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.