Gene therapy on patient-specific stem cell lines with MFRP defect

Abstract

 

Purpose

 

Membrane frizzled-related protein (MFRP) is a newly identified gene that can cause autosomal recessive retinitis pigmentosa (RP). MFRP encodes a retinal pigmented epithelium (RPE) -specific membrane receptor of unknown function. This study focus on modeling MFRP-caused RP using patient-specific induced pluripotent stem cells (iPSCs), applying gene therapy to correct the cellular phenotype in vitro and studying potential role of MFRP in RPE.

 

Methods

 

iPSCs had been created by Yamanaka factors from two patients who carried mutation on MFRP gene and wild-type donor. Patient-specific iPSCs differentiated into RPE and AAV8-MFRP applied on patient-specific iPS-RPE. Immunocytochemistry, transmission electron microscopy (TEM) imaging and measurement of transepithelial resistance (TER) were used to study cellular phenotype. AAV8-CTRP5 applied on autopsy RPE to study the relationship between MFRP and its binding partner CTRP5. Antibodies to MFRP, CTRP5, β-actin, and GAPDH were used to measure the expression level in iPS-RPE and autopsy RPE.

 

Results

 

Patient-specific MFRP deficient iPS-RPE cells presented morphological and functional phenotype, including disorganized actin stress fibers, increased numbers of focal adhesions, loss of apical microvilli and decreased TER. β-actin expression was higher in MFRP deficient iPS-RPE compared with wild-type. Application of AAV-MFRP rescued actin disorganization, recovered apical microvilli and increased TER in MFRP deficient iPS-RPE. In all studied RPE lines, CTRP5 expression opposed MFRP expression. Over-expression of CTRP5 in wild-type RPE phenocopy MFRP deficient RPE.

 

Conclusions

 

A favorable response to gene therapy in patient-specific cell lines suggested that this form of retina degeneration caused by MFRP mutations is a potential target for interventional trials. MFRP and CTRP5, exist in an antagonistic relationship to regulate actin organization in RPE cells.

 

 

TEM images of microvilli. iPS-RPE from Patient 1 and Patient 2 show loss of apical microvilli and microvilli are recovered after AAV-MFRP treatment. Scale bar = 1 μm.

 

 

Overexpression of CTRP5 phenocopies MFRP deficiency. Phalloidin staining on wild-type iPS-RPE (like autopsy RPE) exhibits hexagonal, organized actin filaments around the cell periphery. MFRP deficient iPS-RPE shows disorganized actin stress fibers. Autopsy RPE treated with AAV-CTRP5 shows morphology similar to MFRP deficient RPE. Scale bar = 50 μm.

 

Keywords: 538 gene transfer/gene therapy • 721 stem cells • 696 retinal degenerations: hereditary