Ryan R Klimczak

In Vivo–Directed Evolution of a New Adeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery from the Vitreous

Injection of a new gene therapy vector into the easily accessible vitreous transduced the entire retina and rescued disease phenotypes. New Eye Pod Gene therapy mediated by adeno-associated virus (AAV) vectors has been clinically successful for the treatment of certain inherited diseases of the retina—the light-sensitive structure at the back of the eye that houses the photoreceptor cells (rods and cones). These degenerative disorders arise from mutated genes that either fail to express an essential protein or express harmful proteins that drive structural breakdown, cell death, and, ultimately, blindness. Current gene therapy regimens require damaging injections of gene-carrying vectors into the space between the rod and cone photoreceptors and the retinal pigment epithelium. By this route, the genetic material is delivered to only part of the retina. Now, Dalkara et al. show that delivery of a new vector into the eye’s easily accessible vitreous humour transduces the entire retina and rescues degenerative eye disease phenotypes. The authors used in vivo–directed evolution to fashion an AAV vector that delivers wild-type versions of defective genes throughout the retina after noninjurious injection into the eye’s easily accessible vitreous humour—the gel-like liquid between the lens and the retina. The newly engineered gene therapy systems rescued disease phenotypes in two mouse models of inherited eye diseases (X-linked retinoschisis and Leber’s congenital amaurosis) and transduced photoreceptor cells in nonhuman primates when delivered via the vitreous. Development of these next-generation therapeutic “eye pods” suggests that gene therapy vectors can be designed to penetrate dense tissues, which currently constitute barriers to gene delivery. 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.

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.

Molecular evolution of adeno-associated virus for enhanced glial gene delivery.

The natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, leads to predominant transduction of neurons and epithelia within the central nervous system (CNS) and retina. Despite the clinical relevance of glia for homeostasis in neural tissue, and as causal contributors in genetic disorders such as Alzheimers and amyotrophic lateral sclerosis, efforts to develop more efficient gene delivery vectors for glia have met with limited success. Recently, viral vector engineering involving high-throughput random diversification and selection has enabled the rapid creation of AAV vectors with valuable new gene delivery properties. We have engineered novel AAV variants capable of efficient glia transduction by employing directed evolution with a panel of four distinct AAV libraries, including a new semi-random peptide replacement strategy. These variants transduced both human and rat astrocytes in vitro up to 15-fold higher than their parent serotypes, and injection into the rat striatum yielded astrocyte transduction levels up to 16% of the total transduced cell population, despite the human astrocyte selection platform. Furthermore, one variant exhibited a substantial shift in tropism toward Müller glia within the retina, further highlighting the general utility of these variants for efficient glia transduction in multiple species within the CNS and retina.

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.

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.

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 109 possible human genome locations (P = 4.6 × 10-29). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS.

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.