Experimental gene therapy shows promise for RP

A new gene therapy could preserve vision in people with retinitis pigmentosa and might effectively treat other degenerative nerve disorders, according to researchers from the United States, the United Kingdom, and Australia.

Reporting in the American Society of Gene & Cell Therapy’s Molecular Therapy, the researchers said they had shown for the first time that mice responded to visual stimulation after treatment with genes to produce ciliary neurotrophic factor (CNTF).

The therapy appeared to give life-long protection of photoreceptor cells from a genetic disorder in mice that closely resembles retinitis pigmentosa in humans.

“These findings provide insights into potential novel therapeutic avenues for diseases such as retinitis pigmentosa and amyotrophic lateral sclerosis [ALS], for which CNTF has been evaluated unsuccessfully in clinical trials,” the researchers wrote.

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The researchers were based at the University of Oxford; the University of Florida in Gainesville, Florida, USA; the University of Sheffield, Moorfields Eye Hospital & UCL NIHR Biomedical Research Centre for Ophthalmology, and the University of Western Australia in Perth, Australia.

Previous research showed that CNTF can protect against ganglion cell death in models of oxidative stress and experimental glaucoma as well as retinal diseases.

But these earlier studies noted suppression of physiological function in these photoreceptors as measured by electroretinography.

Additionally, CNTF proved ineffective and toxic when given in large systemic doses to people with ALS.

The researchers in the current study wanted to find other measures of vision loss, measure how long the protection might last, find ways of controlling the CNTF dosing and test the therapy in a model where degeneration had already begun.

Next: Attempting to detect CNTF protein

Thus, they crossed two breeds of mice: one having a genetic defect, knockout of rhodopsin, that simulates human retinitis pigmentosa, and one having enhanced green fluorescent protein in their photoreceptors.

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As in humans with retinitis pigmentosa, rod photoreceptors degenerate first in these mice and the objective is to preserve cone-mediated vision. The absence of rhodopsin protein occurs by the time the mice are 8 weeks old, and is followed by the loss of intrinsically fluorescent cones leading to total photoreceptor degeneration.

The researchers next created a recombinant adeno-associated virus vector to express human CNTF modified with a human nerve growth factor secretion signal.

When the mice were 4 weeks old, the researchers injected the virus into one eye each of three groups of mice. They gave a low dose (2 x 10⁸ genome particles) to 8 mice, a medium dose (2 x 10⁹ genome particles) to 6 mice and a high dose (2 x 10¹⁰ genome particles) to 5 mice. They administered a sham injection in the contralateral eye of each mouse.

Four weeks later, they looked at the retinae of the mice using a confocal scanning laser ophthalmoscope. The excitation wavelength of this laser is proximate to the absorption peak of the enhanced green fluorescent protein, and this allowed the researchers to see the fluorescent cells in the retina.

This imaging showed dots in a pattern corresponding to the expected distribution of the fluorescent cells. Later, the researchers used postmortem lectin staining to confirm that these dots were produced by photoreceptors.

Fluorescent cone receptors were distributed similarly in all the eyes, but in the sham treated group, the number declined rapidly, and reached zero when the mice were 24 weeks old.

The researchers could not detect CNTF protein in the eyes treated with a low dose of genome particles, and the decline of cone photoreceptors in these eyes paralleled the decline in the sham-injected eyes.

Next: Posing questions for the future

Near infrared reflectance imaging showed focal geographic changes in the retinal pigment epithelium consistent with loss of the loss of the overlying photoreceptors. Through histology, the researchers later confirmed the absence of both rod and cone photoreceptors in the sham-treated eyes.

In the eyes treated with the medium dose of genome particles preserved, 45% of their cone photoreceptors, and the eyes treated with the high dose preserved 62% of their cone photoreceptors.

In both the high- and medium-dose groups, the rate of cone photoreceptor loss was nearly zero starting when the mice were 15 weeks old.

The researchers examined the mice’s eyes using electroretinography, a technique that utilises a corneal electrode to indirectly measure the electrical response of the retina to light.

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They found that the greater the presence of CNTF, the less the cones responded. When the mice reached 12 weeks of age, the response dropped below background noise in all the groups. This was consistent with previous experiments, and suggested loss of function in these cones.

Using laser speckle imaging of the visual cortex, the researchers could detect increased movement of red blood cells during light stimulation. There was more response in the eyes treated with both the medium and the high doses compared to the sham-treated eyes.

This was statistically significant for the high-dose eyes.

To investigate further, when the mice were 30 weeks old, the researchers placed the mice into a rotating drum with vertical black and white stripes. They measured the optomotor response of the mice by recording the number of head-tracking movements the mice made in the direction of the drum’s rotation, followed by rapid-phase reorientation to a central position. The head-tracking movements are driven by each eye independently depending on the direction of the drum’s rotation.

The number of these head-tracking movements was not significantly above zero in the sham-treated eyes. But the medium-dose eyes stimulated an average of 2.11 of these movements per minute, and the high-dose eyes stimulated an average of 6.92 of these movements per minute. The difference between these eyes and the sham-treated eyes was statistically significant (p </= 0.001.)

Taken together, these findings suggest that the cone photoreceptors preserved by CNTF remain sensitive to light, the researchers concluded. This protection appeared to continue until the mice were euthanised at 30 weeks of age.

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The findings pose complicated questions for the future of this therapy, because the electroretinography findings suggest that it suppresses at least some elements of visual function at the same time that it preserves other elements.

Next: ALS treatment possibilities

Furthermore, the researchers conducted a transcriptome analysis of the mice’s eyes. They found evidence that overexpression of protease inhibitors underlies cone preservation in retinitis pigmentosa, possibly through direct inhibition of matrix degradation.

This lead the researchers to conclude that this kind of targeted gene therapy, allowing sustained release of CNTF in controlled doses, might effectively treat neurodegenerative diseases such as ALS as well as retinitis pigmentosa.