Gene Therapy for Blind Mice Might Lead to Human Trials


When a mouse sees an owl, it scurries for cover as fast as it can to escape the back-breaking talons of the swooping owl. If the mouse has defective vision and cannot properly see the owl, then the mouse is simply doomed. So ingrained is this escape response into the psyche of mice, that simply showing laboratory mice a video of a swooping owl will send them off into various directions for safety. Consequently, vision scientists can use this behavior to test treatments of blindness.

Rob Lucas of the University of Manchester, UK and his colleagues have played videos of swooping owls to normal and blind laboratory mice, and to blind mice that were treated with an experimental treatment for blindness. Lucas and others showed the owl video to mice that were so blind that they did not respond to the video. Then after giving these blind mice a treatment for blindness, those same mice were shown the same owl video, and they reacted as though they could see the swooping owl just fine. As Lucas explained: “You could say they were trying to escape, but we don’t know for sure. What we can say is that they react to the owl in the same way as sighted mice, whereas the untreated mice didn’t do anything.”

This best evidence acquired by Lucas and others to date that injecting the gene for a pigment that detects light into the eyes of blind mice can help them see again.

The gene therapy used by Lucas and others is meant to treat those types of blindness that are caused by damaged or missing photoreceptor, which are the cells in the neural retina that detect light. There are two types of photoreceptors in the retina: rods and cones. Rods and cones contain a pigment known as an “opsin,” which allows them to respond to light.  Opsin genes encode proteins that contain a vitamin A-based cofactor that helps it respond to light. The amino acid sequence of each opsin gene allows it to specifically respond to a range of frequencies of light. Different types of cones express specific opsins that allow them to specialize in the colors they can detect. Mutations in the opsin genes can cause blindness, and Lucas and his colleagues are interested in replacing the defective opsin genes in the retinas of laboratory mice. The majority of gene therapy experiments to treat blindness to date have concentrated on replacing faulty genes in rarer, specific forms of inherited blindness, such as

When a mouse sees an owl, it scurries for cover as fast as it can to escape the back-breaking talons of the swooping owl. If the mouse has defective vision and cannot properly see the owl, then the mouse is simply doomed. So ingrained is this escape response into the psyche of mice, that simply showing laboratory mice a video of a swooping owl will send them off into various directions for safety. Consequently, vision scientists can use this behavior to test treatments of blindness.

Rob Lucas of the University of Manchester, UK and his colleagues have played videos of swooping owls to normal and blind laboratory mice, and to blind mice that were treated with an experimental treatment for blindness. Lucas and others showed the owl video to mice that were so blind that they did not respond to the video. Then after giving these blind mice a treatment for blindness, those same mice were shown the same owl video, and they reacted as though they could see the swooping owl just fine. As Lucas explained: “You could say they were trying to escape, but we don’t know for sure. What we can say is that they react to the owl in the same way as sighted mice, whereas the untreated mice didn’t do anything.”

This best evidence acquired by Lucas and others to date that injecting the gene for a pigment that detects light into the eyes of blind mice can help them see again.

The gene therapy used by Lucas and others is meant to treat those types of blindness that are caused by damaged or missing photoreceptor, which are the cells in the neural retina that detect light. There are two types of photoreceptors in the retina: rods and cones. Rods and cones contain a pigment known as an “opsin,” which allows them to respond to light. Opsin genes encode proteins that contain a vitamin A-based cofactor that helps it respond to light. The amino acid sequence of each opsin gene allows it to specifically respond to a range of frequencies of light. Different types of cones express specific opsins that allow them to specialize in the colors they can detect. Mutations in the opsin genes can cause blindness, and Lucas and his colleagues are interested in replacing the defective opsin genes in the retinas of laboratory mice. The majority of gene therapy experiments to treat blindness to date have concentrated on replacing faulty genes in rarer, specific forms of inherited blindness, such as Leber congenital amaurosis.

Structures of opsins and of the chromophore retinal. (a) A model of the secondary structure of bovine rhodopsin. Amino-acid residues that are highly conserved in the whole opsin family are shown with a gray background. The retinal-binding site (K296) and the counterion position (E113) are marked with bold circles, as is E181, the counterion in opsins other than the vertebrate visual and non-visual ones. C110 and C187 form a disulfide bond. (b) The chemical structures of the 11-cis and all-trans forms of retinal. (c) The crystal structure of bovine rhodopsin (Protein DataBank ID: 1U19 [PDB:1U19]). The chromophore 11-cis-retinal, K296 and E113 are shown in stick representation in the ringed area. (d) The structure of the Schiff base linkage formed by retinal within the bovine opsin, together with the counterion that stabilizes it.
Structures of opsins and of the chromophore retinal. (a) A model of the secondary structure of bovine rhodopsin. Amino-acid residues that are highly conserved in the whole opsin family are shown with a gray background. The retinal-binding site (K296) and the counter ion position (E113) are marked with bold circles, as is E181, the counter ion in opsins other than the vertebrate visual and non-visual ones. C110 and C187 form a disulfide bond. (b) The chemical structures of the 11-cis and all-trans forms of retinal. (c) The crystal structure of bovine rhodopsin (Protein DataBank ID: 1U19 [PDB:1U19]). The chromophore 11-cis-retinal, K296 and E113 are shown in stick representation in the ringed area. (d) The structure of the Schiff base linkage formed by retinal within the bovine opsin, together with the counter ion that stabilizes it.  Taken from Terakita A, The opsins. Genome Biol 2005; 6(5):213.
The new treatment strategy employed by Lucas and others seek to enable other cells that lie just above the photoreceptors to capture light. Rod and cone cells normally detect light and convert it into an electrochemical signal that is sent to bipolar and then ganglion cells above them, which processing these signals and send them to the brain. By engineering bipolar or even ganglion cells to produce their own light-detecting pigment, they can to some extent compensate for the lost receptors, although the resolution of the vision is poor.

retina_schema

Lucas and others used the human gene for rhodopsin, the pigment used by rod cells to detect light and hooked this gene to a genetic “switch” that would only turn on the gene inside ganglion and bipolar cells. Then they inserted this DNA into a virus that infected the retinal cells of mice whose rods and cones had been destroyed.

After treatment, Lucas and his colleagues found that the mice could distinguish objects by their size quite well, but not as well as sighted mice. “The treated mice could discriminate black and white bars, but only ones that were 10 times thicker than what sighted mice could see,” says Lucas.

In earlier attempts, mice could only tell objects apart under extremely bright light. Therefore, this new finding is crucial. “Our mice could respond in ordinary light, the equivalent of looking at a computer monitor under ordinary office lighting,” says Lucas.

This is also the first time a human gene has been tested this way. The virus they used to deliver the gene therapy to mouse retinal cells has already been approved for use in humans, and Lucas says he hopes to begin trials of a human treatment in about five years.

“This is the most effective example yet of the use of genetic therapy to treat advanced retinal degeneration,” says Robin Ali, whose team at University College London has given gene therapy treatments of people with Leber congenital amaurosis.

But Robert Lanza, chief medical officer at Ocata Therapeutics in Marlborough, Massachusetts, warns that we don’t yet know how long the beneficial effects of the new treatment might last, since it seems that the sight in people with Leber congenital amaurosis who were treated with gene therapy between one and three years ago has begun to wane.

See Current Biology DOI: 10.1016/j.cub.2015.07.029.

Treating Age-Related Blindness with a Stem Cell Replacement Method


A collaboration between German and American scientists in New York City has resulted in the invention of a new method for transplanting stem cells into the eyes of patients who suffer from age-related macular degeneration, which is the most frequent cause of blindness. In an animal test, the implanted stem cells survived in the eyes of rabbits for several weeks.

Approximately 4.5 millino people in Germany suffer from age-related macular degeneration (AMD), which causes gradual loss of visual acuity and affects the ability to read, drive a car or do fine work. The center of the vision field becomes blurry as though covered by a veil. This vision loss is a consequence of the death of cells in the retinal pigment epithelium or RPE, which lies are the back of the eye, underneath the neural retina.

Inflammation within the RPE causes AMD. Increased inflammation prevents efficient recycling of metabolic waste products, and the build-up of toxic wastes causes RPE die off. Without the RPE, the photoreceptors in front of the RPE cells that also depend on the RPE to repair the damage suffered from continuous light exposure, begin to die off too.

RPE

Retinal Pigmented Epithelium

Presently no cure exists for AMD, but scientists at Bonn University, in the Department of Ophthalmology and New York City have tested a new procedure that replaces damaged RPE cells.

In the present experiment, RPE cells made from human stem cells were successfully implanted into the retinas of rabbits.

Boris V. Stanzel, the lead author of this work, said, “These cells have now been used for the first time in research for transplantation purposes.”

The adult RPE stem cells were characterized by Timothy Blenkinsop and his colleagues at the Neural Stem Cell Institute in New York City. Blenkinsop designed methods to isolate and grow these cells. He also flew to Germany to assist Dr. Stanzel with the transplantation experiments.  Blenkinsop obtained his RPE cells from human cadavers, and he grew them on polyester matrices.

These experiments demonstrate that RPE cells obtained from adult stem cells can replace cells destroyed by AMD. This newly developed transplantation method makes it possible to test which stem cells lines are most suitable for transplantation into the eye.

Human Stem Cells Elucidate the Mechanisms of Beta-Cell Failure in Diabetes


Wolfram syndrome is a rare form of diabetes characterized by high blood sugar levels that result from insufficient levels of the hormone insulin.  The chronically high blood sugar levels cause degeneration of the optic nerve, leading to progressive vision loss (optic atrophy).  Wolfram syndrome patients often also have abnormal pituitary glands that release abnormally low levels of the hormone vasopressin (also known as antidiuretic hormone or ADH), which causes hearing loss caused by changes in the inner ear (sensorineural deafness), urinary tract problems, reduced amounts of the sex hormone testosterone in males (hypogonadism), or neurological or psychiatric disorders.

Diabetes mellitus is typically the first symptom of Wolfram syndrome, usually diagnosed around age 6. Nearly everyone with Wolfram syndrome who develops diabetes mellitus requires insulin replacement therapy. Optic atrophy is often the next symptom to appear, usually around age 11. The first signs of optic atrophy are loss of color vision and peripheral (side) vision. Over time, the vision problems get worse, and people with optic atrophy are usually blind within approximately 8 years after signs of optic atrophy first begin.

Mutations in the WFS1 gene cause more than 90 percent of the cases of Wolfram syndrome type 1.  The WFS1 gene encodes a protein called wolframin that regulates the amount of calcium in cells.  A proper calcium balance is important for a whole host of cellular processes, including cell-to-cell communication, the tensing (contraction) of muscles, and protein processing.  Wolframin protein is found in many different tissues, such as the pancreas, brain, heart, bones, muscles, lung, liver, and kidneys.  Inside cells, wolframin is in the membrane of a cell structure called the endoplasmic reticulum that is involved in protein production, processing, and transport. Wolframin is particularly important in the pancreas, where it helps process proinsulin into mature hormone insulin, the hormone that helps control blood sugar levels.

WFS1 gene mutations lead to the production of a sub-functional versions of wolframin.  As a result, calcium levels within cells are not properly regulated and the endoplasmic reticulum does not work correctly.  When the endoplasmic reticulum does not have enough functional wolframin, the cell triggers its own cell death (apoptosis).  In the pancreas, the cells that make insulin (beta cells) die off, which causes diabetes mellitus.  The gradual loss of cells along the optic nerve eventually leads to blindness, and the death of cells in other body systems likely causes the various signs and symptoms of Wolfram syndrome type 1.

A certain mutation in the CISD2 gene also causes Wolfram syndrome type 2. The CISD2 gene provides instructions for making a protein that is in the outer membrane of cell structures called mitochondria,the energy-producing centers of cells.  Even though the function of the CISD2 protein is unknown, CISD2 mutations produce nonfunctional CISD2 protein that causes mitochondria to eventually break down. This accelerates the onset of cell death.  Cells with high energy demands such as nerve cells in the brain, eye, or gastrointestinal tract are most susceptible to cell death due to reduced energy, and people with mutations in the CISD2 gene have ulcers and bleeding problems in addition to the usual Wolfram syndrome features.

Some people with Wolfram syndrome do not have an identified mutation in either the WFS1 or CISD2 gene. The cause of the condition in these individuals is unknown.

Now that you have a proper introduction to Wolfram syndrome, scientists from the New York Stem Cell Foundation and Columbia University Medical Center have produce induced pluripotent stem cells (iPSCs) from skin samples provided by Wolfram syndrome patients.  All of the patients who volunteered for this study were recruited from the Naomi Berrie Diabetes Center and had childhood onset diabetes and required treatment with injected insulin, and all had vision loss.  Control cell lines that did not have mutations in WFS1 were obtained from Coriell Research for Medical Research.

These skin samples contained cells known as fibroblasts and these were reprogrammed into induced pluripotent stem cells.  In order to show that these cells were truly iPSCs, this group implanted them underneath the kidney capsule of immuno-compromised mice, and they formed the teratoma tumors so characteristic of these cells.

When these iPSCs were differentiated into insulin-secreting pancreatic beta cells, Linshan Shang and her colleagues discovered that the beta cells made from cells that did not come from Wolfram syndrome patients secreted normal levels of insulin.  However, those beta cells made from iPSCs derived from Wolfram patients failed to secrete normal quantities of insulin either in culture or when transplanted into the bodies of laboratory animals.  Further investigations of these cells showed these beta cells showed elevated levels of stress in the endoplasmic reticulum as a result of an accumulation of unfolded proteins.

What on earth is endoplasmic reticulum protein-folding stress?  First some cell biology.  When the cell needs to make a protein that will be secreted, embedded in a membrane or vesicle. that protein begins its life on ribosomes (protein synthesis factories of the cell) in the cytoplasm, but later those ribosomes are dragged to a cellular structure called the endoplasmic reticulum.  While on the surface of the endoplasmic reticulum, the ribosome completes the synthesis of the protein and extrudes the protein into the interior of the endoplasmic reticulum or embeds the protein into the endoplasmic reticulum membrane.  From there, the protein is trafficked in a vesicle to another subcellular structure called the Golgi apparatus, were it undergoes further modification, and from the Golgi apparatus, the protein goes to the membrane, secretory vesicle or other places.

If the proteins in the endoplasmic reticulum cannot fold properly, they clump and build up inside the endoplasmic reticulum, and this induces the ERAD or Endoplasmic Reticulum-Associated Protein Degradation response.  The players in the ERAD response are shown below.  As you can see, this response is rather complicated, but if it fails to properly clear the morass of unfolded proteins in the endoplasmic reticulum, then the cell will undergo programmed cell death.

ERAD Response

However, this research team did not stop there.  When they treated the cultured beta cells made from cells taken from Wolfram syndrome patients with a chemical called 4-phenyl butyric acid, the stress on the cells was relieved and the cells survived.  This experiment shows that relieving this unfolded protein stress is a potential target for clinical intervention.

“These cells represent an important mechanism that causes beta-cell failure in diabetes.  This human iPS cell model represents a significant step forward in enabling the study of this debilitating disease and the development of new treatments,” said Dieter Egli, the principal investigator of the study, and senior research fellow at the New York Stem Cell Foundation.

Because all forms of diabetes mellitus ultimately result from an inability of the pancreatic beta cells to provide sufficient quantities of insulin in response to a rise in blood sugar concentrations, this Wolfram patient stem cell model enables an analysis of a more specific pathway that leads to beta-cell failure in more prevalent forms of diabetes.  Furthermore, this strategy enables the testing of strategies to restore beta-cell function that may be applicable to all types of diabetes.

Susan L. Solomon of the New York Stem Cell Foundation, said, “Using stem cell technology, we were able to study a devastating condition to better understand what causes the diabetes syndromes as well as discover possible new drug targets.”

Rudolph L. Leibel, a professor of diabetes research and co-author of this study, said, “This report highlights again the utility of close examination of rare disorders as a path to elucidating more common ones.  Our ability to create functional insulin-producing cells using stem cell techniques on skin cells from patients with Wolfram’s syndrome has helped to uncover the role of ER stress in the pathogenesis of diabetes.  The use of drugs that reduce such stress may prove useful in the prevention and treatment of diabetes.”

The ERAD response seems to play a role in the survival of insulin-producing beta cells in both type 1 and type 2 diabetes.  The ERAD response opposes the stress of the immune assault in type 1 diabetes and the metabolic stress of high blood glucose levels in both types of diabetes.  When the ERAD response fails, cell death ensues and this reduces the number of insulin-producing cells.