Browsing by Subject "Glycopolymer"
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Item Elucidating Glycopolycation Structure-Function Relationships For Improved Gene Therapy(2017-06) Phillips, HaleyThe gene therapy field is devoted to treating disease by adding, altering, or inhibiting gene expression. This type of therapy holds great promise for the treatment and even cure of monogenic diseases such as cystic fibrosis, Duchenne muscular dystrophy, hemophilia A and B, and epidermolysis bullosa. To produce therapeutic effect, nucleic acids must be delivered and expressed in cells of interest. Deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) in many forms can be delivered using viral or non-viral vehicles. Viral vectors provide efficient DNA delivery; however, packaging limitations and occasional safety issues such as immune responses are major issues. In contrast, non-viral vectors are cheaper and easier to mass produce and can package any length of nucleic acid; however, non-viral vectors struggle to deliver genetic cargo at therapeutically beneficial levels. Polymers with the ability to condense and protect genetic material make promising non-viral vectors. They are relatively easy to produce compared to viral vehicles, can safely package various plasmid sizes, and have shown significant uptake in a wide variety of human cell lines. Cationic polymers complex with the negatively charged phosphodiester backbone of DNA or RNA, forming inter-polyelectrolyte complexes termed polyplexes. Herein, we explore using experiments in vitro, ex vivo, and in vivo to probe the structure-function relationships dictating polyplex gene delivery and other glycomaterial applications.Item Glycopolymers for Targeted Gene Delivery and Genome Editing(2017-08) Dhande, YogeshTargeted delivery of therapeutics is of great interest to reduce toxicity and immunogenicity of the treatment. In particular, the liver is an ideal target for nucleic acid therapeutics due to its large size, regenerative capacity, and the role in producing serum proteins. In this work, N-acetyl-D-galactosamine (GalNAc) ligands clustered into a polymeric architecture were studied for enhanced binding to the asialoglycoprotein receptors (ASGPRs) on hepatocytes. A series of cationic glycopolymers based on this architecture was used to encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes) and deliver them to receptor-specific cells. The GalNAc-derived polyplexes were colloidally stable and showed cell type-specific gene expression in cultured cells. This work demonstrated the versatility of glycopolymers in selective delivery of therapeutics to cells of interest. We sought to further understand the role of such gene-delivery vehicles in genome editing applications using the CRISPR/Cas9 system. Our results show that the gene delivery vehicle can play a role in promoting homology-directed repair over nonhomologous end joining based on its gene delivery properties. The frequency of editing correlates with the fraction of cells expressing Cas9 above a certain threshold and higher expression does not contribute to any gains in editing efficiency. Taken together, these observations suggest that future gene-delivery vehicles aimed for genome editing applications should be designed to deliver only a sufficient amount of DNA but to a large fraction of cells.Item Synthesis and Characterization of Polycations with Various Structural Features for Nucleic Acid Delivery(2014-07) Wu, YaoyingCationic polymers have been widely explored as non-viral nucleic acid delivery vectors for gene therapy, as they are able to complex with nucleic acids, protect genetic materials from degradation, and facilitate the internalization of transgene expression process. To understand how various structural elements impact the nucleic acid delivery, several classes of polycations were synthesized and examined for their in vitro transfection performance. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed for the synthesis of several series of diblock glycopolycations with different carbohydrate containing blocks, including poly(2-deoxy-2-methacrylamido glucopyranose) (PMAG) and poly(methacrylamidotrehalose) (PMAT). Amine containing monomers were subsequently polymerized through chain extension to yield cationic blocks for nucleic acid binding, including N-[3-(N, N-dimethylamino) propyl] methacrylamide (DMAPMA), N-(2-aminoethyl) methacrylamide (AEMA), aminoethylmethacrylate (AEMT), N-methyl aminoethylmethacrylate (MAEMT), N,N-dimethyl aminoethylmethacrylate (DMAEMT), and N,N,N-trimethylammoniumethylmethacrylate (TMAEMT). Initially, it was demonstrated that these polymers were all able to complex plasmid DNA into polyplex structures and prevent colloidal aggregation of polyplexes in physiological salt conditions. More importantly, glycopolymers with PMAT block can prevent polyplexes from aggregation, and exhibit the ability to protect polyplexes through the cycle of lyophilization and reconstitution. The role of charge type, block length, and cell type on transfection efficiency and toxicity were studied for the in vitro transfection in both HeLa (human cervix adenocarcinoma) and HepG2 (human liver hepatocellular carcinoma) cells by comparing the polyplexes formulation created with PMAG-b-PAEMA and PMAG-b-PDMAPMA. The glycopolycation vehicles with primary amine blocks and PAEMA homopolymers revealed much higher transfection efficiency and lower toxicity when compared to analogs created with DMAPMA. Block length was also shown to influence cellular delivery and toxicity; as the block length of DMAPMA increased, polyplex toxicity increased while transfection decreased. While the charge block played a major role in delivery, the MAG block length did not affect these cellular parameters. Cell type played a major role in efficiency, these glycopolymers revealed higher cellular uptake and transfection efficiency in HepG2 cells than in HeLa cells, while homopolycations (PAEMA and PDMAPMA) lacking the MAG blocks exhibited the opposite trend signifying that the MAG block could aid in hepatocyte transfection. Lastly, a new class of lipophilic polycations were designed, poly(alkylamidoamine) (PAAA) was synthesized to understand the role of lyophilicity in pDNA transfection. PAAAs with various length of lipophilic linker (C3 to C6) were synthesized via step-growth polymerization, and examined for their in-vitro transfection efficiency and cytotoxicity in multiple cell types, including HDFa (human dermal fibroblasts, adult) cells, HeLa (human cervix adenocarcinoma) cells, HMEC (human mammary epithelial cells), and HUVEC (human umbilical vein endothelial cells). The PAAA vehicles exhibited comparable or even superior transfection efficiency to Lipofectamine 2000, a leading lipid-based transfection reagent. It was revealed that, for the PAAA polymers examined in this study, an increase in lipophilicity leads to higher cytotoxicity, but also raised the transfection efficiency in HeLa cells. Overall, we demonstrated the great potential of carbohydrate containing polymer block, which has potential to serve as a targeting moiety, stealthy coating, and even lyo-protectant for polyplex formulations. Different amine types were examined for their nucleic acid delivery ability, and we conclude that tertiary amine containing monomer, DMAPMA, exhibits high toxicity along with low transfection efficiency while primary amine block shows relatively lower toxicity and higher transfection efficiency. Finally, the lipophilic polycations, PAAAs, were shown to be important for improving transfection efficiency in multiple cell types.