Therapy leads to long-lasting mRNA knockdown and improvements in motor function and behavior.
Scientists have developed therapeutic single-stranded antisense oligonucleotides (ASO) that they claim achieve sustained reversal of Huntington disease (HD) phenotype in rodent models and reduced huntingtin protein levels in the brains of nonhuman primates. The ASOs effectively catalyze RNase H-mediated degradation of huntingtin mRNA, preventing production of the mutant protein. Tests in the animal models showed that transient infusion of the oligonucleotides into the cerebral spinal fluid of symptomatic HD mice delayed disease progression and also led to reversal of disease phenotype that lasted much longer than the mRNA knockdown itself. Importantly, the studies showed that reduction of wild-type huntingtin protein as well as mutant huntingtin produced the same sustained reversal of disease.
The studies are reported in Neuron by Don W. Cleveland, Ph.D. and colleagues at the Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine at the University of California at San Diego, working with scientists at Genzyme, Isis Pharmaceuticals, and the Novartis Institutes for BioMedical Research. The paper is titled “Sustained Therapeutic Reversal of Huntington’s Disease by Transient Repression of Huntingtin Synthesis.”
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in exon 1 of the huntingtin gene. The majority of potential therapies currently under development are aimed at ameliorating symptoms, rather than reversing disease itself.
The ASO developed by the research team was constructed as a 20-mer phosphorothioate modified oligonucleotide complementary to human huntingtin mRNA (HuASO), which contained 2’-O-(-2-methoxy) ethyl modifications on the five nucleotides on the 3’ and 5’ ends to increase its stability, tolerability and potency.
Initial tests showed that continuous infusions of the HuASO for two weeks into the right lateral ventricle of the BACHD mouse model of HD led to significant, dose-dependent decreases in human huntingin mRNA and protein. BACHD mice harbor a full-length mutant human huntingtin gene with an expansion of 97 CAG/CAA repeats, and express the mutant protein at approximately 1.5 times the level of the endogenous mouse huntingtin. Interestingly, the researchers found that treatment led to sustained suppression of human huntingtin mRNA and protein well after HuASO infusion was stopped. In fact levels of the mRNA and protein only returned back to pretreatment levels 16 weeks after the end of the infusion period. These results indicate that “low doses of ASO in the correct cellular compartments are sufficient to be effective and are maintained with long in vivo half lives, particularly in the brain where many of the cells are nondividing,” the researchers state.
To determine whether HuASO infusion would impact on HD-like disease development the team turned initially to YAC128 mice, which express a full-length mutant human huntingtin transgene (including the human huntingtin promoter), to about 75% the level of normal mouse huntingtin. These animals develop a progressive HD phenotype with many of the characteristics of human HD, including motor deficits that start to become evident as early as two months of age.
Three month-old YAC128 mice were treated using a continuous infusion of HuASO into the right lateral ventricle for two weeks. The animals were then assessed over a three month period. As with the initial tests in BACHD animals, HuASO therapy in the YAC128 mice led to significant reductions in human huntingtin mRNA and protein, even at six weeks after treatment had been stopped. The animals also started to show improvements in motor function within a month of HuASP treatment being started, and by two months motor functions appeared restored to nontransgenic control levels .
HuASO Treatment was even effective when administered to 6-month-old YAC128 animals, which had developed a more pronounced HD phenotype. In these mice two weeks’ of HuASO infusion led to improvements in motor function to the extent that “after a 2 month lag, motor function improved and treated animals were no longer significantly different from nontransgenic animals,” the researchers note. Behavioural parameters also improved such that by 9 months of age the treated mice were nearly indistinguishable from control mice. “Thus, while mice transiently treated at this older age (6 months) never reached the improvement achieved in younger animals, therapy initiated in these more phenotypic mice provided sustained suppression of mutant huntingtin synthesis and partial reversal of disease characteristics 3 months after stopping treatment.”
An equivalent experiment was carried out to test two weeks of HuASO infusion in 6-month old BACHD animals, but this time treated mice were followed for six months. Encouragingly, significant improvements in motor performance were evident within eight weeks of treatment, and these persisted for the whole six month follow-up period, a good two months after mutant huntingtin production was back at pretreatment levels. Sustained improvements in behaviour were also evident.
The surprising longevity of therapeutic benefits led the team to repeat their experiment in another cohort of 6-month-old BACHD mice. Again, behavioural improvements including hypoactivity and anxiety, as well as motor coordination improvements were still evident at six and even nine months post treatment.
The in vivo tests thus far had used a HuASO that didn’t impact on native mouse huntingtin mRNA. To see whether ASO therapy that decreased levels of both mutant and normal huntingtin mRNA would affect therapeutic benefits, cohorts of two-month-old BACHD and nontransgenic littermates were treated for two weeks either with an infusion of HuASO, or with an ASO that reduced levels of both the human mutant huntingtin mRNA and the normal mouse huntingtin (MoHuASO) mRNA.
At two months old untreated BACHD animals have already started to display impaired motor coordination, but remarkably, administration of either the HuASO or MoHuASO led to the same degree of improvements in motor coordination that were evident three months after treatment, and were sustained for another five months. In fact both treatments led to an equivalent magnitude and duration of improvements in both motor coordination and hypoactivity, despite the fact that the MoHuASO reduced normal mouse huntingtin mRNA levels by 75%.
In a last set of murine experiments the researchers assessed ASO therapy in the R6/2 mouse model of HD that develops a rapidly progressing disease characterized by motor deficits and a dramatic loss of brain mass that is fatal within about 16 weeks. Infusion an ASO designed to target the mutant R6/2 transgene (HuASOEx1) into the right lateral ventricle of two-month old R6/2 animals for four weeks led to selective suppression of human hunting mRNA. By this stage untreated animals have already developed clear symptoms and gross loss of brain mass, but in the treated animals further brain mass loss was stopped, and lifespan significantly extended.
The researchers finally moved on to evaluate the effects of ASO administration in Rhesus monkeys. Animals received daily infusions of an ASO that is completely complementary to both Rhesus monkey and human huntingtin mRNA (mkHuASO) directly into the cerebral spinal fluid via an intrathecal catheter, for 21 days. Subsequent analyses of brain tissue confirmed that the ASO was distributed throughout most of the brain and spinal cord regions, and that treatment resulted in significant reductions in huntingtin mRNA levels, which only began to rise toward normal levels eight weeks after termination of treatment.
“Our efforts have established what we believe is now a clinically feasible, dose dependent approach for providing long-term disease mitigation and partial phenotypic reversal of Huntington’s disease, as well as establishing the utility of sustained benefit from a transient reduction of mutant huntingtin synthesis and accumulation,” the authors conclude. “For diseases like Huntington’s where a mutant protein product is tolerated for decades prior to disease onset, this finding opens up the provocative possibility that transient suppression of huntingtin can lead to a prolonged effect in patients.”