Preparation Of Oligonucleotide Delivery Agents And Insulin Depot Materials Using Solid Phase Synthesis
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Abstract
Short interfering RNA (siRNA) induced RNA interference (RNAi) is capable of preventing gene translation. In a laboratory setting, various transfection methods have enabled this mechanism to be used for routine gene silencing studies. siRNA can also be used for gene therapy. However, the use of siRNA for this purpose has not matched its initial promise. One reason for this is that an effective delivery method to enable siRNA to reach the systemic circulation is still lacking. Transfection methods that are effective in-vitro have significant toxicity associated with them. The first four chapters of this dissertation outline the attempts to synthesize, characterize and test positively charged single molecular weight materials that could be used for the delivery of siRNA. The materials synthesized were linear oligoarginines and dendritic oligoguanidines. Solid phase techniques were used for the synthesis of these materials. The results of studies conducted on mammalian cell lines with the synthesized materials are described. Also mentioned are the problems faced during the synthesis and attempts to overcome these difficulties. Chapters five through seven deal with insulin delivery. Diabetes is a disease that affects millions. Blood glucose levels in type I diabetes is controlled with insulin therapy. Since a tight control of insulin level throughout the day is essential, a large number of patients carry prefilled insulin syringe to self inject or use insulin pumps. These methods have disadvantages. The studies mentioned were undertaken to provide an alternate option to, and possibly improve upon, previously published work in our laboratory. Insulin was tethered to a biocompatible depot material. A photo cleavable trigger was used between the insulin and the depot material. Light was used was used to control insulin release. The routes tested for linking the insulin to the depot included a diazo method and an imidazole carbamate method. Characterization of the conjugates and light release studies are shown. The final chapter describes experiments performed to cage siRNA. Caging of siRNA is described so that the timing and degree of RNAi activity could be controlled. Data from preliminary studies obtained with LNA and siRNA having a 2’ amine modification are indicated. Time course studies with caged siRNA are also described.
Table of Contents
Introduction and literature review -- Synthesis of single molecular weight linear oligoarginines for siRNA transfeciton -- Synthesis of single molecular weight dendritic amines and dentritic oligoguanidines -- Summary and conclusions -- Introduction and literature review -- Synthesis of photoactivated insulin depots -- Summary and conclusions -- Experiments with modified siRNA and RNA interference
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Ph.D.