Thesis (Ph. D.)--Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2006. / Harish Radhakrishna, Committee Member ; Mark Prausnitz, Committee Co-Chair ; Joseph Le Doux, Committee Chair ; Timothy Wick, Committee Member ; Richard Compans, Committee Member ; Athanassios Sambanis, Committee Member.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Nicholas V. Hud, Committee Chair ; Donald F. Doyle, Committee Member ; Rigoberto Hernandez, Committee Member ; Roger M. Wartell, Committee Member ; Loren D. Willliams, Committee Member.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 126-143). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 71 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 134 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D.)--Baylor University, 2006. / Includes bibliographical references (p. 100-115).

The bestrophinopathies are a set of inherited retinal degenerations caused by mutations in BEST1, and include Best vitelliform macular dystrophy (BVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC) and autosomal recessive bestrophinopathy (ARB). The corresponding protein, bestrophin-1, is localised to the basolateral membrane of the retinal pigment epithelium (RPE), where it is thought to function as a Ca²⁺-activated Cl- channel. Currently, there are no treatments for these conditions. In recent years, gene therapy has emerged as an exciting treatment option for inherited retinal disorders (IRDs). Gene delivery to retinal cells using a recombinant adeno-associated virus (rAAV) has produced positive results in several IRDs. Given the recessive nature of ARB, this thesis proposes that the rAAV-mediated delivery of bestrophin-1 to the RPE could represent a potential therapy. The aims of this thesis were to produce and compare rAAV vectors in vitro and in vivo for protein expression, localisation following transduction, restoration of chloride conductance in vitro and safety following sub-retinal injection in vivo. Following the production of two rAAV vectors expressing bestrophin-1, western blots confirmed bestrophin-1 protein expression following transduction of HEK293 cells in vitro. Immunocytochemistry (ICC) revealed bestrophin-1 expression that was localised to the cytosol. Whole-cell patch-clamping revealed a significant increase in chloride conductance in HEK293 cells transduced with AAV-BEST1 vectors which was then ablated upon the removal of chloride from the buffers. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) indicated that the bestrophin-1 protein was successfully transcribed and translated from the BEST1 coding sequence (CDS). Sub-retinal injections of AAV-BEST1 produced bestrophin-1 expression in the RPE of wild-type C57BL/6 mice however significant retinal thinning was seen at higher doses of vector. In conclusion, rAAV-mediated transfer of bestrophin-1 to the RPE has potential to be a future therapy for ARB, however safety issues need to be addressed and an RPE-specific promoter could be more suitable.

No description available.

Osteoporosis remain major clinical challenges. RNA interference (RNAi) provides a promising approach for promoting osteoblastic bone formation to settle the challenges. However, the major bottleneck for translating RNAi with efficacy and safety to clinical bone anabolic strategy is lack of osteoblast-specific delivery systems for osteogenic siRNAs. Previously, we developed a targeting system involving DOTAP-based cationic liposomes attached to oligopeptides (AspSerSer)6, (also known as (DSS)6), which had good affinity for bone formation surface. Using this system, osteogenic Pleckstrin Homology Domain Containing, Family O Member 1 (Plekho1) siRNA could be specifically delivered to bone formation surface at tissue level and promoted bone formation in osteopenic rodents. However, concerns still exist regarding indirect osteoblast-specific delivery, detrimental retention in hepatocytes, mononuclear phagocyte system (MPS)-induced dose reduction and inefficient nanoparticle extravasation. Aptamers, selected by cell-based Systematic evolution of ligands by exponential enrichment (cell-SELEX), are single-stranded DNA (ssDNA) or RNA which binds to target cells specifically by distinct tertiary structures. By performing positive selection with osteoblasts and negative selection with hepatocytes and peripheral blood mononuclear cells (PBMCs), we aimed to screen an aptamer that could achieve direct osteoblast-specific delivery and minimal hepatocyte and PBMCs accumulation of Plekho1 siRNAs. In addition, lipid nanoparticles (LNPs) have been widely used as nanomaterials encapsulating siRNA due to their small particle size below 90 nm. Polyethylene glycol¡(PEG) as the mostly used hydrophilic polymer, could efficiently prevent LNPs from MPS uptake. So, LNPs with PEG shielding could serve as siRNA carriers to realize efficient extravasation from fenestrated capillaries to osteoblasts and help reduce MPS uptake of the siRNAs. Recently, we screened an aptamer (CH6) by cell-SELEX specifically targeting both rat and human osteoblasts and developed the aptamer-functionalized LNPs encapsulating osteogenic Plekho1 siRNA, i.e., CH6-LNPs-siRNA. Our results demonstrated that CH6-LNPs-siRNA had an average particle size below 90 nm and no significant cytotoxicity in vitro. CH6 aptamer facilitated osteoblast-selective uptake of Plekho1 siRNA and gene silencing in vitro. In this study, we further found that CH6 aptamer facilitated the bone-specific distribution of siRNA by biophotonic imaging and quantitative analysis. Immunohistochemistry results showed that CH6 achieved in vivo osteoblast-specific delivery of Plekho1 siRNA. Dose-response experiment indicated that CH6-LNPs-siRNA achieved almost 80% gene knockdown at the siRNA dose of 1.0 mg/kg and maintained 12 days for over 50% gene silencing. microCT, bone histomorphometry and mechanical testing confirmed that CH6 facilitated bone formation, leading to improved bone micro-architecture, increased bone mass and enhanced mechanical properties in osteoporotic rodents. Furthermore, CH6-LNPs-siRNA achieved better bone anabolic action when compared to the previously developed (AspSerSer)6-liposome-siRNA. There was no obvious toxicity in rats injected with CH6-LNPs-siRNA. All these results indicated that osteoblast-specific aptamer-functionalized LNPs could act as a novel RNAi-based bone anabolic strategy and advance selectivity of targeted delivery for osteogenic siRNAs from tissue level toward cellular level. In addition, the generation of ssDNA from double-stranded PCR products is an essential step in selection of aptamers in SELEX. We found that the size separation derived from unequal primers with chemical modification could be a satisfactory alternative to the classic magnetic separation.

Abstract The progress in recombinant DNA technology has made possible the introduction of exogenous genetic material into cells. Gene therapy aims to correct a defective gene, introduce a therapeutic exogenous gene or a counteracting gene into somatic cells without modification of the germ-cell line. The most important technical interests in the field of gene therapy research have pertained to the development of safe and effective vectors and suitable methods for the delivery of the exogenous gene carrying vectors into the target cells. The aim of this study was to evaluate surgical methods used for gene delivery and to develop an effective gene transfer method for organ-specific gene transfer, primarily into the renal glomeruli. There are genetic and acquired diseases that are candidates for gene therapy. Alport syndrome is an X-chromosome-linked disease caused by a mutation in the type IV collagen α5 chain gene, which causes a defect of the glomerular basement membrane in the kidney, leading to progressive renal failure in males. This manifestation could theoretically be prevented by the transfer of a normal α5 chain gene into the renal glomerular cells. Cystic fibrosis and α1-antitrypsin deficiency are examples of pulmonary diseases and genetic lysosomal storage diseases that are candidates for splenic gene transfer. The gene transfer strategies used so far have proved relatively ineffective. Recombinant adenovirus, retrovirus, adeno-associated virus and liposomes have been previously used as vectors. Direct injection, intra-arterial, intravenous and intratracheal delivery of vectors have been the most extensively studied methods. This preclinical experimental work for marker gene transfer into the kidney, spleen, lung and mammary gland was done by using rabbits, pigs and goats as test animals. The adenoviral vector carrying a β-galactosidase reporter gene was first infused in the renal artery of rabbits and pigs in vivo with or without pharmacological agents. This did not result in any remarkable gene transfer into the kidney. Next, the incubation time between the vector and the target cells was prolonged by ex vivo perfusion of explanted kidneys for 12 hours. Perfusion at room temperature did not improve gene transfer. When the perfusion temperature was raised to 37°C, improved and mostly glomerular gene transfer was observed, with up to 80% of the glomeruli showing β-galactosidase expression in four ex vivo experiments. A closed-circuit organ perfusion method for in vivo gene transfer was developed in this study. The surgical perfusion experiment was tested successfully in ten in vivo perfusions of the kidney, eight of the spleen and eight of the lung in a porcine model. This method led to effective, up to 75% gene transfer into the renal glomeruli as assessed after four days. In the spleen, the perfusion method resulted in relatively effective gene transfer into perifollicular splenic cells, mostly macrophages and endothelial cells. Lung perfusion yielded transgene expression in alveolar epithelial cells, bronchiolar epithelial cells and, to a lesser extent, arteriolar endothelial cells and alveolar macrophages. Perfusion of the goats mammary gland using a retroviral vector in three experiments resulted in growth hormone secretion into the milk. The gene transfer operation was well tolerated by the animals, and no clinical signs of inflammation were observed. No remarkable humoral immunological response against adenovirus or β-galactosidase was elicited in the kidney experiments, but histological signs of inflammation as mononuclear cell clusters in the kidney and lung were seen four and seven days after the experiments. The spleen showed no macroscopic or microscopic pathologic alterations after the perfusion.