Search

ALDH7A1 encodes for the enzyme catalyzing the third step of the lysine degradation pathway. Biallelic pathogenic variants in ALDH7A1 are associated with pyridoxine dependent epilepsy (PDE), of which the c.1279G>C (p.Glu427Gln) variant is the most commonly reported variant and is carried by 30% of PDE patients with European ancestry. In this study, hiPSC lines derived from four PDE patients carrying the c.1279G>C variant in homozygosis in ALDH7A1 were generated and fully characterized. These hiPSC lines can contribute to better understand the molecular mechanism of disease underlying PDE as well as serving as a model system to evaluate new therapeutic strategies.

GCDH encodes for the enzyme catalyzing the sixth step of the lysine degradation pathway. Autosomal recessive variants in GCDH are associated with glutaric aciduria type I (GA1), of which a wide genotypic spectrum of pathogenic variants have been described. In this study, hiPSC lines derived from four GA1 patients with different genotypes were generated and fully characterized. Two patients carry compound heterozygous variants in GCDH, while the other two patients carry a variant in homozygosis. These hiPSC lines can significantly contribute to better understand the molecular mechanism underlying GA1 and provide excellent models for the development of new therapeutic strategies.

Deep-intronic (DI) variants represent approximately 10%–12% of disease-causing genetic defects in ABCA4-associated Stargardt disease (STGD1). Although many of these DI variants are amenable to antisense oligonucleotide-based splicing-modulation therapy, no treatment is currently available. These molecules are mostly variant specific, limiting their applicability to a broader patient population. In this study, we investigated the therapeutic potential of the CRISPR-Cas9 system combined with the amphipathic lipopeptide C18:1-LAH5 for intracellular delivery to correct splicing defects caused by different DI variants within the same intron. The combination of these components facilitated efficient editing of two target introns (introns 30 and 36) of ABCA4 in which several recurrent DI variants are found. The partial removal of these introns did not affect ABCA4 splicing or its expression levels when assessed in two different human cellular models: fibroblasts and induced pluripotent stem cell-derived photoreceptor precursor cells (PPCs). Furthermore, the DNA editing in STGD1 patient-derived PPCs led to a ∼50% reduction of the pseudoexon-containing transcripts resulting from the c.4539+2001G>A variant in intron 30. Overall, we provide proof-of-concept evidence of the use of C18:1-LAH5 as a delivery system for therapeutic genome editing for ABCA4-associated DI variants, offering new opportunities for clinical translation.

Mutations in the lebercilin-encoding gene LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report on the generation of a patient-specific cellular model to study LCA5-associated retinal disease. CRISPR-Cas9 technology was used to correct a homozygous nonsense variant in LCA5 (c.835C>T; p.Q279∗) in patient-derived induced pluripotent stem cells (iPSCs). The absence of off-target editing in gene-corrected (isogenic) control iPSCs was demonstrated by whole-genome sequencing. We differentiated the patient, gene-corrected, and unrelated control iPSCs into three-dimensional retina-like cells, so-called retinal organoids. We observed opsin and rhodopsin mislocalization to the outer nuclear layer in patient-derived but not in the gene-corrected or unrelated control organoids. We also confirmed the rescue of lebercilin expression and localization along the ciliary axoneme within the gene-corrected organoids. Here, we show the potential of combining precise single-nucleotide gene editing with the iPSC-derived retinal organoid system for the generation of a cellular model of early-onset retinal disease.

Pathogenic variants in ABCA4 are associated with Stargardt disease (STGD1), an autosomal recessive macular dystrophy characterized by bilateral central vision loss due to a progressive degeneration of retinal cells. An induced pluripotent stem cell (iPSC) line was generated from late-onset STGD1 patient-derived fibroblasts harboring bi-allelic ABCA4 variants by lentivirus-induced reprogramming. The obtained iPSC line (RMCGENi020-A) showed pluripotent features after the reprogramming process. The generation of this iPSC line facilitates its use to differentiate it into relevant retinal-like cell models, with the aim to adequately evaluate the effects of the ABCA4 variants.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the ''Genome Editing to treat Human Diseases'' (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi’s primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing’s potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Biallelic pathogenic variants in ALDH7A1 are associated with pyridoxine-dependent epilepsy (PDE). ALDH7A1 encodes for the third enzyme of the lysine catabolism pathway. In this study a human isogenic ALDH7A1 knock-out iPSC line was created using CRISPR/Cas9 technology. One clone (SCTCi019-B) with biallelic deletions in ALDH7A1 was obtained and fully characterized, showing expression of pluripotency markers, a normal karyotype and no off-targets. Human-based models derived from this iPSC line will contribute to gain insights in the molecular mechanism of disease underlying PDE.

GCDH encodes for the enzyme catalyzing the sixth step of the lysine catabolism pathway. Biallelic pathogenic variants in GCDH have been associated with glutaric aciduria type 1 (GA1). In this study CRISPR/Cas9 technology was used to create an isogenic GCDH knock-out human iPSC line. One clone with a biallelic deletion (SCTCi019-A) in GCDH was obtained and fully characterized, revealing a normal karyotype, no off-targets detected and expression of pluripotency markers. This iPSC line can contribute to gain insights in the molecular mechanism of disease.

Summary: A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis.One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells.Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment.We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Age-related macular degeneration (AMD) is a common eye disease among the elderly in the Western world. AMD is a multifactorial disease, with a strong association with genetic variation in the complement system. One of the AMD-associated variants is the c.355G>A (p.Gly119Arg) variant in complement factor I (CFI), a central regulator of complement activation. Here, we report the generation of an iPSC line and its isogenic wildtype control derived from peripheral blood mononuclear cells of a male AMD-affected individual carrying the heterozygous variant c.355G>A (p.Gly119Arg). The line can be utilized to study the effects of this variant in disease-specific cell types.

Age-related macular degeneration (AMD) is a common eye disease among the elderly in the Western world. AMD is a multifactorial disease, with a strong association with genetic variation in the complement system. One of the AMD-associated variants is the c.355G>A (p.Gly119Arg) variant in complement factor I (CFI), a central regulator of complement activation. Here, we report the generation of an iPSC line and its isogenic wildtype control derived from peripheral blood mononuclear cells of a female AMD-affected individual carrying the heterozygous variant c.355G>A (p.Gly119Arg). This line can be utilized to study the effects of this variant in disease-specific cell types.

Leber congenital amaurosis (LCA) can be caused by mutations in more than 20 different genes. One of these, RPE65, encodes a protein essential for the visual cycle that is expressed in retinal pigment epithelium cells. In this work, we describe the generation and characterization of the human iPSC line SCTCi16-A. This hiPSC line was generated from peripheral blood mononuclear cells (PBMCs) from a patient affected with LCA caused by the homozygous c.11+5G>A variant in the RPE65 gene. Reprograming was conducted using episomal vectors containing OCT3/4, SOX2, KLF4, L-MYC, and LIN28.

Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.

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. Keywords: retinal dystrophy, oligonucleotide, stem cell, organoid, therapy, QR-110

Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.