Specific tools for targeting and expression in Müller glial cells.

Despite their physiological roles, Müller glial cells are involved directly or indirectly in retinal disease pathogenesis and are an interesting target for therapeutic approaches for retinal diseases and regeneration such as CRB1 inherited retinal dystrophies. In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells. ShH10Y and AAV9 were the most powerful capsids to infect mouse Müller glial cells. Retinaldehyde-binding protein 1 (RLBP1) promoter was the most powerful promoter to transduce Müller glial cells. ShH10Y capsids and RLBP1 promoter targeted human Müller glial cells in vitro. We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors. Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller glial cells. In summary, ShH10Y and AAV9 capsids, and RLBP1 or minimal CMV promoters are of interest as specific tools to target and express in mouse or human Müller glial cells.

In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous.

Inherited retinal degenerative diseases are a clinically promising focus of adeno-associated virus (AAV)-mediated gene therapy. These diseases arise from pathogenic mutations in mRNA transcripts expressed in the eye's photoreceptor cells or retinal pigment epithelium (RPE), leading to cell death and structural deterioration. Because current gene delivery methods require an injurious subretinal injection to reach the photoreceptors or RPE and transduce just a fraction of the retina, they are suitable only for the treatment of rare degenerative diseases in which retinal structures remain intact. To address the need for broadly applicable gene delivery approaches, we implemented in vivo-directed evolution to engineer AAV variants that deliver the gene cargo to the outer retina after injection into the eye's easily accessible vitreous humor. This approach has general implications for situations in which dense tissue penetration poses a barrier for gene delivery. A resulting AAV variant mediated widespread delivery to the outer retina and rescued the disease phenotypes of X-linked retinoschisis and Leber's congenital amaurosis in corresponding mouse models. Furthermore, it enabled transduction of primate photoreceptors from the vitreous, expanding its therapeutic promise.

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.

A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat Müller cells.

BACKGROUND: The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV). Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection. METHODOLOGY/PRINCIPAL FINDINGS: We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6), capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60%) and AAV6. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina.

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.