Biology subjects

Knudsen, N. H.

Publications and source records attributed to Knudsen, N. H..

3 recordsLinked to original sources

bioRxiv2026

A Lenalidomide-Inducible Suicide Switch for Gene- and Cell-Based Therapy

Genetically modified immune effector cells, including chimeric antigen receptor (CAR) T cells, have emerged as transformative anti-cancer agents. However, these autonomous, living therapies are limited by difficult-to-control hyperactivation toxicities. To achieve their full potential as therapies for a broader range of diseases, CAR T cells must target novel antigens and integrate further potency enhancements that raise the possibility of novel and intensified toxicities. High performance safety systems are needed to restrain these highly active next-generation gene- and cell-based therapies. Cell therapy suicide switches remain infrequently used, and are currently limited by inaccessible controller drugs, incomplete cell depletion, fitness cost, and immunogenicity. To address these issues, we engineered a fully human sequence-based cell therapy suicide switch based on the overexpression of Caspase-Activated DNase (CAD) and its stoichiometric chaperone/inhibitor ICAD fused to a lenalidomide-inducible degron. Acute destabilization of ICAD with the broadly available, off-patent molecular glue degrader drug lenalidomide uncaged CAD to induce DNA damage and cell death. By this degradable cage mechanism, the ICAD-CAD suicide switch achieved long-term expression in primary human T cells without detectable leakiness, and deep drug-induced cell depletion. The ICAD-CAD switch was generalizable to alternative acute destabilization systems. Genome-wide CRISPR screening established the necessity of CAD and the lenalidomide-interacting E3 ubiquitin ligase substrate adaptor CRBN for switch function. When co-delivered with a CAR, ICAD-CAD CAR T cells retained effector functions and could be rapidly depleted with lenalidomide.

synthetic biology↗

bioRxiv2026

CRISPR screens identify targets to rescue age-related T cell dysfunction in cancer

Immune aging is being increasingly recognized as a critical barrier to effective cancer immunotherapy, as the aged tumor microenvironment (TME) drives T cell dysfunction and impairs immune control of cancer1-4. However, the key molecular drivers of this process as well as potential targets to rescue T cell dysfunction in aged tumors remain incompletely understood. Therefore, we performed in vivo single-cell CRISPR screens in CD8 T cells within aged tumors and tumor-draining lymph nodes (tdLNs). We identified Dusp5 and Zfp219 as key regulators of T cell persistence and effector differentiation in aged hosts. Loss of Dusp5, a negative regulator of ERK signaling, increased ERK1/2 phosphorylation and enhanced T cell proliferation in both young and aged tumors. In contrast, loss of Zfp219, a transcriptional repressor, induced epigenetic reprogramming of cytotoxic gene programs, thereby increasing granzyme secretion and enhancing antitumor immunity. Moreover, expression of the human ortholog gene ZNF219 is increased within intratumoral CD8 T cells in older cancer patients. High ZNF219 expression correlates with poorer survival following immune checkpoint blockade (ICB) and reduces persistence of human intratumoral T cells. Notably, Zfp219 ablation synergized with anti-PD-1 blockade in mice to expand effector-like CD8 T cells, leading to significantly enhanced anti-tumor immunity and tumor clearance in aged hosts. Together, these findings highlight Dusp5 and Zfp219 as critical drivers of age-related T cell dysfunction and as potential therapeutic targets to rejuvenate T cell antitumor immunity in older cancer patients.

immunology↗

bioRxiv2024

In vivo CRISPR screens identify key modifiers of CAR T cell function in myeloma

Chimeric antigen receptor (CAR) T cells are highly effective in hematologic malignancies. However, loss of CAR T cells can contribute to relapse in a significant number of patients. These limitations could potentially be overcome by targeted gene editing to increase CAR T cell persistence. Here, we performed in vivo loss-of-function CRISPR screens in BCMA-targeting CAR T cells to investigate genes that influence CAR T cell persistence, function and efficacy in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR-library edited T cells in vitro and then at early and late timepoints in vivo to track the performance of gene modified CAR T cells from manufacturing to survival in tumors. The screens revealed several context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, while loss of PTPN2, ZC3H12A, and RC3H1 conferred early selective growth advantages to CAR T cells in vivo. Strikingly, we identified cyclin-dependent kinase inhibitor 1B (CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late timepoints in vivo. CDKN1B ablation increased BCMA CAR T cell proliferation and effector function in response to antigen, significantly enhancing tumor clearance and overall survival. Thus, our findings reveal differing effects of gene-perturbation on CAR T cells over time and in different selective environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma, and underscore the importance of in vivo screening as a tool for identifying genes to enhance CAR T cell function and efficacy.

immunology↗