Conventional and Tropism-Modified High-Capacity Adenoviral Vectors Exhibit Similar Transduction Profiles in Human iPSC-Derived Retinal Organoids.

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however, their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study, we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles, which, with a capacity of up to 36 kb, can potentially accommodate all known retinal gene coding sequences.

Retinal Ciliopathies and Potential Gene Therapies: A Focus on Human iPSC-Derived Organoid Models.

The human photoreceptor function is dependent on a highly specialised cilium. Perturbation of cilial function can often lead to death of the photoreceptor and loss of vision. Retinal ciliopathies are a genetically diverse range of inherited retinal disorders affecting aspects of the photoreceptor cilium.

Characterization and AAV-mediated gene augmentation in human-derived and retinal organoids.

The majority of patients with mutations in develop either early-onset retinitis pigmentosa as young children or Leber congenital amaurosis as newborns. The cause for the phenotypic variability in -associated retinopathies is unknown, but might be linked to differences in CRB1 and CRB2 protein levels in Müller glial cells and photoreceptor cells. Here, and differentiation day 210 retinal organoids showed a significant decrease in the number of photoreceptor nuclei in a row and a significant increase in the number of photoreceptor cell nuclei above the outer limiting membrane.

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy.

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g.

Quality of life in patients with CRB1-associated retinal dystrophies: A longitudinal study.

Purpose: To assess the longitudinal vision-related quality of life among patients with CRB1-associated inherited retinal dystrophies.

Methods: In this longitudinal questionnaire study, the National Eye Institute Visual Function Questionnaire (39 items, NEI VFQ-39) was applied at baseline, two-year follow-up, and 4-year follow-up in patients with pathogenic CRB1 variants. [Correction added on 20 November 2023, after first online publication: The preceding sentence has been updated in this version.

CRB1 is required for recycling by RAB11A+ vesicles in human retinal organoids.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals.

AAV-mediated gene augmentation therapy of CRB1 patient-derived retinal organoids restores the histological and transcriptional retinal phenotype.

Retinitis pigmentosa and Leber congenital amaurosis are inherited retinal dystrophies that can be caused by mutations in the Crumbs homolog 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells. CRB1 patient-derived induced pluripotent stem cells were differentiated into CRB1 retinal organoids that showed diminished expression of variant CRB1 protein observed by immunohistochemical analysis.

Sleep Deprivation Does not Change the Flash Electroretinogram in Wild-type and Mice.

Sleep deprivation reduces the response of neuronal activity in the suprachiasmatic nucleus (SCN) and the phase shift in circadian behaviour to phase shifting light pulses, and thus seems to impair the adaptation of the circadian clock to the external light-dark cycle. The question remains where in the pathway of light input to the SCN the response is reduced. We therefore investigated whether the electroretinogram (ERG) changes after sleep deprivation in wild-type mice and in mutant male mice.

Defining Phenotype, Tropism, and Retinal Gene Therapy Using Adeno-Associated Viral Vectors (AAVs) in New-Born Brown Norway Rats with a Spontaneous Mutation in .

Mutations in the Crumbs homologue 1 () gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of . It has been reported that these mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age.

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier.

Background: Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS).

Methods: To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated.

AAV- protects against vision loss in an inducible retinitis pigmentosa mouse model.

Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology.

Novel Therapeutic Approaches for the Treatment of Retinal Degenerative Diseases: Focus on CRISPR/Cas-Based Gene Editing.

Inherited retinal diseases encompass a highly heterogenous group of disorders caused by a wide range of genetic variants and with diverse clinical symptoms that converge in the common trait of retinal degeneration. Indeed, mutations in over 270 genes have been associated with some form of retinal degenerative phenotype. Given the immune privileged status of the eye, cell replacement and gene augmentation therapies have been envisioned.

Research Models and Gene Augmentation Therapy for Retinal Dystrophies.

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are inherited degenerative retinal dystrophies with vision loss that ultimately lead to blindness. Several genes have been shown to be involved in early onset retinal dystrophies, including and . Gene therapy recently became available for young RP patients with variations in the gene.

Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays.

Inherited retinal dystrophies and optic neuropathies cause chronic disabling loss of visual function. The development of recombinant adeno-associated viral vectors (rAAV) gene therapies in all disease fields have been promising, but the translation to the clinic has been slow. The safety and efficacy profiles of rAAV are linked to the dose of applied vectors.

CLINICAL CHARACTERISTICS AND NATURAL HISTORY OF RHO-ASSOCIATED RETINITIS PIGMENTOSA: A Long-Term Follow-Up Study.

Purpose: To investigate the natural history of RHO-associated retinitis pigmentosa (RP).

Methods: A multicenter, medical chart review of 100 patients with autosomal dominant RHO-associated RP.

Results: Based on visual fields, time-to-event analysis revealed median ages of 52 and 79 years to reach low vision (central visual field <20°) and blindness (central visual field <10°), respectively.

Crumbs2 mediates ventricular layer remodelling to form the spinal cord central canal.

In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination.

-Associated Dystrophies: Clinical, Genetic, and Histopathological Features.

This study describes the clinical, genetic, and histopathological features in patients with -associated retinal dystrophies. Nine male patients from eight unrelated families underwent a comprehensive ophthalmic examination. Additionally, the histopathology of the right eye from a patient with an end-stage cone-rod-dystrophy (CRD)/sector retinitis pigmentosa (RP) phenotype was examined.

Microglial Cell Dysfunction in CRB1-Associated Retinopathies.

Inherited retinal diseases encompass a large group of clinically and genetically heterogeneous diseases estimated to affect two million people worldwide. Among these people, approximately 80,000 are or will become blind in their first decades of life due to mutations in both alleles of the Crumbs homologue-1 (CRB1) gene. Microglia are the resident immune surveyor cells in the retina, and their roles have been heavily studied in several retinal diseases, including retinitis pigmentosa (RP), age-related macular degeneration, and diabetic retinopathy.

Retinogenesis of the Human Fetal Retina: An Apical Polarity Perspective.

The Crumbs complex has prominent roles in the control of apical cell polarity, in the coupling of cell density sensing to downstream cell signaling pathways, and in regulating junctional structures and cell adhesion. The Crumbs complex acts as a conductor orchestrating multiple downstream signaling pathways in epithelial and neuronal tissue development. These pathways lead to the regulation of cell size, cell fate, cell self-renewal, proliferation, differentiation, migration, mitosis, and apoptosis.

CRB2 Loss in Rod Photoreceptors Is Associated with Progressive Loss of Retinal Contrast Sensitivity.

Variations in the Crumbs homolog-1 () gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the -disease.

Human iPSC-Derived Retinas Recapitulate the Fetal CRB1 CRB2 Complex Formation and Demonstrate that Photoreceptors and Müller Glia Are Targets of AAV5.

Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina.