Biology subjects

Glaser, V.

Publications and source records attributed to Glaser, V..

8 recordsLinked to original sources

bioRxiv2025

Repurposing base editors for targeted knock-in and simultaneous knockouts to generate multiplex-edited allogeneic CAR T cells with minimal translocations

The CRISPR-Cas system enables precise genome engineering of cell therapies. For allogeneic applications, multiplex editing is frequently required to improve efficacy, persistence, and safety. However, strategies involving multiple DNA double-strand breaks (DSBs) induce genotoxicity by provoking chromosomal aberrations. Base editors, which enable sequence changes without generating DSBs, are widely used for gene disruption, but their capacity for gene insertion remains unexplored. Here, we developed Base editor-mediated knock-in (BEKI), a non-viral platform that allows targeted transgene insertion in parallel with multiplex gene disruption using a single base editor. Repurposing the Cas9 nickase domain of base editors generates paired nicks, inducing homology-directed repair (HDR). In human T cells, optimized guide RNA orientation and nick distance, together with HDR-enhancing modulators, enabled efficient transgene knock-in at the TRAC, CD3{zeta}, B2M, and CD3{varepsilon} loci. Simultaneous base editing of multiple additional genes produced chimeric antigen receptor (CAR) T cells with increased cytokine secretion, drug resistance, and resistance to allo-rejection. Compared to multiplex editing with Cas9, BEKI markedly reduced chromosomal translocations. BEKI therefore provides a streamlined, scalable strategy for multiplex CAR T-cell engineering with a single enzyme, offering a safer route to clinical-grade manufacturing of off-the-shelf therapies for cancer and autoimmune diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/676172v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f5f6e5org.highwire.dtl.DTLVardef@246411org.highwire.dtl.DTLVardef@125a478org.highwire.dtl.DTLVardef@510278_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Versatile and efficient non-viral integration of large transgenes in human T cells via CRISPR knock-in and engineered integrases

Current gene transfer methods often lack the precision, versatility, or efficiency when integrating large transgenes, limiting the ability to engineer therapeutic T-cells with more complex payloads. Here, we report one-pot PASTA (Programmable and Site-specific Transgene Addition), a non-viral genome engineering strategy for large gene insertion that combines CRISPR-Cas-mediated homology-directed repair (HDR) and site-specific recombination via serine integrases. Using one-pot PASTA with the Bxb1 integrase, we demonstrate efficient integration of transgenes at multiple genomic loci relevant for T-cell engineering (e.g., TRAC, B2M, CD3E, CD3Z, GAPDH). For constructs > 8 kb, one-pot PASTA outperforms conventional HDR by 19-fold on average and prime-editing-assisted site-specific integrase gene editing (PASSIGE) by 5-fold. This enables the delivery of multi-cistronic cargo to generate dual-antigen targeting CAR T-cells with a safety-switch that overcome antigen escape in lymphoma models. Finally, one-pot PASTA can be further optimized with improved integrase enzymes, such as engineered variants of Pa01 or Bxb1, and plasmids with minimized backbones. In summary, one-pot PASTA represents a versatile and scalable platform for precise, non-viral gene insertion in T-cells.

synthetic biology↗

bioRxiv2024

ENTPD3-specific CAR Regulatory T cells for Local Immune Control in T1D

Despite advances in Type 1 Diabetes (T1D) management such as hybrid closed loop systems, patients still face significant morbidity, reduced life expectancy, and impaired glucose regulation compared to healthy individuals or those with pancreas transplants. Here we developed beta cell-specific Chimeric Antigen Receptors (CAR) targeting the antigen ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) using a novel cell-based phage display methodology. ENTPD3 is highly expressed on beta cells of both early and progressed T1D patients. ENTPD3 CAR regulatory T cells (Tregs) homed, expanded and persisted in pancreatic islets in a T1D mouse model (NOD) and completely prevented disease progression. Human ENTPD3 CAR Tregs displayed a stable regulatory phenotype, strong activation, and suppression. Importantly, ENTPD3 CAR T cells recognised and were fully activated by human islets. This approach holds great promise as a durable treatment option for patients with prediabetes, new-onset diabetes, or those undergoing beta cell replacement therapy.

immunology↗

bioRxiv2024

Single-stranded HDR templates with truncated Cas12a binding sequences improve knock-in efficiencies in primary human T cells

Non-viral gene editing via CRISPR-Cas12a offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of editing outcomes is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for inducing homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA (dsDNA), which is toxic when transfected at high doses. Previous studies have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a repair template for Cas9-mediated gene editing can mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a Ultra by exploring PAM orientation and binding requirements of the Cas12a-crRNA complex. Additionally, we rule out in vitro ssDNase activity of AsCas12a Ultra under cell-physiological Mg2+ conditions. Finally, we showcase the advantage of using ssDNA with double-stranded CTS end modifications (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three T-cell receptor-CD3 complex genes (TRAC, CD3{zeta}, CD3{varepsilon}), achieving up to 90% knock-in rates for a 0.8kb insert at the CD3{varepsilon} locus. Overall, AsCas12a Ultra and ssCTS donors represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.

immunology↗

bioRxiv2024

Gene editing of CD3 epsilon gene to redirect regulatory T cells for adoptive T cell transfer

I.Adoptive transfer of regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. Antigen-specific Tregs are more effective in modulating undesired immune reactions, but their low frequency in peripheral blood poses challenges for manufacturing and their clinical application. Chimeric antigen receptors (CARs) have been used to redirect the specificity of Tregs, employing retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral gene editing to redirect Tregs with CARs, using HLA-A2-specific constructs for proof-of-concept studies in transplantation models. We introduce a virus-free CRISPR-Cas12a approach to integrate an antigen-binding domain into the CD3 epsilon (CD3{varepsilon}) gene, generating Tregs expressing a T cell receptor fusion construct (TruC). These CD3{varepsilon}-TruC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TruC-redirected Tregs via repetitive CD3/CD28-stimulation in a GMP-compatible expansion system. Non-viral gene edited CD3{varepsilon}-TruC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3{varepsilon}-TruC Tregs demonstrate superior protection of allogeneic HLA-A2+ skin grafts from rejection compared to polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3{varepsilon}-TruC-based Treg cell products for transplantation immunotherapy and other immunopathologies.

immunology↗

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↗