Mechanisms of functional retinal engraftment by transplanted retinal ganglion cells

About This Grant

Optic neuropathies including glaucoma are the world’s leading cause of irreversible blindness. Though existing treatment for some progressive optic neuropathies slow vision loss, no treatments exist to restore sight lost to these neurodegenerative conditions. This shortcoming relates to the death of retinal ganglion cells (RGCs), which transmit visual information from the eye to the brain via their axons running through the optic nerves and tracts. Though some cold-blooded vertebrates are capable of RGC regeneration, mammals (including humans) lack this capacity. Development of regenerative therapies to repopulate RGCs would improve the quality of life for tens of millions of people suffering from optic neuropathy. Though transplantation of stem cell-derived RGCs has been long considered, challenges to durable graft survival and functional retinal integration have hampered progress. Recently, we overcame these challenges by developing methods to disrupt the retina’s internal limiting membrane (ILM), which dramatically improves the survival, engraftment, and synaptic connectivity of human RGCs in the retina of multiple species. To achieve functional vision restoration, donor RGCs must receive presynaptic input from host bipolar and amacrine cells so that they can encode light stimuli in a manner that is interpretable by the brain. The goal of this project is to augment long-term donor RGC survival and establish physiologic connectivity to functionally relevant inner retinal circuits at translationally relevant levels by transiently suppressing a key cell-intrinsic pathway involved in the transition of neurons from an immature plastic state to a mature non-regenerative state. Our preliminary data show that deletion of the PTEN gene dramatically increases donor RGC survival and dendritogenesis early following transplantation. However, PTEN expression is necessary for neuronal maturation and stability of synapses & neurocircuits. Further, PTEN is a tumor suppressor, and sustained deletion may increase the risk of transformation and tumorigenesis. Therefore, we will leverage inducible CRISPR inhibition to generate human stem cell lines wherein PTEN activity can be suppressed for a specified duration. Using a rat model of glaucoma, we will transplant human RGCs intravitreally and compare the effects of multiple durations of transient PTEN suppression at key early stages of the engraftment process. Using a combination of 1) in vivo adaptive optics scanning laser ophthalmoscopy and 2) volumetric confocal microscopy with AI-based 3D segmentation of donor RGC dendrites within the inner plexiform layer, we will quantify RGC survival, engraftment, and dendritic architecture & localization for up to 16 weeks post-transplantation. Using 3) optical electrophysiology (calcium imaging), 4) transsynaptic circuit tracing, and 5) spatial transcriptomics, we will assess the evolution in and eventual stability of retinal circuit integration, light response properties, and RGC maturation following PTEN re-expression. Successful completion of this project will advance RGC repopulation by determining how transient PTEN suppression of various durations sustains transplant survival & promotes physiologic retinal connectivity to restore vision in optic neuropathy.