👓 Restoring Vision through OptogenEYEsis :
Unlocking the Potential of Microbial Opsins

---

Retinal degeneration stands as a major cause of blindness, characterized by the gradual death of retinal cells. It encompasses various conditions influenced by genetic, environmental, and lifestyle factors. In its advanced stages, retinal degeneration leads to severe visual impairment due to the loss of most light-sensitive cells. A promising recent advancement in therapy is the application of optogenetics to restore vision. This approach involves transforming surviving but non-light-sensitive retinal cells into artificial photoreceptors by introducing microbial opsins (light-sensitive proteins) [1]. Some microbial opsins are currently undergoing clinical trials for visual restoration [2], holding significant potential in the quest to combat retinal degeneration-related blindness.

However, microbial opsins despite having faster kinetics than their animal counterparts have a very low sensitivity to light and necessitating the use of high-intensity light that poses a risk of retinal damage of light that risks toxicity damage to the retina when used in optogenetic therapy [3]. Our project goals are to overcome these challenges, as microbial opsins can be engineered to improve their characteristics and sensitivity to light. This can be achieved by rational design and directed evolution or random mutagenesis. Based on this, we decided to take advantage of the Evolution.T7 system developed by the iGEM Evry Paris-Saclay 2021 team and use it to evolve opsins of interest. For this, we used Escherichia coli as a chassis for the heterologous expression of these microbial opsins, analyzed their properties by spectroscopic analysis [4] and designed screening systems for high throughput opsin activity detection.

Our Motivation and Project Significance

Our project OptogenEYEsis revolves around addressing a major healthcare challenge. Retinal degeneration is a leading cause of blindness. It is characterized by the gradual loss of retinal cells, impacting the lives of countless individuals. We are deeply motivated to find a solution to this problem through synthetic biology approaches.

The Problem and Its Importance

Retinal degeneration is a significant problem for several reasons. It affects a substantial portion of the population, straining healthcare systems and causing a significant reduction in the quality of life for those affected. Moreover, traditional treatment methods often fall short, and the risk of retinal damage associated with high-intensity light in optogenetic therapy poses an added complication. We recognize the importance of addressing this issue for the betterment of those affected by retinal degeneration. Our project has the potential to improve their lives, reduce the burden on healthcare systems, and offer a safer and more effective treatment.

Why We Chose This Project and Addressing It with Synthetic Biology

This year, our team felt a strong call to address a global health issue with a profound impact on individuals and society. The potential of synthetic biology and optogenetics to provide an innovative solution to combat retinal degeneration-related blindness inspired us. We saw the opportunity to be part of a community of like-minded researchers and contribute to the advancement of medical science. We are excited to embark on this journey and hope that our project will bring us closer to a breakthrough in the treatment of retinal degeneration, offering new hope to those who currently face the challenges of living with visual impairment.

The Challenge of Neurodegenerative Diseases and Vision Loss

Neurodegenerative diseases are chronic illnesses characterized by the progressive deterioration of neuronal cells. While Alzheimer's disease, stemming from the degradation of neuronal myelin, and Parkinson's disease, marked by the breakdown of dopamine-producing neurons, are the most well-known examples, there exist other neurodegenerative conditions that specifically target the light-sensitive neurons within the eyes. Notable among these ocular afflictions are age-related macular degeneration (AMD), retinitis pigmentosa, and Stargardt's disease. These diseases cause varying degrees of visual impairment, with some progressing to complete blindness [5].

How Human Vision Works

In humans, sight is made possible by the retina, a neural tissue at the back of our eyes made up of stratified layers of different neuronal cells. When our eyes are open, light penetrates the eye to the back of the retina and reaches the photoreceptor cells (Figure 1). These cells detect the light signal and transform it into a bioelectric signal (or nerve impulse), which is transmitted to the bipolar cells, which then further relay this signal to the ganglion cells. Finally, the ganglion cells transmit the signal via the nerve fibers (or axons) which converge on the optic nerve, to the visual cortex in the brain where the signals are interpreted as an image.

Photoreceptor cells can be divided into 2 categories: rods and cones. Rods allow vision in low light but do not detect colors. The cones, on the other hand, are responsible for high-light vision and enable us to see colors in normal lighting conditions. The conversion of the electromagnetic flow of light into a bioelectric signal (nerve impulse) within the photoreceptor cells is made possible by a photosensitive protein pigment: the opsin. This photosensitive protein has 7 transmembrane alpha helices and binds to a chromophore that changes conformation with light, creating the nerve signal (Figure 2).

In the previously mentioned neurodegenerative diseases, the photoreceptor cells are progressively degraded, eventually leading to vision loss. One example is retinitis pigmentosa, which causes progressive degradation of the rods and then the cones, leading to irreversible blindness [6,7].

Optogenetics to Restore Vision 🩺

There are currently various therapies that are being developed and tested to restore sight or at least limit its loss. These include cell-transplantation therapy, retinal implants and gene therapy techniques such as optogenetics. Optogenetics is a biological technique that combines the principles of bio-photonics (the study of living systems through a light source) and genetic engineering. This technique enables precise and specific control of cellular activity using light [8,9].

The optogenetic approach for vision restoration is based on the expression of a gene coding for a photosensitive protein into non-light sensitive neuronal cells that remain after photoreceptor cell loss such as ganglion cells. The aim is to enable these ganglion cells to emit the action potential needed to transmit the electrical message to the brain.

The opsins present in the human eye are animal opsins ("type 2 opsins"). These opsins are difficult to use in optogenetics because of the signaling cascade required for them to function. Thus, inserting animal opsins into ganglion cells does not guarantee their effectiveness in terms of therapy.

🦠 The Potential of Microbial Opsins in Optogenetics

To overcome the difficulties of animal opsins, optogenetics currently uses microbial opsins instead ("type 1 opsins"). These microbial opsins are found in microorganisms such as bacteria, archaea and microalgae, which use phototrophy to obtain energy, particularly in bacterial photosynthesis, or phototaxis (movement in response to light) [10].

The microbial opsins present indeed several advantages. Firstly, because they are smaller than animal opsins, they can be more easily genetically manipulated and inserted into vectors. In addition, using microbial opsins eliminates the chain reaction necessary for animal opsins to function, because microbial opsins function directly as a channel, without any signaling cascade.

The first microbial opsin was discovered in 1971 by scientists Dieter Oesterhelt and Walther Stoeckenius, in the archaea Halobacterium salinarum [11]. Others have since been discovered since and they are classified in four main families (Figure 4): halorhodopsins [12], channelrhodopsins [13,14], bacteriorhodopsins [15] and sensory rhodopsins [16], each with their own specific function.

Currently, optogenetics mainly uses channelrhodopsins, because these opsins are more stable in cases of retinal degeneration. Studies using channelrhodopsins have shown that inserting them into ganglion cells restored a light response in blind mice [17]. Studies have also been carried out on the retina of non-homosapiens mammals to examine the possibility of restoring light response in patients suffering from degenerative retinal diseases [18]. Using a modified version of channelrhodopsin, called 'CatCh', and a specific promoter derived from the human SNCG gene, researchers succeeded in obtaining a high expression of CatCh in retinal ganglion cells, as well as a safer light intensity [18]. ChrimsonR, a channelrhodopsin derived from Chlamydomonas noctigama, represents another noteworthy addition to the channelrhodopsin family [19]. Besides the aforementioned opsins, optogenetics has witnessed the utilization of various other microbial opsins, each with its unique characteristics. Halorhodopsins such as Jaws and NpHr, for instance, offer an alternative way of controlling neural activity by enabling chloride ion transport. Bacteriorhodopsin, which was one of the first microbial opsins to be discovered, has also been explored for its light-driven proton pump properties. Sensory Rhodopsin II, as NpSRII found in archaea, contributes to signal transduction processes and has been a subject of interest in the field. These diverse microbial opsins open up a spectrum of possibilities for tailored optogenetic applications and continue to be a main point of research and development in pursuing innovative solutions for retinal therapy and vision restoration.

⛔ Limits of Microbial Opsins

Although microbial opsins have great potential in optogenetics [10,20], their application to vision restoration is currently hampered by several limitations

The first major problem of microbial opsins is their reduced spectrum for light sensitivity. Primarily focused on blue light, these opsins have no sensitivity to red light [1]. This is all the more worrying given that exposure to high-intensity blue light is harmful to the human eye. Prolonged or intense exposure to this type of light can lead to eye damage, posing a major problem for any therapy or application based on the use of microbial opsins.

In addition, another inherent limitation of these opsins is their low sensitivity to light. To induce an electrical response characteristic of visual information strong enough to be transmitted to other nerve cells, opsins require a higher light intensity than would be ideal for therapeutic use. This challenge is particularly relevant in the context of optogenetic vision restoration for conditions like retinitis pigmentosa, where patients experience severe visual impairment due to the loss of photoreceptors. The optogenetic approach described in the study indicates that the use of light-stimulating goggles and optogenetic vectors, such as ChrimsonR, can help partially restore vision in these individuals [2]. However, the fact that the patient reported 'vertical vibrations' upon perceiving objects under stimulation suggests that there might still be a need to optimize the sensitivity to light for enhanced and more natural visual perception.

Finally, there is a risk of immunogenicity with this type of opsin that is not to be neglected [21].

These limitations call for further research and development to optimize microbial opsins for use in retinal therapies.

OptogenEYEsis🧬

---

Directed Evolution to Improve Microbial Opsins

To address these issues, our project OptogenEYEsis aims to use synthetic biology to optimize existing microbial opsins already used in gene therapy. We studied and evolved these opsins so that they have an improved light spectrum and greater sensitivity to low light levels. To achieve this, we are using the principle of directed evolution, more specifically with the help of the Evolution.T7 tool developed by the iGEM Evry Paris-Saclay team in 2021. This innovative tool allows mutations to be introduced into target genes using base deaminases, providing a precise and controlled means of modifying and enhancing opsin functionality. To demonstrate the effectiveness of this tool, the project will follow 3 stages: discovery, screening and selection of opsins, according to their absorbance spectrum and sensitivity.

The ultimate aim is to express these microbial opsins and introduce them effectively into retinal cells, first in cell lines, then in primate eyes and finally in human eyes.

🔎 The Search for Improved Opsins in Nature

For searching improved opsins in nature, we explored the omics data of the Tara Oceans project using the Ocean Gene Atlas (OGA) database [22,23], freely accessible on the Internet. This database lists the genomic, transcriptomic and protein sequences of microorganisms from all oceans, derived from the Tara Oceans campaign. As they explored the ocean depths, Tara Oceans researchers identified microorganisms living at different depths, with varying light intensities. Their results revealed that, although microorganisms near the water's surface are more sensitive to the light spectrum, this sensitivity decreases with depth. Their Ocean Gene Atlas database shows organisms adapted to low light levels. Our aim is to exploit this information to find optimal opsins for optogenetics.

Development of Screening Tools for Opsins 🔬

We designed a screening tool for these proteins in order to select the best opsins. This genetic tool uses LEDs to illuminate the bacteria at defined wavelengths and intensities, in order to select the most sensitive opsins (i.e. those which can be activated at low light intensity) and which react at the wavelengths of interest to us (in the human visible range).

🤖 Automating the Screening Process

Automation is a key part of our project. By automating the screening process, we can significantly accelerate our research and improve the efficiency of identifying superior opsins.

To that end, we built a multifunction AI driven lab automation platform, based on digital microfluidics (Figure 6). Digital microfluidics (DMF) is an innovative subfield of microfluidics that focuses on manipulating discrete microdroplets on a substrate using electrical fields. Unlike conventional microfluidics, which utilizes channels and pumps to direct fluid flow, DMF uses electrode arrays to individually control each droplet. This offers a high level of precision and flexibility, making it ideal for a wide range of applications, from biochemical assays to chemical synthesis.

The core principle behind DMF is the use of electrowetting-on-dielectric (EWOD) to control droplet movement. By applying a voltage to a specific electrode, the surface tension of the droplet above it can be altered, causing it to move or merge with other droplets. This allows for intricate droplet manipulations, such as mixing, splitting, and dispensing, all without the need for any moving parts. The droplets can be controlled in a programmable manner, enabling complex sequences of operations to be automated with high repeatability.

Integrating AI with DMF unlocks a multitude of possibilities. An AI-driven lab automation platform can optimize droplet manipulations in real-time, adapting to the specific requirements of a given experiment. For instance, in the context of screening opsins, the platform could automatically adjust conditions based on feedback from previous assays, making the process more efficient and accurate. Moreover, the combination of AI and DMF can facilitate high-throughput screening, leading to faster discovery and validation of superior opsins.

Closing statement 📜

---

In conclusion, our project, OptogenEYEsis, represents a dedicated effort to unlock the potential of microbial opsins in optogenetics, offering a promising solution to restore vision in individuals with neurodegenerative diseases. Through directed evolution, parallel exploration of nature's offerings, advanced screening tools, and innovative hardware devices, we aim to push the boundaries of this field and make strides toward safer and more effective optogenetic therapy for vision restoration.

References

---

• [1] Sakai D, Tomita H, Maeda A. Optogenetic therapy for visual restoration. International Journal of Molecular Sciences (2022) 23: 15041.
• [2] Sahel J-A, Boulanger-Scemama E, Pagot C, Arleo A, Galluppi F, Martel JN, Esposti SD, Delaux A, de Saint Aubert J-B, de Montleau C, Gutman E, Audo I, Duebel J, Picaud S, Dalkara D, Blouin L, Taiel M, Roska B. Partial recovery of visual function in a blind patient after optogenetic therapy. Nature Medicine (2021) 27: 1223–1229.
• [3] Berry MH, Holt A, Salari A, Veit J, Visel M, Levitz J, Aghi K, Gaub BM, Sivyer B, Flannery JG, Isacoff EY. Restoration of high-sensitivity and adapting vision with a cone opsin. Nature Communications (2019) 10: 1221.
• [4] Maresca JA, Keffer JL, Miller KJ. Biochemical analysis of microbial rhodopsins. Current Protocols in Microbiology (2016) 41: 1F.4.1-1F.4.18.
• [5] Thomas CJ, Mirza RG, Gill MK. Age-related macular degeneration. The Medical Clinics of North America (2021) 105: 473–491.
• [6] Narayan DS, Wood JPM, Chidlow G, Casson RJ. A review of the mechanisms of cone degeneration in retinitis pigmentosa. Acta Ophthalmologica (2016) 94: 748–754.
• [7] Song D-J, Bao X-L, Fan B, Li G-Y. Mechanism of cone degeneration in retinitis pigmentosa. Cellular and Molecular Neurobiology (2023) 43: 1037–1048.
• [8] Häusser M. Optogenetics - the might of light. The New England Journal of Medicine (2021) 385: 1623–1626.
• [9] Emiliani V, Entcheva E, Hedrich R, Hegemann P, Konrad KR, Lüscher C, Mahn M, Pan Z-H, Sims RR, Vierock J, Yizhar O. Optogenetics for light control of biological systems. Nature Reviews Methods Primers (2022) 2: 55.
• [10] Zhang F, Vierock J, Yizhar O, Fenno LE, Tsunoda S, Kianianmomeni A, Prigge M, Berndt A, Cushman J, Polle J, Magnuson J, Hegemann P, Deisseroth K. The microbial opsin family of optogenetic tools. Cell (2011) 147: 1446–1457.
• [11] Oesterhelt D, Stoeckenius W. Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nature New Biology (1971) 233: 149–152.
• [12] Engelhard C, Chizhov I, Siebert F, Engelhard M. Microbial halorhodopsins: light-driven chloride pumps. Chemical Reviews (2018) 118: 10629–10645.
• [13] Deisseroth K, Hegemann P. The form and function of channelrhodopsin. Science (New York, N.Y.) (2017) 357: eaan5544.
• [14] Govorunova EG, Sineshchekov OA, Spudich JL. Emerging diversity of channelrhodopsins and their structure-function relationships. Frontiers in Cellular Neuroscience (2021) 15: 800313.
• [15] Li Y-T, Tian Y, Tian H, Tu T, Gou G-Y, Wang Q, Qiao Y-C, Yang Y, Ren T-L. A review on bacteriorhodopsin-based bioelectronic devices. Sensors (Basel, Switzerland) (2018) 18: 1368.
• [16] Hoff WD, Jung KH, Spudich JL. Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annual Review of Biophysics and Biomolecular Structure (1997) 26: 223–258.
• [17] Busskamp V, Duebel J, Balya D, Fradot M, Viney TJ, Siegert S, Groner AC, Cabuy E, Forster V, Seeliger M, Biel M, Humphries P, Paques M, Mohand-Said S, Trono D, Deisseroth K, Sahel JA, Picaud S, Roska B. Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa. Science (New York, N.Y.) (2010) 329: 413–417.
• [18] Chaffiol A, Caplette R, Jaillard C, Brazhnikova E, Desrosiers M, Dubus E, Duhamel L, Macé E, Marre O, Benoit P, Hantraye P, Bemelmans A-P, Bamberg E, Duebel J, Sahel J-A, Picaud S, Dalkara D. A new promoter allows optogenetic vision restoration with enhanced sensitivity in macaque retina. Molecular Therapy: The Journal of the American Society of Gene Therapy (2017) 25: 2546–2560.
• [19] GenSight Biologics. A phase 1/2a, open-label, non-randomized, dose-escalation study to evaluate the safety and tolerability of GS030 in subjects with retinitis pigmentosa. Clinicaltrials.gov (2022).
• [20] Deisseroth K. Optogenetics: 10 years of microbial opsins in neuroscience. Nature Neuroscience (2015) 18: 1213–1225.
• [21] Maimon BE, Diaz M, Revol ECM, Schneider AM, Leaker B, Varela CE, Srinivasan S, Weber MB, Herr HM. Optogenetic peripheral nerve immunogenicity. Scientific Reports (2018) 8: 14076.
• [22] Villar E, Vannier T, Vernette C, Lescot M, Cuenca M, Alexandre A, Bachelerie P, Rosnet T, Pelletier E, Sunagawa S, Hingamp P. The Ocean Gene Atlas: exploring the biogeography of plankton genes online. Nucleic Acids Research (2018) 46: W289–W295.
• [23] Vernette C, Lecubin J, Sánchez P, Tara Oceans Coordinators, Sunagawa S, Delmont TO, Acinas SG, Pelletier E, Hingamp P, Lescot M. The Ocean Gene Atlas v2.0: online exploration of the biogeography and phylogeny of plankton genes. Nucleic Acids Research (2022) 50: W516–W526.