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Compound 1 selectively alleviates disease-associated defects in patient-derived cells. (A) Effect of 1 on poly(GP) abundance in protein lysate extracted from c9ALS patient-derived LCLs (n = 3 C9orf72 LCLs, three replicates per concentration in each line). (B) Effect of 1 on poly(GP) abundance in protein lysate extracted from c9ALS patient-derived iPSCs (n = 4 C9orf72 iPSC lines, three replicates per concentration in each line). (C) Effect of 1 on poly(GP) abundance in protein lysate extracted from patient-derived iPSNs (n = 3 C9orf72 iPSN lines, three replicates per concentration in each line). (D) Effect of 1 on C9orf72 intron 1 abundance, which harbors r(G 4 C 2 ) exp , in c9ALS patient-derived LCLs, as determined by qRT-PCR with intron 1-specific primers (n = 3 C9orf72 LCLs, three replicates per concentration in each line). (E) Effect of 1 on C9orf72 intron 1 abundance in c9ALS patient-derived iPSCs, as determined by qRT-PCR with intron 1-specific primers (n = 4 C9orf72 iPSC lines, three replicates per concentration in each line). (F) Effect of 1 on C9orf72 intron 1 abundance in c9ALS patient-derived iPSNs, as determined by qRT-PCR with intron 1-specific primers primers (n = 3 C9orf72 iPSN lines, three replicates per concentration in each line). (G) Effect of 1 on C9orf72 intron 1 3′ splice in a c9ALS patient-derived LCL, as determined by qRT-PCR with primers spanning the intron 1-exon 2 junction (n = 1 C9orf72 LCL, three replicates per concentration). (H) Effect of 1 on C9orf72 intron 1 3′ splice in a c9ALS patient-derived iPSC line, as determined by qRT-PCR with primers spanning the intron 1-exon 2 junction (n = 1 C9orf72 iPSC line, three replicates per concentration). (I) Effect of 1 on C9orf72 intron 1 3′ splice in a c9ALS patient-derived iPSN, as determined by RT-qPCR with primers spanning the intron 1-exon 2 junction (n = 1 C9orf72 iPSN line, three replicates per concentration). RNA quantification was measured relative to GAPDH. Vehicle indicates 0.1% (v/v) dimethyl sulfoxide (DMSO). *P < 0.05, **P < 0.01, ***P < 0.001 , ****P < 0.0001, as determined by a One Way ANOVA with multiple comparisons. Error bars are reported as SD.
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A hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, or c9ALS/FTD. The RNA transcribed from the expansion, r(G 4 C 2 ) exp , causes various pathologies, including intron retention, aberrant translation that produces toxic dipeptide repeat pro...
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... the molecules emanating from the ReFRAME library screen, we selected 2 for lead optimization due to its structural simplicity and potency (IC 50 = ~300 nM; SI Appendix, Fig. S1B). Derivatives were designed and synthesized to elucidate structure-activity relationships (SAR; Fig. 1B and SI Appendix, Fig. S2A) and improve compound potency and drug-like properties. After a battery of SAR validations including assessing inhibition of RAN translation, toxicity in cellular models and patient-derived cells, and abundance of poly(GP) (one of six DPRs) in patient-derived lymphoblastoid cell lines (LCLs) (SI Appendix, Fig. S2 B-E), compound 1 was ...
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... (SAR; Fig. 1B and SI Appendix, Fig. S2A) and improve compound potency and drug-like properties. After a battery of SAR validations including assessing inhibition of RAN translation, toxicity in cellular models and patient-derived cells, and abundance of poly(GP) (one of six DPRs) in patient-derived lymphoblastoid cell lines (LCLs) (SI Appendix, Fig. S2 B-E), compound 1 was identified as our lead small molecule (Fig. 1B and SI Appendix, Fig. S2). Further evaluation of 1 was carried out through comprehensive Fig. 1. Hallmarks of c9ALS/FTD disease pathology caused by r(G 4 C 2 ) exp and identification of a lead small molecule that alleviates disease-associated pathologies. (A) Schematic ...
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... Cells. We next expanded our studies to evaluate compound 1 comprehensively across several patient-derived cell lines including LCLs, iPSCs and iPSNs. Across four discrete patient-derived LCLs, 1 dose dependently decreased poly(GP) abundance as determined using an electrochemiluminescent immunoassay ( Fig. 2A). This reduction was statistically significant at doses of 50 and 500 nM, which yielded decreases in poly(GP) abundance of 30 ± 6% (P < 0.0001) and 48 ± 6% (P < 0.0001), respectively. In agreement with studies in LCLs, 1 dose dependently reduced poly(GP) in patient-derived iPSCs (n = 4 C9orf72 iPSC lines) with a 48 ± 8% (P < 0.0001) ...
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... of 50 and 500 nM, which yielded decreases in poly(GP) abundance of 30 ± 6% (P < 0.0001) and 48 ± 6% (P < 0.0001), respectively. In agreement with studies in LCLs, 1 dose dependently reduced poly(GP) in patient-derived iPSCs (n = 4 C9orf72 iPSC lines) with a 48 ± 8% (P < 0.0001) decrease in poly(GP) observed upon treatment with 500 nM compound (Fig. 2B). Importantly, despite the reduction of poly(GP), wild-type (WT) C9ORF72 protein levels were unaffected, as determined by Western blotting (SI Appendix, Fig. S7). These results indicate that 1 is selectively inhibiting the aberrant translation of DPRs while having no effect on the canonical translation of C9ORF72 protein. Moreover, in ...
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... as determined by Western blotting (SI Appendix, Fig. S7). These results indicate that 1 is selectively inhibiting the aberrant translation of DPRs while having no effect on the canonical translation of C9ORF72 protein. Moreover, in iPSNs differentiated from three different iPSC lines, 500 nM of 1 reduced poly(GP) by 64 ± 12% (P < 0.0001) (Fig. ...
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... or iPSNs, the following trends were observed upon treatment with 1: intron 1 abundance was dose-dependently reduced (n = 3 patient-derived LCLs; n = 4 patient-derived iPSC lines; n = 3 patient-derived iPSN lines), while the abundance of transcripts amplified with primers spanning the exon 2-exon 3 junction or specific to exon 1b was unchanged (Fig. 2 D-F and SI Appendix, Fig. S8). Importantly, no changes were observed across all transcripts in lymphoblastoid, iPSC or iPSN lines from healthy donors (n = 1 LCL; n = 4 iPSC lines; n = 3 iPSN cell lines), (SI Appendix, Fig. S9). As the abundance of the downstream exon 2-exon 3 junction was unaffected by 1, it suggests selective decay of the repeat-containing intron ...
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... observed in corresponding cells from healthy donors. Similar results were observed when using primers located within intron 1 (SI Appendix, Fig. S10 A and C, and Table S2). Upon treatment with 1 (50 nM), a decrease in intron 1 abundance was observed in c9ALS patient-derived LCLs, iPSCs and iPSNs using primers for the 3′ splice site of intron 1 (Fig. 2 G-I), with treatment at the 500 nM dose showing C9orf72 intron retention (i1-E2 primers) abundance similar to those in cells from healthy donors. Collectively, these results suggest that 1 facilitates intron 1 splicing and promotes its subsequent ...
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... test this hypothesis, we treated c9ALS iPSCs with an siRNA pool targeting hnRNP H or a randomized siRNA as a control. We first confirmed knockdown of hnRNP H by Western blotting and qRT-PCR, both of which were reduced by ~75% upon treatment of 50 nM siRNA pool (SI Appendix, Fig. S12 A-C). Further, this knockdown exacerbated deregulation of the alternative splicing of Trans-Activation-Responsive RNA-Binding Protein 2 (TARBP2) exon 7 (SI Appendix, Fig. S12D), a known substrate processed by hnRNP H (9,14). Additionally, the siRNAs targeting hnRNP H reduced C9orf72 intron 1 abundance by 26 ± 5% (P < 0.001), supporting ...
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... as a control. We first confirmed knockdown of hnRNP H by Western blotting and qRT-PCR, both of which were reduced by ~75% upon treatment of 50 nM siRNA pool (SI Appendix, Fig. S12 A-C). Further, this knockdown exacerbated deregulation of the alternative splicing of Trans-Activation-Responsive RNA-Binding Protein 2 (TARBP2) exon 7 (SI Appendix, Fig. S12D), a known substrate processed by hnRNP H (9,14). Additionally, the siRNAs targeting hnRNP H reduced C9orf72 intron 1 abundance by 26 ± 5% (P < 0.001), supporting the hypothesis that hnRNP H binding prevents splicing and subsequent decay of the intron (Fig. 3A). Thus, we sought to answer if 1's mode of action to reduce intron 1 ...
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... vitro, 1 dose-dependently displaced hnRNP H protein from r(G 4 C 2 ) 8 (SI Appendix, Fig. S12E), as assayed by a previously described time-resolved fluorescence resonance energy transfer assay (16). To demonstrate that 1 displaces hnRNP H from r(G 4 C 2 ) exp in patient-derived cells, we assessed the alternative splicing of TARBP2 exon 7 as well as the formation of toxic RNA foci, which occurs in a subset of CNS cells in c9ALS ...
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... demonstrate that 1 displaces hnRNP H from r(G 4 C 2 ) exp in patient-derived cells, we assessed the alternative splicing of TARBP2 exon 7 as well as the formation of toxic RNA foci, which occurs in a subset of CNS cells in c9ALS patients (12,13). Indeed, 1 (50 nM; co-treated with a randomized siRNA): (i) shifted the TARBP2 exon 7 splicing pattern (increased inclusion) in patient-derived iPSCs toward WT (SI Appendix , Fig. S12D); and (ii) significantly (P = 0.0009) reduced the average number of r(G 4 C 2 ) exp foci per nucleus in patient-derived LCLs (from 0.21 ± 0.01 to 0.10 ± 0.2; Fig. 3B). ...
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... investigations of intron 1 abundance upon treatment with 1. As expected, co-treatment of 1 (50 nM) and a control siRNA resulted in a 42 ± 5% (P < 0.0001) reduction in C9orf72 intron 1 abundance compared to vehicle treatment, as assessed by qRT-PCR using primers specific for C9orf72 intron 1 (Fig. 3A), similar to treatment with 50 nM of 1 alone (Fig. 2D). Interestingly, treatment with the hnRNP H-targeting siRNA alone or combined with 50 nM of 1 reduced intron 1 abundance to a similar extent -by 26 ± 5% and 29 ± 6%, respectively (Fig. 3A). Collectively, these data suggest a mechanism of action in which 1 binds r(G 4 C 2 ) exp , displaces sequestered hnRNP H, and facilitates subsequent ...
Citations
... Other polyGA-specific antibodies have been used in HEK293 cells expressing GA 175 -GFP [9] and in transgenic BAC C9ORF72 mice [37]. A lead molecule screened from ReFRAME small molecule library [38] was further optimized to emerge as a facilitator of degradation of polyGP in patient-derived induced pluripotent stem cells [39]. Since inhibition of GA DPR protein aggregation could provide a therapeutic strategy for C9 ALS-FTD, we have selected specific high affinity aptamers and investigated their role in inhibition of aggregation of polyGA DPRs. ...
Hexanucleotide (GGG GCC) repeat expansion in non-coding region of C9ORF72 is the main genetic cause of amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD). Gain of toxic function, via RNA or proteins, or loss of function via haploinsufficiency, are probable mechanisms of disease progression. Expanded GGG GCC repeat codes for dipeptide repeat (DPR) proteins which form inclusions in the brain. Among all the dipeptides, aggregates formed by polyGA sequence are the most toxic. In this work, inhibition of aggregation of polyGA DPRs using aptamers has been explored as a therapeutic strategy to delay disease progression. Target-specific, high-affinity RNA aptamers were selected against monomeric (GA) 30. Selected aptamers showed significant inhibition of aggregation of (GA) 30 in vitro. Inhibitory RNA sequences were seen to form typical secondary structures which was missing in a non-inhibitory sequence. Some of the RNA aptamers showed increased solubilisation of DPRs formed by (GA) 30 and (GA) 60 in a neuronal cell model of ALS-FTD. Decreased aggregation was accompanied by lower oxidative stress and improved cell survival. Importantly, expression level of one of the markers of autophagy was significantly enhanced in the presence of aptamers, explaining lower aggregation observed in these cells. Thus, aptamers may be developed as potential therapeutic agents in C9 ALS-FTD.
... r(GGGGCC) n , in particular, has previously been shown to arrange into hairpin and G-quadruplex (G4) structures, both of which have exhibited potential involvement in disease progression 19 . Indeed, the potential of these structures as therapeutic targets is demonstrated by using small molecule probes that bind to and stabilise them, which leads to amelioration in disease models 11,[19][20][21][22][23] . In particular, the use of ligands to bind G4s has been shown to ameliorate ALS phenotypes in neuronal cells 10,19,20,22 and targeting of the hairpin with a small molecule inhibited repeat-associated non-ATG (RAN) translation and subsequent generation of toxic dipeptide repeats from the C9orf72 gene mutation 11 . ...
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC) n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC) n , which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD. Amyotrophic lateral sclerosis (ALS) is rapidly progressive, uniformly fatal and untreatable due largely to an incomplete understanding of disease mechanisms. The lifetime risk of ALS is 1:300-1:400, and over an aggressive disease course, patients become paralysed, unable to eat, speak and breathe with an average survival of between 3-5 years 1. Frontotemporal dementia (FTD) is the second most common cause of dementia in patients less than 65 years old and is increasingly recog-nised to share clinical, genetic and pathomechanistic features with ALS, termed ALS/FTD 2. ALS and FTD, much like other neurodegen-erative diseases, are characterised by the presence of pathological aggregates in neurons. Many existing studies have predominantly focused on the protein component as the leading aggregation trigger 3 ,
... Another notable finding is a bloodbrain-penetrant small molecule (compound 1) that selectively binds r(G4C2) expansions in the C9orf72 mutant intron 1 (mi1) (Fig. 2B, small molecule). Compound 1 selectively decreases mi1 retention during splicing, which results in increased mi1 turnover mediated by the nuclear RNA exosome as well as decreases of polyGP in C9-ALS/ FTD cells (LCLs and iPSC-derived neurons) and of polyGP as well as polyGA aggregates in the brains of BAC transgenic C9-ALS/FTD models (Bush et al. 2022). ...
Neurological and neuromuscular diseases resulting from familial, sporadic, or de novo mutations have devasting personal, familial, and societal impacts. As the initial product of DNA transcription, RNA transcripts and their associated ribonucleoprotein complexes provide attractive targets for modulation by increasing wild-type or blocking mutant allele expression, thus relieving downstream pathological consequences. Therefore, it is unsurprising that many existing and under-development therapeutics have focused on targeting disease-associated RNA transcripts as a frontline drug strategy for these genetic disorders. This review focuses on the current range of RNA targeting modalities using examples of both dominant and recessive neurological and neuromuscular diseases.
... r(GGGGCC) n , in particular, has previously been shown to arrange into hairpin and G-quadruplex (G4) structures, both of which have exhibited potential involvement in disease progression 19 . Indeed, the potential of these structures as therapeutic targets is demonstrated by using small molecule probes that bind to and stabilise them, which leads to amelioration in disease models 11,[19][20][21][22][23] . In particular, the use of ligands to bind G4s has been shown to ameliorate ALS phenotypes in neuronal cells 10,19,20,22 and targeting of the hairpin with a small molecule inhibited repeat-associated non-ATG (RAN) translation and subsequent generation of toxic dipeptide repeats from the C9orf72 gene mutation 11 . ...
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.
... An additional small molecule approach used a nuclear RNA exosome to degrade RNA and was designed to be a blood-brain penetrant [127]. This RNA-targeted small molecule is selective in induced pluripotent stem cells and bioactive in multiple mouse models of C9ORF72-FTD/ALS upon delivery by injection. ...
A summit held March 2023 in Scottsdale, Arizona (USA) focused on the intronic hexanucleotide expansion in the C9ORF72 gene and its relevance in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS; C9ORF72-FTD/ALS). The goal of this summit was to connect basic scientists, clinical researchers, drug developers, and individuals affected by C9ORF72-FTD/ALS to evaluate how collaborative efforts across the FTD-ALS disease spectrum might break down existing disease silos. Presentations and discussions covered recent discoveries in C9ORF72-FTD/ALS disease mechanisms, availability of disease biomarkers and recent advances in therapeutic development, and clinical trial design for prevention and treatment for individuals affected by C9ORF72-FTD/ALS and asymptomatic pathological expansion carriers. The C9ORF72-associated hexanucleotide repeat expansion is an important locus for both ALS and FTD. C9ORF72-FTD/ALS may be characterized by loss of function of the C9ORF72 protein and toxic gain of functions caused by both dipeptide repeat (DPR) proteins and hexanucleotide repeat RNA. C9ORF72-FTD/ALS therapeutic strategies discussed at the summit included the use of antisense oligonucleotides, adeno-associated virus (AAV)-mediated gene silencing and gene delivery, and engineered small molecules targeting RNA structures associated with the C9ORF72 expansion. Neurofilament light chain, DPR proteins, and transactive response (TAR) DNA-binding protein 43 (TDP-43)–associated molecular changes were presented as biomarker candidates. Similarly, brain imaging modalities (i.e., magnetic resonance imaging [MRI] and positron emission tomography [PET]) measuring structural, functional, and metabolic changes were discussed as important tools to monitor individuals affected with C9ORF72-FTD/ALS, at both pre-symptomatic and symptomatic disease stages. Finally, summit attendees evaluated current clinical trial designs available for FTD or ALS patients and concluded that therapeutics relevant to FTD/ALS patients, such as those specifically targeting C9ORF72, may need to be tested with composite endpoints covering clinical symptoms of both FTD and ALS. The latter will require novel clinical trial designs to be inclusive of all patient subgroups spanning the FTD/ALS spectrum.
Supplementary Information
The online version contains supplementary material available at 10.1007/s40120-023-00548-8.
... Moreover, the other ASO designated afinersen, which selectively targets C9orf72 V1 and V3 transcript, while sparing V2 transcript, demonstrated reduced poly-GP level in CSF of an C9-ALS patient, validating its target engagement effect (Tran et al., 2022). While ASO facilities degradation of target RNA viaRNase H-dependent pathway, recent RNA technology developed a small molecule binder to G 4 C 2 repeat RNA that recruits RNase L or nuclear RNA exosome and thus selectively degrade G 4 C 2 repeat RNA(Bush et al., , 2022. These RNA-targeting approach would reduce both repeat RNA and DPR, may thus theoretically favorable.The other avenues for the development of treatments for C9-FTD/ ALS include the development of potential RAN translation inhibitors that can reduce DPR toxicity. ...
An hexanucleotide repeat expansion mutation in the non‐coding region of C9orf72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis. This mutation is estimated to be the most frequent genetic cause of these currently incurable diseases. Since the mutation causes autosomal dominantly inherited diseases, disease cascade essentially starts from the expanded DNA repeats. However, molecular disease mechanism is inevitably complex because possible toxic entity for the disease is not just functional loss of translated C9ORF72 protein, if any, but potentially includes bidirectionally transcribed expanded repeat containing RNA and their unconventional repeat‐associated non‐AUG translation products in all possible reading frames. Although the field learned so much about the disease since the identification of the mutation in 2011, how the expanded repeat causes a particular type of fronto‐temporal lobe dominant neurodegeneration and/or motor neuron degeneration is not yet clear. In this review, we summarize and discuss the current understandings of molecular mechanism of this repeat expansion mutation with focuses on the degradation and translation of the repeat containing RNA transcripts. image
