Week in Review: Number 31

CRISPR-Cas9 Treatment for Fuchs' Corneal Dystrophy
Fuchs' endothelial corneal dystrophy (FECD) is the leading cause of corneal transplant in the U.S. and although there is no shortage of donor corneal tissue in this country, corneal transplant is surgery that comes with many risks. Even a successful surgery that restores clear vision with a donor cornea commits the patient to many office visits, co-pays, and post-operative eye drops. A treatment that could be administered prior to or in place of surgery could therefore provide patients with additional options and benefits. As part of an eight-year study, a team of researchers is exploring the use of CRISPR-Cas9 gene editing to knockdown the expression of a mutant protein responsible for a form of Fuchs' endothelial corneal dystrophy. In particular, they are focusing on a single-point mutation in a collagen protein known as COL8A2, or collagen type VIII alpha 2 chain, which causes an early-onset subset of the disease that typically affects patients in their late 30s or early 40s. Notably, previous research in mice showed that silencing the COL8A2  gene did not adversely affect the cornea; rather, it is the mutant variant, a missense mutation, of the protein that causes problems.

The researchers developed a new technique, called start codon disruption, in which they disrupt the initiation site of transcription, the start codon, thereby preventing protein synthesis. While other techniques that disrupt gene expression farther down the gene can also result in termination of protein expression, the farther downstream along the gene, the more likely a viable protein would still be produced that instead has unknown, and possibly unwanted, activity. This research is also novel in the sense that it applies CRISPR gene editing to post-mitotic cells, in this case the corneal endothelium, the cells of which are not replenished as a person ages. Significant loss of corneal endothelial cells leads to loss of pumping function, and in this case results in corneal edema leading to impaired vision. The treatment has so far been tested in mice by intracameral delivery of the gene therapy via adenovirus viral vector. The team reports both preservation of endothelial cells as well as rescue of endothelium pump function with no adverse effects to the surrounding tissues and a safe drug profile for the retina, iris, and other parts of the eye at the maximum tolerated dose. The researchers will continue studies in small animals and non-human primates before moving on to clinical trials. However, the study's senior researcher is hopeful that the therapy will one day reduce the need for corneal transplants for Fuchs’ dystrophy patients, thereby both directly benefiting those patients as well as indirectly helping other patients in need of corneal tissue.

DARC Imaging Predicts Risk of Geographic Atrophy
Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD), the leading cause of visual impairment in individuals over 55 years of age. In an effort to detect early signs of the disease, when interventions and treatments might be more effective, researchers are investigating a retinal imaging technology called Detection of Apoptosing Retinal Cells (DARC), which had previously shown clinical success in predicting the progression of glaucoma and detecting wet AMD. Performed with an intravenous injection of a fluorescent dye (ANX776, fluorescently labelled Annexin A5), DARC detects areas of the retina that are undergoing cellular stress or apoptosis (cellular death), which show up as areas of hypefluorescence when viewed on fundus exam. These areas of damage can be quantified with an AI algorithm, resulting in a "DARC count" that can be monitored over time for progression or prediction. The present phase 2 clinical trial involved 113 participants, 19 of whom had early signs of neovascular AMD and 13 of whom had early signs of geographic atrophy. The research team also recruited healthy volunteers and patients with progressive glaucoma, optic neuritis, and other eye conditions representative of neurodegeneration. All patients were screened with DARC and followed up with ocular coherence tomography (OCT) every six months over three years, the latter condition to assess DARC's validity against OCT. The results showed that patients with a DARC count of more than 10 on initial examination had increased expansion of GA three years later. The researchers plan on larger clinical trials and are exploring a nasal delivery of the fluorescent dye for a less invasive screening tool.

Retinal Organoids as Lab Models for Retinoblastoma
Retinoblastoma is a rare pediatric eye cancer driven by biallelic inactivation of the RB1  gene. Research into the pathophysiology of retinoblastoma, however, has had many limitations. First, rare eye diseases such as retinoblastoma have a small pool of patients to sample from. Genetically engineered mouse models of retinoblastoma do not express the abnormalities seen in humans when the RB1  gene is silenced, and are therefore not always reliable predictors for preclinical drug development. Biopsies in living patients are also contraindicated, since the act of biopsy can further spread the tumor cells. As such, tumor samples have been derived from more advanced stages requiring removal of the eyeball. Scientists are investigating the use of retinal organoid to more accurately model retinoblastoma to study the early stages of the disease as well as to screen for potential therapies. The researchers hope that these organoids could also shed light on other drivers of tumor development beyond the RB1  gene. In this case, the samples were collected from 15 retinoblastoma patients who had germline mutations in RB1, from which 3D cultures of cells were grown from induced pluripotent stem cells (iPSCs). These iPSCs developed into retinal organoids and were then injected into mouse eyes, where retinoblastomas subsequently formed. Whole-genome sequencing, RNA sequencing, and methylation analysis of these retinoblastoma tumors showed that they were indistinguishable from patient samples. The first author of the study comments on the novelty of the research, saying, “The ability of the organoid models to [successfully grow into organoids and later spontaneously developed into tumors] is unique, because cell lines with RB1  mutations do not spontaneously develop into retinoblastoma tumors.” Data from the project is freely available in the Childhood Solid Tumor Network at St. Jude Children's Research Hospital.

Head and Eye Movements & Gaze Tracking in Baseball
Researchers in sports vision conducted a review of film- and lab-based studies pertaining to batters' gaze in baseball to answer the question "Do batters actually keep their eye on the ball?" and if so, whether there is any advantage in strategy with regard to head and eye movements. Their study suggests that while batters do indeed keep their eyes on the ball, they direct their gaze by moving their heads rather than their eyes. Some of the batters were also seen to make what is suspected to be an anticipatory shift in gaze toward home plate. While the two authors of this study found no consensus in head and eye movements among baseball batters, they did uncover a consistent finding that batters move their heads rather than their eyes to direct their gaze at the ball. They speculate that given the time pressure during the half-a-second journey of a pitched ball to home plate, head movement reduces the complexity of negotiating visual and neural signals. However, head movement also leads to an interesting paradox, namely, suppression of the vestibular ocular reflex (VOR). Responsible for turning our eyes in the opposite direction of a head turn, the vestibular ocular reflex allows us to maintain a stabilized view of our surroundings despite head movement. As one of the authors explains, “When it comes to something like batting, when I’m rotating my head to follow the ball, the VOR is telling my eyes to go in the opposite direction. … So that VOR has to be canceled if the eyes are going to stay on the ball.” After confirming that batters do keep their eyes on the ball, the researchers next look to study whether anticipatory saccades away from the ball improved batting success, a pertinent question to sports training. Thus far, they report no conclusive data, but highlight both athletic interest in the topic as well as the need for larger studies under game conditions. While baseball serves as a model situation in this case, the authors posit that questions such as how we organize our thoughts when pressed for time and whether to fix our gaze on the focus of our attention or direct our gaze based on predictions are questions that apply to everyday life. “Ultimately, once we understand how and why the hand, eye and head are coordinated in a certain way, then training these patterns is the next step," one of the researchers concludes.

Myopia and the COVID-19 Pandemic in Hong Kong
The COVID-19 pandemic has brought attention to the increased global incidence of myopia in the pediatric population, as adjustment to lockdown led to less time spent outdoors and more time spent interacting with screens and near work. An often-cited study published in JAMA Ophthalmology  reported overall increased rates of myopia among more than 120,000 homebound children in China. Researchers in Hong Kong expanded upon the topic, as reported in a smaller cohort study involving 1793 children between the ages of 6 and 8, 1084 of whom were recruited before the COVID-19 pandemic and compared with 709 children who were recruited at the beginning of the COVID-19 pandemic. The authors report, "The overall incidence was 19.44% in the COVID-19 cohort, and 36.57% in [the] pre-COVID-19 cohort. During the COVID-19 pandemic, the change in SER [spherical equivalent refraction] and axial length was –0.50±0.51 D and 0.29±0.35 mm, respectively." Note that the incidence is actually reported to be higher in the pre-COVID-19 cohort.

However, because these are two different cohorts, recruited at two different time periods (and over different durations), the authors caution against a direct comparison between them. Instead, they "estimated the risk ratio of incidence of myopia between the two groups using a relative risk regression model (log-binomial model)," and based on that model, they found that "myopia incidence in the COVID-19 cohort was higher than in the pre-COVID-19 cohort (p=0.03) after adjusting for age, gender, follow-up duration, parental myopia, time of outdoor activity and near work." The authors also compared this study to an earlier study from 2004 to further support their data, stating, "The myopia incidence (13.15% over 1 year) in the previous cohort was lower than that of our COVID-19 cohort (19.44% over 8 months, p<0.001) despite having a longer follow-up of 1 year compared with 8 months in the COVID-19 cohort, indicating that the incidence of myopia increased during the COVID-19 pandemic." They attribute this to a decrease in outdoor activities from approximately 1 hour 15 minutes to merely 25 minutes per day and an increase in screen time from 2.5 to nearly 7 hours per day. Despite very indirect methodology in this study, based on comparison with similar studies in Asia, it is reasonable to extrapolate, with caution, that their model captures trends consistent with a global increase in myopia incidence during the COVID-19 pandemic. The researchers conclude with a motivation for the study, saying, "Our results serve to warn eye care professionals, and also policy makers, educators and parents, that collective efforts are needed to prevent childhood myopia—a potential public health crisis as a result of COVID-19."

In Other News
(1) Neuroscientists investigate the cause of Fuchs' corneal dystrophy
(2) A blind paraolympian runner and his guide
(3) Scientists study vision and motion in fruit flies