A novel imaging ligand as a biomarker for mutant huntingtin-lowering in Huntington’s disease

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (PolyQ) tract. While several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, no method is currently available to visualize mHTT levels in the living brain. Here we demonstrate the development of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11C ([11C]CHDI-180R) enables non-invasive monitoring of mHTT pathology in the brain and can track region-and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression. We further show that therapeutic agents that lower mHTT in the striatum have a functional restorative effect that can be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.

programs targeting mHTT expression have advanced to clinical evaluation, no method is 23 currently available to visualize mHTT levels in the living brain. Here we demonstrate the 24 development of a positron emission tomography (PET) imaging radioligand with high affinity 25 and selectivity for mHTT aggregates. This small molecule radiolabeled with 11 C ([ 11 C]CHDI-26 180R) enables non-invasive monitoring of mHTT pathology in the brain and can track region-27 and time-dependent suppression of mHTT in response to therapeutic interventions targeting 28 mHTT expression. We further show that therapeutic agents that lower mHTT in the striatum 29 have a functional restorative effect that can be measured by preservation of striatal imaging 30 markers, enabling a translational path to assess the functional effect of mHTT lowering. 31 Introduction two-tissue compartment model (2TCM) (Extended data Fig. 3f, Supplementary Table 2). The 150 resulting striatal total volume of distribution using IDIF (VT (IDIF) as a surrogate of VT 26 ) in het 151 zQ175DN was significantly increased by 62% compared to wt littermates (Extended Data Fig.  152 3g, P<0.0001) with extremely low coefficients of variation (wt = 2.84%, het = 5.2%; Extended 153 Data Fig. 3g). Scan acquisition could be reduced from 90-min down to 60-min (Extended Data 154 intervention paradigm and that this value is determined by the extent of neuronal transduction 283 and viral distribution in the mouse striatum (38%). This value is in concordance with the 38.9% 284 signal decrease, measured in vivo using PET, with [ 11 C]CHDI-180R. In contrast, in the late 285 intervention paradigm, and consistent with residual pre-existing aggregate pathology that 286 remains after AAV-ZFP administration after disease onset, we only achieved a 23.6% 287 therapeutic effect as measured in vivo with [ 11 C]CHDI-180R at 10 months of age. We employed this model to lower mHtt systemically at 2 or 8 months of age, before and after 303 mHTT inclusion formation and disease onset, and compared them to control mice or 304 LacQ140 I (*) mice with mHtt expressed throughout its life, at 13 months of age (Fig. 5a,b; 305 Supplementary Tables 7). [ 11 C]CHDI-180R VT (IDIF) values were significantly reduced 306 consistently with suppression duration in all brain regions examined following IPTG withdrawal 307 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint distinct restricted distribution patterns, an understanding of the regional effects of HTT lowering 321 agents is fundamental in being able to interpret, and improve upon, clinical trial results. It is in 322 this context that we set out to develop a translational biomarker strategy to identify and 323 characterize potential biomarkers that can help guide the clinical development of HTT lowering 324 agents. Here we extend our prior characterization of CHDI-180 and demonstrate the time-and 325 region-dependent appearance of mHTT pathology in the HD mouse models R6/2, HdhQ80, 326 and zQ175DN. The ligand is suitable to detect genotype and region-specific differences in HTT 327 pathology throughout the brain, allowing for its deployment in therapeutic studies with 328 manageable sample size and a longitudinal manner. We were able to ascertain different 329 regional pathology within the striatum, particularly in HdhQ80 mice, which appears to proceed 330 from a ventral to dorsal trajectory, an observation reminiscent of human pathology that 331 proceeds caudal-to-rostral and dorsal-to-ventral 2,3 . 332 We applied [ 11 C]CHDI-180R in two interventional paradigms when mHTT is lowered in a 333 restricted manner in the striatum of mice, or more broadly throughout the mouse brain, within 334 the range of mHTT suppression expected in clinical studies (~50%). The extent of lowering 335 detected by [ 11 C]CHDI-180R correlates well with the extent of mHTT suppression as measured 336 by quantitative assays for soluble and aggregated forms of mHTT. These studies show that 337 [ 11 C]CHDI-180R can be used irrespective of the regional distribution of the therapeutic agents 338 or the extent of lowering. Furthermore, we verified the extent of lowering by ARG, showing 339 excellent concordance with PET imaging. In the context of the ZFP repressor, the decrease in 340 signals obtained with [ 11 C]CHDI-180R appear rapid (1-month post administration of AAV-ZFP), 341 and are sustained during the duration of the studies (up to 8 months). When administered 342 early, prior to the appearance of pathology, AAV-ZFP prevents mHTT inclusion and 343 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint extranuclear aggregation, and the diminution of the signal detected by [ 11 C]CHDI-180R can be explained by the extent of agent distribution and neuronal transduction (in our case, about 40% 345 of the striatum). 346 While we do not yet have a full understanding of the various species of mHTT that constitute 347 the binding site(s) for CHDI-180, based on recombinant, cell, and tissue protein studies 8,9 , we 348 know this ligand can bind oligomerized and some forms of fibrillar mHTT but not to monomeric 349 soluble HTT. 350 We also investigated potential striatal markers that can serve as markers of functional SPN 351 restoration. Several PET ligands, previously shown to track disease progression in HD 352 individuals, have been shown to track progression in models of HD [33][34][35]41,42 . We show that the 353 response to mHTT lowering in SPNs is fast and durable, and that these effects can be 354 observed even in the context of established disease and aging, at least in the zQ175DN model. 355 During the early intervention paradigm, all striatal markers responded within a month of therapy 356 administration, suggesting an improvement of cellular alterations in indirect-pathway SPNs 357 (expressing both PDE10 and D2R), and direct-pathway neurons (expressing PDE10 and D1R). 358 When AAV-ZFP is administered after disease onset, the response is more muted, but 359 significant for all tracers, particularly as judged by ARG, which has a higher signal to 360 background ratio than microPET. PDE10a and D1R expression appear more responsive to 361 mHTT lowering than D2/3R, arguing that direct pathway neurons (affected later in the disease) 362 might be more amenable to functional restoration. 363 The strong correlation seen in intra-animal comparisons between [ 11 C]CHDI-180R and 364 PDE10a binding across our cohorts strongly support the concept that PDE10a imaging can be 365 a very sensitive translational marker of mHTT lowering. As this marker is one of the earliest 366 markers altered in premanifest individuals, including those far from disease onset 14-16 , PDE10a 367 imaging can be used to track functional responses to HTT lowering in prodromal clinical 368 studies. 369 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. In summary, we demonstrated the development of a small-molecule PET ligand with high 370 affinity and selectivity for mHTT to monitor non-invasively mHTT pathology in the living brain 371 and track region-and time-dependent suppression of mHTT levels in response to therapeutic 372 intervention. We also showed that therapeutic agents, such as AAV-ZFP, can be functionally 373 restorative and their effects can be measured by the preservation of striatal imaging markers. 374 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint (wt, n = 3; R6/2, n = 3; per age). Two-way ANOVA with Tukey's multiple comparison test; mean 381 ± s.d., all points are shown; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Red asterisk 382 denotes signal differences between ages, as indicated for R6/2 mice. One representative study 383 out of n > 15 experiments shown. b, Representative autoradiograms showing total binding of 384 [³H]CHDI-180 in the striatum, cortex, and hippocampus of 12-week-old R6/2 and wt mice; 385 anatomical orientation as indicated. c-d, Binding to knock-in zQ175DN het mouse brains 386 carrying a humanized exon1 Htt sequence with 198 CAG repeats. c, Genotype-specific age-387 dependent increase in [³H]CHDI-180 in striatum, cortex, and hippocampus of 3, 6, 9 months 388 (wt, n = 10; het, n = 10, per age), and 13 months (wt, n = 13; het, n = 17) of age. Two-way 389 ANOVA with Tukey's multiple comparison test; mean ± s.d., all points are shown; * P < 0.05, 390 ** P < 0.01, *** P < 0.001, **** P < 0.0001. Red asterisk denotes signal differences between 391 ages, as indicated for zQ175DN het mice. reference. Coronal and axial planes are shown. c, Regional [ 11 C]CHDI-180R VT (IDIF) 420 quantification in zQ175DN wt and het at 3 months (wt, n = 19; het, n = 21), 6 months (wt, n = 421 15; het, n = 23), 9 months (wt, n = 13; het, n = 20), and 13 months (wt, n = 12; het, n = 17) of 422 age. Repeated measures with linear mixed model analysis with Tukey-Kramer correction; *** 423 P < 0.001, **** P < 0.0001. Data are shown as mean ± s.d., all points shown. Red asterisks 424 denote longitudinal differences within zQ175DN het mice. d, Within zQ175DN het voxel-based 425 analysis of [ 11 C]CHDI-180R VT (IDIF) parametric images. Comparison between 3-6 months (n = 426 21), 6-9 months (n = 20), and 9-13 months (n = 17) of age. Significant (P < 0.001) clusters are 427 co-registered to the MRI template for anatomical reference and shown in the coronal panel. Quantification of inclusions in wt and zQ175DN het mice for mEM48 (f) and 2B4 (h) at 3 months 432 (wt, n = 10; het, n = 10), 6 months (wt, n = 10; het, n = 10), 9 months (wt, n = 10; het, n = 10),  . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021.  zQ175DN wt vehicle, het vehicle, and het ZFP-treated mice at 10 months of age (het ZFP, n 477 = 11; het vehicle, n = 11; wt vehicle, n = 11) following striatal injection at 2 months of age. One-478 way ANOVA with Tukey's multiple comparison test; * P < 0.05, ** P < 0.01, **** P < 0.0001. 479 Data are shown as mean ± s.d., all points shown. 480 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint template for anatomical reference. Coronal and axial planes are shown. A white arrow at 10 490 months of age indicates the ZFP-treated striatal hemisphere with reduced mHTT as well as 491 increased PDE10a, D1R, and D2/3R binding. g-j, Percentage contralateral difference for striatal 492 [³H]Raclopride (D2/3R) (q) in zQ175DN wt vehicle, het vehicle, and het ZFP-treated mice at 10 513 months of age (het ZFP, n = 25; het vehicle, n = 26; wt vehicle, n = 25) following striatal injection 514 at 5 months of age. One-way ANOVA with Tukey's multiple comparison test; * P < 0.05, **** P 515 < 0.0001. Data are shown as mean ± s.d., all points shown. 516 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint LacIR, allowing transcription of the Q140 allele. b, Timeline overview and endpoints in 522 LacQ140 I (*) wt and het mice. c, Mean [ 11 C]CHDI-180R VT (IDIF) parametric images of 523 LacQ140 I (*) wt and het mice at 13 months of age. PET images are co-registered to the MRI 524 template for anatomical reference. Coronal and axial planes are shown. d, Regional [ 11 C]CHDI-525 180R VT (IDIF) quantification in LacQ140 I (*) wt and het at 13 months (het no supp, n = 13; het 8-526 13m, n = 14; het 2-13m, n = 12; wt no supp, n = 13) of age. One-way ANOVA with Tukey's 527 multiple comparison test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0. 13 months of age (het no supp, n = 13; het 8-13m, n = 14; het 2-13m, n = 12; wt no supp, n = 533 13). One-way ANOVA with Tukey's multiple comparison test; *** P < 0.001, **** P < 0.0001. 534 Data are shown as mean ± s.d., all points shown. g, Correlation between striatal mHTT binding 535 measured with microPET and autoradiography in LacQ140 I (*) wt and het mice at 13 months 536 of age. Two-tailed Pearson correlation analysis; R 2 = 0.779; P < 0.0001. j, Correlation between 537 striatal mHTT binding measured with microPET and immunostaining in LacQ140 I (*) wt and het 538 mice at 13 months of age. Two-tailed Pearson correlation analysis; R 2 = 0.794; P < 0.0001. 539 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint

540
In vitro studies using IHC and ARG 541 Brain preparation mouse models of HD and human brain tissue 542 For autoradiography (ARG) and immunohistochemistry (IHC) analysis, fresh frozen whole 543 brain samples from male zQ175DN, HdhQ80, and R6/2 and age-matched wt mice were 544 prepared (Supplementary Table 10). For ARG analyses, mice were either euthanized by 545 cervical dislocation or PBS-perfusion and the brains were snap-frozen in isopentane at -30 to 546 -40°C and stored at -80°C. For IHC analyses, the mice were euthanized by PBS-perfusion 547 followed by 4% paraformaldehyde (PFA). The brains were fixed in 4% paraformaldehyde 548 (PFA) fixation for 24 hours, followed by 48 hours in 30% sucrose at 4°C. Afterwards the brains 549 were embedded in Tissue-Tek® O.C.T. Compound (Sakura, Cat. # 4583) using Peel-A-Way TM 550 embedding molds (Sigma Aldrich, Cat. # E6032-1CS) and stored at -80°C. 551 For ARG and IHC analysis in human brain tissue, post-mortem superior frontal gyrus tissue 552 from HD patients and control donors without any evidence of neurological disease were 553 obtained from Netherlands Brain Bank (NBB). All material has been collected from donors from 554 whom a written informed consent for a brain autopsy and the use of the material and clinical 555 information for research purposes had been obtained. Demographic information is displayed 556 in Supplementary Table 11. 557 558

Autoradiography (ARG) 559
In vitro autoradiography for mHTT was performed using the tritiated ligands (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint Twenty µm-thick sections were prepared from brains of hom, het, and age-matched wt mice 567 by using a cryostat, mounted on superfrost slides, and stored at -80°C for a maximum of 2 568 weeks. On the day of the experiment, slides were adapted to room temperature for 30 min and 569 then equilibrated by immersion into assay buffer (50 mM Tris-HCl pH 7.4; 120 mM NaCl; 5 mM 570 KCl; 2 mM CaCl2; 1 mM MgCl2) for 20 min at room temperature. 571 Radioligand solutions for total binding (TB) (0.5 nM (mouse) or 1 nM (human) for [ 3 H]CHDI-572 180, 1 nM for [ 3 H]SCH23390, or 2 nM for [ 3 H]Raclopride) and non-specific binding (NSB) (0.5 573 nM of + 1 µM unlabeled compound; 1 or 2 nM + 10 µM unlabeled compound) were prepared 574 in assay buffer. Optimal radioligand concentrations were determined in advance based on the 575 signal-to-background ratio obtained in mouse or human brain tissue. Sections were incubated 576 by immersion into assay buffer containing either only radioligand (TB) or radioligand plus the 577 excess concentration of unlabeled compound (NSB) for 60 min at room temperature. 578 Afterwards, slides were washed three times for 10 min with ice-cold washing buffer (50 mM 579 Tris-HCl, pH 7.4) at 4°C and dipped for three seconds in ice-cold distilled water to remove 580 buffer salts. The slides were dried for 2-3 hours at 30°C and exposed to a Tritium Phosphor 581 Screen (GE Healthcare, Fuji BAS-TR 2025 E) together with calibrated tritium standards 582 (American Radiolabeled Chemicals, ART 0123C, and ART 0123B). Slides and commercial 583 tritium activity standard were exposed for 90 hours (  Densitometric data analysis of radioligand binding was performed using the MCID Analysis 7.1 587 software (Interfocus Imaging Ltd.) (Evotec) or ImageJ (National Institute of Health, USA) 588 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint

Immunohistochemistry (IHC) in HdhQ80 mouse brains 592
Twenty-five µm-thick sections were prepared from brains of hom, het, and wt mice by using a 593 cryostat, mounted on 12-well plates (Greiner, Cat. # 665180) and stored at -80°C until further 594 use. The sections were washed three times with DPBS and incubated in 2% H2O2 for 45 min 595 at room temperature, followed by three times washing with TBS. Antigen retrieval was done 596 by incubation in citrate buffer pH 6.0 for 30 min at 80°C. The sections were permeabilized with 597 0.1% Triton-X-100, followed by 10% Mouse-to-Mouse Blocking reagent (SkyTek Laboratories, 598 Cat. # MTM015) in 10% normal goat serum and 0.1% Triton-X-100 in TBS for 45 min at room 599 temperature. Sections were incubated overnight at 4°C with mEM48 (1:500, Merck Millipore, 600 Cat. # MAB5374) in 1% normal goat serum and 0.1% Triton-X-100 in TBS. After washing in (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint in 0.1% Triton-X-100 in TBS. After a brief washing, the sections were allowed to dry and then 615 covered with 400 µl Fluoshield mounting medium (Abcam, Cat. # ab104135), and stored at 616

4°C. 617
Automated image acquisition was conducted using the Opera® High Content Screening 618 system and Opera software 2.0.1 (PerkinElmer Inc.), using a 40x water immersion objective 619 (Olympus, NA 1.15, lateral resolution: 0.32 µm/pixel). Image analysis scripts for single-cell 620 analysis were developed using Acapella® Studio 5.1 (PerkinElmer Inc.) and the integrated 621 Acapella® batch analysis as part of the Columbus® system. 622 623

High-Resolution ARG: Co-registration of [ 3 H]CHDI-180 binding and mEM48 staining 624
Ten µm-thick sections were prepared from PBS-perfused brains of hom, het, and wt mice or 625 human post-mortem brain samples by using a cryostat, mounted on superfrost slides, and 626 stored at -80°C for a maximum of 2 weeks. On the day of the experiment, slides were adapted 627 to room temperature for 30 min. The sections were post-fixed with 4% paraformaldehyde for 628 10 min followed by two times washing with TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl). 629 Epitope retrieval was done with 1% formic acid for 10 min followed by washing with TBS. 630 Slides were equilibrated by immersion into autoradiography assay buffer (50 mM Tris-HCl pH 631 7.4; 120 mM NaCl; 5 mM KCl; 2 mM CaCl2; 1 mM MgCl2) for 20 min at room temperature. 632 Radioligand solutions (3 nM ± 10 µM unlabeled compound) were prepared in assay buffer. 633 Optimal radioligand concentrations were determined in advance based on the signal-to-634 background ratio obtained in mouse or human brain tissue. The solutions were mixed in a 635 Coplin jar (Sigma, S5516-6EA) by gently shaking at 175 rpm for 10 min at room temperature. The slides were allowed to dry and were then covered with NTB emulsion (Kodak/Carestream; 653 8895666). After drying overnight, the slides were exposed for three weeks at 4°C under 654 lightproof conditions. Photographic development was done by applying the developer X-tol 655 (Kodak; KODAK008) and Vario Fix Powder (Tetenal; S32138) according to the manufacturer´s 656 instructions. Slides were washed with distilled water, allowed to dry for a minimum of 5 hours, 657 and covered in Poly-Mount Mounting Media (Polysciences Europe; 08381-120). Image 658 analysis was done using the PreciPoint M8-S microscope with a 40x air objective. 659 660

Immunostaining for Imaging Studies 661
The following primary antibodies were used for immunostaining: monoclonal mouse anti-mHTT 662 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint distinct mHTT species such as inclusion bodies, diffuse nuclear signals, and small nuclear 669 puncta as each antibody may have a higher affinity towards specific species 43 . To assess 670 neuroinflammation/glial reactivity following the intra-striatal injection during the therapy studies, 671 GFAP and IBA1 IHC were executed. For investigating the striatal distribution and efficacy of 672 the ZFP therapy, a co-staining with mEM48 and ZNF-10 was performed. Visualization of the 673 striatal PDE10a levels following ZFP therapy was accomplished by PDE10a staining using the 674 EPR22383 antibody. 675 Sections were air-dried for 5 min, incubated with 4% paraformaldehyde (PFA) for 10 min for 676 tissue post-fixation, and washed using phosphate-buffered saline (PBS). Next antigen retrieval 677 was performed by placing the slides in a container with citrate buffer into a water bath at 80 ºC 678 for 30 min. Then, the slides were cooled at room temperature for 20 min. After rinsing steps 679 with PBS, endogenous peroxidases were inactivated by a 3% H2O2 solution (for colorimetric 680 IHC). Non-specific binding sites were blocked using 5% normal goat serum (NGS) and 0.5% 681 Triton X-100 in PBS for 30 min and goat anti-mouse Fab fragment IgG (26 µg/ml) for 1 hour 682 (for mEM48 or 2B4); 10% NGS and 0.2% Triton X-100 (GFAP, IBA1), 10% normal donkey 683 serum (NDS) and 0.2% Triton X-100 (PDE10a) in PBS for 1 hour; or 10% NDS and 0.5% Triton 684 X-100 (ZNF10/mEM48) in PBS for 1 hour followed by donkey anti-mouse Fab fragment IgG 685 (1:50; 715-006-151; Jackson Immunoresearch) for 1 hour. Next, sections were incubated 686 overnight at room temperature with the primary antibody in PBS: mEM48 with 3% bovine 687 serum albumin (BSA); 2B4 with 0.1% Triton X-100; polyclonal primary antibodies anti-GFAP 688 or anti-IBA1 with 5% NGS; mEM48 and anti-ZNF-10 with 1% NDS and 1% Triton X-100; anti-689 PDE10a with 5% NDS and 0.1% Triton X-100. The next day, sections were rinsed with PBS 690 before a 1-hour incubation with the secondary antibody in PBS: horseradish peroxidase (HRP)-691 conjugated goat anti-mouse (Jackson Immunoresearch, UK) either at 1:500 with 1% NGS for 692 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. procedure. In short, the brain was exposed by drilling a small hole with an electrical drill 787 allowed to recover on a heating pad before returning to their holding. Following the appropriate 793 time to recover, animals were shipped to MICA for imaging studies. 794 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint acquired at each time point of the therapy studies (3, 6, or 10 months) in zQ175DN mice, as 798 well as at 13 months of age in the LacQ140 I (*) mice to generate genotype and age dedicated 799 templates for volume-of-interest (VOI) delineation and co-registration purpose as previously 800 The radioligand was prepared using an automated synthesis module (Carbonsynthon I, 813 Comecer, The Netherlands) adapting the previously described procedure 8 to our system. 814 [ 11 C]CHDI-180R was prepared via single-step carbon-11 labeling starting with 0.5 mg of 815 precursor in 0.5 ml of dimethylformamide (DMF), which was reacted with [ 11 C]MeI, in the 816 presence of Cs2CO3 (2.5-3 mg) for 3.5 min at room temperature. To terminate the reaction and 817 to ensure good retention of the compound of interest on the HPLC column, 0.7 ml of water for 818 injection (WFI) was introduced to the reaction mixture, and the resulting crude product was 819 purified on HPLC using a Waters XBridge C18 5µm, 10 mm × 150 mm column (Waters, 820 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint a flow rate of 4 ml/min. The collected fraction was sterile-filtered through a 0.22 µm Nalgene 822 PES filter and diluted with sterile saline to decrease the ethanol concentration below 10%. The 823 radiochemical purity was greater than 99% as determined using a Kinetex EVO C18, 5µm, 150 824 The Netherlands) adapting the previously described procedure 47 to our system. Briefly, 861 (1 mg) and aqueous NaOH (1 M, 5 µl) in reagent grade acetone (300 µl) at room temperature. 864 Ninety seconds after the end of trapping, the reaction mixture was quenched with water for 865 injection (600 µl) and purified using isocratic semi-preparative reverse phase HPLC (Waters 866 XBridge™ Prep C18 5 µm 10×150 mm, λ = 254 nm; mobile phase: ethanol/phosphate buffer 867 pH 5.5 40/60 V/V, 3 ml/min). The collected fraction was sterile-filtered through a 0.22 µm 868 Nalgene PES filter and diluted with sterile saline to decrease the ethanol concentration below 869 10 %. Radiochemical purity (RCP) was > 99% as determined by analytical isocratic reverse-870 phase HPLC (Waters Symmetry ® C18 3.5 µm 4.6×50 mm, λ = 220 nm; mobile phase: sodium 871 1-heptanesulfonate 2 g/L pH 3.9/acetonitrile 70/30 v/v, 1 ml/min). 872 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint performed on a young (4-month-old) and an aged (10-month-old) cohort of wt and het 876 zQ175DN mice (n = 3-4 for each genotype, time point, and age) adapting the previously 877 described procedure 48,49 . Mice were injected via the lateral tail vein with [ 11 C]CHDI-180R (11.7 878 ± 2.9 MBq in 200 μl) while keeping cold mass within tracers condition (<1.5 µg/kg). Animals 879 were anesthetized, and blood was withdrawn via cardiac puncture and the brain was rapidly 880 removed by dissection at 15, 25, and 40 min p.i. Following centrifugation of blood at 2377 × 881 rcf at 4°C for 5 min, both plasma and brain samples were counted in a gamma counter 882 (Wizard 2 , PerkinElmer) to determine the total radioactivity. Next, equal amounts of ice-cold 883 acetonitrile and 10 µl of cold reference (1 mg/ml) were added to the plasma samples. The brain 884 samples were homogenized in ice-cold acetonitrile (1 ml), 10 µl of cold reference compound 885 (CHDI-180) was added as well. After another centrifugation at 2377 × rcf at 4°C for 5-min, the 886 supernatant was separated from the precipitate and both fractions were counted in the gamma 887 counter to calculate the extraction efficiencies in both plasma and brain samples. Extraction 888 efficiency did not differ among genotypes and ages for plasma (4 months: wt = 94.5 ± 0.4%, 889 het = 94.1 ± 0.6%; 10 months: wt = 94.2 ± 1.2%, het = 94.0 ± 0.8%, mean ± s.d.) and brain 890 samples (4 months: wt = 84.8 ± 1.4%, het = 82.2 ± 4.2%; 10 months: wt = 85.6 ± 2.5%, het = 891 84.2 ± 4.7%, mean ± s.d.). Next, 100 μl of supernatant were loaded onto a pre-conditioned 892 reverse-phase (RP)-HPLC system (Kinetex, 150×4.6 mm, 5 μm HPLC column + Phenomenex 893 security guard pre-column) and eluted using an isocratic system comprised of 0.1% TFA in 894 H2O and acetonitrile: NaOAc 0.05M pH 5.5 (80/20 v/v) buffer at a flow rate of 1 ml/min. RP-895 HPLC fractions were collected at 0.5 min intervals for 8 min and radioactivity was measured in 896 the gamma counter. The radioactivity was expressed as a percentage of the total area of the 897 peaks based on the radiochromatograms. Blood and brain samples spiked in vitro with 37 kBq 898 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. [ 18 F]MNI-659 and [ 11 C]SCH23390. PET scans were followed by a 10 min 80 kV/500 μA CT 920 scan on the same gantry for attenuation correction and coregistration purposes. Acquired PET 921 data were reconstructed into 33 or 39 (for 60 or 90 min acquisition, respectively) frames of 922 increasing length (12 x 10s, 3 x 20s, 3 x 30s, 3 x 60s, 3 x 150s, and 9 or 15 x 300s)  PET images and assessed for accuracy following spatial transformation. Using the VOI 937 template adapted from the Waxholm atlas 51 (as shown in Extended Fig. 3), time-activity curves 938 (TACs) for the striatum, motor cortex, hippocampus, thalamus, and cerebellum were extracted 939 from the dynamic PET images to perform kinetic modeling. 940 Since we previously observed that the use of magnetic resonance imaging (MRI) templates for 941 spatial normalization and VOI definition improves the accuracy of the regional quantification of 942 PET data with focal uptake, the therapeutic and LacQ140 I (*) studies were processed using an 943 MR-based pipeline 34 . VOIs were manually adapted from the Waxholm atlas 51 to match each 944 genotype and age-specific MR template. TACs for the striatum, motor cortex, hippocampus, 945 thalamus, and cerebellum were extracted from the dynamic PET images in order to perform 946 kinetic modeling. 947 For analysis of spinal cord and peripheral tissue (heart, liver, muscle, and adipose tissue), 948 VOIs were manually drawn on the individual CT images, and TACs were extracted from the 949 dynamic scans for regional quantification. 950 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint quantification for [ 11 C]CHDI-180R was performed to calculate the total volume of distribution 954 based on image-derived input function (VT (IDIF)) as a non-invasive surrogate of the VT. Kinetic 955 modeling fitted regional TACs using the Logan model 27 and the image-derived input function 956 (IDIF) with the start of the linear regression (t*) calculated according to the maximum error 957 criterion of 10%. The IDIF was obtained from the whole blood activity derived from the PET 958 images by generating a region-of-interest (threshold-based 50% of max) in the lumen of the 959 left ventricle as previously described 24,48 . Since only negligible metabolism of [ 11 C]CHDI-180R 960 was observed in different genotypes and ages (parent compound >95%), no correction for 961 radiometabolites was applied. 962 Parametric VT (IDIF) maps were generated through voxel-based graphical analysis (Logan) 27 963 using the IDIF as input function, and were then cropped using the brain mask of the MRI 964 template, represented as group averages, and overlaid onto a 3D mouse brain template for potential (BPND) was determined by fitting the regional TACs using the simplified reference 975 tissue modeling (SRTM) 52 . The striatum was selected as the receptor-rich region and the 976 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint generated using SRTM2 53 with the k2' as calculated with SRTM 52 . The individual images were 978 smoothed with an isotropic gaussian filter (0.5 mm in full width at half maximum), cropped 979 using the brain mask of the MRI template, represented as group averages, and overlaid onto 980 each condition-and age-specific 3D brain template for anatomical reference. 981 In the early and late ZFP intervention studies, the therapeutic response of each molecular 982 target was estimated as follows according to Fig. 3b and 4b: was also dissected and sections of 30 µm thickness were collected. 1000 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint frozen or PFA-perfused groups for post-mortem assessment. For the fresh-frozen group, . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint mice were injected with ZFP constructs containing an HA-tag, which allows for better . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. All requests for data will be promptly reviewed by the institutions involved to verify whether the 1272 request is subject to any intellectual property or confidentiality obligations. If deemed 1273 necessary, a material transfer agreement between requestor and institutions involved may be 1274 required for sharing of some data. Any data that can be freely shared will be released. 1275 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021.  hemisphere as well as percentage contralateral difference compared to PDE10a levels (a-d), 1354 D1R (e-h), and D2/3R (i-l) at 10 months of age following early (2 months) and late (5 months) 1355 intervention. Early intervention = het ZFP, n = 10; het vehicle, n = 11; wt vehicle, n = 11. Late 1356 intervention = het ZFP, n = 26; het vehicle, n = 20; wt vehicle, n = 23. Two-tailed Pearson 1357 correlation analyses, all points shown. Related to Figure 3 and 4. 1358 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. vehicle, and het ZFP-treated mice at 8 months (het ZFP, n = 11; het vehicle, n = 10; wt vehicle, 1393 n = 10) of age following striatal injection at 5 months of age. Kruskal-Wallis test with Dunn's 1394 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint comparison test (g,i) and Kruskal-Wallis test with Dunn's correction for multiple comparisons 1399 (h); * P < 0.05, *** P < 0.001, **** P < 0.0001; Data are shown as mean ± s.d., all points are 1400 shown. BLOQ, below limit of quantification. 1401 . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. 100% n = 12 n = 3 n = 3 n = 3 90% n = 15 n = 3 n = 3 n = 3 80% n = 18 n = 3 n = 3 n = 3 70% n = 23 n = 3 n = 3 n = 3 60% n = 31 n = 3 n = 3 n = 3 50% n = 44 n = 4 n = 3 n = 3 40% n = 66 n = 6 n = 3 n = 3 30% n = 113 n = 8 n = 4 n = 3 20% n = 258 n = 16 n = 6 n = 4 Therapeutic efffect (%) Sample size required per experimental arm (n ) . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. 2014-043 Control F 60 8 n/a n/a 0 2017-005 Control F 60 5.5 n/a n/a 0 2017-060 HD F 57 6.5 n/a n/a 0 2008-044 HD M 59 5 n/a n/a n/a . CC-BY-NC 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 11, 2021. ; https://doi.org/10.1101/2021.07.09.451725 doi: bioRxiv preprint