A systemically-delivered stem cell therapy for dry age related macular degeneration

Abstract

Dry age-related macular degeneration (AMD) is a progressive neurodegenerative disorder characterized by geographical atrophy of the retinal pigment epithelium (RPE), causing irreversible central vision loss. Systemically-delivered bone marrow-derived cells (BMDCs), programmed to RPE-like cells via expression of human RPE65, regenerate damaged RPE and preserve vision in murine models of retinal degeneration. RPE65 rapidly activates adenylate cyclase (AC), which then activates endogenous Rpe65 and RPE-associated marker Cralbp. Previous studies expressed RPE65 from an integrating lentiviral vector (ILV), which is an unnecessary safety risk due to the potential for insertional mutagenesis, as long- term expression of RPE65 is not required for BMDC programming. Here, we developed a 3rd generation integrase-defective lentiviral vector (IDLV) for programming both murine and human BMDCs to RPE-like cells, reducing insertional mutagenesis risk and expanding the protocol to include human cells. We enhanced IDLV3-RPE65 infection of murine and human BMDCs by preloading concentrated vector on RetroNectin at MOI 50, and infecting with low-speed centrifugation, increasing RPE65 mRNA levels from ~12-fold to ~25-fold (p<0.05). IDLV3-RPE65 infection initiates expression of endogenous Rpe65 mRNA expression in murine BMDC and Cralbp/CRALBP mRNA in both murine and human BMDCs, indicating programming to RPE-like cells. Inhibiting AC in RPE65infected BMDCs abrogated expression of the endogenous genes, confirming the role of AC activation in programming. Critically, IDLV3-RPE65-infected murine BMDCs are recruited to and incorporate into to the RPE layer, and preserve vision in murine models of retinal degeneration. We conclude that BMDCs programmed with IDLV3-RPE65 successfully prevent retinal degeneration progression and are appropriate for testing in human cells, with a view to move into human clinical trial for the treatment of dry AMD. This approach significantly increases the safety of the therapy and is, to the best of our knowledge, the first application of a single IDLV in the generation of therapeutic cells from adult stem cells.