Here’s a new gene editing strategy to treat genetic blood disorders

If you’re taking a road trip across the country, you have a starting point and an ending point. How you go from point A to point B could be one of a million different routes, but the ultimate outcome is the same: reaching your final destination.

Yesterday scientists from St. Jude Children’s Research Hospital published exciting findings in the journal Nature Medicine on a new gene editing strategy that could offer a different route for treating genetic blood disorders such as sickle cell disease (SCD) and b-thalassemia.

The scientists used a gene editing tool called CRISPR. Unless you’ve been living under a rock, you’ve heard about CRISPR in the general media as the next, hot technology that could possibly help bring cures for serious diseases.

In simple terms, CRISPR acts as molecular scissors that facilitate cutting and pasting of DNA sequences at specific locations in the genome. For blood diseases like SCD and b-thalassemia, in which blood cells have abnormal hemoglobin, CRISPR gene editing offers ways to turn on and off genes that cause the clinical symptoms of these diseases.

Fetal vs. Adult hemoglobin

Before I get into the meat of this story, let’s take a moment to discuss hemoglobin. What is it? It’s a protein found in red blood cells that transports oxygen from the lungs to the rest of the body. Hemoglobin is made up of different subunits and the composition of these hemoglobin subunits change as newborns develop into adults.

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Healthy red blood cell (left), sickle cell (right).

Fetal hemoglobin (HbF) is comprised of a and g subunits while adult hemoglobin (HbA) is typically comprised of a and b subunits. Patients with SCD and b-thalassemia typically have mutations in the b globin gene. In SCD, this causes blood cells to take on an unhealthy, sickle cell shape that can clog vessels and eventually cause premature death. In b-thalassemia, the b-globin gene isn’t synthesized into protein at the proper levels and patients suffer from anemia (low red blood cell count).

One way that scientists are attempting to combat the negative side effects of mutant HbF is to tip the scales towards maintaining expression of the fetal g-globin gene. The idea spawned from individuals with hereditary persistence of fetal hemoglobin (HPFH), a condition where the hemoglobin composition fails to transition from HbF to HbA, leaving high levels of HbF in adult blood. Individuals who have HPFH and are predisposed to SCD or b-thalassemia amazingly don’t have clinical symptoms, suggesting that HbF plays either a protective or therapeutic role.

The current study is taking advantage of this knowledge in their attempt to treat blood disorders. Mitchell Weiss, senior author on the study and chair of the St. Jude Department of Hematology, explained the thought process behind their study:

“It has been known for some time that individuals with genetic mutations that persistently elevate fetal hemoglobin are resistant to the symptoms of sickle cell disease and beta-thalassemia, genetic forms of severe anemia that are common in many regions of the world. We have found a way to use CRISPR gene editing to produce similar benefits.”

CRISPRing blood stem cells for therapeutic purposes

Weiss and colleagues engineered red blood cells to have elevated levels of HbF in hopes of preventing symptoms of SCD. They used CRISPR to create a small deletion in a sequence of DNA, called a promoter, that controls expression of the hemoglobin g subunit 1 (HBG1) gene. The deletion elevates the levels of HbF in blood cells and closely mimics genetic mutations found in HPFH patients.

Weiss further explained the genome editing process in a news release:

Mitchell Weiss

Mitchell Weiss

“Our work has identified a potential DNA target for genome editing-mediated therapy and offers proof-of-principle for a possible approach to treat sickle cell and beta-thalassemia. We have been able to snip that DNA target using CRISPR, remove a short segment in a “control section” of DNA that stimulates gamma-to-beta switching, and join the ends back up to produce sustained elevation of fetal hemoglobin levels in adult red blood cells.”

The scientists genetically modified hematopoietic stem cells and blood progenitor cells from healthy individuals to make sure that their CRISPR gene editing technique was successful. After modifying the stem cells, they matured them into red blood cells in the lab and observed that the levels of HbF increased from 5% to 20%.

Encouraged by these results, they tested the therapeutic potential of their CRISPR strategy on hematopoietic stem cells from three SCD patients. While 25% of unmodified SCD blood stem cells developed red blood cells with a sickle cell shape under low-oxygen conditions (to induce stress), CRISPR edited SCD stem cells generated way fewer sickle cells (~4%) and had a higher level of HbF expression.

Many routes, one destination

The authors concluded that their genome editing technique is successful at switching hemoglobin expression from the adult form back to the fetal form. With further studies and safety testing, this strategy could be one day be developed into a treatment for patients with SCD and b-thalassemia

But the authors were also humble in their findings and admitted that there are many different genome editing strategies or routes for developing therapies for inherited blood diseases.

“Our results represent an additional approach to these existing innovative strategies and compare favorably in terms of the levels of fetal hemoglobin that are produced by our experimental system.”

My personal opinion is the more strategies thrown into the pipeline the better. As things go in science, many of these strategies won’t be successful in reaching the final destination of curing one of these diseases, but with more shots on goal, our chances of developing a treatment that works there are a lot higher.


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Unlocking the secrets of how stem cells decide what kind of cell they’re going to be

Laszlo Nagy, Ph.D., M.D.

Laszlo Nagy, Ph.D., M.D.: Sanford Burnham Prebys Medical Discovery Institute

Before joining CIRM I thought OCT4 was a date on the calendar. But a new study says it may be a lot closer to a date with destiny, because this study says OCT4 helps determine what kinds of cell a stem cell will become.

Now, before we go any further I should explain for people who have as strong a science background as I do – namely none – that OCT4 is a transcription factor, this is a protein that helps regulate gene activity by turning certain genes on at certain points, and off at others.

The new study, by researches at Sanford Burnham Prebys Medical Discovery Institute (SBP), found that OCT4 plays a critical role in priming genes that cause stem cells to differentiate or change into other kinds of cells.

Why is this important? Well, as we search for new ways of treating a wide variety of different diseases we need to find the most efficient and effective way of turning stem cells into the kind of cells we need to regenerate or replace damaged tissue. By understanding the mechanisms that determine how a stem cell differentiates, we can better understand what we need to do in the lab to generate the specific kinds of cells needed to replace those damaged by, say, heart disease or cancer.

The study, published in the journal Molecular Cell, shows how OCT4 works with other transcription factors, sometimes directing a cell to go in one direction, sometimes in another. For example, it collaborates with a vitamin A (aka retinoic acid) receptor (RAR) to convert a stem cell into a neuronal precursor, a kind of early stage brain cell. However, if OCT4 interacts with another transcription factor called beta-catenin then the stem cell goes in another regulatory direction altogether.

In an interview with PhysOrg News, senior author Laszlo Nagy said this finding could help develop more effective methods for producing specific cell types to be used in therapies:

“Our findings suggest a general principle for how the same differentiation signal induces distinct transitions in various types of cells. Whereas in stem cells, OCT4 recruits the RAR to neuronal genes, in bone marrow cells, another transcription factor would recruit RAR to genes for the granulocyte program. Which factors determine the effects of differentiation signals in bone marrow cells – and other cell types – remains to be determined.”

In a way it’s like programming all the different devices that are attached to your TV at home. If you hit a certain combination of buttons you get to one set of stations, hit another combination and you get to Netflix. Same basic set up, but completely different destinations.

“In a sense, we’ve found the code for stem cells that links the input—signals like vitamin A and Wnt—to the output—cell type. Now we plan to explore whether other transcription factors behave similarly to OCT4—that is, to find the code in more mature cell types.”

 

 

Cloning breakthrough: Dolly the sheep has sister clones and they’re healthy

On the topic of famous farm animals, a few come to mind: Babe the pig, Old Yeller, Mr. Ed, and the cast of Charlotte’s Web. Many of us grew up with these fictional characters and hold them near and dear to our heart, but what about real, living farm animals? The first that comes to my mind is Dolly the sheep.

Back in 1996, scientists made a major breakthrough when they cloned a sheep which they named after the famous singer and actress Dolly Parton. This famous sheep was born in a test tube – a product of a scientific process called somatic cell nuclear transfer (SCNT). It involves transferring the nucleus (which contains a cell’s genetic material) from an adult cell – a mammary gland cell in the case of Dolly – into an unfertilized egg cell that has had its own nucleus removed. Much like jumping a car, scientists use an electric shock to trigger the egg cell to divide and develop into an embryo that has the exact genetic makeup as the original organism it was derived from.

Are cloned animals healthy?

SCNT is a very inefficient process with a high failure rate during embryonic and fetal development. Dolly was a huge achievement for scientists as she was the first mammal to be successfully cloned using SCNT. Unfortunately, even though Dolly lived to the age of six and a half years, she wasn’t the healthiest of sheep. She suffered from a severe form of arthritis and tumors in her lungs and was eventually put down to relieve her from pain. Scientists hypothesized that the lung cancer was likely caused by a common virus that infects sheep, but they questioned whether some of Dolly’s other symptoms were caused by accelerated aging resulting from the cloning process.

Whether cloned animals are physically healthy and age normally are questions that have spurred much debate amongst scientists since Dolly’s inception. Further experiments have shown that cloned mammals that survive past their infancy are typically healthy, but some experiments in mice showed that cloned mice tended to be more obese, have diabetic symptoms, and live shorter lives. Concerns about the safety of cloning prompted many countries to ban reproductive cloning in mammals until more was known about the process.

Good news for Dolly’s sisters

Dolly’s 20th anniversary since her birth was earlier this year, and in celebration, many journals and news outlets wrote about the progress of SCNT and cloning over the past two decades. This week, a new study added an exciting new chapter to these recent stories about Dolly.

Published in Nature Communications, scientists from the University of Nottingham in Britain reported that cloned sheep are healthy and live normal lives. They studied 13 cloned sheep, four of which were Dolly’s sisters cloned from the same mammary gland cell line as Dolly. These sheep were between 7-9 years of age which is near the end of a healthy sheep’s average lifespan of 10 years.

Cloned sheep, sisters to the famous Dolly the Sheep. (University of Nottingham)

Cloned sheep, sisters to the famous Dolly the Sheep. (University of Nottingham)

The scientists wanted to know whether cloning had any negative impact on the health and lifespan of these sheep. Lead author on the study, Dr. Kevin Sinclair, explained to the Washington Post:

“When we did the study, these clones were already 2½ years older than Dolly was when she died. And they appeared to be perfectly healthy, but we wanted to see if they might be harboring subtle defects.”

They conducted studies that assessed symptoms typically caused by aging in both humans and sheep. These included tests for blood pressure, insulin sensitivity, arthritis, and heart disease. They also conducted MRI scans and X-rays to look at the integrity of their bones, joints, and muscles.

On the whole, the sheep were healthy and their tests yielded normal results. A few of the cloned sheep had early signs of arthritis, but their conditions were similar to normal non-cloned sheep of the same age. The scientists concluded that there were no obvious signs of premature aging in this group of cloned sheep and that the cloning process did not have negative effects on the health and lifespan of these animals.

“It was quite obvious that the concerns of Dolly just didn’t relate,” Sinclair said. “So you can’t extend beyond the Dolly experience and say this premature aging applies to all clones.”

Cloning breakthrough but questions remain about safety

This study, which many scientists are considering as a “breakthrough in cloning”, has received a lot of attention in the media from major news outlets like the New York Times, Washington Post, Statnews, and NPR.

The New York Times piece does a great job of discussing how the advancements in cloning could have positive impacts on reproductive technology, the farming industry (raising cloned farm animals as a food source), therapeutic development, and saving endangered species. But the article also balances this optimism with caution over the safety and ethics behind reproductive cloning. They posed the cloning safety question to Dr. Sinclair, the lead author on the study, whose response was positive but referenced the remaining issue of cloning being an inefficient process:

“If they [cloned sheep] could speak, they would say ‘yes; it’s perfectly safe. They’re perfectly healthy, and they’re old ladies now, and for them, their whole process worked perfectly. But there are others who struggled to adapt after birth.”

The STATNews piece also made a good point that further scientific studies on the cloned sheep need to be done to test for molecular signs of aging such as shortened telomeres, before the scientists can truly claim that these sheep are living normal healthy lives. The cloned sheep probably will live for another year at which point the scientists said they will conduct further experiments to look for other signs of aging at the cellular level.

CIRM Board targets diabetes and kidney disease with big stem cell research awards

diabetes2

A recent study  estimated there may be more than 500 million people worldwide who have diabetes. That’s an astounding figure and makes diabetes one of the largest chronic disease epidemics in human history.

One of the most serious consequences of untreated or uncontrolled diabetes is kidney damage. That can lead to fatigue, weakness, confusion, kidney failure and even death. So two decisions taken by the CIRM Board today were good news for anyone already suffering from either diabetes or kidney disease. Or both.

The Board awarded almost $10 million to Humacyte to run a Phase 3 clinical trial of an artificial vein needed by people undergoing hemodialysis – that’s the most common form of dialysis for people with kidney damage. Hemodialysis helps clean out impurities and toxins from the blood. Without it waste will build up in the kidneys with devastating consequences.

The artificial vein is a kind of bioengineered blood vessel. It is implanted in the individual’s arm and, during dialysis, is connected to a machine to move the blood out of the body, through a filter, and then back into the body. The current synthetic version of the vein is effective but is prone to clotting and infections, and has to be removed regularly. All this puts the patient at risk.

Humacyte’s version – called a human acellular vessel or HAV – uses human cells from donated aortas that are then seeded onto a biodegradable scaffold and grown in the lab to form the artificial vein. When fully developed the structure is then “washed” to remove all the cellular tissue, leaving just a collagen tube. That is then implanted in the patient, and their own stem cells grow onto it, essentially turning it into their own tissue.

In earlier studies Humacyte’s HAV was shown to be safer and last longer than current versions. As our President and CEO, Randy Mills, said in a news release, that’s clearly good news for patients:

“This approach has the potential to dramatically improve our ability to care for people with kidney disease. Being able to reduce infections and clotting, and increase the quality of care the hemodialysis patients get could have a significant impact on not just the quality of their life but also the length of it.”

There are currently almost half a million Americans with kidney disease who are on dialysis. Having something that makes life easier, and hopefully safer, for them is a big plus.

The Humacyte trial is looking to enroll around 350 patients at three sites in California; Sacramento, Long Beach and Irvine.

While not all people with diabetes are on dialysis, they all need help maintaining healthy blood sugar levels, particularly people with type 1 diabetes. That’s where the $3.9 million awarded to ViaCyte comes in.

We’re already funding a clinical trial with ViaCyte  using an implantable delivery system containing stem cell-derived cells that is designed to measure blood flow, detect when blood sugar is low, then secrete insulin to restore it to a healthy level.

This new program uses a similar device, called a PEC-Direct. Unlike the current clinical trial version, the PEC-Direct allows the patient’s blood vessels to directly connect, or vasularize, with the cells inside it. ViaCyte believes this will allow for a more robust engraftment of the stem cell-derived cells inside it and that those cells will be better able to produce the insulin the body needs.

Because it allows direct vascularization it means that people who get the delivery system  will also need to get chronic immune suppression to stop their body’s immune system attacking it. For that reason it will be used to treat patients with type 1 diabetes that are at high risk for acute complications such as severe hypoglycemic (low blood sugar) events associated with hypoglycemia unawareness syndrome.

In a news release Paul Laikind, Ph.D., President and CEO of ViaCyte, said this approach could help patients most at risk.

“This high-risk patient population is the same population that would be eligible for cadaver islet transplants, a procedure that can be highly effective but suffers from a severe lack of donor material. We believe PEC-Direct could overcome the limitations of islet transplant by providing an unlimited supply of cells, manufactured under cGMP conditions, and a safer, more optimal route of administration.”

The Board also approved more than $13.6 million in awards under our Discovery program. You can see the winners here.

 

CIRM-funded stem cell clinical trial for retinitis pigmentosa focuses on next stage

rp1

How retinitis pigmentosa erodes normal vision

The failure rate for clinical trials is depressingly high. A study from Tufts University in 2010  found that for small molecules – the substances that make up more than 90 percent of the drugs on the market today – the odds of getting from a Phase 1 trial to approval by the Food and Drug Administration are just 13 percent. For stem cell therapies the odds are even lower.

That’s why, whenever a stem cell therapy shows good results it’s an encouraging sign, particularly when that therapy is one that we at CIRM are funding. So we were more than a little happy to hear that Dr. Henry Klassen and his team at jCyte and the University of California, Irvine have apparently cleared the first hurdle with their treatment for retinitis pigmentosa (RP).

jCyte has announced that the first nine patients treated for RP have shown no serious side effects, and they are now planning the next phase of their Phase 1/2a safety trial.

In a news release Klassen, the co-founder of jCyte, said:

“We are pleased with the results. Retinitis pigmentosa is an incurable retinal disease that first impacts people’s night vision and then progressively robs them of sight altogether. This is an important milestone in our effort to treat these patients.”

The therapy involves injecting human retinal progenitor cells into one eye to help save the light sensing cells that are destroyed by the disease. This enables the researchers to compare the treated eye with the untreated eye to see if there are any changes or improvements in vision.

So far, the trial has undergone four separate reviews by the Data Safety Monitoring Board (DSMB), an independent group of experts that examines data from trials to ensure they meet all safety standards and that results show patients are not in jeopardy. Results from the first nine people treated are encouraging.

The approach this RP trial is taking has a couple of advantages. Often when transplanting organs or cells from one person into another, the recipient has to undergo some kind of immunosuppression, to stop their body rejecting the transplant. But earlier studies show that transplanting these kinds of progenitor cells into the eye doesn’t appear to cause any immunological response. That means patients in the study don’t have to undergo any immunosuppression. Because of that, the procedure is relatively simple to perform and can be done in a doctor’s office rather than a hospital. For the estimated 1.5 million people worldwide who have RP that could make getting treatment relatively easy.

Of course the big question now is not only was it safe – it appears to be – but does it work? Did any of those people treated experience improvements in their vision? We will share those results with you as soon as the researchers make them available.

Next step for the clinical trial is to recruit more patients, and treat them with a higher number of cells. There’s still a long way to go before we will know if this treatment works, if it either slows down, stops, or better still helps reverse some of the effects of RP. But this is a really encouraging first step.


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Salk Scientists Unlock New Secrets of Autism Using Human Stem Cells

Autism is a complex neurodevelopmental disorder whose mental, physical, social and emotional symptoms are highly variable from person to person. Because individuals exhibit different combinations and severities of symptoms, the concept of autism spectrum disorder (ASD) is now used to define the range of conditions.

There are many hypotheses for why autism occurs in humans (which some estimates suggest now affects around 3.5 million people in the US). Some of the disorders are thought to be at the cellular level, where nerve cells do not develop normally and organize properly in the brain, and some are thought to be at the molecular level where the building blocks in cells don’t function properly. Scientists have found these clues by using tools such as studying human genetics and animal models, imaging the brains of ASD patients, and looking at the pathology of ASD brains to see what has gone wrong to cause the disease.

Unfortunately, these tools alone are not sufficient to recreate all aspects of ASD. This is where cellular models have stepped in to help. Scientists are now developing human stem cell derived models of ASD to create “autism in a dish” and are finding that the nerve cells in these models show characteristics of these disorders.

Stem cell models of autism and ASD

We’ve reported on some of these studies in previous blogs. A group from UCSD lead by CIRM grantee Alysson Muotri used induced pluripotent stem cells or iPS cells to model non-syndromic autism (where autism is the primary diagnosis). The work has been dubbed the “Tooth Fairy Project” – parents can send in their children’s recently lost baby teeth which contain cells that can be reprogrammed into iPS cells that can then be turned into brain cells that exhibit symptoms of autism. By studying iPS cells from individuals with non-syndromic autism, the team found a mutation in the TRPC6 gene that was linked to abnormal brain cell development and function and is also linked to Rett syndrome – a rare form of autism predominantly seen in females.

Another group from Yale generated “mini-brains” or organoids derived from the iPS cells of ASD patients. They specifically found that ASD mini-brains had an increased number of a type of nerve cell called inhibitory neurons and that blocking the production of a protein called FOXG1 returned these nerve cells back to their normal population count.

Last week, a group from the Salk Institute in collaboration with scientists at UC San Diego published findings about another stem cell model for ASD that offers new clues into the early neurodevelopmental defects seen in ASD patients.  This CIRM-funded study was led by senior author Rusty Gage and was published last week in the Nature journal Molecular Psychiatry.

Unlocking clues to autism using patient stem cells

Gage and his team were fascinated by the fact that as many as 30 percent of people with ASD experience excessive brain growth during early in development. The brains of these patients have more nerve cells than healthy individuals of the same age, and these extra nerve cells fail to organize properly and in some cases form too many nerve connections that impairs their overall function.

To understand what is going wrong in early stages of ASD, Gage generated iPS cells from ASD individuals who experienced abnormal brain growth at an early age (their brains had grown up to 23 percent faster when they were toddlers compared to normal toddlers). They closely studied how these ASD iPS cells developed into brain stem cells and then into nerve cells in a dish and compared their developmental progression to that of healthy iPS cells from normal individuals.

Neurons derived from people with ASD (bottom) form fewer inhibitory connections (red) compared to those derived from healthy individuals (top panel). (Salk Institute)

Neurons derived from people with ASD (bottom) form fewer inhibitory connections (red) compared to those derived from healthy individuals (top panel). (Salk Institute)

They quickly observed a problem with neurogenesis – a term used to describe how brain stem cells multiply and create new nerve cells in the brain. Brain stem cells derived from ASD iPS cells displayed more neurogenesis than normal brain stem cells, and thus were creating an excess amount of nerve cells. The scientists also found that the extra nerve cells failed to form as many synaptic connections with each other, an essential process that allows nerve cells to send signals and form a functional network of communication, and also behaved abnormally and overall had less activity compared to healthy neurons. Interestingly, they saw fewer inhibitory neuron connections in ASD neurons which is contrary to what the Yale study found.

The abnormal activity observed in ASD neurons was partially corrected when they treated the nerve cells with a drug called IGF-1, which is currently being tested in clinical trials as a possible treatment for autism. According to a Salk news release, “the group plans to use the patient cells to investigate the molecular mechanisms behind IGF-1’s effects, in particular probing for changes in gene expression with treatment.”

Will stem cells be the key to understanding autism?

It’s clear that human iPS cell models of ASD are valuable in helping tease apart some of the mechanisms behind this very complicated group of disorders. Gage’s opinion is that:

“This technology allows us to generate views of neuron development that have historically been intractable. We’re excited by the possibility of using stem cell methods to unravel the biology of autism and to possibly screen for new drug treatments for this debilitating disorder.”

However, to me it’s also clear that different autism stem cell models yield different results, but these differences are likely due to which populations the iPS cells are derived from. Creating more cell lines from different ASD subpopulations will surely answer more questions about the developmental differences and differences in brain function seen in adults.

Lastly, one of the co-authors on the study, Carolina Marchetto, made a great point in the Salk news release by acknowledging that their findings are based on studying cells in a dish, not actual patient’s brains. However, Marchetto believes that these cells are useful tools for studying autism:

“It never fails to amaze me when we can see similarities between the characteristics of the cells in the dish and the human disease.”

Rusty Gage and Carolina Marchetto. (Salk Institute)

Rusty Gage and Carolina Marchetto. (Salk Institute)


Related Links

The Spanish Inquisition and a tale of two stem cell agencies

Monty

Monty Python’s Spanish Inquisition sketch: Photo courtesy Daily Mail UK

It’s not often an article on stem cell research brings the old, but still much loved, British comedy series Monty Python into the discussion but a new study in the journal Cell Stem Cell does just that, comparing the impact of CIRM and the UK’s Regenerative Medicine Platform (UKRMP).

The article, written by Fiona Watt of King’s College London and Stanford’s Irv Weissman (a CIRM grantee – you can see his impressive research record here) looks at CIRM and UKRMP’s success in translating stem cell research into clinical applications in people.

It begins by saying that in research, as in real estate, location is key:

“One thing that is heavily influenced by location, however, is our source of funding. This in turn depends on the political climate of the country in which we work, as exemplified by research on stem cells.”

And, as Weissman and Watt note, political climate can have a big impact on that funding. CIRM was created by the voters of California in 2004, largely in response to President George W. Bush’s restrictions on the use of federal funds for embryonic stem cell research. UKRMP, in contrast was created by the UK government in 2013 and designed to help strengthen the UK’s translational research sector. CIRM was given $3 billion to do its work. UKRMP has approximately $38 million.

Inevitably the two agencies took very different approaches to funding, shaped in part by the circumstances of their birth – one as a largely independent state agency, the other created as a tool of national government.

CIRM, by virtue of its much larger funding was able to create world-class research facilities, attract top scientists to California and train a whole new generation of scientists. It has also been able to help some of the most promising projects get into clinical trials. UKRMP has used its more limited funding to create research hubs, focusing on areas such as cell behavior, differentiation and manufacturing, and safety and effectiveness. Those hubs are encouraged to work collaboratively, sharing their expertise and best practices.

Weissman and Watt touch on the problems both agencies ran into, including the difficulty of moving even the best research out of the lab and into clinical trials:

“Although CIRM has moved over 20 projects into clinical trials most are a long way from becoming standard therapies. This is not unexpected, as the interval between discovery and FDA approved therapeutic via clinical trials is in excess of 10 years minimum.”

 

And here is where Monty Python enters the picture. The authors quote one of the most famous lines from the series: “Nobody expects the Spanish Inquisition – because our chief weapon is surprise.”

They use that to highlight the surprises and uncertainty that stem cell research has gone through in the more than ten years since CIRM was created. They point out that a whole category of cells, induced pluripotent stem (iPS) cells, didn’t exist until 2006; and that few would have predicted the use of gene/stem cell therapy combinations. The recent development of the CRISPR/Cas9 gene-editing technology shows the field is progressing at a rate and in directions that are hard to predict; a reminder that that researchers and funding agencies should continue to expect the unexpected.

With two such different agencies the authors wisely resist the temptation to make any direct comparisons as to their success but instead conclude:

“…both CIRM and UKRMP have similar goals but different routes (and funding) to achieving them. Connecting people to work together to move regenerative medicine into the clinic is an over-arching objective and one that, we hope, will benefit patients regardless of where they live.”

Scientists find new stem cell target for regenerating aging muscles

Young Arnold (wiki)

Young Arnold (wiki)

Today I’m going to use our former governor Arnold Schwarzenegger as an example of what happens to our muscles when we age.

One of Arnold’s many talents when he was younger was being a professional bodybuilder. As you can see in this photo, Arnold worked hard to generate an impressive amount of muscle that landed him lead roles in movies Conan the Barbarian and The Terminator.

Older Arnold

Older Arnold

If you look at pictures of Arnold now (who is now 68), while still being an impressively large human being, it’s obvious that much of his muscular bulk has diminished. That’s because as humans age, so do their muscles.

Muscles shrink with age

As muscles age, they slowly lose mass and shrink (a condition called sarcopenia) because of a number of reasons – one of them being their inability to regenerate new muscle tissue efficiently. The adult stem cells responsible for muscle regeneration are called satellite cells. When muscles are injured, satellite cells are activated to divide and generate new muscle fibers that can repair injury and also improve muscle function.

However, satellite cells become less efficient at doing their job over time because of environmental and internal reasons, and scientists are looking for new targets that can restore and promote the regenerative abilities of muscle stem cells for human therapeutic applications.

A study published earlier this week in Nature Medicine, identified a potential new target that could boost muscle stem cell regeneration and improved muscle function in a mouse model of Duchenne muscular dystrophy.

β1-integrin is important for muscle regeneration

Scientists from the Carnegie Institute of Washington found that β1-integrin is important for maintaining the homeostasis (or balance) of the muscle stem cell environment. If β1-integrin is doing its job properly, muscle stem cells are able to go about their regular routine of being dormant, activating in response to injury, dividing to create new muscle tissue, and then going back to sleep.

When the scientists studied the function of β1-integrin in the muscles of aged mice, they found that the integrin wasn’t functioning properly. Without β1-integrin, mouse satellite cells spontaneously turned into muscle tissue and were unable to maintain their regenerative capacity following muscle injury.

Upon further inspection, they found that β1-integrin interacts with a growth factor called fibroblast growth factor 2 (Fgf2) and this relationship was essential for promoting muscle regeneration following injury. When β1-integrin function deteriorates as in the muscles of aged mice, the mice lose sensitivity to the regenerative capacity of Fgf2.

Restoring muscle function in mice with muscular dystrophy

By using an antibody to artificially activate β1-integrin function in the muscles of aged mice, they were able to restore Fgf2 responsiveness and boosted muscle regeneration after injury. When a similar technique was used in mice with Duchenne muscular dystrophy, they observed muscle regeneration and improved muscle function.

Muscle loss seen in muscular dystrophy mice (left). Treatment with beta1 intern boosts muscle regeneration in the same mice (right). (Nature Medicine)

Muscle loss seen in muscular dystrophy mice (left). Treatment with B1-integrin boosts muscle regeneration in the same mice (right). (Nature Medicine)

The authors believe that β1-integrin acts as a sensor of the muscle stem cell environment that it maintains a balance between a dormant and a regenerative stem cell state. They conclude in their publication:

“β1-integrin senses the SC [satellite cell] niche to maintain responsiveness to Fgf2, and this integrin represents a potential therapeutic target for pathological conditions of the muscle in which the stem cell niche is compromised.”

Co-author on the study Dr. Chen-Ming Fan also spoke to the clinical relevance of their findings in a piece by GenBio:

“Inefficient muscular healing in the elderly is a significant clinical problem and therapeutic approaches are much needed, especially given the aging population. Finding a way to target muscle stem cells could greatly improve muscle renewal in older individuals.”

Does this mean anyone can be a body builder?

So does this study mean that one day we can prevent muscle loss in the elderly and all be body builders like Arnold? I highly doubt that. It’s important to remember these are preclinical studies done in mouse models and much work needs to be done to test whether β1-integrin is an appropriate therapeutic target in humans.

However, I do think this study sheds new light on the inner workings of the muscle stem cell environment. Finding out more clues about how to promote the health and regenerative function of this environment will bring the field closer to generating new treatments for patients suffering from muscle wasting diseases like muscular dystrophy.

T cell fate and future immunotherapies rely on a tag team of genetic switches

Imagine if scientists could build microscopic smart missiles that specifically seek out and destroy deadly, hard-to-treat cancer cells in a patient’s body? Well, you don’t have to imagine it actually. With techniques such as chimeric antigen receptor (CAR) T therapy, a patient’s own T cells – immune system cells that fight off viruses and cancer cells – can be genetically modified to produce customized cell surface proteins to recognize and kill the specific cancer cells eluding the patient’s natural defenses. It is one of the most exciting and promising techniques currently in development for the treatment of cancer.

Human T Cell (Wikipedia)

Human T Cell (Wikipedia)

Although there have been several clinical trial success stories, it’s still early days for engineered T cell immunotherapies and much more work is needed to fine tune the approach as well as overcome potential dangerous side effects. Taking a step back and gaining a deeper understanding of how stem cells specialize into T cells in the first place could go a long way into increasing the efficiency and precision of this therapeutic strategy.

Enter the CIRM-funded work of Hao Yuan Kueh and others in Ellen Rothenberg’s lab at CalTech. Reporting yesterday in Nature Immunology, the Rothenberg team uncovered a time dependent array of genetic switches – some with an ON/OFF function, others with “volume” control – that together control the commitment of stem cells to become T cells.

Previous studies have shown that the protein encoded by the Bcl11b gene is the key master switch that when activated sets a “no going back” path toward a T cell fate. A group of other genes, including Runx1, TCF-1 and GATA-3 are known to play a role in activating Bcl11b. The dominant school of thought is that these proteins gradually accumulate at the Bcl11b gene and once a threshold level is achieved, the proteins combine to enable the Bcl11b activation switch to flip on. However, other studies suggest that some of these proteins may act as “pioneer” factors that loosen up the DNA structure and allow the other proteins to readily access and turn on the Bcl11b gene. Figuring out which mechanism is at play is critical to precisely manipulating T cell development through genetic engineering.

To tease out the answer, the CalTech team engineered mice such that cells with activated Bcl11b would glow which allows visualizing the fate of single cells. We reached out to Dr. Kueh on the rationale for this experimental approach:

Hao Yuan Kueh, CalTech

Hao Yuan Kueh, CalTech

“To fully understand how genes are controlled, we need to watch them turn on and off in single, living cells over time.  As cells in our body are unique and different from one another, standard measurement methods, which average over millions of cells, often do not tell us the entire picture.”

The team examined the impact of inhibiting the T cell specific proteins GATA-3 and TCF-1 at different stages in T cell development in single cells. When the production of these two proteins were blocked in very early T cell progenitor (ETPs) cells, activation of Bcl11b was dramatically reduced. But that’s not what they observed when the experiment was repeated in a later stage of T cell development. In this case, blocking GATA-3 and TCF-1 had a much weaker impact on Bcl11b. So GATA-3 and TCF-1 are important for turning on Bcl11b early in T cell development but are not necessary for maintaining Bcl11b activation at later stages.

Inhibition of Runx1, on the other hand, did lead to a reduction in Bcl11b in these later T cell development stages. Making Runx1 levels artificially high conversely led to elevated Bcl11b in these cells.

Together, these results point to GATA-3 and TCF-1 as the key factors for turning on Bcl11b to commit cells to a T cell fate and then they hand off their duties to Runx1 to keep Bcl11b on and maintaining the T cell identity. Dr. Kuhn sums up the results and their implications this way:

“Our work shows that control of gene expression is very much a team effort, where some proteins flip the gene’s master ON-OFF switch, and others set its expression levels after it turns on…These results will help us generate customized T-cells to fight cancer and other diseases.  As T-cells are specialized to recognize and fight foreign agents in our body, this therapy strategy holds much promise for diseases that are difficult to treat with standard drug-based methods.  Also, these intricate gene regulation mechanisms are likely to be in play in other cell types in our body, not just T-cells, and so we believe our results will be widely relevant.”

From flies to mice: Improving stem cell therapy for degenerative eye diseases

Stem cell therapies for degenerative eye diseases sound promising – inject retinal progenitor cells derived from human pluripotent stem cells into the eye where they will integrate and replace damaged retinal tissue to hopefully restore sight. However, a significant road block is preventing these stem cell transplants from doing their job: the transplanted cells are unable to survive and generate healthy retinal tissue due to the unhealthy, degenerative environment they find themselves in.

A retina of a patient with macular degeneration. (Photo credit: Paul Parker/SPL)

A retina of a patient with macular degeneration. (Photo credit: Paul Parker/SPL)

In patients with age-related macular degeneration or retinitis pigmentosa, retinal tissue in the eye is in a state of inflammation initiated by innate immune cells such as macrophage-derived microglia. When activated, microglia can either promote an inflammatory response or resolve inflammation and promote tissue repair and regeneration.

This balance between a pro-inflammation and tissue regeneration is something that scientists are looking to manipulate in order to develop new potential therapeutic strategies for degenerative eye diseases.

Chapter 1: Identifying MANF in flies

In a paper published today in the journal Science, Buck researchers report that they have identified a natural immune system modulator called MANF that improved the success of retinal repair in both fly and mouse models of eye diseases, and enhanced retinal cell transplantation in mouse models of photoreceptor degeneration.

The story of MANF starts with Drosophila fruit flies grown in the lab of Buck Professor Dr. Heinrich Jasper. His lab studies hemocytes, the fly equivalent of blood cells, and the repair factors that they secrete in response to injury. To model retinal damage, Jasper and his lab exposed photoreceptors in the retina of flies to UV light and then screened for secreted proteins that were released by hemocytes in response to UV damage.

They identified a protein called a secreted protein called MANF and hypothesized that this factor could promote tissue regeneration and act as a neuroprotective, “retinal repair factor”.

In a Buck Institute news release, Jasper explained how further experiments showed that MANF was secreted by hemocytes in response to UV induced damage in the retina, and that it shifted these immune cells from promoting inflammation to reducing inflammation and promoting retinal regeneration.

Chapter 2: MANF is neuroprotective in mice

Deepak Lamba and his lab

Deepak Lamba and his lab

Part two of the story involved determining whether MANF had similar neuroprotective and anti-inflammatory properties in mammalian models. Dr. Deepak Lamba, Buck Professor and co-senior author on the study, took the lead and first tested whether MANF could reduce light-induced damage of photoreceptors in mouse models of retinal degeneration.

Injecting MANF protein into the eyes of these mice significantly reduced cell death caused by light exposure. Similarly, injection of fibroblast cells that secreted MANF also had a neuroprotective effect in the damaged retina by recruiting innate immune cells to promote the body’s natural repair mechanisms.

Chapter 3: MANF improves cell transplantation in mice

The final chapter involved testing whether MANF could improve the outcome of transplanted photoreceptor cells in blind mice genetically engineered to have retinal damage. The addition of MANF improved the survival and integration of the transplanted cells in the retinas of the mice and also improved the animals’ visual function.

Lamba concluded in a Buck news release that, “MANF promotes healing and helps create a microenvironment conducive to successful transplantation.”

These preliminary results in flies and mice are encouraging and Jasper believes that the neuroprotective effects of MANF could potentially be applied to other diseases of aging at an early stage that could prevent disease progression.

Heinrich Jasper

Heinrich Jasper

“Our hope is that MANF will be useful for treatment of inflammatory conditions in many disease contexts,” Jasper explained. “Focusing on immune modulation to promote a healthy repair response to tissue damage rather than a deleterious inflammatory response is a new frontier in aging research.”