First-In-Human Clinical Trial Aims to Extend Remission for Children and Young Adults With Leukemia Treated With T-Cell Immunotherapy

Phase 1 pilot study utilizes T-cell antigen presenting cells to prolong the persistence of cancer-fighting chimeric antigen receptor (CAR) T cells, reduce the relapse rate

After phase 1 results of Seattle Children’s Pediatric Leukemia Adoptive Therapy (PLAT-02) trial have shown T-cell immunotherapy to be effective in getting  93 percent of patients with relapsed or refractory acute lymphoblastic leukemia (ALL) into complete initial remission, researchers have now opened a first-in-human clinical trial aimed at reducing the rate of relapse after the therapy, which is about 50 percent. The new phase 1 pilot study, PLAT-03, will examine the feasibility and safety of administering a second T-cell product intended to increase the long-term persistence of the patient’s chimeric antigen receptor (CAR) T cells that were reprogrammed to detect and destroy cancer.

The research team, led by Dr. Mike Jensen at the Ben Towne Center for Childhood Cancer Research at Seattle Children’s Research Institute, is exploring this strategy after discovering that of the patients who relapse in the PLAT-02 trial, about half of them have lost their CAR T cells. Lasting persistence of the CAR T cells is critical in combating a recurrence of cancer cells.

“While it’s promising that we’re able to get these patients who are very sick back into remission, we’re also seeing that the loss of the CAR T cells in some patients may be opening the door for the cancer to return,” said Dr. Colleen Annesley, an oncologist at Seattle Children’s and the lead investigator of the PLAT-03 trial. “We’re pleased to now be able to offer patients who have lost or are at risk of losing their cancer-fighting T cells an option that will hopefully lead to them achieving long-term remission.”

In the PLAT-03 trial, patients will receive “booster” infusions of a second T-cell product, called T antigen-presenting cells (T-APCs). The T-APCs have been genetically modified to express the CD19 target for the cancer-fighting CAR T cells to recognize. Patients will receive a full dose of T-APCs every 28 days for at least one and up to six doses. By stimulating the CAR T cells with a steady stream of target cells to attack, researchers hope the CAR T cells will re-activate, helping to ensure their persistence long enough to put patients into long-term remission.

PLAT-03 is now open to patients who first enroll in phase 2 of Seattle Children’s PLAT-02 trial and who are also identified as being at risk for early loss of their reprogrammed CAR T cells, or those who lose their reprogrammed CAR T cells within six months of receiving them.

The PLAT-03 trial is one of several trials that Seattle Children’s researchers are planning to open within the next year aimed at further improving the long-term efficacy of T-cell immunotherapy. In addition to the current T-cell immunotherapy trial that is open for children with neuroblastoma, researchers also hope to expand this promising therapy to other solid tumor cancers.

“We are pleased to be at a pivotal point where we are now looking at several new strategies to further improve CAR T-cell immunotherapy so it remains a long-term defense for all of our patients,” said Dr. Rebecca Gardner, Seattle Children’s oncologist and the lead investigator of the PLAT-02 trial. “We’re also excited to be working to apply this therapy to several other forms of pediatric cancer beyond ALL, with the hope that T-cell immunotherapy becomes a first line of defense, reducing the need for toxic therapies and minimizing the length of treatment to only weeks.”

To read about the experience of one of the patients in the PLAT-02 trial, please visit Seattle Children’s On the Pulse blog.

The T-cell immunotherapy trials at Seattle Children’s are funded in part by Strong Against Cancer, a national philanthropic initiative with worldwide implications for potentially curing childhood cancers. If you are interested in supporting the advancement of immunotherapy and cancer research, please visit Strong Against Cancer’s donation page.

For more information on immunotherapy research trials at Seattle Children’s, please call (206) 987-2106 or email immunotherapy@seattlechildrens.org.

For Women at Risk of Hereditary Breast Cancer, Multigene Test Could Help Extend Life Expectancy

Value in Health, the official journal of the International Society for Pharmacoeconomics and Outcomes Research (ISPOR), announced today the publication of new research indicating that testing for variants in 7 cancer-associated genes (versus the usual process of testing in just 2 genes) followed by risk-reduction management could cost-effectively improve life expectancy for women at risk of hereditary breast cancer. The report of these findings, A Multigene Test Could Cost-Effectively Help Extend Life Expectancy for Women at Risk of Hereditary Breast Cancer, was published in the April 2017 issue.

Using hypothetical cohorts of women at risk of hereditary breast cancer, the authors used a decision-analytic model to compare the relative cost and effectiveness of two test strategies for detecting pathogenic genetic variants: 1) the usual BRCA1/2 test strategy, and 2) a next-generation 7-gene strategy that tests for variants not only in BRCA1 and BRCA2, but also in TP53, PTEN, CDH1, STK11, and PALB2. The authors then used these test results to select appropriate breast cancer risk reduction treatments / therapies.

In the base-case scenario for 50- and 40-year-old women undergoing genetic testing, the incremental cost-effectiveness ratio (ICER) for the 7-gene test strategy compared with the BRCA1/2 test strategy was $42,067 and $23,734 per life-year gained, or $69,920 and $48,328 per quality-adjusted life-year gained, respectively. At these ICER levels, the 7-gene test strategy would be considered cost effective according to the World Health Organization guidelines.

“Pathogenic variants in the BRCA1 and BRCA2 genes explain only about 15% of the breast cancer familial relative risk,” said lead author Yonghong Li, PhD, Quest Diagnostics, USA, “while pathogenic variants in other genes, including TP53, PTEN, CDH1, and PALB2 contribute further to the familial relative risk. The results of this study,” Dr. Li added, “demonstrate the potential value of newer testing options that allow for the simultaneous analysis of expanded panels of additional genes whose pathogenic variants confer moderate to high risk for breast cancer.”

Success of Sensory Cell Regeneration Raises Hope for Hearing Restoration

In an apparent first, St. Jude Children’s Research Hospital investigators have used genetic manipulation to regenerate auditory hair cells in adult mice. The research marks a possible advance in treatment of hearing loss in humans. The study appears today in the journal Cell Reports.

Loss of auditory hair cells due to prolonged exposure to loud noise, accidents, illness, aging or medication is a leading cause of hearing loss and long-term disability in adults worldwide. Some childhood cancer survivors are also at risk because of hair cells damage due to certain chemotherapy agents. Treatment has focused on electronic devices like hearing aids or cochlear implants because once lost, human auditory hair cells do not grow back.

“In this study, we looked to Mother Nature for answers and we were rewarded,” said corresponding author Jian Zuo, Ph.D., a member of the St. Jude Department of Developmental Neurobiology. “Unlike in humans, auditory hair cells do regenerate in fish and chicken. The process involves down-regulating expression of the protein p27 and up-regulating the expression of the protein Atoh1. So we tried the same approach in specially bred mice.”

By manipulating the same genes, Zuo and his colleagues induced supporting cells located in the inner ear of adult mice to take on the appearance of immature hair cells and to begin producing some of the signature proteins of hair cells.

The scientists also identified a genetic pathway for hair cell regeneration and detailed how proteins in that pathway cooperate to foster the process. The pathway includes the proteins GATA3 and POU4F3 along with p27 and ATOH1. In fact, investigators found that POU4F3 alone was sufficient to regenerate hair cells, but that more hair cells were regenerated when both ATOH1 and POU4F3 were involved.

“Work in other organs has shown that reprogramming cells is rarely accomplished by manipulating a single factor,” Zuo said. “This study suggests that supporting cells in the cochlea are no exception and may benefit from therapies that target the proteins identified in this study.”

The findings have implications for a phase 1 clinical trial now underway that uses gene therapy to restart expression of ATOH1 to regenerate hair cells for treatment of hearing loss.

ATOH1 is a transcription factor necessary for hair cell development. In humans and other mammals, the gene is switched off when the process is complete. In humans, ATOH1 production ceases before birth.

“This study suggests that targeting p27, GATA3 and POU4F3 may enhance the outcome of gene therapy and other approaches that aim to restart ATOH1 expression,” Zuo said.

The research also revealed a novel role for p27. The protein is best known as serving as a check on cell proliferation. However, in this study p27 suppressed GATA3 production. Since GATA3 and ATOH1 work together to increase expression of POU4F3, reducing GATA3 levels also reduced expression of POU4F3. When the p27 gene was deleted in mice, GATA3 levels increased along with expression of POU4F3. Hair cell regeneration increased as well.

“Work continues to identify the other factors, including small molecules, necessary to not only promote the maturation and survival of the newly generated hair cells, but also increase their number,” Zuo said.

Rare Type of Immune Cell Responsible for Progression of Heart Inflammation to Heart Failure in Mice

A new study in mice reveals that eosinophils, a type of disease-fighting white blood cell, appear to be at least partly responsible for the progression of heart muscle inflammation to heart failure in mice.

In a report on the findings, published in The Journal of Experimental Medicine on March 16, researchers found that while eosinophils are not required for heart inflammation to occur, they are needed for it to progress to a condition known as inflammatory dilated cardiomyopathy (DCMi) in mice. The discovery, they say, advances information about the impact of eosinophils on heart function.

“Other studies have shown that people with high levels of eosinophils develop a number of heart diseases. This new work has provided more details about how these immune system cells may lead to deterioration of heart muscle function in mice in a way that lets us draw some parallels to human disease processes,” says Daniela Cihakova, M.D., Ph.D., associate professor of pathology at the Johns Hopkins University School of Medicine and the paper’s senior author.

Heart inflammation, or myocarditis, is rarely diagnosed because it doesn’t always cause severe symptoms and it requires a biopsy to be taken from the patient’s heart. This makes it difficult to study the outcomes of the disease. “We don’t understand why the hearts of some people will heal while those of others develop chronic disease,” says Cihakova.

Different types of myocarditis are distinguished based on the type of immune cell that predominates the inflammation of the heart. For example, in eosinophilic myocarditis, numerous eosinophils infiltrate the heart. It is not known if some types of myocarditis are more likely to progress to DCMi than others. “Our studies show that the presence of eosinophils in the heart makes mice more likely to get DCMi following myocarditis. And if there are a lot of eosinophils, the mice develop even more severe heart failure,” says Nicola Diny, a Ph.D. student in the Bloomberg School of Public Health and the study’s first author. “It will be important to test if the same is true in patients. That way, we may be able to intervene early and prevent DCMi.”

This study, says Cihakova, is the first to examine the role eosinophils play in the development and severity of heart inflammation, and the subsequent progression of inflammation to DCMi. The study addresses a National Institutes of Health-identified need for preclinical models and a clearer understanding of how eosinophils drive heart damage.

For the study, Cihakova and her team first induced myocarditis in two groups of mice: normal mice and a group of mice genetically modified to be eosinophil-deficient. Myocarditis was induced through a process called experimental autoimmune myocarditis, in which mice are immunized with a peptide from heart muscle cells to initiate an immune response against the heart. After 21 days, the researchers found similar levels of acute inflammation in the hearts of both groups by studying the hearts’ tissue. But when the team checked the mice’s hearts later on for evidence of heart failure, the differences between the eosinophil-deficient and the normal mice were striking. The normal mice developed heart failure, while the eosinophil-deficient mice showed no signs of reduced heart function.

“These surprising results told us that it is not the overall severity of inflammation but rather the types of immune cells in the heart that decide whether myocarditis develops into heart failure,” says Diny.

The researchers also examined the hearts for fibrosis, or scar tissue, which develops when mammalian (including human) heart muscles die. This type of scar tissue is also found in DCMi. Although both groups of mice had similar degrees of scar tissue, the eosinophil-deficient mice’s heart functions weren’t negatively affected, while the normal mice developed DCMi.

“This told us that in the absence of eosinophils, heart function can be preserved despite scar tissue formation,” Cihakova says. “It’s also important to note that although eosinophils accounted for just 1 to 3 percent of all heart-infiltrating cells in normal mice, this small percentage can still drive heart failure.”

In another set of experiments, the research team used genetically modified mice, called IL5Tg mice, which have an excess of the protein IL5 that causes the body to make eosinophils. The IL5Tg mice had more inflammation in the atria, or upper chambers of the heart, compared to normal mice in the acute stage and more atrial scar tissue in the chronic stage. IL5Tg mice also had more heart-infiltrating cells in general. Eosinophils accounted for more than 60 percent of heart-infiltrating cells in the IL5Tg mice’s hearts, compared to only 3 percent in normal mice. When the team examined the heart function some 45 days after the start of the experiment, the IL5Tg mice had developed severe DCMi.

To examine whether humans with eosinophil-driven myocarditis also developed inflammation in the atria, the researchers obtained heart tissue samples and cardiac MRI scans from three patients seen at The Johns Hopkins Hospital, all of whom had confirmed eosinophil-driven inflammation.

The images showed that two patients had either inflammation or scar tissue in the atria, which suggests that atrial inflammation and/or scar tissue may also be a feature in humans with eosinophil-driven inflammation, Cihakova says.

To determine whether the IL5 protein is necessary for DCMi development, the research team next examined IL5-deficient mice. The scientists found that they had both inflammation and DCMi severity similar to that of normal mice, suggesting that the IL5 protein is not necessary for DCMi to develop.

Finally, to confirm the differences between the effects of IL5 and eosinophils, the team bred the eosinophil-deficient mice to have excess IL5. Compared to normal mice, these mice showed no decrease in heart function and appeared completely protected from DCMi, which confirms that it is the eosinophils themselves, not high levels of IL5, that are responsible for DCMi development, the investigators say.

To learn more about how eosinophils might drive DCMi progression, the investigators built on the knowledge that eosinophils harbor granules, some of which can kill cells, while others change the function of cells.

“We didn’t see any differences in cell death between the normal mice and those with or without too many eosinophils, so we became interested in the molecules that can change the function of other cells,” says Diny.

In particular, one protein, called IL4, caught the researchers’ attention. Other studies had shown that IL4 made by eosinophils has diverse functions in liver repair and fat tissue. “We wondered if this protein from eosinophils may also be important in the heart,” Cihakova says.

First, the research team used a mouse in which cells that make IL4 turned fluorescent green, thereby allowing researchers to tell where IL4 is made. The team found that eosinophils accounted for the majority of IL4-producing cells. When they used mice that lacked IL4 in all cells, these mice were completely protected from DCMi, just like the eosinophil-deficient mice.

Finally, to determine whether IL4 specifically from eosinophils is necessary for DCMi development, the team used genetically modified mice with no IL4 in their eosinophils but with IL4 in other heart-infiltrating cells. These mice developed less severe DCMi compared to normal mice, which confirms that eosinophils are responsible for DCMi development through IL4.

“The take-home message is that inflammation severity doesn’t necessarily determine long-term disease progression, but specific infiltrating cell types — eosinophils, in this case — do,” says Cihakova. Because eosinophil-driven inflammation is so clinically rare, the percentage of people who develop DCMi is unknown, she notes.

While no drugs are currently available to stop or delay the development of DCMi, the researchers hope their findings will help establish a novel target for IL4-blocking medicines that might be used to treat people with myocarditis, possibly preventing disease progression and the need for heart transplantation.

16 Aplastic Anemia Patients Free Of Disease After Bone Marrow Transplant and Chemo

Physicians at the Johns Hopkins Kimmel Cancer Center report they have successfully treated 16 patients with a rare and lethal form of bone marrow failure called severe aplastic anemia using partially matched bone marrow transplants followed by two high doses of a common chemotherapy drug. In a report on the new transplant-chemo regimen, published online Dec. 22, 2016, in Biology of Blood and Marrow Transplantation, the Johns Hopkins team says that more than a year after their transplants, all of the patients have stopped taking immunosuppressive drugs commonly used to treat the disorder and have no evidence of the disease.

“Our findings have the potential to greatly widen treatment options for the vast majority of severe aplastic anemia patients,” according to Robert Brodsky, M.D., professor of medicine and oncology at the Johns Hopkins Kimmel Cancer Center and an author of the report.

Results of the small clinical trial have already prompted the organization of a larger national trial being led by Amy DeZern, M.D., an assistant professor of oncology and medicine at the Johns Hopkins Kimmel Cancer Center, with plans to involve patients at 25 medical centers across the country.

Diagnosed in about one in 250,000 people each year, aplastic anemia occurs when one’s own immune system damages blood-making bone marrow cells, which gradually stop producing red and white blood cells and platelets.

Patients must receive frequent blood transfusions, take multiple medicines to suppress the autoimmune response that damages the marrow, take other drugs to prevent infections, and limit contact with the outside world to avoid infection and even minor injury. Over the long term, most patients eventually die of infections.

When immunosuppressive therapy fails to keep the disease in check — in as many as 30 to 40 percent of patients — doctors usually prescribe a drug called eltrombopag, which is used in a variety of blood disorders to increase platelets. The drug, according to the Johns Hopkins experts, works only in about 30 percent of patients and usually leads to a partial, not complete, response.

Brodsky and DeZern say that the only curative treatment is a bone marrow transplant, but few patients have donors who are “fully matched” — sharing the same collection of immune-stimulating proteins that decorate every cell in the body.

In an effort to overcome the donor shortage and offer transplant to more patients, DeZern, Brodsky and their colleagues enrolled 16 patients between 11 and 69 years of age in this study from July 2011 through August 2016.

Each of the patients had failed to respond to immunosuppressive therapy or other drug treatments. None had access to a related fully matched bone marrow donor but did have an available and willing donor who was a half match. Three patients used unrelated donors.

After administering a cocktail of drugs designed to suppress their immune system and prevent rejection of the donor marrow, the patients received half-matched bone marrow transplants, some from siblings or parents, and others from unrelated donors.

Three and four days after their transplants, the patients received high doses of the chemotherapy drug cyclophosphamide. For the next year, or slightly longer, they remained on immunosuppressive medications, including tacrolimus, then stopped taking them.

Within weeks of their transplants, tests showed that each of the patients’ red and white blood cell and platelet counts had returned to normal levels without the need for blood transfusions. Once immunosuppressive therapy was stopped, none of the patients required further treatment related to their disease, the Johns Hopkins team reported.

Although 13 patients were able to discontinue immunosuppressive drugs a year after their transplant, three developed mild graft-versus-host disease (GVHD), a common complication of bone marrow transplants that occurs when immune cells in the transplant attack the newly transplanted cells. Two patients had mild GVHD that appeared on their skin, and one patient’s GVHD occurred in the mouth and skin. After a few extra months of immunosuppressive therapy, their GVHD subsided, and they also were able to stop taking these medications.

Ending all therapy related to their disease has been life-changing for the patients, says DeZern. “It’s like night and day,” she says. “They go from not knowing if they have a future to hoping for what they’d hoped for before they got sick. It’s that transformative.”

Successful transplants using partial match donors, Brodsky says, open up the transplant option to nearly all patients with this condition, especially minority patients. Seven of the 16 patients treated at Johns Hopkins self-identified as nonwhite.

A full sibling only has a 25 percent chance of being a full match. However, 100 percent of parents and 50 percent of siblings or half-siblings are half matches, regardless of ethnicity. The average person in the United States has about four half matches or better. “Now, a therapy that used to be available to 25 to 30 percent of patients with severe aplastic anemia is potentially available to more than 95 percent,” says Brodsky.

The idea of using cyclophosphamide after a partial-match transplant was first pioneered decades ago by Johns Hopkins Kimmel Cancer Center experts. Brodsky says the drug destroys patient’s diseased immune system cells but does not harm the donor’s blood stem cells, which create new disease-free blood cells in the patient.

Bone marrow transplants are costly — sometimes exceeding more than $300,000. However, Brodsky and DeZern say that full and half-matched transplants are life-saving for many, and there is cost-saving potential when aplastic anemia patients can avoid a lifetime of immunosuppressive therapy, hospitalizations, medications and blood transfusions.

TapImmune’s T-Cell Cancer Vaccine for Worldwide Eradication of Women’s Cancers

Early proof of efficacy, scalability with cost effective manufacturing, and testability in large Phase III FDA trials are the reasons why TapImmune (TPIV) is the leading T-cell cancer vaccine company and will dominate this space. World class collaborators including the Mayo Clinic, AstraZeneca (AZN), Sloan Kettering and Phase II trial funding from the U.S. Department of Defense prove this point.

Vaccines have eradicated numerous infectious diseases. Cancer is vaccine’s next target, powered by T-cells. White blood cells that are responsible for detecting foreign or abnormal cells including cancerous ones, T-cells specialize in seeking, attaching to, and destroying cancer cells with the help of the rest of the immune system.

Cunning cancer cells often find ways to con the immune system though, making them invisible to T-cells. TapImmune’s effective T-cell cancer vaccine activates cytotoxic T cells and directs them to see and attack specific types of cancer.  TapImmune’s vaccines are superior to other T-cell based cancer treatment candidates due to their powerful and unique combination of proprietary peptide antigens.

Stimulating both killer T-cells (CD8) and helper T-cells (CD4) creates sustained and long lasting tumor cell killing.  Roche’s (ROG:VS) Herceptin, a monoclonal antibody isn’t even designed to kill tumor cells, but in a market desperate for a solution, it has become standard of care by merely slowing down tumor growth. 2015 Herceptin sales exceeded $6.6 billion. If slowing down cancer makes Herceptin a blockbuster, then TapImmune’s cancer killing T-cell vaccine should become an uber-blockbuster and command sales far higher than Herceptin.

Early efficacy results in Phase I trials conducted at the prestigious Mayo Clinic show immune response to TapImmune’s T-cell vaccine in between 75% and 100% of patients treated. These impressive data were published in the Journal of Clinical Oncology and presented at oncology’s top scientific conference, ASCO, as well as at the San Antonio Breast Cancer Symposium.

Beyond efficacy, off-the-shelf platform technology is what sets TapImmune apart in the market and gives its T-cell cancer vaccines the dominant position.

While other cancer vaccines have shown promise, they have for the most part been autologous – meaning doctors take tumor cells from a specific person, process those cells into a vaccine, and reinsert this one-person-specific vaccine into that same person. This kind of personalized medicine is very expensive and difficult to run through large Phase III studies given the way FDA trials are conducted today.

TapImmune’s T-cell vaccines have been specifically designed for high efficacy, high scalability, cost effectiveness, and are ideally suited for large scale FDA testing. This makes them more likely to get FDA approved and reimbursed once on the market.

Today, TapImmune is testing its lead cancer vaccine TPIV-200 in two Phase II studies and is set to begin enrollment any day in two more Phase II studies. Should any of these four trials show promise, TapImmune will charge into larger scale Phase IIIs, armed with an off-the-shelf, consistently manufactured, low cost cancer vaccine that can be used as a therapeutic and prophylactic (preventative).

Indications are ovarian cancer and triple negative breast cancer, two of the hardest women’s cancers to treat. The breast cancer treatment market will top $20 billion by 2024 and ovarian cancer will be $1.4 billion by 2021 according to Decision Resources.

Instead of creating a specific vaccine for each person, TapImmune has selected excellent cancer targets including folate receptor alpha and HER2/neu, which are expressed on the surface of majority of tumor target cells. Deploying TapImmune’s proprietary antigen expression system, these antigens were selected from immune responses in patients to broadly stimulate T-helper cells, T-killer cells and T-memory cells.

Blockbuster Herceptin can only be used in 15-20% of the HER2/neu positive breast cancer population. Besting this by far, TapImmune’s T-cell cancer vaccine is applicable to over 50% of the HER2/neu patient population.

TapImmune vaccines cover about 85% of genotypes, offering broad population coverage from one easy to manufacture vaccine. With this effective formula, TapImmune can mass manufacture cancer vaccines that can be used on any person who needs it. No custom tailoring needed here.

HER2/neu is a valuable cancer target for both ovarian and colorectal cancer, both diseases where there are very few therapeutic options. In breast cancer as well, HER2/neu is overexpressed in ~ 30% of patients.

An equally powerful target is folate receptor alpha, overexpressed in 90% of ovarian cancer cells, over 80% of triple negative breast cancer, and 80% non-small cell lung cancer patients. In the US, approximately 30,000 ovarian cancer and 40,000 triple negative breast cancer patients are newly diagnosed every year. The only treatment options for these patients are surgery, radiation and chemotherapy. With time to recurrence being relatively short, survival prognosis is extremely poor after recurrence.

Fast Track and Orphan Drug designations have been granted by the U.S. FDA to TapImmune’s TPIV 200 in the treatment of ovarian cancer. There is every reason in the world for Fast Track to also be granted for a cancer vaccine to prevent recurrence in the breast cancer population.

Big pharma and the world’s leading medical institutions have independently vetted TapImmune’s technology.

A joint AstraZeneca-TapImmune Phase II ovarian cancer trial is now enrolling patients at the prestigious Sloan Kettering Institute. The trial is testing a combination therapy – TapImmune’s TPIV 200 and AstraZeneca’s durvalumab an anti-PD-L1 antibody, in 40 women who have high-grade ovarian cancer and have not been responsive to platinum chemotherapy, currently the standard of care for advanced ovarian cancer.

Synthetic Stem Cells Could Offer Therapeutic Benefits, Reduced Risks

Researchers from North Carolina State University, the University of North Carolina at Chapel Hill and First Affiliated Hospital of Zhengzhou University have developed a synthetic version of a cardiac stem cell. These synthetic stem cells offer therapeutic benefits comparable to those from natural stem cells and could reduce some of the risks associated with stem cell therapies. Additionally, these cells have better preservation stability and the technology is generalizable to other types of stem cells.

Stem cell therapies work by promoting endogenous repair; that is, they aid damaged tissue in repairing itself by secreting “paracrine factors,” including proteins and genetic materials. While stem cell therapies can be effective, they are also associated with some risks of both tumor growth and immune rejection. Also, the cells themselves are very fragile, requiring careful storage and a multi-step process of typing and characterization before they can be used.

Ke Cheng, associate professor of molecular biomedical sciences at NC State University, associate professor in the joint biomedical engineering program at NC State and UNC and adjunct associate professor at the UNC Eshelman School of Pharmacy, led a team in developing the synthetic version of a cardiac stem cell that could be used in off-the-shelf applications.

Cheng and his colleagues fabricated a cell-mimicking microparticle (CMMP) from poly (lactic-co-glycolic acid) or PLGA, a biodegradable and biocompatible polymer. The researchers then harvested growth factor proteins from cultured human cardiac stem cells and added them to the PLGA. Finally, they coated the particle with cardiac stem cell membrane.

“We took the cargo and the shell of the stem cell and packaged it into a biodegradable particle,” Cheng says.

When tested in vitro, both the CMMP and cardiac stem cell promoted the growth of cardiac muscle cells. They also tested the CMMP in a mouse model with myocardial infarction, and found that its ability to bind to cardiac tissue and promote growth after a heart attack was comparable to that of cardiac stem cells. Due to its structure, CMMP cannot replicate – reducing the risk of tumor formation.

“The synthetic cells operate much the same way a deactivated vaccine works,” Cheng says. “Their membranes allow them to bypass the immune response, bind to cardiac tissue, release the growth factors and generate repair, but they cannot amplify by themselves. So you get the benefits of stem cell therapy without risks.”

The synthetic stem cells are much more durable than human stem cells, and can tolerate harsh freezing and thawing. They also don’t have to be derived from the patient’s own cells. And the manufacturing process can be used with any type of stem cell.

“We are hoping that this may be a first step toward a truly off-the-shelf stem cell product that would enable people to receive beneficial stem cell therapies when they’re needed, without costly delays,” Cheng says.

New stem cell delivery approach regenerates dental pulp-like tissue in a rodent model

When a tooth is damaged, either by severe decay or trauma, the living tissues that comprise the sensitive inner dental pulp become exposed and vulnerable to harmful bacteria. Once infection takes hold, few treatment options–primarily root canals or tooth extraction–are available to alleviate the painful symptoms.

Researchers at Tufts University School of Dental Medicine (TUSDM) now show that using a collagen-based biomaterial to deliver stem cells inside damaged teeth can regenerate dental pulp-like tissues in animal model experiments. The study, published online in the Journal of Dental Research on Dec. 15, supports the potential of this approach as part of a strategy for restoring natural tooth functionality.

“Endodontic treatment, such as a root canal, essentially kills a once living tooth. It dries out over time, becomes brittle and can crack, and eventually might have to be replaced with a prosthesis,” said senior study author Pamela Yelick, PhD, professor at TUSDM and director of its Division of Craniofacial and Molecular Genetics. “Our findings validate the potential of an alternative approach to endodontic treatment, with the goal of regenerating a damaged tooth so that it remains living and functions like any other normal tooth.”

Yelick and her colleagues, including lead study author Arwa Khayat, former graduate student in dental research at TUSDM, examined the safety and efficacy of gelatin methacrylate (GelMA)–a low-cost hydrogel derived from naturally occurring collagen–as a scaffold to support the growth of new dental pulp tissue. Using GelMA, the team encapsulated a mix of human dental pulp stem cells–obtained from extracted wisdom teeth–and endothelial cells, which accelerate cell growth. This mix was delivered into isolated, previously damaged human tooth roots, which were extracted from patients as part of unrelated clinical treatment and sterilized of remaining living tissue. The roots were then implanted and allowed to grow in a rodent animal model for up to eight weeks.

The researchers observed pulp-like tissue inside the once empty tooth roots after two weeks. Increased cell growth and the formation of blood vessels occurred after four weeks. At the eight-week mark, pulp-like tissue filled the entire dental pulp space, complete with highly organized blood vessels populated with red blood cells. The team also observed the formation of cellular extensions and strong adhesion into dentin–the hard, bony tissue that forms the bulk of a tooth. The team saw no inflammation at the site of implantation, and found no inflammatory cells inside implanted tooth roots, which verified the biocompatibility of GelMA.

Control experiments, which involved empty tooth roots or tooth roots with only GelMA and no encapsulated cells, showed significantly less growth, unorganized blood vessel formation, and poor or nonexistent dentin attachment.

The results support GelMA-encapsulated human dental stem cells and umbilical vein endothelial cells as part of a promising strategy to restore normal tooth function, according to the study authors. However, they note that the current study was limited to partial tooth roots and did not examine nerve formation in regenerated dental pulp tissue. They emphasize the need for additional safety and efficacy studies in larger animal models before human clinical trials can be considered.

“A significant amount of work remains to be done, but if we can extend and validate our findings in additional experimental models, this approach could become a clinically relevant therapy in the future,” said Yelick, who is also a member of the Cell, Molecular & Developmental Biology; Genetics; and Pharmacology & Experimental Therapeutics programs at the Sackler School of Graduate Biomedical Sciences at Tufts. “Our work is early stage, but we are excited for the possibility of someday giving patients the option of regenerating their own teeth.” Continue reading “New stem cell delivery approach regenerates dental pulp-like tissue in a rodent model”

Leading Cell Therapy Company Pluristem to Partner with Japan’s Sosei for Commercialization of Regenerative Medicines in Japan

In a major global regenerative medicine deal, Israel-based Pluristem Therapeutics and Japan’s Sosei CVC, the venture capital arm of Japanese biopharma company Sosei Group, agreed to form a joint venture (JV) to commercialize Pluristem’s PLX-PAD cells in Japan. The deal marks Pluristem’s commercial entry into Japan and expands Sosei’s pipeline into regenerative medicines.

Pluristem has been active in the Japan market through discussions with, and applications to, Japan’s health regulatory agency, Pharmaceuticals and Medical Devices Agency (PMDA), seeking accelerated approval pathways for its PLX-PAD cells. The successful result is the PDMA’s acceptance of PLX-PAD cells into the accelerated pathways for regenerative medicine, making possible regulatory approval of PLX-PAD cells in the treatment of Critical Limb Ischemia (CLI) following just one 75-person clinical trial. This JV deal in Japan confirms Pluristem’s strategy of pursuing early approval pathways, as they also achieved in Europe.

Per the terms of the agreement between the two companies, an $11 million investment will be made by Sosei into the JV to fund a clinical trial of PLX-PAD for CLI that may directly lead to early conditional marketing approval and reimbursement based the PMDA’s accelerated regulatory pathway for regenerative medicine.  Pluristem gets 35% of the JV and its future profits. All proprietary rights related to PLX-PAD will be exclusively owned by Pluristem.

Sosei was likely compelled to partner with Pluristem based on the company’s advanced stem cell technology and its regulatory advantages in Japan. In addition to this JV with Pluristem, Sosei has partnerships with Novartis, AstraZeneca, MedIumme, and others. Pluristem and Sosei plan to enter into a definitive agreement no later than March 31, 2017.

The American Society of Nephrology (ASN) Applauds Congress and President Barack H. Obama for Passage and Signing into Law the 21st Century Cures Act

The American Society of Nephrology (ASN) applauds Congress and President Barack H. Obama for passage and signing into law the 21st Century Cures Act today. ASN advocated for specific provisions of the new law to benefit the more than 20 million Americans afflicted with kidney diseases and the 650,000 with kidney failure who rely on a kidney transplant or dialysis to live.

“The 21st Century Cures Act takes dramatic new steps to encourage research and innovation that could benefit the millions of Americans suffering from chronic diseases, including kidney diseases,” said ASN President Raymond C. Harris, MD, FASN.
The new law calls on the National Institutes of Health (NIH) to support prize competitions to improve patients’ health in fields where there is a significant disease burden or that current investment is disproportionately small relative to federal expenditures on that condition. The Medicare program entitles every American suffering from kidney failure—regardless of age—to lifesaving dialysis at a cost of nearly $35 billion annually, more than NIH’s total budget.

“ASN is heartened by this commitment to using the power of prize competitions to foster innovation on behalf of the patients most in need. Last year, the society announced its pledge of the first $7 million dollars toward a prize competition to develop a novel wearable or implantable device that replaces kidney function and improves patient quality of life,” Harris said. “As ASN seeks a partner to launch the prize competition in 2017, I anticipate we will continue close dialogue with NIH and other federal agencies whose expertise and collaboration would be invaluable in making this important endeavor a success.”

ASN applauded the leadership of House Energy and Commerce Chair Fred Upton (R-MI-6th) and Representative Diane DeGette (D-CO-6th), who shepherded the legislation through the House and Senate HELP Committee Chair Lamar Alexander (R-TN) and Ranking Member Patty Murray (D-WA). “There would be no announcement today without their vision and tireless efforts,” Harris commented.

In addition to advocating for prize competitions, ASN encouraged other provisions, including:

1) Permitting patients with kidney failure to enroll in Medicare Advantage Previously, kidney failure was the only pre-existing condition preventing patients from enrolling in Medicare Advantage. This change creates patient choice to selecting a plan that best fits their needs.
The American Society of Nephrology®, ASN®, Kidney Week®, CJASN®, JASN®, NephSAP®, and
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2) Using patient preferences in decision making by the Food and Drug Administration (FDA). The new law clarifies that patients’ own experiences will be incorporated into the FDA decision making for medical products.

3) Increasing funding for NIH research and innovation. The Act bolsters funding for research and innovation that lags behind the costs of caring for patients suffering from chronic diseases.

Rare Obesity Syndrome Therapeutic Target Identified

Columbia University Medical Center (CUMC) researchers have discovered that a deficiency of the enzyme prohormone covertase (PC1) in the brain is linked to most of the neuro-hormonal abnormalities in Prader-Willi syndrome, a genetic condition that causes extreme hunger and severe obesity beginning in childhood. The discovery provides insight into the molecular mechanisms underlying the syndrome and highlights a novel target for drug therapy.

The findings were published online today in the Journal of Clinical Investigation.

“While we’ve known for some time which genes are implicated in Prader-Willi syndrome, it has not been clear how those mutations actually trigger the disease,” said lead author Lisa C. Burnett, PhD, a post-doctoral research scientist in pediatrics at CUMC. “Now that we have found a key link between these mutations and the syndrome’s major hormonal features, we can begin to search for new, more precisely targeted therapies.”

An estimated one in 15,000 people have Prader-Willi syndrome (PWS). The syndrome is caused by abnormalities in a small region of chromosome 15, which leads to dysfunction in the hypothalamus—which contains cells that regulate hunger and satiety—and other regions of the brain. A defining characteristic of PWS is insatiable hunger. People with PWS typically have extreme obesity, reduced growth hormone and insulin levels, excessive levels of ghrelin (a hormone that triggers hunger), and developmental disabilities. There is no cure and few effective treatments for PWS.

Dr. Burnett and her colleagues used stem cell techniques to convert skin cells from PWS patients and unaffected controls into brain cells. Analysis of the stem cell-derived neurons revealed significantly reduced levels of PC1 in the patients’ cells, compared to the controls. The cells from PWS patients also had abnormally low levels of a protein, NHLH2, which is made by NHLH2, a gene that also helps to produce PC1.

To confirm whether PC1 deficiency plays a role in PWS, the researchers examined transgenic mice that do not express Snord116, a gene that is deleted in the region of chromosome 15 that is associated with PWS. The mice were found to be deficient in NHLH2 and PC1 and displayed most of the hormone-related abnormalities seen in PWS, according to study leader Rudolph L. Leibel, MD, professor of pediatrics and medicine and co-director of the Naomi Berrie Diabetes Center at CUMC.

“The findings strongly suggest that PC1 is a good therapeutic target for PWS,” said Dr. Burnett. “There doesn’t seem to be anything wrong with the gene that makes PC1—it’s just not getting activated properly. If we could elevate levels of PC1 using drugs, we might be able to alleviate some of the symptoms of the syndrome.”

“This is an outstanding example how research on human stem cells can lead to novel insight into a disease and provide a platform for the testing of new therapies,” said Dieter Egli, PhD, a stem cell scientist who is an assistant professor of developmental cell biology (in Pediatrics) and a senior author on the paper.

“This study changes how we think about this devastating disorder,” said Theresa Strong, PhD, chair of the scientific advisory board of the Foundation for Prader-Willi Research and the mother of a child with PWS. “The symptoms of PWS have been very confusing and hard to reconcile. Now that we have an explanation for the wide array of symptoms, we can move forward with developing a drug that addresses their underlying cause, instead of treating each symptom individually.”

Following the findings reported in this paper, the Columbia research team began collaborating with Levo Therapeutics, a PWS-focused biotechnology company, to translate the current research into therapeutics.

‘Rewired’ Cells Show Promise for Targeted Cancer Therapy

Human immune cells rationally engineered to sense and respond to tumor signal

A major challenge in truly targeted cancer therapy is cancer’s suppression of the immune system. Northwestern University synthetic biologists now have developed a general method for “rewiring” immune cells to flip this action around.

“Right now, one of the most promising frontiers in cancer treatment is immunotherapy — harnessing the immune system to combat a wide range of cancers,” said Joshua N. Leonard, the senior author of the study. “The simple cell rewiring we’ve done ultimately could help overcome immunosuppression at the tumor site, one of the most intransigent barriers to making progress in this field.”

When cancer is present, molecules secreted at tumor sites render many immune cells inactive. The Northwestern researchers genetically engineered human immune cells to sense the tumor-derived molecules in the immediate environment and to respond by becoming more active, not less.

This customized function, which is not observed in nature, is clinically attractive and relevant to cancer immunotherapy. The general approach for rewiring cellular input and output functions should be useful in fighting other diseases, not just cancer.

“This work is motivated by clinical observations, in which we may know why something goes wrong in the body, and how this may be corrected, but we lack the tools to translate those insights into a therapy,” Leonard said. “With the technology we have developed, we can first imagine a cell function we wish existed, and then our approach enables us to build — by design — a cell that carries out that function.”

Currently, scientists and engineers lack the ability to program cells to exhibit all the functions that, from a clinical standpoint, physicians might wish them to exhibit, such as becoming active only when next to a tumor. This study addresses that gap, Leonard said.

Leonard, who focuses on integrating synthetic biology into medicine, is an associate professor of chemical and biological engineering at the McCormick School of Engineering. He is a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University.

The research comes out of a rich collaboration that Leonard’s team has with clinical oncologists, immunologists and basic cancer researchers at Northwestern University Feinberg School of Medicine as well as other synthetic biologists.

The study, to be published Dec. 12 by the journal Nature Chemical Biology, provides details of the first synthetic biology technology enabling researchers to rewire how mammalian cells sense and respond to a broad class of physiologically relevant cues. Kelly A. Schwarz, a graduate student in Leonard’s research group, is the study’s first author.

“This work is exciting because it addresses a key technical gap in the field,” Schwarz said. “There is great promise for using engineered cells as programmable therapies, and it is going to take technologies such as this to truly realize that goal.”

Starting with human T cells in culture, the research team genetically engineered changes in the cells’ input and output, including adding a sensing mode, and built a cell that is relevant to cancer immunotherapy.

Specifically, the engineered cells sense vascular endothelial growth factor (VEGF), a protein found in tumors that directly manipulates and in some ways suppresses the immune response. When the rewired cells sense VEGF in their environment, these cells, instead of being suppressed, respond by secreting interleukin 2 (IL-2), a protein that stimulates nearby immune cells to become activated specifically at that site. Normal unmodified T cells do not produce IL-2 when exposed to VEGF, so the engineered behavior is both useful and novel.

This work was carried out in cells in culture, and the technology next will be tested in animal studies.

While Leonard’s team has initially focused on the application of this cell programming technology to enabling cancer immunotherapy, it can be readily extended to distinct cellular engineering goals and therapeutic applications. Leonard’s “parts” are also intentionally modular, such that they can be combined with other synthetic biology innovations to write more sophisticated cellular programs.

“To truly accelerate the rate at which we can translate scientific insights into treatments, we need technologies that let us rapidly try out new ideas, in this case by building living cells that manifest a desired biological function,” said Leonard, who also is a founding member of the Center for Synthetic Biology and a member of the Chemistry of Life Processes Institute.

“Our technology also provides a powerful new tool for fundamental research, enabling biologists to test otherwise untestable theories about how cells coordinate their functions in complex, multicellular organisms,” he said.

Researchers Discover a New Gatekeeper Role for Thymic Dendritic Cells in Controlling T Cell Release into the Bloodstream

Better Understanding of Cell’s Role Could Lead to New Strategies to Treat Autoimmune Diseases, Cancer

Newswise — Oakland, CA (December 6, 2016) – A team of scientists led by Julie Saba, MD, PhD at UCSF Benioff Children’s Hospital Oakland, has unveiled a novel role of thymic dendritic cells, which could result in new strategies to treat conditions such as autoimmune diseases, immune deficiencies, prematurity, infections, cancer, and the loss of immunity after bone marrow transplantation.

The study “Dendritic cell sphingosine-1-phosphate lyase regulates thymic egress,” appeared in the ‘Journal of Experimental Medicine’ (October 17, 2016 issue), published by Rockefeller University Press.

T lymphocytes are blood cells that carry out the main functions of our immune system. Dendritic cells and B lymphocytes are other types of immune cells that present foreign substances (such as microbial proteins) and “self” substances from our own tissues to T lymphocytes. In this way, T lymphocytes are “educated” to distinguish between self and non-self, so they can mount an immune response to pathogens but recognize and remain tolerant of one’s own bodily tissues. When this system fails to operate properly, autoimmune disease and immune deficiencies can result.

T lymphocytes undergo maturation in the thymus, a small gland located just above the heart, and are then released into the bloodstream. T lymphocyte egress from the thymus is essential for immune surveillance and to fight various types of infections. Sphingosine-1-phosphate (S1P) is a lipid molecule found at high levels in the blood and low levels in most tissues. Mature T cells produce a cell surface receptor that recognizes S1P, allowing the S1P chemical gradient to attract them into the bloodstream once they have completed their education in the thymus. However, the precise mechanisms that control T lymphocyte egress are not fully understood.

Thymic dendritic cells have a well-established role in antigen presentation and immune tolerance in the body. In addition to this role, dendritic cells also act as metabolic gatekeepers of lymphocyte trafficking. The team showed that thymic dendritic cells take up S1P, a blood borne lipid mediator, and metabolize it through the actions of an enzyme called S1P lyase, generating a localized S1P gradient that facilitates T lymphocyte egress into the blood In light of the fact that dendritic cells are known to continually traffic throughout the body surveying for the presence of infectious agents, the team’s observations raise the possibility that dendritic cells could potentially control the release of T lymphocytes in response to various disease states or conditions. These findings provide a deeper understanding of how the body regulates lymphocyte trafficking.

“T cells are needed to orchestrate the body’s immune response against pathogenic organisms and also against cancer cells,” says UCSF Benioff Oakland’s Dr. Julie Saba, one of the study’s authors. “In addition to natural T cells, genetically engineered T cells are being used in revolutionary ways to treat cancer. However, conditions such as chronic infection, aging, cancer and bone marrow transplantation can reduce T cell output from the thymus, compromising immune function. By learning what controls T cell output, we hope to be able to restore T cell production when it is low and provide more T cells for therapeutic purposes. ”

In addition to Saba, other co-authors are Jesus Zamora-Pineda, Ashok Kumar, Jung H. Suh, and Meng Zhang,

The research is supported by the (CA129438) and Swim Across America funds (to J.D. Saba). Confocal images were acquired at the Children’s Hospital Oakland Research Institute Microimaging Facility supported by an NIH grant (S10RR025472) and the Children’s Hospital Branches, Inc. S1P measurements were obtained using the Children’s Hospital Oakland Research Institute Mass Spectrometry Facility supported by an NIH Health grant (S10OD018070).

About UCSF Benioff Children’s Hospital Oakland
UCSF Benioff Children’s Hospital Oakland (formerly Children’s Hospital & Research Center Oakland) is a premier, not-for-profit medical center for children in Northern California, and is the only hospital in the East Bay 100% devoted to pediatrics. UCSF Benioff Children’s Hospital Oakland affiliated with UCSF Benioff Children’s Hospital San Francisco on January 1, 2014. UCSF Benioff Children’s Hospital Oakland is a national leader in many pediatric specialties including cardiology, hematology/oncology, neonatology, neurosurgery, endocrinology, urology, orthopedics, and sports medicine. The hospital is one of only five ACS Pediatric Level I Trauma Centers in the state, and has one of largest pediatric intensive care units in Northern California. UCSF Benioff Children’s Hospital Oakland is also a leading teaching hospital with an outstanding pediatric residency program and a number of unique pediatric subspecialty fellowship programs.

UCSF Benioff Children’s Hospital Oakland’s research arm, Children’s Hospital Oakland Research Institute (CHORI), is internationally known for its basic and clinical research. CHORI is at the forefront of translating research into interventions for treating and preventing human diseases. CHORI has 250 members of its investigative staff, a budget of about $50 million, and is ranked among the nation’s top ten research centers for National Institutes of Health funding to children’s hospitals. For more information, go to www.childrenshospitaloakland.org and www.chori.org.

TSRI Researchers Show How Circadian ‘Clock’ May Influence Cancer Pathway

A new study led by scientists at The Scripps Research Institute (TSRI) describes an unexpected role for proteins involved with our daily “circadian” clocks in influencing cancer growth.

The research, published recently in the journal Molecular Cell, suggests that disruptions in circadian rhythms might leave levels of an important cancer-linked protein, called cMYC, unchecked. “This appears to have big implications for the connection between circadian rhythms and cancer,” said TSRI biologist Katja Lamia, senior author of the study.

There is growing evidence that shift work and frequent jet lag can raise a person’s risk of cancer, suggesting a link between daily rhythms and cell growth. “We know this connection exists, but we haven’t known why,” said Lamia.
The researchers focused on proteins called cryptochromes, which evolved from bacterial proteins that sense light and repair DNA damage caused by sunlight. In humans, these proteins, called CRY1 and CRY2, regulate our circadian clocks, which influence what times of day we become tired, hungry and much more.

Using cells from mouse models, the researchers demonstrated that deleting the gene that expresses CRY2 reduced the cells’ ability to degrade a protein called cMYC. Without CRY2 keeping cMYC at normal levels, the researchers saw increased cell proliferation—similar to the abnormal growth seen in cancers.

Further studies of protein structures suggested that CRY2 is a key player in a process to “mark” cMYC for degradation. The researchers said it is significant that this process occurs after gene transcription—once the proteins are already produced—rather than during transcription, as in many other cryptochrome functions.
“This is a function of a circadian protein that has never been seen before,” said TSRI Research Associate Anne-Laure Huber, who served as first author of the study.
The researchers say more studies are needed to confirm this connection between circadian clocks and cancer in human tissues.

Long-Sought Genetic Model Of Common Infant Leukemia Described

After nearly two decades of unsuccessful attempts, researchers from the University of Chicago Medicine and the Cincinnati Children’s Hospital Medical Center have created the first mouse model for the most common form of infant leukemia. Their discovery, published in the Nov. 14, 2016, issue of Cancer Cell, could hasten development and testing of new drug therapies.

Pro-B acute lymphoblastic leukemia (ALL) with the (4;11) translocation is responsible for about 70 percent of infant and 10 percent of both childhood and adult acute lymphoblastic leukemias. The new mouse model replicates the human genetic flaw that causes this disease, making it much easier to study.

This subtype of leukemia results from a genetic fusion t(4;11), known as a translocation. This combines parts of two separate genes. One of those genes, MLL (short for mixed-lineage leukemia), comes from chromosome 11. The other fragment, AF4 (short for ALL fused gene) from chromosome 4. The hybrid MLL-AF4 gene results in leukemia.

Children and adults with this disease produce vast numbers of dysfunctional blood cells, which eventually crowd out functional cells. MLL-AF4 leukemia has a dismal prognosis, among the worst of any subset of acute leukemia.

“For 20 years, scientists have repeatedly tried and consistently failed to make a model of MLL-AF4 Pro-B acute lymphoblastic leukemia,” said Michael Thirman, MD, Associate Professor of Medicine at the University of Chicago. “Even though we understood the basic genetic flaw, no one had been able create a mouse model that mimicked the human disease, which is crucial for evaluating potential therapies.”

That frustrated many researchers, who shifted their focus to test alternative hypotheses on the causes of this leukemia or refocused their laboratories to study different aspects of this disease.

Thirman’s team, including longtime colleague Roger Luo, PhD, began working on this problem “years ago,” he said, and stayed with it. They quickly identified two hurdles.

The first was a problem with the retrovirus that scientists used to insert the leukemia-causing gene into mouse cells. That gene, acquired from leukemia patients, consisted of a human gene fragment from MLL linked to the human fragment from AF4.

“We soon discovered that the virus wasn’t working,” Thirman explained. “We knew that certain parts of human DNA can decrease viral titers. So we switched from the human version of AF4 to the mouse version, Af4, which is slightly different. This increased viral titers 30 fold.”

That worked, but it led to hurdle two. The mice injected with virus transporting MLL-Af4 developed leukemia, but it was the wrong kind. They developed acute myeloid instead of acute lymphoblastic leukemia. “Despite the use of lymphoid conditions,” the study authors wrote, “no lymphoid leukemia was observed.”

Next, they collaborated with James Mulloy, PhD, at Cincinnati Children’s Hospital Medical Center, whose graduate student Shan Lin inserted the fused MLL-Af4 gene into human CD34 cells, derived from cord or peripheral blood from volunteer donors. They transferred those cells to mice with immune systems that permit the growth of human cells. This time, the mice developed Pro-B ALL, identical to the leukemia found in humans.

“The model worked perfectly,” Thirman said. Within 22 weeks, all of the mice developed exactly the same type of leukemia as observed in patients.

Expression of MLL-Af4 in human cells “recapitulates the pro-B ALL observed in patient with t(4:11) as shown by immunophenotype, chromatin targeting of the fusion, nuclear complex formation, and gene expression signatures,” the authors wrote. “It mimics the disease found in humans both phenotypically and molecularly.”

“The differences in the type of leukemia that developed using mouse versus human cells were striking,” said Mulloy. “Researchers need to consider these differences carefully when choosing which model to use to mimic human disease. The available evidence now indicates that the approaches are not equivalent.”

They conclude that “our MLL-Af4 model will be a valuable tool to study this most prevalent MLL-fusion leukemia with such a poor prognosis.”

However, there is more work to be done. “MLL fusion disease is not a single genetic entity,” the authors note. “Each has its own genetic and biological features associated with particular fusion partners.” This highlights the need for “more models specific to each fusion. Our MLL-Af4 model will be a valuable tool.”

LJI Scientists Flip Molecular Switches To Distinguish Closely Related Immune Cell Populations

The cornerstone of genetics is the loss-of-function experiment. In short, this means that to figure out what exactly gene X is doing in a tissue of interest—be it developing brain cells or a pancreatic tumor—you somehow cut out, switch off or otherwise destroy gene X in that tissue and then watch what happens. That genetic litmus test has been applied since before people even knew the chemical DNA is what makes up genes. What has changed radically are the tools used by biologists to inactivate a gene.

Until now, scientists wishing to delete a gene in a model organism like a mouse did it by clipping out stretches of DNA encoding entire genes or very big chunks of them from the animal’s genome. This type of gene “knockout” is what La Jolla Institute for Allergy and Immunology (LJI) investigator Catherine C. Hedrick, Ph.D., used in 2011, when her lab discovered that mice without the gene Nr4a1 lack an anti-inflammatory subtype of white blood cells, nicknamed ‘patrolling monocytes’.

Now, the Hedrick group’s latest study reports a next-generation molecular manipulation aimed at inactivating Nr4a1 in a more precise manner. That study, published in the November 15, 2016, edition of Immunity, reports the loss of the same patrolling monocyte population following inactivation of a molecular switch that turns on Nr4a1. “This new work is exciting, because it shows that we can directly target genes within a specific cell type, which is important for targeted therapies,” says Hedrick, a Professor in the Division of Inflammation Biology.

The Hedrick laboratory’s previous demonstration that patrolling monocytes disappear following global Nr4a1 loss proved that the gene is necessary for development of that cell type. Later, her group reported that cancer cells injected into mice lacking Nr4a1 (therefore lacking patrolling monocytes) underwent unchecked metastasis, supporting the idea that patrolling monocytes play anti-cancer roles. But an important experimental question lingered: could the cancer metastasis seen in Nr4a1 knockout mice have anything to do with potential loss of Nr4a1 in a closely related group of cells called macrophages, which use Nr4a1 to control inflammation?

The new paper answers this question by silencing Nr4a1 only in patrolling monocytes. The Hedrick group accomplished this by applying good old-fashioned biochemistry to isolate stretches of DNA that flank the gene and define the on-switch for monocytes. Scientists call tissue-specific gene regulatory elements like this “enhancers.” They then showed that when activated, that DNA region, which they called “enhancer #2” (E2), was capable of switching on Nr4a1 expression only in patrolling monocytes, and not in related cells like macrophages.

The group proved the specificity of the enhancer by engineering mice whose genomes lacked only the E2 enhancer—not the gene itself—and indeed observed a lack of patrolling monocytes. “Until now, we did not have a way to delete a gene only in monocytes without also deleting it in macrophages,” says Graham Thomas, Ph.D., a postdoc in the Hedrick lab and the study’s first author. “Targeting the enhancer allows us to study particular cell types in a highly specific way,” says Thomas. “Also, eliminating enhancers teaches us what turns these genes on in the first place. That knowledge is essential if we are going to design rational targets to go after these cells.”

To confirm that macrophages throw an entirely different molecular switch to turn on Nr4a1, the group exposed mice missing the monocyte E2 switch to a noxious toxin found in bacterial membranes, as a way of seeing whether macrophages can still mount normal inflammatory responses. Indeed, the macrophage response was entirely normal in E2 mutants, unlike the global Nr4a1 “knockout”, showing that macrophages do not use the genetic E2 switch.

Finally, to make sure that E2 enhancer loss mimicked deletion of the entire gene in monocytes the group revisited a tumor model previously used to test Nr4a1’s anti-cancer effect. To do so, they injected melanoma cells into the bloodstream of normal or E2 mutant mice and monitored lung metastasis. Remarkably, outcomes following loss of the switch mirrored what the group had previously observed when they physically removed the gene itself: the lungs of mutant mice contained many more melanoma cells than did lungs of normal mice. This confirmed that the gene regulatory switch is highly specific to one cell type, monocytes and that tumor cell invasion in the absence of this population had nothing to do with deregulated macrophage activity.

Hedrick also thinks the new findings provide new understanding of just how important DNA enhancer regions can be. “Being able to selectively target specific cell types opens up a new world for understanding how to design therapies to treat disease,” she says.

Antibody Breaks Leukemia’s Hold, Providing New Therapeutic Approach

Acute myeloid leukemia (AML) is an aggressive cancer known for drug resistance and relapse. In an effort to uncover new treatment strategies, researchers at University of California San Diego School of Medicine and Moores Cancer Center discovered that a cell surface molecule known as CD98 promotes AML. The study, published October 27 by Cancer Cell, also shows that inhibiting CD98 with the therapeutic antibody IGN523 blocks AML growth in patient-derived cells and mouse models.

“To improve therapeutic strategies for this disease, we need to look not just at the cancer cells themselves, but also at their interactions with surrounding cells, tissues, molecules and blood vessels in the body,” said co-senior author Tannishtha Reya, PhD, professor of pharmacology at UC San Diego School of Medicine and Moores Cancer Center. “In this study, we identified CD98 as a critical molecule driving AML growth. We showed that blocking CD98 can effectively reduce leukemia burden and improve survival by preventing cancer cells from receiving support from the surrounding environment.”

Reya led the study together with Mark Ginsberg, MD, professor of medicine at UC San Diego School of Medicine and Moores Cancer Center. Co-author Edward van der Horst, PhD, senior director at Igenica Biotherapeutics Inc., provided the anti-CD98 antibody IGN523.

AML is a type of cancer in which the bone marrow makes abnormal white blood cells, red blood cells or platelets. Reya’s team and others have previously shown that leukemia cells interact with their surroundings in the body via molecules on their cell surfaces, and that these interactions can help the cancer cells divide, replicate and metastasize.

CD98 is a molecule found on the surface of cells, where it controls how cells stick to one another. CD98 is known to play a role in the proliferation and activation of certain immune cells. CD98 levels are also known to be elevated in some solid tumors, and linked to poor prognosis.

To determine CD98’s role in AML, in this latest study Reya’s team engineered mouse models that lack the molecule. They found that the loss of CD98 blocked AML growth and improved survival. CD98 loss largely spared normal blood cells, which the researchers said indicates a potential therapeutic window. Further experiments revealed that leukemia cells lacking CD98 had fewer stable interactions with the lining of blood vessels — interactions that were needed to fuel AML growth.

Next, the researchers wanted to see what would happen if they blocked CD98 in AML with a deliverable inhibitor. In 2015, Igenica Biotherapeutics Inc. tested IGN523, a humanized antibody that specifically binds and inhibits CD98, in a phase 1 clinical trial at Moores Cancer Center and elsewhere. The trial’s goal was to determine a safe dose for IGN523 administration in AML patients. In this study, Reya and team tested IGN523 in their own AML models.

The researchers found that IGN523 blocks CD98’s AML-promoting activity in both mouse models of AML and human cells in the laboratory. They also transplanted human patient-derived AML cells into mice and treated the recipients soon after with either IGN523, the anti-CD98 antibody, or with a control antibody. Anti-CD98-treatment effectively eliminated AML cells. In contrast, AML in control mice expanded more than 100-fold.

“This study suggests that human AML can’t get established without CD98, and that blocking the molecule with anti-CD98 antibodies could be beneficial for the treatment of AML in both adults and children,” Reya said.

Moving forward, Reya and team are working to further define whether CD98 could be targeted to treat pediatric AML.

“Many of the models we used in this work were based on mutations found in childhood AML,” she said. “While many childhood cancers have become very treatable, childhood AML continues to have a high rate of relapse and death. We plan to work with pediatric oncologists to test if anti-CD98 agents can be effective against pediatric AML, and whether it can improve responses to current treatments. I think this is particularly important to pursue since the anti-CD98 antibody has already been through phase I trials, and could be more easily positioned to test in drug-resistant pediatric AML.”

The American Cancer Society estimates that there will be about 19,950 new cases of AML and about 10,430 deaths from the disease in the United States in 2016, mostly adults. Approximately 500 children are diagnosed with AML in the U.S. each year, and it’s the most common second cancer among children treated for other cancers, according to St. Jude Children’s Research Hospital.

Cytomegalovirus Infection Relies On Human RNA-Binding Protein

Viruses hijack the molecular machinery in human cells to survive and replicate, often damaging those host cells in the process. Researchers at the University of California San Diego School of Medicine discovered that, for cytomegalovirus (CMV), this process relies on a human protein called CPEB1. The study, published October 24 inNature Structural and Molecular Biology, provides a potential new target for the development of CMV therapies.

“We found that CPEB1, one of a family of hundreds of RNA-binding proteins in the human genome, is important for establishing productive cytomegalovirus infections,” said senior author Gene Yeo, PhD, professor of cellular and molecular medicine at UC San Diego School of Medicine.

CMV is a virus that infects more than half of all adults by age 40, and stays for life. Most infected people are not aware that they have CMV because it rarely causes symptoms. However, CMV can cause serious health problems for people with compromised immune systems, or babies infected with the virus before birth. There are currently no treatments or vaccines for CMV.

In human cells, RNA is the genetic material that carries instructions from the DNA in a cell’s nucleus out to the cytoplasm, where molecular machinery uses those instructions to build proteins. CPEB1 is a human protein that normally binds RNAs that are destined to be translated into proteins.

Yeo’s team discovered that CPEB1 levels increase dramatically in human cells infected by CMV. Using genomics technologies, the researchers also found that increased CPEB1 levels in CMV-infected cells leads to abnormal processing of RNAs encoding thousands of human genes. In addition, they were surprised to find that CPEB1 was necessary for proper processing of viral RNAs. Without the host CPEB1 protein, viral RNA did not mature properly and the virus was weakened.

CMV-infected human cells undergo abnormal changes and produce more virus, which ultimately infects other cells. In collaboration with Deborah Spector, PhD, Distinguished Professor at UC San Diego School of Medicine and Skaggs School of Pharmacy and Pharmaceutical Sciences, the team went on to show that suppressing CPEB1 levels during CMV infection reversed these harmful cellular changes and reduced viral production tenfold.

“CPEB1 was previously shown to play a role in neuronal development and function, but this involvement in active viral infections is unexpected,” said first author Ranjan Batra, PhD, a postdoctoral fellow in Yeo’s lab. “This discovery has important implications for many viral infections.”

Yeo said the next steps are to determine the therapeutic value of inhibiting CPEB1 in CMV infections and identify other RNA-binding proteins that may be important in other viral infections.

Researchers Encouraged by Continued Signs of Tolerability and Disease Modification in Gene Therapy Trial for Sanfilippo Syndrome

Researchers from Abeona Therapeutics Inc, a clinical-stage biopharmaceutical company focused on developing therapies for life-threatening rare genetic diseases, provided, an update on clinical results through 30 days post-injection for the completed low-dose cohort (n=3) in the ongoing Phase 1/2 trial for ABO-102 (AAV-SGSH) at the Orphan Drugs & Rare Disease Conference (London, UK). The first-in-man clinical trial utilizes a single intravenous injection of AAV gene therapy for subjects with MPS IIIA (Sanfilippo syndrome type A), a rare autosomal recessive disease affecting every cell and organ in the body causing neurocognitive decline, speech loss, loss of mobility, and premature death in children.

The ongoing Phase 1/2 study is designed to evaluate safety and preliminary indications of efficacy of ABO-102 in subjects suffering from MPS IIIA. Observations 30 days post-injection for the low dose cohort demonstrated:

• ABO-102 is well-tolerated in subjects injected with the low dose of 5E13 vp/kg ABO-102 with no treatment related adverse events or serious adverse events (SAEs). Following favorable review of the safety data by the independent Data Safety Monitoring Board (DSMB), enrollment in the high dose cohort has commenced.

• In the natural history evaluating MPS III subjects it was shown that urine and cerebral spinal fluid GAG (heparan sulfate or “HS”) are significantly elevated in the subject population as a symptom of disease pathology.

• All subjects in the lose dose cohort experienced reductions from baseline in both urinary HS and CSF. At 30 days post-injection, urinary HS reduction was 57.6% +/- 8.2%. Reduction in CSF HS was 25.6% +/- 0.8%, suggesting that ABO-102 crossed the blood brain barrier after intravenous administration.

• The natural history study in 25 subjects with MPS III (Truxal et. al., 2016, Mol. Genet. Metab.) demonstrated that subjects had increased liver and spleen volumes averaging 116% and 88%, respectively, at baseline that did not change over a year of follow up.

• All three subjects demonstrated significant reductions in liver volume (17.1% +/- 1.9%), and spleen volume (17.6% +/- 7.1%) from baseline, as measured by MRI at 30 days post-injection.

Per the design of the clinical trial, subjects in the low-dose cohort received a single, intravenous injection of AB0-102 to deliver the AAV viral vector systematically throughout the body to introduce a corrective copy of the gene that underlies the cause of the MPS IIIA disease. Subjects were evaluated at multiple time points over the initial 30 days post-injection for safety assessments and initial signals of biopotency, which suggest that ABO-102 successfully reached target tissues throughout the body, including the central nervous system, to reduce GAG content that underlies the lysosomal storage pathology central to Sanfilippo syndrome type A (MPSIIIA).

“We remain encouraged by continued signs of tolerability and by early signals demonstrating reduced urinary and CSF GAG,” stated Kevin M. Flanigan, MD, principal investigator with the Center for Gene Therapy at Nationwide Children’s Hospital and Professor of Pediatrics and Neurology at The Ohio State University College of Medicine. “Additionally, we are informed by the natural history study that subjects with MPS IIIA experience hepatosplenomegaly, and are pleased by observations in the low dose cohort of significant reductions in liver and spleen volumes 30 days post-injection as measured by MRI.”

A more complete analysis of these data will be presented from the low-dose cohort and initial high dose cohort at a scientific conference in the first quarter of 2017. The Data Safety Monitoring Board has approved dose escalation of the high dose cohort this quarter.

“The data demonstrate an early and robust systemic delivery of ABO-102, and the reductions in CNS and urinary GAG support our approach for intravenous delivery of ABO-102 for subjects with Sanfilippo syndromes,” stated Timothy J. Miller, Ph. D, President and CEO of Abeona Therapeutics. “We are excited about early biomarker signals in this trial, including the reductions in liver and spleen volumes. Positive impact on hepatosplenomegaly has been an important measure historically in other clinical programs in the lysosomal storage disease space.”
Abeona’s MPS IIIA program, ABO-102, has been granted Orphan Product Designation in the USA and received the Rare Pediatric Disease Designation, and recently announced Orphan Drug Designation has been granted in the European Union.

About ABO-102 (AAV-SGSH): ABO-102 is an adeno-associated viral (AAV)-based gene therapy for subjects with MPS IIIA (Sanfilippo syndrome), that is delivered as a one-time intravenous injection. ABO-102 delivers a functioning, corrective copy of the SGSH gene to cells of the central nervous system (CNS) and other organs with the goal of correcting the underlying deficits caused by the inborn genetic errors that are the cause the disease. ABO-102 has been well tolerated through 30-day post-injection in subjects injected with the low-dose (n=3). The clinical study is supported by neurocognitive evaluations, biochemical assessments and MRI data generated in a 25-subject MPS III Natural History Study, also conducted at Nationwide Children’s Hospital, where subjects continued through one-year of follow up assessments.

Sanfilippo syndromes (or mucopolysaccharidosis (MPS) type III): a group of four inherited genetic diseases each caused by a single gene defect, described as type A, B, C or D, which cause enzyme deficiencies that result in the abnormal accumulation of glycosaminoglycans (GAGs, or sugars) in body tissues. MPS III is a lysosomal storage disease, a group of rare inborn errors of metabolism resulting from deficiency in normal lysosomal function. The incidence of MPS III (all four types combined) is estimated to be 1 in 70,000 births. Mucopolysaccharides are long chains of sugar molecule used in the building of connective tissues in the body. There is a continuous process in the body of replacing used materials and breaking them down for disposal. Children with MPS III are missing an enzyme which is essential in breaking down the used mucopolysaccharides called heparan sulfate. The partially broken down mucopolysaccharides remain stored in cells in the body causing progressive damage. In MPS III, the predominant symptoms occur due to accumulation within the central nervous system (CNS), including the brain and spinal cord, resulting in cognitive decline, motor dysfunction, and eventual death. Importantly, there is no cure for MPS III and treatments are largely supportive care.

Dysfunction In Neuronal Transport Mechanism Linked To Alzheimer’s Disease

Researchers at University of California San Diego School of Medicine have confirmed that mutation-caused dysfunction in a process cells use to transport molecules within the cell plays a previously suspected but underappreciated role in promoting the heritable form of Alzheimer’s disease (AD), but also one that might be remedied with existing therapeutic enzyme inhibitors.

The findings published in the October 11 online issue of Cell Reports.

“Our results further illuminate the complex processes involved in the degradation and decline of neurons, which is, of course, the essential characteristic and cause of AD,” said the study’s senior author Larry Goldstein, PhD, Distinguished Professor in the Departments of Neuroscience and Cellular and Molecular Medicine at UC San Diego School of Medicine and director of both the UC San Diego Stem Cell Program and Sanford Stem Cell Clinical Center at UC San Diego Health. “But beyond that, they point to a new target and therapy for a condition that currently has no proven treatment or cure.”

Alzheimer’s disease is a neurodegenerative disorder characterized by progressive memory loss and cognitive dysfunction. It affects more than 30 million people worldwide, including an estimated 5.4 million Americans. One in 10 persons over the age of 65 has AD; one in three over the age of 85. There are currently no treatments proven to cure or reduce the progression of AD.

Genetically, AD is divided into two groups: the much more common sporadic (sAD) form of the disease in which the underlying primary cause is not known and the rarer familial (fAD) form, produced by inherited genetic mutations. In both forms, the brains of AD patients feature accumulations of protein plaques and neurofibrillary tangles that lead to neuronal impairment and eventual cell death.

The prevailing “amyloid cascade hypothesis” posits that these plaques and tangles are comprised, respectively, of amyloid precursor protein (APP) fragments and tau proteins that fuel cellular stress, neurotoxicity, loss of function and cell death. There has been some evidence, however, of another disease-driver: defects in endocytic trafficking — the process by which cells package large, external molecules into vesicles or membrane-bound sacs for transport into the cell for a variety of reasons or uses.

But previous research focused on non-neuronal cells and did not examine the effects of normal expression levels of AD-related proteins, leaving it unclear to what degree decreased endocytosis and other molecular movement within cells played a causative role.

Goldstein and colleagues analyzed neurons created from induced pluripotent stem cells in which they generated PS1 and APP mutations characteristic of fAD using the emerging genome editing technologies CRISPR and TALEN. In this “disease-in-a-dish” approach, they found that the mutated neurons displayed altered distribution and trafficking of APP and internalized lipoproteins (proteins that combine with or transport fat and other lipids in blood plasma). Specifically, there were elevated levels of APP in the soma or cell body while levels were reduced in the neuronal axons.

In previous work, Goldstein’s team had demonstrated that PS1 and APP mutations impaired the activity of specific cellular enzymes. In the latest work, they found that treating mutated fAD neurons with a beta-secretase inhibitor rescued both endocytosis and transcytosis (molecule movement within a cell) functions.