CEP290 gene addition rescues ciliogenesis in LCA patient cells.

Abstract

Purpose: The purpose of this study was to generate induced plurtipotent stem cell (iPSC)-derived photoreceptor precursor cells from patients with CEP290-associated Leber Congenital Amaurosis (LCA). Additionally, we aimed to produce a lentiviral vector expressing full-length CEP290 to investigate gene replacement strategies in patient-derived cells.

Methods: 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 LCA patient fibroblast cultures. Following serum-starvation and immunolocalization, cilia were counted using confocal microscopy.

Results: A lentiviral vector containing CMV-driven human full length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, RT-PCR analysis revealed vector-derived expression. In cultures derived from LCA patients, fewer cells formed cilia compared to unaffected controls. Cilia that were formed were shorter in patient derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect.

Conclusions: The successful construction and viral transfer of full length CEP290 brings us closer to the goal of providing gene- and cell- based therapies for patients affected with this common form of LCA.

Keywords: 538 gene transfer/gene therapy • 648 photoreceptors • 721 stem cells