Jens Duebel

Genetic Reactivation of Cone Photoreceptors Restores Visual Responses in Retinitis Pigmentosa

Let There Be Light Retinitis pigmentosa, a disease that can result from a wide variety of genetic defects, causes degeneration of photoreceptor cells in the retina and leads to blindness. In the course of the disease, it is generally the rod photoreceptor cells that degenerate first. Cone photoreceptor cells may persist, but in a damaged and nonfunctional state. Busskamp et al. (p. 413, published online 24 June; see the cover; see the Perspective by Cepko) have now applied a gene therapy approach to mouse models of retinitis pigmentosa. Inducing expression of a bacterial light-activated ion pump, halorho dopsin, in the damaged cone cells improved visual responses in the diseased mouse retinas. Thus, it may be possible to rescue cone photoreceptors therapeutically, even after they have already been damaged. A bacterial ion pump rescues visual function in damaged cone-photoreceptor cells in mouse models of retinitis pigmentosa. Retinitis pigmentosa refers to a diverse group of hereditary diseases that lead to incurable blindness, affecting two million people worldwide. As a common pathology, rod photoreceptors die early, whereas light-insensitive, morphologically altered cone photoreceptors persist longer. It is unknown if these cones are accessible for therapeutic intervention. Here, we show that expression of archaebacterial halorhodopsin in light-insensitive cones can substitute for the native phototransduction cascade and restore light sensitivity in mouse models of retinitis pigmentosa. Resensitized photoreceptors activate all retinal cone pathways, drive sophisticated retinal circuit functions (including directional selectivity), activate cortical circuits, and mediate visually guided behaviors. Using human ex vivo retinas, we show that halorhodopsin can reactivate light-insensitive human photoreceptors. Finally, we identified blind patients with persisting, light-insensitive cones for potential halorhodopsin-based therapy.

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.

Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV Restores ON and OFF visual responses in blind mice.

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eyes easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell-specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.

Red‐shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina

Targeting the photosensitive ion channel channelrhodopsin‐2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin‐2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red‐shifted light is vastly lower than that of blue light. Here, we show that a red‐shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV‐ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV‐ and lentivirus‐mediated optogenetic spike responses in ganglion cells of the postmortem human retina.

Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno‐Free and Feeder‐Free Conditions

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two‐step xeno‐free/feeder‐free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self‐forming neuroretinal‐like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation‐compatible CD73+ photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC‐derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73+ photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP‐compliant retinal cell manufacturing protocol allowing large‐scale production and banking of hiPSC‐derived retinal cells and tissues. Stem Cells 2017;35:1176–1188

Fast and accurate spike sorting in vitro and in vivo for up to thousands of electrodes

Understanding how assemblies of neurons encode information requires recording large populations of cells in the brain. In recent years, multi-electrode arrays and large silicon probes have been developed to record simultaneously from hundreds or thousands of electrodes packed with a high density. However, these new devices challenge the classical way to do spike sorting. Here we developed a new method to solve these issues, based on a highly automated algorithm to extract spikes from extracellular data, and show that this algorithm reached near optimal performance both in vitro and in vivo. The algorithm is composed of two main steps: 1) a “template-finding” phase to extract the cell templates, i.e. the pattern of activity evoked over many electrodes when one neuron fires an action potential; 2) a “template-matching” phase where the templates were matched to the raw data to find the location of the spikes. The manual intervention by the user was reduced to the minimal, and the time spent on manual curation did not scale with the number of electrodes. We tested our algorithm with large-scale data from in vitro and in vivo recordings, from 32 to 4225 electrodes. We performed simultaneous extracellular and patch recordings to obtain “ground truth” data, i.e. cases where the solution to the sorting problem is at least partially known. The performance of our algorithm was always close to the best expected performance. We thus provide a general solution to sort spikes from large-scale extracellular recordings.

Gene therapy for the eye focus on mutation-independent approaches.

PURPOSE OF REVIEW This review will discuss retinal gene therapy strategies with a focus on mutation-independent approaches to treat a large number of patients without knowledge of the mutant gene. These approaches rely on the secretion of neurotrophic factors to slow down retinal degeneration and the use of optogenetics to restore vision in late-stage disease. RECENT FINDINGS Success in clinical application of adeno-associated virus (AAV)-mediated gene therapy for Lebers congenital amaurosis established the feasibility of retinal gene therapy. More clinical trials are currently on their way for recessive diseases with known mutations. However, the genetic and mechanistic diversity of the retinal diseases presents an enormous obstacle for the development of gene therapies tailored to each patient-specific mutation. To extend gene therapys promise to a large number of patients, evidence suggests retina-specific trophic factors, such as rod-derived cone viability factor, can be used to slow down loss of cone cells responsible for our high acuity vision. In parallel, it has been shown that microbial opsins are able to restore light sensitivity when expressed in blind retinas. SUMMARY Recent findings imply that using the viral technology that has been demonstrated as well tolerated in patients, there are opportunities to develop widely applicable gene therapeutic interventions in clinical ophthalmology.

Functional rescue of cone photoreceptors in retinitis pigmentosa

In the highly intricate retinal functional anatomy, connectivity and information processing, the photoreceptor cells play a crucial role. In most simple terms, the photoreceptors rods and cones detect the light and transduce it to the brain as electrical signals through a sophisticated network of neurons. Pathologies that affect the photoreceptor structure and function result in impaired vision. Photoreceptor degeneration due to gene mutations causes a large number of blinding disorders known as inherited retinal dystrophies (IRDs). IRDs affect approximately one in every 3,000 individuals and represent the most frequent inherited forms of human visual handicap [1]. Retinitis pigmentosa (RP), a genetic disease that features degeneration of both rod and cone photoreceptors is the most commonly inherited retinal degeneration, affecting approximately 1.5 million people worldwide [2]. Currently, there is no known effective treatment that can prevent or reverse the vision loss in RP.

Noninvasive gene delivery to foveal cones for vision restoration

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.

Restoration of visual function by transplantation of optogenetically engineered photoreceptors

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments (OS) in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). For a RPE-independent treatment approach, we introduced a hyperpolarizing microbial opsin into photoreceptor precursors from new-born mice, and transplanted them into blind mice lacking the photoreceptor layer. These optogenetically transformed photoreceptors were light responsive and their transplantation lead to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generated cone photoreceptors from human induced pluripotent stem cells (hiPSCs), expressing the chloride pump Jaws. After transplantation into blind mice, we observed light-driven responses at the photoreceptor and ganglion cell level. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.