Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. Interleukin-10 (IL-10) is a cytokine that downregulates the proinflammatory response and has a modulatory effect on hepatic fibrogenesis. The aim of this study was to investigate whether IL-10 gene therapy possesses anti-hepatic fibrogenesis in mice. Liver fibrosis was induced by long-term thioacetamide administration in mice. Human IL-10 expression plasmid was delivered via electroporation after liver fibrosis established. IL-10 gene therapy reversed hepatic fibrosis and prevented cell apoptosis in a thioacetamide-treated liver. RT-PCR revealed IL-10 gene therapy could reduce liver transforming growth factor-£]1¡]TGF-£]1¡^, tumor necrosis factor-£\¡]TNF-£\¡^, collagen £\1, cell adhesion molecule, and tissue inhibitors of metalloproteinase¡]TIMPs¡^mRNA upregulation. Following gene transfer, the activation of £\-smooth muscle actin¡]£\-SMA¡^and cyclooxygenase-2¡]COX-2¡^were significantly attenuated. In brief, electroporative IL-10 gene therapy might be an effective therapeutic reagent for liver fibrosis with potential future clinical applications.

The use of gene-based techniques to produce antinociceptive molecules has been actively investigated for treatment of neuropathic pain and trauma of central nervous system. Among the endogenous opioids, b-endorphin (b-EP) is the most potent one, which is derived from pro-opiomelanocortin (POMC). In addition to b-endorphin, POMC is also the precursor of many neuropeptides such as adrenocorticotropin hormone (ACTH), melanocyte-stimulating hormone (a-MSH), ¡Ketc. Appropriate administration of POMC gene is essential for the success of its clinical application. Thus, gene transfer approach seems to be suitable for continuous supply of b-endorphin to alleviate intractable pain. Recombinant adenovirus was used as gene delivery system for POMC because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant adenovirus encoding POMC (Ad-POMC) by PCR and western blot analysis, and detect the presence of opioid peptides including ACTH, a-MSH and b-EP by RIA and chemilluminiscent assay. GH3 cells infected with Ad-POMC showed significantly higher levels of ACTH, b-endorphin, and a¡VMSH comparing with cells of control groups. By using Ad-GFP, the optimal MOI for adenovirus vector to infect neuronal GH3 cells, glial C6 cells, hepatoma Hep3B cells, smooth muscle G8 cells, fibroblast CCD-965K cells, and endothelial EA.hy926 cells was determined at 50, 500, 50, 500, 500, and 200, respectively. The results of determining the efficiency of POMC processing in different types of cells after in vitro cell cultures gene delivery indicated that peripheral cells, though at a lower extent, are capable of cleaving POMC and releasing opioid peptides after POMC gene delivery like neuronal cells of central nervous system. In formalin test, the intrathecal POMC gene delivery significantly decreased the magnitude of the formalin-evoked flinching response phase 1 (P < 0.05) and phase 2 (P < 0.001) when compared with rats receiving saline or Ad-GFP. In conclusion, the intrathecal POMC gene delivery can produce effectively attenuation on the inflammatory pain response. So far, there have been various gene delivery studies confirming the potential role of POMC in antinociception. In the future, more experiments will be needed to characterize the effects of POMC expression on cellular lipid metabolism. This will enable us to evaluate the therapeutic potential of POMC on treatment of obesity.

Obesity is a disorder of energy imbalance and the most prevalent nutritional diseases in developed countries. Besides, obesity is also strongly associated with health problems such as type 2 diabetes (NIDDM), hypertension, hyperlipidaemia, cardiovascular diseases and cancers. However, the defects in lipid metabolism underlying obesity-related disorders are extremely complicated. Thus, extensive studies on the mechanism of endogenous fatty acids synthesis would be one of the keys to elucidate molecular pathogenesis of obesity. In liver or adipose, fatty acid synthase (FAS) utilizes acetyl-CoA, malonyl-CoA and NADPH to synthesize long-chain fatty acids (C16 or C18), which can be converted to triglycerides and stored as fat. During lactation, thioesterase II (TE II) expresses in mammary glands and interacts with FAS to produce medium-chain fatty acid (primarily C10) in milk, which provides immune protection and energy for the newborn. TE II causes premature termination of fatty acid synthesis catalyzed by FAS and releases medium-chain fatty acids. Unlike long-chain fatty acids, medium-chain fatty acids can enter mitochondria directly for beta-oxidation to generate ATP, thus provide energy more efficiently. Since TE II gene expression is under strict regulation, we utilized adenovirus gene transfer techniques to deliver and express TE II in hepatocytes. It was postulated that expression of TE II in hepatocytes might result in the increase of ATP and reduction of long-chain fatty acids, subsequently decrease the fat production. Recombinant adenovirus was used as gene delivery system for TE II because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant Ad-TE II by PCR, western blot analysis, and enzymatic assay, respectively. By using Ad-GFP, we have determined the optimal multiplicity of infection (MOI) for adenovirus to infect HepG2 cells is about 100-200. Adenovirus-mediated TE II expression in hepatocytes was demonstrated by western blot as well as TE II enzymatic assay. We have demonstrated that the adenovirus-mediated TE II expression was slightly cytotoxic to hepatocytes. Besides, an increase of free fatty acids, asparate transaminase, lactate dehydrogenase levels, as well as ATP synthesis was also noted in the TE II-expressed hepatocytes. The enhanced the release of asparate transaminase (AST/GOT) and lactate dehydrogenase (LDH) after TE II expression in the hepatocytes further supported its cytotoxcity to hepatocytes. In the future, we will carry out experiments to further characterize the effects of TE II expression on cellular lipid metabolism through adenovirus gene delivery. We hope that the present studies will not only provide further insights into mammalian lipid metabolism, but also enable us to evaluate the therapeutic potential of TE II on the treatment of obesity and its related disorders.

This work details the attachment of dendrimers to proteins, peptides and single stranded DNA (ssDNA). Dendrimers based on melamine satisfy many of the synthetic demands in the field of bioconjugate chemistry including: monodispersity, synthetic flexibility and scalability. The solution-phase syntheses of both ssDNA-dendrimer and peptide-dendrimer bioconjugates is described, and thorough characterization by matrix-assisted laser desorption ionization/ time-of-flight (MALDI-TOF) mass spectrometry, UV-vis spectroscopy, fluorescence spectroscopy, and polyacrylamide gel electrophoresis is discussed. Non-covalent DNA-dendrimer complexes have been shown to facilitate antisense gene delivery, but are vulnerable to dissociation and subsequent enzymatic degradation within the cell. In an effort to prepare biocompatible antisense agents capable of effectively shielding ssDNA from intracellular nuclease digestion, disulfide-linked ssDNA-dendrimers were prepared and rigorously characterized to rule out the possibility of an electrostatic-based interaction. Hybridization assays were performed to determine if the covalently-attached dendrimer affected the ability of the attached ssDNA strand to anneal with a complementary sequence to form double-stranded DNA (dsDNA)-dendrimers. Results indicate that ssDNA-dendrimer conjugates readily anneal to complementary ssDNA strands either in solution or attached to gold surfaces. Nuclease digestions of conjugates in solution suggested that enzymatic manipulation of dsDNA-dendrimers is possible, offering promise for DNA-based computation and other fields of DNA-nanotechnology. Much larger bioconjugates consisting of dendrimers, proteins and peptides were prepared with the goal of obtaining molecular weights sufficient for enhanced permeability and retention (EPR) in tumors. While the dendrimer provides the advantages of a purely synthetic route for drug delivery, the protein portion of the bioconjugate provides a monodisperse, macromolecular scaffold for the non-covalent self-assembly of the dendrimers. The strategy presented herein is based on the strong interaction between biotin and the 60 kD tetrameric protein streptavidin. Each monomer of streptavidin is capable of binding 1 biotin molecule, thus when biotin functionalized peptide-dendrimers are added to streptavidin they bind to form a cluster of dendrimers, or a megamer. The biotinylated peptides that link the dendrimers to the streptavidin core provide a way to actively target specific cell types for drug delivery. Megamer formation through the addition of tetrameric streptavidin was successful as indicated by MALDI-TOF, UV-vis titration and gel electrophoresis assays.

<p>The study analyzes the ethical implications of human gene transfer technology for enhancement. Although human gene transfer technology is widely accepted on therapeutic grounds the non-therapeutic use of gene transfer technology remains to be a gray zone for moral deliberation. The present discussion addresses several ethical issues concerning the impacts of human gene transfer technology on individuals, the society, and future people. Accordingly, the study examines major ethical issues concerning the use of human gene transfer technology in general and genetic enhancement in particular, and reliability of the putative demarcation between therapy and enhancement, and further proposes ethical guidelines for non-therapeutic application of human gene transfer technology. A special attention is given to three major ethical issues, such as our obligation to future generations, problems concerning justice, fairness, and equality, and the problem of uncertainty.</p>

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 125-139).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 96-104).

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 171-191).

Haemophilia B is a bleeding disorder caused by various mutations of the coagulation Factor IX gene (F9) resulting in qualitative or quantitative Factor IX protein (FIX) deficiency. Factor replacement therapy is the current standard of care. Cure may be possible in the near future by gene therapy — the transfer of normal copies of F9 to patients with haemophilia, causing establishment of FIX production and correction of the bleeding phenotype. Mesenchymal stem cells (MSC) are potential vehicles for gene delivery through ex vivo gene transfer and subsequent transplantation to the patient. Lentiviral vectors can transduce MSC effectively and mediate long term gene expression. However, gene expression may decline with time due to transgene silencing. Ubiquitous Chromatin Opening Element (UCOE) is a set of genetic sequences cloned from housekeeping genes that can maintain a transcriptionally competent, open chromatin structure and was shown to prevent gene silencing by resisting DNA methylation. We tested human F9 expression and FIX protein secretion by transducing MSC with lentiviral vectors that carry the FIX gene under the control of A2UCOE (A2UCOE-hF9). A2UCOE is a 2.2 kb sequence cloned from the HNRPA2B1–CBX3 gene loci that harbour UCOE function. A2UCOE-eGFP, an enhanced Green Fluorescent Protein (eGFP) gene expression construct, was used to assist in vector titration of A2UCOE-hF9 by flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). MSC were transduced at various Multiplicities of Infection (MOIs) by A2UCOE-hF9 lentiviral vector. Upon transduction, F9 mRNA expression and FIX secretion were measured by qRT-PCR and ELISA respectively. Osteogenic and adipogenic differentiation assay were performed to compare differentiation potential before and after transduction at an MOI of 1. F9 mRNA expression and FIX secretion were both undetectable in untransduced MSC. Upon transduction, vector dose-dependent increase in F9 mRNA expression and FIX secretion were detected at MOIs of 1, 2, 4 and 8. The level of secreted FIX ranges from 20 to 150 μIU in 72 hours. Osteogenic and adipogenic differentiation were not affected post-transduction at an MOI of 1. In conclusion, FIX secretion by MSC was detected upon A2UCOE-hF9 lentiviral transduction. However, the level of FIX appeared to be low compared to published studies. Further studies are required to determine the cause of low FIX expression, develop methods to maximize FIX expression and confirm whether A2UCOE can prevent gene silencing and maintain sustainable gene expression. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

Retrovirus gene therapy vectors can deliver therapeutic genes to mammalian cells in a permanent manner by integrating their genome into host chromosomes and therefore provide the potential for long term therapeutic gene expression. Retrovirus integration, however, can be oncogenic. Apart from insertional mutagenesis (IM) genotoxicity may be caused by other factors including DNA damage following infection and integration and epigenetic effects related to incoming viral particles. Thus, using retrovirus and lentivirus infected murine tumour tissue and infected cell lines in vitro this thesis was directed at investigating whether virus infection and integration could cause genotoxicity by alternative route(s) other than IM. Using clonally derived liver tumours that developed in mice, and normal liver and kidney tissues, following EIAV and HIV delivery in utero, comparative genome hybridisation methodology was used to examine for copy number variation. This showed amplification and deletions only in EIAV derived tumours. Real time Q-PCR analysis was then used to measure gene expression changes relating to genes contained within or near to amplifications observed in two tumours of individual mice. The STRING database was then used to find networks linking genes with differential expression profiles and genes in one of these tumours identified with provirus insertions that were also differentially expressed. These data provided preliminary data implicating a role for LV in Hepatocellular carcinoma (HCC). DNA damage is known to cause chromosomal instability that can lead to tumour development. The relationship between double strand breaks (DSB) and virus infection was also investigated in-vitro to find alternative routes to genotoxicity other than IM. Cell viability analysis demonstrated cells with a defective DNA damage response (DDR) have decreased cell viability compared with cells with intact DDR when infected with RV or LV vectors. DSB assays showed RV and LV infection to generate foci over a 6 hour period followed by DDR. Where no viral integrase is present, no DDR appears, however, where the vector is used with or without a genome to infect cells, DDR occurs as shown by the presence of 53BP1 foci indicative of DNA damage. The relationship between DNA damage and methylation was also investigated. Global methylation was found elevated in the genomic DNA of LV and RV infected cells and not in control uninfected cells. In contrast, methylation changes were not found in infected cells lacking the NHEJ repair pathway. These data suggest the DNA damage response is linked to genome methylation. The E2F transcription factor plays a key role in regulating expression of genes known to control oncogenesis and cancer, and E2F is regulated by methylation of its related target gene promoters. Taking into account all genes in the human genome the number of genes that bind E2F is 32.77%. However, using microarray to represent genes differentially expressed after infection, 59% of these were E2F targets. Overall, taking the data obtained in this thesis into account it may be suggested that RV and LV infection causes a number of potentially related changes to cells that include DNA damage and repair and methylation changes that could influence E2F that is an important factor involved in oncogenesis. Combining this with IM, attenuated RV and LV currently in use for gene therapy may cause genotoxicity to infected cells and increase the risk of oncogenesis especially where DNA damage is not correctly repaired. Further work is required to show in greater detail the extent of this genotoxicity, possible by whole genome sequencing of treated host genomes or cell transformation assays linked to the genotoxicity assays presented here. Collectively these data show that alternative factors to IM might exist that could act independently or synergistically to IM.