Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy

Abstract

As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell’s HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.

Gene editing to enhance the efficacy of cancer cell therapies

“…To evade donor HLA-mediated immune rejection, CRISPR-Cas9 T cell editing has been applied to b 2 -microglobulin (B2M) and HLA-II a chain genes (HLA-DRA, DQA, and DPA), showing decreased alloreactivity in vitro. 97 HLA class I, HLA class II, and TCR triple-knockout T cells by CRISPR-Cas9 editing of B2M, class II major histocompatibility complex transactivator (CIITA), and TRAC loci did not induce GVHD in preclinical models. 98 Ongoing clinical trials of gene-edited allogeneic T cell therapies are studying TRAC/B2M-knockout CD19 CAR T cells for B cell leukemia and lymphoma (ClinicalTrials.gov: NCT03166878 and NCT03229876), TRAC-knockout dual CD19/CD22 and CD19/CD20 CAR T cells for B cell leukemia (ClinicalTrials.gov: NCT03398967), and TCR-inserted CD19 CAR T cells for non-Hodgkin lymphoma and B cell ALL (ClinicalTrials.gov: NCT04649112 and NCT03666000).…”

Stem Cell-Based Clinical Trials for Diabetes Mellitus

“…These methods can be classified into two categories: induction of immune tolerance, and gene editing to generate 'cloaked' cells invisible to the immune system. Immune tolerance can be induced with tolerogenic cytokines and immunomodulatory proteins such as CTLA-4, and PD-L1 (107), whereas the generation of 'cloaked' cells is attempted by removal of HLA proteins, mainly through genome editing (108)(109)(110). ViaCyte, in partnership with CRISPR Therapeutics, is currently developing immune-evasive stem cell lines that combine both strategies.…”

Current Perspectives on the Use of off the Shelf CAR-T/NK Cells for the Treatment of Cancer

“…To overcome this the HLA-class 1 molecules can be removed from the T cells [ 117 ]. To remove HLA-class 1 the Beta-2 microglobulin (B2M) gene locus can be disrupted as it is required for HLA-class 1 expression ( Figure 2 ) [ 118 , 119 , 120 ]. Additionally, mechanisms utilised by viruses to evade immune recognition have been investigated.…”