D - Oligonucleotide Therapeutics (including siRNAs, aptamers, antagomirs, miRNAs, shRNA, antisense, and splice switching oligos, plasmids)
337: Therapeutic Oligonucleotides Induce Acute Toxicity in CNS, Preventable by the Addition of Divalent Cations to Formulation
Type: Oral Abstract Session
Presentation Details
Session Title: Oligonucleotide Therapeutics
Oligonucleotide therapeutics (ASOs and siRNAs) have become widely explored for the modulation of gene expression in the central nervous system (CNS). Huntington’s Disease (HD), a neurodegenerative disorder caused by a dominant genetic mutation resulting in the production of toxic mRNA and protein, is an optimal target for oligonucleotide therapeutics. Previous studies show that intracerebroventricular (ICV) injections of siRNAs targeting huntingtin (HTT) protein effectively spread throughout small and large brains and reduce total HTT. Administration of highly concentrated compounds carrying a negative charge to the CNS can induce acute toxicity. We hypothesized that these negatively charged regions promote cation exchange, subsequently chelating divalent cations from the cerebral spinal fluid (CSF) and intracellular stores, which results in abnormal behavioral phenotypes. Electroencephalogram (EEG) and electromyography (EMG) electrophysiology studies in awake mice confirmed these behavioral observations were seizures. Additionally, in vitro ion chromatography studies revealed that each phosphodiester/phosphorothioate (PO/PS) in the oligonucleotide backbone has the capacity to bind divalent cations at molar equivalents. To alleviate this acute toxicity, we developed a method of buffering siRNAs with precise divalent cation concentrations in artificial CSF (aCSF) buffer prior to injection, to prevent this cation exchange and subsequent neurotoxic effects. Here, we demonstrate that adding divalent cations to the injected formulation can mitigate this type of toxicity in a dose-dependent fashion in vivo, without impacting distribution or efficacy. This new formulation enables the safe delivery of oligonucleotides at high doses directly into the CNS, which broadens the opportunities for treating patients with HD and other neurodegenerative diseases with these therapeutic modalities. The data presented here defines the importance of evaluating the safety of therapeutic oligonucleotides for CNS administration and defines the path toward better clinical translation.
Rachael Miller1, Joseph Paquette1, Dimas Echeverria-Moreno1, Ken Yamada1, Ellen Sapp2, Nicholas McHugh1, Brianna Bramato1, Nazomi Yamada1, Alexandra Barker1, Julia Alterman1, Christelle Anaclet3, Marian DiFiglia2, Anastasia Khvorova1, Neil Aronin1
1UMass Chan Medical School, Worcester, MA,2Mass General Institute for Neurodegenerative Disease, Charlestown, MA,3University of California Davis, Davis, CA"
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