Atrophin-1 antisense oligonucleotide provides robust protection from pathology in a fully humanized DRPLA model

Velvet L Smith; Bereket Z Gidi; Robert M Bragg; Jeffrey P Cantle; Aliza Ben-Varon; Briana Noble; Silvia Prades; Andrea Compton; Julie Greenfield; Joanna A Korecka; Anya Gemos; Timothy Yu; Vikram Khurana; Holly B Kordasiewicz; Hien T Zhao; Melissa Barker-Haliski; Daniel D Child; Jeffrey B Carroll

doi:10.1016/j.omtn.2025.102815

. 2025 Dec 31;37(1):102815. doi: 10.1016/j.omtn.2025.102815

Atrophin-1 antisense oligonucleotide provides robust protection from pathology in a fully humanized DRPLA model

Velvet L Smith ¹, Bereket Z Gidi ¹, Robert M Bragg ¹, Jeffrey P Cantle ¹, Aliza Ben-Varon ², Briana Noble ³, Silvia Prades ^4,⁵, Andrea Compton ^4,⁵, Julie Greenfield ^4,⁵, Joanna A Korecka ⁸, Anya Gemos ⁸, Timothy Yu ^10,^11,^12,¹³, Vikram Khurana ^8,⁹, Holly B Kordasiewicz ³, Hien T Zhao ³, Melissa Barker-Haliski ⁷, Daniel D Child ^6,^∗, Jeffrey B Carroll ^1,^4,^6,^∗∗

PMCID: PMC12857545 PMID: 41624332

Abstract

Dentatorubral-pallidoluysian atrophy (DRPLA) is a fatal neurodegenerative disease arising from a CAG repeat expansion in the atrophin-1 (ATN1) gene. Because DRPLA, like many repeat expansion disorders (REDs), arises predominantly from toxic gain-of-function mechanisms, we hypothesized that ATN1 knockdown would have therapeutic potential. To test this, we established the first fully humanized mouse model of a RED, in which one allele of mouse Atn1 is completely replaced by human ATN1, including 112 pure CAG repeats. This novel approach to exploring RED biology provides significant advantages, notably the ability to test sequence-specific therapeutics targeting human sequences, even in introns and untranslated regions of pre-mRNA. We found that our model—the Atn1^Q112/+ mouse—recapitulates key features of human DRPLA, including behavioral alterations, reduced brain size, and aggregate accumulation. We treated Atn1^Q112/+ mice with antisense oligonucleotides (ASOs) targeting mouse Atn1 (to probe for loss of function concerns), human ATN1, or a combination. Treatment with human, but not mouse, ATN1-targeting ASOs provides remarkable protection from a range of disease-related behavioral phenotypes and marked rescue of transcriptional dysregulation in the cerebellum. These results have helped motivate an ongoing human clinical study of ASOs targeting ATN1 for DRPLA.

Keywords: MT: Oligonucleotides: Therapies and Applications, dentatorubral-pallidoluysian atrophy, DRPLA, antisense oligonucleotides, ASOs, atrophin-1, polyglutamine disease, CAG repeat, neurodegeneration, humanized mouse model, mouse models

Graphical abstract

The results demonstrate that knockdown of human ATN1, with antisense oligonucleotides, provides therapeutic benefits in a humanized DRPLA mouse model. These results have helped motivate an ongoing human clinical study of ASOs targeting ATN1 for DRPLA.

Introduction

Dentatorubral-pallidoluysian atrophy (DRPLA) is an invariably fatal neurodegenerative disease caused by expansion of the CAG tract in the atrophin-1 (ATN1) gene.¹^,² In Japan, DRPLA’s prevalence (0.2–0.7/100,000 people)³^,⁴ is comparable with that of Huntington's disease (HD; 0.4–0.5/100,000),³^,⁵^,⁶ the most common repeat expansion disorder (RED) in North America and Europe (HD prevalence in North America is 8.9/100,000).³^,⁴^,⁷ Conversely, DRPLA is vanishingly rare in Western Europe and North America, with several dozen patients described in the literature and ascertained in an ongoing natural history study.⁸^,⁹ The core symptoms of DRPLA are diverse and include ataxia, cognitive decline, myoclonus, chorea, epilepsy, and psychiatric manifestations.¹⁰^,¹¹ Like other REDs, DRPLA shows significant anticipation in the age of onset over generations,¹^,¹⁰^,¹² somatic CAG repeat length instability in the aging brain,¹³ and intranuclear and cytoplasmic aggregates containing the expanded protein.¹⁴ Thus, while DRPLA’s symptoms present a unique challenge compared with other REDs, its underlying mechanisms—and therefore potential approaches to therapy—may be common.

A key challenge in therapeutic development for neurodegenerative diseases is failure of drugs with promising preclinical results to translate into successful human clinical studies.¹⁵^,¹⁶ Inaccurate disease modeling in mice bears some of the culpability, as mouse models of neurodegeneration often insufficiently replicate key features of disease biology, even with purely genetic forms of neurodegeneration such as DRPLA and HD. Ongoing efforts to improve this situation have seen the steady evolution of mouse models, from transgenic to knock-in mice with humanized mutations in the mouse orthologous gene, which has enabled important disease insights in Alzheimer disease (AD),¹⁷^,¹⁸ Parkinson disease,¹⁹^,²⁰^,²¹ HD,²²^,²³^,²⁴ and several polyglutamine spinocerebellar ataxias (SCAs).²⁵^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰ In preclinical studies of AD-associated genetic variants, the need for full genomic contexts has been increasingly recognized, leading to a fully humanized mouse model of Tau.³¹ More recently, the ambitious gene replacement-Alzheimer disease project is generating many humanized mice harboring AD risk genes.³² These mouse models have replaced one of the endogenous murine alleles with the orthologous, full-length human gene, leading to expression of the human gene product in the setting of endogenous murine expression levels and regulatory elements. Full humanization offers clear benefits for oligonucleotide therapeutics, as all potential sequence targets—even those in introns and untranslated regions (UTRs)—can be studied in the mouse context. Furthermore, humanized mice enable targeting phenomena such as alternative splicing and the inclusion of cryptic exons, which often arise from intronic sequences.

Here, we describe the establishment of a novel, fully humanized, DRPLA mouse model (the first fully humanized model of a RED) and the development and preclinical efficacy studies of ATN1-targeting antisense oligonucleotides (ASOs). This work was undertaken as part of a large consortium focused on improving the lives of patients with DRPLA, with several authors (J.G., S.P., J.B.C.) playing roles as the scientific advisors of CureDRPLA, a non-profit advocacy organization focused on advancing treatments for DRPLA. Since launching in 2019, CureDRPLA has funded an array of tools facilitating work in DRPLA, including induced pluripotent stem cell lines, a global natural history study, and an online patient registry.⁹^,³³ These mice, Atn1^Q112/+, are an exciting advancement from existing models of DRPLA³⁴^,³⁵^,³⁶ in that they express the entire human ATN1 gene, allowing ASO targeting of any sequence within the transcript, from 5′ UTR to the 3′ UTR. Another advantage is the ATN1 dosage, as all existing DRPLA models rely on transgenic overexpression of some form of mutant ATN1 (mATN1); thus, our model is the first that allows careful analysis of ATN1 lowering in a genetic context recapitulating individuals with DRPLA.

We hypothesized that reduction of mATN1 is sufficient to alleviate the DRPLA disease phenotype, and we tested this using ASOs in our humanized DRPLA mouse model. Recently, a distinct clinical syndrome associated with missense mutations in ATN1, CHEDDA, has been described (congenital hypotonia, epilepsy, developmental delay, and digital anomalies).³⁷ While CHEDDA likely arises from a distinct, toxic, ATN1 gain of function, the awareness of two diseases arising from ATN1 mutations rendered us sensitive to the potential risks of ATN1 lowering. Thus, we designed ASOs for both human and mouse ATN1 to target each allele individually. We found that treatment with human ATN1 ASO lowers mATN1 and provides remarkably robust protection from DRPLA-relevant signs, including behavior, neuropathology, and transcriptional dysregulation. Similar rescue is not seen in Atn1^Q112/+ mice after silencing WT (wild-type) mouse Atn1, nor are disease-relevant phenotypes evidently exacerbated by treatment with the mouse Atn1 ASO. We believe that our results provide a template for other ultra-rare patient advocacy organizations, particularly those focused on neurological conditions amenable to ASO therapy, as these results supported our decision to pursue ASO therapy for DRPLA patients in clinical studies, which are underway and will be described separately.

Results

Generation of Atn1^Q112/+ mice

To facilitate ASO screening and for maximal construct validity, we generated a mouse in which one endogenous mouse Atn1 locus was replaced with the human ATN1 sequence, from 5′ UTR to 3′ UTR, with expanded sequence-verified pure 112 CAG repeats in exon 5 (Figure 1). Details of the generation and validation of the Atn1^Q112/+ allele are outlined in Figure S1, and in the materials and methods.

Extent of humanization in the *Atn1*^Q112/+ mouse model and location of the ASO target

Schematic of the humanized *Atn1* locus at mouse chromosome 6 with the upstream (*Grcc10*) and downstream (*Eno2*) RefSeq curated genes indicated. The donor human allele included 112 pure, sequence-verified, CAG repeats, indicated as a red bar. The bright blue bar indicates the extent of humanization and CAG repeats.

Development of ATN1 allele-specific ASOs

We developed two ASO compounds—one targeting mouse Atn1 and one targeting human ATN1—by screening ASOs against human or mouse ATN1 pre-mRNA sequence in cell culture and in vivo (using humanized ATN1 knock-in mice without CAG expansion and WT C57BL/6NTac mice). The primary outcome for our screen was ATN1 or Atn1 transcript levels, as quantified by quantitative real-time PCR (qRT-PCR) assays for each screen. Potential lead ASOs were then screened in vivo for their acute and chronic tolerability, as well as their potency and selectivity (Figure S2). We then characterized ASO candidates in a humanized ATN1 line with non-expanded CAG repeat lengths, enabling us to characterize them in vivo without the challenges posed by the severe phenotypes seen in the Atn1^Q112/+ mice. Our chosen mouse- and human-ATN1 ASOs resulted in robust reductions in Atn1 and ATN1 levels, respectively, at 2 and 8 weeks post-intracerebroventricular (ICV) injection as determined by allele-selective qRT-PCR (Figures S3B and S4B). Neither led to any induction of astro- or microglial transcripts or induced any abnormal behaviors when injected in WT or humanized ATN1 mice after ICV injection of ASO (Figures S3C and S4C). We refer to these selected ASOs as mouse ASO (mASO) or human ASO (hASO) throughout this manuscript.

Initial observations and pilot experiment

An initial cohort of Atn1^Q112/+ mice (N = 90) was bred at Taconic and shipped at 8 weeks of age for a pilot study, but we found that many mice died in shipment, and the surviving mice were sufficiently ill that they needed to be sacrificed on arrival. This suggests that Atn1^Q112/+ mice are sensitive to environmental stress by 8 weeks of age. To proceed with our pilot, we conducted in vitro fertilization (IVF) with sperm from Atn1^Q112/+ founders, resulting in pregnant dams, which were shipped to give birth at our facility. The goal of the pilot study was to qualitatively observe the phenotypes of the mice born locally, while testing hASO potency at several time points. Mice were split into three treatment groups: early (postnatal day 1–3), late (7-weeks of age), or early + late (postnatal day 1–3 and 7-weeks of age) (Figure S5A). Within each group, mice were treated with saline or hASO via ICV injection or left untreated as controls (Figure S5A). Early time point treated mice received 50 μg of hASO while mice treated at the late time point received 300 μg of hASO. Mice treated at the early + late time point received both (Figure S5A). We qualitatively observed that behavioral phenotypes in these locally born Atn1^Q112/+ mice progressed rapidly, including incoordination, gait disturbances, and tremors, particularly in response to sudden noise or movement. The mice were sufficiently uncoordinated in that they struggled to eat and required euthanasia by 10 weeks of age. At sacrifice, we examined human ATN1 levels with allele-selective qRT-PCR and confirmed reduction by 75% in the midbrain of Atn1^Q112/+ mice treated with hASO at the early + late time point (p = .002, Figure S5B). We attempted to breed 6 male Atn1^Q112/+ mice with WT females locally with no resulting pups, suggesting that the fertility of Atn1^Q112/+ mice is significantly impaired, as has been observed in other transgenic DRPLA mouse models with very long CAG repeats.³⁵ Empowered with these pilot study results, we used IVF to generate a large cohort of Atn1^Q112/+ mice for a longitudinal efficacy study (Table S1).

Longitudinal study overview

Our human and mouse allele-selective ASOs enabled us to execute a longitudinal characterization of the impact of ATN1 lowering on emergent behavioral phenotypes in our Atn1^Q112/+ mice (Figures 2A and 2B). Twenty pregnant female mice were generated at Taconic using IVF and shipped to our animal facility at the University of Washington. The dams gave birth approximately five days after their arrival, resulting in a large cohort of well-matched Atn1^Q112/+ and WT littermate mice (Tables S2 and S3). Thanks to our pilot study observations (Figure S5), we developed a two-stage ASO treatment strategy based on a published approach in a rapidly developing seizure disorder model.³⁸ All mice in our study received an initial ICV injection at postnatal day 1–3 of life of their allotted treatment (saline, mASO, hASO, or Combo) that delivered 30 μg of ASO (the Combo group received 15 μg of each ASO for a total of 30 μg; Figures 2A and 2B). At 5 weeks of age, all mice received a second ICV injection containing saline or 200 μg of ASO (the Combo group received 100 μg of each ASO for a total of 200 μg; Figures 2A and 2B). We then conducted exhaustive behavioral examinations of the mice from 6 to 8 weeks of age before sacrifice at approximately 63 days of age (range = 58–66). While this study was not powered for a formal survival analysis, we note that, of the 30 Atn1^Q112/+ mice treated with saline or mASO, 10 (33%) died before their planned endpoint. However, of the 27 Atn1^Q112/+ mice treated with hASO (alone or in combination), only 2 died prior to their planned endpoint (7%, Fisher exact test p = .02) (Table S4). At sacrifice, we examined human ATN1 and mouse Atn1 levels in the entire cohort with allele-selective qRT-PCR. In the Atn1^Q112/+ mice, we found hASO induced reduction of human ATN1 by 65% in cortex and 49% in cerebellum. We found mASO induced reduction of mouse Atn1 by 32% in the cortex and 16% in the cerebellum. Additionally, we found no cross-reactive lowering of non-targeted ATN1/Atn1 alleles by the ASOs (Figures 2C and S6).

Trial overview and *Atn1/ATN1* lowering achieved in *Atn1*^Q112/+ mice

(A) Schematic overview of the study design, indicating dates for ASO treatment, behavioral assessments, and sacrifice/tissue collection in weeks. (B) Schematic of the different ASO treatments—the mouse ASO (mASO) only targets mouse *Atn1* and the human ASO (hASO) only targets human *ATN1*. The combination (Combo) of mASO and hASO affects both. (C) Allele selective mouse and human *Atn1/ATN1* primers were used in qRT-PCR to quantify levels of *Atn1/ATN1* in cortex in all mice from the study outlined in A. In the left plot (n = 103), WT *Atn1* in saline- and mASO-treated *Atn1*^Q112/+ mice is significantly reduced compared with controls (WT mice treatment effect [WT Tx] – F_(3,36) = 24.8, p = 6.9^-⁹; *Atn1*^Q112/+ mice treatment effect [*Atn1*^Q112/+ Tx] – F_(3,36) = 4.8, p = 6.6^-³; select Tukey post hoc comparison p-values – WT mASO:saline p = 3.8^-⁸, combo:saline p = 3.9^-⁶, and *Atn1*^Q112/+ mASO:saline p = .03). In the right plot (n = 80), treatment with hASO led to robust reduction in human *ATN1* in *Atn1*^Q112/+ mice when compared with saline-treated *Atn1*^Q112/+ controls (*Atn1*^Q112/+ treatment effect [*Atn1*^Q112/+ Tx] – F_(3,35) = 66.1, p = 1.7^-¹⁴; select Tukey post hoc comparison p-values – *Atn1*^Q112/+ hASO:saline p = 4.0^-¹², combo:saline p = 4.0^-¹⁰).

Human ASO rescues behavioral abnormalities

We weighed mice weekly throughout the study and detected a complex relationship between sex, genotype, and treatment. In brief, saline-treated Atn1^Q112/+ mice were not significantly lighter than sex-matched WT mice (Figure S7). We conducted a compressed assessment of behavior in the entire cohort of mice from 6 to 8 weeks of age. Motor coordination assessed with a fixed-speed rotarod task at 4 and 8 rpm and tapered balance beam task all revealed profound deficits in saline- and mASO-treated Atn1^Q112/+ mice (Figures 3A and 3B). These motor deficits were robustly rescued in Atn1^Q112/+ mice treated with hASO. We used an open field arena to measure activity levels and found that saline-treated Atn1^Q112/+ mice were significantly hypoactive compared with controls, with a striking number of freezing episodes (>200% increase in freezing episodes; Figures 4A, 4C, and S9D). This hypoactive behavior was ameliorated in mice treated with hASO. We also found that Atn1^Q112/+ mice treated with hASO traveled a greater distance during open field testing compared with Atn1^Q112/+ mice treated with saline (Figures 3C, 4B, and 4C). Next, we conducted a modified SHIRPA,³⁹^,⁴⁰ which is a behavioral screening assessment designed to broadly characterize the neurological health of mice (Table S5). This evaluation revealed profound changes in lethargy, tremors, gait, pelvic elevation, and touch escape in saline- and mASO-treated Atn1^Q112/+ mice—all of which were markedly rescued by treatment with hASO (Figures 3D, 3E, S8, and S9A–S9C). We saw similar improvements in circadian behavior in a subset of Atn1^Q112/+ mice treated with hASO compared with saline examined over 67 h (Figure S10). Overall, treatment with the hASO, but not mASO, led to robust protection from deficits in the rotarod (Figure 3A), balance beam (Figure 3B), open field assays (Figures 3C, 4, and S9D), and SHIRPA (Figures 3D and 3E), while partially normalizing circadian rhythms (Figure S10).

Human, but not mouse, *ATN1* ASO treatment improves behavioral deficits in the *Atn1*^Q112/+ mice

(A) Average time to fall in a fixed rotarod test with the average of three testing trials plotted in seconds at 4 rpm (left, n = 101) and 8 rpm (right, n = 99). *Atn1*^Q112/+ mice treated with mASO and saline fell off the rod significantly earlier than WT mice at both speeds, which is rescued by treatment with hASO, alone or in combination (4 rpm: genotype effect [GT] – F_(1,93) = 34.9, p = 5.7⁸, treatment effect [Tx] – F_(3,93) = 5.6, p = .001, interaction effect [GT∗Tx] – F_(3,93) = 9.8, p = 1.12^-⁵; 8 rpm: genotype effect – F_(1,91) = 39.0, p = 1.32^-⁸, treatment effect – F_(3,91) = 2.1, p = .11, interaction effect – F_(3,93) = 12.8, p = 4.6^-⁷, 4 rpm select Tukey post hoc comparison p-values – *Atn1*^Q112/+ saline:WT saline p = 1.21^-⁶, *Atn1*^Q112/+ saline:hASO p = 3.25^-⁴, saline:combo p = 4.86^-⁶, 8 rpm select Tukey post hoc comparison p-values – *Atn1*^Q112/+ saline:WT saline p = 2.67^-⁷*, Atn1*^Q112/+ saline:hASO p = 1.63^-⁴, saline:combo p = .001). (B) Latency to cross tapered balance beam testing with the average of three testing trials plotted in seconds (n = 93). Saline- and mASO-treated *Atn1*^Q112/+ mice are slow to cross an elevated balance beam, which is rescued by treatment with hASO (genotype effect – F_(1,85) = 37.3, p = 2.3^-⁸, treatment effect – F_(3,85) = 11.7, p = 1.7^-⁶, interaction effect – F_(3,85) = 12.9, p = 4.9^-⁷, select Tukey post hoc comparison p-values – WT saline:*Atn1*^Q112/+ saline p = 3.51^-⁴, *Atn1*^Q112/+ mASO:WT saline p = 5.62^-⁶, *Atn1*^Q112/+ saline:hASO p = 8.48^-⁴, saline:combo p = 6.97^-⁷). (C) Total distance traveled in an open field assessment, measured in centimeters (cm; n = 70). *Atn1*^Q112/+ mice treated with saline are hypoactive compared with WT, which is reversed by hASO treatment (genotype effect – F_(1,66) = 12.2, p = 8.4^-⁴, treatment effect – F_(1,66) = 66.6, p = 1.4^-¹¹, interaction effect – F_(1,66) = 15.5, p = 2.0^-⁴, select Tukey post hoc comparison p-values – *Atn1*^Q112/+ saline:WT saline p = 8^-⁷, *Atn1*^Q112/+ saline:hASO p < .00001. (D) Bar plot displaying mean score from SHIRPA observational testing of tremors (n = 103). Error bars indicate standard error. Tremors were significantly elevated in mASO and saline-treated *Atn1*^Q112/+ mice when compared with WT mice, and scores improved with hASO treatment (GLM ANOVA results: genotype effect – X² (1) = 13.4, p = 2.6^-⁴; treatment effect – X² (3) = 8.2 p = 4.1^-²; interaction effect – X² (3) = 14.1, p = 2.7^-³). (E) Bar plot displaying mean score from all SHIRPA observation testing (total score; n = 103). Remaining endpoints not shown here are in Figures S7 and S8C. Total score was calculated by taking the mean of the summed score of all observations. Error bars indicate standard error. The total score of all of the SHIRPA tests were significantly worsened in mASO and saline-treated *Atn1*^Q112/+ mice when compared with WT mice; however, scores improved with hASO treatment (GLM ANOVA results: genotype effect – X² (1) = 101, p = 9.05^-²⁴; treatment effect – X² (3) = 68.9 p = 7.13^-¹⁵; interaction effect – X² (3) = 42.7, p = 2.7^-⁹, select estimated marginal means post hoc comparison p-values – *Atn1*^Q112/+ saline-WT saline p = 1.43^-⁷, *Atn1*^Q112/+ mASO-WT saline p = 2.64^-¹²*, Atn1*^Q112/+ saline-hASO p = 1.28^-⁶, *Atn1*^Q112/+ saline-combo p = 6.82^-⁶).

Human *ATN1* ASO treatment rescues locomotor deficits in the *Atn1*^Q112/+ mice

(A) Representative line graphs of motion over 9 min of open field testing (n = 1 per plot). Freezing is indicated in yellow while movement is in blue. The WT saline-treated mouse (top) displayed a high degree of movement along the full-time course, whereas the saline-treated *Atn1*^Q112/+ mouse (middle) had more freezing episodes. The *Atn1*^Q112/+ mouse treated with hASO displayed a very similar freezing pattern to the WT saline-treated mouse. These data are quantified in Figure S9D for all mice tested. (B) Representative motion traces from 9 min of open field testing (n = 1 per image). The *Atn1*^Q112/+ hASO-treated mouse (bottom) traveled a larger distance than the saline-treated *Atn1*^Q112/+ mouse (middle) and is comparable with the WT saline-treated mouse. These data are quantified in Figure 3C. (C) Representative heatmap displaying the presence of mice in 9 min of open field testing (n = 1 per image). Mouse presence in a certain area of the box increases as color moves from blue to red. The WT saline-treated mouse (left) was present in many different locations within the box, whereas the *Atn1*^Q112/+ saline-treated mouse (middle) spent most of its time in one corner of the box. The *Atn1*^Q112/+ hASO-treated mouse (right) followed a similar pattern to the WT saline-treated mouse.

Human ASO rescues brain weight and aggregate accumulation

We collected terminal plasma (via cardiac puncture), several peripheral organs, and the brains of each animal in the study. We weighed the whole brain and observed a decrease in saline-treated Atn1^Q112/+ mice compared with WT littermates (Figure 5A), which was not observed when normalized to their later in life body weight (Figures 5B and S10). Neurofilament light (NEFL) is an axonal protein whose plasma levels are routinely used as a biomarker of ongoing neural damage.⁴¹^,⁴²^,⁴³ Surprisingly, given the severity of their neurological phenotypes, NEFL levels are normal in our Atn1^Q112/+ mice, and we did not observe any effect of ASO treatment (Figure S11). To examine the impact of ASOs on neuropathological phenotypes, we prepared parasagittal brain sections at the level of the mid-olfactory bulb from 4 to 5 mice per treatment group. These sections were stained with either hematoxylin and eosin (H&E) or by immunohistochemistry for GFAP, IBA1, NeuN, p62, and 1C2. A board-certified neuropathologist (D.D.C.) blindly examined all sections. Consistent with our NEFL observations, there were no signs of neuron loss noted in Atn1^Q112/+ mice or changes in neuron density or distribution (Figure S12A), microvacuolization (Figure S12B), reactive gliosis (Figure S13A), or microglial nodules (Figure S13B). Interestingly, no signs of autophagy adapter p62⁺ aggregates were observed in the regions classically affected in DRPLA, including the globus pallidus, subthalamic nucleus, red nucleus, or deep cerebellar nuclei. However, we observed robust accumulation of p62-immunoreactive inclusions in neurons throughout the cerebral cortex (involving the entire motor, sensory, and visual regions), anterior olfactory nucleus, and anterior thalamus. We observed perinuclear and nuclear puncta only in the brains of saline-treated Atn1^Q112/+ mice, the severity of which was not impacted by mASO treatment (Figure 5C); however, we observed minimal inclusions in hASO-treated brains (Figure 5C). Based on the appearance of our tissue, we devised a semi-quantitative scoring system that incorporated both regional distribution and inclusion burden (Figure 5D). Scoring each brain by this system demonstrated similar inclusion burden within the saline and mASO-treated Atn1^Q112/+ mouse brains, but nearly complete resolution of inclusions in Atn1^Q112/+ mouse brains treated with hASO, which were similar in appearance to WT mice (Figure 5E). To understand if these inclusions were immunoreactive for poly-glutamine expansion specific antibody (1C2), we stained fresh sections from the same mice. We did not observe any 1C2 immunoreactive inclusions anywhere in the brain (Figure S13C).

Human, but not mouse, *ATN1* ASO treatment improves gross neuropathological deficits in the *Atn1*^Q112/+ mice

(A) Raw brain weight (n = 91) is reduced in saline- and mASO-treated *Atn1*^Q112/+ mice, which is rescued by hASO treatment (genotype effect [GT] – F_(1,83) = 70.2, p = 1.15^-¹², treatment effect [Tx] – F_(3,83) = 2.22, p = 9.14^-², interaction effect [GT∗Tx] – F_(3,83) = 21.9, p = 1.53^-¹⁰). (B) When normalized to body weight, more subtle impacts of treatment and genotype remain (n = 84; genotype effect – F_(1,76) = 7.9, p = 6.2^-³, treatment effect – F_(3,76) = 4.4, p = 6.7^-³, interaction effect – F_(3,76) = 1.5, p = .22). (C) Representative light microscopy images of sections stained with p62 antibody, highlighting the presence or absence of cortical neurons inclusions in each treatment group. Scale bars, 20 μm. (D) Semiquantitative scoring system designed to assess neuronal inclusion burden in *Atn1*^Q112/+ mouse brains, encompassing distribution and density of affected neurons. (E) Mean semiquantitative inclusion burden scores across treatment groups. Error bars indicate standard deviation. *Atn1*^Q112/+ mice are annotated with Q112. ∗p < .05, Kruskal-Wallis test followed by Dunn multiple comparison analysis.

Human ASO rescues transcriptional features

While ATN1’s precise cellular roles remain somewhat obscure, it is widely accepted to act as a transcriptional regulator.⁴⁴ Consequently, we conducted bulk RNA sequencing (RNA-seq) of cerebellar tissue from a subset of mice in this study (total N = 30; 5 per arm, excluding Combo). In saline-treated Atn1^Q112/+ animals, we observe widespread transcriptional dysregulation, with a total of 5,423 genes showing evidence of dysregulation at a permissive threshold of FDR <0.1 compared with WT; while 1,041 transcripts are downregulated and 655 upregulated at a more conservative fold change |Log₂FC| > 0.58 and FDR <0.05 threshold (Figure 6A). Unsupervised clustering of differentially expressed genes (DEGs) cleanly separated the genotypes (Figure 6B). Over-representation analysis using {enrichR}⁴⁵^,⁴⁶^,⁴⁷ revealed that DEGs in the Atn1^Q112/+ cerebellum are enriched for functional pathways related to neuronal function, including KEGG pathways for calcium signaling (27/189 overlap, p = .005), glutamatergic synapse (18/114 overlap, p = .007), and axon guidance (25/80 overlap, p = .009; Figure 6C). Of particular interest, given roles for ATN1 in regulating gene expression, DEGs are enriched for PRC2 target genes, with enrichment for genes occupied by SUZ12 (244/1684 overlap with SUZ12 ChEA, p = 4.3^-¹⁸) and EZH2 (34/237 overlap with EZH2 ChEA, p = .002; Figure S14C). We also observe robust enrichment in gene expression studies from HD, suggesting common transcriptional impacts of CAG expansion (Figures S14A, S14B, and S15).

Saline-treated *Atn1*^Q112/+ mice exhibit robust cerebellar transcriptional dysregulation

(A) A volcano plot showing the severity of transcriptional dysregulation in the cerebellum of saline-treated *Atn1*^Q112/+ mice (n = 5) compared with saline-treated WT mice (n = 5), as quantified with bulk RNA sequencing. Dashed horizonal line indicates an FDR corrected p-value of .05, while vertical dashed lines indicate log₂ fold change of ±0.58, or roughly 1.5-fold dysregulation. A majority of transcriptionally dysregulated genes in *Atn1*^Q112/+ mice are downregulated. (B) Unsupervised clustering of the 5,413 DEGs reaching the threshold of FDR < 0.1 in saline-treated *Atn1*^Q112/+ mice compared with saline-treated WT. Each column represents a mouse, while every row indicates a gene. Mice clustered by genotype, indicating that the *Atn1*^Q112/+ transcriptional profile is robust and reproducible. (C) Gene set over-representation analysis on cerebellar DEGs finds enrichment for KEGG pathways related to neuronal function such as calcium signaling, glutamatergic synapse, and axon guidance.

Turning to the impact of ASO treatment, we first noticed that hASO treatment reduced the number of downregulated DEGs at our stringent threshold nearly, FDR <0.05, in half (i.e., from 1,041 in saline-treated Atn1^Q112/+ to 527 in hASO-treated Atn1^Q112/+ compared with saline-treated WT; Figure 7A), with more modest rescue seen in upregulated DEG numbers (from 655 to 529) and only small numbers of DEGs of mASO-treated mice. Overlap analysis determined a core set of dysregulated genes in Atn1^Q112/+ mice that did not respond to treatment, as well as a large set of dysregulated genes shared by saline-treated and mASO-treated Atn1^Q112/+ mice, but not hASO-treated Atn1^Q112/+ mice (Figure 7B). Examples of genes with expression profiles rescued to near WT levels in Atn1^Q112/+ mice following hASO treatment include neuronal signaling genes such as Penk and Gpr63 (Figure 7C). Unsupervised complete linkage clustering of cerebellar DEGs grouped samples primarily by genotype, though we observe treatment-level effects as well (Figure 7D). Using the more stringent FDR and fold change threshold noted above, we visualized enriched KEGG pathways using the compareCluster() function in {clusterProfiler},⁴⁸ finding near-complete loss of enrichment of DEGs in critical neuronal function categories such as calcium signaling pathway, axon guidance, and glutamatergic synapse following hASO treatment (Figure 7E). Plotting the z-score of differential gene expression in saline-treated Atn1^Q112/+ mice versus saline WT (x-axis) against ASO-treated Atn1^Q112/+ mice versus saline-treated WT (y-axis) highlights that hASO treatment restores a larger number of genes to near-WT levels (hASO R = 0.52, Figure 7F) than mASO treatment (mASO R = 0.20; Figure 7F).

Treatment with human *ATN1* ASO leads to normalization of transcriptional deficits in differentially expressed genes

(A) Counts of cerebellar down- (blue) and up-regulated (red) DEGs in saline- or ASO-treated *Atn1*^Q112/+ mice (n = 5 for each treatment) compared with saline-treated WT (n = 5) mice. Lighter shading indicates the DEG count with a more permissive threshold of FDR <0.1, while darker shading indicates a more stringent FDR <0.05 and |LFC| > 0.58. Most DEGs are downregulated with a significant difference in the number of DEGs in *Atn1*^Q112/+ mice treated with hASO. (B) Venn diagram indicating DEGs in *Atn1*^Q112/+ mice with the indicated treatment group compared with saline-treated WT. Darker colors indicate more overlap. (C) Individual gene plots of cerebellar RNA sequencing counts from proenkephalin (*Penk*) and G protein-coupled receptor 63 (*Gpr63*). Saline- or mASO-treated *Atn1*^Q112/+ mice show reduced counts for both genes when compared with WT saline-treated mice (*Penk*: *Atn1*^Q112/+ saline:WT saline *padj* = 1.07^-¹⁸, *Atn1*^Q112/+ mASO:WT saline *padj* = 6.07^-²²; *Gpr63*: *Atn1*^Q112/+ saline:WT saline *padj* = 8.63^-⁶⁰, *Atn1*^Q112/+ mASO:WT saline *padj* = 1.17^-⁸⁴), while *Atn1*^Q112/+ mice treated with hASO have counts significantly restored from *Atn1*^Q112/+ saline-treated mice (*Penk*: *Atn1*^Q112/+ hASO:saline *padj* = 4.33^-¹⁰; *Gpr63*: *Atn1*^Q112/+ hASO:saline *padj* = 1.16^-²⁸), and in the case of *Penk* is indistinguishable from WT saline-treated mice (*Atn1*^Q112/+ hASO:WT saline *padj* = .12). (D) Heatmap with unsupervised clustering shows grouping by genotype. (E) clusterProfiler plot showing enrichment of DEGs in *Atn1*^Q112/+ mice with the indicated treatment group compared with saline-treated WT (FDR <0.05, |LFC| > 0.58). (F) Scatterplots comparing scaled cerebellar differential gene expression (z-scores) between *Atn1*^Q112/+ mice and saline-treated WT (x-axis) to z-scores for *Atn1*^Q112/+ mice treated with hASO (left plot) or mASO (right plot) versus saline-treated WT (y-axis each respective plot). Each point represents one gene, and dashed lines indicate z-scores of ±2.54, encompassing ∼99% of the data range in each dimension. Complete rescue of transcriptional dysregulation would appear as correlation of R = 1 (indicated by a gray line with slope = 1), and genes with expression levels returning to WT levels in treated *Atn1*^Q112/+ mice are expected to be closer to this hypothetical line. Linear regression was performed (blue line), with hASO treatment resulting in R = 0.52, while mASO shows less return to WT-like expression, R = 0.20. Datapoints with z-score ratio 0 < x:y < 2 have been color heatmapped with decreasing red brightness as values diverge from 1. n = 5 per condition for all panels.

Discussion

In this study we established a novel, fully humanized mouse model of DRPLA, developed ASOs targeting mouse and human ATN1, and executed a study of their efficacy in a rescue study. We find that Atn1^Q112/+ mice have early and pronounced neurological impairment including increased ataxia (Figures 3A and 3B), hypoactivity (Figures 3C, 4, and S9D), and gait impairments (Figures 3A, 3B, and S8A). In patients, DRPLA is associated with profoundly debilitating progressive motor, behavioral, and psychiatric symptoms, progressing inevitably toward death.⁹ In qualitative studies we have investigated the most burdensome symptom in DRPLA patients and caregivers and found that motor impairments, notably ataxia and fine motor impairments, present patients and caregivers the most challenges and are reported by virtually every participant.⁴⁹ We are therefore pleased to note that treatment with hASO leads to very pronounced improvements in general activity levels, ataxia, gait, and motor coordination in Atn1^Q112/+ mice.

Consistent with a previously described single-copy transgenic mouse model of DRPLA with very long CAG repeats (“Q129 mice”),³⁵^,⁵⁰^,⁵¹ we find that symptoms in Atn1^Q112/+ mice progress very rapidly to death by approximately 8–10 weeks of age. The Q129 mice also present with pronounced transcriptional dysregulation,³⁵^,⁵⁰ which precedes the development of frank neuropathological symptoms such as neuronal loss and aggregate accumulation. Here, we find that fully humanized Atn1^Q112/+ mice also have very pronounced transcriptional dysregulation in the cerebellum (Figure 6), which excitingly can be rescued with hASO treatment (Figure 7) but is not accompanied at 9 weeks of age by frank neuropathological symptoms such as neuronal loss (Figure S12A), microgliosis (Figure S12B), astrogliosis (Figure S13A) and only subtle accumulation of p62-immunoreactive puncta (Figures 5C–5E). We find it striking that the dramatic behavioral phenotypes we observe in the Atn1^Q112/+ mice occur in the absence of neuronal death (Figure S12A), suggesting that neuronal dysfunction rather than neurodegeneration can evoke very robust DRPLA-relevant phenotypes in mice. A potential confound of both mouse lines is ongoing development, particularly neurodevelopment, that is still occurring at these very early timepoints.⁵²^,⁵³^,⁵⁴

As with any mouse-based study of human neurodegenerative diseases there are limitations to our study. Notably, after many attempts we have been unable to reliably detect mouse or human ATN1 protein from Atn1^Q112/+ mouse brain tissue with either ATN1 or poly-glutamine expansion sensitive antibodies. This is also true histologically, where we see clear accumulation of p62-immunoreactive puncta that are rescued by hASO treatment (Figures 5C–5E), which were not immunoreactive to 1C2,⁵⁵ an antibody sensitive to expanded polyglutamine repeats (Figure S13C). A final limitation of our study is the apparent impact of mASO treatment on body weight in Atn1^Q112/+ mice (Figure S7)—this could be a genotype-selective sensitivity to ATN1 lowering, or modest toxicity of that ASO that is not sufficiently advanced to cause NEFL increases in plasma (Figure S11). Neither of these possibilities can be excluded with our current data.

Our new mouse line adds to the long literature of mouse models of REDs of increasing complexity and fidelity to human genetics. Developments in molecular biology have enabled an evolution from transgenic mice to knock-in mice expressing CAG repeat expansions from orthologous mouse loci to partially humanized mice, such as those available for SCA1⁵⁶ and SCA3.²⁵ We believe that the complete humanization of the target allele achieved here is a significant advancement for modeling REDs, particularly in the context of ASOs, which commonly target intronic sequences.⁵⁷ In fact, after comprehensive empirical screening of ASOs targeting across atrophin-1 (Figure S2), both our mASO and hASO target intronic sequences of the atrophin-1 gene. Our Atn1^Q112/+ mice enable direct testing of ASOs targeting human intronic sequence, which previously relied on transgenic expression of full-length human sequences (for example, in HD).⁵⁸^,⁵⁹ Thus, our work provides a nice evolution of previous mouse ASO studies reported in other forms of CAG expansion disorders, including SBMA,⁶⁰ SCA1,⁶¹ SCA2,⁶² SCA3,⁶³ and SCA7.⁶⁴ In short, we believe complete humanization offers significant benefits for pre-clinical studies in CAG expansion disorders, particularly in the context of oligonucleotide-based therapies.

Materials and methods

Mouse development

Transfection of ES cells to introduce a targeted mutation via CRISPR-Cas9-mediated gene editing

The embryonic stem (ES) cells were grown on a mitotically inactivated feeder layer comprising mouse embryonic fibroblasts in ES cell culture medium containing leukemia inhibitory factor and fetal bovine serum (FBS). The cells were co-transfected with the targeting vector and a plasmid expressing Cas9 as well as the specific sgRNA(s), the latter plasmid contains a selection cassette. One day post transfection the antibiotic was transiently added to the medium to select for transfected cells. ES cell clones were isolated as soon as they showed a distinct morphology and were analyzed by PCR or Southern blotting in a primary screen. Homologous recombinant ES cell clones were expanded and frozen in liquid nitrogen after extensive molecular validation.

Diploid injection

After administration of hormones, superovulated BALB/c females were mated with BALB/c males. Blastocysts were isolated from the uterus at dpc 3.5. For microinjection, blastocysts were placed in a drop of DMEM with 15% fetal calf serum under mineral oil. A flat tip, piezo actuated microinjection-pipette with an internal diameter of 12–15 μm was used to inject 10–15 targeted C57BL/6NTac ES cells into each blastocyst. After recovery, 8 injected blastocysts were transferred to each uterine horn at 2.5 dpc, pseudopregnant NMRI females. Chimerism was measured in chimeras (G0) by coat color contribution of ES cells to the BALB/c host (black/white).

Revitalization of cryopreserved sperm

IVF was performed using oocytes from superovulated C57BL/6NTac females and thawed sperm from previously cryopreserved chimera spermatozoa. After overnight incubation, two-cell embryos were transferred into oviducts of 0.5 dpc pseudopregnant Swiss Webster recipient females. Recipient production colony as well as post embryo transfer animals are regularly monitored for microbial contamination. Germline transmission was identified by the presence of black offspring (strain C57BL/6Ntac) and by genotyping of the black offspring via PCR to prove germline transmission of the inserted mutation.

Genotyping analysis

Genomic DNA was extracted from biopsies and analyzed by PCR. The following templates were used as controls: water (ctrl1), wild-type genomic DNA (ctrl2), and positive DNA sample (ctrl3).

The amplification of the internal control fragment—585 bp (ctrl)—with oligos 1260_1 and 1260_2 confirms the presence of DNA in the PCR reactions (amplification of the CD79b WT allele, nt 17714036–17714620 on Chromosome 11). The amplification of the internal control fragment—333 bp (ctrl)—with oligos 11767_3 and 11767_4 confirms the presence of DNA in the PCR reactions (amplification of the CD79b WT allele, nt 17712493–17712826 on Chromosome 11).

Animal care and surgery

Animal care

Animal experimentation was approved by the University of Washington’s Institutional Animal Care and Use Committee (IACUC) under protocols 4563-01 and 4387-01, as well as by Western Washington University’s IACUC under protocol 22-002.

All mice were housed in cages of 2–5 mice per cage with corn cob bedding and on a 12-h light-dark cycle with access to food and water ad libitum. Male and female mice were used in all experiments and treated equally throughout the study.

Randomization and blinding

Prior to genotyping, whole litters were randomly assigned to saline, hASO, or mASO for the neonatal ICV injections. This treatment assignment was kept the same for the 5-week ICV injection. During behavioral assessments, genotype and treatment were excluded from the data recording documents.

Neonatal ICV injection

Mice were anesthetized by using wet ice to induce hypothermia, which was confirmed via a gentle toe pinch and observed lack of breathing.⁶⁵ The mice were then free hand injected with 2 μL of hASO (30 μg, 50 μg in the pilot), mASO (30 μg), Combo (15 μg of each ASO for a total of 30 μg), or saline using a Hamilton syringe and a 32-gauge needle. The location of the injection was two-fifths of the distance from the lambda suture of one eye with the needle being inserted 2.5–3.0 mm deep. All mice were injected on postnatal day 1–3.

Adult ICV surgery

Mice were anesthetized with vaporized isoflurane and placed on a stereotaxic apparatus where the surgical plane was maintained throughout the procedure. After a small vertical incision was made on the top of the head, the mice were injected with 10 μL of hASO (200 μg), mASO (200 μg), Combo (100 μg of each ASO for a total of 200 μg), or saline using a Hamilton syringe with a 26-gauge needle. The needle was lined up with bregma then moved laterally to the right 1.0 mm and anterior 0.3 mm with the needle being inserted 3.5–3.0 mm deep. The incision was closed with suture glue, and mice were injected with 5 mg/kg of carprofen. All surgeries were performed at five weeks of age except pilot mice, which received surgery at 7 weeks of age.

ASO screening

A-431 cell culture condition

A-431 is a human epidermoid carcinoma cell line purchased from ATCC. A-431 cells were cultured in DMEM growth medium (DMEM 10% FBS, 50 units/mL penicillin, and 50 μg/mL streptomycin) at 37°C and 10% CO₂. Cells were subcultured or trypsinized for plating when they reached 80% confluency. Cells were trypsinized, counted, and diluted to 110,000 cells per mL in room temperature growth medium before adding 100 μL of the cell suspension to the wells of collagen I-coated 96-well culture plate. Immediately after plating the cells, 11 μL of 10× ASO in water was added to the appropriate wells. The culture plate was incubated in a humidified incubator at 37°C and 10% CO₂. After 48 h, the cells were washed once with PBS before lysing with guanidine isothiocyanate-containing buffer for RNA isolation and analysis. For each treatment condition duplicate wells were tested.

mRNA extraction and qRT-PCR

Each cortex and spinal cord sample was homogenized in 1 mL of Trizol reagent (Thermo Fisher Scientific, Waltham, MA), and total RNA was extracted using the Life Technologies mini-RNA purification kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. After purification, the RNA samples were subjected to real-time qRT-PCR analysis using the Life Technologies ABI QuantStudio 7 Flex Sequence Detection System (Applied Biosystems Inc, Carlsbad, CA). Briefly, 10 μL qRT-PCR reactions containing 400 nL of RNA were run with the AgPath-ID One-Step qRT-PCR Kit (Thermo Fisher Scientific, Waltham, MA) reagents and the primer probe sets. This system uses real-time florescence PCR to quantitatively determine mRNA expression levels. The assay is based on a target-specific probe labeled with a fluorescent reporter and quencher dyes at opposite ends. The probe is hydrolyzed through the 5′-exonuclease activity of Taq DNA polymerase, leading to an increasing fluorescence emission of the reporter dye that can be detected during the reaction. All qRT-PCR reactions were run in triplicate. Outliers in triplicates due to technical issues were eliminated. Target mRNA was then normalized to Ppia, a ubiquitously expressed housekeeping gene, and this was further normalized to the level measured in control animals that were administered PBS. mRNA levels are reported as percent control.

Behavior

Balance beam

The balance beam apparatus consisted of a metal rod (length: 100 cm, diameter: 12 mm and 6 mm) set up at a tapered angle with a dark box at one end and 60-watt lamp on the other. The dark box had nestling and bedding from the mouse’s home cage. The dark box and rod were cleaned with 70% ethanol between cages. Mice were trained for two days with three trials on both rods. The mice were tested on the third day with three trials on the 6 mm rod. All mice were tested and trained from 6 to 7 weeks of age.

Fixed rotarod

Motor coordination was tested using a fixed rotarod apparatus (Harvard Apparatus, 76-0770). Each mouse went through three 5-min training trials at two speeds (4 and 8 rpm). Mice were then tested for three trials at both speeds with a maximum time of 5 min. All mice were tested and trained from 6 to 7 weeks of age.

Open field

Mice were placed in the center of a 41 × 41 cm closed box with a transparent acrylic lid. A GoPro camera, mounted in the lid, recorded a 12-min video of each individual mouse. Quantitative analysis was performed using Cai Lab’s ezTrack script to measure total distance traveled and the freezing to motion ratio.⁶⁶ Open field data presented here are from a cohort of mice from a later study that utilized the same mouse model and hASO treatment; however, the mice did receive multiple intraperitoneal injections of saline (Table S3).

Modified SHIRPA

This screening test is a modified version of SHIRPA (SmithKline Beecham Pharmaceuticals; Harwell, MRC Mouse Genome Centre and Mammalian Genetics Unit; Imperial College School of Medicine at St Mary’s; Royal London Hospital, St Bartholomew’s and the Royal London School of Medicine; Phenotype Assessment).⁶⁷ In our modified SHIRPA we observed mice inside of their home cage to assess their level of lethargy, gait issues, tremors, pelvic and tail elevation, hindlimb and forelimb grasping, piloerection, and touch escape. Each phenotype that was assessed had an individual scale that was used to measure the behavior (Table S5). Overall, the higher the mouse’s score for any phenotype equated to worsening of the phenotype in the mouse. All mice were assessed at 4 weeks of age.

Analysis of circadian patterns of home cage behavior

A subset of male Atn1^Q112/+ mice (n = 4) aged eight weeks were followed in a behavioral activity monitored home cage using the Noldus PhenoTyper system (Noldus Information Technology, Wageningen, The Netherlands) as described previously.⁶⁸ Plexiglas cages measuring 30 × 30 cm included corncob bedding, with cameras recording overhead for the entirety of the monitoring period. All cages were recorded simultaneously for 67 h, beginning at 12 p.m. on day 1 and ending at 7 a.m. Mice were able to freely enter a plastic enclosure (termed “hidden shelter”) with two entry/exit points. EthoVision XT software (Noldus Information Technology, Wageningen, The Netherlands) was used to track the movement of the mice for the duration of the experiment. Utilizing this software, the 67 h of behavioral tracking data were analyzed offline in an automated manner by a blinded investigator by dividing the cage recording into various zones (arena center, arena perimeter, hidden shelter) and the duration within each zone (seconds) determined for each mouse over the monitoring period. Relative measurements of the arena areas were as follows: arena center 293 cm², arena perimeter 347 cm², and hidden shelter 106 cm². Behavioral activity measurements were binned into 1-h sessions and activity tracked across all zones, with average activity of each bin taken for the light periods (Light-1, -2, and -3) in comparison with the dark periods (Dark-1, -2, and -3). Two saline-treated Atn1^Q112/+ mice died during the monitoring period (at 31- and 47-h time points) and behavioral analysis until death was included for analysis; all other animals survived the entirety of the monitoring period. Mice were 8 weeks of age during testing. Total activity for each 1-h bin and average activity in each light phase was analyzed by mixed-effects model (time x treatment), with p < .05 considered significant (using GraphPad Prism version 9.0 or later).

ATN1 and NEFL quantification

RNA extraction and cDNA synthesis

Cortical tissue (30–50 mg) was homogenized in 500 μL of QIAzol Lysis Reagent using the Bead Blaster 24 (Benchmark). RNA was extracted using the RNeasy Lipid Tissue Mini kit (Qiagen 74804) according to the manufacturer’s instructions. RNA quality was tested using the NanoDrop Spectrophotometer 2000 (Thermo Fisher Scientific, ND-2000). cDNA synthesis was carried out using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) according to the manufacturer’s instructions.

qRT-PCR

qRT-PCR used 10 μL TaqMan Universal Master Mix II with UNG (Thermo Fisher Scientific, 4440046) along with 5 μL molecular biology grade water, 3 μL cDNA, and 2 μL of probes. The reaction was quantified on the QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific, 4485688). β-Actin (Thermo Fisher, Mm02619580_g1) was used as the reference gene to determine the relative expression of ATN1 (Thermo Fisher Scientific, Hs01073465_m1) and Atn1 (Thermo Fisher Scientific, Mm01185029_m1). This expression was compared with the average expression of control samples. Fold change was given by 2^(−ΔΔCt).

NEFL quantification

NEFL was quantified using an electrochemiluminescent ELISA measured with a MESO QuickPlex SQ120MM (Meso Scale Discovery; MSD). Ninety-six well plates (MSD, L45SA-1) were coated in capture antibody (MSD, F217X-3) that was diluted in Diluent 100 (MSD, R50AA-2) and incubated for one hour at room temperature with shaking at 700 rpm. The plates were rinsed with wash buffer (PBS with 0.2% Tween 20) three times. NEFL calibrator (MSD, F217X-3) was serially diluted in Diluent 12 (MSD, R50JA-2), and plasma samples were diluted at a 1:10 dilution in Diluent 12. The diluted calibrator and samples were then loaded into the plate(s) and incubated at room temperature for one hour with shaking at 700 rpm. Plates were rinsed again with wash buffer three times then coated in sulfo-tag conjugated detection antibody (MSD, F217X-3) that was diluted to 1× with Diluent 11 (MSD, R55BA-3). Plates were incubated at room temperature for one hour while shaking at 700rpm then rinsed again with wash buffer three times. Finally, 150 μL of Read Buffer B (MSD, R60AM-1) was added to the plates, which were then read with MESO QuickPlex SQ120MM.

Neuropathology

Tissue preparation and histochemical staining

At nine weeks of age mice were sacrificed via CO₂. Half of the brain was drop fixed in 10% non-buffered formalin. Hemibrains were removed after 12 h and transferred into 1× PBS with 0.02% sodium azide.

Parasagittal sections were prepared from 32 fixed hemibrains by embedding in paraffin and sectioning at 5 μm along the sagittal plane using a standard microtome to the level of the mid-olfactory bulb. Four brains were mounted on each slide for further evaluation. H&E staining was performed on several of these sections according to standard protocol.

Immunohistochemistry

Immunohistochemistry was performed on parasagittal brain sections using a Leica Bond III Fully Automated IHC and ISH Staining System (Leica Biosystems, Wetzlar, Germany). The sections were immunostained with mouse monoclonal antibody against glial fibrillary acidic protein (GFAP, 1:600; Biocare Medical CM065A), Iba1 (1:1000; Wako 019-19741), NeuN (1:500; EMD Millipore MAB377), p62 (1:500; Abcam ab56416), and 1C2 (1:10,000, Sigma MAB1574). Appropriate positive and negative controls were included with each antibody and each run.

Bulk RNA sequencing

cDNA libraries were constructed at Azenta (South Plainfield, NJ) using the Illumina TruSeq RNA Sample Prep Kit with ERCC spike-in and sequenced on a HiSeq 2000 (2 × 150 bp) to an average read depth of 2.9⁷. Sequence reads were trimmed to remove adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. Reads were aligned to the Mus musculus GRCm38 ERCC reference genome assembly (ENSEMBL) using STAR aligner v.2.5.2b. Unique gene hit counts were calculated using featureCounts() from the {Subread} package v.1.5.2. Differential gene expression analysis was conducted in R using {edgeR},⁶⁹ with {voom}.⁷⁰ Unsupervised clustering was performed using package {pheatmaps}. For correlation plots in Figure 6B, Log2FC for the indicated comparison was converted to Z scores for each gene. The GO enrichment plots from Figure S15A were generated with package {clusterProfiler}.⁷¹ Because of the important role of the dentate gyrus in DRPLA neuropathology, the cerebellum was chosen for RNA-seq analysis prior to receiving any results from neuropathology.

Statistics

Behavioral and molecular data were recorded and stored in a Carroll Lab cloud storage solution (Google Drive). Raw data were read into an R project for processing, statistical comparisons, and generating graphs, which were finalized in Adobe Illustrator. Analysis of molecular and behavioral data was done using one or multi-factor ANOVAs, with Tukey Honestly Significant Difference post hoc tests. Count data (e.g., SHIRPA) were analyzed in R using a general linear model (GLM) with a Poisson log-link function. Neuropathological data were analyzed with Kruskal-Wallis tests followed by Dunn multiple comparison analysis. All R scripts used for analysis and figure generation are available on the linked Dryad data repository https://doi.org/10.5061/dryad.1vhhmgr7k. Raw RNA-seq FASTQs were deposited to GEO under accession GSE296072.

Data availability

All R scripts used for analysis and figure generation are available on the linked Dryad data repository https://doi.org/10.5061/dryad.1vhhmgr7k. RNA-seq results are available at gene expression omnibus (GSE296072).

Acknowledgments

All work was funded by CureDRPLA, which is a US-based non-profit, founded by Paul and Andrea Compton. We acknowledge the contributions of Seongheon Leo Rho and Kevin M. Knox for their technical assistance with the mouse phenotyping and circadian experiments.

Author contributions

V.L.S. and B.Z.G. designed research studies, conducted experiments, acquired and analyzed data, and contributed to manuscript writing. R.M.B. and J.P.C. analyzed data and contributed to writing the manuscript. A.B.-V. contributed in research study design, acquiring and analyzing data. B.N. conducted experiments, acquired and analyzed data, and contributed to manuscript writing. S.P., A.C., J.G., T.Y., V.K., H.B.K., and H.T.Z. provided advising and contributed to manuscript writing. J.A.K. conducted experiments, acquired and analyzed data, and contributed to manuscript preparation. A.G. conducted experiments and acquired and analyzed data. D.D.C. acquired and analyzed data, provided advice, and contributed to manuscript writing. J.B.C. designed research studies, provided advising, acquired and analyzed data, and contributed to manuscript writing.

All authors have read and approved the final manuscript.

Declaration of interests

B.N., H.B.K., and H.T.Z. are employees of Ionis Pharmaceuticals where ASOs are under development for neurological indications.

V.K. is a co-founder of and senior advisor to DaCapo Brainscience, a company focused on CNS drug discovery.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.omtn.2025.102815.

Contributor Information

Daniel D. Child, Email: dchild@uw.edu.

Jeffrey B. Carroll, Email: jeffcarr@uw.edu.

Supplemental information

Document S1. Figures S1–S15 and Tables S1–S5

mmc1.pdf^{(2.8MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(31MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S15 and Tables S1–S5

mmc1.pdf^{(2.8MB, pdf)}

Document S2. Article plus supplemental information

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Data Availability Statement

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PERMALINK

Atrophin-1 antisense oligonucleotide provides robust protection from pathology in a fully humanized DRPLA model

Velvet L Smith

Bereket Z Gidi

Robert M Bragg

Jeffrey P Cantle

Aliza Ben-Varon

Briana Noble

Silvia Prades

Andrea Compton

Julie Greenfield

Joanna A Korecka

Anya Gemos

Timothy Yu

Vikram Khurana

Holly B Kordasiewicz

Hien T Zhao

Melissa Barker-Haliski

Daniel D Child

Jeffrey B Carroll

Abstract

Graphical abstract

Introduction

Results

Generation of Atn1Q112/+ mice

Figure 1.

Development of ATN1 allele-specific ASOs

Initial observations and pilot experiment

Longitudinal study overview

Figure 2.

Human ASO rescues behavioral abnormalities

Figure 3.

Figure 4.

Human ASO rescues brain weight and aggregate accumulation

Figure 5.

Human ASO rescues transcriptional features

Figure 6.

Figure 7.

Discussion

Materials and methods

Mouse development

Transfection of ES cells to introduce a targeted mutation via CRISPR-Cas9-mediated gene editing

Diploid injection

Revitalization of cryopreserved sperm

Genotyping analysis

Animal care and surgery

Animal care

Randomization and blinding

Neonatal ICV injection

Adult ICV surgery

ASO screening

A-431 cell culture condition

mRNA extraction and qRT-PCR

Behavior

Balance beam

Fixed rotarod

Open field

Modified SHIRPA

Analysis of circadian patterns of home cage behavior

ATN1 and NEFL quantification

RNA extraction and cDNA synthesis

qRT-PCR

NEFL quantification

Neuropathology

Tissue preparation and histochemical staining

Immunohistochemistry

Bulk RNA sequencing

Statistics

Data availability

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Contributor Information

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

Generation of Atn1^Q112/+ mice