BioOphthalmology

BioOphthalmology Novel Gene Therapy for Stargardt Disease

Ateam at Radboud University Medical Centre, Nijmegen, the Netherlands, reported a novel antisense oligonucleotide strategy for the treatment of Stargardt disease. The research team developed an RNA therapy to rescue splice defects within a particular gene, ABCA4, with a corrective strategy to remove aberrant transcripts that aim to restore normal biological function.

Stargardt disease (STGD1) develops through mutations in the photoreceptor-specific ABCA4 gene which encodes the adenosine triphosphate-binding cassette, subfamily A, member 4. The ABCA4 gene encodes a transporter protein within retinal photoreceptor cells facilitating the active transport of potentially toxic retinoid compounds removing toxic by-products from the visual cycle. A number of pathogenic variants within specific introns of the ABCA4 gene may result in aberrant splicing of the gene—including pseudogenes—essentially non-functional segments of DNA.

Splicing defects within pseudogenes of ABCA4 can insert aberrant “pseudoexons” (PEs) into the ABCA4 pre-mRNA.These PEs can result in the disruption of the reading frame and also lead to nonsense-mediated decay (NMD).

One way to address this problem is through the use of antisense oligonucleotides (AON), which aim to block pseudoexon insertion and develop therapeutic possibilities to overcome the splicing defects. Patients studied from derived cultured cells use these AON agents to block the PEs from splicing into the ABCA4 mRNA and restore normal splicing. A recent publication on these patient-derived cells, including 3D retinal organoids, reported that several AONs, “appear to be a promising tool to correct splicing defects associated with the pathogenic variants identified in this study, warranting further development of these molecules toward clinical trials in order to halt the progression of this disease.” (doi.org/10.1016/j.omtn.2020.06.007)

Stargardt disease, also referenced as fundus flavimaculatus, was first discovered by German ophthalmologist Karl Stargardt in 1909. His report described juvenile macular dystrophy in seven patients from two separate families, presenting in the first or second decade of life with a reduction in central visual acuity. Today, it is the most common form of inherited macular dystrophy, affecting 1 in 10,000 individuals worldwide. Not dissimilar to retinitis pigmentosa with a high degree of clinical heterogeneity, Stargardt patients may develop severe visual impairment or even complete blindness due to photoreceptor death and the retinal pigment epithelium (RPE) cells.

ABCA4-associated retinopathy develops through biallelic variants in ABCA4 identifying the transmembrane protein of 2,273 amino acids. Allelic heterogeneity presents with more than 1,000 pathogenic mutations to date, and there is a strong correlation between the clinical phenotype with the severity of the ABCA4 genotype. Over recent decades, ABCA4 variants have been identified outside the protein-coding exons. A majority of deepintronic pathogenic variants result in the activation of a splice site within an intron. Insertion of an aberrant pseudoexon into the ABCA4 pre-mRNA leads to disruption of the normal protein.

One mechanical way to correct this challenge is to use relatively small RNA molecules of 18–25 nucleotides binding complementarily to their target pre-mRNA, preventing the inclusion of the PE into the final mRNA transcript upon splicing. AONs represent a new class of genetic therapies that exert their action by modulating target gene expression, a potentially viable therapeutic approach for the treatment of numerous inherited retinal diseases.

Single-stranded oligodeoxynucleotides can be readily designed to hybridize with a target pre-mRNA. While poor stability has hampered the clinical viability of such oligos, significant advances have been enormously improved on the modified chemistry of these AONs.

EARLY CLINICAL TRIALS Clinical trial reports from phases one and two of an intravitreally administered AON to correct a splicing defect with the common deep-intronic mutation in CEP290-Leber congenital amaurosis (LCA10) have demonstrated safety and early efficacy in improved visual function. Moreover, AON-based therapies are also in early trials for common mutations found in RHO-associated autosomal dominant retinitis pigmentosa (ADRP) and Usher syndrome type 2. Beyond the AON approach, a virus-based design could use AAV with engineered nucleotide sequence to target the relevant splice sites, avoiding continual repeat injection administrations.

The research group has announced a start-up company, “Astherna”1, to advance their work and develop proposed clinical trial studies. The group’s main objective is overcoming the current unmet treatment of Stargardt disease.

Astherna is the first biotech company founded by the Radboud University Medical Centre for eye diseases. One founder of the research team, Professor Carrel Hong commented, “I am delighted and proud that Astherna has been founded. We can now really mean something for the many patients with Stargardt disease. [At] the Radboud medical centre, we see people with this condition [the most], so we can make our research directly applicable to patients. I see young patients in my outpatient clinic who know that they are going to lose their sight at some point. It has a huge impact on the life choices they need to make: what education can they do, what kind of job lies ahead for them? The earlier we can help them, the less sight they will lose. These are the people we do it for.”

a

b

Pre-mRNA

Exon 1

mutation

Exon 2

mRNA

Exon 1 Exon 2 Exon 3 Exon 4

cryptic exon containing stop codon or frameshift nonsense-mediated decay cryptic splice donor site

Exon 3 Exon 4

AON

Exon 1 Exon 2 Exon 3 Exon 4

normal splicing

Pre-mRNA Exon 1

mRNA

Exon 1 Exon 2

Premature termination nonsensemediated decay or truncated protein Mutation frameshift

Exon 2 Exon 3

AON Exon 4

Exon 1 Exon 2 Exon 4

In-frame skipping of exon 3 (3n nucleotides) partially functional protein

c

Pre-mRNA 5’ UTR

AON Exon 1

Toxic gain-of-function mutation or triplet repeat expansion

Exon 2 Exon 3 Exon 4

AON AON

Blockage of ribosome binding reduced translation Exon 1 Exon 3 Exon 4

Out-of-frame skipping of exon 2 (3n+l or 3n+2 nucleotides frameshift protein knockdown RNA-DNA hybrid RNase H-mediated degradation

Kanmin Xue & Robert E. MacLaren (2020) Antisense oligonucleotide therapeutics in clinical trials for the treatment of inherited retinal diseases, Expert Opinion on Investigational Drugs, 29:10, 1163–1170, DOI: 10.1080/13543784.2020.1804853