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A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo

Abstract

In many repeat diseases, such as Huntington’s disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSβ. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats.

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Fig. 1: NA binds to long CAG slip-outs.
Fig. 2: NA specifically inhibits repair of long CAG slip-outs by human (HeLa) cell extracts.
Fig. 3: NA cellular distribution, non-toxicity and effects on repeat instability in HD patient cells.
Fig. 4: NA induces CAG contractions independent of proliferation, dependent on rCAG transcription.
Fig. 5: NA affects interaction of DNA repair proteins on long CAG slip-outs.
Fig. 6: NA induces CAG contractions in R6/2 mouse striatum.
Fig. 7: NA induces a reduction in mHTT aggregates in R6/2 mice.
Fig. 8: Schematic of the plausible mechanisms through which NA may induce contractions of expanded CAG tracts.

Data availability

Raw sequencing data have been deposited at the Sequence Read Archive (SRA) under accession numbers SRR10532698, SRR10532697, SRR10532700 and SRR10532699. Source data for Figs. 18 and Extended Data Figs. 1 and 4 are provided online.

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Acknowledgements

This work was partially supported by the Canadian Institutes of Health Research (FRN388879, J.-Y.M. and FRN148910, C.E.P.), Natural Sciences and Engineering Research Council (RGPIN-2016-08355, C.E.P.), Muscular Dystrophy Canada (C.E.P.), Tribute Communities (C.E.P.), The Petroff Family Fund (C.E.P.), The Kazman Family Fund (C.E.P.), The Marigold Foundation (C.E.P.), The National Center of Neurology and Psychiatry (29-4, M.N.), a JSPS KAKENHI Grant-in-Aid for Young Scientists (Start-up A, 24890110 and 25713034, M.N.), Scientific Research (B, 16H05321, M.N.) and Specially Promoted Research (26000007, K.N.), the Cancer Research UK Catalyst Award (C569/A24991, R.G.), the US National Institutes of Health (2 R01 ES014737, M.Y.W.T.L.) and a US National Institutes of Health (NIH) grant (HG010169 to E.E.E.). E.E.E. is an investigator of the Howard Hughes Medical Institute, A. Shlien holds the Canada Research Chair in Childhood Cancer Genomics, J.-Y.M. holds the Fonds de Recherche de Santé Québec Chair in Genome Stability, and C.E.P. holds the Canada Research Chair in Disease-Associated Genome Instability. We acknowledge the technical support of R. Manabe, K. Hayashi, P. Wang, L. Yu, M. Mirceta, N. Thakkar, I. Panigrahi, D. Ripsman, H. Adhikary, S. Bérubé, Y. Coulombe, A. Couturier, M. Scofield Sorensen and K. Hoekzema. We acknowledge TCAG (SickKids) for expedited sequencing.

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Authors

Contributions

M.N. performed repeat length analysis, and cell and mouse treatments. G.B.P. performed replication, repair, footprinting and R-loop processing. S.L. performed NA binding, repeat instability analysis, protein interactions, polδ extension assay and instability index assessment. H.H., H.T., M.P.T. and H.M. performed mouse treatments. T.O., J.Li, A. Sakata, A.M. and K.N. synthesized, purified and characterized NA. J.Luo and T.P. performed cell treatments. T.G.-D. performed assessment of NA on mHTT aggregates, HTT translation and TUNEL assay. M.-C.C., N.J., J.-Y.M., M.S.W., X.W. and M.Y.W.T.L. performed protein purification. S.L., G.B.P. and K.C. performed in vitro repair/binding assays. J.H., K.M.M. and E.E.E. performed single-molecule sequencing and bioinformatic analysis. R.G. and M.S.-K. performed MSI assay. S.D., M.L., L.-M.E. and A. Shlien performed whole-genome sequencing and mutation signature analysis. C.E.P., M.N., K.N., G.B.P. and S.L. conceived experiments, analyzed data and wrote the manuscript. M.N., G.B.P. and S.L. contributed equally to the study. All authors discussed the results and commented on the manuscript.

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Correspondence to Christopher E. Pearson.

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Extended data

Extended Data Fig. 1 NA does not affect replication efficiency or replication fork progression.

Three circular plasmids containing the SV40 origin of replication, and an expanded (CAG)79•(CTG)79 repeat tract (pDM79EF and pDM79HF) or no repeats (pKN16), were replicated in vitro by human (HeLa) cell extracts without or with NA (7.5 µM or 15 µM) treatment. The location of SV40-ori determines the replication direction and which strand will be used as the leading or the lagging strand template. pDM79HF uses the CAG strand as the lagging strand template, while pDM79EF uses the CTG strand as the lagging strand template (schematic on the top of the gel panel). Replication products were purified and linearized with BamHI. An equal portion of the reaction material was also digested with BamHI and DpnI as DpnI digests un-replicated and partially-replicated material, as shown in the schematic (top figure). The digestion products were electrophoresed on a 1% agarose gel to resolve completely replicated and un-replicated material (bottom figure). Equal amount of unreplicated plasmid DNA was digested with DpnI and stained with Ethidium Bromide to show the complete digestion of unreplicated plasmid DNA (Bottom panel). Panel I, ethidium bromide stained, Panel II, autorad: marker (lane 1); DpnI undigested plasmid DNA (lane 2); DpnI digested unreplicated plasmid DNA (lane 3-4); replicated plasmid DNA, DpnI resistant (lane 5). No difference in DpnI resistant material is observed between replication in the presence or absence of NA, in all the three templates tested (panel III, IV, V). Blots have been cropped and the corresponding full blots are available in the Source Data files.

Source data

Extended Data Fig. 2 NA does not affect non-mutant genetically stable repeats.

a, b, Representative data showing small-pool PCR (spPCR) for the non-expanded CAG/CTG repeat length of CASK and Mdf15 in HD primary fibroblast cells (a) and spPCR for the non-expanded CAG/CTG repeat length of CASK, Mdf15, ATXN8 and the non-expanded HTT allele genes in HT1080-(CAG)850 cells (b). Even under the sensitive mutation detection capacity of spPCR, length variation was not observed in either NA treated- and untreated-cells. Notably, some reactions did not show any product as is typical of the low genomic DNA template dilutions used in spPCR. c, The repeat-tract lengths of the CASK, ATXN8, and Mfd15 loci in HT1080-(CAG)850 cells (initial clone and cells after 30 days incubation with or without NA). Length variation was not observed at any of these repeats of normal length loci in HT1080-(CAG)850 cells (after 30 days incubation with or without NA). Three independent experiments were performed. d, spPCR for the non-expanded CAG tracts in TBP alleles in HD patient fibroblasts treated with or without NA for 40 days. e, spPCR for the non-expanded CAG tracts in TBP alleles in HD R6/2 mouse striatum with four injections of NA or saline. f, Microsatellite instability assay. Assay scores >1.3 indicate increased MSI relative to a control sample set from peripheral blood leukocytes. Both NA positive and NA negative HD cells with (CAG)43 or (CAG)180 scored <1.3, indicating no effect of treatment on MSI. Eight known CMMRD-negative controls and 3 known CMMRD-positive controls were included in the assay.

Extended Data Fig. 3 NA is not a general mutagen.

Towards assessing whether NA-treatment acted as a general mutagen to sequences other than CAG slip-outs, we harnessed the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS). Single-molecule sequencing was done on the HPRT1 gene – widely used as a surrogate indicator of the global effect of induced genetic variation. For each replicate, we calculated the relative mutation rate between NA- and saline-treated cells as the mutation rate for NA-treated cells minus the rate for saline-treated cells and identified excess mutation rates based on an absolute relative rate >0.5%. a, Schematic of HPRT1 sequencing for mutation detection. Briefly, cells were grown under identical conditions differing only by the addition of NA (50 μM) or saline, DNAs were isolated, HPRT1 exons 2 and 3 PCR amplified and sequenced. b, Quality control for our analysis. c,d, Comparison of sequence variations between NA-treated and saline treated is presented. We chose to compare the single-molecule sequence reads of individual X chromosome-linked HPRT1 alleles (exons 2 and 3) from our male HD patient-derived cells (c), and our male R6/2 mice (d), that had been NA- or saline-treated. Each read represents a single cell (Supplementary Note). Graphs show the distribution of sequence variants by relative mutation rate between three experimental replicates of NA-treated and saline-treated cells sequenced with PacBio single-molecule long reads.

Extended Data Fig. 4 NA does not affect HTT transcription or translation.

a, NA does not affect transcription across expanded repeats in HTT in HD patient cells, determined by quantitative real-time reverse transcriptase (qRT)-PCR and normalized to U6 RNA. Data are indicated as the mean ± s.d. of independent triplicates. b, Western blot showing that NA does not affect HTT translation in HD patient cells with (CAG)43. Western blots were repeated 4 times with similar results. Blots have been cropped and the corresponding full blots are available in the Source Data files. c, Extraction of NA from DNA by solvents.

Source data

Extended Data Fig. 5 NA induces contractions during R-loops processing.

a, Schematic of R-loop formation, processing, and analysis. Pre-formed double-R-loops were processed by terminally differentiated (retinoic acid) human neuron-like cell extracts (SH-SY5Y) in the absence or presence of NA (50 μM), as described and DNA repeat lengths scored as expansions, contractions, or stable, by the STRIP assay (Methods). b, Representative example of STRIP analysis. Transcription products were isolated, processed and transformed in E. coli cells, previously shown to stably maintain the (CAG)79•(CTG)79 lengths (Methods). Plasmids isolated from individual bacterial colonies were digested with restriction enzymes to release the repeat containing fragment, resolved on 4% polyacrylamide gels and scored for instability. c, Graphical analysis of STRIP results. Two-sided χ2 test was performed to compare 191 untreated colonies vs. 100 NA-treated colonies.

Extended Data Fig. 6 Dosing regimen.

A single drug administration involved six separate stereotactic injections (three injections of drug in saline or saline into three different striatal regions of either the left or right striatum, respectively). At the onset mice were 6-weeks old.

Extended Data Fig. 7 Instability Index calculation.

Instability index determination was as described77,78, using a relative peak height threshold, with modifications. To quantify the levels of instability from GeneMapper traces peak height was used to determine a relative threshold of 20% based upon the main peak in the shorter mode of the control striatum (see points 1 & 2 in the figure). We used a conservative threshold factor (20%) as this detects peaks with good signal intensity and is more resistant to amplification variation than lower thresholds. Lower thresholds (10%, 5%) can provide more sensitive quantification. Peaks falling below his threshold were excluded from analysis. Peak heights were scored (see point 3) and normalized to the total of all peak heights in a given scan (see point 4). Since we are comparing the effect of NA versus saline upon instability in the striatum, the CAG length distribution in tail is not a factor in this comparison, but is for determining absolute instability, as in previous studies62,66. So as to facilitate comparison between NA and saline-treated striatum, these were normalized by multiplying the values by the change in CAG length of each peak relative to the highest peak in saline-treated striatum (see point 5), as opposed to the highest peak in the tail, as previously done77,78. These normalized values (see point 6) were summed to generate the instability index (see point 7). Striatum analysis for mouse vi is shown as an example R6/2, 6-weeks treated with four injections spanning 4 weeks of saline (red) or NA (blue). Peaks of the main allele in the saline-treated striatum, NA-treated striatum and tail of the same mouse, are indicated by triangle-brackets at the top (see point 1).

Extended Data Fig. 8 A total of ten HD mice revealed consistent NA-induced contractions of expanded CAG repeats.

Instability Indices in striatum of ten mice (iv-xiii) treated four times with saline in the right striatum and NA in the left striatum. Indices in NA-treated striatum were significantly different from the control saline-treated striatum (Mann-Whitney, P= 0.00035). Instability Indices for mouse v and xi are positive for both NA and saline as there are less data points to the left of the highest peak compared to the points to the right. Still, after NA treatment there is a reduction in the index.

Extended Data Fig. 9 NA does not induce cell death in the CNS and cell proliferation, and does not affect transcription across the Htt locus.

a, Histological study, mouse striatum with saline, NA in saline, or no injection, followed by H&E staining. Three independent experiments were performed. b, NeuN staining showing that NA does not induce cell death. Quantification of NeuN positive cells below. Data are indicated as mean ± s.d. of triplicates. c, Doublecortin staining showing that NA does not induce cell proliferation. Three independent experiments were performed. d, The effect of NA on TUNEL signal as assessed via fluorescent microscopy and immunohistochemistry. Representative 40x magnification confocal images of striatal medium spiny neurons (MSNs) of R6/2 mice treated with saline (right striata) and 50 μM NA (left striata) stained for TUNEL (red, staining apoptotic cells), and DARPP32 (green, staining MSNs). Panel locations (i-vi) correspond to the locations outlined in Fig. 7 (middle panels). e, NA does not affect transcription across expanded repeats in HTT in HD patient cells and mouse striatum, determined by quantitative real-time reverse transcriptase (qRT)-PCR and normalized to U6 RNA, expressed as the ratio of NA-treated vs. PBS-treated R6/2 striatum. Data are indicated as mean ± s.d. of independent triplicates.

Supplementary information

Supplementary Information

Supplementary Note, Figs. 1–6 and Table 2

Reporting Summary

Supplementary Table 1

Summary of the instability data from analysis of HD patient fibroblasts GM09197 with (CAG)180 and GM02191 with (CAG)43, and the cell model HT1080, and complete spPCR datasets for each repetition of each experiment and the associated graphs.

Source data

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Nakamori, M., Panigrahi, G.B., Lanni, S. et al. A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo. Nat Genet 52, 146–159 (2020). https://doi.org/10.1038/s41588-019-0575-8

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