Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is ...safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.
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.
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.
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Khan et al. report on the clinical characterization of patients harboring a recurrent deep-intronic variant in ABCA4 underlying retinal disease and demonstrate that this, and other variants close by, lead to splicing defects that can be rescued by antisense oligonucleotides.
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.
SGIP1 encodes a protein Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1. It is involved in the regulation of clathrin-mediated endocytosis along with having ...a role in energy homeostasis in neuronal systems. We generated an isogenic human induced pluripotent stem cell (iPSC) line with a biallelic frameshift variant in SGIP1. This exon has been shown to be subject to alternative splicing, leading to an isoform lacking 24 amino acids that are present in the longest SGIP isoform. The newly generated iPSC line will be helpful to dissect the differential properties of the two SGIP isoforms.
Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. AMD is multifactorial eye disease with a strong genetic contribution. Here, we report ...the generation and characterization of induced pluripotent stem cells (iPSCs) derived from peripheral blood mononuclear cells of three patients with AMD carrying rare variants in the complement factor H (CFH) gene. These cell lines were generated for cellular studies investigating the disease mechanisms and developing therapeutic interventions for AMD.
Intellectual disability (ID) is a diverse neurodevelopmental condition and almost half of the cases have a genetic etiology. SGIP1 acts as an endocytic protein that influences the signaling of ...receptors in neuronal systems related to energy homeostasis through its interaction with endophilins. This study focuses on the generation and characterization of induced pluripotent stem cells (iPSC) from two unrelated patients due to a frameshift variant (c.764dupA, NM_032291.4) and a splice donor site variant (c.74 + 1G > A, NM_032291.4) in the SGIP1 gene.
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.
CACNA1A encodes a P/Q-type voltage-gated calcium channel. Heterozygous loss-of-function variants in this gene have been associated with episodic ataxia type 2. In this study, we used CRISPR/Cas9 to ...generate isogenic human induced pluripotent stem cell lines with a gene-dosage dependent deficiency of CACNA1A. We obtained one clone with monoallelic (UCSFi001-A-60) and two clones with biallelic (UCSFi001-A-61; UCSFi001-A-62) frameshift variants in CACNA1A. All three lines showed expression of pluripotency markers and a normal karyotype.
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