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Optical imaging of retinal function for gene and cell therapies

Periodic Reporting for period 1 - OPTORETINA (Optical imaging of retinal function for gene and cell therapies)

Reporting period: 2021-04-01 to 2022-09-30

Evaluating single cell function in the living eye is a major challenge for biotherapies. Gene and cell therapies have recently begun to emerge in the clinic – with the exciting potential to save or even restore sight in people with very low vision. However a major obstacle to their advancement is a lack of adequate tools to measure short-term benefit of the therapy in the difficult environment of the living eye. The Optoretina project proposes to fill that gap by developing novel imaging tools capable of resolving individual cells in patients retinas and of non invasively probing the function of these cells in the living eye. We’ve recently achieved non invasive all optical measurement of cell activity by developing a dynamic live imaging technique based on the movements of intracellular mitochondria and vesicles inside cell cytoplasm. We’ve been using this dynamic technique in retinal organoids which are stem cell derived human tissues used in therapy validation and we’ve been able to show that we can distinguish different cell types and behaviors in a completely label free way based on this dynamic profile alone. The main objective of Optoretina is to bring functional cell mapping into the clinic. We have pioneered both cell-scale retinal imaging tools and methods of measuring an optical response to a visible stimulus. If we can now adapt methods of measuring function to living eyes, we would produce a non invasive map of cell activity. This new technology should lift the major imaging obstacles to gene and cell therapies and boost them into the clinic to potentially save patient’s sight.
Work has been carried out in three main areas:
1) Live imaging in retinal organoids. In vitro functional dynamic full field optical coherence tomography (D-FFOCT). A D-FFOCT module has been designed that can be interfaced to a commercial microscope (patent filed 04/22) and allows long term non invasive 3D imaging of retinal organoids and cell cultures under controlled culture conditions. Automation of image acquisition in multiwell plates has been implemented computationally, and methods of reproducibly targeting the exact same imaging plane in different organoids in the plate are currently being explored. Previous limitations on imaging at shallow depths have been overcome with a new interferometer design (patent field 09/22). We have also advanced in automating cell classification in organoids based on learning methods. A new excitation source has been integrated into the D-FFOCT microscope setup to allow photostimulation of the sample. Several tests on light sensitive samples have allowed us to refine the best optical design for accurate photostimulation and detection. The appropriate stimulation protocol will now be devised to detect cell activity changes in response to incident light. Working closely with biologists, we have several batches of both naturally and optogenetically light sensitive organoids currently under test for this purpose.
2) Imaging in patients and healthy controls, for phenotyping and therapy follow-up. In vivo imaging with adaptive optics technology. Projects have been initiated in three main directions: i) measurement of subjective perception in patients was carried out on an adaptive optics flood illumination ophthalmoscope for the first time, with first promising results obtained on adaptive optics-assisted microperimetry ii) imaging of retinal structure in patients with retinal disease undergoing gene therapy has been carried out with AOSLO-OCT in a multimodal protocol allowing comparison with clinical imaging. The imaging results in these patients have been invaluable in creating links with key clinicians who are now convinced of the merit of our methods and their motivated patients who will be included in the Optoretina study for the functional imaging stage. We also have a better idea of the potential structural biomarkers to follow during therapy. Iii) technology upgrade to allow subjective and objective functional imaging on two adaptive optics devices: flood illuminated and scanning laser ophthalmoscopes. These adaptations of devices developed in collaboration with industrial partners now puts us in a ready position to begin exploring photostimulation protocols in healthy volunteers and patients in the next stage of the project.
1) In vivo functional OCT technology: In vivo functional imaging with optical coherence tomography. Since submission of the Optoretina project, others in the community have shown advances in this area which encouraged us to explore multiple solutions. Two projects have hence been launched in parallel to ensure the greatest chances of success in this aim. Initial tests on the existing FFOCT retinal imaging device on patients showed a promising functional imaging optoretinography (ORG) response in the standard OCT channel, but also indicated that a more accurate tracking would be necessary to obtain results with the FFOCT channel. In consequence, we have now built an FFOCT device with adaptive optics for high resolution and high sensitivity measurements. This novel AO-FFOCT device will be used for the first optoretinography tests in the coming year. Meanwhile, we are also exploring an alternative method to record ORG signal using a holographic OCT approach. Once the best OCT method for ORG measurement is evaluated, we will tackle the final challenge of detecting dynamic OCT signals in vivo, which will require the ultimate performance in terms of sensitivity and stability.
We have filed patents for two innovations in dynamic OCT imaging, for both the DFFOCT module, and for the novel method for imaging at shallow depth without artefact.

We now have some of the most extensive data worldwide on adaptive optics imaging of gene therapy trials, thanks to the links established with key clinicians since the beginning of the project.

We have demonstrated adaptive optics assisted microperimetry in patients with retinal degeneration with a flood illumination system for the first time.

We now have two adaptive optics devices useable in the clinic that are equipped with photostimulation capacity, either at or beyond state of the art of other teams.

We have developed or are developing three novel OCT methods: FFOCT applied to clinical retinal imaging, adaptive optics FFOCT for high sensitivity ORG, and holographic OCT for ORG.

By the end of the project, we expect to achieve i) recording of a photostimulation induced signal in photosensitive retinal organoids ii) recording of retinal function with adaptive optics and OCT devices iii) imaging of dynamic behavior in vivo with OCT based methods. With the help of these new techniques, we hope to detect therapeutic effect in gene therapy trials ongoing in our research and clinical centers and define new potential clinical endpoints for such trials which can be detected in the short term thanks to the high resolution and sensitivity of our measurement methods.

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