Efficacy, biodistribution and safety comparison of chemically modified antisense oligonucleotides in the retina.

Antisense oligonucleotides (AONs) are a versatile tool for treating inherited retinal diseases. However, little is known about how different chemical modifications of AONs can affect their biodistribution, toxicity, and uptake in the retina. Here, we addressed this question by comparing splice-switching AONs with three different chemical modifications commonly used in a clinical setting (2'O-methyl-phosphorothioate (2-OMe/PS), 2'O-methoxyethyl-phosphoriate (2-MOE/PS), and phosphorodiamidite morpholino oligomers (PMO)). These AONs targeted genes exclusively expressed in certain types of retinal cells. Overall, studies in vitro and in vivo in C57BL/6J wild-type mouse retinas showed that 2-OMe/PS and 2-MOE/PS AONs have comparable efficacy and safety profiles. In contrast, octa-guanidine-dendrimer-conjugated in vivo PMO-oligonucleotides (ivPMO) caused toxicity. This was evidenced by externally visible ocular phenotypes in 88.5% of all ivPMO-treated animals, accompanied by severe alterations at the morphological level. However, delivery of unmodified PMO-AONs did not cause any toxicity, although it clearly reduced the efficacy. We conducted the first systematic comparison of different chemical modifications of AONs in the retina. Our results showed that the same AON sequence with different chemical modifications displayed different splicing modulation efficacies, suggesting the 2'MOE/PS modification as the most efficacious in these conditions. Thereby, our work provides important insights for future clinical applications.

Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4.

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters the pre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

Understanding and Rescuing the Splicing Defect Caused by the Frequent ABCA4 Variant c.4253+43G>A Underlying Stargardt Disease.

Pathogenic variants in ABCA4 are the underlying molecular cause of Stargardt disease (STGD1), an autosomal recessive macular dystrophy characterized by a progressive loss of central vision. Among intronic ABCA4 variants, c.4253+43G>A is frequently detected in STGD1 cases and is classified as a hypomorphic allele, generally associated with late-onset cases. This variant was previously reported to alter splicing regulatory sequences, but the splicing outcome is not fully understood yet. In this study, we attempted to better understand its effect on splicing and to rescue the aberrant splicing via antisense oligonucleotides (AONs). Wild-type and c.4253+43G>A variant-harboring maxigene vectors revealed additional skipping events, which were not previously detected upon transfection in HEK293T cells. To restore exon inclusion, we designed a set of 27 AONs targeting either splicing silencer motifs or the variant region and screened these in maxigene-transfected HEK293T cells. Candidate AONs able to promote exon inclusion were selected for further testing in patient-derived photoreceptor precursor cells. Surprisingly, no robust splicing modulation was observed in this model system. Overall, this research helped to adequately characterize the splicing alteration caused by the c.4253+43G>A variant, although future development of AON-mediated exon inclusion therapy for ABCA4 is needed.

Antisense Oligonucleotide-Based Rescue of Complex Intronic Splicing Defects in ABCA4.

The ABCA4 gene, involved in Stargardt disease, has a high percentage of splice-altering pathogenic variants, some of which cause complex RNA defects. Although antisense oligonucleotides (AONs) have shown promising results in splicing modulation, they have not yet been used to target complex splicing defects. Here, we performed AON-based rescue studies on ABCA4 complex splicing defects. Intron 13 variants c.1938-724A>G, c.1938-621G>A, c.1938-619A>G, and c.1938-514A>G all lead to the inclusion of different pseudo-exons (PEs) with and without an upstream PE (PE1). Intron 44 variant c.6148-84A>T results in multiple PE inclusions and/or exon skipping events. Five novel AONs were designed to target these defects. AON efficacy was assessed by in vitro splice assays using midigenes containing the variants of interest. All screened complex splicing defects were effectively rescued by the AONs. Although varying levels of efficacy were observed between AONs targeting the same PEs, for all variants at least one AON restored splicing to levels comparable or better than wildtype. In conclusion, AONs are a promising approach to target complex splicing defects in ABCA4.

QR-1011 restores defective ABCA4 splicing caused by multiple severe ABCA4 variants underlying Stargardt disease.

Stargardt disease type 1 (STGD1), the most common form of hereditary macular dystrophy, can be caused by biallelic combinations of over 2200 variants in the ABCA4 gene. This leads to reduced or absent ABCA4 protein activity, resulting in toxic metabolite accumulation in the retina and damage of the retinal pigment epithelium and photoreceptors. Approximately 21% of all ABCA4 variants that contribute to disease influence ABCA4 pre-mRNA splicing. This emphasizes the need for therapies to restore disrupted ABCA4 splicing and halt STGD1 progression. Previously, QR-1011, an antisense oligonucleotide (AON), successfully corrected splicing abnormalities and restored normal ABCA4 protein translation in human retinal organoids carrying the prevalent disease-causing variant c.5461-10T>C in ABCA4. Here, we investigated whether QR-1011 could also correct splicing in four less common non-canonical splice site (NCSS) variants flanking ABCA4 exon 39: c.5461-8T>G, c.5461-6T>C, c.5584+5G>A and c.5584+6T>C. We administered QR-1011 and three other AONs to midigene-transfected cells and demonstrate that QR-1011 had the most pronounced effect on splicing compared to the others. Moreover, QR-1011 significantly increased full-length ABCA4 transcript levels for c.5461-8T>G and c.5584+6T>C. Splicing restoration could not be achieved in the other two variants, suggesting their more severe effect on splicing. Overall, QR-1011, initially developed for a single ABCA4 variant, exhibited potent splice correction capabilities for two additional severe NCSS variants nearby. This suggests the possibility of a broader therapeutic impact of QR-1011 extending beyond its original target and highlights the potential for treating a larger population of STGD1 patients affected by multiple severe ABCA4 variants with a single AON.

Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease.

Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient.