Construction And Application Of Gene Delivery Vector Targeting The Endoplasmic Reticulum

Gene therapy,the delivery of therapeutic nucleic cargos into cells to modify or correct genetic information,has been proven to be a promising method used in biological research and therapeutics for conditions such as cancer,diabetes,autoimmune diseases,and infectious diseases.However,naked nucleic acids cannot achieve desired outcomes because of their rapid clearance and short duration,non-specific biodistribution and low cellular internalization.Hence,the primary challenge for gene therapy is to develop safe and effective vectors.Compared with viral vectors,non-viral vectors have the advantages of low immunogenicity,low toxicity,easy structural modification and large-scale production.However,their application is still inhibited by the low transfection efficiency due to their poor membrane penetration capacity,high lysosome retention,hard nuclear entrance and cargo release.The retention of exogenous genes in the lysosome is a primary barrier due to the serious degradation of the genes.Lysosomes contain lots of acid hydrolases,which will degrade nucleic acids,resulting in serious damage to the integrity and quantity of nucleic acids.At present,the commonly used strategy is to endow the gene vectors with the ability to induce lysosome rupture by means of"proton sponge effect","photochemical internalization"or the addition of lysosome-tending agents,so that the loaded nucleic acid can escape from the lysosome.However,recent works have suggested that lysosome rupture causes protease and other hydrolases in lysosomes released into the cytosol,which results in cytotoxicity and degradation of nucleic acids.Hence,many strategies have been implemented to find alternate trafficking patterns for vectors to prevent their capture by lysosomes after internalization,such as by enhancing their mediated uptake by caveolae,in an effort to reduce the loss of gene cargo resulting from lysosomal degradation and to promote the transfer of the genes to the perinuclear region.Increasing nuclear membrane penetration of exogenous genes poses another challenge for gene therapy.The nucleus is surrounded by a nuclear envelope(NE)that is composed of two membranes-an inner and an outer nuclear membrane.Embedded in these membranes are hundreds to thousands of nuclear pore complexes(NPC)that allow selective transport of proteins between the two compartments.The outer nuclear membrane is continuous with the rest of the endoplasmic reticulum(ER)and they share many of the same proteins.Recent studies have revealed that ER components are redistributed into the NE,suggesting that ER could provide the precursor membrane for NE reconstruction.Based on the close connection between the ER and the nuclear membrane,the transport via the ER-nucleus could be an alternative strategy for increasing the number of exogenous genes that enter the nucleus.Based on these backgrounds,we constructed a cationic liposome PAR-Lipo with ER targeting function modified by the cationic peptide Pardaxin.Pardaxin,a single peptide chain composed of 33 amino acids,could be inserted into the phosphobicity and target the ER directly.Previous studies of our group have shown that small molecules modified by Pardaxin could transport through non-lysosomal pathways and finally localize to the ER.Therefore,this study focused on whether the delivery efficiency of exogenous genes could be improved by targeting the ER.In the first part,DSPE-PEG₂₀₀₀-PAR was synthesized by the condensation reaction of an amino group in DSPE-PEG-NH₂with a carboxyl group on pardaxin.By adjusting the ratio of DOTAP,DOPE and DSPE-PEG₂₀₀₀,the cationic liposome PAR-Lipo targeting the ER and the Non-Lipo transporting through lysosomal pathway were prepared by thin film dispersion method.By examining the physicochemical properties,stability and safety,it is proved that PAR-Lipo has the ability to compound nucleic acid and has high biological safety.Compared to Non-Lipo and Lipofectamine?2000(Lipo2000),PAR-Lipo mediated remarkably higher gene delivery efficiency in vitro and better antitumor efficacy in vivo.Fluorescence colocalization revealed that PAR-Lipo and its mediated DNA have strong fluorescent colocalization signals with the ER,but only a small amount of colocalization with lysosomes,indicating that the enhanced gene transfection efficiency stems from a special endocytosis mechanism and intracellular transport pathways.The above studies have solved the two key problems about lysosome retention and nuclear barrier,and greatly improved the efficiency of gene transfection.However,during the in vivo study,we found that hypoxia is an important factor limiting the effect of gene therapy.Malignant cellular proliferation and aberrant neovascularization cause tumor hypoxia inevitably,which is a common characteristic in rapidly growing solid tumors.As one of the most energy-consuming processes in cells,translation requires enormous amounts of ATP,and the production of ATP is extremely oxygen-dependent.Gene translation and protein synthesis are blocked under hypoxia,which greatly hinders gene therapy.Here,we proposed a staged therapeutic strategy.To alleviate hypoxia,the PFOB liposomes loaded oxygen(expressed as O₂@PL)were applied to deliver oxygen to the hypoxic tumor sites.The dissolved oxygen experiment confirmed that O₂@PL had the highest increase in oxygen content and the slowest oxygen release.Both in vitro and in vivo transfection results demonstrated that O₂@PL effectively improved the transfection efficiency of PAR-Lipo under hypoxia.Finally,P53 was selected as the therapeutic gene.Obtained results indicated that our strategy effectively broke the restriction of hypoxia on gene therapy and greatly delayed tumor growth.The unique intracellular transport behavior of PAR-Lipo can enhance the protection to DNA payload,prolong their residence time in the cells,and promote their entry into the nucleus relying on the intimate relationship between ER and nuclear membrane,which is the explanation for enhanced gene-therapy effect mediated by PAR-Lipos.What's more,this work was the first attempt to utilize an oxygen nanocarrier to assist the therapeutic effect of gene therapy under hypoxia,providing a new reference for gene therapy in malignant tumors.