Study advances gene therapy for glaucoma

While testing genes to treat glaucoma by reducing pressure inside the eye, University of Wisconsin-Madison scientists stumbled onto a problem: They had trouble getting efficient gene delivery to the cells that act like drains to control fluid pressure in the eye.

Genes can’t work until they enter a cell.

Glaucoma, one of the most common blinding diseases, is caused by excess pressure inside the eye, usually due to a clog in the fluid drain. “Most glaucoma can be treated with daily drug treatment,” says Paul Kaufman, professor of ophthalmology and visual sciences at the University of Wisconsin-Madison. “Replacement genes could, theoretically, restore normal fluid flow for years on end, without requiring daily self-administration of eye drops that is inconvenient and may have local or even systemic side effects.”

In a study published today in the scientific journal Investigative Ophthalmology and Visual Science, Kaufman and Curtis Brandt, a fellow professor of ophthalmology and visual sciences at UW-Madison, showed an improved tactic for delivering new genes into the drain, called the trabecular meshwork.

The colleagues have been testing a vector based on feline immunodeficiency virus (FIV) to deliver the genes. Like the related c, FIV can insert genes into the host’s DNA. The eye’s innate defenses against FIV, however, interfered with the delivery.

Virus particles contain genes wrapped in a protein coat and then a lipid membrane. After the virus enters the cell and sheds its membrane, defensive molecules from the host can “drag the virus particle to the cell’s garbage disposal, called the proteasome, where it is degraded,” Brandt says. “We wanted to know if temporarily blocking the proteasome could prevent the destruction of the gene delivery vector and enhance delivery.”

In the current study, FIV virus carrying a marker protein was placed on cells of the trabecular meshwork, with or without a chemical that blocks proteasomes.

Above a dosage threshold, the treatment roughly doubled the transfer of genes entering the target cells, Brandt says. The new genes also spread more uniformly across the meshwork tissue. Delivering more copies of the gene should give a greater therapeutic effect, opening the meshwork drain and reducing pressure inside the eye.

The present study concerns the tools for transferring genes, not the genes themselves, Brandt says. But even before the current study, he says he and Kaufman “have already identified at least two genes that could unplug the drain.”

In the long struggle to replace genes and cure disease, “eyes have been one of the big success stories,” Brandt says. A blinding eye disease called Leber’s congenital amaurosis damages the function of cells that keep the light-sensitive cells healthy; replacing the mutated genes has preserved and even improved vision in young patients. Approval for this gene therapy is now pending at the Food and Drug Administration.

To forestall danger from injecting a virus, “We take out pretty much all of the virus’ genes, so it has no chance to replicate and spread from where it’s initially injected,” says Brandt.

Although the technique does interfere with the anti-viral defense in the eye, the effect is temporary. “You encounter the drug once, then it is metabolized, and the innate inhibition is lost,” Brandt says.

“We have shown that this strategy does work in eye organ culture,” Brandt says. “Once we do further work on efficiency and identify which gene to deliver, then we are probably ready to move toward clinical trials.”

NIH Discovery Brings Stem Cell Therapy for Eye Disease Closer to the Clinic

Scientists at the National Eye Institute (NEI), part of the National Institutes of Health, report that tiny tube-like protrusions called primary cilia on cells of the retinal pigment epithelium (RPE)—a layer of cells in the back of the eye—are essential for the survival of the retina’s light-sensing photoreceptors. The discovery has advanced efforts to make stem cell-derived RPE for transplantation into patients with geographic atrophy, otherwise known as dry age-related macular degeneration (AMD), a leading cause of blindness in the U.S.  The study appears in the January 2 Cell Reports.

“We now have a better idea about how to generate and replace RPE cells, which appear to be among the first type of cells to stop working properly in AMD,” said the study’s lead investigator, Kapil Bharti, Ph.D., Stadtman Investigator at the NEI. Bharti is leading the development of patient stem cell-derived RPE for an AMD clinical trial set to launch in 2018.

In a healthy eye, RPE cells nourish and support photoreceptors, the cells that convert light into electrical signals that travel to the brain via the optic nerve. RPE cells form a layer just behind the photoreceptors. In geographic atrophy, RPE cells die, which causes photoreceptors to degenerate, leading to vision loss.

Bharti and his colleagues are hoping to halt and reverse the progression of geographic atrophy by replacing diseased RPE with lab-made RPE. The approach involves using a patient’s blood cells to generate induced-pluripotent stem cells (iPSCs), cells capable of becoming any type of cell in the body. iPSCs are grown in the laboratory and then coaxed into becoming RPE for surgical implantation.

Attempts to create functional RPE implants, however, have hit a recurring obstacle: iPSCs programmed to become RPE cells have a tendency to get developmentally stuck, said Bharti. “The cells frequently fail to mature into functional RPE capable of supporting photoreceptors. In cases where they do mature, however, RPE maturation coincides with the emergence of primary cilia on the iPSC-RPE cells.”

The researchers tested three drugs known to modulate the growth of primary cilia on iPSC-derived RPE. As predicted, the two drugs known to enhance cilia growth significantly improved the structural and functional maturation of the iPSC-derived RPE. One important characteristic of maturity observed was that the RPE cells all oriented properly, correctly forming a single, functional monolayer. The iPSC-derived RPE cell gene expression profile also resembled that of adult RPE cells. And importantly, the cells performed a crucial function of mature RPE cells: they engulfed the tips of photoreceptor outer segments, a pruning process that keeps photoreceptors working properly.

By contrast, iPSC-derived RPE cells exposed to the third drug, an inhibitor of cilia growth, demonstrated severely disrupted structure and functionality.

As further confirmation of their observations, when the researchers genetically knocked down expression of cilia protein IFT88, the iPSC-derived RPE showed severe maturation and functional defects, as confirmed by gene expression analysis. Tissue staining showed that knocking down IFT88 led to reduced iPSC-derived RPE cell density and functional polarity, i.e., cells within the RPE tissue pointed in the wrong direction.

Bharti and his group found similar results in iPSC-derived lung cells, another type of epithelial cell with primary cilia. When iPSC-derived lung cells were exposed to drugs that enhance cilia growth, immunostaining confirmed that the cells looked structurally mature.

The report suggests that primary cilia regulate the suppression of the canonical WNT pathway, a cell signaling pathway involved in embryonic development. Suppression of the WNT pathway during RPE development instructs the cells to stop dividing and to begin differentiating into adult RPE, according to the researchers.

The researchers also generated iPSC-derived RPE from a patient with ciliopathy, a disorder that causes severe vision loss due to photoreceptor degeneration. The patient’s ciliopathy was associated with mutations of cilia gene CEP290. Compared to a healthy donor, iPSC-derived RPE from the ciliopathy patient had cilia that were smaller. The patient’s iPSC-derived RPE also had maturation and functional defects similar to those with IFT88 knockdown.

Further studies in a mouse model of ciliopathy confirmed an important temporal relationship: Looking across several early development stages, the RPE defects preceded the photoreceptor degeneration, which provides additional insights into ciliopathy-induced retinal degeneration.

The study’s findings have been incorporated into the group’s protocol for making clinical-grade iPSC-derived RPE. They will also inform the development of disease models for research of AMD and other degenerative retinal diseases, Bharti said.

This work was supported by the NEI intramural research program and the NIH Common Fund’s Regenerative Medicine Program.

FDA Panel Approves Gene Therapy For A Form Of Childhood Blindness

An advisory board at the  Food and Drug Administration today endorsed the first gene therapy for an inherited disorder — a rare condition that causes a progressive form of blindness that usually starts in childhood.

The recommendation came in a unanimous 16-0 vote after a day full of hearings that included emotional testimonials by doctors, parents of children blinded by the disease and from children and young adults helped by the treatment.

The treatment will now progress to a final decision from the FDA and, if approved, will be the first gene therapy legally available in the United State for an inherited disorder. The FDA is under no obligation to follow the advisory board’s recommendation but usually does.

The treatment, which will be marketed as Luxturna, fixes a mutation in the RPE65 gene. It involves a single treatment to each eye, which introduces genetically engineered virus particles carrying a corrected version of the mutated gene. Spark Therapeutics, the treatment’s developer, estimates that 6,000 people around the world could benefit from this treatment. More than 90 percent of the patients treated in the study showed some improvement in eyesight within just a few days of treatment.

This is a huge step forward for the field of gene therapeutics. “[O]n multiple fronts, it’s a first and ushers in a new era of gene therapy,” assistant professor of ophthalmology at the Oregon Health and Science University, Paul Yang, told NPR.

Alone, this treatment could also be applied to other formally incurable genetic eye diseases. “There are a lot of retinal diseases like this, and if you added them together it’s a big thing because they are all incurable,” says lead researcher Albert Maguire in an interview with NPR before the hearing.

Sources: NPR