Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter

In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (∼100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use

XIAP Protects Retinal Ganglion Cells in the Mutant ND4 Mouse Model of Leber Hereditary Optic Neuropathy

Leber hereditary optic neuropathy (LHON) is a genetic form of vision loss that occurs primarily owing to mutations in the nicotinamide adenine dinucleotide dehydrogenase (ND) subunits that make up complex I of the electron transport chain. LHON mutations result in the apoptotic death of retinal ganglion cells. We tested the hypothesis that gene therapy with the X-linked inhibitor of apoptosis (XIAP) would prevent retinal ganglion cell apoptosis and reduce disease progression in a vector-induced mouse model of LHON that carries the ND4 mutation. Adeno-associated virus (AAV) encoding full length hemagglutinin-tagged XIAP (AAV2.HA-XIAP) or green fluorescent protein (AAV2.GFP) was injected into the vitreous of DBA/1J mice. Two weeks later, the LHON phenotype was induced by AAV delivery of mutant ND4 (AAV2.mND4FLAG) to the vitreous. Retinal function was assessed by pattern electroretinography. Optic nerves were harvested at 4 months, and the effects of XIAP therapy on nerve fiber layer and optic nerve integrity were evaluated using immunohistochemistry, transmission electron microscopy and magnetic resonance imaging. During LHON disease progression, retinal ganglion cell axons are lost. Apoptotic cell bodies are seen in the nuclei of astrocytes or oligodendrocytes in the optic nerve, and there is thinning of the optic nerve and the nerve fiber layer of the retina. At 4 months after disease onset, XIAP gene therapy protects the nerve fiber layer and optic nerve architecture by preserving axon health. XIAP also decreases nuclear fragmentation in resident astrocytes or oligodendrocytes and decreases glial cell infiltration. XIAP therapy improves optic nerve health and delays disease progression in LHON

Genetically timed, activity-sensor and rainbow transsynaptic viral tools.

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions