Sue Fletcher

Heterogeneous and predominantly sporadic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), remain highly challenging to model. Patient-derived induced pluripotent stem cell (iPSC) technologies offer great promise for these diseases; however, large-scale studies demonstrating accelerated neurodegeneration in patients with sporadic disease are limited. Here we generated an iPSC library from 100 patients with sporadic ALS (SALS) and conducted population-wide phenotypic screening. Motor neurons derived from patients with SALS recapitulated key aspects of the disease, including reduced survival, accelerated neurite degeneration correlating with donor survival, transcriptional dysregulation and pharmacological rescue by riluzole. Screening of drugs previously tested in ALS clinical trials revealed that 97% failed to mitigate neurodegeneration, reflecting trial outcomes and validating the SALS model. Combinatorial testing of effective drugs identified baricitinib, memantine and riluzole as a promising therapeutic combination for SALS. These findings demonstrate that patient-derived iPSC models can recapitulate sporadic disease features, paving the way for a new generation of disease modeling and therapeutic discovery in ALS.

Duplications of one or more dystrophin exons that disrupt the reading frame account for about 15% of all Duchenne cases, and like the more common genomic deletions, most pathogenic duplications of single or multiple dystrophin exons are also amenable to targeted exon skipping. However, additional considerations must be taken into account: (i) Skipping of all duplicated exons, and flanking exons as necessary, will frequently be required to restore the reading frame and generate an in-frame Becker muscular dystrophy-like mRNA, (ii) the phosphorodiamidate morpholino oligomer chemistry is more effective than the 2′-O-methyl modified oligonucleotides at inducing multiple exon skipping, and (iii) the apparent efficiency of exon skipping can be confounded by the choice of RT-PCR system. Standard RT-PCR systems can preferentially amplify the shorter amplicons, implying more efficient exon skipping than may actually be induced. Unless high fidelity RT-PCR systems are used, strand slippage during annealing/elongation steps will generate normal length transcripts that are artifacts of the amplification.

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases, including inclusion body myositis (IBM), Sjögren’s syndrome, and systemic lupus erythematosus. While they have diagnostic relevance for IBM, little is known about the autoreactive B cells that produce these antibodies. To address this, we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in
Escherichia coli
. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase, preserving the protein’s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation, this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A, the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases.

Accurate exon selection and processing of pre-messenger RNA are crucial for normal gene expression. Mutations that alter splicing disrupt pre-mRNA processing and can have diverse effects on transcript structure, making the consequences of many such mutations difficult to predict. While next-generation sequencing technologies have transformed genetic diagnosis for many patients, deep intronic variants generally evade detection and characterisation. Of all the known types of splicing mutations, the most elusive to predict are those that activate pseudoexons. Because transcripts that contain pseudoexons are otherwise generally intact, exclusion (or ‘skipping’) of the pseudoexon during processing of the pre-mRNA is likely to generate a normal, functional mRNA. Characterisation of pseudoexon mutations will open opportunities for the development of antisense oligonucleotide strategies to overcome these disease-causing mutations.

Purpose: A phase 1 single ascending dose first-in-human study was conducted to evaluate the safety and tolerability of VP-001, an antisense oligonucleotide designed to reduce CNOT3 expression thus allowing upregulation of PRPF31.

Methods: Twelve participants with PRPF31-associated rod-cone dystrophy (RP11) were recruited in four cohorts. One eye of each participant received an intravitreal injection of VP-001 (3, 10, 30, and 75 μg) and followed for 48 weeks for adverse events. Dose escalation was determined by a safety review committee. Visual acuity, slit lamp examination, microperimetry, fundus autofluorescence imaging, optical coherence tomography, full-field sensitivity threshold and clinical chemistry parameters were collected.

Results: No significant drug-related adverse events or intraocular inflammation were observed during the 12-week follow-up period for the 30 μg cohort, and for at least 24 weeks in the 3 and 10 μg cohorts. Results for the fourth cohort receiving a 75 μg dose will be presented. One subject who received a 30 μg dose demonstrated >7 dB retinal sensitivity improvement in 6 of the 10-2 microperimetry grid loci in the treated eye compared to only one locus in the untreated fellow eye at eight weeks post-dosing, and the effect in the treated eye persisted for 15 weeks. OCT showed no significant change in central subfield thickness.

Conclusions: A single intravitreal injection of VP-001 was safe and well tolerated at 3, 10 and 30 μg doses. One case demonstrated significant retinal sensitivity improvement. Longer-term data from all four cohorts will be presented.

Purpose : There are no therapeutic options for Retinitis Pigmentosa type 11 (RP11), a blinding inherited retinal disease caused by haploinsufficiency of the PRPF31 (pre-mRNA processing factor 31) gene. VP-001 has been developed to upregulate PRPF31 protein by downregulating CNOT3 protein expression. CNOT3 negatively regulates PRPF31 by interacting with the PRPF31 promoter region. VP-001 successfully downregulated CNOT3 protein by skipping exon 17 of CNOT-3 pre-mRNA, and upregulated PRPF31 gene expression and the protein in patient derived cellular models. No observed adverse effect level (NOAEL) of VP-001 was established in rabbits and monkeys. Subsequently, a single ascending dose first in human study was started to evaluate initial safety and tolerability of VP-001.

Methods : An open-label, single ascending dose study, recruited participants with genetically confirmed PRPF31 mutation in 3 cohorts. Study eyes received an intravitreal injection of VP-001 (3 µg, 10 µg, 30 µg) and are then followed for 24-week and 48-week time period for incidence, severity and relatedness of ocular treatment-emergent adverse events and treatment-emergent serious adverse events. Three subjects are enrolled for each cohort, and the dose escalation is based on review by a safety review committee (SRC) using 4-week and incremental data collected after each cohort dosing. Safety and tolerability are evaluated based on adverse event (ocular and non-ocular) reporting, that includes clinical chemistry parameters, best corrected visual acuity, perimetry, microperimetry, slit lamp and fundus examination, fundus autofluorescence imaging, and spectral-domain optical coherence tomography.

Results : Six participants (3 µg and 10 µg cohorts) were enrolled till date. No drug related adverse events and no intraocular inflammation were observed at 4 weeks for 10 µg cohort, and at 4 weeks and 12 weeks for 3 µg cohort. Third cohort will receive 30 µg dose, and the safety data collected at day 2, day 14 and week 4 will be reviewed by the SRC. Safety of all the participants will be followed for 24 and 48 weeks.

Conclusions : A single intravitreal injection of VP-001 was safe and well tolerated at 3 µg and 10 µg doses. A 30-µg dose is also administered, and if shown safe at week 4, a 75-µg dose cohort may be added to the study based on recommendation by the SRC. Data from all 3 cohorts will be presented.

Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31. The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.

Vascular Ehlers–Danlos syndrome or Ehlers–Danlos syndrome type IV (vEDS) is a connective tissue disorder characterised by skin hyperextensibility, joint hypermobility and fatal vascular rupture caused by COL3A1 mutations that affect collagen III expression, homo-trimer assembly and secretion. Along with collagens I, II, V and XI, collagen III plays an important role in the extracellular matrix, particularly in the inner organs. To date, only symptomatic treatment for vEDS patients is available. Fibroblasts derived from vEDS patients carrying dominant negative and/or haploinsufficiency mutations in COL3A1 deposit reduced collagen III in the extracellular matrix. This study explored the potential of an antisense oligonucleotide (ASO)-mediated splice modulating strategy to bypass disease-causing COL3A1 mutations reported in the in-frame exons 10 and 15. Antisense oligonucleotides designed to redirect COL3A1 pre-mRNA processing and excise exons 10 or 15 were transfected into dermal fibroblasts derived from vEDS patients and a healthy control subject. Efficient exon 10 or 15 excision from the mature COL3A1 mRNA was achieved and intracellular collagen III expression was increased after treatment with ASOs; however, collagen III deposition into the extracellular matrix was reduced in patient cells. The region encoded by exon 10 includes a glycosylation site, and exon 15 encodes hydroxyproline and hydroxylysine-containing triplet repeats, predicted to be crucial for collagen III assembly. These results emphasize the importance of post-translational modification for collagen III homo-trimer assembly. In conclusion, while efficient skipping of target COL3A1 exons was achieved, the induced collagen III isoforms generated showed defects in extracellular matrix formation. While therapeutic ASO-mediated exon skipping is not indicated for the patients in this study, the observations are restricted to exons 10 and 15 and may not be applicable to other collagen III in-frame exons.

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.