Enhancing efficacy of virus-specific T cell therapy following hematopoietic stem cell transplantation by CD52 knockout

Viral infections are common and can be severe in immunocompromised patients, including those undergoing hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT). Virus-specific T cells (VSTs) have been effective in prevention and treatment of viral infections in patients post HSCT or SOT, but are inactivated or eliminated by immunosuppressive therapies including alemtuzumab, which limits their application and efficacy. We have demonstrated that CD52 knockout via CRISPR/Cas9 can be performed in CMV-specific T cells with maintenance of antiviral specificity. The overarching goal of this proposal is to study the impact of CD52-knockout on the function of multivirus-specific T cells, including antiviral specificity, cytokine profile, and cytotoxicity, as well as the persistence and safety of CD52-KO VSTs in vivo. In this study, we will address the following specific aims: 1) To establish the biological activity of multivirus-specific CD52-KO T cells in comparison with non-edited virus-specific T cells, and 2) To determine if CD52-KO enables T cell persistence and efficacy in the presence of alemtuzumab in vivo. To evaluate CD52-KO as a preventative cellular therapy, we propose to optimize CRISPR/Cas9 editing of rapidly-expanded multiviral T cells (targeting cytomegalovirus, Epstein-Barr virus (EBV), and adenovirus) from healthy donors, followed by extensive characterization of the phenotype and function of CD52-KO VSTs versus mock-edited VSTs, including surface markers, cytokine profile, single cell RNA-sequencing, and T cell receptor sequencing. Using a mouse model, we will evaluate the persistence and safety of CD52-KO VSTs in the presence of alemtuzumab. To assess function in vivo, we will test CD52-KO or mock-edited VSTs against EBV-lymphoblastoid tumors in mice, in the presence or absence of alemtuzumab. Finally, we will perform off-targeting analyses of the lead single guide RNA candidates by in silico and orthogonal testing of CD52-KO and mock-edited VSTs. We hypothesize that CD52-KO will have negligible impact on VST phenotype and function, and will enable persistence and activity in the presence of alemtuzumab in vivo. Completion of this study would provide a novel antiviral therapy which could reduce virus-associated morbidity in HSCT, and will develop a pipeline for production of a next generation of gene-modified VSTs to enhance efficacy in immunocompromised patients.