Ianthe Pitout

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.

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.

Nucleic acids drugs have been proven in the clinic as a powerful modality to treat inherited and acquired diseases. However, key challenges including drug stability, renal clearance, cellular uptake, and movement across biological barriers (foremost the blood–brain barrier) limit the translation and clinical efficacy of nucleic acid–based therapies, both systemically and in the central nervous system. In this study we provide an overview of an emerging class of nucleic acid therapeutic, called DNAzymes. In particular, we review the use of chemical modifications and carrier molecules for the stabilization and/or delivery of DNAzymes in cell and animal models. Although this review focuses on DNAzymes, the strategies described are broadly applicable to most nucleic acid technologies. This review should serve as a general guide for selecting chemical modifications to improve the therapeutic performance of DNAzymes.

The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T > C; 5603A > T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13–50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects.

Purpose : Retinitis pigmentosa 11 (RP11) is an inherited degenerative retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 (PRPF31) for which there is currently no effective treatment available. RP11 features incomplete penetrance within affected families; the level of PRPF31 expression from the healthy allele determines whether mutation carriers develop symptoms. The CCR4-NOT transcription complex subunit 3 (CNOT3) is a major disease modifier that regulates PRPF31 levels via transcription inhibition. Higher CNOT3 levels are observed in RP11 cases. In this study, we aim to lower CNOT3 expression and function to indirectly upregulate functional PRPF31 from the healthy allele and rescue RP11 disease phenotypes. Methods : Seventy-four antisense oligomers (ASO) were designed to target exonic splice enhancers to mediate exclusion of selected CNOT3 exons to (i) induce translational frameshift and mRNA decay or (ii) produce truncated low/non-functional CNOT3 isoform(s). PRPF31 expression and function were assessed in RP11 iPSC-derived retinal pigment epithelial (RPE) cells. Results : We observed an inverse correlation between CNOT3 and PRPF31 mRNA levels in healthy fibroblasts (n=20). In iPSC-RPE cells, we found 10% higher expression of CNOT3 with 17% lower PRPF31 expression in a patient compared to an asymptomatic relative, both carrying a PRPF31 c.1205 C>A nonsense mutation. Fewer and shorter primary cilia were observed in the symptomatic patient RPE compared to asymptomatic, healthy RPE. Lowering CNOT3 levels and function with ASOs demonstrated a 1.7-fold increase in PRPF31 expression in patient RPE and significantly improved cilia number and length. Healthy cilia play an integral role in phagocytosis and are crucial for normal retinal function. Conclusions : Subtle changes in CNOT3 and PRPF31 levels in retinal cells determine disease penetrance in PRPF31 mutation carriers within an affected family. Modulating expression levels of these proteins can reverse the cellular disease phenotype in RP11. ASOs are effective modulators of CNOT3 expression and function with the ability to increase PRPF31 transcription from the unaffected allele to an expected therapeutic level. Future studies will assess the restoration of transcriptional profiles linked to improved PRPF31 levels in patient derived iPSC-retinal organoids and RPE compared to healthy controls.

The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species.

Objective As structural variations may underpin susceptibility to complex neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the objective of this study was to investigate a structural variant (SV) within sequestosome 1 (SQSTM1). Methods A candidate insertion/deletion variant within intron 5 of the SQSTM1 gene was identified using a previously established SV evaluation algorithm and chosen according to its subsequent theoretical effect on gene expression. The variant was systematically assessed through PCR, polyacrylamide gel fractionation, Sanger sequencing, and reverse transcriptase PCR. Results A reliable and robust assay confirmed the polymorphic nature of this variant and that the variant may influence SQSTM1 transcript levels. In a North American cohort of patients with familial ALS (fALS) and sporadic ALS (sALS) (n = 403) and age-matched healthy controls (n = 562), we subsequently showed that the SQSTM1 variant is associated with fALS (p = 0.0036), particularly in familial superoxide dismutase 1 mutation positive patients (p = 0.0005), but not with patients with sALS (p = 0.97). Conclusions This disease association highlights the importance and implications of further investigation into SVs that may provide new targets for cohort stratification and therapeutic development.

Recent approvals of oligonucleotide analogue drugs to alter gene expression have been welcomed by patient communities but not universally supported. These compounds represent a class of drugs that are designed to target a specific gene transcript, and they include a number of chemical entities to evoke different antisense mechanisms, depending upon the disease aetiology. To date, oligonucleotide therapeutics that are in the clinic or at advanced stages of translation target rare diseases, posing challenges to clinical trial design, recruitment and evaluation and requiring new evaluation paradigms. This review discusses the currently available and emerging therapeutics that alter exon selection through an effect on pre-mRNA splicing and explores emerging concerns over safety and efficacy. Although modification of synthetic nucleic acids destined for therapeutic application is common practice to protect against nuclease degradation and to influence drug function, such modifications may also confer unexpected physicochemical and biological properties. Negatively charged oligonucleotides have a strong propensity to bind extra- and intra-cellular proteins, whereas those analogues with a neutral backbone show inefficient cellular uptake but excellent safety profiles. In addition, the potential for incorporation of chemically modified nucleic acid monomers, yielded by nuclease degradation of exogenous oligonucleotides, into biomolecules has been raised and the possibility not entirely discounted. We conclude with a commentary on the ongoing efforts to develop novel antisense compounds and enhance oligonucleotide delivery in order to further improve efficacy and accelerate implementation of antisense therapeutics for human disease.