CRISPR Gene Editing Tool Linked to Unexpected Collateral DNA Damage

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Photo Credit: Genetic Literacy Project

 

CRISPR–Cas9 has been widely hailed as the gene editing tool of the future. But research, published in the journal Nature Biotechnology,  about the effects of CRISPR/Cas9, have found it can cause unexpected genetic damage which could lead to dangerous changes in some cells.

Scientists have also learned there may be some safety implications for gene therapies that are being developed using CRISPR/Cas9.

These results come on the heels of a few studies published last month which suggested the CRISPR gene editing tool may inadvertently increase cancer risk in some cells.

“We found that changes in the DNA have been seriously underestimated before now,” said Allan Bradley, a professor at Britain’s Wellcome Sanger Institute who co-led the research published on Monday.

CRISPR/Cas9 can alter sections of DNA in cells by cutting at specific points and introducing changes at that location and is seen by many as a promising way to create treatments for diseases such as HIV or cancer.

Bradley’s team carried out a full systematic study in both mouse and human cells and discovered that CRISPR/Cas9 frequently caused extensive mutations including large genetic rearrangements such as DNA deletions and insertions.

These could lead to important genes being switched on or off – as intended by the therapies – but could also have major unexpected implications, the scientists said.

While experts say treatments like these could inactivate a disease-causing gene, or correct a genetic mutation, much more research is still needed to ensure techniques are safe.

Headline: Stem Cell Roundup: Here are some stem cell stories that caught our eye this past week.

In search of a miracle

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Luane Beck holds Jordan in the emergency room while he suffers a prolonged seizure. Jordan’s seizures sometimes occur one after another with no break, and they can be deadly without emergency care. Photo courtesy San Francisco Chronicle’s Kim Clark

One of the toughest parts of my job is getting daily calls and emails from people desperate for a stem cell treatment or cure for themselves or a loved one and having to tell them that I don’t know of any. You can hear in their voice, read it in their emails, how hard it is for them to see someone they love in pain or distress and not be able to help them.

I know that many of those people may think about turning to one of the many stem cell clinics, here in the US and in Mexico and other countries, that are offering unproven and unapproved therapies. These clinics are offering desperate people a sense of hope, even if there is no evidence that the therapies they provide are either safe or effective.

And these “therapies” come with a big cost, both emotional and financial.

The San Francisco Chronicle this week launched the first in a series of stories they are doing about stem cells and stem cell research, the progress being made and the problems the field still faces.

One of the biggest problems, are clinics that offer hope, at a steep price, but no evidence to show that hope is justified. The first piece in the Chronicle series is a powerful, heart breaking story of one mother’s love for her son and her determination to do all she can to help him, and the difficult, almost impossible choices she has to make along the way.

It’s called: In search of a miracle.

A little turbulence, and a French press-like device, can help boost blood platelet production

Every year more than 21 million units of blood are transfused into people in the US. It’s a simple, life-saving procedure. One of the most important elements in transfusions are  platelets, the cells that stop bleeding and have other healing properties. Platelets, however, have a very short shelf life and so there is a constant need to get more from donors. Now a new study from Japan may help fix that problem.

Platelets are small cells that break off much larger cells called megakaryocytes. Scientists at the Center for iPS Cell Research and Application (CiRA) created billions of megakaryocytes using iPS technology (which turns ordinary cells into any other kind of cell in the body) and then placed them in a bioreactor. The bioreactor then pushed the cells up and down – much like you push down on a French press coffee maker – which helped promote the generation of platelets.

In their study, published in the journal Cell, they report they were able to generate 100 billion platelets, enough to be able to treat patients.

In a news release, CiRA Professor Koji Eto said they have shown this works in mice and now they want to see if it also works in people:

“Our goal is to produce platelets in the lab to replace human donors.”

Stem Cell Photo of the Week 

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Students at the CIRM Bridges program practice their “elevator pitch”. Photo Kyle Chesser

This week we held our annual CIRM Bridges to Stem Cell Research conference in Newport Beach. The Bridges program provides paid internships for undergraduate and masters-level students, a chance to work in a world-class stem cell research facility and get the experience needed to pursue a career in science. The program is training the next generation of stem cell scientists to fill jobs in California’s growing stem cell research sector.

This year we got the students to practice an “elevator Pitch”, a 30 second explanation, in plain English, of what they do, why they do it and why people should care. It’s a fun exercise but also an important one. We want scientists to be able to explain to the public what they are doing and why it’s important. After all, the people of California are supporting this work so they have a right to know, in language they can understand, how their money is changing the face of medicine.

Starving stem cells of oxygen can help build stronger bones

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J. Kent Leach: Photo courtesy UC Davis

We usually think that starving something of oxygen is going to make it weaker and maybe even kill it. But a new study by J. Kent Leach at UC Davis shows that instead of weakening bone defects, depriving them of oxygen might help boost their ability to create new bone or repair existing bone.

Leach says in the past the use of stem cells to repair damaged or defective bone had limited success because the stem cells often didn’t engraft in the bone or survive long if they did. That was because the cells were being placed in an environment that lacked oxygen (concentration levels in bone range from 3% to 8%) so the cells found it hard to survive.

However, studies in the lab had shown that if you preconditioned mesenchymal stem cells (MSCs), by exposing them to low oxygen levels before you placed them on the injury site, you helped prolong their viability. That was further enhanced by forming the MSCs into three dimensional clumps called spheroids.

Lightbulb goes off

In the  current study, published in Stem Cells, Leach says the earlier spheroid results  gave him an idea:

“We hypothesized that preconditioning MSCs in hypoxic (low oxygen) culture before spheroid formation would increase cell viability, proangiogenic potential (ability to create new blood vessels), and resultant bone repair compared with that of individual MSCs.”

So, the researchers placed one group of human MSCs, taken from bone marrow, in a dish with just 1% oxygen, and another identical group of MSCs in a dish with normal oxygen levels. After three days both groups were formed into spheroids and placed in an alginate hydrogel, a biopolymer derived from brown seaweed that is often used to build cellular cultures.

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Brown seaweed

The team found that the oxygen-starved cells lasted longer than the ones left in normal oxygen, and the longer those cells were deprived of oxygen the better they did.

Theory is great, how does it work in practice?

Next was to see how those two groups did in actually repairing bones in rats. Leach says the results were encouraging:

“Once again, the oxygen-deprived, spheroid-containing gels induced significantly more bone healing than did gels containing either preconditioned individual MSCs or acellular gels.”

The team say this shows the use of these oxygen-starved cells could be an effective approach to repairing hard-to-heal bone injuries in people.

“Short‐term exposure to low oxygen primes MSCs for survival and initiates angiogenesis (the development of new blood vessels). Furthermore, these pathways are sustained through cell‐cell signaling following spheroid formation. Hypoxic (low oxygen) preconditioning of MSCs, in synergy with transplantation of cells as spheroids, should be considered for cell‐based therapies to promote cell survival, angiogenesis, and bone formation.”

CIRM & Dr. Leach

While CIRM did not fund this study we have invested more than $1.8 million in another study Dr. Leach is doing to develop a new kind of imaging technology that will help us see more clearly what is happening in bone and cartilage-targeted therapies.

In addition, back in March of 2012, Dr. Leach spoke to the CIRM Board about his work developing new approaches to growing bone.

 

For the first time, scientists entirely reprogram human skin cells to iPSCs using CRISPR

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CRISPR iPSC colony of human skin cells showing expression of SOX2 and TRA-1-60, markers of human embryonic pluripotent stem cells

Back in 2012, Shinya Yamanaka was awarded the Nobel Prize in Physiology or Medicine for his group’s identification of “Yamanaka Factors,” a group of genes that are capable of turning ordinary skin cells into induced pluripotentent stem cells (iPSCs) which have the ability to become any type of cell within the body. Discovery of iPSCs was, and has been, groundbreaking because it not only allows for unprecedented avenues to study human disease, but also has implications for using a patient’s own cells to treat a wide variety of diseases.

Recently, Timo Otonkoski’s group at the University of Helsinki along with Juha Kere’s group at the Karolinska Institutet and King’s College, London have found a way to program iPSCs from skin cells using CRISPR, a gene editing technology. Their approach allows for the induction, or turning on of iPSCs using the cells own DNA, instead of introducing the previously identified Yamanka Factors into cells of interest.

As detailed in their study, published in the journal Nature Communications, this is the first instance of mature human cells being completely reprogrammed into pluripotent cells using only CRISPR. Instead of using the canonical CRISPR system that allows the CAS9 protein (an enzyme that is able to cut DNA, thus rendering a gene of interest dysfunctional) to mutate any gene of interest, this group used a modified version of the CAS9 protein, which allows them to turn on or off the gene that CAS9 is targeted to.

The robustness of their approach lies in the researcher’s identification of a DNA sequence that is commonly found near genes involved in embryonic development. As CAS9 needs to be guided to genes of interest to do its job, identification of this common motif allows multiple genes associated with pluripotency to be activated in mature human skin cells, and greatly increased the efficiency and effectiveness of this approach.

In a press release, Dr. Otonkoski further highlights the novelty and viability of this approach:

“…Reprogramming based on activation of endogenous genes rather than overexpression of transgenes is…theoretically a more physiological way of controlling cell fate and may result in more normal cells…”

 

Gene-editing Technique in Mice Shows Promise for Genetic Disorder in Utero

 

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New research presents a promising new avenue for research into treating genetic conditions during fetal development.
Credit: © llhedgehogll / Fotolia

Each year roughly 16 million parents receive the heartbreaking news that their child is likely to be born with a severe genetic disorder or birth defect. And while these genetic conditions can often be detected during pregnancy, using amniocentesis, there haven’t been any treatment options to correct these genetic conditions before birth. Well – thanks to a group of researchers at Carnegie Mellon University and Yale University that could one day change and offer alternative treatment options for children with genetic disorders while they are still in the womb.

For the first time ever, according to a Carnegie Melon press release, scientists used a gene editing technique to successfully cure a genetic condition in a mouse in utero. Their findings, published in Nature Communications, not only present a promising new avenue for research into treating genetic conditions, but they also open the doors for additional treatment options in the future.

In this study, the researchers used a synthetic molecule called a peptide nucleic acid (PNA) as the basis for a gene editing technique. They had previously used this method to cure beta-thalassemia, a genetic blood disorder that results in the reduced production of hemoglobin, in adult mice. Their technique uses an FDA-approved nanoparticle to deliver PNA molecules, paired with donor DNA, to the site of a genetic mutation. When the PNA-DNA complex identifies a designated mutation, the PNA molecule binds to the DNA and unzips its two strands. The donor DNA then binds with the faulty DNA and spurs the cell’s DNA repair pathways into action, correcting the error.

The researchers believe that their technique might even be able to achieve higher success rates if they can administer it multiple times during gestation. They also hope to see if their technique can be applied to other conditions.

While this research is promising there is a long way to go before the team will be ready to test it in people. However, one CIRM-supported project has already reached that milestone. Dr. Tippi MacKenzie and her team at UCSF are using in utero blood stem cell transplants from the mother to the fetus to help treat alpha thalassemia major, a blood disorder that is almost always fatal.

We recently blogged about this research and how it helped one couple deliver a healthy baby.

https://blog.cirm.ca.gov/2018/06/04/cirm-funded-study-results-in-the-first-ever-in-utero-stem-cell-transplant-to-treat-alpha-thalassemia/

 

 

Stem cell gene therapy combination could help children battling a rare genetic disorder

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A child with Hunter Syndrome

Hunter syndrome is devastating. It’s caused by a single enzyme, IDS, that is either missing or malfunctioning. Without the enzyme the body is unable to break down complex sugar molecules and as those build up they cause permanent, progressive damage to the body and brain and, in some instances, result in severe mental disabilities. There is no cure and existing treatments are limited and expensive.

But now researchers at the University of Manchester in England have developed an approach that could help children – the vast majority of them boys – suffering from Hunter syndrome.

Working with a mouse model of the disease the researchers took some blood stem cells from the bone marrow and genetically re-engineered them to correct the mutation that caused the problem. They also added a “tag” to the IDS enzyme to help it more readily cross the blood brain barrier and deliver the therapy directly to the brain.

In a news release Brian Bigger, the lead researcher of the study published in EMBO Molecular Medicine, said the combination therapy helped correct bone, joint and brain disease in the mice.

“We expected the stem cell gene therapy approach to deliver IDS enzyme to the brain, as we have shown previously for another disease: Sanfilippo types A and B, but we were really surprised to discover how much better the tag made the therapy in the brain. It turns out that the tag didn’t only improve enzyme uptake across the blood brain barrier, but also improved uptake of the enzyme into cells and it appeared to be more stable in the bloodstream – all improvements on current technology.”

While the results are very encouraging it is important to remember the experiment was done in mice. So, the next step is to see if this might also work in people.

Joshua Davies has made a video highlighting the impact Hunter syndrome has on families: it’s called ‘Living Beyond Hope’

CIRM Supported Scientist Makes Surprising Discovery with Parasitic Gut Worms

 

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Image of gut lining and parasites.  Photo courtesy of UCSF/ Michael Fortes

 

It’s no secret that researchers have long believed adult stem cells could contribute to wound healing in the gut and skin, but in a new paper in Nature — a group of scientists at UC San Francisco made a surprising discovery.

Through several experiments using parasitic worms in the mouse gut, they found that as parasites dug into the intestinal walls of mice, the gut responded in an unexpected way – by reactivating a type of cell growth previously seen in fetal tissues.

So why is this important?

Simply put, it gives scientists new targets to go after. According to UCSF CIRM supported scientist Ophir Klein, MD, Ph.D., this discovery could be paradigm-shifting in terms of our understanding of how the mammalian body can repair damage and could help scientists develop more ways to enhance the body’s natural healing abilities.

Adult stem cells in the intestines are vital for maintaining the digestive status quo. The gut lining is made up of epithelial cells which absorb nutrients and produce protective mucus. These cells are replaced every few days by the stem cells at the base of crypts — indentations in the gut lining. Researchers expected that the same stem cells could also help repair tears in the gut.

How did they do it?

Larvae from parasites like H. polygyrus invade the gut lining in a mouse’s intestine, burying themselves to develop in the tissue. Based on prevailing ideas in the field, the scientists predicted that, in response, nearby stem cells would increase their productivity and patch up the worm-created wounds, but that is not what happened.

Instead, signs of the stem cells in worm-infected areas disappeared entirely; fluorescent markers that should have been expressed by one of the genes in the regular stem cell program completely vanished. And yet, even with no identifiable stem cells in the area, the wounded tissue regenerated more quickly than ever.

Researchers spent years trying to resolve this mystery and after a number of false starts and dead ends, the team eventually noticed the recurrence of a different gene, known as Sca-1.

Using antibody staining for the Sca-1 protein, the researchers realized that where the stem cell genes had disappeared, a different gene program was expressed instead: one that resembled the way that mouse guts develop in utero.

Upon their discovery, the researchers wondered whether the reactivation of this fetal program was a specific response to parasite infections, or if it could be a general strategy for many kinds of gut injury. Additional experiments showed that shutting down gut stem cells with irradiation or genetically targeting them for destruction triggered aspects of the same response: despite an absence of detectable stem cell activity, undifferentiated tissue grew rapidly nonetheless.

Later, once the acute injury is repaired, the gut may return to the normal stem cell program of producing differentiated cells that perform specific functions.

Many other injured tissues could benefit from the ability to quickly and efficiently make generalized repairs before returning to specialized adult cell production, opening up therapeutic opportunities. For example, developing treatments that bestow an ability to control the change between adult and fetal genetic programs might offer new strategies to manage conditions such as inflammatory bowel disease (IBD).

Early CIRM support helps stem cell pioneer develop promising new therapy for cancer

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Irv Weissman, Ph.D., Photo: courtesy Stanford University

When you get praise from someone who has been elected to the National Academy of Sciences and has been named California Scientist of the Year you know you must be doing something right.

That’s how we felt the other day when Irv Weissman, Director of the Stanford Institute of Stem Cell Biology and Regenerative Medicine, issued a statement about how important the support of CIRM was in advancing his research.

The context was the recent initial public offering (IPO) of Forty Seven Inc.. a company co-founded by Dr. Weissman. That IPO followed news that two Phase 2 clinical trials being run by Forty Seven Inc. were demonstrating promising results against hard-to-treat cancers.

Dr. Weissman says the therapies used a combination of two monoclonal antibodies, 5F9 from Forty Seven Inc. and Rituximab (an already FDA-approved treatment for cancer and rheumatoid arthritis) which:

“Led to about a 50% overall remission rate when used on patients who had relapsed, multi-site disease refractory to rituximab-plus-chemotherapy. Most of those patients have shown a complete remission, although it’s too early to tell if this is complete for life.”

5F9 attacks a molecule called CD47 that appears on the surface of cancer cells. Dr. Weissman calls CD47 a “don’t eat me signal” that protects the cancer against the body’s own immune system. By blocking the action of CD47, 5F9 strips away that “don’t eat me signal” leaving the cancer vulnerable to the patient’s immune system. We have blogged about this work here and here.

The news from these trials is encouraging. But what was gratifying about Dr. Weissman’s statement is his generosity in sharing credit for the work with CIRM.

Here is what he wrote:

“What is unusual about Forty Seven is that not only the discovery, but its entire preclinical development and testing of toxicity, etc. as well as filing two Investigational New Drug [IND] applications to the Food and Drug Administration (FDA) in the US and to the MHRA in the UK, as well as much of the Phase 1 trials were carried out by a Stanford team led by two of the discoverers, Ravi Majeti and Irving Weissman at Stanford, and not at a company.

The major support came from the California Institute of Regenerative Medicine [CIRM], funded by Proposition 71, as well as the Ludwig Cancer Research Foundation at the Ludwig Center for Cancer Stem Cell Research at Stanford. CIRM will share in downstream royalties coming to Stanford as part of the agreement for funding this development.

This part of the state initiative, Proposition 71, is highly innovative and allows the discoverers of a field to guide its early phases rather than licensing it to a biotech or a pharmaceutical company before the value and safety of the discovery are sufficiently mature to be known. Most therapies at early-stage biotechs are lost in what is called the ‘valley of death’, wherein funding is very difficult to raise; many times the failure can be attributed to losing the expertise of the discoverers of the field.”

Dr. Weissman also had praise for CIRM’s funding model which requires companies that produce successful, profitable therapies – thanks to CIRM support – to return a portion of those profits to California. Most other funding agencies don’t have those requirements.

“US federal funds, from agencies such as the National Institutes of Health (NIH) similarly support discovery but cannot fund more than a few projects to, and through, early phase clinical trials. And, under the Bayh-Dole Act, the universities keep all of the equity and royalties derived from licensing discoveries. In that model no money flows back to the agency (or the public), and nearly a decade of level or less than level funding (at the national level) has severely reduced academic research. So this experiment of funding (the NIH or the CIRM model) is now entering into the phase that the public will find out which model is best for bringing new discoveries and new companies to the US and its research and clinical trials community.”

We have been funding Dr. Weissman’s work since 2006. In fact, he was one of the first recipients of CIRM funding.  It’s starting to look like a very good investment indeed.

 

Video: Behind the scenes of a life-saving gene therapy stem cell treatment

“We were so desperate. When we heard about this treatment were willing to do anything to come here.”

In the above quote from Zahraa El Kerdi, “here” refers to UCLA, a world away from her hometown in Lebanon. In September 2015, Zahree gave birth to a son, Hussein, who appeared perfectly healthy. But by six months, he was barely clinging to life due to an inherited blood disorder, ADA-SCID, also called Bubble Baby disease. The disorder left Hussein without a functioning immune system so even a common cold could prove deadly. In fact, SCID babies rarely survive past one year of age. Up until now, no treatment options existed for the disease.

But Zahraa and her husband Ali heard about a CIRM-funded clinical trial, led by Donald Kohn, M.D. at UCLA, that could modify Hussein’s blood stem cells to fix the gene problem that’s causing his disease. The El Kerdi’s 7500-mile journey to save Hussein’s life is captured in a wonderful, five-minute video produced by UCLA’s Broad Stem Cell Research Center.

With before and after scenes of Hussein’s treatment as well as animation describing how the therapy works, the short documentary is equal parts heart wrenching, uplifting and educational. Basically, what I’m trying to say is, it’s a must-see and available to view above.

CIRM Board invests in new approaches to brain cancer and Parkinson’s disease

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Parkinson’s disease and glioblastoma are very different diseases of the brain but neither has very good treatment options and both clearly represent an unmet medical need. With that in mind, the governing Board of the California Institute for Regenerative Medicine (CIRM), the state’s stem cell agency, yesterday voted to invest almost $9.5 million in developing new approaches to both conditions.

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John Zaia, M.D., at City of Hope, was awarded $3.68 million to do the late stage preclinical research needed to apply to the US Food and Drug Administration (FDA) to run a clinical trial targeting glioblastoma.

There is no cure for glioblastoma, the deadliest form of brain cancer. The standard treatment involves surgery, chemotherapy and radiation but even then, the median survival time is only around 15 months (meaning half the patients survive this long). Because chemotherapy can cause toxic side effects in the blood the strength of the dose that patients get is often limited. The City of Hope team plans to both genetically engineer the patient’s own blood stem cells to protect them from chemotherapy and sensitize the tumor cells to make them more vulnerable to the chemotherapy. This will hopefully enable patients to get a higher dose of chemotherapy and improve survival time and quality of life.

Dr. Maria Millan, CIRM’s President & CEO, says even people who have access to the best treatment rarely do well with this form of cancer:

“Glioblastoma is the most common, and the most aggressive, form of brain cancer that led to the death of U.S. Senator Ted Kennedy and former Vice President Joe Biden’s son Beau Biden. CIRM has supported a variety of stem cell-based approaches to target this devastating and currently untreatable condition.  The project approved by our Board today is unique in that it seeks to use gene modified stem cells to allow patients to tolerate the high doses of chemotherapy while also making these tumors more susceptible to the chemotherapy.”

The CIRM Board also awarded $5.8 million to Krystof Bankiewicz, M.D., Ph.D., at the University of California, San Francisco (UCSF).  In collaboration with Clive Svendsen, Ph.D. at Cedars-Sinai, this team is testing the potential for neural progenitor cells, engineered to express the growth factor GDNF, to impact Parkinson’s disease. With CIRM funding, the investigators will perform pre-clinical research that is aimed at enabling them to file an application with the FDA to test this approach in a clinical trial.

Dr. Millan noted that the cells used in this project are already being used in a CIRM-funded clinical trial:

“CIRM is currently funding a Phase 1 clinical trial at Cedars-Sinai with this neural progenitor cell product for the treatment of ALS, another devastating neurodegenerative disorder for which there is no cure.”

David Higgins

David Higgins, PhD, the CIRM Board Patient Advocate for Parkinson’s disease says there is a real need for something that can have a big impact on the disease:

“One of the big frustrations for people with Parkinson’s, and their families and loved ones, is that existing therapies only address the symptoms and do little to slow down or even reverse the progress of the disease. That’s why it’s important to support any project that has the potential to address Parkinson’s at a much deeper, longer-lasting level.”