US-Controlled CRISPR/Cas9 Nanosystem For Precise Knockdown Of Nrf2 Gene To Augment Sonodynamic Therapy

Objective: Hepatocellular carcinoma(HCC)is one of the leading causes of morbidity and mortality,with poor prognosis,ranking as the third cause of cancer-related deaths worldwide.Sonodynamic therapy(SDT),sonosensitizer activated under low-intensity ultrasound irradiations to generate highly toxic reactive oxygen species(ROS),has been regarded as a promising pre-clinical non-invasive therapeutic modality in the last decade.The scientific principle underlying SDT-mediated cytotoxicity mainly roots in the generation of ROS,which disturbs the intracellular redox homeostasis to damage crucial components of the cancer cells.However,there is a comprehensive antioxidant defense system to regulate the levels of ROS and prevent the accumulation of damage induced by ROS,in which nuclear factor erythroid 2-related factor 2(Nrf2 or NFE2L2)is an important transcription factor that targets the antioxidant response element in the upstream regulatory regions.Therefore,Nrf2 possibly inhibits the efficacy of ROSbased SDT and would be a potentially vulnerable target in HCC.Compared to RNAi technology,the CRISPR/Cas9 system has the following features and advantages:including higher targeting accuracy,complete knocking down of target gene,and no species restriction.Notably,it is still elusive to efficiently translocate the CRISPR/Cas9 system into target tissues/cells for biomedical applications due to numerous extracellular and intracellular barriers,which considerably restricts the widespread utilization of CRISPR/Cas9 in vivo.On this basis,it is hypothesized that the combination of SDT and CRISPR/Cas9-mediated gene editing technology would address the current challenges in the treatment of HCC and the delivery of CRISPR/cas9 system.Methods: At the beginning of this study,we initially found that the Nrf2 was rapidly activated after SDT,which dissociated from Kelch-1ike ECH-associated protein l(Keap1)and rapidly translated into the nucleus,and then bond to antioxidant response elements to maintain intracellular redox homeostasis.Subsequently,Nrf2 overexpression and interference experiments showed that Nrf2 promoted tumor cell growth and proliferation,which was positively correlated with tumor size.Thus,to address this issue,an ultrasound(US)-responsive CRISPR/Cas9 nanodelivery system(HMME@Lip-Cas9)was constructed to knock down Nrf2 and enhance the efficiency of SDT.US Food and Drug Administration(FDA)-approved DLin-MC3-DMA lipid nanoparticles(Lips)were used as carriers for loading the sonosensitizers hematoporphyrin monomethyl ether(HMME)and Cas9/sg RNA ribonucleoprotein(RNP).Results: The as-obtained US-responsive CRISPR/Cas9 system(HMME@Lip-Cas9)produced ROS,which effectively disrupted the structure of lysosomal membrane,thus achieving efficient escape of Cas9/sg RNA RNP from lysosomes,releasing into the cytoplasm and transporting to the nucleus to exert their therapeutic effect.Unlike the Lips can only mediate the release of 1–4% of RNA into the cytoplasm,this system can efficiently release RNP to activate Cas9 under the spatial control of US stimulation,avoiding gene mutations in nontarget regions.Conclusions: This research not only systematically elucidates the cellular selfprotection mechanism against ROS-induced oxidative stress and address the challenges on the development of SDT,but also a highly biocompatible and transfection-efficient non-viral vector has been explored for the spatiotemporally controlled transmission of the CRISPSR/Cas9 system,which has a broad scope in clinical translation.