Gene therapy has the potential to treat most inherited and acquired genetic disorders by either knocking down the expression of a pathological gene product or by replacing a missing or nonfunctional protein. However, for nucleic acids, such as short interfering RNA (siRNA), messenger RNA (mRNA) and plasmid DNA (pDNA), to elicit a desired biological response they require delivery into the appropriate cellular compartment of target cells. Unfortunately, the delivery process is extremely complex and challenged by numerous physiological barriers, especially circulatory stability, receptor targeting, and endosomal escape. In order to overcome these barriers sophisticated nonviral delivery systems are required. The work presented in this thesis sought to mainly to develop methodology to improve the efficacy of PEGylated polyacridine peptide-mediated pDNA and ds mRNA delivery to the liver of mice in vivo. Specifically, we developed a synthetic strategy to incorporate highly branched N-Glycan oligosaccharides onto the ends of PEGylated polyacridine peptides for liver specific targeting. At optimal zeta potential, PEGylated Glycopeptides provided a statistically significant increase in luciferase expression in comparison to an untargeted polyplex control.
Next, we explored the concept of endosomal escape. Although PEGylated glycopeptides produce stabilized pDNA polyplexes, they lack a mechanism for endosomal escape. We hypothesized that we could improve in vivo efficacy of PEGylated glycopeptides by co-administration of co-targeted potent endosomal escape agents. Three separate strategies were followed to create three unique and novel targeted endosomal escape agents. During experimentation around this concept, we discovered a unique mechanism to create reversibly glycosylated TRI-PLA2 enzyme, through disulfide exchange reaction with phospholipase A2 disulfide bridges.
Following extensive characterization, the efficacy of the novel endosomal escape agents was evaluated in vitro using miniaturized 384 luciferase transfections with both primary hepatocytes and HepG2 cells. Unfortunately, numerous technical difficulties with the assay prevented us from advancing this project and drawing conclusions from the produced preliminary data.
Finally, we sought to advance the development of ds mRNA. ds mRNA is produced by hybridization of a translationally competent Forward mRNA with a reverse complement RNA. Given the inherent inactivity of the reverse stand of ds mRNA, we hypothesized that it could be used a scaffold to incorporate novel chemical modifications into ds mRNA, potentially leading to an increase in efficacy. Preliminary structure activity relationships revealed that although ds mRNA expression is diminished by the incorporation of chemically modified 5’aminoallyl nucleotide triphosphates, activity can be fully recovered by incorporation of a biologically well tolerated chemical moiety.
It is our hope that the novel compounds introduced in this thesis, the synthetic pathways utilized in their synthesis, and the knowledge acquired from their biological testing will aid in the development of novel vectors or adjuvants for non-viral delivery.
|Advisor:||Rice, Kevin G|
|Commitee:||Kerns, Robert, Roman, David, Doorn, Jonathan, Fuentes, Ernesto|
|School:||The University of Iowa|
|Department:||Pharmaceutical Sciences and Experimental Therapeutics|
|School Location:||United States -- Iowa|
|Source:||DAI-B 81/8(E), Dissertation Abstracts International|
|Keywords:||ds mRNA, Endosomal escape, Gene expression, Glycoconjugates, Nonviral gene delivery, Targeting|
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