Andrew G.L. Douglas

Splicing therapy for neuromuscular disease

Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision.

RNA splicing: disease and therapy.

The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. The mechanism of pre-mRNA splicing is highly complex, requiring multiple interactions between pre-mRNA, small nuclear ribonucleoproteins and splicing factor proteins. Regulation of this process is even more complicated, relying on loosely defined cis-acting regulatory sequence elements, trans-acting protein factors and cellular responses to varying environmental conditions. Many different human diseases can be caused by errors in RNA splicing or its regulation. Targeting aberrant RNA provides an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. Antisense oligonucleotide therapies show particular promise in this area and, if coupled with improved delivery strategies, could open the door to a multitude of novel personalized therapies.

C9orf72 Hexanucleotide Expansions Are Associated with Altered Endoplasmic Reticulum Calcium Homeostasis and Stress Granule Formation in Induced Pluripotent Stem Cell-Derived Neurons from Patients with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC‐derived motor neurons, decreased cell survival is correlated with dysfunction in Ca2+ homeostasis, reduced levels of the antiapoptotic protein Bcl‐2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC‐derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063–2078

C9orf72 Hexanucleotide Expansions are Associated with Altered ER Calcium Homeostasis and Stress Granule Formation in iPSC-Derived Neurons from Patients with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC‐derived motor neurons, decreased cell survival is correlated with dysfunction in Ca2+ homeostasis, reduced levels of the antiapoptotic protein Bcl‐2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC‐derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063–2078

C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

Evolutionary constraint helps unmask a splicing regulatory region in BRCA1 exon 11.

Background Alternative splicing across exon 11 produces several BRCA1 isoforms. Their proportion varies during the cell cycle, between tissues and in cancer suggesting functional importance of BRCA1 splicing regulation around this exon. Although the regulatory elements driving exon 11 splicing have never been identified, a selective constraint against synonymous substitutions (silent nucleotide variations that do not alter the amino acid residue sequence) in a critical region of BRCA1 exon 11 has been reported to be associated with the necessity to maintain regulatory sequences. Methodology/Principal Findings Here we have designed a specific minigene to investigate the possibility that this bias in synonymous codon usage reflects the need to preserve the BRCA1 alternative splicing program. We report that in-frame deletions and translationally silent nucleotide substitutions in the critical region affect splicing regulation of BRCA1 exon 11. Conclusions/Significance Using a hybrid minigene approach, we have experimentally validated the hypothesis that the need to maintain correct alternative splicing is a selective pressure against translationally silent sequence variations in the critical region of BRCA1 exon 11. Identification of the trans-acting factors involved in regulating exon 11 alternative splicing will be important in understanding BRCA1-associated tumorigenesis.

Oligonucleotide-Based Therapy for FTD/ALS Caused by the C9orf72 Repeat Expansion: A Perspective

Amyotrophic lateral sclerosis (ALS) is a progressive and lethal disease of motor neuron degeneration, leading to paralysis of voluntary muscles and death by respiratory failure within five years of onset. Frontotemporal dementia (FTD) is characterised by degeneration of frontal and temporal lobes, leading to changes in personality, behaviour, and language, culminating in death within 5–10 years. Both of these diseases form a clinical, pathological, and genetic continuum of diseases, and this link has become clearer recently with the discovery of a hexanucleotide repeat expansion in the C9orf72 gene that causes the FTD/ALS spectrum, that is, c9FTD/ALS. Two basic mechanisms have been proposed as being potentially responsible for c9FTD/ALS: loss-of-function of the protein encoded by this gene (associated with aberrant DNA methylation) and gain of function through the formation of RNA foci or protein aggregates. These diseases currently lack any cure or effective treatment. Antisense oligonucleotides (ASOs) are modified nucleic acids that are able to silence targeted mRNAs or perform splice modulation, and the fact that they have proved efficient in repeat expansion diseases including myotonic dystrophy type 1 makes them ideal candidates for c9FTD/ALS therapy. Here, we discuss potential mechanisms and challenges for developing oligonucleotide-based therapy for c9FTD/ALS.

Recurrent duplications of 17q12 associated with variable phenotypes

The ability to identify the clinical nature of the recurrent duplication of chromosome 17q12 has been limited by its rarity and the diverse range of phenotypes associated with this genomic change. In order to further define the clinical features of affected patients, detailed clinical information was collected in the largest series to date (30 patients and 2 of their siblings) through a multi‐institutional collaborative effort. The majority of patients presented with developmental delays varying from mild to severe. Though dysmorphic features were commonly reported, patients do not have consistent and recognizable features. Cardiac, ophthalmologic, growth, behavioral, and other abnormalities were each present in a subset of patients. The newly associated features potentially resulting from 17q12 duplication include height and weight above the 95th percentile, cataracts, microphthalmia, coloboma, astigmatism, tracheomalacia, cutaneous mosaicism, pectus excavatum, scoliosis, hypermobility, hypospadias, diverticulum of Kommerell, pyloric stenosis, and pseudohypoparathryoidism. The majority of duplications were inherited with some carrier parents reporting learning disabilities or microcephaly. We identified additional, potentially contributory copy number changes in a subset of patients, including one patient each with 16p11.2 deletion and 15q13.3 deletion. Our data further define and expand the clinical spectrum associated with duplications of 17q12 and provide support for the role of genomic modifiers contributing to phenotypic variability.

Extending the phenotypic spectrum of CHARGE syndrome: a case with preaxial polydactyly

We report the case of a boy born with multiple congenital abnormalities. These included chorioretinal coloboma, ventricular septal defect (confirmed on echocardiography), 11 pairs of ribs, preaxial polydactyly (Figs 1 and 2), tracheo-oesophageal fistula, choanal atresia, genital hypoplasia and dysmorphic features including epicanthic folds and bilateral low-set ears. He also had gastrooesophageal reflux. His birthweight was 2.2 kg. A clinical diagnosis of CHARGE syndrome was made.

Increasing success and evolving barriers in the hepatitis C cascade of care during the direct acting antiviral era

Barriers remain in the hepatitis C virus (HCV) cascade of care (CoC), limiting the overall impact of direct acting antivirals. This study examines movement between the stages of the HCV CoC and identifies reasons why patients and specific patient populations fail to advance through care in a real world population. We performed a single-center, ambispective cohort study of patients receiving care in an outpatient infectious diseases clinic between October 2015 and September 2016. Patients were followed from treatment referral through sustained virologic response. Univariate and multivariate analyses were performed to identify factors related to completion of each step of the CoC. Of 187 patients meeting inclusion criteria, 120 (64%) completed an evaluation for HCV treatment, 119 (64%) were prescribed treatment, 114 (61%) were approved for treatment, 113 (60%) initiated treatment, 107 (57%) completed treatment, and 100 (53%) achieved a sustained virologic response. In univariate and multivariate analyses, patients with Medicaid insurance were less likely to complete an evaluation and were less likely to be approved for treatment. Treatment completion and SVR rates are much improved from historical CoC reports. However, linkage to care following referral continues to be a formidable challenge for the HCV CoC in the DAA era. Ongoing efforts should focus on linkage to care to capitalize on DAA treatment advances and improving access for patients with Medicaid insurance.