Regulated control of gene therapies by drug-induced splicing.

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.

SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

Genetic circuitry of Survival motor neuron, the gene underlying spinal muscular atrophy.

The clinical severity of the neurodegenerative disorder spinal muscular atrophy (SMA) is dependent on the levels of functional Survival Motor Neuron (SMN) protein. Consequently, current strategies for developing treatments for SMA generally focus on augmenting SMN levels. To identify additional potential therapeutic avenues and achieve a greater understanding of SMN, we applied in vivo, in vitro, and in silico approaches to identify genetic and biochemical interactors of the Drosophila SMN homolog. We identified more than 300 candidate genes that alter an Smn-dependent phenotype in vivo. Integrating the results from our genetic screens, large-scale protein interaction studies, and bioinformatic analysis, we define a unique interactome for SMN that provides a knowledge base for a better understanding of SMA.

CSF Proteomics in Patients With Progressive Supranuclear Palsy.

BACKGROUND AND OBJECTIVES: Identification of fluid biomarkers for progressive supranuclear palsy (PSP) is critical to enhance therapeutic development. We implemented unbiased DNA aptamer (SOMAmer) proteomics to identify novel CSF PSP biomarkers. METHODS: This is a cross-sectional study in original (18 clinically diagnosed PSP-Richardson syndrome [PSP-RS], 28 cognitively healthy controls]), validation (23 PSP-RS, 26 healthy controls), and neuropathology-confirmed (21 PSP, 52 non-PSP frontotemporal lobar degeneration) cohorts. Participants were recruited through the University of California, San Francisco, and the 4-Repeat Neuroimaging Initiative. The original and neuropathology cohorts were analyzed with the SomaScan platform version 3.0 (5026-plex) and the validation cohort with version 4.1 (7595-plex). Clinical severity was measured with the PSP Rating Scale (PSPRS). CSF proteomic data were analyzed to identify differentially expressed targets, implicated biological pathways using enrichment and weighted consensus gene coexpression analyses, diagnostic value of top targets with receiver-operating characteristic curves, and associations with disease severity with linear regressions. RESULTS: A total of 136 participants were included (median age 70.6 ± 8 years, 68 [50%] women). One hundred fifty-five of 5,026 (3.1%), 959 of 7,595 (12.6%), and 321 of 5,026 (6.3%) SOMAmers were differentially expressed in PSP compared with controls in original, validation, and neuropathology-confirmed cohorts, with most of the SOMAmers showing reduced signal (83.1%, 95.1%, and 73.2%, respectively). Three coexpression modules were associated with PSP across cohorts: (1) synaptic function/JAK-STAT (ß = -0.044, corrected p = 0.002), (2) vesicle cytoskeletal trafficking (ß = 0.039, p = 0.007), and (3) cytokine-cytokine receptor interaction (ß = -0.032, p = 0.035) pathways. Axon guidance was the top dysregulated pathway in PSP in original (strength = 1.71, p < 0.001), validation (strength = 0.84, p < 0.001), and neuropathology-confirmed (strength = 0.78, p < 0.001) cohorts. A panel of axon guidance pathway proteins discriminated between PSP and controls in original (area under the curve [AUC] = 0.924), validation (AUC = 0.815), and neuropathology-confirmed (AUC = 0.932) cohorts. Two inflammatory proteins, galectin-10 and cytotoxic T lymphocyte-associated protein-4, correlated with PSPRS scores across cohorts. DISCUSSION: Axon guidance pathway proteins and several other molecular pathways are downregulated in PSP, compared with controls. Proteins in these pathways may be useful targets for biomarker or therapeutic development.

Large-scale network analysis of the cerebrospinal fluid proteome identifies molecular signatures of frontotemporal lobar degeneration.

The pathophysiological mechanisms driving disease progression of frontotemporal lobar degeneration (FTLD) and corresponding biomarkers are not fully understood. We leveraged aptamer-based proteomics (> 4,000 proteins) to identify dysregulated communities of co-expressed cerebrospinal fluid proteins in 116 adults carrying autosomal dominant FTLD mutations (C9orf72, GRN, MAPT) compared to 39 noncarrier controls. Network analysis identified 31 protein co-expression modules. Proteomic signatures of genetic FTLD clinical severity included increased abundance of RNA splicing (particularly in C9orf72 and GRN) and extracellular matrix (particularly in MAPT) modules, as well as decreased abundance of synaptic/neuronal and autophagy modules. The generalizability of genetic FTLD proteomic signatures was tested and confirmed in independent cohorts of 1) sporadic progressive supranuclear palsy-Richardson syndrome and 2) frontotemporal dementia spectrum syndromes. Network-based proteomics hold promise for identifying replicable molecular pathways in adults living with FTLD. 'Hub' proteins driving co-expression of affected modules warrant further attention as candidate biomarkers and therapeutic targets.

An orally available, brain penetrant, small molecule lowers huntingtin levels by enhancing pseudoexon inclusion.

Huntington's Disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the huntingtin (HTT) gene. The mutant HTT (mHTT) protein causes neuronal dysfunction, causing progressive motor, cognitive and behavioral abnormalities. Current treatments for HD only alleviate symptoms, but cerebral spinal fluid (CSF) or central nervous system (CNS) delivery of antisense oligonucleotides (ASOs) or virus vectors expressing RNA-induced silencing (RNAi) moieties designed to induce mHTT mRNA lowering have progressed to clinical trials. Here, we present an alternative disease modifying therapy the orally available, brain penetrant small molecule branaplam. By promoting inclusion of a pseudoexon in the primary transcript, branaplam lowers mHTT protein levels in HD patient cells, in an HD mouse model and in blood samples from Spinal Muscular Atrophy (SMA) Type I patients dosed orally for SMA (NCT02268552). Our work paves the way for evaluating branaplam's utility as an  HD therapy, leveraging small molecule splicing modulators to reduce expression of dominant disease genes by driving pseudoexon inclusion.

Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs.

Branaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects, we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMNΔ7), and in juvenile Wistar Hannover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image-analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.

Use of Intramolecular 1,5-Sulfur-Oxygen and 1,5-Sulfur-Halogen Interactions in the Design of N-Methyl-5-aryl-N-(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,4-thiadiazol-2-amine SMN2 Splicing Modulators.

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by low levels of functional survival motor neuron protein (SMN) resulting from a deletion or loss of function mutation of the survival motor neuron 1 (SMN1) gene. Branaplam (1) elevates levels of full-length SMN protein in vivo by modulating the splicing of the related gene SMN2 to enhance the exon-7 inclusion and increase levels of the SMN. The intramolecular hydrogen bond present in the 2-hydroxyphenyl pyridazine core of 1 enforces a planar conformation of the biaryl system and is critical for the compound activity. Scaffold morphing revealed that the pyridazine could be replaced by a 1,3,4-thiadiazole, which provided additional opportunities for a conformational constraint of the biaryl through intramolecular 1,5-sulfur-oxygen (S···O) or 1,5-sulfur-halogen (S···X) noncovalent interactions. Compound 26, which incorporates a 2-fluorophenyl thiadiazole motif, demonstrated a greater than 50% increase in production of full-length SMN protein in a mouse model of SMA.

Plasma Tau and Neurofilament Light in Frontotemporal Lobar Degeneration and Alzheimer Disease.

OBJECTIVE: To test the hypothesis that plasma total tau (t-tau) and neurofilament light chain (NfL) concentrations may have a differential role in the study of frontotemporal lobar degeneration syndromes (FTLD-S) and clinically diagnosed Alzheimer disease syndromes (AD-S), we determined their diagnostic and prognostic value in FTLD-S and AD-S and their sensitivity to pathologic diagnoses. METHODS: We measured plasma t-tau and NfL with the Simoa platform in 265 participants: 167 FTLD-S, 43 AD-S, and 55 healthy controls (HC), including 82 pathology-proven cases (50 FTLD-tau, 18 FTLD-TDP, 2 FTLD-FUS, and 12 AD) and 98 participants with amyloid PET. We compared cross-sectional and longitudinal biomarker concentrations between groups, their correlation with clinical measures of disease severity, progression, and survival, and cortical thickness. RESULTS: Plasma NfL, but not plasma t-tau, discriminated FTLD-S from HC and AD-S from HC. Both plasma NfL and t-tau were poor discriminators between FLTD-S and AD-S. In pathology-confirmed cases, plasma NfL was higher in FTLD than AD and in FTLD-TDP compared to FTLD-tau, after accounting for age and disease severity. Plasma NfL, but not plasma t-tau, predicted clinical decline and survival and correlated with regional cortical thickness in both FTLD-S and AD-S. The combination of plasma NfL with plasma t-tau did not outperform plasma NfL alone. CONCLUSION: Plasma NfL is superior to plasma t-tau for the diagnosis and prediction of clinical progression of FTLD-S and AD-S. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that plasma NfL has superior diagnostic and prognostic performance vs plasma t-tau in FTLD and AD.

Plasma Neurofilament Light for Prediction of Disease Progression in Familial Frontotemporal Lobar Degeneration.

OBJECTIVE: We tested the hypothesis that plasma neurofilament light chain (NfL) identifies asymptomatic carriers of familial frontotemporal lobar degeneration (FTLD)-causing mutations at risk of disease progression. METHODS: Baseline plasma NfL concentrations were measured with single-molecule array in original (n = 277) and validation (n = 297) cohorts. C9orf72, GRN, and MAPT mutation carriers and noncarriers from the same families were classified by disease severity (asymptomatic, prodromal, and full phenotype) using the CDR Dementia Staging Instrument plus behavior and language domains from the National Alzheimer's Disease Coordinating Center FTLD module (CDR+NACC-FTLD). Linear mixed-effect models related NfL to clinical variables. RESULTS: In both cohorts, baseline NfL was higher in asymptomatic mutation carriers who showed phenoconversion or disease progression compared to nonprogressors (original: 11.4 ± 7 pg/mL vs 6.7 ± 5 pg/mL, p = 0.002; validation: 14.1 ± 12 pg/mL vs 8.7 ± 6 pg/mL, p = 0.035). Plasma NfL discriminated symptomatic from asymptomatic mutation carriers or those with prodromal disease (original cutoff: 13.6 pg/mL, 87.5% sensitivity, 82.7% specificity; validation cutoff: 19.8 pg/mL, 87.4% sensitivity, 84.3% specificity). Higher baseline NfL correlated with worse longitudinal CDR+NACC-FTLD sum of boxes scores, neuropsychological function, and atrophy, regardless of genotype or disease severity, including asymptomatic mutation carriers. CONCLUSIONS: Plasma NfL identifies asymptomatic carriers of FTLD-causing mutations at short-term risk of disease progression and is a potential tool to select participants for prevention clinical trials. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT02372773 and NCT02365922. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that in carriers of FTLD-causing mutations, elevation of plasma NfL predicts short-term risk of clinical progression.

Plasma phosphorylated tau 217 and phosphorylated tau 181 as biomarkers in Alzheimer's disease and frontotemporal lobar degeneration: a retrospective diagnostic performance study.

BACKGROUND: Plasma tau phosphorylated at threonine 217 (p-tau217) and plasma tau phosphorylated at threonine 181 (p-tau181) are associated with Alzheimer's disease tau pathology. We compared the diagnostic value of both biomarkers in cognitively unimpaired participants and patients with a clinical diagnosis of mild cognitive impairment, Alzheimer's disease syndromes, or frontotemporal lobar degeneration (FTLD) syndromes. METHODS: In this retrospective multicohort diagnostic performance study, we analysed plasma samples, obtained from patients aged 18-99 years old who had been diagnosed with Alzheimer's disease syndromes (Alzheimer's disease dementia, logopenic variant primary progressive aphasia, or posterior cortical atrophy), FTLD syndromes (corticobasal syndrome, progressive supranuclear palsy, behavioural variant frontotemporal dementia, non-fluent variant primary progressive aphasia, or semantic variant primary progressive aphasia), or mild cognitive impairment; the participants were from the University of California San Francisco (UCSF) Memory and Aging Center, San Francisco, CA, USA, and the Advancing Research and Treatment for Frontotemporal Lobar Degeneration Consortium (ARTFL; 17 sites in the USA and two in Canada). Participants from both cohorts were carefully characterised, including assessments of CSF p-tau181, amyloid-PET or tau-PET (or both), and clinical and cognitive evaluations. Plasma p-tau181 and p-tau217 were measured using electrochemiluminescence-based assays, which differed only in the biotinylated antibody epitope specificity. Receiver operating characteristic analyses were used to determine diagnostic accuracy of both plasma markers using clinical diagnosis, neuropathological findings, and amyloid-PET and tau-PET measures as gold standards. Difference between two area under the curve (AUC) analyses were tested with the Delong test. FINDINGS: Data were collected from 593 participants (443 from UCSF and 150 from ARTFL, mean age 64 years [SD 13], 294 [50%] women) between July 1 and Nov 30, 2020. Plasma p-tau217 and p-tau181 were correlated (r=0·90, p<0·0001). Both p-tau217 and p-tau181 concentrations were increased in people with Alzheimer's disease syndromes (n=75, mean age 65 years [SD 10]) relative to cognitively unimpaired controls (n=118, mean age 61 years [SD 18]; AUC=0·98 [95% CI 0·95-1·00] for p-tau217, AUC=0·97 [0·94-0·99] for p-tau181; pdiff=0·31) and in pathology-confirmed Alzheimer's disease (n=15, mean age 73 years [SD 12]) versus pathologically confirmed FTLD (n=68, mean age 67 years [SD 8]; AUC=0·96 [0·92-1·00] for p-tau217, AUC=0·91 [0·82-1·00] for p-tau181; pdiff=0·22). P-tau217 outperformed p-tau181 in differentiating patients with Alzheimer's disease syndromes (n=75) from those with FTLD syndromes (n=274, mean age 67 years [SD 9]; AUC=0·93 [0·91-0·96] for p-tau217, AUC=0·91 [0·88-0·94] for p-tau181; pdiff=0·01). P-tau217 was a stronger indicator of amyloid-PET positivity (n=146, AUC=0·91 [0·88-0·94]) than was p-tau181 (n=214, AUC=0·89 [0·86-0·93]; pdiff=0·049). Tau-PET binding in the temporal cortex was more strongly associated with p-tau217 than p-tau181 (r=0·80 vs r=0·72; pdiff<0·0001, n=230). INTERPRETATION: Both p-tau217 and p-tau181 had excellent diagnostic performance for differentiating patients with Alzheimer's disease syndromes from other neurodegenerative disorders. There was some evidence in favour of p-tau217 compared with p-tau181 for differential diagnosis of Alzheimer's disease syndromes versus FTLD syndromes, as an indication of amyloid-PET-positivity, and for stronger correlations with tau-PET signal. Pending replication in independent, diverse, and older cohorts, plasma p-tau217 and p-tau181 could be useful screening tools to identify individuals with underlying amyloid and Alzheimer's disease tau pathology. FUNDING: US National Institutes of Health, State of California Department of Health Services, Rainwater Charitable Foundation, Michael J Fox foundation, Association for Frontotemporal Degeneration, Alzheimer's Association.

Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth.

Myelin inhibitors of axonal regeneration, like Nogo and MAG, block regrowth after injury to the adult CNS. While a GPI-linked receptor for Nogo (NgR) has been identified, MAG's receptor is unknown. We show that MAG inhibits regeneration by interaction with NgR. Binding of and inhibition by MAG are lost if neuronal GPI-linked proteins are cleaved. Binding of MAG to NgR-expressing cells is GPI dependent and sialic acid independent. Conversely, NgR binds to MAG-expressing cells. MAG, but not a truncated MAG that binds neurons but does not inhibit regeneration, precipitates NgR from NgR-expressing cells, DRG, and cerebellar neurons. Importantly, NgR antibody, soluble NgR, or dominant-negative NgR each prevent inhibition of neurite outgrowth by MAG. Also, MAG and Nogo66 compete for binding to NgR. These results suggest redundancy in myelin inhibitors and indicate therapies for CNS injuries.

PKC mediates inhibitory effects of myelin and chondroitin sulfate proteoglycans on axonal regeneration.

Successful axon regeneration in the mammalian central nervous system (CNS) is at least partially compromised due to the inhibitors associated with myelin and glial scar. However, the intracellular signaling mechanisms underlying these inhibitory activities are largely unknown. Here we provide biochemical and functional evidence that conventional isoforms of protein kinase C (PKC) are key components in the signaling pathways that mediate the inhibitory activities of myelin components and chondroitin sulfate proteoglycans (CSPGs), the major class of inhibitors in the glial scar. Both the myelin inhibitors and CSPGs induce PKC activation. Blocking PKC activity pharmacologically and genetically attenuates the ability of CNS myelin and CSPGs to activate Rho and inhibit neurite outgrowth. Intrathecal infusion of a PKC inhibitor, Gö6976, into the site of dorsal hemisection promotes regeneration of dorsal column axons across and beyond the lesion site in adult rats. Thus, perturbing PKC activity could represent a therapeutic approach to stimulating axon regeneration after brain and spinal cord injuries.

Discovery of Small Molecule Splicing Modulators of Survival Motor Neuron-2 (SMN2) for the Treatment of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.

Synthetic transcription elongation factors license transcription across repressive chromatin.

The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich's ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.

Identification of novel genes and pathways regulating SREBP transcriptional activity.

BACKGROUND: Lipid metabolism in mammals is orchestrated by a family of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control the expression of genes required for the uptake and synthesis of cholesterol, fatty acids, and triglycerides. SREBPs are thus essential for insulin-induced lipogenesis and for cellular membrane homeostasis and biogenesis. Although multiple players have been identified that control the expression and activation of SREBPs, gaps remain in our understanding of how SREBPs are coordinated with other physiological pathways. METHODOLOGY: To identify novel regulators of SREBPs, we performed a genome-wide cDNA over-expression screen to identify proteins that might modulate the transcription of a luciferase gene driven from an SREBP-specific promoter. The results were verified through secondary biological assays and expression data were analyzed by a novel application of the Gene Set Enrichment Analysis (GSEA) method. CONCLUSIONS/SIGNIFICANCE: We screened 10,000 different cDNAs and identified a number of genes and pathways that have previously not been implicated in SREBP control and cellular cholesterol homeostasis. These findings further our understanding of lipid biology and should lead to new insights into lipid associated disorders.

Phosphorylated FTY720 promotes astrocyte migration through sphingosine-1-phosphate receptors.

Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.