Fabrication Of Polycationic Gene Vectors With Abundant Hydroxyl Groups Based Onsmall Biological Molecules

Gene therapy is one promising therapeutic method for the treatment of criticaldiseases including genetic diseases,cardiovascular diseases and cancers.Gene therapy relies on the delivery of therapeutic genes into patients'cells to replace or supplement the mutated gene,or inhibit the expression of pathological proteins to fix a genetic problem.To achieve these objectives,propergene vectors with high transfection efficacy and safety are in demand.The conventional viral vectors are often risky because of their immunogenicity.To address this issue,the development of non-viral vectors has attracted much attention in the field of biomaterials.In particular,a variety of polycationic gene vectors based on ethanolamine(EA)-functionlizedpoly(glycidyl methacrylate)(PGMA)(named PGEA)have been reported,exhibiting good transfection efficacy,low cytotoxicity,and long blood circulation time due to the abundant hydroxyl groups in PGEA.However,the current PGEA-based gene vectorsare still unsatisfactory due to lackin functionalities.Lipids aremajor constituents in cell membranes,and have been widely used forbiomedical applicationsbecause of their excellent biological properties.The incorpoartion of membrane lipid molecules into genevectors would confer better biocompatibility,cellular uptake and transfection efficiency.In Chapter 2,a series of polycations containing cholesterol(CHO)-and phosphatidylinositol(PI)-moieties(namely CHO-PGEAs andPI-PGEAs,respectively)were synthesized based onatom transfer radical polymerization(ATRP).Both CHO-PGEAs and PI-PGEAs demonstrated better transfectionefficacy compared to linear PGEA and "gold standard"branched polythylenimine(PEI,25 kDa).In addition,both CHO-PGEA and PI-PGEA polycations complexed with tumor suppressor p53 gene exhibited potent tumor-inhibiting effects.Compared with the control group,the quality of the tumors could be reduced to below 0.15 g on average after treatments.Encouraged by the good performances of lipid-containing polycations in tumor therapy,we explored the application of CHO-PGEA in the treatment ofcardiovascular diseases.CRISPR/Cas9 systems have emerged as a powerful tool in gene editing for the treatment of genetic diseases.However,the delivery of CRISPR/Cas9 systems into vasculature cells are still challenging due to the endothelialbarrier in the treatment of genetic vascular diseases especially inaorta.In Chapter 3,we demonstrated that CHO-PGEA can successfully deliver plasmid based pCas9-sgFbn1 system for the knockoutof exon 10 in Fbn1 genein vitro.More importantly,we also demonstrated that the combination of CHO-PGEA/pCas9-sgFbn1 nanosystems with theadministration of Ang II provided a promising approach forin vivo gene editing in aorta.The diameter of edited aorta increased approximately 0.01 mm,which was consistent with the expected result.In this chapter,we also demonstrated that the CHO-PGEA could be employed as gene vector for the treatment of persistent cardiac hypertrophy,which would cause heart failure and sudden death.In an in vivo study,the CHO-PGEA vectors were able to deliver miR-182-in into cardiomyocytes,blocking the functions of overexpressed miR-182 and upregulate the factor FOXO3.Compared with control group which HW/BW of mice were 1.5 times than normal,with this treatment,cardiac hypertrophycould be significantly suppressed.The CHO-PGEA vectors had two important features:(1)lipophilic cholesterol groupsenhanced transfection efficiency in cardiomyocytes;(2)abundanthydroxyl groups endowed superior biocompatibility.Thoracic aortic dissection(TAD)is a critical and life-threatening vascular disease which requires early diagnosis and effective treatment.However,there are no effective drug delivery systems for the therapy of TAD due to the complexity and narrowness in the localized vascular structure.In Chapter 4,one multifunctional delivery nanosystem(TP-Gd/miRNA-ColIV),containing gadolinium-chelated tannic acid with pendant PGEA arms(TP-Gd),complexed miRNA,and type IV collagen targeting peptide(ColIV),was synthesized for targeted nucleic acid therapy,early diagnosis and noninvasive monitoring of TAD development.MiR-145 can be effectively delivered using TP-Gd/miR-145-ColIV for the treatment of TAD.After the treatment with TP-Gd/miR-145-ColIV,the incidence of dissection decreased to less than 20%with TAD model.Moreover,TP-Gd/miRNA-ColIV also demonstrated good magnetic resonance imaging(MRI)ability and can be used to monitor the development of TAD.In conclusion,we have developeda variety ofpolycationic gene delivery systemswith abundant hydroxyl groups based on small biological molecules.These advancedgene vectors exhibited high transfection efficiency and goodbiocompatibility in both in vitro and in vivo studies,which shed light on the development of next-generation genetherapy for the treatment of cancer and critical cardiovascular diseases.