April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Gene therapy on patient-specific stem cell lines with MFRP defect
Author Affiliations & Notes
  • Yao Li
    Ophthalmology, Columbia University, New York, NY
  • Wen-Hsuan Wu
    Ophthalmology, Columbia University, New York, NY
  • Yi-Ting Tsai
    Ophthalmology, Columbia University, New York, NY
  • Haiqing Hua
    Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, NY
  • Takayuki Nagasaki
    Ophthalmology, Columbia University, New York, NY
  • Irene H Maumenee
    Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, IL
  • Lawrence A Yannuzzi
    Ophthalmology, Columbia University, New York, NY
  • Quan V Hoang
    Ophthalmology, Columbia University, New York, NY
  • Dieter Egli
    New York Stem Cell Foundation, New York, NY
  • Stephen H Tsang
    Department of Pathology and Cell Biology, Columbia University, New York, NY
  • Footnotes
    Commercial Relationships Yao Li, None; Wen-Hsuan Wu, None; Yi-Ting Tsai, None; Haiqing Hua, None; Takayuki Nagasaki, None; Irene Maumenee, None; Lawrence Yannuzzi, None; Quan Hoang, None; Dieter Egli, None; Stephen Tsang, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 834. doi:https://doi.org/
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      Yao Li, Wen-Hsuan Wu, Yi-Ting Tsai, Haiqing Hua, Takayuki Nagasaki, Irene H Maumenee, Lawrence A Yannuzzi, Quan V Hoang, Dieter Egli, Stephen H Tsang; Gene therapy on patient-specific stem cell lines with MFRP defect. Invest. Ophthalmol. Vis. Sci. 2014;55(13):834. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.
 
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.
 
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  
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