Synthesis, biodistribution and efficacy of monomeric and multimeric antisense oligonucleotides in visceral tissue and the central nervous system

Abstract

Synthesis, biodistribution and efficacy of monomeric and multimeric
antisense oligonucleotides in visceral tissue and the central nervous system
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
immunofluorescence, 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.

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