The in situ vaccine effect of radiation therapy (RT) has been shown to be limited in both preclinical and clinical settings, possibly due to the inadequacy of RT alone to stimulate in situ vaccination in immunologically "cold" tumor microenvironments (TMEs) and the mixed effects of RT in promoting tumor infiltration of both effector and suppressor immune cells. To address these limitations, we combined intratumoral injection of the radiated site with IL2 and a multifunctional nanoparticle (PIC). The local injection of these agents produced a cooperative effect that favorably immunomodulated the irradiated TME, enhancing the activation of tumor-infiltrating T cells and improving systemic anti-tumor T cell immunity. In syngeneic murine tumor models, the PIC +IL2+RT combination significantly improved the tumor response, surpassing the single or dual combinations of these treatments. Furthermore, this treatment led to the activation of tumor-specific immune memory and improved abscopal effects. Our findings suggest that this strategy can be used to augment the in situ vaccine effect of RT in clinical settings.
Genome editing mediated by the CRISPR-Cas system holds great promise for the treatment of genetic diseases. However, safe and efficient in vivo delivery of CRISPR genome editing machinery remains a challenge. Here, we report a lipopeptide-based nanoparticle (LNP) that can efficiently deliver the CRISPR Cas9/sgRNA ribonucleoprotein (RNP) and enable efficient genome editing both in vitro and in vivo. An artificial lipopeptide, GD-LP, was constructed by linking a hydrophilic guanidinium-rich head to an oleic acid-based hydrophobic tail via a disulfide bond. LNP formed by the self-assembly of GD-LP can easily form a complex with RNP with a loading content of up to 20 wt %. The resulting RNP-LNP nanocomplex led to 72.6% gene editing efficiency in GFP-HEK cells with negligible cytotoxicity. The LNP also showed significantly higher transfection efficiencies than Lipofectamine 2000 for the delivery of mRNA in NIH 3T3 and RAW 264.7 and the delivery of plasmid DNA in B78 cells. In vivo studies showed that intramuscular injection of the RNP-LNP nanocomplex in Ai14 mice induced efficient gene editing in muscular tissues. Moreover, the delivery of Cas9 RNP and donor DNA by LNP (i.e., RNP/ssODN-LNP nanocomplex) restored dystrophin expression, reduced skeletal muscle fibrosis, and significantly improved muscle strength in a Duchenne muscular dystrophy (DMD) mouse model.
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