CRISPR-Cas9 gene editing has emerged as a powerful therapeutic technology, but the lack of safe and efficient in vivo delivery systems, especially for tissue-specific vectors, limits its broad clinical applications. Delivery of Cas9 ribonucleoprotein (RNP) owns competitive advantages over other options; however, the large size of RNPs exceeds the loading capacity of currently available delivery vectors. Here, we report a previously unidentified genome editing delivery system, named exosome ^RNP , in which Cas9 RNPs were loaded into purified exosomes isolated from hepatic stellate cells through electroporation. Exosome ^RNP facilitated effective cytosolic delivery of RNP in vitro while specifically accumulated in the liver tissue in vivo. Exosome ^RNP showed vigorous therapeutic potential in acute liver injury, chronic liver fibrosis, and hepatocellular carcinoma mouse models via targeting p53 up-regulated modulator of apoptosis ( PUMA ), cyclin E1 ( CcnE1 ), and K (lysine) acetyltransferase 5 ( KAT5 ), respectively. The developed exosome ^RNP provides a feasible platform for precise and tissue-specific gene therapies of liver diseases.

Stimuli-responsive nano-immunochemotherapy modulates the tumor microenvironment to enhance antitumor immunity.

Cancer cells appear to depend heavily on antiapoptotic proteins for survival and so targeted inhibition of these proteins has therapeutic potential. One innovative strategy is to inhibit the cyclin‐dependent kinases (CDKs) responsible for the regulation of RNA polymerase II (RNAPII). In our study, we investigated the detailed cellular mechanism of a novel small‐molecule CDK inhibitor (CDKI‐71) in cancer cell lines, primary leukemia cells, normal B ‐ & T‐ cells, and embryonic lung fibroblasts and compared the cellular and molecular responses to the clinical CDK inhibitor, flavopiridol. Like flavopiridol, CDKI‐71 displayed potent cytotoxicity and caspase‐dependent apoptosis induction that were closely associated with the inhibition of RNAPII phosphorylation at serine‐2. This was caused by effective targeting of cyclinT–CDK9 and resulted in the downstream inhibition of Mcl‐1. No correlation between apoptosis and inhibition of cell‐cycle CDKs 1 and 2 was observed. CDKI‐71 showed a 10‐fold increase in potency in tumor cell lines when compared to MRC‐5 human fibroblast cells. Significantly, CDKI‐71 also demonstrated potent anti‐chronic lymphocytic leukemia activity with minimal toxicity in normal B‐ and T‐cells. In contrast, flavopiridol showed little selectivity between cancer and normal cells. Here, we provide the first cell‐based evidence that flavopiridol induces DNA double‐strand breaks: a fact which may explain why flavopiridol has such a narrow therapeutic window in preclinical and clinical settings. Taken together, our data provide a rationale for the development of selective CDK inhibitors as therapeutic agents and CDKI‐71 represents a promising lead in this context.

Cancer fibrosis serves as a major therapeutic barrier in desmoplastic tumors. Relaxin (RLN; a systemic hormone) is efficacious to decrease fibrosis, but the in vivo mechanism of action is not clear. Considering the localization of relaxin family peptide receptor type 1 (RXFP1), the receptor for RLN, on macrophages, we hypothesize that macrophages can be modulated by RLN to ameliorate cancer fibrosis. Using KPC mouse model of pancreatic ductal adenocarcinoma (PDAC), here, we report locally expressed RLN with targeted gene delivery induces increased F4/80⁺CD206⁺ macrophages originating from Ly6C⁺ monocytes, promoting fibrosis depletion and cytotoxic T cell infiltration. Moreover, RLN gene delivery synergizes with PD-L1 blockade for tumor inhibition by enhancing T cell–mediated tumor cell killing and macrophage phagocytosis. Collectively, our results reveal previously unidentified insights into the modulation of macrophages to regulate tumor-associated fibrosis, providing a feasible strategy to reverse the immunosuppressive environment and improve the therapeutic outcome of checkpoint immunotherapies.