ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability.
By locus-specific ...analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4.
We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells.
Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.
Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To ...date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.
Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.
Inherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the ...past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs.
This open access volume gathers a variety of models, delivery systems, and approaches that can be used to assess RNA technology for exploiting antisense as a therapeutic intervention. Beginning with ...a section on the design of antisense technology and their delivery, the book continues by covering model systems developed to evaluate efficacy, both in vivo and in vitro, as well as methods to evaluate preclinically the toxicity associated with these new potential drugs, and intellectual property considerations. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Antisense RNA Design, Delivery, and Analysis provides basic knowledge and a large collection of methods to facilitate the work of newcomers to this vibrant and expanding field. This book was conceived thanks to the network DARTER (Delivery of Antisense RNA Therapeutics). DARTER is funded by the EU Cooperation of Science and Technology (COST), which aims to enhance interaction and collaborations between researchers in Europe and other countries.
Inherited retinal diseases (IRDs) are both genetically and clinically highly heterogeneous and have long been considered incurable. Following the successful development of a gene augmentation therapy ...for biallelic
-associated IRD, this view has changed. As a result, many different therapeutic approaches are currently being developed, in particular a large variety of molecular therapies. These are depending on the severity of the retinal degeneration, knowledge of the pathophysiological mechanism underlying each subtype of IRD, and the therapeutic target molecule. DNA therapies include approaches such as gene augmentation therapy, genome editing and optogenetics. For some genetic subtypes of IRD, RNA therapies and compound therapies have also shown considerable therapeutic potential. In this review, we summarize the current state-of-the-art of various therapeutic approaches, including the pros and cons of each strategy, and outline the future challenges that lie ahead in the combat against IRDs.
Age-related macular degeneration (AMD) is the main cause of vision loss among the elderly in the Western world. While AMD is a multifactorial disease, the complement system was identified as one of ...the main pathways contributing to disease risk. The strong link between the complement system and AMD was demonstrated by genetic associations, and by elevated complement activation in local eye tissue and in the systemic circulation of AMD patients. Several complement inhibitors have been and are being explored in clinical trials, but thus far with limited success, leaving the majority of AMD patients without treatment options to date. This indicates that there is still a gap of knowledge regarding the functional implications of the complement system in AMD pathogenesis and how to bring these towards clinical translation. Many different experimental set-ups and disease models have been used to study complement activation in vivo and in vitro, and recently emerging patient-derived induced pluripotent stem cells and genome-editing techniques open new opportunities to study AMD disease mechanisms and test new therapeutic strategies in the future. In this review we provide an extensive overview of methods employed to understand the molecular processes of complement activation in AMD pathogenesis. We discuss the findings, advantages and challenges of each approach and conclude with an outlook on how recent, exciting developments can fill in current knowledge gaps and can aid in the development of effective complement-targeting therapeutic strategies in AMD.
The discovery of novel intronic variants in the
locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting ...pre-mRNA splicing makes
a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.
Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation ...in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.
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The eye is the organ in charge of vision and, given its properties, has become an excellent organ to test genetic therapies, including antisense oligonucleotide (AON) technology. In fact, the first ...AON receiving FDA and EMA approval was meant to treat an eye condition. Currently, dozens of clinical trials are being conducted for a variety of subtypes of inherited retinal disease. Although most of them are based on gene augmentation therapies, a phase 3 and two phase 1/2 clinical trials using AONs are ongoing. Since the retina is a layered structure of nondividing cells, obtaining human retinal tissue and expanding it in the lab is not possible, unless induced pluripotent stem cell technology is used. Mouse models have helped to elucidate the function of many genes, and the retinal structure is quite similar to that of humans. Thus, drug delivery to the mouse eye can provide valuable information for further optimization of therapies. In this chapter, the protocol for intravitreal injections of AONs is described in detail.
To elucidate the functional effect of the ABCA4 variant c.5461-10T→C, one of the most frequent variants associated with Stargardt disease (STGD1).
Case series.
Seventeen persons with STGD1 carrying ...ABCA4 variants and 1 control participant.
Haplotype analysis of 4 homozygotes and 11 heterozygotes for c.5461-10T→C and sequence analysis of the ABCA4 gene for a homozygous proband. Fibroblasts were reprogrammed from 3 persons with STGD1 into induced pluripotent stem cells, which were differentiated into photoreceptor progenitor cells (PPCs). The effect of the c.5461-10T→C variant on RNA splicing by reverse-transcription polymerase chain reaction was analyzed using PPC mRNA. In vitro assays were performed with minigene constructs containing ABCA4 exon 39. We analyzed the natural history and ophthalmologic characteristics of 4 persons homozygous for c.5461-10T→C.
Haplotype and rare variant data for ABCA4, RNA splice defects, age at diagnosis, visual acuity, fundus appearance, visual field, electroretinography (ERG) results, fluorescein angiography results, and fundus autofluorescence findings.
The frequent ABCA4 variant c.5461-10T→C has a subtle effect on splicing based on prediction programs. A founder haplotype containing c.5461-10T→C was found to span approximately 96 kb of ABCA4 and did not contain other rare sequence variants. Patient-derived PPCs showed skipping of exon 39 or exons 39 and 40 in the mRNA. HEK293T cell transduction with minigenes carrying exon 39 showed that the splice defects were the result of the c.5461-10T→C variant. All 4 subjects carrying the c.5461-10T→C variant in a homozygous state showed a young age of STGD1 onset, with low visual acuity at presentation and abnormal cone ERG results. All 4 demonstrated severe cone-rod dystrophy before 20 years of age and were legally blind by 25 years of age.
The ABCA4 variant c.5461-10T→C is located on a founder haplotype lacking other disease-causing rare sequence variants. In vitro studies revealed that it leads to mRNA exon skipping and ABCA4 protein truncation. Given the severe phenotype in persons homozygous for this variant, we conclude that this variant results in the absence of ABCA4 activity.
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