Personalized CRISPR knock-in cytokine gene therapy to remodel the tumor microenvironment and enhance CAR T cell therapy in solid tumors
The immunosuppressive tumour microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumours. We present a gene-therapeutic strategy that enables localized remodelling of the TME via tumour-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumour-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma as a model, CancerPAM analysis of tumour sequencing data identifies optimal knock-in sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG), and CancerPAM rankings correlate strongly with target-site specificity and knock-in efficiency, validating its predictive performance. CRISPR-mediated CXCL10 knock-in enhances CAR T cell infiltration and antitumour efficacy in vitro and in vivo, including humanized CD34⁺ HuNOG mice, where CXCL10-expressing tumours show stronger immune infiltration and prolonged tumour control within a reconstituted human immune microenvironment. Our findings establish a framework for safe and effective CRISPR-based cytokine delivery, integrating localized TME remodelling with cellular immunotherapies to enhance CAR T cells and other treatments in immune-refractory solid tumours.
Authors
- Dimitrios L. Wagner (ORCID: https://orcid.org/0000-0002-2189-3579)
- Anton George Henssen (ORCID: https://orcid.org/0000-0003-1534-778X)
- Kathleen Anders (ORCID: https://orcid.org/0000-0002-0429-200X)
- Annette Künkele (ORCID: https://orcid.org/0000-0002-8406-5412)
- Maria Stecklum (ORCID: https://orcid.org/0009-0001-7951-5730)
- Casper F. T. van der Ven (ORCID: https://orcid.org/0000-0001-7947-6105)
- Marc L Appis (ORCID: https://orcid.org/0000-0002-0155-1918)
- Marvin Jens (ORCID: https://orcid.org/0000-0002-6660-8743)
- John G. Hohmann (ORCID: https://orcid.org/0009-0001-5374-1350)
- Jonas Kath (ORCID: https://orcid.org/0000-0001-7721-7978)
- Felix Zirngibl (ORCID: https://orcid.org/0000-0003-1764-4841)
- Lena Andersch
- Angelika Eggert (ORCID: https://orcid.org/0000-0003-3476-8184)
- Elias Rodríguez-Fos (ORCID: https://orcid.org/0000-0002-2555-0178)
- Stefanie Beez
- Ralf Kühn (ORCID: https://orcid.org/0000-0003-1694-9803)
- Emily Ohlendorf
- Michael Launspach (ORCID: https://orcid.org/0000-0001-5337-5605)
- Sajal Afzal (ORCID: https://orcid.org/0009-0009-5490-6098)
- Julia Macos (ORCID: https://orcid.org/0009-0008-9196-3975)
- Maximilian Pilgram (ORCID: https://orcid.org/0009-0002-3423-5249)
- Chahrazad Lachiheb
- Karin Töws
Institutions
- German Cancer Research Center (DE)
- Baylor College of Medicine (US)
- Max Delbrück Center (DE)
- Heidelberg University (DE)
- Humboldt-Universität zu Berlin (DE)
- Essen University Hospital (DE)
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin (DE)
- Charité - Universitätsmedizin Berlin (DE)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-12-09
- DOI
- https://doi.org/10.1038/s41467-025-67328-w
- Citations
- 1
- Primary Topic
- CAR-T cell therapy research
- Type
- article
- Field-Weighted Citation Impact
- 1.05
Funders
- Berlin Institute of Health