Antisense oligonucleotide therapy for spinocerebellar ataxia type 2

SCA2 is caused by DNA CAG repeat expansion leading to an increase in the polyglutamine (polyQ) domain in the N-terminal part of the ATXN2 protein. Although the disease was initially described as a cerebellar degeneration, it is now known to involve multiple neuronal subtypes outside the cerebellum including presentation as amyotrophic lateral sclerosis (ALS) or Parkinson disease^5,6. Neurodegenerative diseases caused by dominant mutations in human genes have been difficult to approach therapeutically, and few studies have targeted disease genes directly as the first step in pathogenesis (discussed below). Developing therapeutics for SCAs can be complicated by the occurrence of repeat-associated non-AUG translation (RAN) translation, which appears to be irrelevant for SCA2⁷. Although targeting the mutation itself appears attractive, this approach has proven to be technically difficult in polyQ diseases. We have used SCA2 as a model to develop an approach to use ASOs to directly target ATXN2 mRNA.

ASOs have undergone significant recent development². Modification of DNA backbone chemistry has reduced toxicity, increased target engagement, and improved destruction of DNA-RNA hybrids. We designed multiple chimeric 2’-O-methoxyethyl (MOE)/DNA gapmer ASOs targeting ATXN2 for an in vitro screen and screened for ATXN2 RNA reduction in a cell based assay, identifying several ASOs that reduced ATXN2 expression. After testing of ASOs for in vivo tolerability, we tested one ASO in detailed behavioral, biochemical and physiological studies. This ASO targets exon 11 of human ATXN2 and is designated ASO7.

Prior studies indicated that dosage of the polyQ expanded ATXN2 is important in pathology as homozygote animals developed more severe disease manifestations⁸. On the other hand, reducing ATXN2 dosage did not affect survival or CNS morphology in heterozygous or homozygous knock-out animals⁹, suggesting ATXN2 reduction by ASOs as a viable strategy for SCA2-mediated neurodegeneration.

Our in vitro screen included 152 ASOs designed in silico for targeting human ATXN2. The top 8 lead ASOs lowered ATXN2 expression by ≥85% (Extended Data Fig. 1, Supplementary Table 1). In vivo testing revealed ASO7 and two other ASOs among the most effective for lowering ATXN2 expression after 7 d treatment (Extended Data Fig. 2a–c). To assess astroglial and microglial activation we measured cerebellar Gfap and Aif1 expression by qPCR. We tested several doses of ASO7 and observed significant reduction of ATXN2 in cerebella after 14 days treatment with 210 μg ASO7 without activation of Gfap or Aif1 (Extended Data Fig. 2a–c, and 3). Some ASOs were not further pursued owing to activation of astroglial or microglial responses in > 10 wk treatments. We verified that ASO7 as well as other lead ASOs were taken up by PCs by IHC (Extended Data Fig. 2d, e–i).

ATXN2-Q127 mice have a human ATXN2 cDNA transgene with 127 CAG repeats. We treated ATXN2-Q127 mice ICV at 8 wks of age, coinciding with the age of motor phenotype onset⁴, with ASO7 and tested motor performance on the accelerating rotarod at weeks 5, 9, and 13 after ICV injection. Consensus criteria for the conduct of preclinical trials were closely followed (see Supplementary Discussion). Progression of motor dysfunction in saline treated mice was as previously observed⁴. Mice injected with ASO7 had significantly improved motor performance compared to saline-treated control mice (Fig. 1a), and human ATXN2 and mouse Atxn2 mRNA expression was reduced by 75% and 20%, respectively, after motor phenotype testing demonstrating target engagement (Fig. 2a–c).

In preclinical studies, it is important to test compounds in multiple models of the human disease. We therefore developed a mouse model that replicated the human disease more closely by expressing a bacterial-artificial-chromosome (BAC) transgene incorporating the human promoter as well as all exons and introns and flanking regions. The BAC resulted in expression of ATXN2 with 72 glutamines in multiple neuronal tissues including spinal cord³. The BAC-Q72 model approximates human disease not only with regard to disease manifestations and less dramatic progression, but also because it contains the entire human gene in its proper intronic-exonic context. We conducted an additional preclinical trial in this mouse model with treatment initiated at 8 wks of age, as for the experiment using ATXN2-Q127 mice. Again, the mutated human ATXN2 and mouse Atxn2 were engaged by ASO7 with mRNA levels reduced by 60% and 26%, respectively (Fig. 2d), and human ATXN2 was not detected in cerebellar lysates from treated mice by Western blotting (Fig. 2e, f). Ten weeks after ICV injection, significant improvement in motor behavior compared to saline-ICV injected mice was seen (Fig. 1b). However, the rotarod performance of the ASO7 treated mice did not perfectly mirror that of wildtype mice. This might indicate that additional treatment time would be necessary to more completely restore cerebellar function. Rotarod results observed were not impacted by different mouse weights between the experimental groups (Extended Data Fig. 4).

Previously, we established transcriptome and protein profiles characterized by progressive changes in the course of degeneration in both mouse models³. To evaluate the effect of ASO7 on steady-state mRNA and protein levels we chose the top 6 mRNAs that showed the most significant changes in both mouse models³. Given that the ATXN2-Q127 model expresses the transgene specifically in PCs, we investigated genes significantly altered by SCA2 progression that are highly and specifically expressed in PCs: Rgs8, Pcp2, Pcp4, Cep76, Homer3 and Fam107b³. RGS proteins inhibit mGlur1¹⁰, which is a positive regulator of Ca²⁺ release from internal stores¹¹. Because mGlur1 signaling is disrupted in SCA2 PCs leading to abnormal Ca²⁺ release, mGlur1 regulation may also be a function of Rgs8^3,12. mGlur1 is also regulated in PCs by Homer3¹³. Pcp2 is a regulator of G(i/o)-coupled receptors including the P-type Ca²⁺ channel, important for sensorimotor control¹⁴. Pcp4 loss causes abnormal PC parallel fiber synapses¹⁵, and Cep76 is required for normal centriole function¹⁶. Fam107b and Rgs8 were identified as cerebellar hub genes with reduced expression in SCA1 mice¹⁷. Although significant increases at the mRNA level occurred for half of the genes after ASO7 treatment, we observed restored protein expression for all six of the genes on Western blots (Fig. 2, uncropped Western blots in Supplemental Fig. 1). This observation is consistent with an emerging role of polyglutamine expanded ATXN2 in translational dysregulation^3,18.

PCs are spontaneously firing neurons with a resting firing frequency of 40–50 Hz in mammals. The circuits underlying cerebellar learning and functioning are well understood^12,19,20 with PCs integrating signals received from parallel and climbing fibers and providing the only output from cerebellar cortex to deep cerebellar nuclei (DCN). In acute slice preparations, we had previously shown that mutant ATXN2 caused a slowing of PC firing. This change had an onset and progression that closely mirrored that of motor deficits with onset at 6–8 weeks and an eventual slowing to 20 Hz at 24 weeks of age⁴. It has been suggested that the change in PC physiology is tightly linked to Ca²⁺ homeostasis and subsequent cell death^12,21. This tight linkage between behavior and physiology led us to hypothesize that ASO treatment would restore normal PC firing. We injected ATXN2-Q127 mice at 8 wks and analyzed PC physiology 6 and 14 wks later in acute cerebellar slices. Similarly, we injected BAC-Q72 mice at 30 wks, >12 weeks after motor phenotype onset, and analyzed PC firing 10 wks later. In both models, a single injection of ASO7 resulted in near complete restoration of normal PC firing frequency (Figs. 3–4). Mice in these experiments were subsets of those in Figs. 1a and b with established rotarod phenotypes. These results suggest that ATXN2 ASO treatment directly affects PC physiology and at least in part explains the amelioration of motor behavior.

SCA2 and other polyglutamine diseases are characterized by toxic gains of function, suggesting that therapeutics that lower expression of the disease causing genes would be beneficial. ASOs represent an ideal approach for directly lowering ATXN2 expression because ASOs are sequence-specific, well tolerated, can reach CNS targets by intrathecal delivery. Feasibility is further supported by an early-phase human clinical trial to develop an ASO therapy for SOD1-ALS²², and most recently by the FDA approval of the ASO drug nusinersin for spinal muscular atrophy (SMA). Various approaches to ASO therapeutics for polyglutamine diseases have been developed including targeting wildtype and mutant alleles as in this study or attempting to target mutant alleles via the expanded CAG repeat or via SNPs in linkage disequilibrium with the mutation^23,24. DCN injected adeno-associated viruses (AAVs) targeting shRNAs to PCs were also effective for SCA1, reducing target expression and improving motor and cerebellar phenotypes in SCA1 transgenic mice^25,26. At least for SCA2 and with the caveats of preclinical mouse studies, the strategy of targeting both mutant and WT alleles is showing promise. One potential advantage of ASOs over current viral vectors is that they distribute through the nervous system whereas adenoviral vectors are primarily transported transynaptically (also, gene transfer comes with more complex safety concerns). Thus for SCA2, AAVs injected into DCN neurons would reach PCs, but may not reach other neuronal groups.

In conclusion, we rigorously screened ASOs to identify a potent and well tolerated ASO, ASO7, that lowered cerebellar ATXN2 expression, and verified in two SCA2 models that ASO7 delayed SCA2 motor and electrophysiological phenotypes in replicate experiments. Cerebellar degenerative diseases provide excellent model systems to study the interaction of dysfunction in a neuronal network, behavior and neurodegeneration. Our studies suggest that reduction of mutant ATXN2 effects all three domains including endophenotypic markers of cell death common in ataxias^3,17,27. Multiple lines of evidence suggest that ASOs may have applications in human patients. ASO7 lowered expression of wildtype and mutant ATXN2 in SCA2 patient-derived fibroblasts (Extended Data Fig. 5). ASO7 delivered by ICV injection localized throughout the spinal column and lowered ATXN2 expression not only in brain, but also in spinal cord (data not shown). Notably, lowering of ATXN2 by ASO treatment or genetic knockout delays dysfunction and greatly extends survival²⁸. ASO7 or ASOs with further modifications in ASO chemistry may provide an effective human drug for SCA2, ALS caused by ATXN2 mutation and other forms of ALS.