Self-assembled Nucleic Acids Nanomaterials For Gene Delivery Through Endocytosis-independent Pathway

Gene therapy,as a promising way to regulate or replace abnormal genes,has made remarkable achievements in many clinical trials.However,the development of gene therapy has been greatly limited by the difficulties in delivering gene therapeutics into target cells.Although technologies to protect and deliver gene therapeutics have been continuously developed,the currently developed delivery strategies often rely on the endocytosis-dependent pathway,resulting in endosomal entrapment and low delivery efficiency.In this thesis,we aim to develop strategies based on endocytosis-independent pathway for gene therapeutics delivery to achieve efficient gene therapy.This thesis is divided into five chapters and the main contents are listed as follows:In chapter 1,difficulties faced by nucleic acids delivery and drawbacks of commonly used delivery strategies,including viral vector and nonviral vector,were introduced.Then,virous endocytosis-independent deliery strategies were introduced,the mechanism of action and design of these systems were major concerns.Meanwhile,the development of thiol-mediated delivery strategies was described in detail including the development,mechanism,and resent advance.The topic ideas and research contents of this thesis were summarized.In chapter 2,we developed an oligonucleotide(ON)-template-assisted polymerization approach to construct ON nanoparticles for effective gene delivery through endocytosis-independent pathway.The size of these nanoparticles could be precisely controlled by varying the length of the ON templates.Notablely,the nanoparticles can directly enter into the cytoplasm through thiol-mediated cellular uptake,and further depolymerize in the reductive environment of cytoplasm to release the ONs,achieving in efficient gene silencing.The ON nanoparticles thus hold broad application prospects in gene therapy.In chapter 3,we developed self-assembled RNA nanoparticles(RNA NPs)as universal vehicles to achieve efficient RNA delivery.Importantly,the nanoparticles possess unique and desirable features including improved resistance to nuclease degradation,directly cytoplasmic delivery through thiol-mediated cellular uptake and cytosolic environment-responsive release,which greatly enhanced the bioavailability of RNA therapeutics.Benefiting from these advantages,the RNA NPs exhibited efficient gene silencing by delivering either siRNA or miRNA in various cell lines.These results demonstrate the broad potential of RNA NPs to serve as a universal vehicle for delivery of RNA therapeutics.In chapter 4,we applied siRNA NPs to realizing gene delivery and gene silence in vivo.Using in vitro penetration model and distribution analysis in zebrafish,we proved that siRNA NPs could efficiently cross the blood vessel barrier,and further accumulate in target tissue.With xenograft pancreatic tumour model in zebrafish embryos,the gene silencing capability of RNA NPs in vivo was examined.The results verified that RNA NPs had a remarkable gene silencing effect,which was significantly higher than PEI.These results demonstrated the broad potential of RNA NPs to serve as a universal vehicle for delivery of RNA therapeutics both in vitro and in vivo.In chapter 5,we have constructed cicular DNA(cDNA)probe and combined it with graphene oxide(GO)for highly efficient intracellular mRNA imaging and gene therapy.The cDNA/GO complexes were superior to single-stranded DNA/GO complexes in many respects.First,the cDNA/GO complexes improved the efficiency of recognizing target mRNA,including sensitivity,selectivity and anti-interference ability.As an intracellular sensing platform,the cDNA/GO complexes reduced false-positive response and background signals.In addition,the low endo/ lysosome colocalization makes cDNA/GO complexes effectively inhibiting the translation of mRNA.Therefore,it can not only effectively inhibit the expression of target protein,but also significantly inhibit cell proliferation.