Atrophin-1 antisense oligonucleotide provides robust protection from pathology in a fully humanized DRPLA model methods
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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 ( ). 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 a...
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) ( A). Within each group, mice were treated with saline...
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 ( A 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 ( and ). Thanks to our pilot study observations ( ), we developed a two-stage ASO treatment strategy based on a published approach in a rapidly developing seizure disorder model. 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...
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. 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.
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 ( ). 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 ( A 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; A, 4C, and D). This hypoactive behavior was ameliorated in...
Human ASO rescues behavioral abnormalities
(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 D 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 C. (C) Representative heatmap displaying the presence of mice in 9 min of open field testing ( n = 1 per image...
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).
Animal care and surgery
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.
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Evidence quotes (8)
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 ( ). 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 ( B and B). 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 ( C and C). We refer...
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) ( A). Within each group, mice were treated with saline or hASO via ICV injection or left untreated as controls ( A). 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 ( A). We qualitatively observed that behavioral p...
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 ( A 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 ( and ). Thanks to our pilot study observations ( ), we developed a two-stage ASO treatment strategy based on a published approach in a rapidly developing seizure disorder model. 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; A 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; A and 2B). We then conducted exhaustive behavioral examinations of the mice from 6 to 8 weeks of...
Mice were anesthetized by using wet ice to induce hypothermia, which was confirmed via a gentle toe pinch and observed lack of breathing. 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.
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 ( ). 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 ( A 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; A, 4C, and D). 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 ( C, B, and 4C). Next, we conducted a modified SHIRPA,, which is a behavioral screening assessment designed to broa...
(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 D 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 C. (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/+...
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).
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.
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