Across scientific disciplines, 3-D organoid culture systems offer platforms to integrate basic research findings with clinical care. The National Eye Institute mounted a $1.1 million 3-D Retina Organoid Challenge. Organoids developed through the Challenge will be valuable resources for drug screening, disease modeling, and precision and regenerative medicine.

Retinoschisis is an X-linked recessive genetic disease that leads to vision loss in males. X-linked retinoschisis (XLRS) typically affects young males; however, progressive vision loss continues throughout life. Although discovered in 1898 by Haas in two brothers, the underlying biology leading to blindness has become apparent only in the last 15 years with the advancement of human genetic analyses, generation of XLRS animal models, and the development of ocular monitoring methods such as the electroretinogram and optical coherence tomography. It is now recognized that retinoschisis results from cyst formations within the retinal layers that interrupt normal visual neurosignaling and compromise structural integrity. Mutations in the human retinoschisin gene have been correlated with disease severity of the human XLRS phenotype. Introduction of a normal human retinoschisin cDNA into retinoschisin knockout mice restores retinal structure and improves neural function, providing proof-of-concept that gene replacement therapy is a plausible treatment for XLRS.

Purpose

The b-wave of the cone ERG increases in amplitude and speed during the first few minutes of adaptation to a rod-suppressing background light. Earlier studies implicate rod pathway input to the cone pathway in these changes.

Methods

The timing and amplitude of the cone b-wave and isolated oscillatory potentials (OP) during the first 10 minutes of light adaptation in wild-type (WT) mice and two mutant lines without functional rods was examined: rhodopsin knockout (Rho-/-), lacking rod outer segments, and NRL knockout (Nrl-/-), in which rods are replaced by S-cones. Expression of the immediate-early gene c-fos, which is increased in the inner retina by light-induced activity, was evaluated by immunohistochemistry in dark- and light-adapted retinas.

Results

WT b-wave and OP amplitudes increased, and implicit times decreased during light adaptation. Subtracting OP did not alter b-wave changes. Rho-/- b-wave and OP amplitudes did not increase during adaptation. B-wave timing and amplitude and the timing of the major OP at 1 minute of adaptation were equivalent to WT at 10 minutes. The light-adapted ERG b-wave in Nrl-/- mice, which originates in both the rod and cone pathways, changed in absolute amplitude and timing similar to WT. C-fos expression was present in the inner retinas of dark-adapted Rho-/- but not WT or Nrl-/- mice.

Conclusions

Activity in the distal rod pathway produces changes in the cone ERG during light adaptation. Rods in Rho-/- mice constitutively activate this rod-cone pathway interaction. The rod pathway S-cones in Nrl-/- mice may maintain the WT interaction.

To understand RS1 gene interaction networks in the X-linked retinoschisis (XLRS) mouse retina (Rs1^-/y), we analyzed the transcriptome by RNA sequencing before and after in vivo expression of exogenous retinoschisin (RS1) gene delivered by AAV8. RS1 is a secreted cell adhesion protein that is critical for maintaining structural lamination and synaptic integrity of the neural retina. RS1 loss-of-function mutations cause XLRS disease in young boys and men, with splitting ("schisis") of retinal layers and synaptic dysfunction that cause progressive vision loss with age. Analysis of differential gene expression profiles and pathway enrichment analysis of Rs1-KO (Rs1^-/y) retina identified cell surface receptor signaling and positive regulation of cell adhesion as potential RS1 gene interaction networks. Most importantly, it also showed massive dysregulation of immune response genes at early age, with characteristics of a microglia-driven proinflammatory state. Delivery of AAV8-RS1 primed the Rs1-KO retina toward structural and functional recovery. The disease transcriptome transitioned toward a recovery phase with upregulation of genes implicated in wound healing, anatomical structure (camera type eye) development, metabolic pathways, and collagen IV networks that provide mechanical stability to basement membrane. AAV8-RS1 expression also attenuated the microglia gene signatures to low levels toward immune quiescence. This study is among the first to identify RS1 gene interaction networks that underlie retinal structural and functional recovery after RS1 gene therapy. Significantly, it also shows that providing wild-type RS1 gene function caused the retina immune status to transition from a degenerative inflammatory phenotype toward immune quiescence, even though the transgene is not directly linked to microglia function. This study indicates that inhibition of microglial proinflammatory responses is an integral part of therapeutic rescue in XLRS gene therapy, and gene therapy might realize its full potential if delivered before microglia activation and photoreceptor cell death. Clinical Trials. gov Identifier NTC 02317887.

Purpose

To describe the motivations, expectations, and other factors men with X-linked retinoschisis (XLRS) consider when making decisions to participate in an early phase ocular gene therapy clinical trial.

Design

Qualitative interview study.

Methods

Men with XLRS who were considering participation in a phase I/IIa ocular gene therapy clinical trial at the National Eye Institute were eligible for this study. Trial participants (n = 9) were interviewed prior to receiving the gene transfer and then at 3 and 12 months later. Trial participation decliners (n = 2) were interviewed at an initial visit and 12 months later. Those screened for the trial and found ineligible (n = 2) were interviewed at an initial visit only. Interviews were transcribed, coded, and analyzed thematically.

Results

Interview participants described decision making factors as risk-benefit assessments, personal intuition, trust in the study team, and religious faith. Altruism and the potential for therapeutic benefit were the main motives for trial participation, whereas the uncertainty of risks and benefits was the reason 2 men declined participation. Although most participants hoped for direct benefit, no one expected to benefit. Almost all interview participants considered their decision straightforward and were satisfied with their decision when interviewed over time. Meaningful relationships with the study team and perceived secondary benefits to participation contributed to positive trial experiences.

Conclusions

Engaging prospective research participants in a discussion about their hopes, expectations, and personal factors provides a more complete understanding of patient decision making and may help support informed choices to participate in clinical trials for XLRS.

Purpose

To test the effects of rearing light intensity on retinal function and morphology in the retinoschisis knockout (Rs1-KO) mouse model of X-linked retinoschisis, and whether it affects functional outcome of RS1 gene replacement.

Methods

Seventy-six Rs1-KO mice were reared in either cyclic low light (LL, 20 lux) or moderate light (ML, 300 lux) and analyzed at 1 and 4 months. Retinal function was assessed by electroretinogram and cavity size by optical coherence tomography. Expression of inward-rectifier K+ channel (Kir4.1), water channel aquaporin-4 (AQP4), and glial fibrillary acidic protein (GFAP) were analyzed by Western blotting. In a separate study, Rs1-KO mice reared in LL (n = 29) or ML (n = 27) received a unilateral intravitreal injection of scAAV8-hRs-IRBP at 21 days, and functional outcome was evaluated at 4 months by electroretinogram.

Results

At 1 month, no functional or structural differences were found between LL- or ML-reared Rs1-KO mice. At 4 months, ML-reared Rs1-KO mice showed significant reduction of b-wave amplitude and b-/a-wave ratio with no changes in a-wave, and a significant increase in cavity size, compared to LL-reared animals. Moderate light rearing increased Kir4.1 expression in Rs1-KO mice by 4 months, but not AQP4 and GFAP levels. Administration of scAAV8-hRS1-IRBP to Rs1-KO mice showed similar improvement of inner retinal ERG function independent of LL or ML rearing.

Conclusions

Rearing light conditions affect the development of retinal cavities and post-photoreceptor function in Rs1-KO mice. However, the effect of rearing light intensity does not interact with the efficacy of RS1 gene replacement in Rs1-KO mice.

Objective

To compare subjective and objective clinical tests used in the screening for hydroxychloroquine retinal toxicity to multifocal electroretinography (mfERG) reference testing.

Design

Prospective, single-center, case control study.

Participants

Fifty-seven patients with a previous or current history of hydroxychloroquine treatment of more than 5 years' duration.

Methods

Participants were evaluated with a detailed medical history, dilated ophthalmologic examination, color fundus photography, fundus autofluorescence (FAF) imaging, spectral-domain (SD) optical coherence tomography (OCT), automated visual field testing (10-2 visual field mean deviation [VFMD]), and mfERG testing. We used mfERG test parameters as a gold standard to divide participants into 2 groups: those affected by hydroxychloroquine-induced retinal toxicity and those unaffected.

Main outcome measures

We assessed the association of various imaging and psychophysical variables in the affected versus the unaffected group.

Results

Fifty-seven study participants (91.2% female; mean age, 55.7±10.4 years; mean duration of hydroxychloroquine treatment, 15.0±7.5 years) were divided into affected (n = 19) and unaffected (n = 38) groups based on mfERG criteria. Mean age and duration of hydroxychloroquine treatment did not differ statistically between groups. Mean OCT retinal thickness measurements in all 9 macular subfields were significantly lower (<40 μm) in the affected group (P < 0.01 for all comparisons) compared with those in the unaffected group. Mean VFMD was 11 dB lower in the affected group (P < 0.0001). Clinical features indicative of retinal toxicity were scored for the 2 groups and were detected in 68.4% versus 0.0% using color fundus photographs, 73.3% versus 9.1% using FAF images, and 84.2% versus 0.0% on the scoring for the perifoveal loss of the photoreceptor ellipsoid zone on SD-OCT for affected and unaffected participants, respectively. Using a polynomial modeling approach, OCT inner ring retinal thickness measurements and Humphrey 10-2 VFMD were identified as the variables associated most strongly with the presence of hydroxychloroquine as defined by mfERG testing.

Conclusions

Optical coherence tomography retinal thickness and 10-2 VFMD are objective measures demonstrating clinically useful sensitivity and specificity for the detection of hydroxychloroquine toxicity as identified by mfERG, and thus may be suitable surrogate tests.

Adeno-associated virus (AAV) vector-mediated gene delivery is a promising approach for therapy, but implementation in the eye currently is hampered by the need for delivering the vector underneath the retina, using surgical application into the subretinal space. This limits the extent of the retina that is treated and may cause surgical injury. Vector delivery into the vitreous cavity would be preferable because it is surgically less invasive and would reach more of the retina. Unfortunately, most conventional, non-modified AAV vector serotypes penetrate the retina poorly from the vitreous; this limits efficient transduction and expression by target cells (retinal pigment epithelium and photoreceptors). We developed a method of applying a small and safe electric current across the intact eye in vivo for a brief period following intravitreal vector administration. This significantly improved AAV-mediated transduction of retinal cells in wild-type mice following intravitreal delivery, with gene expression in retinal pigment epithelium and photoreceptor cells. The low-level current had no adverse effects on retinal structure and function. This method should be generally applicable for other AAV serotypes and may have broad application in both basic research and clinical studies.

Purpose

Ciliary neurotrophic factor (CNTF) was recently shown to augment cone function in CNGB3 mutant achromat dogs. However, testing CNTF-releasing implant in human CNGB3 achromats failed to show benefit. We evaluated the effects of CNTF protein on the retinal function in an additional achromatopsia model, the CNGB3-/- mouse.

Methods

Fifty-nine CNGB3-/- mice (postnatal day [PD] ± SD = 30 ± 7) received a unilateral intravitreal injection of 1 or 2 μg CNTF protein, and 15 wild-type (WT) mice (PD = 34 ± 3) received 1 μg CNTF. Retinal function was evaluated by flash ERG and photopic flicker ERG (fERG) at 7 and 14 days after treatment.

Results

Seven days post CNTF, the photopic b-wave Vmax was significantly increased in CNGB3-/- mice (P < 0.01), whereas it was reduced in WT mice (P < 0.05). Ciliary neurotrophic factor significantly increased the amplitude of photopic fERG and the photopic oscillatory potentials (OPs) in CNGB3-/- mice. Ciliary neurotrophic factor did not alter the scotopic a-wave in either CNGB3-/- or WT mice, but it increased the scotopic b-wave k (P < 0.01) in CNGB3-/- mice, indicating diminished scotopic sensitivity, and reduced the scotopic b-wave Vmax in WT mice (P < 0.05). No difference was found in ERG parameters between 1 or 2 μg CNTF. Fourteen days after CNTF injection the ERG changes in CNGB3-/- mice were lost.

Conclusions

Intravitreal bolus CNTF protein caused a small and transient improvement of cone-mediated function in CNGB3-/- mice, whereas it reduced rod-mediated function. The increase in photopic OPs and the lack of changes in scotopic a-wave suggest a CNTF effect on the inner retina.

Purpose

Dominant-active RAC1 rescues photoreceptor structure in Drosophila rhodopsin-null mutants, indicating an important role in morphogenesis. This report assesses the morphogenetic effect of activated RAC1 during mammalian rod photoreceptor development using transgenic mice that express constitutively active (CA) RAC1.

Methods

Transgenic mice were generated by expressing CA RAC1 under control of the Rhodopsin promoter, and morphological features of the photoreceptors were evaluated by histology, immunohistochemistry, and transmission electron microscopy. Function was evaluated by electroretinography. Potential protein partners of CA RAC1 were identified by co-immunoprecipitation of retinal extracts.

Results

Constitutively active RAC1 expression in differentiating rods disrupted outer retinal lamination as early as postnatal day (P)6, and many photoreceptor cell nuclei were displaced apically into the presumptive subretinal space. These photoreceptors did not develop normal inner and outer segments and had abnormal placement of synaptic elements. Some photoreceptor nuclei were also mislocalized into the inner nuclear layer. Extensive photoreceptor degeneration was subsequently observed in the adult animal. Constitutively active RAC1 formed a complex with the polarity protein PAR6 and with microtubule motor dynein in mouse retina. The normal localization of the PAR6 complex was disrupted in CA RAC1-expressing rod photoreceptors.

Conclusions

Constitutively active RAC1 had a profound negative effect on mouse rod cell viability and development. Rod photoreceptors in the CA RAC1 retina exhibited a defect in polarity and migration. Constitutively active RAC1 disrupted rod morphogenesis and gave a phenotype resembling that found in the Crumbs mutant. PAR6 and dynein are two potential downstream effectors that may be involved in CA RAC1-mediated defective mouse photoreceptor morphogenesis.