Retinal degeneration are blinding eye diseases that impact millions of lives around the world. Age-related macular degeneration alone is predicted to affect over 250 million people around 2040 as the population ages. The most prevalent form of inherited retinal degeneration, retinitis pigmentosa, has been found to have more than 200 different causative mutations, showing that treatments for retinal degeneration may not have a one-size-fit-all solution and require different approaches and concerted efforts from scientists around the world.

Gene therapy is an emerging and effective treatment option in restoring the vision and the quality of life of a subset of retinal degenerative patients. However, the success of gene therapy faces the limitations of low vector transduction efficiency, low vector carrying capacity, and immutable disease progression. Stem cell therapies face similar problems of immunogenicity and low cell integration efficiency. We show here that, through a combination of these two treatment modalities, endogenous regeneration through the genetic reprogramming of glia may be another effective and more broadly applicable approach in treating retinal degeneration.

Müller cells, the primary glia of the retina, have been demonstrated to possess neuroprotective as well as retinal progenitor cell-like regenerative properties in cases of retinal damage in zebrafish. The helix-loop-helix transcription factor Ascl1 is a key factor in determining neuronal cell fate in the nervous system, and it has also been shown to be capable of transdifferentiating astrocytes as well as Müller glia into neurons in vitro. The Let7 siRNA regulator Lin28 likewise seems to influence the zebrafish Müller glia cell fate through an Ascl1-dependent manner. While damage-dependent regeneration of the retina in zebrafish relies on the expression of Ascl1 and Lin28, no large scale proliferation and differentiation of the Müller glia have been observed in the mammalian retina, and as such, the role of Ascl1 and Lin28 in determining the mammalian Müller glia cell fate is currently unknown.

In chapter 2, we demonstrate that Müller glia in zebrafish and mice are capable of proliferation and fate change in vivo through the approach of forced expression of both Ascl1 and Lin28. Lin28 enhances the Ascl1-dependent proliferation of the Müller glia in zebrafish and mice when retinal damage is induced via NMDA. Notch suppression increases this proliferation in zebrafish further, but curiously not in mice. In chapter 3 we look at the different conditions involved in this Ascl1/Lin28 dependent proliferation through the exploration of different retinal damage models such as cobalt chloride induced hypoxic response and UV light induced photoreceptor apoptosis. We also attempt to induce Müller glia proliferation in retinal degenerative mouse models. Interestingly, while we find some success in inducing Müller glia proliferation in the retinal damage models, we find that genetic mouse models of retinal degeneration do not seem to respond to Ascl1 and Lin28 manipulation.

The retina contributes to the first steps in processing visual information. Rods and cone photoreceptors are the two main retina cells contributing to the initial steps in sensing light and translating it to an electrical signal to the downstream neuronal cells finally to be processed in the primary visual cortex. Many cells and hundreds of genes are involved in the light response and keeping retinal cells metabolically active. This high degree of activity makes the retina more vulnerable to mutations and degeneration. The majority of mutations identified in patients affect genes involved in either the photoreceptor structural integrity or in the phototransduction cascade. Many inherited retinal degenerative (IRDs) diseases, such as retinitis pigmentosa, lead to blindness as a result of initial rod photoreceptor cell death followed by cones and remodeling of the retina.

Gene therapy has been a growing field in the past decade and has proven to be an efficient and safe way of treating single-gene mutations leading to blindness by providing therapeutic DNA to targeted cells in the retina. The clinical trials for Leber congenital amaurosis type 2 (LCA) were the first to validate the proof-of-concept for gene therapy in the retina, after the successful use of adeno-associated viruses (AAVs) to deliver a healthy copy of the RPE65 gene to the affected cells. AAV, a small and nonpathogenic virus, has been used widely in many clinical trials, due to its safety profile and efficiency at targeting a wide range of tissues and cell types.

However, many obstacles remain from the optimization of delivery and design of viral vectors to elucidating molecular mechanisms behind the heterogenous genetic complexity in retinal diseases. While over 250 gene-causing diseases have been identified, more than 30-40% of genes involved remain unknown or outside of canonical coding sequences. For these patients, a gene replacement is not yet possible or applicable and therefore requires novel approaches.

My dissertation focuses on the use and optimization of engineered AAV vectors to achieve cell-selective targeting of retinal cells and organelles, to design novel mutation-independent gene therapies adapted for progressive degenerative diseases such as retinitis pigmentosa. It also aims at elucidating and characterizing molecular mechanisms underlying non-coding region mutations, with the use of genome editing tools to engineer mouse models of untranslated region mutations found in LCA patients. My thesis provides new knowledge and AAV toolkit to better tackle patients’ unmet need for novel gene therapy strategies addressing undiagnosed and noncanonical mutations in inherited retinal diseases.

Inherited retinal degenerations are genetically heterogeneous conditions affecting roughly 1:3000 people and are characterized by the loss of photoreceptors. Progressive retinal degenerative disease is the leading cause of vision loss in industrialized countries, and is the result of a wide range of mutations, mostly in rod-specific transcripts. Over 140 disease-causing genes have been identified to date. As the genetic mechanisms underlying inherited forms of retinal degeneration are identified, gene therapy is becoming a promising approach for the treatment of many inherited blinding diseases.

Indeed, the recent success of three clinical trials using adeno-associated virus (AAV) to deliver a normal copy of the RPE65 gene to the retinas of Leber congenital amaurosis (LCA) patients illustrates the potential of gene therapy in the retina. AAV has been shown safe and effective especially in a younger cohort of patients. Some important obstacles remain, however, for AAV-mediated gene therapy to become widely applicable across the range of existing retinal degenerative diseases. It will be essential to carefully evaluate the method used to deliver therapeutic genetic material to the retina, as this will determine the success of the treatment.

The serotype of vector used, the promoter chosen to drive expression and the method of injection are important components of the gene delivery system. A wide variety of AAV serotypes exist with different tropisms for cell populations in the retina, potentially allowing treatments to be targeted to specific cell types. The retinal cell types AAV can infect differs, however, depending on whether the vector is delivered into the vitreous cavity or the subretinal space. Subretinal injections, which were used in the LCA trials, result in the creation of a retinal detachment and localized injury to the retina while delivering high concentrations of transgene to only a limited area. An intravitreal approach has the potential to transduce panretinally and is less invasive, and therefore preferable, but naturally occurring serotypes of AAV transduce photoreceptors poorly from the vitreous, as a result of structural barriers that exist on the inner surface of the retina.

Recent advances in the understanding of AAV and the production of viral vectors have shown the flexibility of this virus, indicating that its function can be altered and tailored to the requirements of retinal gene therapy. A directed evolution approach has been used to select, out of a highly diverse library of AAV capsid variants, a novel variant with improved tropism for Müller glia. And in a parallel approach, residues on the capsid surface have been mutated to avoid ubiquitination and altering the nuclear trafficking of the virus.

This dissertation examines the use of engineered viral vectors for gene therapy in the retina. The creation of a novel variant of AAV, called 7m8, which is characterized by increased transduction of photoreceptors from the vitreous, is described below. 7m8 was derived from an AAV2 peptide insertion library and contains a 7mer motif. Injected intravitreally, 7m8 transduces cells throughout the retina, including photoreceptors in the outer retina, significantly more efficiently compared to the parental serotype. Expression was restricted to photoreceptors using a rhodopsin promoter. This virus, as well as the previously described Müller-specific variant ShH10, was used to deliver a wild-type copy of the retinoschisin gene to mice lacking this protein. Retinoschisin is secreted from photoreceptors, and retinas deficient in this protein are severely structurally impaired. Subretinal injections, which are damaging in nature, are therefore suboptimal because they are likely to cause additional injury. We show that 7m8 is able to efficiently target photoreceptors via intravitreal injection in this mouse model, leading to high levels of retinoschisin protein production, as well as structural and functional rescue. This rescue is longer lasting than that seen using ShH10, indicating the importance of targeting photoreceptors in this disease model.

AAV9 has been shown to transduce the murine retina when injected intravenously through the tail vein. We used two surface tyrosine-to-phenylalanine mutations to improve the retinal expression of AAV9, and demonstrated that these mutations lead to higher infectivity of all retinal layers, most dramatically in photoreceptors and the inner nuclear layer, but also including the retinal pigment epithelium and ganglion cells. This novel vector was then used to explore the bifunctionality of the Nxnl1 gene, which encodes two isoforms of the rod-derived cone viability factor (RdCVF). The short form of RdCVF is secreted and has been shown to support cone survival, while the long isoform is retained intracellularly and has been implicated in redox signaling. AAV92YF and 7m8 were used to express the two isoforms of RdCVF in the rd10 mouse model of retinitis pigmentosa. RdCVF rescued cone survival when injected intravenously or intravitreally, but had little effect on rod survival. Early expression of RdCVFL in dark-reared rd10 mice delayed rod, and subsequently cone death.

Optogenetics, the ability to control cells with light, has revolutionized neuroscience by endowing researchers with hyper precise tools which can dissect neural circuits. However, the use of optogenetic effectors is not limited to academic studies on neurons. This dissertation examines two alternative applications of optogenetics: to study glia-neuron interactions and to therapeutically restore light sensitivity to degenerate retinas. In order express these optogenetic effectors, Adeno-Associated Virus (AAV) was used to deliver transgenes.

Over the course of these studies, the ability to specifically target certain types of cells became paramount. Specifically, special AAV capsid variants had to be used in order to transduce Müller glia, the principal glia cell of the retina. The capsid variants, 7m8 and ShH10, are not specific for Müller cells, so different glial promoters were investigated. Two promoters evaluated, GLAST and gfaABC1D, had unexpected expression profiles. Although GLAST is a Müller cell specific protein, the GLAST promoter was unable to restrict transgene expression to Müller glia. However, the smaller the gfaABC1D promoter was able to completely restrict transgene expression to retinal glia in both health and disease.

With new methods to selectively transduce Müller glia, their role in glutamate uptake was investigated. The electrogenic transporters used to transport glutamate against its concentration gradient require a hyperpolarized membrane potential made possible through high expression of potassium channels. If glutamate uptake was dependent on Müller cell membrane potential, then transient depolarization of the Müller glia should inhibit glutamate uptake. To transiently depolarize Müller cells, they were targeted to express a bistable channelrhodopsin2 (ChR2) mutant (C128S/D156A, “BiChR2”). The effect of BiChR2 induced Müller glia depolarization on the retinal light response was investigated by examining the electroretinogram (ERG). While there were no significant changes to the ON-bipolar cell generated ERG B-wave, subtle temporal changes in arose that should be investigated further.

Optogenetics have a therapeutic potential to restore vision in degenerate retinas. People with late stage retinal degenerative disease lose sight as a consequence of photoreceptor death. However, other retinal cells remain. Endowing the surviving retinal cells with the ability to sense light through optogenetics could restore sight. The effectiveness of optogenetics tools used for vision restoration were analyzed based on their light and temporal sensitivity.

Abstract

Development and Assessment of Gene Therapies for Inherited Blinding Diseases

By

Kathleen Durgin Kolstad

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor John Flannery, Committee Chair

There are two therapeutic approaches for inherited retinal disease addressed in

this dissertation: we sought to slow retinal degeneration and reverse visual loss after

complete photoreceptor apoptosis. In the first approach, by viral gene transfer to the

support cells of the retina, Müller glia (RMCs), we achieved sustained secretion of

human glial derived neurotrophic factor (hGDNF) (Chapter 3). We hypothesized that

hGDNF production by retinal glia will enhance the protective affects of RMCs in the

diseased retina and help slow photoreceptor degeneration. Furthermore, this method avoids extra photoreceptor stress caused by direct hGDNF gene transfer to cells that are already stressed. We were able to optimize gene transfer to RMCs and observe the beginnings of functional rescue in an animal model of autosomal dominant retinitis pigmentosa with this technique. One major advantage of this therapeutic approach is that it is applicable to multiple retinal disease genotypes.

The second approach to ocular gene therapy presented in this dissertation was to

re-introduce photosensitivity to the retina after complete photoreceptor degeneration

(Chapter 4). To this end, we employed the engineered light activated glutamate receptor (LiGluR) to confer light sensitivity on retinal ganglion cells (RGCs) in the diseased retina. We first showed LiGluR mediated RGC photo-activation in in vitro retinal tissue preparation. We then characterized in vivo cell population responses (visually evoked potentials, VEPs) in V1 when retinal input was limited to LiGluR induced activity in the retina. VEPs driven by LiGluR are approximately 50% of the amplitude of full field light flash driven responses in the wild type animal. LiGluR driven cortical responses in blind animals suggest that it is a promising therapy for restoring visual function and processing in the late stages of retinal degeneration.

In the third part of this dissertation (Chapter 2a and 2b), the goal was to develop

and assess methods of making ocular gene therapies safer and more efficacious. Current gene therapies for retinal degenerative diseases rely on subretinal delivery of viral vectors carrying therapeutic DNA. However, this method of delivery limits the viral transduction profile to the region of injection and seriously compromises the retina during detachment. We have identified natural barriers to viral vector delivery to the outer retina from the vitreous. Furthermore, we have developed artificial methods and characterized disease states that allow these barriers to be overcome. The understanding of and the ability to manipulate barriers to vitreal delivery of viral vectors will help avoid the limitations, risks, and damage associated with subretinal injections.

Current retinal gene therapies using adeno-associated viral (AAV) vectors are limited in their efficacy due to their dependence on an invasive subretinal delivery route to achieve sufficient gene delivery. Additionally, the genetic heterogeneity of inherited retinal degenerative diseases makes gene replacement and antisense therapeutic strategies impractical for treating a broad array of patients. This dissertation sought to address these limitations by creating intravitreally-permissive AAV vectors through directed evolution and examining their therapeutic potential using a mutation-independent approach.

Firstly, we examined natural barriers to intravitreal vector delivery. We identified the inner limiting membrane (ILM) as the main obstacle to efficient intravitreal gene delivery and found mild digestion with a nonspecific protease, Pronase E, substantially enhanced retinal transduction for several AAV serotypes, most notably for AAV serotype 5 (AAV5). Secondly, we evolved more efficient glial-permissive AAV vectors in the CNS and retina using an in vitro selection methodology. We isolated an AAV variant, ShH10, closely related to AAV6, capable of efficient, selective Müller cell infection through intravitreal injection. Thirdly, we utilized this variant to examine the efficacy of intravitreally targeting Müller cells to secrete glial-derived neurotrophic factor (GDNF) as a mutation-independent therapy for delaying photoreceptor degeneration in a rat model of retinitis pigmentosa (RP). We achieved functional and histological rescue in treated animals at five months post injection, with an average approximate rescue of 50% in ERG a and b-wave amplitudes. Lastly, we evolved AAV variants to intravitreally transduce photoreceptors using an in vivo selection approach in a transgenic line of mice bearing a rhodopsin-GFP fusion. We isolated an AAV variant, 7M8, derived from an AAV2 peptide display library, capable of significant intravitreal photoreceptor transduction relative to AAV2.

The creation of intravitreally-permissive AAV vectors as well as the development of mutation-independent gene therapies will enable safer and more effective therapies for treating inherited retinal degenerative diseases.

Inherited retinal degenerations affect millions of people worldwide, with few or no treatment options. The most prevalent form of progressive retinal degeneration is retinitis pigmentosa, a heterogenous disease affecting over one million people around the world. Over 70 genes have been shown to cause retinitis pigmentosa, mainly affecting rod photoreceptors. As genetic mechanisms are identified, gene therapy becomes a promising potential treatment through gene replacement.

However, gene replacement therapy is not a viable option for several of these diseases, where autosomal dominant forms such as the P23H mutation of rhodopsin require gene silencing instead of replacement. Since retinitis pigmentosa patients mostly present mutations in genes necessary for correct function of rod photoreceptors, leading to subsequent loss of cones, it is possible to target cone survival and provide a wide-ranging solution. Rod-derived cone viability factor (RdCVF) has been shown to be essential for cone survival, as it is produced and secreted by rods and promotes glucose uptake by cones. Chapter 2 of this dissertation examines the delivery of RdCVF through an AAV viral vector to a transgenic pig model of autosomal dominant P23H retinitis pigmentosa. Previous studies have shown cone rescue through delivery of RdCVF in mice, and here we explore therapeutic delivery to a large animal model that more closely resembles humans. In this study we report that RdCVF rescue is dose-dependent, as well as dependent on retinal regions reached by AAV. While electroretinogram results are not conclusive, immunohistochemistry indicates improvements in cone morphology and even rod retention in the retinal tissue.

For more common recessive retinal diseases, such as the wide variety involving neuronal ceroid lipofuscinoses (NCL), gene replacement therapy is desirable. Of the 14 genes responsible for NCL, all but one are recessive, causing lysosomal dysfunction that leads to cerebellar atrophy, seizures, and in most cases retinopathies and vision loss. As gene therapies are developed targeting the brain, it is also advantageous to target the retina, as patients would require both forms of treatment. In chapter 3 of this dissertation, a gene therapy for CLN11, caused by mutations to the progranulin gene, is developed. Progranulin is a protein secreted mainly by microglia in the brain and retina, and is then endocytosed by neurons, thus regulating their lysosomal function. Absence of progranulin causes cerebellar atrophy, seizures, ataxia, dementia and vision loss. In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. While mice that receive AAV vectors carrying the progranulin gene at post-natal day 3 or 4 show reduction in retina thinning, mice intravitreously injected at months 1 and 6 show no improvement, and mice injected at 12 months of age show increased retinal thinning in comparison to their controls. Thus, delivery of progranulin proves to be time sensitive, requiring early delivery. This is potentially due to its secretion, although further studies are required to elucidate this phenomenon.

There are currently over 300 genetic causes for inherited retinal disease (IRD). Retinitis pigmentosa is part of the many diseases under the IRD umbrella, where rod photoreceptors progressively degenerate, followed by the outer segments of cone photoreceptors. The increasing number of genes found to be responsible for IRDs suggest the need to design a mutation agnostic approach to treat a wider range of IRD patients. Adeno-associated viral (AAV) optogenetic gene therapy is a promising mutation-independent treatment that aims to restore light-sensitivity to the degenerating retina. Studies directing ectopic expression of opsins to add a light-receptive function to surviving retinal neurons have been successful and currently there are four AAV-delivered optogenetic gene therapies in clinical trials. These clinical studies report restored perception of shape, color, contrast, and more. However, many new optogenetic tools have been engineered and have the potential to improve the vision restored after optogenetic gene therapy treatment.

There are many factors to consider when designing AAV optogenetic gene therapies. An ideal optogenetic protein should combine both light sensitivity over a broad range of intensities and response speed to permit vision with motion. To this end, we examined the characteristics of the candidate opsins for optogenetic gene therapy, such as response kinetics, light-sensitivity, and peak excitation wavelength within the visible light spectrum. The current study explores targeting retinal ganglion cells (RGCs) to become photoreceptors using novel channelrhodopsins developed by structure-guided mutagenesis and never previously tested for vision restoration: 1) ChRmine, from the algae Rhodomonas lens, 2) T119A-ChRmine, a more sensitive ChRmine variant, and 3) ChroME2S, a second-generation ChroME-based opsin. We use behavioral tests to show that treated rd1 mice recover high-sensitivity vision and use binocular vision to perceive depth. Using these opsins, we demonstrate the feasibility of using new and improved opsins to enhance vision in patients. We additionally show that AAV constructs designed with opsins tagged with a membrane-targeting sequence are more light-sensitive than opsins without the tag. We thus demonstrate that level of membrane localization may be important for a sensitive treatment. Further, we tested multiple treatment titers and found mice with diluted titers are more light-responsive and have better binocular vision than the highest titer.

A major concern in the degenerating retina is spontaneous activity due to deafferented bipolar cells. RGCs have discrete bursts of activity, which can manifest as visual hallucinations in IRD patients. Most optogenetic therapies choose to target the optical control of RGCs, which may restore vision, but spontaneous activity may affect visual acuity. These oscillations block any remnant light responses from residual photoreceptors. We wanted to understand if functional behavior would recover if we target inhibitory opsin expression in a portion of RGCs. We used a promoter that not only transfects RGCs, but also transfects upstream neurons that are likely cone cell bodies left after outer segment degeneration. We show that expression of inhibitory opsins in cones and RGCs restored an important and complex aspect of vision, binocular depth perception. We may have inadvertently suppressed spontaneous activity by also forcing cone hyperpolarization in response to light, but this study additionally confirms that visual pathways in late-stage retinal disease are still functional. The field of optogenetics has consistently discovered or engineered increasingly potent and fast opsins, some of which we have used in this study. Our most sensitive candidate opsin drove the most light-responsive behaviors. When we used a hyperpolarizing opsin to reduce spontaneous activity in RGCs, additional off-target expression in upstream neurons caused recovery of depth perception in treated animals. This not only showed restoration of binocular vision that suggests correlated brain activity but revealed that the retinal connectome is well preserved and can function even in late-stage degeneration. Together, these results demonstrate that optogenetic proteins must be carefully considered when designing effective AAV optogenetic gene therapies in order to translate the visual scene most accurately.

Inherited retinal degenerations, effecting about 1:3000 people, have historically been challenging to treat. A large number of mutations have been identified in these patients, which share a similar phenotype of a gradual loss of photoreceptor cells leading to blindness. In recent decades, a promising new form of treatment called gene therapy has been developed to address patient needs. Encouragingly, there have been several successful clinical trials using adeno-associated virus (AAV) to deliver genetic cargo to the necessary retinal cells. Viral vectors based on AAV have seen success because of the ability of the virus to achieve high transfection efficiency and a strong safety profile. The seminal gene therapy clinical trial utilizing AAV was the delivery of RPE65 to the retinas of patients with Leber congenital amaurosis (LCA), which proved to have long-term safety and showed significant efficacy. But, challenges still remain for broad application of AAV. It is therefore necessary to develop AAV variants with novel properties in order to address the wide range of retinal degenerations.

There are a number of natural AAV serotypes that exhibit different tropisms for different retinal cells allowing for the potential of treatments targeted to different cell types. Importantly, the route of delivery strongly affects the cells targeted and depends on whether the virus is administered subretinally or intravitreally. Natural AAV serotypes perform poorly when delivered intravitreally due to structural barriers, such as the inner limiting membrane. Therefore, initial clinical trials chose to deliver AAV subretinally, which has the advantage of local delivery to the photoreceptors or retinal pigment epithelium and limited immune response. But, expression is restricted to only a portion of the retina and there is a risk of damage from retinal detachment in a tissue that is already degenerating. Intravitreal injection would be preferred and would allow for pan-retinal expression and would not cause retinal detachment. Importantly, recent evidence has shown species specificity for AAV variants necessitating future AAV engineering approaches to take into account the specific challenges of the human retina. One method of AAV engineering, directed evolution, has yielded many novel AAV variants with advantageous properties. Directed evolution is a biomimetic process that emulates how viruses naturally evolve. Large libraries of highly diverse AAV variants are iteratively selected utilizing pressures for a desired attribute.

This dissertation focuses on the directed evolution of novel variants that are able to intravitreally deliver cargo in large animal models. Canines serve as a strong preclinical model for retinal degenerations and contain a visual streak for high acuity vision, which is lacking in rodents. Directed evolution was performed in a canine model and variants were selected utilizing deep sequencing of each round of evolution resulting in AAVs that had high outer nuclear layer or bipolar cell tropism when delivered intravitreally. Directed evolution was also performed in non-human primates (NHPs) because the retina shares many similarities to the human retina, including a cone-rich macula and fovea, and has the highest chance of yielding the most clinically relevant AAVs.

To generate a NHP variant, several new approaches and tools were developed. First, novel AAV libraries were generated including AAV4-, AAV5-, and Ancestral-based peptide insertion libraries. Multiple plasmid backbones with unique restriction and primer binding sites were created to allow for tuning of different pools of libraries throughout the directed evolution process. A packaging strategy that mitigated the possibility of replication and immune response during NHP directed evolution was developed leading to a 10-fold increase in safety. Serum screening for animals without pre-existing antibodies to AAV also increased safety. Implementing these tools and strategies for directed evolution in a NHP model led to an AAV2-based peptide insertion variant that showed increase cone targeting and decreased ganglion cell targeting when intravitreally delivered. For both the canine and NHP directed evolution studies, deep sequencing of each round of selection revealed “hidden” variants and elucidated the biases in the selection process due to initial overrepresentation of particular variants. An additional study to increase the packaging capacity of AAV was performed and demonstrated the size limits of AAV cargo. The research performed in this dissertation led to AAV variants with increased therapeutic properties and insights into the mutability of AAV and performance of individual variants throughout the directed evolution process.