Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

Abstract

Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

Chitosan-Mangafodipir nanoparticles designed for intranasal delivery of siRNA and DNA to brain

“…5). This Cy3-hsiRNA was designed to a) allow quantitative measurement of its levels in brain tissue over time and b) to silence the Huntington’s Disease (htt) gene [15] (See Supplementary Material describing sequence of hsiRNA). Future studies are planned to test the anti -htt mNPs-Cy3-hsiRNA in a mouse model of HD.…”

“…Hydrophobically modified Cy3-tagged siRNA (hsiRNA) directed against the huntingtin gene (htt) mRNA was prepared in the Khvorova lab using methods previously described [15] (See Supplemental material). …”

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

“…5). As a test model, we used hsiRNA HTT , a compound for which efficacy and distribution studies have been performed following direct brain administration [24]. Eight mice per group were unilaterally injected in the right hemisphere with 2 mL of PBS, a NTC (hsiRNA NTC ), or two different doses of hsiRNA targeting the huntingtin mRNA (hsiRNA HTT ).…”

“…HTT is most likely due to the sharp gradient of diffusion observed following direct injection into the striatum, accounting for the higher degree of variability observed [24]. Additionally, the reduction in Htt expression resulting from hsiRNA HTT affects the signal-to-noise ratio.…”

“…hsiRNAs are asymmetric compounds consisting of a 20-nucleotide antisense stand and a 15-nucleotide sense strand carrying a 3¢-cholesterol bioconjugate and several 2¢-O-methyl or 2¢-fluoro sugar modifications. In addition, the 5¢ and 3¢-ends of these strands are modified with phosphorothioate backbone substitutions to promote exonuclease resistance [24,25].…”