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Erin Burnight, Emily Kaalberg, Mari Eyestone, Jeremy Hoffman, Christine Haas, Robert Mullins, Edwin Stone, Budd Tucker; Gene Therapy for CEP290-associated LCA in Patient-Derived Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2730.
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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 for which there is currently no cure. Autosomal recessive CEP290-associated LCA is a good candidate for gene-replacement therapy and reprogrammed somatic cell technologies provide researchers with the ability to study human disease and therapeutic gene correction in vitro. The purpose of this study was to develop lentiviral vectors containing human CEP290 and to use these vectors to deliver wild type CEP290 to induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells (PRPCs) from mice and humans with CEP290-associated LCA.
Fibroblast-derived iPSCs were generated from the retinal degenerative mouse model CEP290rd16 and patients with molecularly confirmed CEP290-associated LCA. Mouse and human iPSCs were differentiated into photoreceptor precursor cells using our previously developed step-wise differentiation protocol. An HIV-1 lentiviral vector containing human CEP290 under the control of the CMV promoter was packaged and used to transduce PRPCs from rd16 mice and LCA patients. Vector-derived mRNA and protein were detected via RT-PCR and western blot, respectively.
To evaluate CEP290 gene transfer we initially transduced a surrogate cell line, JK1, derived from murine testicular stromal cells that do not express the human CEP290 transcript. RT-PCR analysis using human-specific primers demonstrated a dose-dependent increase in CEP290 expression post-transduction. However, cell viability was significantly reduced in cultures transduced with the highest dose. Using results from these pilot experiments, we delivered a lower dose of CEP290 vector to iPSC-derived murine and human PRPCs. RT-PCR analysis and western blotting indicated vector-derived expression in these cells.
We show successful gene transfer of human CEP290 to iPSC-derived photoreceptor precursor cells. Cell viability assays suggest that overexpression of CEP290 is cytotoxic. Thus, it will be important to carefully titrate vector dosage when developing gene replacement strategies for this disease. This work will contribute to our overall goal of vision restoration in patients with LCA through gene and patient-specific photoreceptor cell replacement.
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