Construction Of Therapeutic Nucleic Acid Delivery Systems And The Application In Biomedicine

Therapeutic nucleic acids are a class of DNAs or RNAs with therapeutic functions that can block or activate the expression of functional genes and the transmission of genetic information through a series of processes,including gene activating,gene silencing,gene splicing and gene editing.These molecules offer the possibility to specifically intervent or even cure a variety of indications.However,their applications in vivo are highly dependent on delivery platforms to improve their physiological stability,cellular internalization efficiency,etc.However,achieving safe and efficient nucleotide delivery,particularly to extrahepatic tissues still requires in-depth studies to overcome a series of extracellular and intercellular barriers,includin g renal clearance,reticuloendothelial interference,traverse the plasma membrane and escape the endolysosomal system.Currently,nanoplatforms derived from polymers,lipids,lipidoids,and conjugates have provided great opportunities to overcome these challenges,owing to their unique physical and chemical properties,and facile functionalization.In this thesis,we have developed a series of multifunctional nanodelivery platforms for effective and targeted delivery of therapeutic nucleic acids including small interfering RNA(siRNA),microRNA(miRNA),antisense oligonucleotides and G-quadruplicates,and regulation of the expression of intracellular RNA or DNA.Their in vivo performance and molecular mechanisms in the treatment of diseases such as cancer or neurodegenerative diseases have been systemically explored.The specific research contents of this research paper are as follows:(1)Although the lipid nanoplatforms were widely used for nucleic acid delivery,they still have limiations such as complex c omposition,low loading efficiency,and poor stability.To this end,we design and synthesize a delivery platform composed of a peptide-branched polymer conjugate and a dendritic cationic lipid.After targeting delivery siRNA of PD-L1,an innovative immune checkpoint nanoblocker based on RNA interferenceis successfully constructed,which could achieve efficient loading of siPD-L1,maintain high stability in serum and keep good blood circulation performance.Its potency has been evaluated in non-small cell lung cancer.Unlike traditional immune checkpoint blocker based on antibodies,this engineered nanoblocker can effiently knockdown the expression of PD-L1,and more importantly,trigger cell apoptosis of H460 cells independent of immune T cells.Besides,th is nonoblocker has good biocompatibility.We expect it to provide a new therapeutic strategy for the field of immune checkpoint blockade.(2)The change of intracellular signaling pathways and induction of the generation of cell heterogeneity represent an adverse effect during gene therapy.Aiming at overcoming cell senescence-related heterogeneous changes caused by gene therapy,we construct an aptamer AS1411-functionalized lipid nanoparticle targeted delivery of Antagomir-515 to ovarian cancer cells.Our findings demonstrate that this system can efficiently kockdown the expression of microRNA 515 in SKOV3 cells,promoting cell cycle arrest in G1 phase,and triggering evident cell apoptosis and senescence.Basesd on these findings,a synergistic therapeuti c effect has been achived in combination with a senolytic agent.This combination therapy is expected to solve the potential problems of cancer metastasis and recurrence induced by therapy-induced cell senescence.(3)Owing to the lack of responsiveness in tranditional nucleic acid delivery systems often have relatively low effective concentrations on the target site after administration,resulting in limited therapeutical efficacy.To reduce the dosage and improve the biosafety of delivery systems,it is v ery important to develop intelligent bioresponsive nucleic acid delivery systems.Therefore,we take advantage of the high programmability and precise assembling properties of DNAs,and construct an ions-gated DNA framework channel system.This system can effectively anchor on the cell membrane for specific transmembrane transport of K~+ions.This plug-and-play transmembrane DNA framework is expected to enable precise regulation of important biological processes related to chemoionic environments,in a mann er independent of intracellular trafficking.(4)To achieve the regulation of gene expression at the genome level,therapeutic nucleic acids usually need to be safely delivered into nucleus,which further increases the difficulty of nucleic acid delivery.In an attempt of overcoming this issue,we use the gene promoter G-quadruplex(G4)as a gene expression regulatory element and utilize the ion transport properties of the previously constructed transmembrane DNA framework system,to regulate the stability of the genomic promoter G4 structure in cells.In an ischemic stress-induced cell damage model,this system allows effective regulation of the expression of anti-apoptotic gene by dissociating the endogenous promoter G4,thereby reducing the ischemia-induced cell apoptosis in neuron cells.This strategy holds the potential to offer a new way for the therapeutic nucleic acid-based disease treatment.