Figure 3. (A) Effect of the ON length on the HTT mRNA knockdown efficiency. LNA/DNA CAG 10- to 19-mer PS ONs were transfected at 100 nM concentration into.

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Figure 3. (A) Effect of the ON length on the HTT mRNA knockdown efficiency. LNA/DNA CAG 10- to 19-mer PS ONs were transfected at 100 nM concentration into GM04281 human HD fibroblasts using RNAiMAX. Four days post treatments, cells were lysed and RNA was isolated and analyzed by qRT-PCR using primers and probes for HTT and for HPRT as a housekeeping reference gene. HTT mRNA levels after treatment with CAG-12, -13, -14, -15, -16, 18- and -19 were all significantly reduced as compared to CAG-10PS and to all the mismatched and scrambled controls (significance levels between each ON and the scrambled ON1 are shown in the figure). CAG-15PS differed significantly as compared to CAG-12PS, CAG-14PS and CAG-18PS. CAG-16 PS differed also significantly as compared to CAG-14PS and CAG-18PS. No other significant differences were found between the ONs from 12 to 19-mer in HTT mRNA knockdown levels. (B) CAG ONs down-regulate HTT mRNA more potently as compared to control CTG ONs. The 13- and 19-mer ONs of either CAG or CTG sequence were transfected into HD fibroblasts GM04281 for 4 days and the remaining HTT mRNA levels were assessed using q RT-PCR. A significant difference was found between the remaining HTT mRNA levels after CAG-19PS versus control CTG-19PS treatment (P < 0.01) and they were both significantly different from the control ON (mismatched 1) (P < 0.001 and P < 0.001). CAG-13PS effect was significantly different from mismatched1 ON (P < 0.00001). CTG-13PS and CTG-13PS control ONs did not show any significant difference compared to the control mismatched ONs used in the study. From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 4. Effect of the ON length on the HTT protein knockdown efficiency and allele selectivity. LNA/DNA CAG 10- to 19-mer PS ONs were transfected at 100 nM concentration into GM04281 human HD fibroblasts using RNAiMAX. Four or seven days post treatments, cells were lysed and western blots were performed to detect the HTT protein with its two isomers: muHTT and wtHTT. Importin 7 was used as a housekeeping control. (A) Shows a representative western blot gel for the 4 days-time point, while (C) shows a representative gel for 7 days. Results from three replicate experiments were quantified and plotted in (B) and (D) for the 4 and 7 days data, respectively. Total HTT protein levels remained after treatments with ONs (from 12 to 19-mers) were significantly different from all control ONs tested in the study. Significance levels between each CAG ON and the control ON (mismatched 1) are shown in panel (B) and (D). No significant difference was found between the control ONs used in the study. A significant difference was found between the remaining muHTT and wtHTT protein levels 4 and 7 days after treatment with CAG-19PS (P < 0.01 and P < 0.05, respectively). From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 5. CAG ONs versus control CTG ONs in HTT protein down-regulation efficiency. The 13- and 19-mer ONs of either CAG or CTG sequence were transfected into HD fibroblasts GM04281 for 4 days or 7 days followed by cell lysis and western blots. (A) and (C) show representative western blot gels for the 4 and 7 days data, respectively. Quantification of western blot gels is shown in (B) for the 4 days and (D) for the 7 days results. Significant difference was found between the remaining muHTT and wtHTT protein levels 4 and 7 days after treatment with CAG-19PS (P <0.01 and P < 0.05) and with control CTG-19PS (P < 0.01and P < 0.05). No other significant difference was found between muHTT and wtHTT after treatment with any other ON. Total HTT protein was significantly reduced after treatment with CAG-19PS, control CTG-19PS and CAG-13PS as compared to the mismatched controls (significance levels between each ON and mismatched 1 are shown in the panels B and D). Control CTG-13PS and 13PSgap were not significant compared to the mismatched control ONs. From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 6. UV melting profiles of CAG, CTG and target RNA ONs Figure 6. UV melting profiles of CAG, CTG and target RNA ONs. All graphs show the Tm curves (marked ‘Tm’ in the figure) together with the second derivative in the background. (A) target RNA alone, (B) CTG13 alone, (C) RNA+CTG13, (D) CAG13 alone, (E) RNA+CAG13, (F) CAG19 alone and (G) RNA+CAG19. Only CTG13 is capable of binding the target RNA and shift the Tm above the baseline of the RNA ON alone. From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 7. Binding of CAG ONs to target plasmid DNA visualized by restriction digestion and PAGE. Panel (A) shows the gel lanes with control and ON-hybridized plasmids digested with HindIII and AvrII, as indicated. * Denotes bands believed to originate from a subclone containing a truncated repeat region. Panel (B) schematically illustrates the mechanism by which the ONs hinder the cleavage by AvrII. Upper panel shows the cleavage pattern when no part of the binding ON is interfering with AvrII cleavage. Lower panel shows that longer ON binding randomly increases the probability of steric blocking of the Avr-site. From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 8. Serine 2 phosphorylated RNA Pol II occupancy across the HTT gene is reduced upon transfection with CAG-19PS (A) UCSC genome browser image demonstrating RNA Pol II ser2 occupancy across the HTT gene. Primer positions at exon 1 upstream of the CAG repeat and at exons 30 and the last exon, 67, are indicated. (B) RNA pol II occupancy is significantly increased at exon 1 of the HTT gene immediately upstream of the CAG repeat (*P < 0.05) and reduced at the 3΄ exons 30 and 67 (*P < 0.05, ***P < 0.001). (C) No difference is observed at control loci in the last exons of the PRDM1 or ACTB genes. From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 9. CAG ONs can be delivered to the patient fibroblasts by peptide and gymnotic delivery. (A) LNA/DNA CAG 10- to 19-mer PS ONs were transfected at 100 nM into GM04281 human HD fibroblasts using RNAiMAX or PF14 at molar ratio 1:5 (ON: peptide) or were added to the cells at 1 μM without transfection reagents (gymnosis). Control CTG-19PS, mismatched, scrambled ONs and siRNA were used as controls. Four days post treatments, the cells were lysed and RNA was isolated and analyzed by q RT-PCR using primers and probes for HTT and HPRT as a housekeeping reference gene. Significant differences were found between CAG-19PS and CTG-19PS using all delivery methods (RNAiMAX (P < 0.01), PF14 (P < 0.01), gymnosis (P < 0.0001)). All ONs ranged from 12 to 19-mer delivered by gymnosis significantly reduced HTT mRNA (significance levels are shown in the graph). (B) LNA/DNA CAG 12-, 13- or 14-mer ONs with either PS or PO backbone were transfected at 100 nM concentration after formulation with RNAiMAX or with PF14 at molar ratio 1:5 (ON: peptide) into human HD patient fibroblasts GM04281. There was no significant (ns) difference between RNAiMAX and PF14 formulations with CAG-13PS and CAG-14PS. For PF14 formulations, the mismatched controls differed significantly from all treatments except for CAG-12PS, CAG-12PO and CAG-13PO (significance levels for all ONs compared to mismatched 1 are shown in the figure). From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 1. CAG phosphorothioate (PS) oligonucleotides (ONs) reach the nucleus and significantly down-regulate Huntington (HTT) gene expression on mRNA level. LNA/DNA CAG 12-, 13- or 14-mer ONs, with either PS or PO backbone, were transfected at 100 nM concentration after formulation with the commercial cationic lipid RNAiMAX into human HD patient fibroblasts GM04281. Four days post treatment, the cells were lysed and RNA was isolated and further analyzed by quantitative reverse transcriptase multiplex PCR (qRT-PCR). (A) Shows the HTT mRNA levels after each treatment normalized to HPRT as a housekeeping reference gene. Significant differences were found between CAG-12PS versus CAG-12PO (P < 0.05), CAG-13PS versus CAG-13PO (P < 0.0001) and CAG-14PS versus CAG-14PO (P < 0.01). Mismatched ON1 and mismatched ON2 were used as control ONs and were significantly different from all constructs except for the 13PO and 14PO (significance levels shown in the figure). (B) Shows the uptake behavior of Cy5 labelled ONs (PS or PO backbone) after transfection into GM04281 fibroblasts as detected by fluorescence microscopy of live cells. From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 2. CAG PS ONs significantly down-regulate HTT gene expression on protein level. LNA/DNA CAG 12-, 13- or 14-mer ONs, with either PS or PO backbone, were transfected at 100 nM concentration after formulation with RNAiMAX into human HD patient fibroblasts GM04281. (A and B) Four days or (C and D) seven days post treatment, cells were lysed and western blots were performed to detect the HTT protein with its two isomers: mutant HTT (muHTT) and wild-type (wtHTT). Importin 7 was used as a housekeeping control. (A) Shows a representative western blot gel for the 4 days-time point, while (C) shows a representative gel for 7 days. Results from three replicate experiments were quantified and plotted in (B) and (D) for the 4 and 7 days data, respectively. There were significant differences between the total (mutant + wild-type) remaining HTT protein amounts after the treatment with: CAG-12PS versus CAG-12PO (P <0.01 and P < 0.01, for 4 and 7 days), CAG-13PS versus CAG-13PO (P < 0.0001 and P < 0.05, for 4 and 7 days), CAG-14PS versus CAG-14PO (P < 0.01 and P < 0.05, for 4 and 7 days). Mismatched ON1 and ON2 were used as controls and there was no significant difference between them, whereas total HTT protein levels obtained by all PS ONs were significantly different from that obtained by mismatched ON1 (significance levels shown in the figure). From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 10. Effect of ON chemical modifications on efficiency Figure 10. Effect of ON chemical modifications on efficiency. (A) CAG-14PS, CAG-14PS-OMe and CAG-14PS-gly were transfected at 100 nM into GM04281 human HD fibroblasts using RNAiMAX, or were added to the cells at 1 μM concentration without transfection reagents (gymnosis). Four days post treatments, the cells were lysed and RNA was isolated and analyzed by q RT-PCR using primers and probes for HTT and HPRT. Significant differences were found between mRNA levels after treatment with: CAG-14PS versus CAG-14PS-OMe (P < 0.05), CAG-14PS versus CAG-14PS-gly (P < 0.01). Significance levels of all ONs compared to mismatched 1 ON are shown in the figure. (B) A dose response curve for the three ONs after gymnosis with increasing concentrations of each ON in GM04281. From: CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression Nucleic Acids Res. 2017;45(9):5153-5169. doi:10.1093/nar/gkx111 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com