The Exploration Of Gene Therapy Of Retinitis Pigmentosa Caused By Mutation Of Human CRB1

Purpose:Degeneration of photoreceptor cells is a common cause of blindness, which could brought serious damage to human health, and by now, there is still no good treatment for these kinds of disease. So far, gene therapy and stem cell therapy are the most promising method. Among those many human genes associated with retinal degenerative diseases, mutations of CRB1 can cause different kinds of Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), but there is not any report about gene therapy ofCRBl, one of the most important reason is that the CRB1 gene is far too large, which absolutely beyond the length limit of adeno-associated virus (AAV), and at the same time, the structure of CRB1 is complex, so the function of each domain is not very clear. In view of this, we aim to use zebrafish as model, analyze the function of its Crb2a domains, and on the basis of this, we could explore gene therapy of RP and LCA that caused by the defect of CRB1.Methods:First, by bioinformatic analyses, we compared different Crb isomers in zebrafish with mammals CRB1, and we found that crb2a is the homologous genes of CRB1 in zebrafish. Then, we predicted each protein domain of zebrafish Crb2a, combined with the analysis of naturally existed different isomers, we cloned some of the single domain or newly organized domain group. We try to do further experiment in zebrafish and cells, so to detect if they could rescue the phenotype of the fish which have crb2a mutation, and to observe its ability of mediating protein-protein adhesion, so that we could understand the function of each Crb2a domain. And based on this, we try to find single domain or domain group that may keep the full-length Crb2a gene function, and then test the feasibility of gene therapy of CRB1 mutant in zebrafish through transgenic technology.Results:Bioinformatic analysis showed that crb2a has three Laminin G domians and 16 EGF domains. Although the Crb protein that have its function is considered to be composed of all domains above, cDNA cloning showed that crb2a has many different forms of transcription splicing. On this basis, we restructuring eight different crb2a constructs through genetic engineering technology. After microinjection the mRNA that transcribed in vitro into zebrafish embryos, we observed whether they can rescue the phenotype of crb2a mutation. Each domain of Crb2a has been proved to have relatively independent functions, and different domain showed different rescue effect in different organs.Conclusion:This study preliminary proved multiple crb2a domains has relatively independent functions, by recombining crb2a genes, it may possible for us to obtain a construct not only with a size appropriate to AAV vectors, but also can retain full-length crb2a function. Thus provide a new advantage for gene therapy of human disease associated with CRB1.