In vivo-directed evolution of adeno-associated virus in the primate retina.

Efficient adeno-associated virus-mediated (AAV-mediated) gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution - guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library - to create AAV viral capsids with the capability to deliver genes to the outer retina in primates. A replication-incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates - involving intravitreal library administration, recovery of genomes from outer retina, and extensive next-generation sequencing of each round - resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These viral vectors expand the toolbox of vectors available for primate retina, and they may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

Mutation-Independent Gene Therapies for Rod-Cone Dystrophies.

The clinical success of gene replacement therapies in recent years has served as a proof of concept for the treatment of inherited retinal degenerations using adeno-associated virus (AAV) as viral vector. However, inherited retinal degenerative diseases showcase a broad genetic and mechanistic heterogeneity, challenging the development of mutation-specific therapies for each specific mutation. Mutation-independent approaches must be developed to slow down retinal degeneration regardless of the underlying genetic mutation and onset of the disease. New understanding of cell death mechanisms in rod-cone dystrophies have led to promising rescue of photoreceptor cell death by virally mediating expression of anti-apoptotic factors and secretion of retinal neurotrophic factors. Optogenetic therapies are also able to restore light sensitivities in blind retinas.

Neuron-glia signaling in developing retina mediated by neurotransmitter spillover.

Neuron-glia interactions play a critical role in the maturation of neural circuits; however, little is known about the pathways that mediate their communication in the developing CNS. We investigated neuron-glia signaling in the developing retina, where we demonstrate that retinal waves reliably induce calcium transients in Müller glial cells (MCs). During cholinergic waves, MC calcium transients were blocked by muscarinic acetylcholine receptor antagonists, whereas during glutamatergic waves, MC calcium transients were inhibited by ionotropic glutamate receptor antagonists, indicating that the responsiveness of MCs changes to match the neurotransmitter used to support retinal waves. Using an optical glutamate sensor we show that the decline in MC calcium transients is caused by a reduction in the amount of glutamate reaching MCs. Together, these studies indicate that neurons and MCs exhibit correlated activity during a critical period of retinal maturation that is enabled by neurotransmitter spillover from retinal synapses.

Visual stimulation switches the polarity of excitatory input to starburst amacrine cells.

Direction-selective ganglion cells (DSGCs) are tuned to motion in one direction. Starburst amacrine cells (SACs) are thought to mediate this direction selectivity through precise anatomical wiring to DSGCs. Nevertheless, we previously found that visual adaptation can reverse DSGCs's directional tuning, overcoming the circuit anatomy. Here we explore the role of SACs in the generation and adaptation of direction selectivity. First, using pharmacogenetics and two-photon calcium imaging, we validate that SACs are necessary for direction selectivity. Next, we demonstrate that exposure to an adaptive stimulus dramatically alters SACs' synaptic inputs. Specifically, after visual adaptation, On-SACs lose their excitatory input during light onset but gain an excitatory input during light offset. Our data suggest that visual stimulation alters the interactions between rod- and cone-mediated inputs that converge on the terminals of On-cone BCs. These results demonstrate how the sensory environment can modify computations performed by anatomically defined neuronal circuits.