Molecular and cellular requirements for Crb1 gene function in the onset and therapeutic rescue of an inherited retinal degeneration

ABSTRACT Mutations that impair function of the CRB1 gene are among the most prevalent causes of inherited retinal de- generation. The events leading to degeneration when CRB1 is lost are unknown. To answer this question it is essential to understand the function of CRB1 at each site where it is expressed within developing and mature retina. This knowledge will reveal the functions of CRB1 that are most responsible for retinal pathology, thereby pinpointing therapeutic strategies to prevent pathology. The objective here is to determine how distinct CRB1 isoforms and CRB1-expressing cell types contribute to disease pathology in the Crb1null mouse model of CRB1 disease. The central hypothesis is that a photoreceptor specific isoform, known as CRB1-B, needs to be present and functional to prevent outer limiting membrane pathology and visual impairment. The rationale for this work is that systematically evaluating CRB1 function at each time and place where it is expressed will es- tablish a rational strategy for gene replacement therapy with a high likelihood of effectively halting degenera- tion. To test this hypothesis three Specific Aims are proposed. Aim 1: Determine the contribution of specific Crb1 isoforms to disease-related phenotypes. Preliminary studies defined 3 major mRNA isoforms of the mouse Crb1 and human CRB1 gene: CRB1-A, -B, and -C. To systematically test their function the PI's labora- tory has generated mouse mutant strains that remove specific isoforms, either individually or in combination. These mutants will be subjected to anatomical, electrophysiological, and behavioral analysis, thereby estab- lishing the contribution of each isoform to specific aspects of the CRB1 mutant phenotype. Aim 2: Determine how each Crb1-expressing cell type contributes to disease-related phenotypes. Conditional Crb1null mu- tant mice will be generated that allow removal or re-expression of all isoforms in a cell type-specific manner. These mice will undergo the battery of phenotypic tests noted for Aim 1 above, thereby establishing the role of each CRB1-expressing cell type in distinct aspects of disease pathology. Aim 3: Rescue disease-related phenotypes using isoform- and cell type-specific Crb1 re-expression. Preliminary data suggest a critical role for the photoreceptor-specific CRB1-B isoform in onset and rescue of disease pathology. This Aim will test the working hypothesis that restoring CRB1-B to photoreceptors will produce functional rescue in the Crb1null disease model. This work will be significant because it will clarify the molecular and cellular aspects of CRB1 expression that are required to prevent retinal pathology. Once we know the consequences for retinal pathol- ogy when specific sites of CRB1 expression are lost or restored, it will become clear which of these sites would be most beneficial to target for CRB1 replacement therapy. Thus, the proposal is expected to yield innovative new strategies for gene therapy which could be rapidly deployed to pre-clinical testing.