Periodic Reporting for period 2 - OPTORETINA (Optical imaging of retinal function for gene and cell therapies)
Reporting period: 2022-10-01 to 2024-03-31
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). A new excitation source has been integrated into the D-FFOCT microscope setup to allow photostimulation of the sample. Tests on light sensitive samples are allowing us to refine the best optical design for accurate photostimulation and detection. Longitudinal imaging on retinal organoids generated from human iPS cells from patients with degenerative disease has revealed modifications in the photoreceptor layer, thus validating the disease model.
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. iii) technology upgrade to allow subjective and objective functional imaging on two adaptive optics devices: line scan adaptive optics and scanning laser ophthalmoscopes. Optoretinography (ORG) signals have now been recorded in a large cohort (n=40) of healthy subjects of various ages, thus establishing baseline data which will be compared with subjects with pathologies. We have explored the influence of age, eccentricity and source coherence length on the ORG signals detected.
3) 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. Three 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 ORG tests in the coming year. Also, the standard OCT channel of our FFOCT device is being upgraded with a new design to allow routine ORG recordings. Meanwhile, we are also exploring an alternative method to record ORG signal using a high-speed holographic OCT approach.
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). A new excitation source has been integrated into the D-FFOCT microscope setup to allow photostimulation of the sample. Tests on light sensitive samples are allowing us to refine the best optical design for accurate photostimulation and detection. Longitudinal imaging on retinal organoids generated from human iPS cells from patients with degenerative disease has revealed modifications in the photoreceptor layer, thus validating the disease model.
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. iii) technology upgrade to allow subjective and objective functional imaging on two adaptive optics devices: line scan adaptive optics and scanning laser ophthalmoscopes. Optoretinography (ORG) signals have now been recorded in a large cohort (n=40) of healthy subjects of various ages, thus establishing baseline data which will be compared with subjects with pathologies. We have explored the influence of age, eccentricity and source coherence length on the ORG signals detected.
3) 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. Three 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 ORG tests in the coming year. Also, the standard OCT channel of our FFOCT device is being upgraded with a new design to allow routine ORG recordings. Meanwhile, we are also exploring an alternative method to record ORG signal using a high-speed holographic OCT approach.
We have filed patents for three innovations in dynamic OCT imaging, for the DFFOCT module, for the novel method for imaging at shallow depth without artefact, and recently for a new method of image acquisition that allows smoother operation.
We have also filed a patent on an improved clinical FFOCT retinal imaging approach, inspired by the first use of the device in patients in the course of this project, and are currently filing a new patent regarding adaptive optics technology.
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, with which we have acquired extensive baseline data in healthy volunteers and are now beginning patient imaging.
We have developed or are developing three novel OCT approaches to functional imaging: FFOCT applied to clinical retinal imaging with simultaneous ORG recording provided by SD-OCT, 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 samples in the lab ii) recording of retinal function with adaptive optics and OCT devices in patients in comparison to healthy controls 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.
We have also filed a patent on an improved clinical FFOCT retinal imaging approach, inspired by the first use of the device in patients in the course of this project, and are currently filing a new patent regarding adaptive optics technology.
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, with which we have acquired extensive baseline data in healthy volunteers and are now beginning patient imaging.
We have developed or are developing three novel OCT approaches to functional imaging: FFOCT applied to clinical retinal imaging with simultaneous ORG recording provided by SD-OCT, 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 samples in the lab ii) recording of retinal function with adaptive optics and OCT devices in patients in comparison to healthy controls 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.