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Alona O Cramer, Mandeep S Singh, Michelle McClements, Robert E MacLaren, Nuffield Laboratory of Ophthalmology, University of Oxford; Transplantation of Ex-Vivo Genetically Modified Photoreceptor Precursors. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1446.
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Retinitis pigmentosa (RP) is one of the primary causes of inherited retinal blindness, affecting more than 15 million people worldwide. In this disease an initial loss of rod photoreceptors leads to a cellular cascade that promotes a secondary degeneration of cone photoreceptors. Pluripotent stem cells and rod photoreceptor progenitors have hence been widely researched as candidates for cell replacement in RP. Patient-specific induced pluripotent stem cells (iPSc) could provide an autologous expandable source of cells for transplantation. However, the use of patient-derived iPSc would require that the disease-causing mutation be repaired in vitro before cells are transplanted. Genetic modification and subretinal transplantation of photoreceptor precursor cells (PPC) are here studied in an endeavour to develop ex-vivo gene therapy for cell replacement in retinal degeneration.
By optimizing early postnatal retinal culture conditions, rod PPC dissociated from Nrl.GFP+ mice were maintained in vitro. In this model, green fluorescent protein (GFP) is expressed specifically in rod photoreceptors. Cultured PPCs were transfected using DsRed florescent reporter adeno-associated virus (AAV) vector carrying the human Rhodopsin gene. Magnetic assisted cell sorting was performed to enrich rod PPC via the cell surface antigen CD73 and repeated washing steps were performed to remove free AAV particles. Cells were transplanted into adult mice with retinal degeneration (Rho-/- and Rd1).
A reliable cell culture system was achieved by use of supplemented neuronal growth media and incubation at 34 degrees Celsius. Rod PPC numbers significantly increased ex-vivo in the first 14 days of culture and maintained high viability for 31 days. Transfection of PPC was achieved by a florescent reporter AAV8 and confirmed ex-vivo. Dissociated retinal cell cultures were enriched to over 85% Nrl.GFP+ positive rod PPC prior to transplantation, and survival of transplanted Nrl.GFP+ PPCs in the subretinal space was observed within 14 days.
Prolonged survival of rod PPC in vitro will allow for a sufficient period of time for ex vivo assessment of gene therapies before transplantation. Our results show successful transplantation of genetically modified, rod cells in murine models of retinal disease and afford a foundation for ex vivo gene therapy in human photoreceptor precursors derived from autologous iPSc.
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