An involuntary yelp told him that his wife was trying to roll over in bed. For weeks, he had watched helplessly as the infection and her immune system fought their battle, with her as the central casualty.
Although the fever had long broken, the pain remained and intensified. Every movement was calculated and every plan failed as her skin recognized everything as an enemy.
The blankets that kept off the scalding chill were themselves ribbons of lava leaving invisible welts on every square inch that they touched. The simple act of breathing, her inflating lungs expanding her chest, felt like it would tear her skin open.
The over-the-counter medications she had always relied on to soothe tired muscles and headaches had become little more than water in their efficacy, and a source of constipation rather than relief. And the prescription analgesics that effectively shut off her brain, leaving her drowsy and mentally cloudy, weren’t working as well as they had at first, as her threshold for pain—psychological and physiological—plummeted. Eventually, for fear of addiction or overdose, her physician refused to order a refill.
Another weakened cry, yet more tears of agony and frustration. And her husband sat at her bedside, helpless.
Agonizing challenges
According to the American Academy of Pain Medicine, about 100 million Americans are dealing with chronic pain, making the condition four to ten times more frequent than diabetes (26 million), coronary heart disease (16 million) or cancer (12 million). And when you combine treatment costs with those related to lost wages and productivity, pain represents an economic burden of approximately $600 billion.
And although there are shades of grey in every medical condition, pain seems to be the murkiest, where the only true metric is a patient’s answer to the question “How much does it hurt?”
Part of the challenge in understanding pain, according to Paul Karila, vice president of discovery services at Cellectricon, is that pain itself is not a disease but is rather a “spectrum or palette of different states.”
“Some [patients] share the same phenotype but there are probably many [different] underlying mechanisms,” he suggests.
And in some cases, he continues, the phenotype can be diffuse. He offers the example of lower back pain, where it can be difficult to point to the origin of the pain.
These sentiments were echoed in an editorial written by Lundbeck’s Gordon Munro for a special issue of CNS Neuroscience & Therapeutics dedicated to pain.
“Despite being classed as one condition, patients with, for example, diabetic neuropathic pain can have differing pathophysiological mechanisms that contribute to their signs and symptoms,” Munro suggested. “And this situation is probably manifest throughout the whole spectrum of chronic pain conditions to some degree or another. Accordingly, no one drug can be expected to work effectively in all patients.”
And yet, despite this acknowledgement and years of research, the armamentarium remains limited.
“There are really two classes of drugs for pain that are used again and again,” says Daniel Burch, vice president and therapeutic area head for neuroscience at PPD, suggesting the biggest are non-steroidal anti-inflammatory drugs (NSAIDs) and opiates.
“A lot of the focus on opiates, frankly, the last five or 10 years has been in tamper- and diversion-proof types of presentations; making them safer to use and not as much of a threat to society, and that’s a whole other can of worms. That’s a big problem; opiate abuse and addiction in our country.”
The numbers tell the tale.
In 2012 alone, according to the CDC, physicians wrote 259 million prescriptions for opioid pain relievers, approximately one per citizen. And in 2013, they reported that about 2 million Americans aged 12 and over either abused or were dependent on these drugs, with more than 16,000 dying from overdose.
The other challenge with these categories, says Burch, is that whereas they may work well in treating acute pain, they are less efficacious in chronic situations like neuropathic pain. Thus, clinicians have sought to repurpose other central nervous system (CNS)-relevant drugs in the hope of ameliorating pain.
Neuropathic pain, for example, has been called the epilepsy of the peripheral nervous system, Burch continues, so antiepileptic drugs have been tried with some success.
“Probably one of Pfizer’s most successful drugs, Neurontin (gabapentin), worked in this condition,” he explains. “But these drugs have side effects. Neurontin’s pretty safe except it causes sedation and it’s not 100-percent effective.”
People have also tried antidepressants for chronic pain.
“Probably the most significant from a commercial point-of-view in the last 10 years was Lilly’s [dual reuptake inhibitor] Cymbalta (duloxetine),” Burch enthuses, also adding tricyclic antidepressants to the list.
But even this armamentarium of drugs for chronic neuropathic pain has challenges.
“Most of them have mechanisms that tend to suppress the CNS like the anticonvulsants and so forth, or maybe have unwanted side effects or just don’t work that well,” he states.
From his perspective, the drugs simply need to be better targeted. But that may not be so easy.
“The biology of pain is complex and, because it is a primary survival mechanism, has considerable redundancy and overlap with other sensory functions,” offered the University of Arizona’s Frank Porreca and colleagues in Science Translational Medicine in 2014. “Consequently, modulation of single proteins, the strategy most common in drug discovery, may not produce the desired effects in the general patient population.”
Tackling targets
Despite this acknowledged complexity, the workhorse platform for elucidating and characterizing targets that might be involved in pain has been patch-clamp experiments.
Almost 40 years old, the technology relies on a micropipette that isolates a small portion of the cell surface and its resident ion channels. This pipette acts as an electrode, while a grounding electrode sits in the cell medium.
By monitoring transitions between conductance states across these ion channels under varying conditions, researchers can learn more about their involvement in electrophysiological functions such as action potentials and nerve activity. They can then compare this activity in the presence of potential drugs.
Cellectricon’s Karila reflects on his early days at AstraZeneca’s pain unit, which he explains had a very strong focus on molecular biology, expressing ion channels into over-expressing cell lines like HEK cells for high-throughput patch-clamp experiments using equipment like the IonWorks from Molecular Devices.
But again, using this technique to screen drugs was a very systematic approach, relying on an inherent knowledge of the involvement of a given channel in a given pain phenotype.
This approach therefore made complete sense, he says, for situations like the sodium channel Nav 1.7, where lack-of-function and gain-of-function mutations produce altered pain phenotypes. But there are very few known cases where a chronic pain state is caused by a single mutation.
“It is more likely that it is the sum or perhaps changes in or gradients of growth factors, inflammatory factors, etc.,” he explains. “You have a cocktail of inflammatory mediators that are produced probably not by neurons but by surrounding cells, and invading cells to the area where you have a neuropathic nerve injury.”
For this reason, Cellectricon took a more phenotypic approach to screening compounds for their impact on action potentials and other functional and morphological changes in neuronal tissues. The result was the Cellaxess Elektra platform.
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An involuntary yelp told him that his wife was trying to roll over in bed. For weeks, he had watched helplessly as the infection and her immune system fought their battle, with her as the central casualty.
Although the fever had long broken, the pain remained and intensified. Every movement was calculated and every plan failed as her skin recognized everything as an enemy.
The blankets that kept off the scalding chill were themselves ribbons of lava leaving invisible welts on every square inch that they touched. The simple act of breathing, her inflating lungs expanding her chest, felt like it would tear her skin open.
The over-the-counter medications she had always relied on to soothe tired muscles and headaches had become little more than water in their efficacy, and a source of constipation rather than relief. And the prescription analgesics that effectively shut off her brain, leaving her drowsy and mentally cloudy, weren’t working as well as they had at first, as her threshold for pain—psychological and physiological—plummeted. Eventually, for fear of addiction or overdose, her physician refused to order a refill.
Another weakened cry, yet more tears of agony and frustration. And her husband sat at her bedside, helpless.
Agonizing challenges
According to the American Academy of Pain Medicine, about 100 million Americans are dealing with chronic pain, making the condition four to ten times more frequent than diabetes (26 million), coronary heart disease (16 million) or cancer (12 million). And when you combine treatment costs with those related to lost wages and productivity, pain represents an economic burden of approximately $600 billion.
And although there are shades of grey in every medical condition, pain seems to be the murkiest, where the only true metric is a patient’s answer to the question “How much does it hurt?”
Part of the challenge in understanding pain, according to Paul Karila, vice president of discovery services at Cellectricon, is that pain itself is not a disease but is rather a “spectrum or palette of different states.”
“Some [patients] share the same phenotype but there are probably many [different] underlying mechanisms,” he suggests.
And in some cases, he continues, the phenotype can be diffuse. He offers the example of lower back pain, where it can be difficult to point to the origin of the pain.
These sentiments were echoed in an editorial written by Lundbeck’s Gordon Munro for a special issue of CNS Neuroscience & Therapeutics dedicated to pain.
“Despite being classed as one condition, patients with, for example, diabetic neuropathic pain can have differing pathophysiological mechanisms that contribute to their signs and symptoms,” Munro suggested. “And this situation is probably manifest throughout the whole spectrum of chronic pain conditions to some degree or another. Accordingly, no one drug can be expected to work effectively in all patients.”
And yet, despite this acknowledgement and years of research, the armamentarium remains limited.
“There are really two classes of drugs for pain that are used again and again,” says Daniel Burch, vice president and therapeutic area head for neuroscience at PPD, suggesting the biggest are non-steroidal anti-inflammatory drugs (NSAIDs) and opiates.
“A lot of the focus on opiates, frankly, the last five or 10 years has been in tamper- and diversion-proof types of presentations; making them safer to use and not as much of a threat to society, and that’s a whole other can of worms. That’s a big problem; opiate abuse and addiction in our country.”
The numbers tell the tale.
In 2012 alone, according to the CDC, physicians wrote 259 million prescriptions for opioid pain relievers, approximately one per citizen. And in 2013, they reported that about 2 million Americans aged 12 and over either abused or were dependent on these drugs, with more than 16,000 dying from overdose.
The other challenge with these categories, says Burch, is that whereas they may work well in treating acute pain, they are less efficacious in chronic situations like neuropathic pain. Thus, clinicians have sought to repurpose other central nervous system (CNS)-relevant drugs in the hope of ameliorating pain.
Neuropathic pain, for example, has been called the epilepsy of the peripheral nervous system, Burch continues, so antiepileptic drugs have been tried with some success.
“Probably one of Pfizer’s most successful drugs, Neurontin (gabapentin), worked in this condition,” he explains. “But these drugs have side effects. Neurontin’s pretty safe except it causes sedation and it’s not 100-percent effective.”
People have also tried antidepressants for chronic pain.
“Probably the most significant from a commercial point-of-view in the last 10 years was Lilly’s [dual reuptake inhibitor] Cymbalta (duloxetine),” Burch enthuses, also adding tricyclic antidepressants to the list.
But even this armamentarium of drugs for chronic neuropathic pain has challenges.
“Most of them have mechanisms that tend to suppress the CNS like the anticonvulsants and so forth, or maybe have unwanted side effects or just don’t work that well,” he states.
From his perspective, the drugs simply need to be better targeted. But that may not be so easy.
“The biology of pain is complex and, because it is a primary survival mechanism, has considerable redundancy and overlap with other sensory functions,” offered the University of Arizona’s Frank Porreca and colleagues in Science Translational Medicine in 2014. “Consequently, modulation of single proteins, the strategy most common in drug discovery, may not produce the desired effects in the general patient population.”
Tackling targets
Despite this acknowledged complexity, the workhorse platform for elucidating and characterizing targets that might be involved in pain has been patch-clamp experiments.
Almost 40 years old, the technology relies on a micropipette that isolates a small portion of the cell surface and its resident ion channels. This pipette acts as an electrode, while a grounding electrode sits in the cell medium.
By monitoring transitions between conductance states across these ion channels under varying conditions, researchers can learn more about their involvement in electrophysiological functions such as action potentials and nerve activity. They can then compare this activity in the presence of potential drugs.
Cellectricon’s Karila reflects on his early days at AstraZeneca’s pain unit, which he explains had a very strong focus on molecular biology, expressing ion channels into over-expressing cell lines like HEK cells for high-throughput patch-clamp experiments using equipment like the IonWorks from Molecular Devices.
But again, using this technique to screen drugs was a very systematic approach, relying on an inherent knowledge of the involvement of a given channel in a given pain phenotype.
This approach therefore made complete sense, he says, for situations like the sodium channel Nav 1.7, where lack-of-function and gain-of-function mutations produce altered pain phenotypes. But there are very few known cases where a chronic pain state is caused by a single mutation.
“It is more likely that it is the sum or perhaps changes in or gradients of growth factors, inflammatory factors, etc.,” he explains. “You have a cocktail of inflammatory mediators that are produced probably not by neurons but by surrounding cells, and invading cells to the area where you have a neuropathic nerve injury.”
For this reason, Cellectricon took a more phenotypic approach to screening compounds for their impact on action potentials and other functional and morphological changes in neuronal tissues. The result was the Cellaxess Elektra platform.
Using high-content-analysis-compatible multiwell plate, researchers grow neuronal cultures such as dorsal root ganglia (DRG), which they then treat with a fluorescent probe and the compounds to be assayed. After applying electric field stimulation (EFS) to all the wells simultaneously, they use digital imaging from below the plate to quantify differences in fluorescence signal.
The company co-developed their DRG assay with Pfizer, and validated the assay against a library of 500 compounds that were either prescribed pain medications or part of the pharma company’s pain discovery effort.
“We could pick up the activity of most of them in our system,” Karila proclaims. “Having said that, there were some compound classes that we did not pick up.”
Applying the platform, Cellectricon helps clients identify compounds or combinations of conditions with capacity to alter neuronal function or morphology even in the absence of knowing the precise mechanism by which this happens. This not only allows researchers to see the desired effects but also potentially highlights secondary and possibly unwanted effects that might have been missed in more target-focused approaches.
This isn’t to say, however, that the Cellaxess Elektra won’t work for targeted experiments.
In an internal development effort, Karila says, the company is transfecting siRNAs against targets that it knows are relevant in pain, like different sodium channel subtypes and the nerve growth factor receptor TrkA.
They’ve also started to use a genetically encoded calcium sensor called GCaMP, developed by The Howard Hughes Medical Institute’s Janelia Research Campus.
“The beauty of this system is that rather than incubating the entire culture with a calcium indicator, including all of the non-neuronal cells in the culture, we are actually only looking at the effect of those cells that have been electroporated,” he says. “And that will then give an increased sensitivity in our assay to be able to detect the effect of siRNAs that we have co-transfected.”
Companies like Axion Biosystems, meanwhile, are providing similar support using microelectrode arrays (MEAs) for high-throughput, label-free analysis of electrically active cells such as DRGs. With multiple electrodes per well, MEAs allow researchers to monitor not only the electrophysiology of populations of cells, but also variations between cells within a population.
As suggested by Pfizer’s Sharan Bagal and colleagues in a review paper on sodium channel modulators, the breadth of techniques for identifying and characterizing targets does have its drawbacks.
“Although developments in the field of automated electrophysiology now allow for its use in higher throughput screening cascades through to more in depth biophysical characterization of channel interactions, the biggest limitation remains with how to interpret this electrophysiology data,” the authors wrote in Bioorganic & Medicinal Chemistry Letters. “The range of platforms available and subtle differences in the protocol designs are so varied that comparing information across screening platforms is an additional significant challenge.”
It is at this point that compounds move into animal models, of which there are many, but these too have their limitations, not the least of which is a certain degree of subjectivity and questions of quantification. Unlike animal models of many other diseases, which have measurable physiological outcomes such as blood enzymes or respiration rates or tumor mass, animal models of pain are fundamentally behavioral, relying on the frequency of a tail flick or licking of a paw.
“I wish that there were more opportunities to do more in vitro testing as a model system to get away from animal models,” says KPI Therapeutics CEO and President Chuck Magness. “As you know, animals and people may experience pain differently on the one hand, and animals can’t really tell you even what pain they may be experiencing.
“The problem is even when new approaches can come into the scientific domain and be validated, it can be some time for the whole regulatory system to accept them and allow their use for approving drugs. It doesn’t move as quickly as you’d like.”
That validation will require connecting the dots between the in-vitro experiments and the clinic.
“The real breakthrough will come as more clinical data becomes available to interpret which in-vitro assays or protocols are the most predictive of clinical outcomes,” suggested Bagal and colleagues. “Recent advances in current clamp technology on the automated platforms should help with this in-vitro to clinical in-vivo translation by allowing measurement of effects on excitability.”
As well, there is the general question about injecting chemicals into animal paws or sitting them on hot plates.
“More and more, scientists within various therapeutic areas of drug discovery R&D are making fantastic efforts to address animal welfare issues within their working environment and embrace the principle of adhering to the 3R’s guidelines,” continued Munro in his editorial. “Accordingly, bridging methods that involve the use of, for example, isolated spinal cord, or novel approaches such as Cellectricon’s proprietary technology ... provide us with a tantalizing glimpse of the future of pain drug discovery efforts.”
Regardless, suggested Porreca and colleagues, “Validation only occurs in humans.”
Painfully slow progress
The Holy Grail in terms of new pain therapeutics is to be targeted at the molecular level, offers PPD’s Burch. And a survey of ongoing clinical trials would indicate that many companies are doing just that (see table Progress in chronic pain graphic below).
But targeted at the molecular level does not necessarily imply targeted to the site of pain.
“Since the same molecular mechanisms and pathways may be present in the CNS, maybe you can find a drug or an approach that is targeted to the periphery and doesn’t cross the blood-brain barrier (BBB).”
Such is the case for KPI Therapeutics, which is looking to the venom of marine cone snails for clues to analgesia.
“We got introduced through our network collaborators to Baldomero ‘Toto’ Olivera and his group at the University of Utah,” explains Magness, who in addition to his KPI leadership role is a co-founder of the Seattle-based biotech Kineta. “Toto’s been working on cone snails and their peptides for decades, and has really been a leader in that field.”
It was Olivera’s group, he adds, that did the research that led to the commercialization of Jazz Pharmaceutical’s Prialt, another cone snail peptide.
“It’s a great opportunity to bring something that’s really novel, with a different mechanism, into this marketspace where we may be able to deal with both the actual pain issues that patients are having but also do it in a way that doesn’t lead to some of these other problems like uncomfortable side effects that may have to be medically treated on their own or most importantly, to avoid the addictive potential and the cognitive impact of opioid-based drugs.”
The peptides act peripherally at the site of damage rather than via the CNS, and in fact, were engineered not to cross the BBB.
“It works by blocking this α9α10 nicotinic acetylcholine receptor (nAChR) channel, which is involved in the transmission of the pain signal in the nerve fibers,” Magness explains, adding that the compound also exhibits some neuroprotective and anti-inflammatory effects. These latter facets could be important factors in the efficacy of the peptide.
“By being able to reduce or minimize that inflammatory state, it helps to take some of the pressure off of those nerve fibers, which is probably part of the overall response that we see in our model systems.”
Although the candidate is still being developed at the preclinical stage, the company is looking to bring it into the clinic by 2017.
With a similar goal, Cara Therapeutics has dipped into the opioid analgesic well with its lead pain candidates. But whereas earlier opioids were directed centrally, triggering the side effects and addictive challenges mentioned earlier, CR845 and CR665 are kappa opioid receptor agonists that specifically target sensory nerves that predominantly occur in the peripheral nervous system.
When compared in animal models to oth
er kappa agonists such as enadoline, CR665 showed a much wider safety margin when examining the doses that produce analgesia versus those that produce CNS impairment. CR845, meanwhile, has completed three Phase 2 trials in acute post-operative pain as well as showing efficacy in a Phase 2 trial in pain resulting from osteoarthritis of the hip or knee.
Not everyone is ready to give up on centrally acting analgesics, however.
In 2014, a group led by Angiochem’s Michel Demeule described their efforts to specifically target neurotensin to the brain by conjugating it to a peptide known as Angiopep-2. By binding to a receptor on brain capillary endothelial cells, Angiopep-2 and its conjugate partner are transported across the BBB.
Presenting their findings in the Journal of Clinical Investigation, the researchers showed that not only was neurotensin able to bind its receptor target in vitro when conjugated to Angiopep-2, but the transporter peptide even increased neurotensin’s half-life in rat plasma from 30 minutes to 7 hours. More importantly, they demonstrated that the conjugate compound offered marked improvements in animal models of persistent, neuropathic and bone cancer pains.
But as with preclinical testing on animal models, subjectivity both of patients experiencing pain and of pain measurement continues to be a challenge (see also the sidebar article Placebo perils below just after the end of this main article).
“A lot of the traditional clinical approaches are based upon patient questionnaires, which are based on subjective questions,” says Magness. “We’d like to get away from subjective numbers to things that are more quantifiable.”
“We are big on developing biomarkers and will continue to do that as we’re advancing the drug into the clinic,” he enthuses. “And ultimately, the first human trials will give us the best data to actually develop a statistically significant biomarker story.”
Echoing Bagal’s earlier comments, he suggests that researchers can only do so much of that in cell models and the like in vitro without real treatment data and responses in signaling pathways to close the loop in identifying good markers of activity.
“While we are advancing the program from a more-or-less traditional drug development/regulatory acceptance perspective, we are looking for some of these other methodologies like biomarkers and surrogates for how we can measure pain that gets it to be less subjective,” Magness says.
Likewise, there is also a significant need to better understand the baseline of a patient population to provide some assurance that the changes noted in drug trials reflect true efficacy.
As Cellectricon’s Karila suggests, person-to-person variation within the healthy population is sometimes larger than the difference between normal and diseased patient groups in certain phenotypes. This is where he sees another opportunity for Cellaxess Elektra and iPS-derived neuronal cells.
“We could be a means of helping characterize a fairly broad base of control patients,” he explains. “And then that could be extended to pain patients having a known phenotype and perhaps also a known genotype to see if these controlled patients have a phenotype that is significantly different from the normal population.”
This approach could then be extended to patient stratification in clinical trials, helping to improve the odds of seeing a truly significant effect of treatment versus placebo.
“Having that data set from a fairly large portion of both diseased and healthy individuals, we would be able to see if cells derived from a person who should or should not be included in a clinical trial will have the disease phenotype or not,” Karila enthuses. “Then we could offer a functional way of screening these people as part of the inclusion criteria.”
Thus, while the next generation of analgesics is still experiencing some growing pains in its progress to market, opportunities continue on all fronts to ease those pains.
Placebo perils
Like the pain from the abscessed molar that disappears on the way to the dentist, how people experience pain can vary not only from person-to-person, but also within an individual depending on his or her environment. And in some cases, patients simply feel better because someone is listening to them.
A lot of chronic pain studies fail, says Daniel Burch, vice president and therapeutic area head for neuroscience at PPD, either because the response within the placebo group was too high or because there was too much variability.
“Clinical trials ... often group patients with heterogeneous pain phenotypes, and probably different underlying pain mechanisms, into broad pain classifications and indications, and then evaluate responses subjectively,” explained University of Arizona’s Frank Porreca and colleagues in Science Translational Medicine. “Together, these factors can produce large statistical variance, potentially masking clinical activity in subpopulations of patients and thus potential efficacy in these subgroups.”
Meanwhile, Burch suggests that the placebo response is a great thing in the hands of a practicing clinician; you want people to get better. And some people will get better just because you’ve “laid hands on them” or talked to them or they’ve been in the clinical environment.
“But when you have a high placebo response, it makes it difficult to tell whether the drug really worked or not.”
There are a number of ways to manage this problem, including simply making sure that recruited patients understand that they’re research subjects; that they should tell the investigator what’s really going on rather than try to anticipate what the investigators really want to hear.
Unfortunately, this is where the lack of objective clinical metrics can be quite problematic in pain research. Simply stated, the trials rely heavily on patient diaries and questionnaires.
Trying to minimize the impact of placebo responders, however, PPD has licensed a trial methodology—the sequential parallel comparison design (branded as Trimentum)—from collaborators at Massachusetts General Hospital that separates pain trials into two phases.
“You run the first part of the trial like you normally would, except you load up the placebo group disproportionately because you’re interested in placebo non-responders,” explains Burch. “And then in stage two, you re-randomize placebo non-responders, which is presumably a very informative group of patients. These are patients who didn’t get a response from hanging around in the doctor’s office and going through the other study procedures.”
The idea is that the placebo non-responders are more likely to give you a sense of the true drug response rate. But while the design allows you to run trials with 30 to 40 percent fewer patients, the statistical analysis of the trial is a bit more complicated because you are counting some subjects twice.
“The jury is still out on whether this trial design works in chronic pain, but if it works in depression, it should work in chronic pain,” Burch adds. “We’ll find out more in the coming few years.”
Getting into their heads: A neuroscience roundup
By Jeffrey Bouley
Of course, while pain is the focus of this month’s Special Report on Neuroscience, the world of neuroscience and neurology consists of so much more. So, in an effort not to get too much tunnel vision, let’s look at some recent research and news in a wide range of neurological arenas.
TSRI Scientists clone mouse neurons and find brain cells with 100+ unique mutations
LA JOLLA, Calif.—In a new study published in March in the journal Neuron, scientists from The Scripps Research Institute (TSRI) say they are the first to sequence the complete genomes of individual neurons and to produce live mice carrying neuronal genomes in all of their cells.
Use of the technique revealed surprising insights into these cells’ genomes, according to TSRI—including the findings that each neuron contained an average of more than 100 mutations and that these neurons accumulated more mutations in genes they used frequently.
“Neuronal genomes have remained a mystery for a long time,” said TSRI Associate Professor Kristin Baldwin, senior author of the new study and member of the Dorris Neuroscience Center at TSRI. “The findings in this study, and the extensive validation of genome sequencing-based mutation discovery that this method permits, open the door to additional studies of brain mutations in aging and disease, which may help us understand or treat cognitive decline in aging, neurodegeneration and neurodevelopmental diseases such as autism.”
Our individual genomes are inherited from our parents and make us unique in our behavior, appearance and susceptibility to disease, TSRI points out. While new mutations in genomes of individual cells are known to cause cancer, only recently have researchers begun to appreciate how different the genomes within normal cells of the body may be. Several lines of research have suggested cells in the brain may be particularly unique—and prone to accumulating new mutations of various sorts, including “jumping” genes called transposons.
Many of these mutations may not be harmful; however, collecting too many mutations, or having them build up in genes needed for a cell’s function, might lead to loss of neurons or incorrect brain wiring, which are suspected causes of diseases such as Alzheimer’s and autism.
“We need to know more about mutations in the brain and how they might impact cell function,” said TSRI Research Associate Jennifer Hazen, co-first author of the new study with Gregory Faust of the University of Virginia School of Medicine.
However, studying mutations in single neurons has presented a challenge: A single cell doesn’t contain enough genetic material for analysis, yet these mutations only exist in single cells. Unfortunately, current single-cell analysis approaches introduce new DNA errors and also destroy the only copy of the cell’s DNA in the process, making it impossible to go back and check to see if the mutations were really there. Scientists can’t generate copies of neurons because, unlike other cell types, neurons don’t divide in cell culture.
“There has been no easy way to get more copies of a neuron,” explained TSRI Research Assistant William Ferguson, a co-author of the paper.
The new study helps solve this problem. The team took a mouse neuron’s nucleus, which houses its DNA, and inserted it into an egg cell, which then divided and copied the mutation. The cloned cells then developed into thousands, or even millions, of stem cells with enough DNA for genomic analysis. The researchers repeated the process to create several lines of cloned neurons.
“We worked to get the egg itself to copy the genomes of brain cells using cloning,” said Baldwin.
“We’re tricking the neuron into thinking it’s not a neuron,” added Hazen. “This gives us a renewable source of copies of these genomes.”
To confirm that the cloned cells were indeed neurons, rather than other brain cells, the researchers tagged the cells with bright fluorescent markers. “When you see the marker, it’s a sigh of relief—it worked,” said TSRI Research Assistant Alberto Rios Rodriguez, a co-author of the study. Genomic analysis of the cloned cells provided further evidence that the neuron’s unique mutations were indeed being passed along.
For the first time, the team was even able to make cloned stem cell line neurons from mice older than eight weeks. This allowed the researchers to see mutations that build up over time. Even more strikingly, several of these stem cell lines could be grown into fertile adult mice which were clones of a single mouse neuron and carried the neuronal mutations in every cell on top of the rest of the DNA from the original mouse.
Sergey Kupriyanov, director of the Mouse Genetics Core at TSRI and co-author of the study, called the project “technically challenging.” The researchers discovered that not every mutated neuron could be developed into a stem cell line, although more research is needed to explain why.
The stem cell lines that did develop, however, provided some surprising insights into the brain.
The researchers found that neurons accumulate more mutations in the genes they use, which contrasts with other cell types that seem to protect their commonly used genes.
“Even more surprisingly,” said Baldwin. “We found that every neuron we looked at was unique—carrying more than 100 DNA changes or mutations that were not present in other cells.”
The researchers aren’t sure why this diversity is so common—there’s no evidence that neurons rearrange their DNA like blood cells do—but Baldwin said that if this phenomenon holds true in humans, our brains could hold 100 billion unique genomes.
Next, the researchers plan to use their technique to study neuronal genomes of very old mice and those with neurologic diseases. They hope this work will lead to new insights and therapeutic strategies for treating brain aging and neurologic diseases caused by neuronal mutations.
In addition to Baldwin, Hazen, Faust, Ferguson, Rodriguez and Kupriyanov, authors of the study, “The Complete Genome Sequences, Unique Mutational Spectra, and Developmental Potency of Adult Neurons Revealed by Cloning,” were Svetlana Shumilina and Royden A. Clark of the University of Virginia School of Medicine; Michael J. Boland, Greg Martin, Pavel Chubukov and Ali Torkamani of TSRI; Rachel K. Tsunemoto of TSRI and the University of California, San Diego; and Ira M. Hall of the Washington University School of Medicine.
GenSight enrolls first patients in Phase 3 studies of GS010 in Leber’s Hereditary optic neuropathy
PARIS—Toward the end of February, GenSight Biologics S.A., a clinical-stage biotechnology company discovering and developing novel gene therapies for neurodegenerative retinal diseases—and in the future of the central nervous system—enrolled its first patients in both RESCUE and REVERSE, two parallel pivotal Phase 3 trials with the company’s lead product candidate GS010 for the treatment of Leber’s hereditary optic neuropathy (LHON).
Dr. Bernard Gilly, chairman and CEO of GenSight, commented: “With the first patient recruited, we are now entering the last mile of GS010’s development, which we hope will demonstrate GS010’s ability to durably stop, if not restore, the brutal vision loss caused by LHON. GenSight continues to deliver on its strategy to develop novel approaches against blinding diseases.”
The pivotal trials are intended to determine whether GS010 can halt or reverse vision loss associated with LHON due to the NADH dehydrogenase 4 (ND4) mutation or be effective as prophylaxis for vision loss in an eye not yet affected. The trials will also seek to identify the therapeutic window of opportunity for treatment after onset of disease.
As early intervention is potentially a major factor in maximizing therapeutic success, the two clinical trials will focus on treating patients who have manifested visual decline for up to one year. RESCUE is expected to enroll 36 patients with an onset of vision loss up to 6 months in duration, while REVERSE is expected to enroll 36 patients with an onset of vision loss ranging from 7 to 12 months in duration.
GS010 will be administered as a single intravitreal injection to one eye of each subject, while the fellow eye will receive a sham procedure. At the end of the initial 48-week study period, a minimal three-year long term follow-up period will be initiated to determine the sustainability of efficacy outcomes and long-term safety of treatment.
“After three decades of study, I’m thrilled that we have reached this point where we may be able to offer effective treatment in LHON,” noted Dr. Alfredo A. Sadun, of the Doheny Eye Institute in the University of California, Los Angeles (UCLA) Department of Ophthalmology. “We now have good reason to hope for a solution to this devastating bilateral cause of congenital blindness in young adults.”
The first patient in the entire program was injected at the Doheny Eye Institute at UCLA as part of the REVERSE trial, while the first RESCUE trial patient was injected at Wills Eye Hospital, Philadelphia.
The trials will be conducted in parallel in seven centers across the United States, the United Kingdom, France, Germany and Italy. Topline results at 48 weeks are expected by the end of 2017.
Retrotope advances RT001 in clinical trials to treat Friedreich’s ataxia
LOS ALTOS, Calif.—Retrotope, a privately held clinical-stage pharmaceutical company, announced in February the successful completion of the first dose cohort and the opening of patient enrollment for the highest dose cohort in its ongoing 28-day study of orally dosed RT001 in Friedreich’s ataxia (FA) patients. RT001 was well tolerated and no serious adverse events or dose limiting toxicities were observed.
The RT001 is a chemically stabilized form of a natural fatty acid that confers resistance to lipid peroxidation in mitochondrial and cellular membranes via a novel mechanism. In FA, free iron is a catalyst for lipid peroxidation of exactly the type that can be mitigated with the drug, RT001. FA is a debilitating, life-shortening neurodegenerative disorder that affects approximately 6,000 people in the United States. A progressive loss of coordination and muscle strength leads to motor incapacitation, the full-time use of a wheelchair and ultimately early death from cardiac complications. There are currently no approved treatments for FA.
Dr. Robert De Jager, chief medical officer of Retrotope, commented: “We are very pleased that RT001 appears to be safe and well tolerated in this ongoing first-in-human study in FA patients. Primary endpoints are safety, tolerability, and the pharmacokinetic profile of orally dosed RT001. Secondary endpoints are the disease-related Friedreich’s Ataxia Rating Scale neurological score, the timed 25-foot walk and various exploratory measures and biomarkers.”
Retrotope is conducting the study at two sites: the University of South Florida Ataxia Research Center and the Collaborative Neuroscience Network in Long Beach, Calif.
UCLA study throws support behind cutting-edge genetic test
LOS ANGELES—April saw UCLA researchers report that they have found that a state-of-the-art molecular genetic test greatly improves the speed and accuracy with which they can diagnose neurogenetic disorders in children and adults. The discovery could lead directly to better care for people with rare diseases like spinocerebellar ataxia, leukodsystrophy, spastic paraplegia and many other conditions.
The exome sequencing test involves determining the order of all of the genes in a person’s genome. When used in concert with a complete patient evaluation and family medical history, the approach can help doctors identify disorders that may have gone undiagnosed for years, said Dr. Brent Fogel, first author of a review that appears in the April issue of Neurology Clinical Practice.
Exome sequencing is more efficient and less costly than the type of genetic testing that has been more commonly used, Fogel said, and a proper diagnosis can end what for many patients is an agonizing journey just to find a name for their conditions.
The growing body of evidence supporting the use of the test, and the demonstrated benefits to patients, should lead to greater insurance coverage of the test, said Fogel, who is director of the UCLA Neurogenetics Clinic and an associate professor of neurology and human genetics.
“Despite extensive literature supporting the use of this technology, many insurance companies still consider it to be investigational and may refuse coverage,” he said. “Our article outlines the appropriate use, benefits and limitations of exome sequencing that these companies need to consider when making coverage decisions.”
UCLA has been a leader in using the test as a diagnostic tool since 2012. Fogel and his team were among the first to adopt the technology for routine neurological practice, and he has been a strong advocate for wider use.
Fogel and colleagues wrote a 2014 study about exome sequencing that was published in the Journal of the American Medical Association Neurology. That research found that 20 percent of a group of people with spinocerebellar ataxia could be diagnosed immediately using the technique, and useful genetic information could be identified in more than 60 percent of the subjects, regardless of their age when the disease began or their family history.
OXiGENE receives European orphan drug designation for CA4P to treat neuroendocrine tumors
SOUTH SAN FRANCISCO, Calif.—OXiGENE Inc., a biopharmaceutical company developing vascular disrupting agents (VDAs) for the treatment of cancer, announced in late March that the European Commission has granted a designation of orphan drug status to CA4P for the treatment of gastro-entero-pancreatic neuroendocrine tumors (NETs). The designation provides for 10 years of marketing exclusivity in European Union (EU) member countries following product approval. Earlier this year, OXiGENE announced that CA4P received orphan drug designation from the U.S. Food and Drug Administration for NETs, which provides for seven years of marketing exclusivity after approval.
“I am pleased that the EU has provided the orphan designation to CA4P for neuroendocrine tumors,” stated Dr. William D. Schwieterman, president and CEO of OXiGENE. “This designation represents another successful step as we execute on our strategy of bolstering the proprietary position of CA4P in the potential indications in which we are most interested. Separately, we continue to expect final data from our phase 2a clinical trial of CA4P in NETs to be available later in 2016.”
