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Erin R. Burnight, Emily E. Kaalberg, Bonita L. Moses, Jennifer A. Halder, Heather T. Daggett, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker; LCA Gene Therapy In Somatic-Cell-Derived Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1897. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the CEP290 gene are major contributors to Leber Congenital Amaurosis (LCA), the most severe form of inherited retinal degenerative disease. CEP290-associated LCA is in herited in an autosomal recessive manner and is thus a good candidate for gene-replacement therapy. Reprogrammed somatic cell technologies provide researchers with the ability to study human disease and therapeutic correction in vitro. The purpose of this study is to generate iPSCs and subsequently photoreceptor precursor cells from a mouse model of and patients with LCA. These cells will be used for the study of therapeutic gene correction in iPSCs.
Fibroblast-derived iPSCs were generated from the retinal degenerative mouse model CEP290rd16 and patients with molecularly confirmed CEP290-associated LCA using a lentiviral reprogramming vector. To determine if cells were fully reprogrammed, iPSCs were examined for the presence of pluripotency marker transcripts and proteins. Mouse and human iPSCs were differentiated into photoreceptor precursors using our previously developed step-wise differentiation protocol. Lentiviral vectors expressing GFP under the control of retina-specific promoters were developed to generate reagents for cell-specific therapeutic transgene expression.
CEP290-associated LCA mouse and human iPSCs were generated and assessed for pluripotency marker expression. rt-PCR demonstrated that iPSCs expressed the pluripotency markers Nanog, c-Myc, Klf4, Sox2, and Oct4. Immunocytochemical analysis confirmed pluripotency marker expression. Immunocytochemistry with antibodies targeted against OTX2 and Blue Opsin showed successful generation of retinal progenitor cells (RPCs) and photoreceptor precursors, respectively. Lentiviral vectors expressing GFP from the retinal-specific promoters OTX2, CRX, NRL, and RhoK were generated and tested in primary human RPCs.
We show successful somatic cell reprogramming and differentiation of mouse and human CEP290-associated LCA iPSCs into photoreceptor precursors. Lentiviral vectors expressing human CEP290 from retina-specific promoters have been generated and will be used in subsequent gene replacement therapies and transplantation studies. This work will contribute to our overall goal of using both gene and cell based therapies to restore vision in patients with LCA.
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