Personalized CRISPR Knock-In Cytokine Gene Therapy to Remodel the Tumor Microenvironment and Enhance CAR T Cell Therapy in Solid Tumors

Launspach, M.; Macos, J.; Afzal, S.; Hohmann, J.; Beez, S.; Pilgram, M.; van der Ven, C.; Lachiheb, C.; Andersch, L.; Jens, M.; Appis, M. L.; Zirngibl, F.; Kath, J.; Stecklum, M.; Anders, K.; Wagner, D. L.; Kuehn, R.; Eggert, A.; Kuenkele, A.

doi:10.1101/2025.04.25.650703

bioRxiv · 10.1101/2025.04.25.650703

Personalized CRISPR Knock-In Cytokine Gene Therapy to Remodel the Tumor Microenvironment and Enhance CAR T Cell Therapy in Solid Tumors

Launspach, M.·Macos, J.·Afzal, S.·Hohmann, J.·Beez, S.·Pilgram, M.·van der Ven, C.·Lachiheb, C.·Andersch, L.·Jens, M.·Appis, M. L.·Zirngibl, F.·Kath, J.·Stecklum, M.·Anders, K.·Wagner, D. L.·Kuehn, R.·Eggert, A.·Kuenkele, A.

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Abstract

The immunosuppressive tumor microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumors. To address this, we developed a novel gene therapeutic strategy that enables localized remodeling of the TME via tumor-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumor-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma--a pediatric solid tumor with a suppressive TME--as a model, we applied CancerPAM to sequencing data from cell lines and patients to identify optimal integration sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG). CRISPR-mediated CXCL10 knock-in into tumor cells significantly enhanced CAR T cell infiltration and antitumor efficacy both in vitro and in vivo. In vivo, CXCL10-expressing tumors showed significantly increased early CAR T cell infiltration and prolonged survival compared to controls. CancerPAM rankings correlated strongly with target-site specificity and knock-in efficiency, validating its predictive performance. Our findings establish CancerPAM as a powerful tool for safe and effective CRISPR-based interventions and provide a conceptual framework for integrating cytokine-driven TME remodeling with cellular immunotherapies. This personalized strategy holds promise for enhancing CAR T cells and other immunotherapies across immune-refractory solid tumors.

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Launspach, M., Macos, J., Afzal, S., Hohmann, J., Beez, S., Pilgram, M., van der Ven, C., Lachiheb, C., Andersch, L., Jens, M., Appis, M. L., Zirngibl, F., Kath, J., Stecklum, M., Anders, K., Wagner, D. L., Kuehn, R., Eggert, A., Kuenkele, A.. 2025-04-27. Personalized CRISPR Knock-In Cytokine Gene Therapy to Remodel the Tumor Microenvironment and Enhance CAR T Cell Therapy in Solid Tumors. https://doi.org/10.1101/2025.04.25.650703

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