Design and In Vitro Use of Antisense Oligonucleotides to Correct Pre-mRNA Splicing Defects in Inherited Retinal Dystrophies

2017 ◽  
pp. 61-78 ◽  
Author(s):  
Alejandro Garanto ◽  
Rob W. J. Collin
Keyword(s):  
Antisense Oligonucleotides ◽  
Mrna Splicing ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Splicing Defects

scholarly journals Bestrophinopathies: perspectives on clinical disease, Bestrophin-1 function and developing therapies

2021 ◽  
Vol 13 ◽  
pp. 251584142199719
Author(s):  
Simranjeet Singh Grewal ◽  
Joseph J. Smith ◽  
Amanda-Jayne F. Carr
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Incomplete Penetrance ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
High Acuity ◽  
Induced Pluripotent

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that typically affect the macular region, an area synonymous with central high acuity vision. This spectrum of disorders is caused by mutations in bestrophin1 ( BEST1), a protein thought to act as a Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE) of the eye. Although bestrophinopathies are rare, over 250 individual pathological mutations have been identified in the BEST1 gene, with many reported to have various clinical expressivity and incomplete penetrance. With no current clinical treatments available for patients with bestrophinopathies, understanding the role of BEST1 in cells and the pathological pathways underlying disease has become a priority. Induced pluripotent stem cell (iPSC) technology is helping to uncover disease mechanisms and develop treatments for RPE diseases, like bestrophinopathies. Here, we provide a comprehensive review of the pathophysiology of bestrophinopathies and highlight how patient-derived iPSC-RPE are being used to test new genomic therapies in vitro.

scholarly journals Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52

2021 ◽  
Vol 22 (7) ◽  
pp. 3479
Author(s):  
Jessica M. Cale ◽  
Kane Greer ◽  
Sue Fletcher ◽  
Steve D. Wilton
Keyword(s):  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Exon Skipping ◽  
Mrna Splicing ◽  
Immunofluorescent Staining ◽  
Proof Of Concept ◽  
Connective Tissue Disorders ◽  
Fibrillin 1 ◽  
Healthy Control

Marfan syndrome is one of the most common dominantly inherited connective tissue disorders, affecting 2–3 in 10,000 individuals, and is caused by one of over 2800 unique FBN1 mutations. Mutations in FBN1 result in reduced fibrillin-1 expression, or the production of two different fibrillin-1 monomers unable to interact to form functional microfibrils. Here, we describe in vitro evaluation of antisense oligonucleotides designed to mediate exclusion of FBN1 exon 52 during pre-mRNA splicing to restore monomer homology. Antisense oligonucleotide sequences were screened in healthy control fibroblasts. The most effective sequence was synthesised as a phosphorodiamidate morpholino oligomer, a chemistry shown to be safe and effective clinically. We show that exon 52 can be excluded in up to 100% of FBN1 transcripts in healthy control fibroblasts transfected with PMO52. Immunofluorescent staining revealed the loss of fibrillin 1 fibres with ~50% skipping and the subsequent re-appearance of fibres with >80% skipping. However, the effect of exon skipping on the function of the induced fibrillin-1 isoform remains to be explored. Therefore, these findings demonstrate proof-of-concept that exclusion of an exon from FBN1 pre-mRNA can result in internally truncated but identical monomers capable of forming fibres and lay a foundation for further investigation to determine the effect of exon skipping on fibrillin-1 function.

scholarly journals Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4

2021 ◽  
Vol 22 (9) ◽  
pp. 4621
Author(s):  
Tomasz Z. Tomkiewicz ◽  
Nuria Suárez-Herrera ◽  
Frans P. M. Cremers ◽  
Rob W. J. Collin ◽  
Alejandro Garanto
Keyword(s):  
Antisense Oligonucleotide ◽  
Mrna Splicing ◽  
Stargardt Disease ◽  
Aberrant Splicing ◽  
Suitable Candidate ◽  
Pathogenic Variants ◽  
Splice System ◽  
Splicing Defects ◽  
Intronic Variants

The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 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.

scholarly journals Pre-mRNA Splicing by the EssentialDrosophila Protein B52: Tissue and Target Specificity

2000 ◽  
Vol 20 (1) ◽  
pp. 181-186 ◽  
Author(s):  
Bryan E. Hoffman ◽  
John T. Lis
Keyword(s):  
Mrna Splicing ◽  
Larval Brain ◽  
Sr Protein ◽  
In Vitro System ◽  
Nuclear Extracts ◽  
S100 Extract ◽  
Z Ring ◽  
Splicing Defects ◽  
The Brain

ABSTRACT B52, an essential SR protein of Drosophila melanogaster, stimulates pre-mRNA splicing in splicing-deficient mammalian S100 extracts. Surprisingly, mutant larvae depleted of B52 were found to be capable of splicing at least several pre-mRNAs tested (H. Z. Ring and J. T. Lis, Mol. Cell. Biol. 14:7499–7506, 1994). In a homologous in vitro system, we demonstrated that B52 complements a Drosophila S100 extract to allow splicing of a Drosophila fushi tarazu(ftz) mini-pre-mRNA. Moreover, Kc cell nuclear extracts that were immunodepleted of B52 lost their ability to splice thisftz pre-mRNA. In contrast, splicing of this sameftz pre-mRNA occurred in whole larvae homozygous for theB52 deletion. Other SR protein family members isolated from these larvae could substitute for B52 splicing activity in vitro. We also observed that SR proteins are expressed variably in different larval tissues. B52 is the predominant SR protein in specific tissues, including the brain. Tissues in which B52 is normally the major SR protein, such as larval brain tissue, failed to produce ftzmRNA in the B52 deletion line. These observations support a model in which the lethality of the B52 deletion strain is a consequence of splicing defects in tissues in which B52 is normally the major SR protein.

scholarly journals Application of CRISPR Tools for Variant Interpretation and Disease Modeling in Inherited Retinal Dystrophies

Genes ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 473
Author(s):  
Carla Fuster-García ◽  
Belén García-Bohórquez ◽  
Ana Rodríguez-Muñoz ◽  
José M. Millán ◽  
Gema García-García
Keyword(s):  
Vision Loss ◽  
Disease Modeling ◽  
Functional Studies ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Molecular Imprint ◽  
Large Rearrangements

Inherited retinal dystrophies are an assorted group of rare diseases that collectively account for the major cause of visual impairment of genetic origin worldwide. Besides clinically, these vision loss disorders present a high genetic and allelic heterogeneity. To date, over 250 genes have been associated to retinal dystrophies with reported causative variants of every nature (nonsense, missense, frameshift, splice-site, large rearrangements, and so forth). Except for a fistful of mutations, most of them are private and affect one or few families, making it a challenge to ratify the newly identified candidate genes or the pathogenicity of dubious variants in disease-associated loci. A recurrent option involves altering the gene in in vitro or in vivo systems to contrast the resulting phenotype and molecular imprint. To validate specific mutations, the process must rely on simulating the precise genetic change, which, until recently, proved to be a difficult endeavor. The rise of the CRISPR/Cas9 technology and its adaptation for genetic engineering now offers a resourceful suite of tools to alleviate the process of functional studies. Here we review the implementation of these RNA-programmable Cas9 nucleases in culture-based and animal models to elucidate the role of novel genes and variants in retinal dystrophies.

scholarly journals Gene editing preserves visual function in a mouse model of retinal degeneration

2019 ◽  
Author(s):  
Paola Vagni ◽  
Laura E. Perlini ◽  
Naïg A. L. Chenais ◽  
Tommaso Marchetti ◽  
Martina Parrini ◽  
...  
Keyword(s):  
Mouse Model ◽  
Retinitis Pigmentosa ◽  
Gene Editing ◽  
Point Mutations ◽  
Loss Of Function ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Function Point

AbstractInherited retinal dystrophies are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favourable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. We used a tailored CRISPR/Cas9-based gene editing system to prevent retinal photoreceptor death in the Rd10 mouse model of retinitis pigmentosa. We tested the gene editing toolin vitroand then usedin vivosubretinal electroporation to deliver it to one of the retinas of mouse pups at different stages of photoreceptor differentiation. Three months after gene editing, the treated eye exhibited a higher visual acuity compared to the untreated eye. Moreover, we observed preservation of light-evoked responses both in explanted retinas and in the visual cortex of treated animals. Our study validates a CRISPR/Cas9-based therapy as a valuable new approach for the treatment of retinitis pigmentosa caused by autosomal recessive loss-of-function point mutations.

scholarly journals Functional assays of non-canonical splice-site variants in inherited retinal dystrophies genes

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Ana Rodriguez-Muñoz ◽  
Alessandro Liquori ◽  
Belén García-Bohorquez ◽  
Teresa Jaijo ◽  
Elena Aller ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Splice Site ◽  
In Silico ◽  
In Vitro Assays ◽  
Aberrant Splicing ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Progressive Degeneration ◽  
Next Generation Sequencing Ngs

AbstractInherited retinal dystrophies are a group of disorders characterized by the progressive degeneration of photoreceptors leading to loss of the visual function and eventually to legal blindness. Although next generation sequencing (NGS) has revolutionized the molecular diagnosis of these diseases, the pathogenicity of some mutations casts doubts. After the screening of 208 patients with a panel of 117 genes, we obtained 383 variants that were analysed in silico with bioinformatic prediction programs. Based on the results of these tools, we selected 15 variants for their functional assessment. Therefore, we carried out minigene assays to unveil whether they could affect the splicing of the corresponding gene. As a whole, seven variants were found to induce aberrant splicing in the following genes: BEST1, CACNA2D4, PRCD, RIMS1, FSCN2, MERTK and MAK. This study shows the efficacy of a workflow, based on the association of the Minimum Allele Frequency, family co-segregation, in silico predictions and in vitro assays to determine the effect of potential splice site variants identified by DNA-based NGS. These findings improve the molecular diagnosis of inherited retinal dystrophies and will allow some patients to benefit from the upcoming gene-based therapeutic strategies.

In vitro evaluation of hexitol nucleic acid antisense oligonucleotides

1999 ◽  
Author(s):  
Arthur Van Aerschot ◽  
Mark Vandermeeren ◽  
Johan Geysen ◽  
Walter Luyten ◽  
Marc Miller ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Antisense Oligonucleotides ◽  
In Vitro Evaluation

scholarly journals NRL S50T mutation and the importance of ‘founder effects’ in inherited retinal dystrophies

2000 ◽  
Vol 8 (10) ◽  
pp. 783-787 ◽  
Author(s):  
David AR Bessant ◽  
Annette M Payne ◽  
Catherine Plant ◽  
Alan C Bird ◽  
Anand Swaroop ◽  
...  
Keyword(s):  
Founder Effects ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies

scholarly journals Carbon Dot Nanoparticles: Exploring the Potential Use for Gene Delivery in Ophthalmic Diseases

Nanomaterials ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 935
Author(s):  
Manas R. Biswal ◽  
Sofia Bhatia
Keyword(s):  
Gene Delivery ◽  
Therapeutic Option ◽  
Retinal Dystrophy ◽  
Retinal Cells ◽  
Adeno Associated Virus ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Efficient Gene ◽  
New Type ◽  
Viral Nanoparticles

Ocular gene therapy offers significant potential for preventing retinal dystrophy in patients with inherited retinal dystrophies (IRD). Adeno-associated virus (AAV) based gene transfer is the most common and successful gene delivery approach to the eye. These days, many studies are using non-viral nanoparticles (NPs) as an alternative therapeutic option because of their unique properties and biocompatibility. Here, we discuss the potential of carbon dots (CDs), a new type of nanocarrier for gene delivery to the retinal cells. The unique physicochemical properties of CDs (such as optical, electronic, and catalytic) make them suitable for biosensing, imaging, drug, and gene delivery applications. Efficient gene delivery to the retinal cells using CDs depends on various factors, such as photoluminescence, quantum yield, biocompatibility, size, and shape. In this review, we focused on different approaches used to synthesize CDs, classify CDs, various pathways for the intake of gene-loaded carbon nanoparticles inside the cell, and multiple studies that worked on transferring nucleic acid in the eye using CDs.

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