Efficient correction of ABCA4 variants by CRISPR-Cas9 in hiPSCs derived from Stargardt disease patients

Siles, Laura; Ruiz-Nogales, Sheila; Navinés-Ferrer, Arnau; Méndez-Vendrell, Pilar; Pomares, Esther

doi:10.1016/j.omtn.2023.02.032

Cited by 11 publications

(3 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nevertheless, the use of AONs to treat the effects of c.6480-35A>G could potentially result in its binding to the region upstream of the canonical SAS that may disrupt regulatory motifs and the binding of auxiliary splice proteins. Additionally, recent studies have shown the efficiency of the CRISPR/Cas9 system in correcting variants in the ABCA4 gene without off-target genomic alterations ( De Angeli et al, 2022 ; Siles et al, 2023 ). These are promising areas of research that could potentially lead to effective treatments for ABCA4 -associated retinopathy, but more research is required to determine their safety and effectiveness in clinical trials.…”

Section: Discussionmentioning

confidence: 99%

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease

Rodríguez-Hidalgo,

de Bruijn,

Corradi

et al. 2023

Front. Genet.

View full text Add to dashboard Cite

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone–rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants.Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases.Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

show abstract

Section: Discussionmentioning

confidence: 99%

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease

Rodríguez-Hidalgo,

de Bruijn,

Corradi

et al. 2023

Front. Genet.

View full text Add to dashboard Cite

show abstract

“…105 Recently, gene editing via CRISPR/Cas9 has been employed to correct pathogenic variants of ABCA4 in human iPSCs (hiPSC) for STGD1 patients. 106 However, there are potential safety concerns associated with gene editing methods being developed for STGD1, namely the introduction of double-stranded breaks (DSBs) in the genome during editing. Gene editing systems such as CRISPR/ Cas9 create DSBs, which run the risk of triggering error-prone endogenous DNA repair mechanisms that could otherwise cause unwanted effects.…”

Section: Reviewmentioning

confidence: 99%

Stargardt macular dystrophy and therapeutic approaches

Fujinami,

Waheed,

Laich

et al. 2023

Br J Ophthalmol

View full text Add to dashboard Cite

Stargardt macular dystrophy (Stargardt disease; STGD1; OMIM 248200) is the most prevalent inherited macular dystrophy. STGD1 is an autosomal recessive disorder caused by multiple pathogenic sequence variants in the largeABCA4gene (OMIM 601691). Major advances in understanding both the clinical and molecular features, as well as the underlying pathophysiology, have culminated in many completed, ongoing and planned human clinical trials of novel therapies.The aims of this concise review are to describe (1) the detailed phenotypic and genotypic characteristics of the disease, multimodal imaging findings, natural history of the disease, and pathogenesis, (2) the multiple avenues of research and therapeutic intervention, including pharmacological, cellular therapies and diverse types of genetic therapies that have either been investigated or are under investigation and (3) the exciting novel therapeutic approaches on the translational horizon that aim to treat STGD1 by replacing the entire 6.8 kbABCA4open reading frame.

show abstract

“…Another future treatment will probably rely on CRISPR-Cas9-mediated genome editing in human induced pluripotent stem cells derived from patients and subsequent subretinal transplantation of differentiated retinal cells. Successful results in cell cultures were achieved for several types of retinal dystrophies such as Stargardt disease [ 21 ] and LCA [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

Optimised, Broad NGS Panel for Inherited Eye Diseases to Diagnose 1000 Patients in Poland

Matczyńska,

Beć-Gajowniczek,

Sivitskaya

et al. 2024

Biomedicines

View full text Add to dashboard Cite

Advances in gene therapy and genome editing give hope that new treatments will soon be available for inherited eye diseases that together affect a significant proportion of the adult population. New solutions are needed to make genetic diagnosis fast and affordable. This is the first study of such a large group of patients with inherited retinal dystrophies (IRD) and inherited optic neuropathies (ION) in the Polish population. It is based on four years of diagnostic analysis using a broad, targeted NGS approach. The results include the most common pathogenic variants, as well as 91 novel causative variants, including frameshifts in the cumbersome RPGR ORF15 region. The high frequency of the ABCA4 complex haplotype p.(Leu541Pro;Ala1038Val) was confirmed. Additionally, a deletion of exons 22–24 in USH2A, probably specific to the Polish population, was uncovered as the most frequent copy number variation. The diagnostic yield of the broad NGS panel reached 64.3% and is comparable to the results reported for genetic studies of IRD and ION performed for other populations with more extensive WES or WGS methods. A combined approach to identify genetic causes of all known diseases manifesting in the posterior eye segment appears to be the optimal choice given the currently available treatment options and advanced clinical trials.

show abstract

Efficient correction of ABCA4 variants by CRISPR-Cas9 in hiPSCs derived from Stargardt disease patients

Cited by 11 publications

References 68 publications

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease

Stargardt macular dystrophy and therapeutic approaches

Optimised, Broad NGS Panel for Inherited Eye Diseases to Diagnose 1000 Patients in Poland

Contact Info

Product

Resources

About