Oral Microto-Nano\nGenome-Editing System Enabling Targeted\nDelivery and Conditional Activation of CRISPR-Cas9 for Gene Therapy\nof Inflammatory Bowel Disease

The potent CRISPR-Cas9 technology can correct genes in\nhuman mutated\ncells to achieve the treatment of multiple diseases, but it lacks\nsafe and effective delivery systems. Herein, we proposed an oral microto-nano\ngenome-editing system aiming at the enteric excessive level of TNF-α\nfor specific gene therapy of inflammatory bowel disease (IBD). This\nediting system facilitated the assembly of Cas9/sgRNA ribonucleoprotein\n(RNP) into nanoclusters (NCs) through the bridging of disulfide bonds.\nRNP-NCs were subsequently encapsulated within inflammatory cell-targeted\nlipopolysaccharide-deleted outer membrane vesicles (dOMVs) sourced\nfrom Escherichia coli Nissle 1917,\nwhich were further shielded by an outer layer of calcium alginate\nmicrospheres (CAMs). By leveraging the protection effect of CAMs,\nthe oral administration system withstood gastric acid degradation\nupon entry into the stomach, achieving targeted delivery to the intestines\nwith high efficiency. As the pH gradually rose, the microscale CAMs\nswelled and disintegrated, releasing nanoscale RNP-NCs encapsulated\nin dOMVs into the intestines. These RNP-NCs@dOMVs could traverse the\nmucosal barrier and target inflammatory macrophages where conditionally\nactivated Cas9/sgRNA RNPs effectively perform genomic editing of TNF-α\nwithin the nucleus. Such oral microto-nano genome-editing systems\nrepresent a promising translational platform for the treatment of\nIBD.