Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain

Abstract

The use of siRNA-based therapies for the treatment of neurodegenerative disease requires efficient, nontoxic distribution to the affected brain parenchyma, notably the striatum and cortex. Here, we describe the synthesis and activity of a fully chemically modified siRNA that is directly conjugated to docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in the mammalian brain. DHA conjugation enables enhanced siRNA retention throughout both the ipsilateral striatum and cortex following a single, intrastriatal injection (ranging from 6–60 μg). Within these tissues, DHA conjugation promotes internalization by both neurons and astrocytes. We demonstrate efficient and specific silencing of Huntingtin mRNA expression in both the ipsilateral striatum (up to 73%) and cortex (up to 51%) after 1 week. Moreover, following a bilateral intrastriatal injection (60 μg), we achieve up to 80% silencing of a secondary target, Cyclophilin B, at both the mRNA and protein level. Importantly, DHA-hsiRNAs do not induce neural cell death or measurable innate immune activation following administration of concentrations over 20 times above the efficacious dose. Thus, DHA conjugation is a novel strategy for improving siRNA activity in mouse brain, with potential to act as a new therapeutic platform for the treatment of neurodegenerative disorders.

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin

“…When injected directly in mouse striatum, DHA-siRNAs induced efficient Htt mRNA silencing and had no measurable impact on neuronal integrity or innate immune activation (Nikan et al, 2016). Mice were directly injected with 4 nmol DHA-siRNA NTC , DHA- siRNA HTT (Nikan et al, 2016), ASO NTC , and ASO HTT (Hung and Leeds, 2007) in the right striatum (n = 3 animals per group; Figure 4A). After 7 days, levels of nuclear and cytoplasmic Htt mRNA foci were assessed by FISH (Figures 4B and4C).…”

The chemical evolution of oligonucleotide therapies of clinical utility

“…Modulating the identity of the hydrophobic conjugate can be used to alter the tissue distribution profile or modulation of diffusion for the site of injection 123 . Notably, changing the hydrophobic moiety to a polyunsaturated fatty acid derivative supports a wider therapeutic index, thus enabling another direction in systemic conjugate-based gene modulation 123 .…”

“…Both single-stranded and double-stranded oligomers can be effectively targeted to the liver by GalNAc modification, and both are actively in development (Table 1). But for other tissues (e.g., the brain or spinal cord), single-stranded character provides a big delivery advantage 122, 123 with three ASOs in clinical trials for central nervous system (CNS) indications (see p. WahlestedtXXX in this issue).…”

“…The single-stranded fully-phosphorothioate tail resembles ssASOs and in part confers PK/PD behavior characteristic of conventional ASOs. The fully phosphorothioate single-stranded region works in combination with different conjugates to enhance in vivo delivery and cellular uptake, demonstrating properties that cannot be achieved with the conjugate alone, including promising activity in CNS tissues 122, 123 .…”

Tapping the RNA world for therapeutics

“…Some of the most attractive approaches in mouse models include siRNA conjugation to RNA or DNA aptamers that recognize cell surface receptors 83–85 , siRNA conjugation to CpG nucleotides that bind to an innate immune receptor on dendritic cells or macrophages 86 , antibody fusion protein complexed siRNAs 87,88 , antibody-coated LNPs 89–91 and siRNA conjugation to a fatty acid constituent of neuronal membranes for nerve cell delivery 92 (Fig. 3).…”