A deep learning approach to identify gene targets of a therapeutic for human splicing disorders.

Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of ELP1 exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in CFTR, LIPA, MLH1 and MAPT. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.

MCOLN1 gene therapy corrects neurologic dysfunction in the mouse model of mucolipidosis IV.

Mucolipidosis IV (MLIV) is an orphan disease leading to debilitating psychomotor deficits and vision loss. It is caused by loss-of-function mutations in the MCOLN1 gene that encodes the lysosomal transient receptor potential channel mucolipin1, or TRPML1. With no existing therapy, the unmet need in this disease is very high. Here, we showed that AAV-mediated CNS-targeted gene transfer of the human MCOLN1 gene rescued motor function and alleviated brain pathology in the MLIV mouse model. Using the AAV-PHP.b vector in symptomatic mice, we showed long-term reversal of declined motor function and significant delay of paralysis. Next, using self-complementary AAV9 clinical candidate vector, we showed that its intracerebroventricular administration in post-natal day 1 mice significantly improved motor function, myelination and reduced lysosomal storage load in the MLIV mouse brain. Based on our data and general advancements in the gene therapy field, we propose scAAV9-mediated CSF-targeted MCOLN1 gene transfer as a therapeutic strategy in MLIV.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

The MeCP2/YY1 interaction regulates ANT1 expression at 4q35: novel hints for Rett syndrome pathogenesis.

Rett syndrome is a severe neurodevelopmental disorder mainly caused by mutations in the transcriptional regulator MeCP2. Although there is no effective therapy for Rett syndrome, the recently discovered disease reversibility in mice suggests that there are therapeutic possibilities. Identification of MeCP2 targets or modifiers of the phenotype can facilitate the design of curative strategies. To identify possible novel MeCP2 interactors, we exploited a bioinformatic approach and selected Ying Yang 1 (YY1) as an interesting candidate. We demonstrate that MeCP2 interacts in vitro and in vivo with YY1, a ubiquitous zinc-finger epigenetic factor regulating the expression of several genes. We show that MeCP2 cooperates with YY1 in repressing the ANT1 gene encoding a mitochondrial adenine nucleotide translocase. Importantly, ANT1 mRNA levels are increased in human and mouse cell lines devoid of MeCP2, in Rett patient fibroblasts and in the brain of Mecp2-null mice. We further demonstrate that ANT1 protein levels are upregulated in Mecp2-null mice. Finally, the identified MeCP2-YY1 interaction, together with the well-known involvement of YY1 in the regulation of D4Z4-associated genes at 4q35, led us to discover the anomalous depression of FRG2, a subtelomeric gene of unknown function, in Rett fibroblasts. Collectively, our data indicate that mutations in MeCP2 might cause the aberrant overexpression of genes located at a specific locus, thus providing new candidates for the pathogenesis of Rett syndrome. As both ANT1 mutations and overexpression have been associated with human diseases, we consider it highly relevant to address the consequences of ANT1 deregulation in Rett syndrome.

Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regulates EGR-1 and REST expression in neuroblastoma cells.

Neurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase-transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1-M4 subtypes and the activation of both IP(3) and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor-1 (EGR-1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR-1 induction. The role of EGR-1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR-1. EGR-1 clones show increased neurite extension and a decrease in Repressor Element-1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc-finger domain, a dominant negative inhibitor of EGR-1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR-1 and REST transcription factors.

Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9.

The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na(+) channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.

Readthrough acetylcholinesterase expression remains minor after stress or exposure to inhibitors.

The gene of mammalian acetylcholinesterase (AChE) generates multiple molecular forms, by alternative splicing of its transcripts and association of the tailed variant (AChET) with structural proteins. In the mammalian brain, the major AChE species consists of AChET tetramers anchored to the cell membrane of neurons by the PRiMA protein (Perrier et al., 2002). Stress and anticholinesterase inhibitors have been reported to induce rapid and long-lasting expression of the readthrough variant (AChER) in the mouse brain (Kaufer et al., 1998). In the readthrough transcript, there is no splicing after the last exon encoding the catalytic domain, so that the entire alternatively spliced 3' region is maintained. It encodes a C-terminal peptide with no specific interaction properties: COS cells transfected with AChER produce a soluble, nonamphiphilic monomeric form. We quantified AChER and total AChE expression in the mouse brain after an immobilization stress and after heat shock in neuroblastoma cells, and compared the observed effects with those induced by irreversible AChE inhibition (Perrier et al., 2005).

The readthrough variant of acetylcholinesterase remains very minor after heat shock, organophosphate inhibition and stress, in cell culture and in vivo.

Acetylcholinesterase (AChE) exists in various molecular forms, depending on alternative splicing of its transcripts and association with structural proteins. Tetramers of the 'tailed' variant (AChE(T)), which are anchored in the cell membrane of neurons by the PRiMA (Proline Rich Membrane Anchor) protein, constitute the main form of AChE in the mammalian brain. In the mouse brain, stress and anticholinesterase inhibitors have been reported to induce expression of the unspliced 'readthrough' variant (AChE(R)) mRNA which produces a monomeric form. To generalize this observation, we attempted to quantify AChE(R) and AChE(T) after organophosphate intoxication in the mouse brain and compared the observed effects with those of stress induced by swimming or immobilization; we also analyzed the effects of heat shock and AChE inhibition on neuroblastoma cells. Active AChE molecular forms were characterized by sedimentation and non-denaturing electrophoresis, and AChE transcripts were quantified by real-time PCR. We observed a moderate increase of the AChE(R) transcript in some cases, both in the mouse brain and in neuroblastoma cultures, but we did not detect any increase of the corresponding active enzyme.