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“There is a compelling need to explore gene therapy in the eye,” according to Paul A. Sieving, MD, PhD.
"There is a compelling need to explore gene therapy in the eye," according to Paul A. Sieving, MD, PhD.
His current work is in X-linked retinoschisis (XLRS) with the goal of understanding it and to advance gene therapy. During delivery of the Gertrude Pyron Award Lecture, he described this work and provided a rationale for pursuing gene therapy for XLRS.
“We now know that XLRS is a retinal synaptic disease that can readily be treated in adult mice,” said Dr. Sieving, director, National Eye Institute, Bethesda, MD. “The question remains about whether treatment will work in humans.”
The eye is a good place to explore gene therapy because it is a small, closed compartment. The vector would carry a low risk of systemic toxicity, he noted.
“It also is worth noting that the cell types in the eye are evolutionarily conserved from mouse to human,” he said, citing the study of RPE65 gene therapy that began with gene discovery in 1993 and resulted in gene therapy in humans for Leber’s congenital amaurosis over 15 years.
“We now understand that RPE65 is the most critical factor in retinoid cycling in the retinal pigment epithelium,” he said.
Dr. Sieving focused on two components of XLRS, i.e., structure and function. Regarding the former, optical coherence tomography shows cavitation throughout the retinal layers, particularly in the deeper retina and the plexiform layer. Another facet is the electronegative response, which is key to what happens in the disease.
At the outset of Dr. Sieving’s interest in XLRS, he found a pedigree of 119 family members, but his cloning of the gene was pre-empted by a German investigator, Bernard Weber.
The next step was to determine the gene function by disabling it in a mouse model, which, in turn, showed that the B-wave was missing and normally there was copious protein in the rod inner retinal segments and in the synaptic zone. In both regions in the knockout mice there was disruption in the outer plexiform layer (OPL).
Dr. Sieving showed the presence of retinoschisin protein on the outer membrane of the inner segment, not the outer segment. He showed when two inner segments were side by side, in the cleft between them there is a large amount of retinoschisin protein, the function of which is as yet unclear.
When the investigators examined the protein itself using cryoelectronmicroscopy, they found collections of the retinoschisin protein that were lined up like a frisbee, with two such configurations coupled back to back.
It is interesting, Dr. Sieving pointed out, that higher-resolution images showed that nothing embeds this molecular complex in a membrane. He hypothesized that when looking at two cells, one on top and the other below, the protein complex attaches to one membrane and to a second membrane and when they are in proximity, they stick together.
“This could be important in some parts of the retina and might explain partly why the retina does not self-adhere in the disease,” he said. “Even more probable is that the electrostatic forces of the molecular complex hold receptors or channels that are important for cell function.”
The retinoschisis protein is also present in the OPL in the synaptic region. The rod pre-synaptic element releases glutamate normally, but the post-synaptic structure in the dendritic tips are abnormal.
The glutamate receptors are normal, but the necessary TRPM1 channels are missing in the knockout mouse causing the membrane to become hyperpolarized and the B-wave becomes defective.
Dr. Sieving noted that adding the vector resulted in relocalization of the TRPM1 channels to the dendritic tips. They were structurally normal in the treated animals and the B-wave was restored.
The vector was applied to the mouse eye by intravitreal injection rather than placed in the sub-retinal space.
A single-center study at the National Institutes of Health involved 9 patients with XLRS who received one of three dose levels of the vector by intravitreal injection showed retinal structural improvement with the highest dose. Two weeks after vector dosing, the parafoveal schisis was closed in one patient, but inflammation developed.
“The dose range is being extended in our trial, and we are learning to manage the acute ocular inflammation and learn what is driving the inflammation, which is self-limiting,” Dr. Sieving said. “The field in general needs better vectors to, extend the duration of the treatment benefit, and must develop intravitreal vectors. However, each disease has unique features. The outcome metrics must be tailored specifically for each condition.”
Paul Sieving, MD
Dr. Sieving has no financial interest in any aspect of this report. He delivered the Gertrude Pyron Award Lecture at the 2017 meeting of the American Society of Retina Specialists in Boston.
Physicians and potential participants can email Dr. Sieving directly.
Details of the study are available at ClinicalTrials.Gov #NCT02317887