Using induced Pluripotent Stem Cells to discover advanced therapies for Pre-mRNA processing factor linked Retinitis Pigmentosa

Abstract

Retinitis pigmentosa (RP) is an inherited retinal disease that affects 1 in 4000 people. Over one-third of cases are caused by autosomal dominant inheritance. Of these, ~20% are caused by mutations in pre-mRNA processing factors (PRPFs). This retinal phenotype is interesting as PRPFs are ubiquitously expressed and are core components of the spliceosome. Studies of mice harbouring RP-causing PRPF mutations identified retinal pigmented epithelial (RPE) cells as the most affected cell type. Each RPE cell possesses a primary cilium which is necessary for RPE function, this includes the diurnal phagocytosis of photoreceptor outer segments and the formation of a tight blood-retina-barrier. Both functions are recapitulated by RPE cells derived from induced pluripotent stem cells (iPSCs). Moreover, previous work from the Lako lab has shown iPSC-RPE cells that harbour PRPF31 mutations exhibit reduced phagocytosis and barrier function, as well as ciliary abnormalities. This makes iPSC derived RPE cells a useful tool during pre-clinical evaluation of new therapies. There are currently no treatments for PRPF31 or PRPF8 linked RP. However, there is evidence to suggest that PRPF31-linked RP is caused by a loss of wild type PRPF31 protein expression. There is no evidence to suggest that PRPF8 mutations reduce protein expression. This study aims to generate an iPSC-RPE cell model of PRPF31- and PRPF8-linked RP and evaluate the potential of gene-therapies for the treatment of these diseases. To achieve this, we generated iPSC-RPE cells from a donor with PRPF31 mutations and supplemented PRPF31 transcript using an adeno-associated virus (AAV). In parallel, iPSCs from three donors with PRPF8-linked RP underwent CRISPR/Cas9 mediated gene editing to correct the pathogenic mutation before differentiation to iPSC-RPE. Efficacy of these treatments was assessed using a combination of techniques including flow cytometry, transepithelial electrical resistance (TEER), immunofluorescent microscopy and electron microscopy. This thesis shows that PRPF31 supplementation can rescue phagocytic function of iPSC-RPE cells that were derived from a patient with PRPF31-linked RP. Cilia length was also rescued but the reduced cilia incidence and barrier function persisted following PRPF31-AAV transduction. Next, the effect of PRPF8 gene editing is presented. Phagocytosis and barrier function of iPSC-RPE cells was not affected by PRPF8 mutation. Quantitative ARL13B immunofluorescent microscopy showed cilia length is increase by PRPF8 mutation but cilia incidence is unaffected. Quantitative ultrastructural analysis revealed ciliary swelling and fewer mitochondria in iPSC-RPE cells harbouring pathogenic PRPF8 mutations. Furthermore, immunofluorescent microscopy of markers of both apical and basal epithelial polarity suggests epithelial cell polarity is reduced by RP-causing PRPF8 mutations. This thesis identifies several features that are caused by both PRPF31 and PRPF8 mutations. These include altered cilia length, cilia swelling, and reduced epithelial polarity. Additional evidence suggests PRPF31- and PRPF8¬-linked RP can be treated through gene supplementation and gene editing, respectively. These results highlight the potential of such therapies in the future treatment of RP and more broadly, retinal disease.