Noninvasive gene delivery to foveal cones for vision restoration

Khabou, Hanen; Garita-Hernández, Marcela; Chaffiol, Antoine; Reichman, Sacha; Jaillard, C.; Brazhnikova, Elena; Bertin, Stéphane; Forster, Valérie; Desrosiers, Mélissa; Winckler, Céline; Goureau, Olivier; Picaud, Serge; Duebel, Jens; Sahel, José‐Alain; Dalkara, Deniz

doi:10.1172/jci.insight.96029

Cited by 100 publications

(109 citation statements)

References 52 publications

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“…The use of AAV in human ROs is relatively limited at the moment, but studies have reported varying efficiency of transduction, which could be attributed to age of treatment, time of harvesting, vector tropisms, viral titre, the promotor used and sensitivity of detection of the transgene, e.g. antibody versus intrinsic fluorescence of a reporter (Gonzalez-Cordero et al, 2018, Tornabene et al, 2019, Quinn et al, 2019, Khabou et al, 2018. Here we demonstrate highly efficient transduction using an AAV2/5 vector with RP2 under the control of a CAG promoter.…”

Section: Discussionmentioning

confidence: 82%

Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

Lane¹,

Jovanović²,

Shortall

et al. 2020

Preprint

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Mutations in RP2 lead to a severe form of X-linked retinitis pigmentosa (XLRP). RP2 functions as a GTPase activating protein (GAP) for the small GTPase ARL3, which is essential for cilia function and for photoreceptor development and maintenance. The mechanisms of RP2 associated retinal degeneration in humans are poorly understood, and genetically engineered animal models of RP2 XLRP present with differing retinal phenotypes and slow degeneration suggesting potential species differences. Here, we developed CRISPR gene edited isogenic RP2 knock-out (RP2 KO) induced pluripotent stem cells (iPSC) and RP2 patient derived iPSC to produce 3D retinal organoids as a human retinal disease model. The isogenic RP2 KO retinal organoids and two unrelated RP2 patient iPSC lines produced retinal organoids with a defined photoreceptor cell layer (ONL) containing rod and cone photoreceptors. Strikingly the RP2 KO and RP2 patient derived organoids showed a thinning of the ONL by 180 days (D180) of culture, which was associated with a spike in cell death in the ONL at D150 of differentiation.RNA sequencing confirmed induction of cell death pathways in the RP2 null organoids at this stage. Photoreceptor cell death and ONL thinning was attributed to cells undergoing terminal rod cell differentiation characterized by reduced RHO expression and fewer rhodopsin immunoreactive photoreceptors. Gene augmentation with a human RP2 transgene in an AAV2/5 vector efficiently transduced RP2 KO organoids and led to high levels of RP2 expression in both rods and cones. Importantly, the viral transduction significantly increased ONL thickness and restored rhodopsin expression, suggesting rescue of the RP2 degeneration phenotype. These data show that 3D retinal organoids can be used to model molecular defects associated with inherited retinal disease, photoreceptor cell death and also to test potential therapies targeted to prevent photoreceptor degeneration.

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Section: Discussionmentioning

confidence: 82%

Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

Lane¹,

Jovanović²,

Shortall

et al. 2020

Preprint

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“…However, Wiley et al upon exploring AAV serotype tropism observed differences in the transduction efficiency between human and porcine retinal explant models . Even within human retina, considerable difference in the AAV‐mediated viral transduction was observed between foveal cones and peripheral cones in a study aiming to transduce foveal cone photoreceptors . Despite these differences, AAV‐mediated viral transduction was achieved in retinal tissues obtained from live donors post‐surgery …”

Section: Successful Applications Of Human Retinal Explants In Culturementioning

confidence: 99%

“…Less useful and often unhealthy human retinal tissues can also be obtained from live donors undergoing retinectomy or retinotomy procedures as part of a retinal surgical procedure. These tissues are usually discarded as clinical waste or used for exploratory studies to understand retinal pathology, however, with the combined effort of the surgeon and the researcher, these dissected tissues can be retained for in‐vitro culture to study the pathophysiology of human ocular diseases . Depending on the source of the tissue, the time from death to enucleation can vary from a few minutes to a few hours or days, which influences the retinal viability.…”

Section: Human Retinal Explant Culture Conditionsmentioning

confidence: 99%

“…These tissues are usually discarded as clinical waste or used for exploratory studies to understand retinal pathology, however, with the combined effort of the surgeon and the researcher, these dissected tissues can be retained for in-vitro culture to study the pathophysiology of human ocular diseases. 105 Depending on the source of the tissue, the time from death to enucleation can vary from a few minutes to a few hours or days, which influences the retinal viability. Kim et al has reported that enucleation within 6 hours post-mortem exhibited improved retinal survival in culture as opposed to enucleation 8 to 24 hours post-mortem.…”

Section: Source Of the Tissuementioning

confidence: 99%

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Retinal explant culture: A platform to investigate human neuro‐retina

Murali

Ramlogan‐Steel

Andrzejewski

et al. 2018

Clinical Exper Ophthalmology

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Funding information Layton Vision FoundationThe retina is the tissue responsible for light detection, in which retinal neurons convert light energy into electrical signals to be transported towards the visual cortex. Damage of retinal neurons leads to neuronal cell death and retinal pathologies, compromising visual acuity and eventually leading to irreversible blindness. Models of retinal neurodegeneration include 2D systems like cell lines, disassociated cultures and co-cultures, and 3D models like organoids, organotypic retinal cultures and animal models. Of these, ex vivo human retinal cultures are arguably the most suitable models for translational research as they retain complex intercellular interactions of the retina and precisely mimic in-situ responses. In this review, we summarize the distinguishing features of the human retina which are important to preserve in experimental culture, the historical development of human retinal culture systems, the factors affecting ex vivo human retinal culture and the applications and challenges associated with current methods of human retinal explant culture. K E Y W O R D S explant, human, neurodegeneration, post-mortem, retina

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“…Cone photoreceptors are responsible for high acuity daylight vision in humans, and are therefore the preferred choice for transplantation. To render these cones light sensitive, we used the hyperpolarizing chloride pump Jaws, based on its enhanced expression level and improved membrane trafficking in human tissue, compared to NpHR 22,23 . By using an AAV vector, encoding Jaws-GFP under the control of CAR promoter, we delivered the microbial opsin to the hiPSC-derived cone photoreceptors (Fig.…”

mentioning

confidence: 99%

Restoration of visual function by transplantation of optogenetically engineered photoreceptors

Garita-Hernández

Lampic

Chaffiol

et al. 2018

Preprint

Self Cite

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A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments (OS) in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). For a RPE-independent treatment approach, we introduced a hyperpolarizing microbial opsin into photoreceptor precursors from new-born mice, and transplanted them into blind mice lacking the photoreceptor layer. These optogenetically transformed photoreceptors were light responsive and their transplantation lead to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generated cone photoreceptors from human induced pluripotent stem cells (hiPSCs), expressing the chloride pump Jaws. AfterWe thank Thierry Leveillard for providing the rd1 mice and Cheryl Craft for providing the hCAR antibody. We are thankful to Romain Caplette, Olivier Marre and Stéphane Deny for their help with the MEA recording and analysis. We thank Abhishek Sengupta for the construction of the light/dark box. We are grateful to Mélissa Desrosiers and Camille Robert for AAV productions and the fundraising department of the Vision Institute. MG optimized AAV mediated transduction of hiPSC-derived retinal organoids, performed culture, imaging, qPCR and histology, designed experiments and wrote the manuscript. ML performed in vivo injections, cell transplantation, MEA recordings, behavioral experiments, confocal microscopy, histology, designed experiments and wrote the manuscript. AC performed patch-clamp recordings and 2-photon imaging. LG generated hiPSC-derived retinal organoids, performed in vivo injections, cell transplantations, behavioral experiments, imaging, and histology. FR performed behavioral experiments. TF contributed to cell transplantation. GG and SR helped to optimize hiPSC cultures and differentiation protocols. SP and JAS provided scientific input, financial and administrative support. OG provided hiPSCs and gave feedback on the manuscript. MA provided Cpfl1/Rho -/mice, contributed to cell transplantation and gave feedback on the manuscript. DD and JD designed experiments and wrote the manuscript.

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Noninvasive gene delivery to foveal cones for vision restoration

Cited by 100 publications

References 52 publications

Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

Retinal explant culture: A platform to investigate human neuro‐retina

Restoration of visual function by transplantation of optogenetically engineered photoreceptors

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