K. D. Kolstad

Remote Control of Neuronal Activity with a Light-Gated Glutamate Receptor

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.

Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous

Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy.

LiGluR Restores Visual Responses in Rodent Models of Inherited Blindness

Inherited retinal degeneration results from many different mutations in either photoreceptor-specific or nonphotoreceptor-specific genes. However, nearly all mutations lead to a common blinding phenotype that initiates with rod cell death, followed by loss of cones. In most retinal degenerations, other retinal neuron cell types survive for long periods after blindness from photoreceptor loss. One strategy to restore light responsiveness to a retina rendered blind by photoreceptor degeneration is to express light-regulated ion channels or transporters in surviving retinal neurons. Recent experiments in rodents have restored light-sensitivity by expressing melanopsin or microbial opsins either broadly throughout the retina or selectively in the inner segments of surviving cones or in bipolar cells. Here, we present an approach whereby a genetically and chemically engineered light-gated ionotropic glutamate receptor (LiGluR) is expressed selectively in retinal ganglion cells (RGCs), the longest-surviving cells in retinal blinding diseases. When expressed in the RGCs of a well-established model of retinal degeneration, the rd1 mouse, LiGluR restores light sensitivity to the RGCs, reinstates light responsiveness to the primary visual cortex, and restores both the pupillary reflex and a natural light-avoidance behavior.

Intravitreal injection of AAV2 transduces macaque inner retina

PURPOSE Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans. METHODS In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters. RESULTS AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-β-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters--human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)--AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis. CONCLUSIONS The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.

AAV Mediated GDNF Secretion From Retinal Glia Slows Down Retinal Degeneration in a Rat Model of Retinitis Pigmentosa

Mutations in over 80 identified genes can induce apoptosis in photoreceptors, resulting in blindness with a prevalence of 1 in 3,000 individuals. This broad genetic heterogeneity of disease impacting a wide range of photoreceptor functions renders the design of gene-specific therapies for photoreceptor degeneration impractical and necessitates the development of mutation-independent treatments to slow photoreceptor cell death. One promising strategy for photoreceptor neuroprotection is neurotrophin secretion from Müller cells, the primary retinal glia. Müller glia are excellent targets for secreting neurotrophins as they span the entire tissue, ensheath all neuronal populations, are numerous, and persist through retinal degeneration. We previously engineered an adeno-associated virus (AAV) variant (ShH10) capable of efficient and selective glial cell transduction through intravitreal injection. ShH10-mediated glial-derived neurotrophic factor (GDNF) secretion from glia, generates high GDNF levels in treated retinas, leading to sustained functional rescue for over 5 months. This GDNF secretion from glia following intravitreal vector administration is a safe and effective means to slow the progression of retinal degeneration in a rat model of retinitis pigmentosa (RP) and shows significant promise as a gene therapy to treat human retinal degenerations. These findings also demonstrate for the first time that glia-mediated secretion of neurotrophins is a promising treatment that may be applicable to other neurodegenerative conditions.

Changes in adeno-associated virus-mediated gene delivery in retinal degeneration.

Gene therapies for retinal degeneration have relied on subretinal delivery of viral vectors carrying therapeutic DNA. The subretinal injection is clearly not ideal as it limits the viral transduction profile to a focal region at the injection site and negatively affects the neural retina by detaching it from the supportive retinal pigment epithelium (RPE). We assessed changes in adeno-associated virus (AAV) dispersion and transduction in the degenerating rat retina after intravitreal delivery. We observed a significant increase in AAV-mediated gene transfer in the diseased compared with normal retina, the extent of which depends on the AAV serotype injected. We also identified key structural changes that correspond to increased viral infectivity. Particle diffusion and transgene accumulation in normal and diseased retina were monitored via fluorescent labeling of viral capsids and quantitative PCR. Viral particles were observed to accumulate at the vitreoretinal junction in normal retina, whereas particles spread into the outer retina and RPE in degenerated tissue. Immunohistochemistry illustrates remarkable changes in the architecture of the inner limiting membrane, which are likely to underlie the increased viral transduction in diseased retina. These data highlight the importance of characterizing gene delivery vectors in diseased tissue as structural and biochemical changes can alter viral vector transduction patterns. Furthermore, these results indicate that gene delivery to the outer nuclear layer may be achieved by noninvasive intravitreal AAV administration in the diseased state.