highlight its clinical promise.