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.
Dr. Karnowski developed expertise in fiber imaging probes during his postdoctoral research at the University of Western Australia. He continued this line of research through the NAWA Polish Returns project, which was integrated into the broader ICTER research initiatives.
Our team proposed an innovative design for imaging probes that strategically combines the advantages of previous probe technologies. These GRIN-Ball-lens probes (GBLP) demonstrate superior performance, particularly in terms of working distance, when compared to traditional GRIN-fiber probes (GFP) and ball-lens probes (BLP). A key breakthrough of this design is the ability to fabricate probes that are at least twice smaller in diameter without compromising optical performance.
Through meticulous research and development, we have cultivated expertise in probe fabrication that offers unprecedented flexibility. This includes creating probes with varying ball lens sizes and exploring different ball lens materials with distinct refractive indices. This versatility in probe design opens up a wide range of potential applications across multiple research domains.
The adaptability of our GRIN-Ball-lens probes suggests promising future investigations in fields such as:
Biomedical imaging
Microscopy
Endoscopic technologies
Minimally invasive diagnostic techniques
We are particularly excited about exploring these potential applications in the near future, anticipating that our novel probe design could significantly advance imaging capabilities in various scientific and medical contexts.
Text: Karol Karnowski, PhD, Acting IDoc Leader
Project team:
Karol Karnowski
Related funding: NAWA – Polish Returns, NCN – Miniatura.
References:
K. Karnowski, G. Untracht, M. Hackmann, O. Cetinkaya, D Sampson, “Superior Imaging Performance of All-Fiber, Two-Focusing-Element Microendoscopes,” IEEE Photonics Journal, 14 (5), 1-10 (2022)
G. Untracht, K. Karnowski D. D. Sampson, “Imaging the small with the small: Prospects for photonics in “micro-endomicroscopy for minimally invasive cellular-resolution bioimaging,” APL Photonics, 6 (6), pp. 060901, (2021)
M. J. Hackmann, A. Cairncross, J. G. Elliot, S. Mulrennan, K. Nilsen, B. R. Thompson, Q. Li, K. Karnowski, D. D. Sampson, R. A. McLaughlin, B. Cense, A. L. James, P. B. Noble, “Quantification of smooth muscle in human airways by polarization-sensitive optical coherence tomography requires correction for perichondrium,” American Journal of Physiology-Lung Cellular and Molecular Physiology, 326:3, L393-L408 (2024)
The anterior chamber of the eye plays a critical role in our visual mechanism, serving dual purposes of protection and optical functionality. This vital anatomical region acts not only as a protective barrier but also as a crucial optical pathway that focuses light onto the retina, enabling our visual perception of the world.
Our comprehensive research project is dedicated to developing advanced imaging methodologies for examining the structure and function of the anterior eye chamber, leveraging cutting-edge Fourier domain Optical Coherence Tomography (OCT) techniques. Our approach transcends traditional imaging methods based solely on light scattering, exploring innovative systems capable of capturing complex physiological characteristics including polarization and mechanical properties of ocular tissues.
The primary objective of our research is to develop diagnostic technologies with significant potential for early detection of anterior segment disorders and identifying retinal conditions that manifest through anterior segment changes, such as glaucoma. A notable sub-project involved designing a clinical system for multi-spot measurements of corneal deformation induced by air pulses. Through collaborative research with an ophthalmology clinic in Bydgoszcz, we conducted an extensive study involving nearly 100 patients, utilizing detailed quantitative analyses to identify and characterize corneal deformation parameters that could serve as potential biomarkers for keratoconus.
Text: Karol Karnowski, PhD, Acting IDoc Leader
Project team:
Karol Karnowski
Jadwiga Milkiewicz
Piotr Nalewajko
Related financing: IMCUSTOMEYE, IRAP (MAB) by FNP
References:
1. D. Alonso-Caneiro, K. Karnowski, B. Kaluzny, A. Kowalczyk, and M. Wojtkowski, “Assessment of corneal dynamics with high-speed swept source Optical Coherence Tomography combined with an air puff system”, Optics Express, Vol. 19, Issue 15, pp. 14188-14199 (2011)
2. S. Marcos, C. Dorronsoro, K. Karnowski, M. Wojtkowski, „Corneal biomechanics From Theory to Practice: OCT with air puff stimulus”, Kugler Publications 2016, edited by C.J. Roberts, J. Liu
3. K. Karnowski, E. Maczynska, M. Nowakowski, B. Kaluzny, I. Grulkowski, M. Wojtkowski, “Impact of diurnal IOP variations on the dynamic corneal hysteresis measured with air-puff swept-source OCT”, Photonics Letters of Poland, (2018)
4. E. Maczynska, K. Karnowski, K. Szulzycki, M. Malinowska, H. Dolezyczek, A. Cichanski, M. Wojtkowski, B. Kaluzny and I. Grulkowski, “Assessment of the influence of viscoelasticity of cornea in animal ex vivo model using air-puff optical coherence tomography and corneal hysteresis”, J Biophotonics, 2019; 12:e201800154 (2019)
5. A. Curatolo, J. S. Birkenfeld, E. Martinez-Enriquez, J. A. Germann, G. Muralidharan, J. Palací, D. Pascual, A. Eliasy, A. Abass, J. Solarski, K. Karnowski, Maciej Wojtkowski, Ahmed Elsheikh, and Susana Marcos, “Multi-meridian corneal imaging of air-puff induced deformation for improved detection of biomechanical abnormalities,” Biomed. Opt. Express 11, 6337-6355 (2020).
Two-photon excitation fluorescence (TPEF) is emerging as a powerful imaging technique with superior penetration power in scattering media, allowing for functional imaging of biological tissues at a subcellular level. TPEF is commonly used in cancer diagnostics, as it enables the direct observation of metabolism within living cells. The technique is now widely used in various medical fields, including ophthalmology. The eye is a complex and delicate organ with multiple layers of different cell types and tissues. Although this structure is ideal for visual perception, it generates aberrations in TPEF eye imaging. However, adaptive optics can now compensate for these aberrations, allowing for improved imaging of the eyes of animal models for human diseases. The eye is naturally built to filter out harmful wavelengths, but these wavelengths can be mimicked and thereby utilized in diagnostics via two-photon (2Ph) excitation. Recent advances in laser-source manufacturing have made it possible to minimize the exposure of in vivo measurements within safety, while achieving sufficient signals to detect functional images, making TPEF a viable option for human application. Recent advances in wavefront-distortion correction in animal models and in the safety of the use of TPEF on human subjects, make TPEF a potentially powerful tool for ophthalmological diagnostics.
Two-photon imaging. (A) Jablonski diagram of fluorophore excitation by single, (B) two-photon, and (C) secondary-harmonic generation (SHG). (D) comparison of one and two-photon (yellow arrow) excitation profiles. (E) focal plane range by single, (F) two-photon, and (G) SHG. (H) FLIM histogram of photon counts versus arrival time after the laser pulse. (I) two-photon excited fluorescence and SHG are isotopically emitted. (J) experimental TPEF-SLO setup, and its (K) imaging processing. (L) experimental SHG setup. (M) relative TPEF intensity as a function of pulse repetition frequency. (N) anatomy of the human eye.
Team:
Dr. Humberto Fernandes
Dr. Vineeta Kaushik
Luca Gessa
Nelam Kumar
Dr. Michał Dąbrowski (former POB member)
Reference:
“Two-photon excitation fluorescence in ophthalmology: safety and improved imaging for functional diagnostics” (2024) V Kaushik, M Dąbrowski, L Gessa, N Kumar, H Fernandes, Frontiers in Medicine 10, 1293640.
Diabetic retinopathy (DR) is a severe disease with a growing number of afflicted patients, which places a heavy burden on society, both socially and financially. While there are treatments available, they are not always effective and are usually administered when the disease is already at a developed stage with visible clinical manifestation. However, homeostasis at a molecular level is disrupted before visible signs of the disease are evident. There is evidence that early detection and prompt disease control are effective in preventing or slowing DR progression. Thus, there has been a constant search for effective biomarkers that could signal the onset of DR. As a possible new biomarker, we focus part of our research on retinol binding protein 3 (RBP3). We argue that it displays unique features that make it a very good biomarker for non-invasive, early-stage DR detection. Linking chemistry to biological function and focusing on new developments in eye imaging and two-photon technology test a new potential diagnostic tool that would allow rapid and effective quantification of RBP3 in the retina. Moreover, this tool would also be useful in the future to monitor therapeutic effectiveness if levels of RBP3 are elevated by DR treatments.
Homeostasis disruption upon DR progression, with decreased levels of RPB3 in the IPM with advances in the severity of DR.
Team:
Dr. Humberto Fernandes
Dr. Vineeta Kaushik
Luca Gessa
Nelam Kumar
Reference:
“Towards a new biomarker for diabetic retinopathy: exploring RBP3 structure and retinoids binding for functional imaging of eyes in vivo” (2023) V Kaushik, L Gessa, N Kumar, H Fernandes, International Journal of Molecular Sciences 24 (5), 4408.
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
Manage Cookie Consent
We use cookies to optimize our website and our service.
Functional
Always active
The technical storage or access is strictly necessary for the legitimate purpose of enabling the use of a specific service explicitly requested by the subscriber or user, or for the sole purpose of carrying out the transmission of a communication over an electronic communications network.
Preferences
The technical storage or access is necessary for the legitimate purpose of storing preferences that are not requested by the subscriber or user.
Statistics
The technical storage or access that is used exclusively for statistical purposes.The technical storage or access that is used exclusively for anonymous statistical purposes. Without a subpoena, voluntary compliance on the part of your Internet Service Provider, or additional records from a third party, information stored or retrieved for this purpose alone cannot usually be used to identify you.
Marketing
The technical storage or access is required to create user profiles to send advertising, or to track the user on a website or across several websites for similar marketing purposes.
18.12.2024 
