Search

Huntington's disease (HD) is an autosomal dominant disease caused by the expansion of cytosine-adenine-guanine (CAG) repeats in one copy of the HTT gene (mutant HTT, mHTT). The unaffected HTT gene encodes wild-type HTT (wtHTT) protein, which supports processes important for the health and function of the central nervous system. Selective lowering of mHTT for the treatment of HD may provide a benefit over nonselective HTT-lowering approaches, as it aims to preserve the beneficial activities of wtHTT. Targeting a heterozygous single-nucleotide polymorphism (SNP) where the targeted variant is on the mHTT gene is one strategy for achieving allele-selective activity. Herein, we investigated whether stereopure phosphorothioate (PS)- and phosphoryl guanidine (PN)-containing oligonucleotides can direct allele-selective mHTT lowering by targeting rs362273 (SNP3). We demonstrate that our SNP3-targeting molecules are potent, durable, and selective for mHTT in vitro and in vivo in mouse models. Through comparisons with a surrogate for the nonselective investigational compound tominersen, we also demonstrate that allele-selective molecules display equivalent potency toward mHTT with improved durability while sparing wtHTT. Our preclinical findings support the advancement of WVE-003, an investigational allele-selective compound currently in clinical testing (NCT05032196) for the treatment of patients with HD., Competing Interests: N.I., Y.L., M.F.-K., A.M., W.C.T., K.T., N.K., A.A., K.B., J.D.S., A.L., X.S.H., H.G.J., P.K., F.L., K.L., R.L., M., J.M., Q.P., E.P.-E., M.S., P.S.P., S.S., H.Y., A.Z., C.F., M.B., E.D., and C.V. were employees of Wave Life Sciences during the completion of this work. M. is currently an employee of Stoke Therapeutics. G.L.V. is on the board of directors and is a consultant and shareholder for Wave Life Sciences., (© 2024 The Author(s).)

Herein, we report the systematic investigation of stereopure phosphorothioate (PS) and phosphoryl guanidine (PN) linkages on siRNA-mediated silencing. The incorporation of appropriately positioned and configured stereopure PS and PN linkages to N-acetylgalactosamine (GalNAc)-conjugated siRNAs based on multiple targets (Ttr and HSD17B13) increased potency and durability of mRNA silencing in mouse hepatocytes in vivo compared with reference molecules based on clinically proven formats. The observation that the same modification pattern had beneficial effects on unrelated transcripts suggests that it may be generalizable. The effect of stereopure PN modification on silencing is modulated by 2'-ribose modifications in the vicinity, particularly on the nucleoside 3' to the linkage. These benefits corresponded with both an increase in thermal instability at the 5'-end of the antisense strand and improved Argonaute 2 (Ago2) loading. Application of one of our most effective designs to generate a GalNAc-siRNA targeting human HSD17B13 led to ∼80% silencing that persisted for at least 14 weeks after administration of a single 3 mg/kg subcutaneous dose in transgenic mice. The judicious use of stereopure PN linkages improved the silencing profile of GalNAc-siRNAs without disrupting endogenous RNA interference pathways and without elevating serum biomarkers for liver dysfunction, suggesting they may be suitable for therapeutic application., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro. In vivo, AIMers targeted to hepatocytes with N-acetylgalactosamine achieve up to 50% editing with no bystander editing of the endogenous ACTB transcript in non-human primate liver, with editing persisting for at least one month. These results support further investigation of the therapeutic potential of stereopure AIMers., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Modestly increased expression of transactive response DNA binding protein (TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed ~30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43., Competing Interests: The authors have declared that no competing interests exist.

Expansions of a G 4 C 2 repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Using C9-ALS/FTD patient-derived cells and C9ORF72 BAC transgenic mice, we generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G 4 C 2 repeat-containing transcripts and effectively suppress tissue levels of poly(GP) dipeptides. ASOs with reduced phosphorothioate content showed improved tolerability without sacrificing efficacy. In a single patient harboring mutant C9ORF72 with the G 4 C 2 repeat expansion, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of cerebrospinal fluid poly(GP). This report provides insight into the effect of nucleic acid chemistry on toxicity and, to our knowledge, for the first time demonstrates the feasibility of clinical suppression of the C9ORF72 gene. Additional clinical trials will be required to demonstrate safety and efficacy of this therapy in patients with C9ORF72 gene mutations., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)