Biology subjects

Glaser, V.

Publications and source records attributed to Glaser, V..

3 recordsLinked to original sources

bioRxiv2023

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells

I.Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3{zeta} (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3{zeta} gene as the CARs activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCR{gamma}/{delta} T cells, regulatory T cells, and NK cells. In T cells, CD3{zeta} in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3{zeta}-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3{zeta}-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC-edited CAR-T cells, integration of a Her2-CAR into CD3{zeta} conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3{zeta} gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3{zeta} gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key pointsO_LIIntegration of {zeta}-deficient CARs into CD3{zeta} gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use C_LIO_LICD3{zeta}-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions C_LI

immunology↗

bioRxiv2023

Matching or genetic engineering of HLA Class I and II facilitates successful allogeneic 'off-the-shelf' regulatory T cell therapy

The potential to harness regulatory T cells (Tregs) for the treatment of autoimmune diseases and transplant rejection has been restricted by several barriers: donor variability, manufacturing complications, and time-consuming expansion processes. These issues further complicate the use of autologous Tregs during acute disease phases or when Tregs are low in number or dysfunctional. Here we explore the potential of off-the-shelf allogeneic Tregs, from healthy donors or universal sources, to provide a more practical solution. We discover that the efficacy of these cells is undermined by the recipients immune response, and that that rigorous matching of HLA classes I and II overcomes this barrier. Importantly, genetically manipulating HLA expression enables the use of unmatched allogeneic Tregs with in vivo efficacy. Our findings underscore the transformative potential of HLA-engineered Tregs, offering a novel, ready-to-use therapeutic avenue for treating a wide array of inflammatory diseases. One-Sentence SummaryMatching or engineering of HLA-I and HLA-II facilitates allogeneic off-the-shelf regulatory T cells for immunoregulation.

immunology↗

bioRxiv2022

Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

I.Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out (KO) or targeted transgene knock-in (KI). However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted KI and Cas9-derived base editing technology for DSB free KOs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two KOs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduced translocations to 1.5% of edited cells. Small insertions and deletion at the base editing target sites indicated guide RNA exchange between the editors. This was overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR KI and a Cas9-derived base editor enabled the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting T cell receptor- (TCR-) and MHC-negative CAR T cells resisted allogeneic T cell targeting in vitro. Thus, we demonstrate a solution for safer multiplex-edited cell products and a path towards off-the-shelf CAR therapeutics.

bioengineering↗