To date, various approaches have been used to restore some visual functions in affected patients, primarily human derived retinal organoids and gene therapies. First directly administered Adeno-associated virus (AAV) based gene therapy approved by U.S. Food and Drug Administration (FDA), Luxturna, significantly improved the vision of patients with retinal dystrophy at low light levels. Nevertheless, there are still difficulties with restoring high-resolution vision that need to be overcome.This project aims to develop a novel proof-of-concept approach to therapeutic gene delivery using a modified Rabies virus as a vector specific to bipolar cells. We propose that specific targeting of the surviving cell population within the degenerated retina, especially bipolar cells (BC) by our modified Rabies virus (RV), pseudotyped with LRIT3 (RV-LRIT3), may constitute a new strategy of gene therapy in retinal diseases. We hypothesize that the proposed viral approach can reach many BCs thanks to the interactions with the TRPM1 channels and deliver high levels of light-gated opsins to restore vision. The main innovation of this project is use of the RV, which can transfect up to 4 genes. Therefore, it can deliver a few opsins with different excitation spectrums and increase overall light sensitivity. We suggest this technique will restore selective responses in the mice’s visual cortex and superior colliculus. We perform single-neuron recordings and behavioral visual discrimination tasks to estimate visual network selectivity in response to complex and moving stimuli.
Image: The strategy of the vision assessment after LRIT3-RV treatment. A. the whole mount retina shows cells infected with the RV-mCherry vector carrying three opsins Bar: 100 um. B. scheme of the simultaneous recordings from the Superior colliculus (SC) and the visual cortex (V1) with representative evoked potentials (VEP) in response to a flash of light. C. Two examples of cells expressing two contradictory opsins. Blue laser caused activation of the cell expressing channelrhodopsin. The second cell expressed the ArchT opsin causing suppression of the activity while yellow laser illumination. D, VEPs recorded in single healthy (black), treated Rd12 with gene therapy (green), and diseased Rd12 (magenta) mice.
P. Węgrzyn, W. Kulesza, M. Wielgo, S. Tomczewski, A. Galińska, B. Bałamut, K. Kordecka, O. Cetinkaya, A. Foik, R. J. Zawadzki, D. Borycki, M. Wojtkowski, A. Curatolo “In vivo volumetric analysis of retinal vascular hemodynamics in mice with spatio-temporal optical coherence tomography” Neurophotonics. 11(4):0450031-4500322. (2024) https//doi: 10.1117/1.NPh.11.4.045003.
Visual impairments are affecting millions of people worldwide, and the number of visually disabled people is increasing due to the aging of the population and chronic diseases. By 2050, 61 million people will be blind, and 474 million will have moderate and severe vision impairment. Decrease or loss of vision impacts the lives of those affected, making daily activities difficult, leading to a loss of independence, and increasing the risk of mental diseases. That is why it is crucial to look for effective therapies that will slow disease progression and restore vision. Recently, viral gene therapies providing defective genes or optogenetic tools appeared to be the most effective ways to restore vision. It is mainly related to privileged immune status and easy accessibility for treatment delivery of the eye. The First AAV-based gene therapy that improves the vision of patients with retinal dystrophy was approved by the US FDA in 2017. There are also multiple AAV-based gene therapies focused on delivering different variants of microbial Channelrhodopsin 2 (Chr2) subjected to clinical trials. As recent clinical trials focus on opsins that perform their function in high light intensities, there is still a need to design an optogenetic tool that would be successfully activated by relatively low light stimulation. In a healthy retina, signals received from light-activated photoreceptors are processed from bipolar cells to amacrine and ganglion cells. However, in the degenerated retina, irreversible changes and mutations cause profound loss of light-detecting rods and cones. Despite the loss of photoreceptors and structural changes, surviving cells remain functional. This project aims to develop novel proof-of-concept chimeric opsins with exceeded functionality to mimic natural circuit mechanisms and information processing within a degenerated retina. We propose that chimeric RecRho variants may constitute a new gene therapy strategy in retinal diseases. We hypothesize that moderate light levels can activate chimeric RecRho variants delivered to surviving cell populations within the degenerated retina, will transform transduced cells into direct light detectors, and restore high-level vision. Besides converting transduced cells into direct light detectors, we believe that our chimeric proteins will also positively impact structure and synaptic plasticity on a large scale through the whole retina. For testing we utilize single neuron recordings and behavioral visual discrimination tasks to estimate visual network selectivity in response to complex and moving stimuli. Such an approach of synthetic protein with multiple functions, based on the mGluR1 transduction pathway, wasn’t proposed and used before.
Image: On the left: Retinal cross-section with cells non-specifically infected with AAV-CAG-mNeon virus. (ONL- outer nuclear layer, INLinner nuclear layer, RGC – retinal ganglion cells). AAV-CAG-mNeon (green), anti-Rhodopsin antibody (red), DAPI (nuclei marker, blue). On the right: Example of a Patch-Clamp experiment performed on HEK293t cells expressing Channelrhodopsin-2. Cell was stimulated with blue light (473 nm), and light-induced evoked potentials were recorded.
There is a strong need to understand how our brain processes visual information and how different brain centers interact with each other. Vision is an essential sense in our life. Lack of such interaction disable performance of an attentive task or to memorize visual information. One of the structures heavily involved in cognitive processes and general brain activity is a Basal Forebrain (BF). This project is designed to investigate the Basal Forebrain’s role in visual information processing. The BF is the primary source of cholinergic inputs into the visual cortex, and therefore the main source of the acetylcholine neurotransmitter. We propose that cholinergic modulation coming from BF significantly impacts single neuronal responses of the primary visual cortex (V1) in Long-Evans rats. Precisely, we hypothesize that the BF cholinergic modulation will change single cells’ stimulus preference like optimal size, orientation selectivity. Furthermore, we assume that it has an even stronger impact on the visual system activity by changing the oscillations carrying information through the sensory systems. We attempt to investigate if only cholinergic inputs directly affect single cells in the V1 or there is needed additional involvement of GABAergic cells in the Basal Forebrain. In this aim, we use newly developed viral tools to target GABAergic or cholinergic cells in the BF specifically. By using opsins, that can be activated by particular light color, we manipulate basal forebrain activity and correlate it with encoding in the V1. We also hypothesize that such manipulations will significantly impact behavioral performance and pattern recognition due to more robust activity while performing attentive tasks.
Image: Cartoon visualization of the viral injection into the V1 and retrogradely targeting specifically GABAergic or cholinergic cells in the BF. Labeled cells in the thalamus (LGN or LP) will be proof that Rabies viral tracing works appropriately.
P. Węgrzyn, W. Kulesza, M. Wielgo, S. Tomczewski, A. Galińska, B. Bałamut, K. Kordecka, O. Cetinkaya, A. Foik, R. J. Zawadzki, D. Borycki, M. Wojtkowski, A. Curatolo „In vivo volumetric analysis of retinal vascular hemodynamics in mice with spatio-temporal optical coherence tomography” Neurophotonics. 11(4):0450031-4500322. (2024) https//doi: 10.1117/1.NPh.11.4.045003
R. Hołubowicz, S. W. Du, J. Felgner, R. Smidak, E. H Choi, G. Palczewska, C. Rodrigues Menezes, Z. Dong, F. Gao, O. Medani, A. L. Yan, M. W. Hołubowicz, P. Z. Chen, M. Bassetto, E. Risaliti, D. Salom , J. N. Workman, P. D. Kiser, A. T. Foik, D. C. Lyon, G. A. Newby, D. R. Liu, P. L Felgner, K. Palczewski “Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations” Nat Biomed Eng. (2024) https://www.nature.com/articles/s41551-024-01296-2
Eye, and particularly the retina, is a “window into the brain”. It reveals sufficient information about patient condition, and for humans, it is the main organ responsible for world perception. The retina is a very fragile and complex structure, located on the back of the eyes and is exposed to many environmental hazards leading to its defects or even retinal degeneration (RD). In modern society, an increasing number of people suffering from visual impairment are being observed. The predominant causes of visual impairments are irreversible changes in the retinal structure and mutations leading to photoreceptor (our light detectors) death in the retina. Two main diseases related to photoreceptor death are retinitis pigmentosa and age-related macular degeneration which are causing blindness/vision loss in ~200 million people globally. These conditions significantly reduce the quality of life and, therefore, drive a strong need for developing new, easy to use and effective techniques for not only slowing down the disease progression but also restoring vision. There have been a large number of attempts aiming to prevent complete loss of photoreceptors or restoring vision by either opsin delivery or retinal transplantation. The viral gene therapy based on delivering light-sensitive ion channels to surviving retinal cells seems the most direct way of curing blindness. However, a number of issues remain to be solved in order to create an effective therapy. The general aim of this project is to develop a new approach to deliver light-sensitive ion channels in order to restore selective neuronal responses to low light stimuli in the visual system of a blind animal. To do so we use a modified Rabies virus (RV) tracing technique that allows not only for efficient gene expression but also cell-type-specific network tracing to precisely deliver proteins of interest to the desired retinal circuit. This approach is crucial to obtain adequate light sensitivity and restore selective visually evoked responses in the brain. It will also create a specific functional network that will mimic the natural processing of the retina. The Rabies virus isused for opsin delivery into the degenerated retina to restore visual responses in the blind animals. We test visual responses in the primary visual cortex for not only sensitivity to flash of light but also for selectivity to certain stimulus parameters like contrast, spatial frequency, orientation/direction, and size. It is crucial to investigate whether viral/gene therapy can restore selective neuronal responses in the visual system. We take full advantage of the hierarchical connectivity of the retinal network to target different functional circuits and information processing channels by infecting different retinal layers through monosynaptic viral tracing.
This unique method will reveal the extent to which the visual system will respond to complex stimuli like moving spots, drifting gratings, pattern motion, and natural stimuli after viral treatment.
Figure. The strategy of an opsin delivery and targeting specific cell-type networks in the retina using pseudotyped Rabies virus in wild-type mice and those affected by P23H gene mutation, which leads to rod photoreceptors degeneration and vision loss.
F. Gao, E. Tom, C. Rydz, W. Cho, A. V. Kolesnikov, Y. Sha, A. Papadam, S. Jafari, A. Joseph, A. Ahanchi, N. Balalaei Someh Saraei, D. Lyon, A. Foik, Q. Nie, F. Grassmann, V. J. Kefalov, D. Skowronska-Krawczyk “Polyunsaturated Fatty Acid – mediated Cellular Rejuvenation for Reversing Age-related Vision Decline” bioRxiv., 2024.07.01.601592. (2024) https://www.biorxiv.org/content/10.1101/2024.07.01.601592v1
H. Leinonen, J. Zhang, L. M. Occelli, U. Seemab, E. H. Choi, L. Felipe L P Marinho, J. Querubin, A. V. Kolesnikov, A. Galinska, K. Kordecka, T. Hoang, D. Lewandowski, T. T. Lee, E. E. Einstein, D. E. Einstein, Z. Dong, P. D. Kiser, S. Blackshaw, V. J. Kefalov, M. Tabaka, A. Foik, S. M. Petersen-Jones, K. Palczewski “A combination treatment based on drug repurposing demonstrates mutation-agnostic efficacy in pre-clinical retinopathy models” Nat Commun., 15(1):5943. (2024) https://www.nature.com/articles/s41467-024-50033-5
R. Hołubowicz, S. W. Du, J. Felgner, R. Smidak, E. H Choi, G. Palczewska, C. Rodrigues Menezes, Z. Dong, F. Gao , O. Medani, A. L. Yan, M. W. Hołubowicz, P. Z. Chen, M. Bassetto, E. Risaliti, D. Salom , J. N. Workman, P. D. Kiser, A. T. Foik, D. C. Lyon, G. A. Newby, D. R. Liu, P. L Felgner, K. Palczewski “Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations” Nat Biomed Eng. (2024) https://www.nature.com/articles/s41551-024-01296-2
P. Węgrzyn, W. Kulesza, M. Wielgo, S. Tomczewski, A. Galińska, B. Bałamut, K. Kordecka, O. Cetinkaya, A. Foik, R. J. Zawadzki, D. Borycki, M. Wojtkowski, A. Curatolo „In vivo volumetric analysis of retinal vascular hemodynamics in mice with spatio-temporal optical coherence tomography” Neurophotonics. 11(4):0450031-4500322. (2024) https//doi: 10.1117/1.NPh.11.4.045003
Z. J. Engfer, D. Lewandowski, Z. Dong, G. Palczewska, J. Zhang, K. Kordecka, J. Płaczkiewicz, D. Panas, A. T. Foik, M. Tabaka, and Krzysztof Palczewski „Distinct mouse models of Stargardt disease display differences in pharmacological targeting of ceramides and inflammatory responses” Proc Natl Acad Sci U S A, 120(50):e2314698120., (2023) https://www.pnas.org/doi/10.1073/pnas.2314698120
E. H. Choi, S. Suh, A. T. Foik, H. Leinonen, G. A. Newby, X. D. Gao, S, Banskota, T. Hoang , S. W. Du, Z. Dong, A. Raguram, S. Kohli, S. Blackshaw, D. C. Lyon, D. R. Liu, K. Palczewski “In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration” Nat Commun. 13(1):1830, (2022), https://doi.org/10.1038/s41467-022-29490-3
H. Leinonen, D. C Lyon, K. Palczewski, A. T. Foik “Visual System Hyperexcitability and Compromised V1 Receptive Field Properties in Early-Stage Retinitis Pigmentosa in Mice” eNeuro.9(3):ENEURO.0107-22.2022., (2022). https://www.eneuro.org/content/9/3/ENEURO.0107-22.2022
J.C. Frankowski, A. T. Foik, A. Tierno, J. R. Machhor, D. C. Lyon, R. F. Hunt “Traumatic brain injury to primary visual cortex produces long-lasting circuit dysfunction” Commun Biol (1):1297, (2021). https://www.nature.com/articles/s42003-021-02808-5
Retinal pigment epithelium (RPE) located at the back of the eye is essential for vision. It supports the photoreceptors, providing molecules required for their function. One of the main proteins produced by the RPE and indispensable for vision is the RPE65 enzyme, which is responsible for chemical signaling at the initial step of visual processing. De novo nonsense mutations in the Rpe65 gene underlie inherited genetic disorders of the eyes, resulting in blindness. To address this problem, we have harnessed the power of adenine base editors (ABEs) with Cas9 – single-guide RNA machinery to target the mutations in the Rpe65 gene for their repair. We delivered genes coding for ABEs and the Cas9 system subretinally via a lentiviral vector. Our therapeutic manipulation corrected the pathogenic mutation in a mouse model with up to 29% efficiency and with minimal formation of indel and off-target mutations. The ABE-treated mice displayed restored RPE65 expression and its activity in the visual cycle. Moreover, we have observed near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.
Authors:
dr Andrzej Foik, e-mail: afoik@ichf.edu.pl & dr Anna Posłuszny, e-mail: aposluszny@ichf.edu.pl
Pertinent published article:
Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing
Susie Suh, Elliot H. Choi, Henri Leinonen, Andrzej T. Foik, Gregory A. Newby, Wei-Hsi Yeh, Zhiqian Dong, Philip D. Kiser, David C. Lyon, David R. Liu & Krzysztof Palczewski, Nat Biomed Eng. 2021 Feb;5(2):169-178.
One of the methods for evoking plasticity in the visual system is repeated stimulation with appropriate visual stimuli. Repeated exposure to sensory stimuli can induce neuronal network changes in the cortical circuits and improve the perception of these stimuli in the primary visual cortex (V1). The aim of our studies was to investigate the effect of repetitive visual training on the magnitude of visual responses in the primary visual cortex and in the superior colliculus (SC), the subcortical structure of the extrageniculate visual pathway in rats. Our study showed that a three-hour, passive visual training with light flashes enhanced visual responses both at the cortical level and in the superior colliculus. The next part of our study focused on distinguishing which input projection is responsible for the observed training effect in the SC, especially whether the increase of collicular response depends on the enhancement in the V1. The SC receives information both from the retina and from layer 5 of the V1. The experiment with pharmacological blocking of V1 did not suppress training-related plasticity in the SC. These results for the first time identified the superior colliculus as a possible target for training strategies to improve the efficiency of the visual process; e.g., in the case of primary visual cortex injuries.
Author:
dr Katarzyna Kordecka, e-mail: kkordecka@ichf.edu.pl
Publication
Cortical Inactivation Does Not Block Response Enhancement in the Superior Colliculus
Katarzyna Kordecka, Andrzej T. Foik, Agnieszka Wierzbicka and Wioletta J. Waleszczyk
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18.12.2024 
