PROJECT SUMMARY
Huntington’s Disease (HD) is caused by a CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (HTT)
gene that produces mutant HTT mRNA and protein. Expression of mutant HTT leads to progressive
neurodegeneration via mechanisms that are poorly understood. Because HD is a genetically-defined disease, it
is an ideal candidate for study and therapeutic intervention by small interfering RNAs (siRNAs) – which
incorporate into the RNA-induced silencing complex (RISC) to target and degrade disease-causing genes. With
the development of a divalent (di)-siRNA chemical architecture, siRNAs can be delivered throughout the central
nervous system (CNS) of rodents and non-human primates. To ensure stability in the CNS, di-siRNAs require
chemical modifications on every nucleotide. However, chemical modifications can affect siRNA activity and
cellular localization – limiting the utility and flexibility of di-siRNAs in the CNS.
The most common nucleotide modifications in siRNA replace the 2'OH of the ribose with 2'-Fluoro (2'F) or 2'-O-
Methyl (2'OMe). Recent work suggests that incorporation of 2'OMe and 2'F at certain nucleotide positions may
hinder siRNA loading into RISC or target binding/cleavage by RISC, and may alter the nuclear-to-cytoplasmic
localization of siRNAs. Yet, the limited scope of this work has made it difficult to identify general design
parameters for efficacious, compartment-specific siRNA. With guidance from Drs. Anastasia Khvorova (siRNA
chemistry), Neil Aronin (HD), Phillip Zamore (RNA biochemistry) and Athma Pai (RNA sequencing), this
proposal will systematically assess the impact of modification patterns on siRNA efficacy and cellular localization
in the CNS to optimize siRNAs as an HD therapy and research tool for dissecting HD pathology.
Aim 1 will characterize how siRNA chemical modifications impact RISC loading in vivo and target binding and
cleavage in vitro. To measure how modifications alter RISC loading, a pool of differentially-modified siRNAs will
be injected into the CNS of mice, RISC will be pulled down and loaded siRNAs will be sequenced. To determine
the effect of siRNA chemical modifications on RISC-target interactions, target binding and cleavage kinetics will
be measured for a panel of modified siRNAs using single-molecule total internal reflection fluorescence
microscopy. Mechanistic insight into how modifications impact siRNA efficacy in the CNS will provide a
framework with which to design optimized siRNAs to treat HD and other CNS diseases. Aim 2 will use the same
modified siRNA pool from Aim 1 to identify optimal modification patterns for enhanced nuclear localization of
siRNA in the CNS. These data will be used to design efficacious chemically-modified di-siRNAs targeting nuclear
or cytoplasmic-only HTT RNA. These di-siRNA will then be injected into YAC128 HD mice and the effect on
motor deficits, neurodegeneration, and striatal mRNA expression will be assessed. These results will provide
valuable insight into the biology of HD and determine the potential of nuclear RNA-targeting siRNAs as a
therapeutic paradigm for repeat expansion disorders with underlying RNA toxicity.
