Frankfurt (Main), Universiẗat, Diss., 2007.

Thesis (Ph.D. in Biostatistics) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 237-239). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Zugl.: Regensburg, Univ., Diss., 2008

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 121-140).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (p. 105-112).

Universiẗat, Diss., 2002--Dortmund.

Les oligonucléotides sont de courtes séquences d’acide nucléique qui ont la capacité d’inhiber ou de moduler l’expression d’un gène cible par différents mécanismes. Cependant, leur potentiel thérapeutique est limité par leur faible internalisation. Pour pallier ce problème de vectorisation, il a été envisagé de conjuguer les oligonucléotides à des molécules biocompatibles. Ce travail de thèse porte sur des bioconjugués composés d’un nucléolipide à l’extrémité 5’ ou 3’ des oligonucléotides. Tout d’abord, les propriétés physico-chimique et d’hybridation de ces nouveaux composés ont été évaluées. Des études biologiques ont ensuite été réalisées in vitro et in vivo. Les résultats obtenus ont permis de mettre en évidence le mécanisme d’internalisation cellulaire mais aussi de prouver l’efficacité de transfection et d’inhibition de ces conjugués. En outre, le caractère amphiphile de ce type de composé rend possible leur auto-assemblage pour la formulation de substances actives. Dans ce cadre, différentes formulations ont été investiguées. Ainsi, dans ce travail de thèse une nouvelle technologie d’oligonucléotides conjugués a été développée. La séquence de ces molécules peut être modulée et maitrisée de manière à l’adapter à la cible thérapeutique visée. Actuellement, ce système est appliqué sur treize projets différents à l’échelle nationale. L’effet thérapeutique est ainsi évalué dans différentes pathologies telles que des cancers hormono-dépendants, des leucémies, des maladies neurologiques chroniques ou encore la résistance aux antibiotiques. / Oligonucleotides are short nucleic acid molecules able to modulate or inhibit gene expression. However the main drawback of oligonucleotides lies in their poor cellular internalization, which limit their therapeutic applications. Herein, to overcome this limitation, oligonucleotides were conjugated to biocompatible molecules as a nucleolipid to either the 5'- or the 3'-end. First, physico-chemical properties and binding behaviour of this newly compound were investigated. Then in vitro and in vivo biological assays were performed to characterize but also understand the cellular internalization pathways and their biological activities. Finally, the amphiphilic nature of the oligonucleotide-nucleolipid confers spontaneously self-assembling properties for drugs loading and vectorization purposes. This Ph.D. thesis focuses on new oligonucleotide bioconjugates for various biological applications. Sequences of nucleotides can also be modulated to specifically bind to the therapeutic target. This tuneable technology is actually used in 13 different projects, including hormone-dependent cancers, leukemia, chronic neurological disorders and antibiotic resistance, for example.

abstract: The need of organs for transplantation has become an increasing medical need due to a limited donor organ supply. Many organs fail within 10 years due to acute and chronic rejection. Acute or antibody mediated rejection leads to decreased long term graft survival and increases the need for a repeat transplant. In prior work, reducing endothelial heparan sulfation and blockade of chemokine-glycosaminoglycan (GAG) interaction with Myxomavirus-derived protein, M-T7, reduced aortic and renal graft vascular inflammation and rejection. Conditional endothelial Ndst1 deficiency and inhibition of chemokine-GAG interaction reduces early allograft damage and suggest new therapeutic options for graft rejection. Here acute renal rejection was examined in grafts with conditional endothelial N-deacetylase-N-sulfotransferase-1 knockout (Ndst1-/-) and in wildtype (WT) C57Bl6/J grafts treated with saline, M-T7, antisense oligonucleotides (ASO) for Ndst1 or a scrambled ASO control. Viruses have a highly adaptive ability to evade hosts defense and immune response. The immunomodulatory proteins derived from viruses provide potential therapeutic uses to alleviate this need for organs. The Myxoma virus derived protein M-T7 is a promising therapeutic for reducing kidney transplant rejection. Orthotopic transplantations in mice are extremely difficult and costly because they require a highly trained microsurgeon. This kidney to kidney subcapsular and subcutaneous transplant model is a practical and simpler method that requires fewer mice, one kidney can be used for transplants in 6 or more mice and there is much lower morbidity, pain and mortality. Heterotopic transplantation of allografts is a simple model for preliminary testing of treatments for early inflammation, ischemia, and graft rejection. Subcapsular kidney transplantation provides a first step approach to test virus-derived proteins as potential treatments to reduce transplant rejection and inflammation. This project reports on a broadly applicable platform on which to rapidly and conveniently test new treatments for transplant rejection. This finding will significantly lower the barrier to entry for labs which are interested in translating their laboratory findings to animal models of organ transplantation which is a complex surgical procedure, and thus accelerate the bench-to-bedside translation of novel, putative treatments for transplant rejection as an initial screening tool. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2020

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a monogenic, autosomal dominantly inherited, neurodegenerative disorder resulting in loss of pain and temperature sensation in the distal limbs. HSAN1 is caused by point mutations in a single allele of serine palmitoyltransferase long chain base 1 (SPTLC1), resulting in production of neurotoxic deoxysphingolipids (dSLs). Oligonucleotide therapeutics (ONTs) can be used to downregulate the mutant allele and/or the wild type allele and thus are viable treatment strategies. We investigated the ability of two classes of ONTs, short interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), to downregulate SPTLC1 in an in vitro model of HSAN1 derived from the C133W mouse model overexpressing mutant hamster SPTLC1. We screened a panel of siRNAs and ASOs targeting mutant hamster SPTLC1 and identified four lead compounds. We demonstrated these compounds’ ability to reduce mutant hamster SPLTC1 and/or wild type mouse SPTLC1 mRNA in CHO cells and C57BL/6J embryonic mouse primary cortical neurons. We then showed that these compounds downregulate hamster and mouse SPTLC1 mRNA and protein in embryonic primary cortical neuron cultures derived from C133W mice. These compounds demonstrate therapeutic potential and should be developed further in vivo.

Non-coding RNA (ncRNA) therapeutics are based on short oligonucleotides, both naturally occurring and artificial, which target RNA in a site-specific way to modulate gene expression. As of today, 12 synthetically produced ncRNA-based drugs are available on the market in the US and Europe, and there is a possibility of more to be approved in the near future. This project is ordered by Cytiva, a global life science company, with the aim to present an overview of the current ncRNA therapeutics field. The aim is to give Cytiva a clear indication of what type of products for oligonucleotide synthesis are requested by their clients in the pharmaceutical industry. ncRNAs were examined extensively for their potential as therapeutic agents during our literature study. Based on the number of approved drugs, clinical trials, and our overall impression of future potential, we selected the following four ncRNAs; Antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), and small nuclear RNA (snRNA). Among these, the most promising ncRNAs for therapeutic use are siRNA and ASO which usually are 20-30 nucleotides long. The most common modifications to improve drug-like properties are modifications to the backbone, sugar modifications at position 2, and methylation of nucleobases at position 5 of the oligonucleotide. In addition, ncRNA-based drugs on the market today are delivered either through non-viral mechanisms or without a delivery system. The conclusion that can be drawn from our report is the importance of being able to synthesize chemically modified ASOs and siRNA on a large scale to meet the future demand of the pharmaceutical industry.