Developing Potential Treatments with Patient-specific Stem Cells and Personalized Gene Therapy

NEW YORK, NY — Columbia University Medical Center (CUMC) researchers have created a way to develop personalized gene therapies for patients with retinitis pigmentosa (RP), a leading cause of vision loss. The approach, the first of its kind, takes advantage of induced pluripotent stem (iPS) cell technology to transform skin cells into retinal cells, which are then used as a patient-specific model for disease study and preclinical testing.

Columbia University Medical Center

Using this approach, researchers led by Stephen H. Tsang, MD, PhD, showed that a form of RP caused by mutations to the gene MFRP (membrane frizzled-related protein) disrupts the protein that gives retinal cells their structural integrity. They also showed that the effects of these mutations can be reversed with gene therapy. The approach could potentially be used to create personalized therapies for other forms of RP, as well as other genetic diseases. The paper was published recently in the online edition of Molecular Therapy, the official journal of the American Society for Gene & Cell Therapy.

“The use of patient-specific cell lines for testing the efficacy of gene therapy to precisely correct a patient’s genetic deficiency provides yet another tool for advancing the field of personalized medicine,” said Dr. Tsang, the Laszlo Z. Bito Associate Professor of Ophthalmology and associate professor of pathology and cell biology.

While RP can begin during infancy, the first symptoms typically emerge in early adulthood, starting with night blindness. As the disease progresses, affected individuals lose peripheral vision. In later stages, RP destroys photoreceptors in the macula, which is responsible for fine central vision. RP is estimated to affect at least 75,000 people in the United States and 1.5 million worldwide.

More than 60 different genes have been linked to RP, making it difficult to develop models to study the disease. Animal models, though useful, have significant limitations because of interspecies differences. Researchers also use human retinal cells from eye banks to study RP. As these cells reflect the end stage of the disease process, however, they reveal little about how the disease develops. There are no human tissue culture models of RP, as it would dangerous to harvest retinal cells from patients. Finally, human embryonic stem cells could be useful in RP research, but they are fraught with ethical, legal, and technical issues.

The use of iPS technology offers a way around these limitations and concerns. Researchers can induce the patient’s own skin cells to revert to a more basic, embryonic stem cell–like state. Such cells are “pluripotent,” meaning that they can be transformed into specialized cells of various types.

In the current study, the CUMC team used iPS technology to transform skin cells taken from two RP patients—each with a different MFRP mutation—into retinal cells, creating patient-specific models for studying the disease and testing potential therapies.

By analyzing these cells, the researchers found that the primary effect of MFRP mutations is to disrupt the regulation of actin, the protein that makes up the cytoskeleton, the scaffolding that gives the cell its structural integrity. “Normally, the cytoskeleton looks like a series of connected hexagons,” said Dr. Tsang. “If a cell loses this structure, it loses its ability to function.”

The researchers also found that MFRP works in tandem with another gene, CTRP5, and that a balance between the two genes is required for normal actin regulation.

In the next phase of the study, the CUMC team used adeno-associated viruses (AAVs) to introduce normal copies of MFRP into the iPS-derived retinal cells, successfully restoring the cells’ function. The researchers also used gene therapy to “rescue” mice with RP due to MFRP mutations. According to Dr. Tsang, the mice showed long-term improvement in visual function and restoration of photoreceptor numbers.

“This study provides both in vitro and in vivo evidence that vision loss caused by MFRP mutations could potentially be treated through AAV gene therapy,” said coauthor Dieter Egli, PhD, an assistant professor of developmental cell biology (in pediatrics) at CUMC, who is also affiliated with the New York Stem Cell Foundation.

Dr. Tsang thinks this approach could also be used to study other forms of RP. “Through genome-sequencing studies, hundreds of novel genetic spelling mistakes have been associated with RP,” he said. “But until now, we’ve had very few ways to find out whether these actually cause the disease. In principle, iPS cells can help us determine whether these genes do, in fact, cause RP, understand their function, and, ultimately, develop personalized treatments.”

- MFP News Services
- 8/19/14

New Compound Treats Blindness and Diabetes in Rats, Mice

In a new study led by UC San Francisco (UCSF) scientists, a chemical compound designed to precisely target part of a crucial cellular quality-control network provided significant protection, in rats and mice, against degenerative forms of blindness and diabetes.

University of California - San Francisco

In addition to opening a promising drug-development path for the wide range of diseases caused by cell loss, the new research offers a new view of the workings of the unfolded protein response (UPR), a cellular “life-or-death” signaling network: When cells are under stress, the UPR works to ensure that they produce properly configured proteins, but those cells not up to this task are quickly prompted by the UPR to self-destruct.

A component of the UPR known as the IRE1 pathway has generally been thought to handle the protective aspects of this response, promoting cell survival by providing cells with the biological resources they need to cope with stress, while a complementary pathway, called PERK, has been associated with cell death.

But in the new research, published in the July 10, 2014 edition of Cell, when researchers used KIRA6, a small-molecule kinase inhibitor they designed to inhibit the actions of IRE1 alpha —the molecular sensor that triggers the IRE1 pathway—they blocked cell death and preserved function in experimental models of two human diseases.

In two rat models of retinitis pigmentosa, a disease in which light-sensing cells in the eye progressively die off, causing blindness, KIRA6 preserved both the number of these cells and visual function. And in mice from a strain known as Akita, which carry a genetic mutation that causes diabetes in early life as stressed insulin-producing beta cells of the pancreas degenerate, KIRA6 protected beta cells from cell death, leading to a two-fold increase in insulin production and improving blood glucose control.

“This is a huge advance in our field,” said co-senior author Scott A. Oakes, MD, associate professor of pathology at UCSF. “On the surface these would seem to be two very different diseases, but IRE1-induced cell death is at the root of both of them.”

The results are the culmination of “a gigantic project,” first to establish that the IRE1 pathway could drive degenerative disease, and then to design and test compounds to head off the damage, said UCSF’s Feroz Papa, MD, PhD, associate professor of medicine and co-senior author, and a member of the California Institute for Quantitative Biosciences. “It took four years, over a hundred separate experiments in various contexts—not counting replications—and involved 24 researchers working in seven labs labs across four cities.”

KIRA6 is the latest in a series of compounds (the acronym stands for “Kinase-Inhibiting RNase Attenuators) that were originally designed and synthesized in the labs of study co-authors Dustin J. Maly, PhD, associate professor of chemistry at The University of Washington, Seattle, and Bradley J. Backes, PhD, associate professor of medicine at UCSF.

“While KIRA6 showed efficacy in animals,” said Papa, “it is important to stress that more optimization through medicinal chemistry efforts is needed to develop this class of compounds to the stage where they could be tested for efficacy in humans through clinical trials.”

Oakes and Papa said that support from the Cleveland, Ohio-based Harrington Discovery Institute was crucial to sustaining this complex collaboration. Both scientists were 2013 winners of Scholar-Innovator Awards from the Institute, which is part of The Harrington Project for Development and Discovery a $250 million national model to accelerate the development of medical breakthroughs by physician-scientists into medicines that benefit patients. Other critical support for the work came from the National Institutes of Health, the Juvenile Diabetes Research Foundation, the Burroughs Wellcome Fund, the American Cancer Society, and the Howard Hughes Medical Institute.

- MFP News Services
- 8/18/14

Brain Activity for Patients with Sex Addiction Mirrors Patients with Drug Addiction

Pornography triggers brain activity in people with compulsive sexual behaviour – known commonly as sex addiction – similar to that triggered by drugs in the brains of drug addicts, according to a University of Cambridge study published in the journal PLOS ONE. However, the researchers caution that this does not necessarily mean that pornography itself is addictive.

University of Cambridge

Although precise estimates are unknown, previous studies have suggested that as many as one in 25 adults is affected by compulsive sexual behaviour, an obsession with sexual thoughts, feelings or behaviour which they are unable to control. This can have an impact on a person’s personal life and work, leading to significant distress and feelings of shame. Excessive use of pornography is one of the main features identified in many people with compulsive sexual behaviour. However, there is currently no formally accepted definition of diagnosing the condition.

In a study funded by the Wellcome Trust, researchers from the Department of Psychiatry at the University of Cambridge looked at brain activity in nineteen male patients affected by compulsive sexual behaviour and compared them to the same number of healthy volunteers. The patients started watching pornography at earlier ages and in higher proportions relative to the healthy volunteers.

“The patients in our trial were all people who had substantial difficulties controlling their sexual behaviour and this was having significant consequences for them, affecting their lives and relationships,” explains Dr Valerie Voon, a Wellcome Trust Intermediate Clinical Fellow at the University of Cambridge. “In many ways, they show similarities in their behaviour to patients with drug addictions. We wanted to see if these similarities were reflected in brain activity, too.”

The study participants were shown a series of short videos featuring either sexually explicit content or sports whilst their brain activity was monitored using functional magnetic resonance imaging (fMRI), which uses a blood oxygen level dependent (BOLD) signal to measure brain activity.

The researchers found that three regions in particular were more active in the brains of the people with compulsive sexual behaviour compared with the healthy volunteers. Significantly, these regions – the ventral striatum, dorsal anterior cingulate and amygdala – were regions that are also particularly activated in drug addicts when shown drug stimuli. The ventral striatum is involved in processing reward and motivation, whilst the dorsal anterior cingulate is implicated in anticipating rewards and drug craving. The amygdala is involved in processing the significance of events and emotions.

The researchers also asked the participants to rate the level of sexual desire that they felt whilst watching the videos, and how much they liked the videos. Drug addicts are thought to be driven to seek their drug because they want – rather than enjoy – it. This abnormal process is known as incentive motivation, a compelling theory in addiction disorders.

As anticipated, patients with compulsive sexual behaviour showed higher levels of desire towards the sexually explicit videos, but did not necessarily rate them higher on liking scores. In the patients, desire was also correlated with higher interactions between regions within the network identified – with greater cross-talk between the dorsal cingulate, ventral striatum and amygdala – for explicit compared to sports videos.

Dr Voon and colleagues also found a correlation between brain activity and age – the younger the patient, the greater the level of activity in the ventral striatum in response to pornography. Importantly, this association was strongest in individuals with compulsive sexual behaviour. The frontal control regions of the brain – essentially, the ‘brakes’ on our compulsivity – continue to develop into the mid-twenties and this imbalance may account for greater impulsivity and risk taking behaviours in younger people. The age-related findings in individuals with compulsive sexual behaviours suggest that the ventral striatum may be important in developmental aspects of compulsive sexual behaviours in a similar fashion as it is in drug addictions, although direct testing of this possibility is needed.

“There are clear differences in brain activity between patients who have compulsive sexual behaviour and healthy volunteers. These differences mirror those of drug addicts,” adds Dr Voon. “Whilst these findings are interesting, it’s important to note, however, that they could not be used to diagnose the condition. Nor does our research necessarily provide evidence that these individuals are addicted to porn – or that porn is inherently addictive. Much more research is required to understand this relationship between compulsive sexual behaviour and drug addiction.”

Dr John Williams, Head of Neuroscience and Mental Health at the Wellcome Trust, says: “Compulsive behaviours, including watching porn to excess, over-eating and gambling, are increasingly common. This study takes us a step further to finding out why we carry on repeating behaviours that we know are potentially damaging to us. Whether we are tackling sex addiction, substance abuse or eating disorders, knowing how best, and when, to intervene in order to break the cycle is an important goal of this research.”