The CRB1 Complex: Following the Trail of Crumbs to a Feasible Gene Therapy Strategy

Abstract

Once considered science fiction, gene therapy is rapidly becoming scientific reality, targeting a growing number of the approximately 250 genes linked to hereditary retinal disorders such as retinitis pigmentosa and Leber's congenital amaurosis. Powerful new technologies have emerged, leading to the development of humanized models for testing and screening these therapies, bringing us closer to the goal of personalized medicine. These tools include the ability to differentiate human induced pluripotent stem cells (iPSCs) to create a “retina-in-a-dish” model and the self-formed ectodermal autonomous multi-zone, which can mimic whole eye development. In addition, highly specific gene-editing tools are now available, including the CRISPR/Cas9 system and the recently developed homology-independent targeted integration approach, which allows gene editing in non-dividing cells. Variants in the CRB1 gene have long been associated with retinopathies, and more recently the CRB2 gene has also been shown to have possible clinical relevance with respect to retinopathies. In this review, we discuss the role of the CRB protein complex in patients with retinopathy. In addition, we discuss new opportunities provided by stem cells and gene-editing tools, and we provide insight into how the retinal therapeutic pipeline can be improved. Finally, we discuss the current state of adeno-associated virus-mediated gene therapy and how it can be applied to treat retinopathies associated with mutations in CRB1.

Mouse Models of Inherited Retinal Degeneration with Photoreceptor Cell Loss

“…A primary defect of a disruption in CRB1 is the fragmentation of the outer limiting membrane [345,346]. As reviewed previously [344,347], the outer limiting membrane consists of adherens/tight junctions formed in part by the CRB1 complexes between Müller glia and the rod or cone IS that form a diffusion barrier. Loss of components of the CRB1 complexes (CRB1-MPP5-PATJ, CRB1-MPP5-MPDZ, and CRB1-PARD6A-MPP5-MPP3/MPP4) leads to lamination defects with formation of rosettes and a progressive loss of PRs.…”

“…CRB1 and its interacting partners, such MPP3, MPP5, and PARD6A, have been shown to be important in establishing proper retinal lamination presumably through their essential roles in establishing cellular apical basal polarity [344]. A primary defect of a disruption in CRB1 is the fragmentation of the outer limiting membrane [345,346].…”

Clinically-guided mutation screening of two families with hereditary retinal disease

“…CRB1 is a protein localized in the inner segment of the photoreceptor cells 38 . It is the orthologue of the Crumbs protein in the fly.…”

AAV-Mediated Gene Therapy for CRB1-Hereditary Retinopathies

“…Both the CRB1 and CRB2 have a large extracellular domain with epidermal growth factor-like and laminin-A globular domains, a single transmembrane domain and a short intracellular C-terminal domain. The C-terminal domain of 37 amino acids has a single FERM-protein-binding motif juxtaposed to the transmembrane domain and a single C-terminal PDZ protein-binding motif [53][54][55]. While CRB3, the third family member, contains the transmembrane and C-terminal domain but is very short in length since it lacks the large extracellular domain.…”