RD3 is a 23 kDa protein implicated in the stable expression of guanylate cyclase in photoreceptor cells. Truncation mutations are responsible for photoreceptor degeneration and severe early-onset vision loss in Leber congenital amaurosis 12 (LCA12) patients, the rd3 mouse and the rcd2 collie. To further investigate the role of RD3 in photoreceptors and explore gene therapy as a potential treatment for LCA12, we delivered adeno-associated viral vector (AAV8) with a Y733F capsid mutation and containing the mouse Rd3 complementary DNA (cDNA) under the control of the human rhodopsin kinase promoter to photoreceptors of 14-day-old Rb(11.13)4Bnr/J and In (5)30Rk/J strains of rd3 mice by subretinal injections. Strong RD3 transgene expression led to the translocation of guanylate cyclase from the endoplasmic reticulum (ER) to rod and cone outer segments (OSs) as visualized by immunofluorescence microscopy. Guanylate cyclase expression and localization coincided with the survival of rod and cone photoreceptors for at least 7 months. Rod and cone visual function was restored in the In (5)30Rk/J strain of rd3 mice as measured by electroretinography (ERG), but only rod function was recovered in the Rb(11.13)4Bnr/J strain, suggesting that the latter may have another defect in cone phototransduction. These studies indicate that RD3 plays an essential role in the exit of guanylate cyclase from the ER and its trafficking to photoreceptor OSs and provide a 'proof of concept' for AAV-mediated gene therapy as a potential therapeutic treatment for LCA12.

Purpose Subretinal transplantation of stem-cell-derived photoreceptor precursor cells (PPCs) is a promising and innovative approach to treating a range of blinding diseases. However, common barriers to efficient preclinical transplantation comes in the form of suboptimal graft architecture, limited graft survival, and immune-rejection, each of which cannot be assessed using conventional in vivo imaging (ie, rodent ophthalmoscopy). With the majority of PPCs reported to die within the first few weeks after transplantation, understanding the mechanisms of graft failure, and ultimately devising preventative methods, currently relies on lengthy end point histology.To address these limitations, we hypothesized that combining two imaging modalities, optical coherence tomography (OCT) and fluorescence confocal scanning laser ophthalmoscopy (fcSLO), could provide a more rapid and comprehensive view of PPC engraftment. Methods Human ESC-derived PPCs were transplanted into 15 retinal dystrophic rats that underwent bimodal imaging at 0, 8, and 15 days posttransplant. Results Bimodal imaging provided serial detection of graft: placement, architecture, and survival; each undetectable under ophthalmoscopy. Bimodal imaging determined graft placement to be either: subretinal (n = 7), choroidal (n = 4), or vitreal (n = 4) indicating neural retinal perforation. Graft architecture was highly variable at the time of transplantation, with notable redistribution over time, while complete, or near complete, graft loss was observed in the majority of recipients after day 8. Of particular importance was detection of vitreal aggregates overlying the graft-possibly an indicator of host-site inflammation and rejection. Conclusion Early real-time feedback of engraftment has the potential to greatly increase efficiency of preclinical trials in cell-based retinal therapeutics.

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The emergency distance learning methods adopted to face the pandemic are connected to some inherent problems which concern the lack of social aspects, interactions, and motivation. These and other frequent issues threaten to stop the opportunities offered by the widespread use of educational technology. This paper examines how the Higher Education Institutions adapted to the evolution of the pandemic, compared to relevant insights from University of Pisa gathered during the academic year 2020/21.Focusing on the communicative and socio-emotional aspects of the didactic event, we propose engagement tools – real-time interaction applications designed to increase active learning and motivation – as a feasible solution that might tackle some of the emerged teaching-learning issues. In this context, it appears that the potential of these versatile and easy-to-apply tools has not been fully exploited. Specific teacher training actions are therefore suggested.