259. Targeted Gene Silencing by U1 Adaptor Oligonucleotides in Preclinical Models of Parkinson's Disease and Huntington's Disease
Authors: Eric Richfield, Kavita Prasad, Mark Brenneman, Rafal Goraczniak, Samuel Gunderson
Journal: Molecular Therapy (2016)

Many candidate genes are implicated in neurodegenerative disease, but to study potential therapeutic effects of modifying their expression in the central nervous system of animal models has been difficult, often requiring slow, expensive transgenic methods. Transient gene silencing with synthetic oligonucleotides can be a fast, inexpensive alternative to making new transgenic animal models, and a complimentary technique to extend the utility of existing ones. For genes with products that have been validated as therapeutic targets, but are not amenable to small molecule drugs, gene silencing may also be the therapeutic modality of choice. U1 Adaptors are a third generation of oligonucleotide-mediated gene silencing technology, mechanistically distinct from antisense or siRNA. U1 Adaptors act by selectively interfering with a key step in mRNA maturation: the addition of a 3' polyadenosine (polyA) tail. Nearly all protein-coding mRNAs require a polyA tail, and failure to add one results in rapid degradation of the nascent mRNA inside the nucleus, preventing expression of a protein product. U1 Adaptor oligonucleotides are well suited to in vivo applications because they can accept extensive chemical modifications to improve nuclease resistance and the attachment of bulky groups, such as tags for imaging or ligands for receptor-mediated uptake by target cells, without loss of silencing activity. To explore the feasibility of U1 Adaptor technology for CNS targets, we designed panels of candidate U1 Adaptor oligos for mouse Scna (alpha-synuclein) and human HTT (Huntingtin), and screened them in cell culture. We identified U1 Adaptors that robustly suppress mouse Scna mRNA and reduce alpha-synuclein protein levels in mouse cells. Similarly, we identified U1 Adaptors that suppress the predominant, full length human HTT mRNA and reduce HTT protein levels in human cells. We also identified U1 Adaptors that suppress the HTT exon-1 truncation isoform recently implicated in HD pathogenesis. For in-vivo PK/PD studies, U1 Adaptors were delivered into the CNS of mice, by intracerebroventricular (ICV) injection or by direct stereotaxic injections into the striatum. To examine distribution, cellular uptake and persistence over time, fluorescently tagged U1 Adaptors were administered, then visualized by confocal microscopy in brain sections. ICV injection achieved broad distribution of fluorescent U1 Adaptors throughout the brain, with uptake visible in most cells. Subcellular distribution 24 hours after injection was diffuse in both cytoplasmic and nuclear compartments, but became more punctate and perinuclear by 48 hours. U1 Adaptor oligonucleotides were detected on northern blots of small RNA recovered from brain tissue specimens. Their levels in tissue were estimated by comparison to a standard loading curve, and correlated well with ICV-injected dose. After direct stereotaxic injection to the striatum, U1 Adaptors diffused rapidly and widely, and were taken up by all striatial cells, though preferentially by neurons. Adaptors persisted in tissue for at least five days (the last time point assayed) and reached the nuclei of striatial cells. We then did a series of studies with U1 Adaptors specific for mouse Scna mRNA. RNAScope was used to visualize relative levels of mRNA in situ. Injection of U1 Adaptors directly into the striatum resulted in clearly reduced expression of Scna mRNA and also of mRNA for synaptophysin, known to be down-regulated when α-synuclein expression is reduced.