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Mechanism of Pathogenesis and Recent Developments in Its Therapeutic Strategies

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Mechanism of Pathogenesis and Recent Developments in Its Therapeutic Strategies

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It was first hypothesized that Huntington disease is caused by a misfolded huntingtin protein interrupting the neurological functioning of the brain due to a amplification of adenosine-guanine chain in the huntingtin's amino terminal end. However. studies have recently shown that opposed to this first believed hypothesis, that change in the mRNA of the protein gives rise to misfolded protein and the fragments accumulate, by making interaction with the other elements in cells resulting in the problems associated with HD. This experiment highlights the possible cause, pathogenesis, and therapy treatments that can reduce and down-regulate the expression of huntingtin, lowering the aggregation of the huntingtin protein.

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Huntington disease is caused by a mutation in the gene for a protein called huntingtin. This defect causes the DNA amino acids cytosine, adenine, an guanine to abnormally repeat, causing an expanded hHtt protein. Most people have fewer than 27 CAG repeats in their HD gene, so they are not at risk for the disease (NIH).

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HD is an autosomal dominant disorder, which means one you need one copy of the non typical gene to be inherited. Each child has a 50% chance of inheriting a copy of chromosome 4 that carries the HD mutation (Mayo).

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Huntington disease affects an estimated 3 to 7 per 100,000 people of European ancestry. The disorder appears to be less common in some other populations, including people of Japanese, Chinese, and African descent (MedlinePlus).

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There is currently no cure for Huntington's Disease. However, there are treatments to alleviate symptoms such as:

Medications to lessen the mental and physical effects of the disease like anti psychotics to control hallucinations, delusions, and violent outbursts.

Therapy for depression and anxiety.

Physical therapies to help movement problems.

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The most effective and accurate method of testing for Huntington disease—called the direct genetic test—counts the number of CAG repeats in the Huntington gene, using DNA taken from a blood sample. The presence of 36 or more repeats supports a diagnosis of Huntington disease . A test result of 26 or fewer repeats rules out Huntington's Disease (NIH) .
Testing for Huntington disease informs the family if they are at risk of developing it later on in life. However, many choose to not know until symptoms appear.

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RNA-targeting CRISPR–Cas13d system has shown to improve motor coordination, attenuated striatal atrophy, and reduction of mutant HTT protein aggregates in patients with no adverse effects and minimal off-target transcriptomic effects. Demonstrating a therapeutic approach for HD, and a strategy with implications for the treatment of other dominantly inherited disorders.

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Main

HD is an autosomal dominant neurodegenerative disorder caused by a CAG short tandem repeat (STR) expansion in exon 1 of the huntingtin ( HTT) gene1. This trinucleotide sequence codes for the amino acid glutamine (Q), placing HD in a broader class of neurological disorders known as polyglutamine (polyQ) diseases. Motor symptoms can manifest from childhood to old age, with onset inversely correlated with CAG repeat length in mutant HTT, with the longer the CAG repeat, the earlier symptoms arise2,3. The therapies currently available to patients with HD offer only moderate symptomatic relief and affected individuals typically die 15-20 years post-diagnosis due to complications2,4. Therefore, a major focus of HD therapeutic development has shifted toward targeting the root of the disease through depletion of mutant HTT 5,6. Besides the toxicity of the mutated HTT protein, an increasing body of evidence indicates that mutant HTT mRNA also contributes to disease pathogenesis7,8. Consequently, strategies to suppress both HTT transcripts and protein levels would be most beneficial as a treatment. RNA interference (RNAi) and antisense oligonucleotide (ASO) strategies have shown preclinical efficacy and are being tested in clinical trials6. However, most of these approaches do not precisely differentiate mutant HTT from the normal allele9.

Most patients with HD are heterozygous for the CAG expansion and rely on their normal HTT allele to play important roles during brain development as well as in adult central nervous system (CNS) function10. In the adult brain, HTT helps regulate intracellular vesicle trafficking, transcriptional regulation14,15 and synaptic connectivity. While partial reduction of normal HTT levels is tolerable in multiple preclinical animal models of HD19,20, long-term ramifications of reductions in humans are unclear given its involvement in a myriad of biological functions21. Sustained reduction of normal HTT levels may even exacerbate HD pathogenesis10,22. Indeed, the recent termination of the phase III clinical trial ( NCT03761849) of a non-allele-selective HTT-lowering ASO developed by Roche in patients with HD underscores the urgency to develop an allele-selective strategy that can selectively and effectively suppress mutant HTT mRNA expression23,24,25,26.

The RNA-guided, RNA-targeting subtype of VI-D CRISPR-Cas, Cas13d, has been recently identified as an efficient and specific RNA-targeting approach that can be applied in mammalian cells27. Cas13d, with a size of only approximately 930 amino acids, can be packaged and delivered with its single-guide RNAs (sgRNAs) to target cells via a single adeno-associated virus (AAV) capsid. Cas13d possesses dual RNase activities and is capable of processing CRISPR arrays and cleaving target RNAs in a protospacer flanking sequence-independent manner. In cell-based screenings and side-by-side comparisons to short-hairpin RNA (shRNA), nuclear localized sequence-fused Cas13d showed a strong ability to cleave target RNA with high efficiency (approximately 96% knockdown by Cas13d compared to approximately 65% by shRNA) and high specificity with minimal off-target effects in mammalian cell culture28. These attributes indicate that CRISPR-Cas13d is a promising platform for RNA targeting in clinical applications such as selective mHTT depletion. Unlike gene therapies engineered from DNA-targeting CRISPR systems-which can cause irreversible nonspecific and unintended genetic modifications in the patient's genome, that can be inherited in subsequent generations-targeting RNA appears a safer, viable alternative.

In this study, we tested a CRISPR-Cas13d-based gene therapy approach that silences mutant toxic CAG-expanded (CAG EX) HTT RNA in both human and mouse models of HD. Our CAG EX-targeting Cas13d system (Cas13d-CAG EX) selectively reduces CAG EX HTT RNA in neurons derived from patients with HD with CAG expansions ranging from 66 to 109 STRs. AAV-mediated delivery of Cas13d-CAG EX to the striatum of premanifest zQ175/+ HD mice resulted in allele-selective suppression of mutant HTT mRNA and protein aggregates while maintaining normal HTT mRNA and protein levels, significantly improved motor function and attenuated striatal atrophy. Our data provide the first evidence, to our knowledge, that CRISPR-Cas13d-mediated elimination of mutant mRNA and protein is a promising allele-selective therapeutic strategy.

Results

Development of a Cas13d system that targets CAGEX

Although the etiology of HD is complex, many proposed mechanisms arise from the transcription and subsequent translation of CAG EX HTT, ultimately causing disease through a toxic gain-of-function mechanism. Therefore, therapeutic approaches that suppress mutant HTT at the RNA level are actively being pursued. We recently pioneered the repurposing of the Cas9 system to target and eliminate toxic repeat RNAs in vitro30 and in vivo31 delivered by a two-vector AAV system, demonstrating that RNA-targeting CRISPR approaches are effective with RNA repeat expansions. In this study, due to the smaller size and natural RNA-targeting capacity of Ruminococcus flavefaciens XPD3002 (Rfx) CRISPR-Cas13d28, we developed a CAG EX RNA-targeting Cas13d (Cas13d-CAG EX) system that we packaged into a single vector in both lentiviral and AAV delivery vehicles to evaluate its therapeutic potential in multiple established preclinical models of HD. These included fibroblasts from patients with HD, differentiated neurons with striatal characteristics from a panel of induced pluripotent stem cell (iPSC) lines from patients with HD and a full-length mutant HTT knock-in mouse model expressing a human mutant exon-1 with the expanded CAG repeat (approximately 220 repeats) within the native mouse huntingtin gene, zQ175/+ (Fig. 1a).

a, Treatment scheme of our single gene therapy that expresses Cas13d and a gRNA designed to eliminate CAG-expanded HTT HTTRNA in both human striatal neuronal cultures derived from patient iPSCs and in the striatum of an established mouse model of HD, zQ175/+. b, Diagram of a series of CAG-expanded, RNA-targeting vectors that consists of (1) Cas13d tagged with an HA epitope and (2) one of three U6 promoter-driven Rfx CRISPR-Cas13d gRNAs (denoted as CAG EX gRNA 1-3). c, Western blot analysis of polyQ protein from protein lysates isolated from HEK293 cells transfected with a CAG ¹⁰⁵ repeat plasmid and each candidate Cas13d vector. d, e, RNA dot blot analysis ( d) and quantification ( e) of CAG-expanded RNA within HEK293 cells transfected with a CAG ¹⁰⁵ repeat plasmid along with a nontargeting control (NT) or CAG EX-2 vector (one-way ANOVA, Tukey post hoc test, **** P < 0.0001; n = 1 technical replicates, n = 3 biological replicates). f, g, RNA dot blot analysis ( f) and quantification ( g) of CUG-expanded RNA within HEK293 cells transfected with a CUG ¹⁰⁵ repeat plasmid along with a NT or CAG EX-2 vector (one-way ANOVA, Tukey post hoc test, n = 3 technical replicates, n  = 3 biological replicates).
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First, to optimize knockdown of CAG EX HTT RNA by Cas13d, we engineered three distinct RNA-targeting vectors that consist of (1) Cas13d tagged with a human influenza hemagglutinin (HA) epitope and (2) one of three U6 promoter-driven Cas13d gRNAs (denoted as CAG EX gRNA 1-3). All three gRNAs are complementary to the CAG EX RNA sequence with each guide targeting a different codon within the repeat expansion: CAG EX gRNA-1 (GTC), CAG EX gRNA-2 (TCG) and CAG EX gRNA-3 (CGT) (Fig. 1b). For an initial assessment of the capability of each CAG EX gRNA to knockdown CAG EX RNA, HEK293 cells were cotransfected with a repeat expansion plasmid with 105 CAG STRs along with a Cas13d-containing vector with a nontargeting gRNA designed to target a sequence from the λ bacteriophage (Cas13d-NT) or one of the three CAG EX-targeting guides as described previously30. Since aggregation of toxic polyQ protein translated from CAG EX RNA is well documented as one of the primary hallmarks of HD neuropathology, we determined if and to what extent Cas13d in conjunction with each CAG EX-targeting gRNA can eliminate polyQ protein in live human cells. We observed that polyQ protein produced by this plasmid was dramatically reduced in cells cotransfected with Cas13d and CAG EX gRNA-2 compared to cells only transfected with the CAG ¹⁰⁵-expressing plasmid via western blot with a polyQ-specific antibody (MAB1574)30 (Fig. 1c). RNA blots performed with RNA isolated from cells cotransfected with our CAG repeat expression plasmid and Cas13d with the CAG EX-2 gRNA also showed a significant (85.4%) reduction of CAG EX RNA compared with cells transfected with our nontargeting Cas13d vector (one-way analysis of variance (ANOVA), **** P < 0.001) (Fig. 1d, quantified in Fig. 1e). Importantly, expression studies using CUG expansion (CUG EX) RNA-expressing plasmids and a semiquantitative RNA dot blot were performed to determine if Cas13d with the CAG EX-2 gRNA specifically eliminates RNA transcripts that contain CAG expansions without degrading similar GC-rich transcripts such as those with CUG expansions. Excitingly, we observed that Cas13d with the CAG EX-2 gRNA specifically targets and degrades CAG EX transcripts while leaving CUG EX transcripts intact (Fig. 1f, quantified in Fig. 1g). Taken together, these data confirm that our Cas13d system, which includes the CAG EX-2 gRNA, now referred to as 'Cas13d-CAG EX', can effectively eliminate CAG EX RNA and subsequent polyQ protein in human cells possibly without targeting other trinucleotide repetitive elements such as CUG EX.

Cas13d-CAGEX reduces mHTT in cells from patients with HD

To evaluate Cas13d-CAG EX in a human preclinical model, a panel of neuronal cultures enriched for striatal characteristics was generated from three previously validated iPSC lines derived from individual patients with HD. These independent lines contained repeats in the HTT locus ranging from 66 to 109 CAGs (referred to as HD 66, 77 and 109) and were compared to 3 non-isogenic, neurotypical iPSC lines isolated from three different individuals with <25 CAG repeats in exon 1 of HTT. Although not age- or sex-matched to our lines from patients with HD, the control samples (controls 1-3) used to evaluate the effects of different lengths of CAG expansions on human neurons have been characterized in previous publications and checked for aberrant genomic alterations via karyotype and copy number variation (CNV) arrays to avoid line-specific confounds in downstream analyses. Each HD and control neuronal culture was transduced with a constitutive, lentiviral system supplying either Cas13d-CAG EX or nontargeting Cas13d-NT at 16 days post-differentiation, when most of each culture consisted of neuroprogenitor cells (Fig. 2a). At day 32 post-differentiation, both HD and control cultures consisted of neurons positive for striatal markers, dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) and COUP-TF1-interacting protein 2 (CTIP2) (Fig. 2b, quantified in Extended Data Fig. 1a,b). A high percentage of DARPP-32 (65.5%) and CTIP2 (82.3%) was observed indicating that our human cell cultures consisted of neurons enriched for striatal characteristics. Widespread transduction was confirmed with Cas13d-HA immunofluorescence with 78% or more (average 83.4%) of neurons showing Cas13d expression (Extended Data Fig. 1c-f). RNA slot blot hybridization detected that CAG EX RNA was significantly reduced by Cas13d-CAG EX with an 84.1% reduction in HD 66, a 79.8% reduction in HD 77 and a 56.2% reduction in HD 109 (Fig. 2c, quantified in Fig. 2d). Since protein aggregates are pathological hallmarks of HD, we determined the effect of Cas13d-CAG EX on mutant HTT aggregation, immunolabeled by the EM48 antibody. Cas13d-CAG EX-treated HD lines had significantly reduced mutant HTT aggregates compared with those treated with Cas13d-NT (Fig. 2e, representative images in Fig. 2f,g), indicating that Cas13d-CAG EX can selectively suppress mutant HTT aggregates.

a, Schematic of the differentiation protocol used for iPSC-derived neurons treated with a lentiviral system expressing Cas13d-CAG EX. b, Representative immunofluorescence images of cells at day 32 in control neuronal cells, showing that the cells are positive for striatal markers, DARPP-32 (top) and CTIP2 (bottom). Scale bar, 20 μm. n = 1 technical replicate. c, d, RNA dot blot analysis ( c) and quantification ( d) of CAG-expanded RNA within MSN cells transduced with Cas13d-NT- or Cas13d-CAG EX-expressing lentiviral vectors (one-way ANOVA, Tukey post hoc test, *** P < 0.001). HD 66 Cas13d-CAG EX P = 0.00037, HD 77 Cas13d-CAG EX P = 0.00054, HD 109 Cas13d-CAG EX P = 0.00067; n = 3 technical replicates, n = 3 biological replicates. Data are presented as mean values ± s.e.m. e- g, Quantification of mHTT aggregates within MSN cells transduced with Cas13d-NT- or Cas13d-CAG EX-expressing lentiviral vectors ( e) (one-way ANOVA, Tukey post hoc test, **** P < 0.0001, n = 3 technical replicates, n = 4 differentiations, 200 cells per experimental group). Representative images of the analysis are in f and g (scale bar, 20 μm). Data are presented as mean values ± s.e.m. h, i, Quantification of mutant and WT HTT RNA via allele-specific RT-qPCR in GM04723 ( h) and GM02151 ( i) fibroblasts (two-way ANOVA, Tukey post hoc test, **** P < 0.0001, n  = 5 biological replicates). DAPI, 4,6-diamidino-2-phenylindole.
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To evaluate the allele-selectivity of Cas13d-CAG EX on shorter repeats more common in adult-onset patients, we utilized two human fibroblast lines from patients with HD with (67/15) CAG repeats (GM04723) and (46/18) CAG repeats (GM02151). Allele-specific quantitative PCR with reverse transcription (RT-qPCR) using patient-specific single-nucleotide polymorphism (SNP)-based primers showed a significant and specific knockdown of mutant HTT RNA in each patient fibroblast line treated with Cas13d-CAG EX-expressing lentiviral vectors compared to those transfected with Cas13d-NT (Fig. 2h,i). These data support the broad utility of Cas13d-CAG EX for patients with adult- or juvenile-onset HD-relevant CAG repeat lengths in mutant HTT.

Cas13d-CAGEX reduces specific molecular biomarkers in patients with HD

Transcriptome-wide RNA sequencing (RNA-seq) analysis utilizing the DESeq2 package35 identified 988 differentially expressed genes (DEGs) (false discovery rate (FDR)-adjusted P < 0.0001) that distinguish our HD neuronal lines from controls (Fig. 3a-c and Supplementary Table 1). Gene ontology (GO) biological process analyses revealed that these DEGs were enriched for terms associated with neurodegenerative disorders including 'Rho GTPases', 'translation' and the 'Vascular endothelial growth factor A-vascular endothelial growth factor receptor 2 signaling pathway'. The term 'Huntington disease' was also enriched and included genes associated with known HD-mediated pathological pathways such as cytosolic and mitochondrial calcium overload, endoplasmic reticulum stress through proteasomal dysfunction and impaired autophagy function36,37,38,39,40,41,42,43,44 (Fisher's exact test with FDR-adjusted P < 0.05; Extended Data Fig. 2a and Supplementary Table 2). Therefore, we opine that these gene expression changes are a useful measure of the efficacy of our Cas13d-based approach. In comparing individual HD neuronal cultures treated with Cas13d-CAG EX or Cas13d-NT to all 3 control lines treated with Cas13d-NT, we determined that 61.2% (605 out of 988 DEGs), 77.1% (762 out of 988 DEGs) and 57.5% (568 out of 988 DEGs) of HD DEG signatures were partially reversed in the HD 109, HD 77 and HD 66 lines, respectively, treated with Cas13d-CAG EX, with evident reversal defined as a fold change of 50% or more in the expression of each DEG toward wild-type (WT) levels satisfying an FDR-adjusted P < 0.01 (Supplementary Table 1). Importantly, reversed DEGs were enriched for each HD-associated pathological pathway mentioned above, suggesting that our gene therapy can alleviate previously reported downstream effects of m HTT (Extended Data Fig. 2a and Supplementary Table 2). We also made the critical observation that 48% (HD 109), 56% (HD 77) and 45% (HD 66) of the most robust HD DEGs (twofold change, P < 0.00001) were partially reversed (Fig. 3d-f). In addition to the common 988 DEGs, excitingly, we also determined that patient-specific DEGs (FDR-adjusted P < 0.0001) were also partially reversed by 76.7% (4,582 out 5,973) in HD 109 and 81.3% (4,870 out of 5,991) in HD 77 and to a lesser degree (36.2%; 1,830 out of 5,055) in HD 66 (which harbored only approximately 80% of the extent of changes in HD 77 and HD 109) by Cas13d-CAG EX treatment (Supplementary Table 3). Our results demonstrate that our gene therapy approach reduces the most prominent disease markers common in both groups and individual patients.

a- f, Scatter plots of a common list of 988 upregulated and downregulated DEGs within HD 109, HD 77 and HD 66 treated with either Cas13d-NT (HD 109 ( a), HD 77 ( b), and HD 66 ( c)) or Cas13d-CAG EX (HD 109 ( d), HD 77 ( e), and HD 66 ( f)) lentivirus vector compared to controls 1-3 referred to as 'control'. The most significant HD-associated DEGs defined by a twofold change from control and a FDR-adjusted P < 0.00001 are distinguished from total HD-associated DEGs with a broken vertical black line on the x axis (log ₂ fold change) and a solid gray line on the y axis (−log ₁₀( P)), showing partial reversal of HD-mediated changes in the human transcriptome defined in a- c by Cas13d-CAG EX in d- f.

Importantly, neither quantification of HTT mRNA by transcript expression levels (transcripts per million) nor qPCR could detect a significant change in total HTT mRNA levels in any of the 3 control lines treated with Cas13d-CAG EX compared to Cas13d-NT or left untreated (one-way ANOVA, Tukey's post hoc test, P < 0.05) (Extended Data Fig. 2b,c). To assess the potential off-target effects of Cas13d-CAG EX on other transcripts with non-disease-associated CAG repeats, we next examined the expression levels of protein-coding transcripts with five or more CAG, AGC or GCA tandem repeats, which could be inadvertently targeted by the CAG EX gRNA. Of 92 transcripts with five or more repeats, seven statistically significant (twofold change, FDR-adjusted P < 0.01) DEGs were detected ( ZNF853, MN1, IRS1, MSH4, VASH2, LMX1A, ST6GALNAC5; Extended Data Fig. 2d and Supplementary Table 4). Considering that most of these genes are involved in neurodevelopment, including transcriptional regulation of developmental genes ( ZNF8585, MN1, LMX1A) and axon development ( VASH2), it is uncertain how their dysregulation will affect the adult brain. However, none of these DEGs are associated with adult-onset neurological disorders. These data suggest that other CAG-containing genes may be minimally affected by our Cas13d-based gene therapy approach.

Therapeutic efficacy and safety of Cas13d-CAGEX in vivo

To determine if our approach can prevent or halt the progression of neurodegenerative phenotypes in vivo in zQ175/+ mice, we packaged Cas13d-CAG EX or Cas13d-NT into single-stranded AAV serotype 9 (AAV9) viral vectors and conducted bilateral intrastriatal injection of Cas13d-CAG EX or Cas13d-NT to an equal number of two-month-old zQ175/+ HD mice and age-matched WT littermate controls. At three weeks postinjection, we observed successful expression of both Cas13d-HA fusion constructs in mouse striatum, indicated by red fluorescent signals of HA protein with an anti-HA antibody (Extended Data Fig. 3a,b). Motor function and body weight were monitored longitudinally over eight months postinjection as illustrated (Fig. 4a) and mHTT aggregates, HTT mRNA and protein levels were determined at the end of the study. We evaluated the effect of treatment on motor function on a balance beam. While zQ175/+ HD mice exhibited clear motor deficits at eight months of age and after; those treated with Cas13d-CAG EX showed significantly improvements in motor performance, indicated by shorter traverse times, compared with zQ175/+ mice treated with Cas13d-NT ( P < 0.001) or left untreated ( P < 0.01) (Fig. 4b and Extended Data Fig. 4d-f). Importantly, Cas13d-CAG EX had no effect on motor function in WT mice (Fig. 4c) and did not alter body weight in zQ175/+ (Fig. 4d) or WT mice (Fig. 4e), implying that Cas13d-CAG EX does not produce gross adverse effects in mice.

a, Timeline of experimental design and outcome measures. b- e, Motor function and body weight. Mice were tested on a 5-mm balance beam and time crossing the beam (traverse time) was recorded at the indicated ages in zQ175/+ HD ( b) or WT mice ( c) with the indicated treatments. *** P = 0.0004, ** P = 0.0057, two-way ANOVA with Bonferroni post hoc tests. Body weight in zQ175/+ HD mice ( d) or WT mice ( e) with the indicated treatments ( n = 10, 5 males and 5 females per group). Data are presented as mean values ± s.e.m. f, Representative MRI images of the zQ175/+ HD and WT mice injected with the indicated AAVs. g, h, Striatal ( g) and neocortex ( h) volume was quantified from 3D structural MRI in the indicated groups at 9 months of age (7 months after AAV injections). * P < 0.05, *** P < 0.001, one-way ANOVA with Tukey analysis. n = 6 mice per group, 3 females and 3 males per group, 9 months of age. g, P (Q175 + Cas13d-NT versus Q175 + Cas13d-CAG EX) = 0.0196; P(Q175 + Cas13d-NT versus WT + Cas13d-NT) = 0.0241. h, P(Q175 + Cas13d-NT versus Q175 + Cas13d-CAG EX) = 0.0451; P(Q175 + Cas13d-NT versus WT + Cas13d-NT) = 0.0005.
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Next, we examined whether Cas13d-CAG EX-mediated mHTT silencing could improve neuropathology in zQ175/+ mice. Using human-translatable high-resolution structural magnetic resonance imaging (MRI), we delineated regional brain volumes accurately using an automatic segmentation, large deformation diffeomorphic metric mapping (LDDMM) including the striatum and neocortex. We performed MRI scans of nine-month-old zQ175/+ mice, when these HD mice display striatal atrophy as we reported previously47. Our behavioral results indicate that Cas13d-NT has no significant effect compared to the untreated control mice; thus, we focused on four groups in the MRI study: WT or HD mice injected with Cas13d-NT or Cas13d-CAG EX. We demonstrate that zQ175/+ mice injected with Cas13d-NT exhibit significantly reduced striatal volume and neocortex volume compared to WT mice with the same injection, while Cas13d-CAG EX-injected zQ175/+ mice have preserved striatal volume and neocortex volume compared to HD mice injected with Cas13d-NT (by group mean analysis; Fig. 4f-h and Supplementary Table 5). In addition, we performed correlation analysis between striatal volume and traverse time or body weight in zQ175/+ mice treated with either Cas13d-NT or Cas13d-CAG EX and found that there was no significant correlation between striatal volume and traverse time in zQ175/+ mice ( P = 0.2625; Extended Data Fig. 4g) or body weight ( P = 0.3755; Extended Data Fig. 4h). We note that the brain volume data comparison is between two groups of HD mice treated with either Cas13d-CAG EX or Cas13d-NT. By considering the brain volumetric measurement of individual zQ175/+ mice, our results suggest that the individual variability in striatal volume of zQ175/+ mice in each treatment group is mainly due to natural variation rather than different responders to the treatment.

Selective mHTT knockdown by Cas13d-CAGEX in zQ175/+ mice

We next determined the effect of Cas13d-CAG EX on mHTT aggregation in zQ175/+ mouse striatum. The Cas13d-CAG EX-injected striatum of zQ175/+ mice harbored significantly reduced EM48⁺ mutant aggregates compared with those injected with Cas13d-NT (Fig. 5a-c), indicating that Cas13d-CAG EX can effectively suppress mutant HTT aggregates in vivo. Importantly, we observed a significant reduction of mHTT HTT and protein levels in the striatum of Cas13d-CAG EX -injected zQ175/+ mice. WT mouse HTT mRNA and protein levels, which could be distinguished from the mutant human exon-containing HTT allele, were preserved in both zQ175/+ and WT mice treated with Cas13d-CAG EX (Fig. 5d-f quantified in Fig. 5g-k). This once again demonstrates the potential for allele-selective targeting of pathogenic HTT mRNA using Cas13d-CAG EX.

a, b, Representative mHTT aggregates detected by immunostaining with EM48 antibody in zQ175/+ mice injected with Cas13d-NT ( a) or Cas13d-CAG EX ( b). Red, EM48 immunostaining; blue, DAPI nuclear staining. Scale bar, 10 µm. c, Quantification of mHTT aggregates in the zQ175 mice injected with AAV9-Cas13d-NT or AAV9-Cas13d-CAG EX. * P < 0.05 by two-sided Student's t-test. n = 4 mice per group, 2 females and 2 males per group, 10 months of age. P = 0.0261. d, Quantification of mutant and WT mRNA via allele-specific RT-qPCR in Q175/+ mice treated with either Cas13d-NT or Cas13d-CAG EX (two-way ANOVA, Tukey post hoc test, **** P < 0.0001, n = 4 per experimental group). e, f, Western blot analysis of mutant HTT, WT HTT and HA (Cas13d constructs containing HA tag) protein levels in zQ175/+ ( e) and WT ( f) mice treated with either Cas13d-NT or Cas13d-CAG EX, n = 3 mice/group. g- k, Quantification of mutant HTT ( g), WT HTT ( h, i) and HA ( j, k) protein levels in WT and zQ175/+ mice treated with either Cas13d-NT or Cas13d-CAG EX ( n = 3 biological replicates per experimental group). One-sided Student's t-test. NS, not significant; * P < 0.05. P = 0.03 in Fig. 5g. No adjustment was made for multiple comparisons.
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We next performed transcriptome-wide analyses on striatal samples isolated from zQ175/+ and WT control mice treated with Cas13d-CAG EX or Cas13d-NT. In doing so, we identified widespread transcriptome dysregulation caused by m HTT with a total of 2,413 downregulated and 2,368 upregulated DEGs in the Cas13d-NT-treated zQ175/+ mice compared to WT littermates (FDR-adjusted P < 0.01) (Supplementary Table 6). GO analysis showed that many of our DEGs in zQ175/+ mice are involved in 'cAMP signaling', 'protein phosphorylation' and 'negative regulation of cell cycle transition'. These results resonate with previous reports48,49 and include a large panel of established striatum-specific markers of HD (Fisher's exact test with FDR-adjusted P < 0.05; Extended Data Fig. 5a and Supplementary Table 7). Indeed, we observed statistically significant overlaps between our DEGs from our Q175/+ studies and those previously reported48,49 (hypergeometric P < 0.0001; Extended Data Fig. 5b and Supplementary Tables 8 and 9).

We next determined that approximately 56% of these m HTT-mediated transcriptional changes identified in Q175/+ mice treated with Cas13d-NT (2,677 out of 4,781 DEGs) were reversed by Cas13d-CAG EX treatment with a fold change of 50% or more toward WT levels (satisfying an FDR-adjusted P threshold of <0.01; Supplementary Table 6). Excitingly, we also found that 60.2% of the most robust HD DEGs (twofold change, FDR-adjusted P < 0.001) were partially to fully reversed, including most of previously reported striatum-specific HD disease markers identified in our control-treated zQ175/+ mice (59 out of 74; 79.7%; Fig. 6a,b, Supplementary Table 10 and Extended Data Fig. 5a-o). The DEGs that were corrected were evenly enriched for each HD-associated pathological pathway identified in our zQ175/+ mice treated with Cas13d-NT, supporting that our approach can mitigate some known downstream effects of m HTT (Extended Data Fig. 6a and Supplementary Table 8). Furthermore, when comparing zQ175/+ mice treated with Cas13d-CAG EX to ones treated with Cas13d-NT, 66.5% (189 out of 284) of upregulated DEGs and 54.0% (238/441) of downregulated DEGs partially to fully reversed to resemble WT levels, that is, if they exhibited a significant reduction in difference between their zQ175/+ and WT levels (FDR-adjusted P < 0.01, difference of more than 20% and depicted in Fig. 6c; Supplementary Table 11). Excitingly, some genes achieved almost complete rescue ( P < 10 −4, Wilcoxon two-sample paired signed-rank test; Fig. 6d,e).

Cas13d-CAG EX or Cas13d-NT was injected into the striatum of two-month-old HD zQ175 or WT mice and the striatum was collected at ten months of age. Total RNA was extracted and quantified by RNA-seq. n = 4 or 5 mice per experimental group. a, b, Scatter plots of upregulated ( a) and downregulated ( b) DEGs in zQ175/+ mice treated with either Cas13d-NT or Cas13d-CAG EX AAV9 showing partial reversal of HD-mediated transcriptome changes in HD mouse striatum by Cas13d-CAG EX. c, Volcano plot for DEGs between Q175 + Cas13d-CAG EX versus Q175 + Cas13d-NT. Most DEGs correspond to a partial to full reversal of HD-associated DEGs (green). Fold change is relative to Q175 + Cas13d-NT. Significance cutoffs are fold change greater ±20% and FDR-adjusted P < 0.05. d, e, Averaged expression of HD-associated upregulated ( d) or downregulated ( e) DEGs that were significantly reversed by Cas13d-CAG EX treatment. The red dashed line at 0 indicates full rescue to WT levels. **** P < 0.0001, two-sample paired Wilcoxon signed-ranks test ( n = 4 mice per experimental group). The box extends from the first quartile to the third quartile of the data with a line at the median. The whiskers extend from the box by 1.5× the interquartile range. Flier points are those past the end of the whiskers. f, Scatter plot of upregulated and downregulated DEGs in WT mice treated with either Cas13d-NT or Cas13d-CAG EX AAV9 showing limited off-targets. g- i, Quantification of CNS-specific markers of immunogenicity via qPCR (NeuN ( g), GFAP ( h), and IBA1 ( i)) (one-way ANOVA, Tukey post hoc test, n = 3 per experimental group; n  = 1). Data are presented as mean values ± s.e.m.
Source data

We next evaluated the safety profile of Cas13d-CAG EX based on our RNA-seq data. We observed that approximately 80 genes are differentially expressed when comparing Cas13d-NT-treated and untreated zQ175/+ mice, which may be a result of injection, the AAV9 viral delivery vector, Cas13d, the gRNA or a combination of these factors (Extended Data Fig. 7a). Nevertheless, when comparing WT mice treated with Cas13d-CAG EX or Cas13d-NT, we found only 18 statistically significant (FDR-adjusted P < 0.01) changes in gene expression (Fig. 6f). We then examined the expression levels of protein-coding transcripts with five or more non-disease-associated CAG, AGC or GCA tandem repeats, which could be inadvertently targeted by the CAG EX gRNA. Of 344 transcripts with 5 or more repeats, only 3 significant DEGs (FDR-adjusted P < 0.01) were detected (Extended Data Fig. 7b and Supplementary Table 12). Furthermore, qPCR also confirmed that Cas13d-CAG EX did not disrupt CNS-specific markers of immunogenicity including neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (IBA1) (Fig. 6g-i). Taken together, these results support the efficacy and safety of Cas13d-CAG EX in HD preclinical models.

Discussion

We packaged R. flavefaciens XPD3002 Cas13d, an RNA-targeting enzyme 2.4 kilobases of DNA in size, and a CAG EX RNA-targeting gRNA in a single viral delivery vehicle. We determined that Cas13d-CAG EX selectively reduces CAG EX HTT RNA in striatal neurons derived from the iPSC lines of three patients with HD with CAG expansions ranging from 66 to 109 STRs and patient fibroblasts with 46 and 67 CAG repeats in HTT, suggesting potential therapeutic benefit for both adult and juvenile patient populations with HD. Viral transduction of Cas13d-CAG EX in premanifest zQ175/+ HD mice resulted in allele-selective suppression of mutant HTT mRNA and protein with minimal off-targets. Lastly, we demonstrated significantly improved motor function, reduced m HTT aggregates and attenuated striatal atrophy in Cas13d-CAG EX-treated zQ175/+ HD mice.

Approximately, 2.7 out of 100,000 individuals worldwide currently manifest symptoms of HD50. Unfortunately, to date, neither curative nor disease-modifying treatments for HD exist. Current therapeutic strategies target different aspects of HD pathology including excitotoxicity, the dopamine pathway, caspases, mHTT protein aggregation, mitochondrial dysfunction and transcriptional dysregulation51. Unfortunately, the only therapeutics currently approved by the U. S. Food and Drug Administration (FDA) only alleviate symptoms and do not modify disease progression. Because the molecular etiology of HD is complex, one accepted therapeutic approach for all patient populations suffering from HD includes the selective elimination of mutant HTT (RNA and protein) produced by this locus52. Current strategies to attenuate gene expression via targeted destruction of mHTT RNA include ASOs, RNAi compounds and small molecule splicing modulators. The most promising of these include the Roche phase III ASO candidate tominersen and the Wave Therapeutics phase I/II ASO candidates WVE-120101 and WVE-120102 (ref. 53). However, each failed to show adequate efficacy and safety in their respective clinical trials. Preliminary results from clinical trial NCT03761849 indicated that two independent dosing regimens of tominersen in patients performed slightly worse than placebo on the primary outcome measures of the Composite Unified Huntington Disease Rating Scale and Total Functional Capacity26,54. Revised clinical trials will be conducted to determine if these outcomes resulted from either the repeated high dose used or interference with WT HTT protein levels26,54. Wave's ASO candidates that target patient-specific single-nucleotide polymorphisms on mHTT pre-mRNA showed limited target specificity, which led to low efficacy.

To identify an optimal gRNA against CAG expansions, we profiled three different Cas13d gRNAs that target the three possible sequence frames in the CAG repeat tract. One directly in-frame to CAG expansions (CTG-) and two with one or two bases out of frame were constructed and screened for CAG EX RNA knockdown efficiency with our TGC-targeting gRNA surprisingly yielding the most effective depletion of the CAG. This was a critical optimization step since knockdown efficiency of RNA transcripts by each individual RNA-targeting CRISPR system is highly dependent on gRNA-specific features and target site context. Since Rfx-Cas13d is still a newly characterized RNA-targeting system, the molecular engineering field is in the process of understanding the targeting parameters and sequence constraints for optimal gRNA design, which is especially important for double-stranded RNA structures since hairpins form from CAG expansions. Indeed, mRNA stability largely depends on the mRNA nucleotide sequence, which affects the secondary and tertiary structures of mRNAs and the accessibility of various RNA-binding proteins to the mRNA55. Abnormal expansions of CAG repetitive elements within RNA form stable secondary structures that consists of a base, a hairpin structure forming the stem and a terminal loop with the overall thermodynamic stability of CAG-expanded mRNA transcripts increasing with CAG repeat length56,57,58,59,60. Such stable GC-rich RNA hairpin structures have been widely known to sequester RNA-binding proteins that have an increased binding affinity for hairpin loops formed from expanded STRs compared to their canonical targets8,61, suggesting that Cas13d may also have a similar binding preference. Interestingly, the observed specificity of our Cas13d system for long, pathogenic repeats that form RNA hairpins has also been observed in recent studies that involve RNA-targeting CRISPR-Cas9 systems programmed to degrade microsatellite repeat expansions in vitro30 and in vivo31 with a similar gRNA design.

Excitingly, we demonstrate that administration of Cas13d-CAG EX in premanifest zQ175/+ HD mice resulted in allele-selective suppression of mutant HTT mRNA and protein while maintaining normal mRNA and protein levels with limited off-target effects on the mouse transcriptome. This is an improvement over current RNA-targeting approaches, including small molecules with limited target specificity compared to Watson-Crick base pairing and RNAi compounds that may deplete the endogenous RNAi machinery and cause undesired global alterations in gene expression. We also showed safe and long-lasting therapeutic efficacy eight months postinjection from AAV9-mediated delivery of Cas13d-CAG EX to the striatum of zQ175/+ HD mice. AAV9 is an FDA-approved gene therapy delivery method that affords sustained transgene expression and is currently being used in several clinical trials. On the other hand, ASOs and other small molecules must be continually readministered for the lifetime of the patient, which can pose safety issues in the affected CNS tissues. However, such transient therapeutic effects of ASOs and small molecules can be seen as an advantage over AAV drug delivery because their administration can be halted if negative side effects are observed at any point during a patient's lifetime and such adverse effects are likely to be reversible.

Taken together, we demonstrate a proof of principle for a CAG EX RNA-targeting CRISPR-Cas13d system as a potential allele-sensitive therapeutic approach for HD, a strategy with broad implications for the treatment of other neurodegenerative disorders.

Methods

Animals

Heterozygous zQ175 (zQ175/+) mice and WT littermates (both sex- and age-matched in each group) were used in the study. The zQ175 line in C57BL/6 background strain was obtained from The Jackson Laboratory and bred and maintained in our laboratory. Genotyping and CAG repeat size were determined at Laragen by PCR of tail snips. The CAG repeat length was 223 ± 3 in male zQ175/+ mice and 225 ± 3 in female zQ175 mice/+ used in the study. All mice were housed under specific pathogen-free conditions with a reversed 12-h light-dark cycle maintained at 23 ˚C with 30-70% humidity and provided with food and water ad libitum. All behavioral tests and longitudinal MRI measures were done in the mouse dark phase (active). The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Animal Care and Use Committee at Johns Hopkins University. A rigorous study design was implemented. Mice were randomized into groups to avoid bias. A balanced sex ratio was considered in all experiments. Data were collected using animal IDs and analyzed by investigators who were blinded to genotype and treatment. Data from different sexes were analyzed grouped when there was no sex-dependent difference in the outcome measures.

Cell lines

Human iPSC lines derived from individuals with HD and neurotypical controls have been previously characterized elsewhere. HD stem cell lines 66, 77 and 109 were a gift from the L. Thompson's laboratory at UC Irvine whereas our neurotypical controls were a gift from the Goldstein laboratory at UCSD. iPSC colonies were expanded on Matrigel-coated dishes (BD Biosciences) with mTeSR1 medium (STEMCELL Technologies). Cells were routinely checked by karyotype and CNV arrays to avoid genomic alterations in the culture. They were purchased from the American Type Culture Collection and were not further authenticated. HEK293T cells were obtained from Millipore Sigma (catalog no. 12022001). HD human fibroblast lines GM04723 (CAG15/67) and GM02151 (CAG18/46) were obtained from the Coriell Cell Repository.

Cas13d-CAGEX design strategy

We designed Rfx CRISPR-Cas13d gRNAs adapted from Konermann et al.28 to be complementary to CAG EX and packaged the RNA-targeting Cas13d system into a single AAV9 vector or lentiviral vector termed 'Cas13d-CAG EX '. Three gRNAs, one directly in-frame to CAG expansions (CTGCTG....) and the other two 1 or 2 bases out of frame where (TGC.... and GCT....) were constructed and screened for expanded-CAG RNA knockdown efficiency.

The sequence of gRNAs was: CAG EX-1 gRNA GTCGTCGTCGTCGTCGTCGTC; CAG EX-2 gRNA TCGTCGTCGTCGTCGTCGTCG; and CAG EX-3 gRNA CGTCGTCGTCGTCGTCGTCGT.

Transfections

HEK293 cells were plated at a density of 3 × 10 ⁵ cells per well of a 6-well plate for RNA isolation. For RNA isolation, 150 ng of a CAG ¹⁰⁵ plasmid that expressed 105 CAG repeats was used with 500 ng each of plasmids containing both Cas13d and 1 of the 3 CAG-targeting guides. Transfection was carried out using Lipofectamine 3000 according to the manufacturer's instructions. Cells were incubated for 72 h at 37 °C and 5% CO ₂.

PolyQ western blot

Protein lysates were resolved on 4-15% Mini-PROTEAN Tris-Glycine gels (Bio-Rad) and transferred to an Invitrolon and Immobilon-P polyvinylidene fluoride membrane. Membranes were blocked with 5% skimmed milk in TBST (1× Tris-buffered saline, 0.1% Tween 20) and incubated overnight with primary antibodies: anti-polyQ disease protein antibody, 1:1,000, Merck Millipore MAB1574 (clone 5TF1-1C2) and anti-beta tubulin, 1:5,000, catalog no. ab6046 (Abcam) diluted in blocking solution at 4 °C. After three 10-min washes in TBST, the blots were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG (catalog no. NEF812001) and anti-mouse IgG (catalog no. NEF822001EA); PerkinElmer) diluted in blocking solution. After three 10-min washes in TBST, the blots were developed using Western Lightning Plus-ECL enhanced chemiluminescence substrate (PerkinElmer). Phosphorylation of protein was detected via the addition of alkaline phosphatase to the protein lysate (1:1,000, catalog no. 10567752001; Merck Millipore) before to SDS-polyacrylamide gel electrophoresis.

Allele-specific RT-qPCR in the fibroblasts of patients with HD

The Huntington's disease fibroblast lines GM04723 (CAG15/67) and GM02151 (CAG18/46) were obtained from the Coriell Cell Repository and maintained in complete minimal essential medium with 20% FCS. CAG repeat lengths were confirmed by sequencing.

mRNA transfection was performed using a 4D-Nucleofector X Kit (Lonza). We transfected 1 µg Cas13d-NT or Cas13d-CAG EX per 5 × 10 ⁵ cells. Seventy-two hours after transfection, cells were collected for gene expression analysis by RT-qPCR. RNA was purified using RNeasy columns (QIAGEN) and converted to complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Allele-specific detection of human HTT expression in HD fibroblasts GM04723 (CAG15/67) and GM02151 (CAG18/46) was performed with the PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) as well as Integrated DNA Technologies PrimeTime qPCR assays adapted from Zeitler et al.62 with a probe to a primer ratio of 2:1 based on SNP rs363099-C/T (exon 29): 363099C-forward: AGTTTGGAGGGTTTCTC; 363099T-forward: AGTTTGGAGGGTTTCTT; 363099T-blocker: AGGGTTTCTCCGCTCAGC/phos/; 363099-common reverse: TCGACTAAAGCAGGATTTCAGG.

Differentiation of iPSCs into two-dimensional neuronal cultures containing enrichment for striatal neurons

Differentiations were performed as follows: iPSC colonies were washed with PBS, pH 7.4 (Gibco) and then switched to neural induction medium (advanced DMEM/F12 (1:1) supplemented with 2 mM GlutaMAX (Gibco), 2% B27 without vitamin A (Thermo Fisher Scientific), 10 μM SB431542 and 1 μM LDN 193189 (both STEMCELL Technologies) and 1.5 μM IWR1 (Tocris)) with daily medium changes; this was day 0. On day 4 cells were passaged 1:2 with Stempro Accutase (Invitrogen) for 5 min at 37 °C and replated on human embryonic stem cell (hESC)-qualified Matrigel. At day 8, cells were passaged again 1:2 with Stempro Accutase for 5 min at 37 °C and replated on hESC-qualified Matrigel in a different medium (advanced DMEM/F12 (1:1) supplemented with 2 mM GlutaMAX, 2% B27 without vitamin A, 0.2 μM LDN 193189, 1.5 μM IWR1, 20 ng ml −1 Activin A; PeproTech). At day 16, cells were plated for neuronal differentiation in SCM1 medium (advanced DMEM/F12 (1:1) supplemented with 2 mM GlutaMAX, 2% B27 (Invitrogen), 10 μM DAPT, 10 μM Forskolin, 300 μM γ-aminobutyric acid, 3 μM CHIR99021, 2 μM PD 0332991 (all Tocris), to 1.8 mM CaCl ₂, 200 μM ascorbic acid (Sigma-Aldrich), 10 ng ml −1 brain-derived neurotrophic factor (BDNF) (PeproTech), medium was 50% changed every 2-3 d. On day 23, there was a full medium change to SCM2 medium (advanced DMEM/F12 (1:1): neurobasal A (Gibco) (50:50) supplemented with 2 mM GlutaMAX), 2% B27, to 1.8 mM CaCl ₂, 3 μM CHIR99021, 2 μM PD 0332991, 200 μM ascorbic acid, 10 ng ml −1 BDNF) and medium was 50% changed every 2-3 d until day 32 for collection.

Mycoplasma testing

All iPSC and striatal neuron cultures were routinely tested for Mycoplasma by PCR. Media supernatants (with no antibiotics) were collected, centrifuged and resuspended in saline buffer. Ten microliters of each sample were used for PCR with the following primers: forward: GGCGAATGGGTGAGTAAC; reverse: CGGATAACGCTTGCGACCT. Only negative samples were used in the study.

Lentivirus production

Both Cas13-CAG EX and Cas13d-NT constructs were cotransfected with psPAX2 (packaging plasmid) (Addgene plasmid no. 12260) and VSV-G (viral envelope) into Lenti-X HEK293T cells (Clontech) at a mass ratio of 5:4:3 using polyethyleneimine. Viral-containing supernatants were collected and concentrated with Lenti-X Concentrator reagent (Clontech) according to the manufacturer's protocol (100× concentration) and aliquoted for storage.

Immunocytochemistry

iPSC-derived neurons were permeabilized with 0.3% Triton X (Sigma-Aldrich) in PBS for 10 min and then blocked for 30 min in 5% goat serum 2.5% BSA (Gibco) 0.1% Triton X in PBS. Samples were incubated with primary antibodies overnight at 4 °C (DARPP-32, 1:500, catalog no. ab40801 (EP720Y) (Abcam); CTIP2, 1:500, catalog no. ab18465 (25B6) (Abcam); anti-huntingtin protein antibody 1:200, catalog no. MAB5347 (mEM48) (Merck Millipore)) and then washed 3 times with PBS before an overnight 4 °C incubation in secondary antibody (Alexa Fluor rabbit 555 IgG (catalog no. A-31572; Invitrogen), rat 488 IgG (catalog no. A48262; Invitrogen) and mouse 488 IgG (catalog no. A32723; Invitrogen) for 1 h at 1:500 and subsequently incubated with Hoechst 33342 (Sigma-Aldrich) and mounted with VECTASHIELD. Samples were imaged with a Zeiss LSM 780 confocal microscope at 20×.

Human moesin bulk RNA-seq library generation

RNA was isolated from approximately 100,000 moesin (MSN) cells from 4 independent differentiations per experimental group according to the manufacturer's protocol and purified using RNeasy columns. A total of 72 mRNA libraries for Illumina sequencing were prepared using the KAPA mRNA HyperPrep Kit (catalog no. KK8581; KAPA Biosystems) from 250 ng of total RNA. Libraries were sequenced on an Illumina NovaSeq instrument to a minimum of 3 × 10 ⁷ paired-end 100-base pair (bp) reads.

Human MSN RNA-seq data processing and analysis

RNA-seq reads were adapter-trimmed using Cutadapt v.1.14 and mapped to human-specific repetitive elements from RepBase v.18.05 by STAR v.2.4.0i63. Repeat-mapping reads were removed and remaining reads were mapped to the human genome assembly (hg38) with STAR. Read counts for all genes annotated in GENCODE v.24 (hg38) were calculated using the read summarization program featureCounts (v. 2.0.2)64 (coding sequence regions only). Differential expression analysis between different experimental groups (HD untreated, HD Cas13d-NT, HD Cas13d-CAG EX, control 1-3 untreated, control 1-3 Cas13d-NT, control 1-3 Cas13d-CAG EX) was performed using DESeq2 (v.1.38.1)35. Differentially expressed mRNAs were defined as having an FDR-adjusted P < 0.01 as determined by the Wald test, the standard statistical test used by DESeq235. We used the Unix grep search tool to scan the human genome for all protein-coding transcripts with five or more consecutive CAG, AGC and GCA tandem repeats.

RNA dot blot

HEK293 cell and human MSN RNA quality and concentrations were estimated using the NanoDrop spectrophotometer. Then, 5 μg of total RNA was resuspended in 1 mM EDTA, pH 8.0 to a final volume of 50 μl followed by the addition of 30 μl 20× SSC and 20 μl 37% formaldehyde to denature the RNA. The RNA was incubated for 30 min at 60 °C followed by a 2-min incubation on ice. The Bio-Rad Bio-Dot apparatus was assembled according to the manufacturer's protocol with Hybond N+ nylon membrane (GE Healthcare) on the top. The membrane was equilibrated by passing 100 μl of 20× SSC through the slots using a vacuum. The denatured RNA was passed through the slots and the membrane was washed with 20× SSC. The membrane was crosslinked at the auto setting in the UV Stratalinker (Stratagene). ULTRAhyb ultrasensitive hybridization buffer prewarmed at 37 °C was used to pretreat the membrane for 10 min at 37 °C. A custom biotinylated probe (CTGCTGCTGCTGCTGCTGCTGCTG-3′biotin (Integrated DNA Technologies)) was added to the prehybridization solution (5 μl ml −1). Hybridization was conducted for 5 h at 37 °C and the membrane was washed once with 1× SSC for 10 min at 37 °C. The membrane was exposed using the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific).

AAV packaging

Cas13d-CAG EX (no. 1457 Cas13d-CAG EX: 3.9 × 10 ¹² vg ml −1) and Cas13d-NT (no. 1457 Cas13d-NT: 4.4 × 10 ¹² vg ml −1) were cloned into an AAV vector provided by the UCSD Viral Vector Core. Each vector was packaged into single-stranded AAV9 by both the UCSD Viral Vector Core and the Emory Viral Vector Core (Emory University) using standard procedures and quality controls established at each independent core.

Stereotaxic injection

The mice were anesthetized with 1.5% isoflurane inhalation and stabilized in a stereotaxic instrument (David Kopf Instruments). Mice were injected into the striatum using the following stereotaxic coordinates: 0.62 mm rostral to bregma; ±1.75 mm lateral to midline; and 3.5 mm ventral to the skull surface. Then, 1 μl of a Cas13d-CAG EX-containing AAV9 titer (3.9 × 10 ¹² vg ml −1) or Cas13d-NT-containing AAV9 titer (4.4 × 10 ¹² vg ml −1, diluted to 3.9 × 10 ¹² vg ml −1) were injected into the striatum using a Hamilton syringe infusion pump (World Precision Instruments) at a perfusion speed of 200 nl min −1.

Immunohistochemistry and quantification

Mice were anesthetized and perfused transcardially with PBS followed by 4% paraformaldehyde. Brains were postfixed overnight followed by immersion in 30% sucrose for 24 h. Frozen sections (thickness, 10-15 μm) of coronal brain were immunostained with the following antibody: EM48 (1:200, catalog no. MAB5347; Merck Millipore). Briefly, the sections were washed three times with PBS, then permeabilized by incubating with 0.3% Triton X-100 for 5 min, followed by incubation with blocking solution containing 5% donkey serum or 3% goat serum and 0.3% Triton X-100 for 1 h. The sections were then incubated with primary antibody at 4 °C overnight. After three washes with PBS, the sections were incubated with fluorescence-labeled secondary antibody (Alexa Fluor mouse 555 IgG (catalog no. A32727, Invitrogen)) for 2 h at room temperature. Sections were mounted onto Superfrost slides (Thermo Fisher Scientific) dried and then covered with antifade mounting solution. Fluorescence images were acquired with a Keyence BZ-X700 All-in-One florescence microscope.

For image analysis, the samples were coded with ID and images were analyzed by investigators who were blinded to the genotypes and treatment. The results of the analysis were then calculated statistically and decoded by different investigators at the end. The results from three microscopy fields per slide and three sections per mouse were calculated for each mouse brain. EM48-positive puncta quantities were determined using the ImageJ (version 1.53t) (Fiji) analyze particles plugin function. The numbers of EM48-positive puncta per mm ² were determined at a constant threshold for each stain using ×20 images for quantifications.

Mouse mHtt RT-qPCR

For qPCR, RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantification of mRNA in mouse tissue samples was completed with the PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) and Integrated DNA Technologies PrimeTime qPCR assays adapted from Kingwell54.

The forward primer for the WT Htt allele was CAGGTCCGGCAGAGGAACC. The forward primer for the Q175 mHtt allele was GCCCGGCTGTGGCTGA. The reverse primer for both the WT and Q175 Htt alleles was TTCACACGGTCTTTCTTGGTGG. The probe for both the WT and Q175/+ Htt alleles was TGCACCGACCAAAGAAGGAACTCT.

Total mouse Htt was quantified with the Integrated DNA Technologies assay Mm.PT.58.12088552 while the following Integrated DNA Technologies PrimeTime qPCR assay were used to quantify striatal-specific markers of HD in Extended Data Fig. 6: Drd2: Mm.PT.58.7811767; Adcy5: Mm.PT.58.37585706; Adora2a: Mm.PT.58.29505675; Ppp1r1b: Mm.PT.58.8281675; Pde10a: Mm.PT.58.17658388; Penk: Mm.PT.58.45924319; Ace: Mm.PT.58.43658045; Rasd2: Mm.PT.58.33380601; Mchr1: Mm.PT.58.10781964; Drd1: Mm.PT.58.43576955.g; Dock4: Mm.PT.58.11402098; rt9: Mm.PT.58.8514559; Gpr83: Mm.PT.58.9029332; Rgs2: Mm.PT.58.41856362; Pdyn: Mm.PT.58.29316720.

Mouse Actn2 Integrated DNA Technologies PrimeTime qPCR assay Mm.PT.58.28953379 was used as an internal housekeeping control.

Western blotting

Striatal samples were homogenized in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% (w/v) SDS, 1.0% NP-40, 0.5% sodium deoxycholate and 1% (v/v) protease inhibitors. Then, 30 μg of proteins were separated in a 4-20% gradient gel and transferred to a nitrocellulose membrane. The membrane was blotted with the following primary antibodies: anti-HTT, 1:1,000, catalog no. MA2166 (clone 1HU-4C8) (Merck Millipore); anti-polyQ, 1:1,000, catalog no. MABN2427 (clone MW1) (Merck Millipore); and β-actin, 1:5,000, catalog no. A5316 (clone AC-74) (Sigma-Aldrich). After incubation with HRP-conjugated secondary antibodies (anti-rabbit IgG NEF812001 and anti-mouse IgG NEF822001EA) bands were visualized by chemiluminescence.

In vivo structural MRI acquisition

In vivo MRI was performed on a vertical 9.4 Tesla MR scanner (Bruker Biospin) with a triple-axis gradient and a physiological monitoring system (electrocardiogram, respiration and body temperature). Mice were anesthetized with isoflurane (1%) mixed with oxygen and air at 1:3 ratios via a vaporizer and a facial mask and scanned longitudinally (the same mice were imaged repeatedly over a 12-month period). We used a 20-mm diameter volume coil as the radiofrequency transmitter and receiver. Temperature was maintained by a heating block built into the gradient system. Respiration was monitored throughout the entire scan.

High-resolution anatomical images were acquired by using a three-dimensional (3D) T2-weighted fast spin echo sequence with the following parameters: echo time (TE)/repetition time (TR) = 40/700 ms; resolution = 0.1 × 0.1 × 0.25 mm; echo train length = 4; number of average = 2; and flip angle = 40°. Multislice T2-weighted images of the mouse brain were acquired by the rapid acquisition with refocused echoes (RARE) sequence with the following parameter-echo time (TE)/repetition time (TR) = 40 ms/1,500 ms, RARE factor = 8, in-plane resolution = 0.125 × 0.125 mm, slice thickness = 1 mm, total imaging time <2 min)-and used for high-resolution anatomical imaging. Total imaging time was about 50 min per mouse. Mice recovered quickly once the anesthesia was turned off and all mice survived the imaging sessions.

Structural MRI image analysis

Images were first rigidly aligned to a template image from an adult control mouse which was selected from one of the images acquired from age-matched littermate control mice (mouse had the medium brain volume among the control group), which had been manually adjusted to the orientation defined by the Paxinos atlas with an isotropic resolution of 0.1 × 0.1 × 0.1 mm per pixel. After rigid alignment, images had the same position and orientation as the template image and image resolution was also adjusted to an isotropic resolution of 0.1 × 0.1 × 0.1 mm per pixel. Signals from non-brain tissue were removed manually (skull stripping). Skull-stripped, rigidly aligned images were analyzed using DiffeoMap ( https://www.mristudio.org/). The registration accuracy was accessed by independent sets of labmarks that were manually placed at the brain contours. Intensity values of the gray matter, white matter and cerebrospinal fluid were normalized to the values in the template images by using a piece-wise linear function. This procedure ensured that subject and template images have similar intensity histograms. The intensity-normalized images were submitted by Landmarker software to a Linux cluster, which runs large deformation diffeomorphic metric mapping (LDDMM). The transformations were then used for quantitative measurement of changes in local tissue volume among different mouse brains by computing the Jacobian values of the transformations generated by LDDMM. There are 29 different brain regions segmented automatically.

Behavioral tests

The 5-mm balance beam testing was conducted on an 80-cm-long and 5-mm-wide square-shaped balance beam that was mounted on supports 50-cm-high. A bright light illuminated the start platform and a darkened enclosed 1,728 cm ³ escape box (12 × 12 × 12 cm ³) was situated at the end of the beam. Mice were trained to walk across the beam twice at least 1 h before testing. The time for each mouse to traverse the balance beam was recorded with a 125-s maximum cutoff and falls were scored as 125 s.

Mouse RNA isolation and RNA-seq library preparation

RNA was isolated from snap-frozen striatal mouse tissue from two independent cohorts of mice according to the manufacturer's protocol and purified using RNeasy columns. A total of 13 mRNA libraries for Illumina sequencing were prepared using the KAPA mRNA HyperPrep Kit from 250 ng total RNA. Libraries were sequenced on an Illumina NovaSeq instrument to a minimum of 2 × 10 ⁷ paired-end 100-bp reads.

Mouse RNA-seq data processing and analysis

To correct for batch effects between library preparations, ComBat-seq was applied to the raw read counts65. RNA-seq reads were adapter-trimmed using Cutadapt v.1.14 and mapped to mouse-specific repetitive elements from RepBase v.18.05 by STAR v.2.4.0i64. Repeat-mapping reads were removed and remaining reads were mapped to the mouse genome assembly (mm10) with STAR. Read counts for all protein-coding genes annotated in GENCODE v.M20 were calculated using the read summarization program featureCounts v.1.38.166. Differential expression analysis was performed using DESeq2 v. 2.0.265. Differentially expressed mRNAs were defined as having an FDR-adjusted P  < 0.01 as determined by the Wald Test in the DESeq2 program. We used the Unix grep search tool to scan the mouse genome for all protein-coding transcripts with five or more consecutive CAG repeats.

Human and mouse GO analysis

PANTHER v.16 was used for GO analysis67. All human or mouse protein-coding genes with at least five reads in any sample were used as the background set. Significant GO terms were determined by Fisher's exact test after FDR correction at P  < 0.05 and sorted by fold enrichment.

Statistical analysis

Data are expressed as the mean ± s.e.m. unless otherwise noted. A Student's t-test was used to measure the significant levels between WT or control and zQ175/+ or human HD groups at each given time point. Two-way repeated ANOVA (genotype and treatment) was performed to examine the statistical difference between different groups along with age and treatment. Statistical analysis was performed with Prism (GraphPad Software version 8.0.2). P values less than 0.05 were considered statistically significant ( n is reported in the figure legends). Based on the means and s.d. from a previous study with Q175/+ mice66, a power analysis revealed that <3 mice reliably distinguish mutant and control populations ( P < 0.05). To have 95% confidence in detecting an improvement with inhibition of the mutant HTT allele, 4 animals per genotype and treatment group are required (power analysis: two-way ANOVA, genotype × treatment, P ≤ 0.05). No statistical methods were used to predetermine sample sizes for the in vitro experiments but our sample sizes are similar to those reported in previous publications30,33. Data distribution was assumed to be normal but this was not formally tested for all experiments. Investigators were blinded to genotype and treatment both in vitro and in vivo experiments. No animals nor data points were excluded from the analyses for any reason. Neuronal cultures with <70% Cas13d transduction were eliminated from the study before outcome measures were taken. Mice were randomized into groups to avoid bias. Balanced sex ratio was considered in all experiments. Randomization was not available for the in vitro studies due to limited availability of patient lines and established neurotypical controls. All replications were successful and included in our outcome measures. Technical replication is represented by n in the figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Acknowledgements

We thank the Sequencing Core Facilities of the La Jolla Institute for Immunology and the IGM Genomics Center, University of California San Diego (UCSD), for use of their Illumina sequencing platforms supported by National Institutes of Health (NIH) Shared Instrumentations grant nos. OD025052 and OD026929, respectively. This work was supported by NIH grant nos. EY029166, NS103172, MH107367, AI132122, HG004659 and HG009889 to G.W.Y.; the Bev Hartig Huntington's Disease Foundation; and NIH grant nos. NS099397 and NS124084 awarded to W.D. An NIH NS112654 postdoctoral fellowship and a University of California President's Postdoctoral Fellowship were awarded to K.H.M. An Allen Distinguished Investigator Award, a Paul G. Allen Frontiers Group-advised grant of the Paul G. Allen Foundation, supports G.W.Y. Q.W. was a visiting PhD student from the Beijing University of Chinese Medicine, People's Republic of China (January 2019 to December 2020); her visit to the Johns Hopkins University School of Medicine was sponsored by a scholarship from the China Scholarship Council and the National Natural Science Foundation of China (no. 82174278 and no. 81973748 to her PhD mentor J.C.). The Hereditary Disease Foundation supported C.S.G. A T32 institutional award to the UCSD Genetics Training Program from NIH (no. GM008666) and an NIH predoctoral fellowship (no. NS111859) supported R.J.M. who is now a postdoctoral fellow at Harvard Medical School in the department of Biological Chemistry and Molecular Pharmacology. A National Science Foundation Graduate Research Fellowship (grant no. DGE-2038238), a Myotonic Dystrophy Foundation Doctoral Research Fellowship, and an Association for Women in Science Scholarship were awarded to M.L.G. A Triton Research and an Experiential Learning Scholarship by a Eureka! Research Scholarship were awarded to A.L. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. GO plots were completed by http://www.bioinformatics.com, an online platform for data analysis and visualization. All schematics (Fig. 1a; Fig. 2a; Fig. 4a) were created using BioRender ( https://biorender.com/).

Author information

Author notes

These authors contributed equally: Kathryn H. Morelli, Qian Wu.

Authors and Affiliations

1. Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA

Kathryn H. Morelli, Maya L. Gosztyla, Ryan J. Marina, Kari Lee, Krysten L. Jones, Megan Y. Huang, Allison Li & Gene W. Yeo

2. Division of Neurobiology, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Qian Wu, Hongshuai Liu, Minmin Yao, Chuangchuang Zhang & Wenzhen Duan

3. School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China

Qian Wu & Jiaxu Chen

4. Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA, USA

Charlene Smith-Geater & Leslie M. Thompson

5. Department of Neurobiology and Behavior, University of California Irvine, Irvine, CA, USA

Leslie M. Thompson

6. The Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Wenzhen Duan

7. Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Wenzhen Duan

8. Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA

Gene W. Yeo

9. Stem Cell Program, University of California San Diego, La Jolla, CA, USA

Gene W. Yeo

Contributions

W.D., K.H.M. and G.W.Y. conceptualized the study. W.D., Q.W., K.H.M., L.M.T., C.S. and G.W.Y. devised the methodology. Q.W., K.H.M., M.H., A.L., H.L., M.Y., C.Z., J.C., R.J.M., K.L. and K.L.J. carried out the experimental work. Q.W., K.H.M. and M.L.G. carried out the formal analysis. K.H.M. and Q.W. wrote the original manuscript draft. W.D., K.H.M. and G.W.Y. reviewed and edited the manuscript. W.D. and G.W.Y. acquired the funding. W.D. and G.W.Y. supervised the study.

Corresponding authors

Correspondence to Wenzhen Duan or Gene W. Yeo.

Ethics declarations

Competing interests

G.W.Y. is a scientific advisory board (SAB) member of Jumpcode Genomics and a cofounder, member of the board of directors, on the SAB, equity holder, and paid consultant for Locanabio and Eclipse BioInnovations. G.W.Y. is a Distinguished Visiting Professor at the National University of Singapore. G.W.Y.'s interests have been reviewed and approved by UCSD in accordance with its conflict-of-interest policies. The authors declare no other competing interests.

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Morelli, K.H., Wu, Q., Gosztyla, M.L. et al. An RNA-targeting CRISPR-Cas13d system alleviates disease-related phenotypes in Huntington's disease models. Nat Neurosci 26, 27-38 (2023). https://doi.org/10.1038/s41593-022-01207-1
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DOI: https://doi.org/10.1038/s41593-022-01207-1

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An RNA-targeting CRISPR-Cas13d system alleviates disease-related phenotypes in Huntington's disease models - Nature Neuroscience

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Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Since the reduction of pathogenic mutant HTT messenger RNA is therapeutic, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR-Cas13d system (Cas13d-CAGEX) that eliminates toxic CAGEX RNA in fibroblasts derived from patients with HD and induced pluripotent stem cell-derived neurons.

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Huntington's disease (HD) is a progressive brain disorder caused by a defective gene. This disease causes changes in the central area of the brain, which affect movement, mood and thinking skills.

About Huntington's disease

Huntington's disease is a progressive brain disorder caused by a single defective gene on chromosome 4 - one of the 23 human chromosomes that carry a person's entire genetic code.

This defect is "dominant," meaning that anyone who inherits it from a parent with Huntington's will eventually develop the disease. The disorder is named for George Huntington, M.D., the physician who first described it in the late 1800s.

The defective gene codes the blueprint for a protein called huntingtin. This protein's normal function isn't yet known, but it's called "huntingtin" because scientists identified its defective form as the cause of Huntington's disease. Defective huntingtin protein leads to brain changes that cause abnormal involuntary movements, a severe decline in thinking and reasoning skills, and irritability, depression and other mood changes.

Symptoms

Symptoms of Huntington's disease usually develop between ages 30 and 50, but they can appear as early as age 2 or as late as 80. The hallmark symptom of Huntington's disease is uncontrolled movement of the arms, legs, head, face and upper body. Huntington's disease also causes a decline in thinking and reasoning skills, including memory, concentration, judgment, and ability to plan and organize.

Huntington's disease brain changes lead to alterations in mood, especially depression, anxiety, and uncharacteristic anger and irritability. Another common symptom is obsessive-compulsive behavior, leading a person to repeat the same question or activity over and over.

Diagnosis

Scientists identified the defective gene that causes Huntington's disease in 1993. A diagnostic genetic test is now available. The test can confirm that the defective gene for huntingtin protein is the cause of symptoms in people with suspected Huntington's disease and can detect the defective gene in people who don't yet have symptoms but are at risk because a parent has Huntington's.

Experts strongly recommend professional genetic counseling both before and after genetic testing for Huntington's disease.

Causes and risks

The defective gene identified in 1993 causes virtually all Huntington's disease.

The huntingtin gene defect involves extra repeats of one specific chemical code in one small section of chromosome 4. The normal huntingtin gene includes 17 to 20 repetitions of this code among its total of more than 3,100 codes. The defect that causes Huntington's disease includes 40 or more repeats. Genetic tests for Huntington's disease measure the number of repeats present in an individual's huntingtin protein gene.

Scientists don't yet understand the normal function of huntingtin protein or how a few dozen extra repeats in its genetic blueprint lead to the devastating symptoms of Huntington's disease. Researchers are eager to solve these mysteries to find the answer to Huntington's. These solutions also may offer important insights into a wide range of other brain disorders, including Alzheimer's, Parkinson's disease and amyotrophic lateral sclerosis (ALS). Sign up for our e-news to receive updates about Alzheimer's and dementia care and research.

Treatment and outcomes

There is currently no cure for Huntington's disease and no way to slow or stop the brain changes it causes. Treatments focus on managing symptoms. A group of international experts recommended the following treatments as first-line strategies for three of the disease's most troubling symptoms:

Chorea (involuntary movements): Some experts believe beginning treatment with an atypical antipsychotic drug, such as olanzapine, is best. Others start with another type of drug recently approved by the U.S. Food and Drug Administration (FDA) specifically for Huntington's, called tetrabenazine.

Irritability: For severe anger and threatening behavior, experts agree that an atypical antipsychotic drug is the preferred approach. For less severe, nonthreatening irritability, experts recommend first trying a selective serotonin reuptake inhibitor (SSRI), which is a type of antidepressant.

Obsessive-compulsive thoughts and actions: Experts also recommend SSRIs as the standard treatment for these symptoms.

Other Huntington's symptoms, such as anxiety, depression and insomnia, also should be treated according to generally accepted guidelines. Experts encourage people with Huntington's to keep all their medical appointments and not to get discouraged if it takes their health care team some time to find the best drugs and the most effective doses.

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Huntington's disease - learn about HD symptoms, diagnosis, causes and treatments and how this disorder relates to Alzheimer's and other dementias.

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National Institute of Neurological Disorders and Stroke

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Huntington’s disease (HD) is an inherited disorder that causes nerve cells (neurons) in parts of the brain to gradually break down and die. The disease attacks areas of the brain that help to control voluntary (intentional) movement, as well as other areas.

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Overview

Huntington's disease is a rare, inherited disease that causes the progressive breakdown (degeneration) of nerve cells in the brain. Huntington's disease has a wide impact on a person's functional abilities and usually results in movement, thinking (cognitive) and psychiatric disorders.

Huntington's disease symptoms can develop at any time, but they often first appear when people are in their 30s or 40s. If the condition develops before age 20, it's called juvenile Huntington's disease. When Huntington's develops early, symptoms are somewhat different and the disease may progress faster.

Medications are available to help manage the symptoms of Huntington's disease. But treatments can't prevent the physical, mental and behavioral decline associated with the condition.

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Symptoms

Huntington's disease usually causes movement, cognitive and psychiatric disorders with a wide spectrum of signs and symptoms. Which symptoms appear first varies greatly from person to person. Some symptoms appear more dominant or have a greater effect on functional ability, but that can change throughout the course of the disease.

Movement disorders

The movement disorders associated with Huntington's disease can include both involuntary movement problems and impairments in voluntary movements, such as:

Involuntary jerking or writhing movements (chorea)

Muscle problems, such as rigidity or muscle contracture (dystonia)

Slow or unusual eye movements

Impaired gait, posture and balance

Difficulty with speech or swallowing

Impairments in voluntary movements - rather than involuntary movements - may have a greater impact on a person's ability to work, perform daily activities, communicate and remain independent.

Cognitive disorders

Cognitive impairments often associated with Huntington's disease include:

Difficulty organizing, prioritizing or focusing on tasks

Lack of flexibility or the tendency to get stuck on a thought, behavior or action (perseveration)

Lack of impulse control that can result in outbursts, acting without thinking and sexual promiscuity

Lack of awareness of one's own behaviors and abilities

Slowness in processing thoughts or ''finding'' words

Difficulty in learning new information

Psychiatric disorders

The most common psychiatric disorder associated with Huntington's disease is depression. This isn't simply a reaction to receiving a diagnosis of Huntington's disease. Instead, depression appears to occur because of injury to the brain and subsequent changes in brain function. Signs and symptoms may include:

Feelings of irritability, sadness or apathy

Social withdrawal

Insomnia

Fatigue and loss of energy

Frequent thoughts of death, dying or suicide

Other common psychiatric disorders include:

Obsessive-compulsive disorder, a condition marked by recurrent, intrusive thoughts and repetitive behaviors

Mania, which can cause elevated mood, overactivity, impulsive behavior and inflated self-esteem

Bipolar disorder, a condition with alternating episodes of depression and mania

In addition to the above disorders, weight loss is common in people with Huntington's disease, especially as the disease progresses.

Symptoms of juvenile Huntington's disease

The start and progression of Huntington's disease in younger people may be slightly different from that in adults. Problems that often present early in the course of the disease include:

Behavioral changes

Difficulty paying attention

Rapid, significant drop in overall school performance

Behavioral problems

Physical changes

Contracted and rigid muscles that affect gait (especially in young children)

Tremors or slight involuntary movements

Frequent falls or clumsiness

Seizures

When to see a doctor

See your health care provider if you notice changes in your movements, emotional state or mental ability. The signs and symptoms of Huntington's disease can be caused by a number of different conditions. Therefore, it's important to get a prompt, thorough diagnosis.

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Causes

Huntington's disease is caused by an inherited difference in a single gene. Huntington's disease is an autosomal dominant disorder, which means that a person needs only one copy of the nontypical gene to develop the disorder.

With the exception of genes on the sex chromosomes, a person inherits two copies of every gene - one copy from each parent. A parent with a nontypical gene could pass along the nontypical copy of the gene or the healthy copy. Each child in the family, therefore, has a 50% chance of inheriting the gene that causes the genetic disorder.

Complications

After Huntington's disease starts, a person's functional abilities gradually worsen over time. The rate of disease progression and duration varies. The time from the first symptoms to death is often about 10 to 30 years. Juvenile Huntington's disease usually results in death within 10 years after symptoms develop.

The clinical depression associated with Huntington's disease may increase the risk of suicide. Some research suggests that the greatest risk of suicide occurs before a diagnosis is made and in the middle stages of the disease when a person starts to lose independence.

Eventually, a person with Huntington's disease requires help with all activities of daily living and care. Late in the disease, the person will likely be confined to a bed and unable to speak. Someone with Huntington's disease is generally able to understand language and has an awareness of family and friends, though some won't recognize family members.

Common causes of death include:

Pneumonia or other infections

Injuries related to falls

Complications related to the inability to swallow

Prevention

People with a known family history of Huntington's disease are understandably concerned about whether they may pass the Huntington gene on to their children. These people may consider genetic testing and family planning options.

If an at-risk parent is considering genetic testing, it can be helpful to meet with a genetic counselor. A genetic counselor will discuss the potential risks of a positive test result, which would indicate that the parent will develop the disease. Also, couples will need to make additional choices about whether to have children or to consider alternatives, such as prenatal testing for the gene or in vitro fertilization with donor sperm or eggs.

Another option for couples is in vitro fertilization and preimplantation genetic diagnosis. In this process, eggs are removed from the ovaries and fertilized with the father's sperm in a laboratory. The embryos are tested for presence of the Huntington gene, and only those testing negative for the Huntington gene are implanted in the mother's uterus.

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Huntington's disease - Symptoms and causes

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This condition causes a progressive breakdown of nerve cells in the brain. Find out about what signs to watch for and how treatments may help.

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Description

Huntington's disease is a progressive brain disorder that causes uncontrolled movements, emotional problems, and loss of thinking ability (cognition).

Adult-onset Huntington's disease, the most common form of this disorder, usually appears in a person's thirties or forties. Early signs and symptoms can include irritability, depression, small involuntary movements, poor coordination, and trouble learning new information or making decisions. Many people with Huntington's disease develop involuntary jerking or twitching movements known as chorea. As the disease progresses, these movements become more pronounced. Affected individuals may have trouble walking, speaking, and swallowing. People with this disorder also experience changes in personality and a decline in thinking and reasoning abilities. Individuals with the adult-onset form of Huntington's disease usually live about 15 to 20 years after signs and symptoms begin.

A less common form of Huntington's disease known as the juvenile form begins in childhood or adolescence. It also involves movement problems and mental and emotional changes. Additional signs of the juvenile form include slow movements, clumsiness, frequent falling, rigidity, slurred speech, and drooling. School performance declines as thinking and reasoning abilities become impaired. Seizures occur in 30 percent to 50 percent of children with this condition. Juvenile Huntington's disease tends to progress more quickly than the adult-onset form; affected individuals usually live 10 to 15 years after signs and symptoms appear.

Frequency

Huntington's disease affects an estimated 3 to 7 per 100,000 people of European ancestry. The disorder appears to be less common in some other populations, including people of Japanese, Chinese, and African descent.

Causes

Variants (also called mutations) in the gene cause Huntington's disease. The HTT gene provides instructions for making a protein called huntingtin. Although the function of this protein is unclear, it appears to play an important role in nerve cells (neurons) in the brain.

The HTT variant that causes Huntington's disease involves a DNA segment known as a CAG trinucleotide repeat. This segment is made up of a series of three DNA building blocks (cytosine, adenine, and guanine) that appear multiple times in a row. Normally, the CAG segment is repeated 10 to 35 times within the gene. In people with Huntington's disease, the CAG segment is repeated 36 to more than 120 times. People with 36 to 39 CAG repeats may or may not develop the signs and symptoms of Huntington's disease, while people with 40 or more repeats almost always develop the disorder.

An increase in the size of the CAG segment leads to the production of an abnormally long version of the huntingtin protein. The elongated protein is cut into smaller, toxic fragments that bind together and accumulate in neurons, disrupting the normal functions of these cells. The dysfunction and eventual death of neurons in certain areas of the brain underlie the signs and symptoms of Huntington's disease.

Inheritance

This condition is inherited in an autosomal dominant pattern, which means one copy of the altered gene in each cell is sufficient to cause the disorder. An affected person usually inherits the altered gene from one affected parent. In rare cases, an individual with Huntington's disease does not have a parent with the disorder.

As the altered gene is passed from one generation to the next, the size of the CAG trinucleotide repeat often increases in size. A larger number of repeats is usually associated with an earlier onset of signs and symptoms. This phenomenon is called anticipation. People with the adult-onset form of Huntington's disease typically have 40 to 50 CAG repeats in the HTT gene, while people with the juvenile form of the disorder tend to have more than 60 CAG repeats.

Individuals who have 27 to 35 CAG repeats in the HTT gene do not develop Huntington's disease, but they are at risk of having children who will develop the disorder. As the gene is passed from parent to child, the size of the CAG trinucleotide repeat may lengthen into the range associated with Huntington's disease (36 repeats or more).

Other Names for This Condition

Huntington chorea

Huntington chronic progressive hereditary chorea

Huntington disease

Huntington's chorea

Additional Information & Resources

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Genetic and Rare Diseases Information Center

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Catalog of Genes and Diseases from OMIM

Scientific Articles on PubMed

References

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Huntington disease is a progressive brain disorder that causes uncontrolled movements, emotional problems, and loss of thinking ability (cognition). Explore symptoms, inheritance, genetics of this condition.

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There are two ways to answer the question "how many people have Huntington disease (HD)?" The first is by determining the prevalence (i.e., the proportion of a population that has a given condition at a particular point in time), and the second is by determining the incidence (i.e., the number of people who will develop a condition over a defined period of time) of HD. Prevalence answers the question of "how many people currently have HD?" and incidence answers the question of "how frequently are people being diagnosed with HD?" Research suggests that the answers to both of these questions are changing and perhaps increasing. These changes will have important biological, economic, and social implications for the future.

The prevalence of HD has been reported to vary with ethnicity and geographical location, confirming that demographic differences influence the number and composition of individuals with HD. Since HD is believed to have major origins in Northern Europe, 2 it is not surprising that populations of Northern European descent are recorded as having the highest prevalence of HD in theworld. ³ In the 1970's and 1980's, a number of studies around the globe examined the prevalence of HD. In Europe it was estimated that an average of 4-7 persons per 100,000 were affected with the disease. 4,5,6,7 Canadian studies suggested 2.4-8.4 persons per 100,000 had HD. 8,9 In the United States, it was estimated that 4.1 - 5.2 persons per 100,000 were living with thedisorder. 10,11 Figure 1 reviews additional global prevalence estimates for HD. While a number of studies have examined the prevalence of HD, very few studies have investigated the disease's incidence. Findings from Minnesota, USA suggested that 3 individuals per million were diagnosed with HD each year between 1950-1989. ¹² In British Columbia, Canada, 6.9 individuals per million were diagnosed each year between 1987-1999. ¹³

In recent years, the accuracy of published prevalence estimates for HD has been called into question. The majority of prevalence estimates for HD were conducted prior to the identification of the genetic mutation underlying the disease; with a genetic test now available, diagnosing HD has become more precise. Patients with atypical symptoms, late disease onset, and negative family histories are more likely to be diagnosed, reflecting a likely increase in the number of persons living with the disease. However, fear of experiencing genetic discrimination from health insurers, among family members, or in social settings ¹⁴ may prevent some individuals from disclosing that they are either at-risk or premanifest for HD, thus making it difficult to accurately identify every patient in a given population.

A brief review of the number of patients cared for by the Huntington Disease Association in the United Kingdom (UK) indicated that approximately 6,700 individuals in the UK are currently living with HD. This means that 12.4 per 100,000 persons or 1 out of every 8,065 individuals may be affected with HD ¹⁵.Although these numbers at best provide a minimum prevalence estimate in the UK, since the Association does not care for all HD patients in England and Wales, the numbers do demonstrate that previous prevalence figures are likely underestimated. Fortunately, recent increases in HD awareness and access to information and support, developments of legislations dedicated to preventing genetic discrimination, and continuations of promising basic and clinical HD research have had impact on the willingness of individuals to involve themselves in the HD community. More patients and families are reaching out to medical service providers and becoming involved in HD research, and their increased involvement in the HD community provides an ideal opportunity to reassess the global prevalence of HD.

While the need to establish new baseline prevalence estimates for HD is apparent, these figures must be frequently revised to determine how the prevalence of HD changes over time. Factors such as the projected demographic shifts in median age and our increasing longevity will likely impact the prevalence of HD in the coming years. With an aging population that is expected to live longer, more individuals who have late onset HD will be diagnosed, thus increasing the number of affected individuals. Accurate prevalence estimates are vital to ensuring that there is appropriate access to clinical services and support for patients and families. Additionally, since medical and research funding is often allocated on the basis of the disease burden on society, it is imperative to know the true prevalence of the disease in order to secure proportional financial support for HD research and care. With a combined global effort from HD patients, families, researchers, and caregivers, we will finally be able to accurately answer the question: "How many people have Huntington disease?"

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3 Caro AJ. Huntington's Chorea; a clinical problem in East Anglia. PhD thesis, University of East Anglia.

4 Quarrell OWJ, Tyler A, Jones MP, et al. Population Studies of Huntington's disease in Wales. Clin Genet 1988;33(3):189-95.

⁵ Simpson A, Johnston AW. The prevalence and patterns of care of Huntington's disease in Grampian. Br J Psychiatry 1989; 155:799-804.

6 Harper, PS. The epidemiology of Huntington's disease. Human Gen 1992; 89:365-376.

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How Many People Have Huntington Disease?

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There are two ways to answer the question "how many people have Huntington disease (HD)?" The first is by determining the prevalence (i.e., the proportion of a population that has a given condition at a particular point in time), and the second is by determining the incidence (i.e., the number of people who will develop a condition over a defined period of time) of HD.

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Huntington's disease is a condition that damages nerve cells in the brain causing them to stop proper functioning. This incapacitates patients by affecting their ability to move, think, and behave normally.

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Due to lack of community knowledge on Huntington's disease, suffers of the disease may experience discrimination, ignorance, and social isolation. Leading to depression, anxiety, and an overall degradation of life.