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Title: Synthesis, biodistribution and efficacy of monomeric and multimeric antisense oligonucleotides in visceral tissue and the central nervous system
Author: Moazami, Michael
ISNI:       0000 0004 7234 2325
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2018
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Antisense oligonucleotides (ASOs) are a promising class of therapeutics for treating diseases of a genetic origin. ASOs are able to modulate the expression of disease-causing RNA, interact with the immune system and affect the endogenous regulation of genes. ASOs are, however, poorly taken up into some tissue types which limits their potential therapeutic benefit. This work aimed to improve the biodistribution and efficacy of antisense oligonucleotides by synthesising them as dendrimers - discrete structures comprising multiple oligonucleotides around a central core. Hence, the various synthetic approaches are described for attempts to access these molecules, their effects on in vitro and in vivo distribution and silencing of the long non-coding RNA MALAT1. It is demonstrated that larger dendrimers are more rapidly taken up into cultured cells than monomeric oligomers and that the compounds distribute differently in the mouse. Nevertheless, silencing efficacy both in vitro and in vivo seems to peak with monomeric or dimeric structures. It is anticipated that the rapid uptake of these dendrimers may be able to be translated into improved silencing by modifying their chemistry in the future. To adequately assay the biodistribution of these oligonucleotides, the available techniques discussed in the literature are compared. Dissatisfied with the current state of the art, the process of preparing a polyclonal antibody directed against the chemically modified backbone found in most therapeutic oligonucleotides is described. It is shown that the antibody generated is able to be used for immunohistochemistry and immunouorescence, and that it shows promise for use as a quantitative assay for determining oligonucleotide concentration in tissue. Finally, efforts in improving oligonucleotide chemistry and structure for silencing disease-causing RNA in the CNS are described. Using C9ORF72 repeat-expanded Amyotrophic Lateral Sclerosis as a target disease, it is show that some oligonucleotides are intrinsically and surprisingly toxic on central administration and that this toxicity can be ameliorated by reduction of the phosphorothioate content of the backbone. The generation of a preclinical lead compound is described that will, building on this work, be screened for toxicity in non-human primates and advanced to a compassionate care trial in two patients, pending FDA approval.
Supervisor: Watts, Jonathan K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available