Abstract

Expanding the Chemical Diversity of Therapeutic Oligonucleotides

Anastasia Khvorova, Professor, RNA Therapeutics Institute, University of Massachusetts Medical School

RNA interference has revolutionized human functional genomics and therapeutics, but its impact on neuroscience research has been limited by the lack of simple and efficient methods to deliver oligonucleotides to primary neurons and the brain. We show that primary neurons rapidly internalize fully stabilized, hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium and that hsiRNAs induce potent and long-lasting silencing in vitro. A single injection of unformulated hsiRNA (cholesterol-conjugated) into mouse brain silences locally with great potency and longevity. Limited distribution from the site of administration precludes direct use of this type of chemistry for modulation of gene expression in larger brains and potential therapeutic development. Using fully chemically modified siRNA scaffolds, we systematically screened a wide range of bioactive conjugates and demonstrated that the chemical nature of the conjugation modality has a major impact on brain tissue retention, distribution and cellular internalization. We have identified several novel chemical classes of conjugates that demonstrate markedly improved brain distribution and robust in vivo efficacy. Direct conjugation of a fully chemically modified siRNA to docosahexaenoic acid (DHA), the most abundant poly-unsaturated fatty acid in the brain, results in improved tissue retention with wide distribution and robust efficacy in the striatum and cortex after single injection. Most importantly, DHA-hsiRNA conjugates do not induce neural cell death or measurable innate immune activation following administration of concentrations 20-fold over the efficacious dose, establishing a new approach toward development of RNAi-based therapeutics for a wide range of neurodegenerative disorders.