Mechanistic Study On The Novel Nano Lipid Particles Of Modified Antisense Oligonucleotides Against Hepatocellular Carcinoma

Antisense oligonucleotides（ASOs）are an emerging gene therapy method,which can specifically inhibit the expression of genes by complementary binding to target m RNA sequences.ASOs have many advantages such as clear sequence structure,clear therapeutic target and simple preparation and synthesis process.This technology has been applied to the research and treatment of many diseases.At present,the US Food and Drug Administration and the European Medicines Agency have approved 9 ASO drugs,which are mainly used to treat genetic diseases and neurological diseases caused by single-gene genetic mutations.However,ASO still has many problems such as poor stability,difficult transmembrane transportation,high dose,etc.These problems restrict the clinical application and therapeutic effect of ASOs,so further research and exploration are needed.Hepatocellular carcinoma（HCC）is a malignant tumor whose incidence is increasing year by year and has become a global public health problem.Traditional treatment methods include surgical resection and local treatment of metastatic HCC.However,due to the aggressive and metastatic nature of HCC,the efficacy of these traditional treatments is limited,and new therapeutic strategies need to be explored.In hepatocellular carcinoma,the abnormal expression of multiple key genes leads to the proliferation and metastasis of tumor cells,such as insulin growth factor type I receptor（IGF1R）and vascular endothelial growth factor（VEGF）genes.In addition,targeting c-Myc and STAT3,two important transcription factors associated with HCC,can inhibit the occurrence and development of HCC through multiple pathways.Therefore,using ASOs to target and silence the expression of these genes may become one of the effective methods for the treatment of HCC.In this study,the neutral nucleosyl lipid DNCA and the cationic lipid CLD designed and synthesized by our group were firstly used to realize the effective encapsulation and delivery of the ASO CT102 targeting IGF1R m RNA.The composition of the formulation was further optimized.The molar ratio of DNCA/CLD/single nucleotide in the optimal formulation is 1/1/1（N/P ratio is 4/1）.Subsequently,the biochemical stability of ASO was improved through chemical modifications such as 2′-OMOE,2′-OMe combined with PS.In vitro experiments show that different modification structures and modification patterns have a great influence on the activity of ASO.Through in vitro cell activity evaluation,the modified sequence CT102_MOE5with the optimal activity was determined,and its proliferation inhibition rate on Hep G2 cells and Huh-7 cells was about 2.4 times and 1.6 times that of CT102,respectively.Based on CT102_MOE5,a variety of terminal conjugates of Gal NAc-derived structures were constructed,and the Gal NAc conjugates with better silencing effect on target m RNA and tumor suppression effect in vivo than unconjugated sequences were screened.Through multiple rounds of in vivo and in vitro efficacy experiments,it was finally determined that Glu-CT102_MOE5 was the optimal candidate structure,whose in vitro activity was comparable to that of unconjugated CT102_MOE5,and in vivo efficacy was significantly better than that of CT102_MOE5.Secondly,in this study,antisense and RNAi technologies were used to design multiple ASO and si RNA sequences targeting IGF1R m RNA.Cell activity screening was conducted to obtain N-02 and N-07 sequences with optimal activity.These sequences could effectively knock down 60%of the target m RNA expression.The proliferation of HCC cells was inhibited by 80%（ASO:100 n M）.In addition,the two si RNA sequences had the same silencing activity on target genes as N-04 and N-06,but the inhibitory activity on cell proliferation was only about 20%,which was significantly lower than that of ASOs.In contrast to si RNA,which is almost impossible to observe in the nucleus,it takes 2 hours for the ASOs delivered by mixed lipid to enter the nucleus,and reaches its maximum accumulation in the nucleus in about 12 hours.At the same time,CT102 and N-02 can promote the early and late apoptosis in approximately 35%of Hep G2 cells,and the late apoptosis accounts for 90%of the total apoptotic cells,suggesting that ASOs targeting IGF1R m RNA encapsulated in mixed lipids can cause irreversible late apoptosis of Hep G2 cells.Finally,this study conducted a transcriptomic analysis of CT102 and its derivatives delivered by mixed lipids.The results showed that differential genes were enriched in a variety of tumor-related signaling pathway protein genes.In addition to the known target gene IGF1R which acts as tyrosine kinase receptor mediated signal transduction,there are also significant differences in extracellular receptor interactions and calcium ion signaling pathways.Combined proteomics data analysis showed that about 36.6%of the differential proteins were localized in the nucleus,focusing on functional proteins such as transcription,protein phosphorylation,phosphatase activation,DNA/RNA binding.Among the significantly differentially expressed m RNAs and proteins,GAS2,POLA2,LGALS2,and IGFBP1 all had functional roles in tumor development.The q RT-PCR experiment verified that after the administration of CT102 and its derivatives,the m RNA expressions of GAS2,POLA2,and LGALS2 in Hep G2 cells all significantly decreased,while the IGFBP1 m RNA expression is significantly increased.The results of pathway enrichment analysis showed that differential genes and proteins were mainly enriched in PPAR signaling pathway,cell aging,DNA replication,mismatch repair,P53signaling pathway,TGF-βsignaling pathway,etc.All of these pathways and processes are crucially involved in the onset and progression of liver cancer.It is further suggested that although both ASOs and si RNAs are effective RNA silencing techniques,ASOs act on more signaling pathways and links through multiple mechanisms,and thus exhibit better efficacy in inhibiting tumor development.In summary,new ASO sequences targeting hepatocellular carcinoma-related genes were designed and screened by using the neutral cytidinyl/cationic lipid delivery system.On this basis,2′-chemical modification and Gal NAc conjugation strategies were employed.The efficacy of ASOs targeting IGF1R m RNA for the treatment of HCC in vitro and in vivo were verified,and the mechanism by which the formulation regulates multiple targets in the cytoplasm and nucleus to exert high-efficiency anti-HCC effect was explored.It laid a preliminary theoretical foundation for in-depth research on the biological mechanism and drug development of ASO against HCC.Through transcriptomic and proteomic analysis,this study also revealed that CT102 and its derivatives inhibit the development of HCC by acting on protein genes in various tumor-related signaling pathways and functional proteins in the nucleus.These results are of great significance for the further development and clinical application of ASO drugs.