SummaryTransplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.

Leber congenital amaurosis is a group of inherited retinal dystrophies that cause severe sight impairment in childhood; RPE65-deficiency causes impaired rod photoreceptor function from birth and progressive impairment of cone photoreceptor function associated with retinal degeneration. In animal models of RPE65 deficiency, subretinal injection of recombinant adeno-associated virus (AAV) 2/2 vectors carrying RPE65 cDNA improves rod photoreceptor function, and intervention at an early stage of disease provides sustained benefit by protecting cone photoreceptors against retinal degeneration. In affected humans, administration of these vectors has resulted to date in relatively modest improvements in photoreceptor function, even when retinal degeneration is comparatively mild, and the duration of benefit is limited by progressive retinal degeneration. We conclude that the demand for RPE65 in humans is not fully met by current vectors, and predict that a more powerful vector will provide more durable benefit. With this aim we have modified the original AAV2/2 vector to generate AAV2/5-OPTIRPE65. The new configuration consists of an AAV vector serotype 5 carrying an optimized hRPE65 promoter and a codon-optimized hRPE65 gene. In mice, AAV2/5-OPTIRPE65 is at least 300-fold more potent than our original AAV2/2 vector.

Alzheimer’s disease, characterized by brain deposits of amyloid-β plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood–brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer’s disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood–brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-β levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood–brain barrier function and decreasing amyloid-β and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood–brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-β load, due to increased clearance and degradation of amyloid-β by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood–brain barrier breakdown early in Alzheimer’s disease can be restored by hrANXA1 as a potential therapeutic approach.

The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network.