Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

Abstract

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10-15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a "molecular tape measure" approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.

Fig. 1.

Zinc finger arrays to bind CAG repeats. (A) A twelve-finger array shows recognition helices contacting 5′-GCT-3′ bases on the lower DNA strand. Similar arrays of 4, 6, 12, or 18 zinc fingers were built (ZF4xHunt, ZF6xHunt, ZF12xHunt, and ZF18xHunt). Nuclear localization signals (NLS) and effectors (e.g., Kox-1 transcription repression domain) were added to N and C termini, respectively. (B) Gel shift assays show 4-, 6-, or 12-finger arrays binding poly-CAG DNA and forming distinct complexes. TNT is a negative control. (C) A hybrid zinc finger design recognizes 5′-GC(A/T)-3′, allowing binding to either the (GCA)_n or (GCT)_n complementary strand of the CAG repeat. A gel shift assay shows equal binding to GCA or GCT triplets in mixed sequences. (D) Specificity gel shift assay. Zinc fingers bind preferentially to CAG repeats (CAG₇) compared with degenerate mutant sequences (DAG₇, CSG₇, or CAH₇; D = A, G, T; S = C, G; H = A, C, T).

Fig. 2.

Episomal poly-CAG reporter repression by ZFxHunt with 0–18 fingers. (A) pEH reporter plasmids contain EGFP, fused to the N-terminal CAG repeats of the human HTT gene, expressing different-length polyQ coding sequences under an SV40 promoter. A control HcRed gene, under a CMV promoter, measures off-target repression. (B) FACS assay to measure the fold reduction in EGFP and HcRed fluorescent cells, in response to different zinc fingers. A 10-fold repression is equivalent to a 90% reduction. Results are the mean ± SEM of three independent experiments. (C) EGFP Western blot for ZFP repression of pEH-Qx targets. (D) qRT-PCR assay to measure fold repression of EGFP or HcRed mRNA by ZFP. Results are the mean ± SEM of four independent experiments.

Fig. 3.

ZFP competition assay against pairs of different-length CAG repeats. Each small square represents one transfection experiment, where cells simultaneously receive two reporter plasmids, polyQ-EGFP and polyQ-mCherry, of different lengths (Q0 to Q104). ZFxHunt constructs with 4, 6, 11, or 18 fingers were tested for their ability to reduce the number of detectable green and red cells in FACS assays (%). Longer polyQ constructs are repressed preferentially, resulting in the shorter green or shorter red polyQ constructs dominating expression.

Fig. 4.

Expression of chromosomal CAG-repeat genes 20 d after retroviral ZFP delivery. Assays were carried out in wt mouse STHdh cells (Q7/Q7), in polyQ STHdh mutants (Q111/Q111), and in human HEK293T cells, as indicated. Mouse and human genes are prefixed by “m” and “h,” respectively. (A) Repression of endogenous HTT by ZF6xHunt and ZF11xHunt, with and without Kox-1 repressor domain. Western blots for HTT (Top) were controlled with β-actin staining and quantified using ImageJ (protein fold repression; Middle). qRT-PCR was used to compare HTT mRNA levels (RNA fold repression; Bottom). The experiment was repeated independently three times with similar results; one experiment is displayed. (B) mRNA levels of other wt CAG-repeat genes are broadly unaffected in STHdh cells (pooled samples: 3 Q7/Q7 and 3 Q111/Q111). Seven genes were tested by qRT-PCR (ATN1, atrophin1; ATXN1–3, 7, ataxin-1–3, 7; CACNA1A, calcium channel α1A subunit; TBP, TATA binding protein). CAG-repeat numbers are in brackets; the first number corresponds to pure CAG repeats, and the second number corresponds to broken CAG repeats (containing CAA or CAT). Two genomic neighbors of HTT [G protein-coupled receptor kinase 4 (GRK4), ∼7 kb upstream; G protein signaling 12 (Rgs12), ∼188 kb downstream] were also unaffected in STHdh cells. (C) mRNA levels of the seven wt human CAG genes and HTT (21 repeats) were also broadly unaffected in HEK293T cells. (D) ZF11xHunt–Kox-1 represses only HTT in a heterozygous human patient-derived cell line (mutant Q45/wt Q21). RNA was extracted from FACS-sorted transduced cells 7 d after infection. (CACNA1A is not expressed in HEK293T cells or the Q45/Q21 patient cell line.)

Fig. 5.

Gene delivery by stereotaxis. (A) Cross-section of mouse brain, injected in one hemisphere with AAV2/1-CAG-GFP-WPRE, reveals widespread green fluorescence in the striatum. (B) Similar distributions are seen when injecting the GFP construct in both hemispheres (as for the behavioral assay). The zinc finger construct AAV2/1–CAG–ZF11xHunt–Kox-1–WPRE was injected at an identical titer in one or both hemispheres as described. (C) Schematic drawings show the maximum and minimum volume covered by GFP expression in mice injected in both hemispheres (n = 4). AP levels are as in the Paxinos and Watson atlas of the mouse brain (54). aca, anterior comissure; Acb, nucleus accumbens; AP, anteroposterior; lo, olfactory tract; St, striatum; Tu, olfactory tubercle. (Magnification, A, B: 2.5× objective; Scale bar, C: 1 mm.)

Fig. 6.

Zinc finger repression in vivo. (A) qRT-PCR data quantify mRNA levels in mouse samples injected striatally with ZF11xHunt–Kox-1, compared with the control hemisphere. The mutant HTT (mut HTT; n = 6) is repressed ∼40% by the zinc finger construct in the striatum, whereas it is unaffected in the noninjected cerebellum. The wt HTT (n = 9) is unaffected in all samples. Control groups contain both untreated and GFP-treated mice (the groups are similar; Fig. S6B). n.s., not significant. (B) Linear regression analysis of zinc finger expression (ZF11xHunt–Kox-1) vs. mut HTT expression shows a significant negative correlation (P = 0.02). Data are from the treated hemispheres (n = 6) and the corresponding untreated hemispheres (mean ± SEM). a.u., arbitrary units. (C) Anti-HTT immunostaining of brain samples reveals a reduction of mutant aggregates in injected striatum with ZF11xHunt–Kox-1 treatment. (Scale bars: 100 μm.) (D) Quantifying HTT-positive aggregates by automatic counting of mut HTT-positive particles with ImageJ software, as previously described (52) (Fig. S7). The data are from three mice and represent comparisons between their injected and noninjected hemispheres in striatum and cortex.

Fig. 7.

Rescue of HD phenotypes with in vivo zinc finger treatment. (A) HD mice show a characteristic clasping behavior (diseased) corresponding to neurological pathology (43). (B) Clasping assay shows a significant improvement after zinc finger treatment in both hemispheres (P = 0.03). Only 1 in 8 zinc finger-treated mice displays symptoms by week 7, compared with 6 in 12 control mice. (C) Performance in the accelerating rotarod shows a clear decline with respect to presurgery levels in the GFP-injected R6/2 mice, whereas zinc finger-treated mice do not show a significant decline compared with wt mice.

Fig. P1.

ZF repressors (4-, 6-, 11-, 12-, and 18-fingers) preferentially bind longer CAG repeats. (A) A 12-finger array has recognition helices that contact 5′-GCT-3′ bases on the lower DNA strand. (B) AAV2/1 viral delivery of GFP and ZF constructs to the mouse striatum. A cross-section of the mouse brain, with a GFP-expressing AAV2/1 viral construct injected in one hemisphere, reveals widespread green fluorescence in the striatum. (C) Similar results were observed following the injection of constructs in both hemispheres (as for subsequent behavioral assays). (Field width, B and C: 8 mm.) (D) HD mice exhibit characteristic clasping behavior (diseased) corresponding to neurological pathology. (E) A clasping assay shows a significant improvement after ZF AAV treatment in both hemispheres (P = 0.03). Compared with 6 of 12 control mice, only 1 of 8 ZF-treated mice exhibited symptoms by week 7.