Huntingtin’s disease (HD) is a neurologic disease that results in impaired cognitive and behavioral functions, which lead to decreased life expectancy. This autosomal dominant disorder is characterized by an expanded CAG-repeat in the huntingtin (HTT) gene that results in an expanded polyglutamine tract in the HTT protein, which is believed to cause toxicity in neurons. Currently, there are no cures or treatments to slow the progression of the disease, but recent advances in oligonucleotide (ON) based approaches show great promise.
Until now, ON-based strategies for potential HD treatment have fallen into three categories: i) non-allele selective silencing of both the mutant (mu) or wildtype (wt) HTT using RNA-targeting ONs, ii) allele selective silencing of muHTT by targeting the expanded CAG repeat with ONs that act via steric block or miRNA pathways, and iii) allele selective silencing of muHTT by directing antisense oligonucleotides (ASOs) or siRNA to SNPs associated with the CAG repeats. Concerns over the potential effect of decreased wtHTT expression, high drug doses and pharmacokinetic challenges, have prompted Isis Pharmaceuticals to take an alternative approach.
In a recently published “Top 5%” Nucleic Acid Research article (DOI:10.1093/nar/gkt1725), Michael Østergaard and coworkers describe a new strategy for allele selective inhibition of muHTT expression, which is based on a detailed understanding of human RNase H enzymology. Gapmer ASOs are targeted toward a SNP site in an intronic region of HTT RNA. The resulting heteroduplexes are fully complementary when muHTT is targeted, but contain a central GT wobble base pair when wtHTT is targeted. The presence of the GT wobble influences the cleavage pattern of RNase H, leading to preferential cleavage of the muHTT RNA. Through clever optimization of the lead gapmer – using different probe architectures and chemically modified nucleotides – the Isis team successfully developed constructs that allowed for efficient reduction of muHTT RNA in HD human fibroblast cells (IC50 = 0.15 µM), with no reduction of wtHTT RNA at the highest evaluated dose (>133-fold selectivity).
Remarkably, the enhancement in allele selectivity was also observed in a transgenic mouse model of HD following a single intracerebral-ventricular bolus injection, both at the RNA and protein level (ED50 = 110 mg) throughout the mouse brain with no apparent toxicity even after prolonged treatment.
Isis Pharmaceuticals has partnered with Roche to pursue development of ASOs for treatment of HD, whereby it will gain access to Roche’s delivery platform (“brain shuttle”). Implications of allele selective ASO technology extend far beyond HD and can be envisioned as an approach to treat any autosomal dominant disease.
Patrick J. Hrdlicka
Co. Author – Dale C. Guenther is currently pursuing her Ph.D-degree under the guidance of Prof. Hrdlicka working on biophysical and biological characterization of DNA-targeting Invader probes.