Identification and Allele-Specific Silencing of the Mutant Huntingtin Allele in Huntington's Disease Patient-Derived Fibroblasts

Abstract

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by the expression of mutant huntingtin protein (Htt). Suppression of Htt expression, using RNA interference, might be an effective therapy. However, if reduction of wild-type protein is not well tolerated in the brain, it may be necessary to suppress just the product of the mutant allele. We present a small interfering RNA (siRNA) that selectively reduces the endogenous mRNA for a heterozygous HD donor's pathogenic allele by approximately 80% by specifically targeting a single-nucleotide polymorphism (SNP) located several thousand bases downstream from the disease-causing mutation. In addition, we show selective suppression of endogenous mutant Htt protein, using this siRNA. We further present a method, using just a heterozygous patient's own mRNA, to determine which SNP variants correspond to the mutant allele. The method may be useful in any disorder in which a targeted SNP is far downstream from the pathogenic mutation. These results indicate that allele-specific treatment for Huntington's disease may be clinically feasible and practical.

Using non-coding small RNAs to develop therapies for Huntington's disease

“…Ongoing research aims to identify additional SNP sites in the HD gene that could be used for siRNA silencing directed specifically to alleles with the CAG expansion. 16,19 As the HD locus is large and both isoforms are over 10 kb, SNPs in that gene are usually distant from the CAG repeat at the 5¢-terminal. For this reason, it is difficult to identify SNPs that are linked to the harmful triplet expansion in mutated HD alleles.…”

“…A second round of PCR is conducted on the resulting cDNA, using primers spanning the CAG repeat sequence. 16 Using this method, 11 known SNP heterozygote sites in fibroblast cells from 21 different HD patients were allele-specifically determined. 16 For example, fibroblasts from one patient had the rs363125 SNP with cytosine at that position in the mutant HD allele, but adenine in its wild-type counterpart (Figure 1).…”

“…16 Using this method, 11 known SNP heterozygote sites in fibroblast cells from 21 different HD patients were allele-specifically determined. 16 For example, fibroblasts from one patient had the rs363125 SNP with cytosine at that position in the mutant HD allele, but adenine in its wild-type counterpart (Figure 1). On the basis of this information, siRNA guide strand targeting at mutant allele with rs363125 SNP can silence the mutant HD while preserving expression of the wild-type one.…”

“…On the basis of this information, siRNA guide strand targeting at mutant allele with rs363125 SNP can silence the mutant HD while preserving expression of the wild-type one. 16 Another method used the circularization method to link the SNP with CAG repeats. 19 A primer flanking the SNP with a KasI site at each end was used to amplify DNA ranging from SNP to CAG expansion.…”

“…Allele-specific silencing of the mutant species has been achieved in these cells with both endogenous wild-type and mutant HD (Figure 1). [15][16][17][18][19][20][21][22][23][24][25][26] APPLICATION OF RNAi IN HD TRANSGENIC ANIMAL MODELS To date, all published studies on non-coding RNAs therapy in HD transgenic mice have used animals expressing short fragments of human huntingtin protein (instead of the full-length huntingtin) with an expanded CAG repeat. These mice exhibit a rapidly progressing syndrome reminiscent of HD in humans.…”