UC research examines lung cell turnover as risk factor & target for treatment of influenza pneumonia

Influenza is a recurring global health threat that, according to the World Health Organization, is responsible for as many as 500,000 deaths every year, most due to influenza pneumonia, or viral pneumonia. Infection with influenza most typically results in lung manifestations limited to dry cough and fever, and understanding how the transition to pneumonia occurs could shed light on interventions that reduce mortality. Research led by University of Cincinnati (UC) scientists takes a different approach to investigating how influenza spreads through the lungs by focusing on how resistant or susceptible cells lining the airway are to viral infection.

The work published today in the Proceedings of the National Academy of Sciences (PNAS) shows how stimuli that induce cell division in the lung promote spread of influenza from the airway to the gas exchanging units of the lung, known as the alveoli. The UC study also demonstrates that interventions that prevent alveolar cells from dividing reduce influenza mortality in animal models, suggesting a potential prophylactic and/or therapeutic strategy for influenza pneumonia.

“Almost all research into susceptibility or resistance to influenza focuses on host immune responses,” says Nikolaos Nikolaidis, PhD, research scientist in the Division of Pulmonary, Critical Care and Sleep Medicine in the Department of Internal Medicine at the UC College of Medicine and lead author on the paper. “Our approach was to examine factors that influence the vulnerability of alveolar cells to influenza infection, separate from how the immune system is dealing with the virus.”

“Less than 1 percent of alveolar cells are actively dividing at any given time in the healthy lung, rendering it naturally resistant to influenza infection,” says Frank McCormack, MD, Gordon and Helen Hughes Taylor Professor of Internal Medicine and director of the Division of Pulmonary, Critical Care and Sleep Medicine and senior author on the paper. “Recovery from lung injury due to supplemental oxygen therapy, cigarette smoke or scarring lung diseases is associated with expression of growth factors that result in multiplication of lung cells. Our work demonstrated that these mitogenically stimulated cells are rich targets for influenza infection while they are dividing.”

The researchers found that when sirolimus, which is FDA-approved for use as an anti-growth agent for the rare lung disease, lymphangioleiomyomatosis (LAM), was given to influenza-infected animal models, it prevented alveolar cells from dividing, and as a result, protected the mice from viral pneumonia and death.

“Although sirolimus also has off target immunosuppressive properties that could potentially pose added risks of side effects in virus-infected patients, trials of inhaled sirolimus could lead to approaches that do not entail systemic exposure,” says McCormack.

The McCormack lab expressed optimism that this observation has the potential to ultimately inform understanding of other unexplained risk factors for influenza, including very young age and pregnancy, and perhaps even to change medical management, such as more judicious use of supplemental oxygen in patients admitted with suspected viral pneumonia. Further, the team has hopes that the research could lead to a paradigm shift in the approach to therapy.

Nikolaidis says the next step in this research is to further explore why the multiplying alveolar epithelial cell is a better target for influenza. “Is it because the virus gets into the dividing cell more easily, because multiplying stimuli expand the pool of cellular machinery used by the virus to replicate, or because proliferation is associated with a reduction in innate cellular defenses? We are anxious to explore these and other potential mechanisms of viral susceptibility,” he adds.

CAR T-Cell Therapy for Leukemia Leads to Remissions in Clinical Trial

In an early-phase clinical trial of an experimental immunotherapy, researchers achieved durable molecular remissions in patients with chronic lymphocytic leukemia who had failed other treatments

Researchers at Fred Hutchinson Cancer Research Center showed about 70 percent of patients with the most common adult leukemia had their tumors shrink or disappear following an experimental chimeric antigen receptor (CAR) T-cell immunotherapy.

The researchers also found that measuring genetic traces of cancer cells taken from bone marrow biopsies might be a better indicator of prognosis than the standard lymph node scan.

The Journal of Clinical Oncology published the results online July 17 of the Phase 1/2 clinical trial, which included 24 patients with chronic lymphocytic leukemia (CLL) who had failed other treatments. Most of the patients had seen their cancer progress despite treatment with ibrutinib, a targeted cancer drug approved in 2014 for CLL by the U.S. Food and Drug Administration.

This history placed them in a high-risk group that was found in previous studies to have short survival with standard therapies.

“It was not known whether CAR T-cells could be used to treat these high risk CLL patients,” said lead author Dr. Cameron Turtle, an immunotherapy researcher at Fred Hutch. “Our study shows that CD19 CAR T-cells are a highly promising treatment for CLL patients who have failed ibrutinib.”

CD19 CAR T-cells are a type of immunotherapy in which a patient’s T cells are extracted from their blood and modified in a lab to recognize CD19, a target on the surface of leukemia cells. The engineered T cells are then infused back into the patient where they multiply and hunt down and kill cancer cells.

In CLL, bone marrow makes too many abnormal lymphocytes, which are a type of white blood cell. The American Cancer Society estimates that in the U.S., there will be about 20,000 new cases and 4,600 deaths from CLL in 2017. Tests of blood, bone marrow and lymph nodes—where lymphocytes congregate to fight infection—reveal the disease.

The 24 patients participating in the study ranged in age from 40 to 73 years, with a median age of 61. They had received a median of five other therapies with as few as three and as many as nine.

Researchers found that 17 out of 24 (71 percent) of patients saw their tumors shrink or disappear following CAR T-cell therapy using the standard measure of lymph node size by CT scans four weeks after treatment.

Of side effects of CAR-T cell therapy, 20 of the 24 patients—83 percent—experienced cytokine release syndrome (grade 1-2, 18 patients; grade 4, one patient; grade 5, one patient) and 8 patients (33 percent) developed neurotoxicity (grade 3, five patients; grade 5, one patient). For the most part the side effects were reversible, but two patients had side effects severe enough to require being admitted to the intensive care unit and one of those patients died.

 (An earlier report on trial results was presented by Turtle in December at the American Society of Hematology annual meeting.)

The new paper expands on the measures used to indicate whether the CAR T-cell treatment is working.

To take a closer look to see if any cancer cells remained after treatment, the research team analyzed samples taken from some of the patients’ bone marrow four weeks after the CAR T-cell infusion. The team used a genetic test called IGH deep sequencing, which is akin to a bar code and enables researchers to track cancer cells in the body.

Turtle and his collaborators did the sequencing analysis in 12 of the patients. Seven of the 12 patients had no malignant copies. All patients without malignant copies were alive and free of disease at a median follow-up of 6.6 months after CAR T-cell infusion.

Compared with the CT scans, having no malignant gene sequences in bone marrow following CAR T-cell therapy was a better predictor of the cancer staying at bay—known as “progression-free survival,” the researchers found.

The study is the first to suggest that deep sequencing might be a superior measure for predicting outcomes four weeks after CAR T-cell therapy for CLL.

The immunotherapy team at Fred Hutch is still enrolling eligible patients with CLL, acute lymphoblastic leukemia and non-Hodgkin lymphoma for treatment on CD19 CAR T-cell trials. The patients are seen at Seattle Cancer Care Alliance, the clinical care partner for Fred Hutch.

Fred Hutch co-authors of the paper are Kevin Hay, Laila-Aicha Hanafi, Shelly Heimfeld, Stanley R. Riddell and David G. Maloney. Other co-authors are Daniel Li, Juno Therapeutics; Sindhu Cherian, Xueyan Chen and Brent Wood, University of Washington; and Arletta Lozanski and John C. Byrd, The Ohio State University.

Funding for the project came from Juno Therapeutics, National Cancer Institute, National Institute of Diabetes and Digestive and Kidney Diseases, Life Science Discovery Fund, the Bezos family, and the University of British Columbia.

Turtle, Maloney and Riddell receive research funding from Juno Therapeutics and are named as inventors on one or more patents or patent applications related to this work. Riddell is a co-founder of Juno Therapeutics and has equity interest in Juno Therapeutics. Li is an employee of and has equity interests in Juno Therapeutics. Fred Hutch receives research funding from Juno Therapeutics.

Largest study of malaria gene function reveals many potential drug targets

The malaria parasite’s success is owed to the stripping down of its genome to the bare essential genes, scientists at the Wellcome Trust Sanger Institute and their collaborators have found. In the first ever large-scale study of malaria gene function, scientists analysed more than half of the genes in the parasite’s genome and found that two thirds of these genes were essential for survival — the largest proportion of essential genes found in any organism studied to date.

The results, published today (13 July) in Cell, identify many potential targets for new antimalarial drug development, which is an important finding for this poorly understood parasite where drug resistance is a significant problem.

Nearly half of the world’s population is at risk of malaria and more than 200 million people are infected each year. The disease caused the deaths of almost half a million people globally in 2015*.

The genetics of the parasite that causes malaria, Plasmodium, have been tricky to decipher. Plasmodium parasites are ancient organisms and around half their genes have no similar genes — homologs — in any other organism, making it difficult for scientists to find clues to their function. This study provides the first ever experimental evidence of function for most of the genes.

Scientists studied the genes in one species of malaria, Plasmodium berghei, which were expressed in a single blood stage of its complicated, multi-stage life cycle. In the study, scientists designed a new method to decipher the function of the malaria parasite’s genes. The team switched off, or knocked out, 2,578 genes — more than half of the genome — and gave each knockout a unique DNA barcode**.

The team then used next generation genome sequencing technology to count those barcodes, and hence measure the growth of each genetically modified malaria parasite. If the switched-off gene was not essential, the parasite numbers shot up, but if the knocked out gene was essential, the parasite disappeared.

Dr Oliver Billker, joint lead author from the Wellcome Trust Sanger Institute, said: “This work was made possible by a new method that enabled us to investigate more than 2,500 genes in a single study — more than the entire research community has studied over the past two decades. We believe that this method can be used to build a deep understanding of many unknown aspects of malaria biology, and radically speed up our understanding of gene function and prioritisation of drug targets.”

The team systematically showed that the malaria parasite can easily dispose of the genes which produce proteins that give away its presence to the host’s immune system. This poses problems for the development of malaria vaccines as the parasite can quickly alter its appearance to the human immune system, and as a result the parasite can build resistance to the vaccine.

Dr Julian Rayner, joint lead author from the Wellcome Trust Sanger Institute, said: “We knew from previous work that on its surface the malaria parasite has many dispensable parts. Our study found that below the surface the parasite is more of a Formula 1 race car than a clunky people carrier. The parasite is fine-tuned and retains the absolute essential genes needed for growth. This is both good and bad: the bad news is it can easily get rid of the genes behind the targets we are trying to design vaccines for, but the flip side is there are many more essential gene targets for new drugs than we previously thought.”

Dr Francisco Javier Gamo, Director of the Malaria Unit at GlaxoSmithKline, said: “This study of unprecedented scale has resulted in many more, unique drug targets for malaria. The Holy Grail would be to discover genes that are essential across all of the parasite lifecycle stages, and if we could target those with drugs it would leave malaria with nowhere to hide. The technology that the Sanger Institute has developed gives us the potential to ask those questions systematically for the first time.”

Breathing in a New Gene Therapy to Treat Pulmonary Hypertension

Mount Sinai has partnered with Theragene Pharmaceuticals, Inc. to advance a novel airway-delivered gene therapy for treating pulmonary hypertension (PH), a form of high blood pressure in blood vessels in the lungs that is linked to heart failure. If the therapy succeeds in human clinical trials, it will provide patients for the first time with a way to reverse the damage caused by PH.

This gene therapy technique comes from the research of Roger J. Hajjar, MD, Professor of Medicine and Director of the Cardiovascular Research Center at the Icahn School of Medicine at Mount Sinai, and has been proven effective in rodent and pig animal models. PH is a deadly disease that disproportionately affects young adults and women; 58 percent of cases are found in young adults and 72 percent are women. There is currently no effective cure for PH, and about 50 percent of people who are diagnosed will die from the disease within five years.

PH is a rare (15-50 cases per million people), rapidly progressing disease that occurs when blood pressure is too high in vessels leading from the heart to the lungs. The high pressure is caused by abnormal remodeling of the lung blood vessels, characterized by a proliferation of smooth muscle cells and a thickening and narrowing of these vessels, and can lead to failure of the right ventricle of the heart and premature death. Abnormalities in calcium cycling within the vascular cells play a key role in the pathophysiology of pulmonary hypertension, along with deficiencies in the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) protein which regulates intracellular calcium within these vascular cells and prevents them from proliferating within the vessel wall. Downregulation of SERCA2a leads to the proliferative remodeling of the vasculature. This gene therapy, delivered via an inhaled aerosolized spray, aims to increase the expression of SERCA2a protein, and has been shown in rodents and pigs to improve heart and lung function, as well as reduce and even reverse cellular changes caused by PH.

Genetic modifier for Huntington’s disease progression identified

A team led by UCL and Cardiff University researchers has developed a novel measure of disease progression for Huntington’s disease, which enabled them to identify a genetic modifier associated with how rapidly the disease progresses.

“We’ve identified a gene that could be a target for treating Huntington’s disease. While there’s currently no cure for the disease, we’re hopeful that our finding could be a step towards life-extending treatments,” said Dr Davina Hensman Moss (UCL Huntington’s Disease Centre, UCL Institute of Neurology), one of the lead authors of the Lancet Neurology study.

Huntington’s disease (HD) is a fatal neurological disease caused by a genetic mutation. Larger mutations are linked to rapidly progressing disease, but that does not account for all aspects of disease progression. Understanding factors which change the rate of disease progression can help direct drug development and therapies.

The research team used the high quality phenotypic data from the intensively studied TRACK-HD cohort of people with the HD gene mutation. They established that different symptoms of disease progress in parallel, so they were able to combine the data from 24 cognitive, motor and MRI brain imaging variables to generate their progression score for genetic analysis.

They then looked for areas of the genome associated with their progression measure, and found a significant result in their sample of 216 people, which they then validated in a larger sample of 1773 people from a separate cohort, the European Huntington’s Disease Network (EHDN) REGISTRY study.

The genetic signal is likely to be driven by the gene MSH3, a DNA repair gene which has been linked to changes in size of the HD mutation. The researchers identified that a variation in MSH3 encodes an amino acid change in the gene. MSH3 has previously been extensively implicated in the pathogenesis of HD in both mouse and cell studies. The group’s findings may also be relevant to other diseases caused by repeats in the DNA, including some spinocerebellar ataxias.

Dr Hensman Moss said: “The gene variant we pinpointed is a common variant that doesn’t cause problems in people without HD, so hopefully it could be targeted for HD treatments without causing other problems.”

Professor Lesley Jones (Cardiff University), who co-led the study, said: “The strength of our finding implies that the variant we identified has a very large effect on HD, or that the new progression measure we developed is a much better measure of the relevant aspects of the disease, or most likely, both.”

The researchers say their study demonstrates the value of getting high quality data about the people with a disease when doing genetic studies.

Professor Sarah Tabrizi (UCL Huntington’s Disease Centre), who co-led the study said: “This is an example of reverse translation: these novel findings we observed in people with HD support many years of basic laboratory work in cells and mice. Now we know that MSH3 is critical in the progression of HD in patients, we can focus our attention on it and how this finding may be harnessed to develop new therapies that slow disease progression.”

Altered virus may expand patient recruitment in human gene therapy trials

For many patients, participating in gene therapy clinical trials isn’t an option because their immune system recognizes and fights the helpful virus used for treatment. Now, University of Florida Health and University of North Carolina researchers have found a solution that may allow it to evade the body’s normal immune response.

The discovery, published May 29 in the Proceedings of the National Academy of Sciences, is a crucial step in averting the immune response that prevents many people from taking part in clinical trials for various disorders, said Mavis Agbandje-McKenna, Ph.D., a professor in the University of Florida College of Medicine department of biochemistry and molecular biology and director of the Center for Structural Biology.

During gene therapy, engineered viruses are used to deliver new genes to a patient’s cells. While the recombinant adeno-associated virus, or AAV, is effective at delivering its genetic cargo, prior natural exposure to AAV results in antibodies in some people. As many as 70 percent of patients have pre-existing immunity that makes them ineligible for gene therapy clinical trials, Agbandje-McKenna said.

The findings provide a road map for designing virus strains that can evade neutralizing antibodies, said Aravind Asokan, Ph.D., an associate professor in the department of genetics at the University of North Carolina, who led the study. At UF Health, the structural “footprints” where pre-existing antibodies interact with the virus were identified using cryo-electron microscope resources provided by the UF College of Medicine and the UF Office of Research’s Division of Sponsored Programs. The UNC researchers then evolved new viral protein shells. Using serum from mice, rhesus monkeys and humans, the researchers showed that the redesigned virus can slip past the immune system.

“This is the blueprint for producing AAV strains that could help more patients become eligible for human gene therapy. Now we know how to do it,” Agbandje-McKenna said.

While the findings prove that one variation of AAV can be evolved, further study in preclinical models is needed before the approach can be tested in humans. Next, the immune profile of one particularly promising virus variant will need to be evaluated in a larger number of human serum samples, and dose-finding studies are needed in certain animal models. Researchers may also need to study whether the same virus-manipulating technique can be used in a broader range of gene therapy viruses, Agbandje-McKenna said.

Although human gene therapy remains an emerging field and has yet to reach patients on a wide scale, researchers elsewhere have used AAV therapy to successfully treat hemophilia, a blood-clotting disorder, in a small trial. It has also been or is now being studied as a way to treat hereditary blindness, certain immune deficiencies, neurological and metabolic disorders, and certain cancers.

The latest findings are the result of more than 10 years of studying the interactions between viruses and antibodies and a long-standing collaboration with Asokan, who heads the synthetic virology group at the UNC Gene Therapy Center, according to Agbandje-McKenna.

One gene closer to regenerative therapy for muscular disorders

A detour on the road to regenerative medicine for people with muscular disorders is figuring out how to coax muscle stem cells to fuse together and form functioning skeletal muscle tissues. A study published June 1 by Nature Communications reports scientists identify a new gene essential to this process, shedding new light on possible new therapeutic strategies.

Led by researchers at the Cincinnati Children’s Hospital Medical Center Heart Institute, the study demonstrates the gene Gm7325 and its protein – which the scientists named “myomerger” – prompt muscle stem cells to fuse and develop skeletal muscles the body needs to move and survive. They also show that myomerger works with another gene, Tmem8c, and its associated protein “myomaker” to fuse cells that normally would not.

In laboratory tests on embryonic mice engineered to not express myomerger in skeletal muscle, the animals did not develop enough muscle fiber to live.

“These findings stimulate new avenues for cell therapy approaches for regenerative medicine,” said Douglas Millay, PhD, study senior investigator and a scientist in the Division of Molecular Cardiovascular Biology at Cincinnati Children’s. “This includes the potential for cells expressing myomaker and myomerger to be loaded with therapeutic material and then fused to diseased tissue. An example would be muscular dystrophy, which is a devastating genetic muscle disease. The fusion technology possibly could be harnessed to provide muscle cells with a normal copy of the missing gene.”

Bio-Pioneering in Reverse

One of the molecular mysteries hindering development of regenerative therapy for muscles is uncovering the precise genetic and molecular processes that cause skeletal muscle stem cells (called myoblasts) to fuse and form the striated muscle fibers that allow movement. Millay and his colleagues are identifying, deconstructing and analyzing these processes to search for new therapeutic clues.

Genetic degenerative disorders of the muscle number in the dozens, but are rare in the overall population, according to the National Institutes of Health. The major categories of these devastating wasting diseases include: muscular dystrophy, congenital myopathy and metabolic myopathy. Muscular dystrophies are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. The most common form is Duchenne MD.

Molecular Sleuthing

A previous study authored by Millay in 2014 identified myomaker and its gene through bioinformatic analysis. Myomaker is also required for myoblast stem cells to fuse. However, it was clear from that work that myomaker did not work alone and needed a partner to drive the fusion process. The current study indicates that myomerger is the missing link for fusion, and that both genes are absolutely required for fusion to occur, according to the researchers.

To find additional genes that regulate fusion, Millay’s team screened for those activated by expression of a protein called MyoD, which is the primary initiator of the all the genes that make muscle. The team focused on the top 100 genes induced by MyoD (including GM7325/myomerger) and designed a screen to test the factors that could function within and across cell membranes. They also looked for genes not previously studied for having a role in fusing muscle stem cells. These analyses eventually pointed to a previously uncharacterized gene listed in the database – Gm7325.

Researchers then tested cell cultures and mouse models by using a gene editing process called CRISPR-Cas9 to demonstrate how the presence or absence of myomaker and myomerger – both individually and in unison – affect cell fusion and muscle formation. These tests indicate that myomerger-deficient muscle cells called myocytes differentiate and form the contractile unit of muscle (sarcomeres), but they do not join together to form fully functioning muscle tissue.

Looking Ahead

The researchers are building on their current findings, which they say establishes a system for reconstituting cell fusion in mammalian cells, a feat not yet achieved by biomedical science.

For example, beyond the cell fusion effects of myomaker and myomerger, it isn’t known how myomaker or myomerger induce cell membrane fusion. Knowing these details would be crucial to developing potential therapeutic strategies in the future, according to Millay. This study identifies myomerger as a fundmentally required protein for muscle development using cell culture and laboratory mouse models.

The authors emphasize that extensive additional research will be required to determine if these results can be translated to a clinical setting.

Novel Gene Therapy Provides Significant Wound Healing in Severe Form of Epidermolysis Bullosa

Recent data presented at the Society for Investigative Dermatology (SID) conference demonstrated that EB-101, a gene therapy provided significant wound healing in patients with Recessive Dystrophic Epidermolysis Bullosa (RDEB), a severe form of epidermolysis bullosa (EB).

RDEB is a subtype of an inherited genetic skin disorder characterized by chronic skin blistering, open and painful wounds, joint contractures, esophageal strictures, pseudosyndactyly, corneal abrasions and a shortened life span. Patients with RDEB lack functional type VII collagen owing to mutations in the gene COL7A1 that encodes for C7 and is the main component of anchoring fibrils, which stabilize the dermal-epidermal basement membrane

EB-101 is an autologous, ex-vivo gene therapy in which COL7A1 is transduced into autologous keratinocytes for the treatment of Recessive Dystrophic Epidermolysis Bullosa (RDEB).

The therapy provided healing (defined as greater than 50% healed) in 100% of Treated Wounds (36/36) at 3 Months; 89% (32/36) at 6 months, 83% (20/24) at 12 months, 88% (21/24) at 24 Months and 100% (6/6) at 36 months Post-Administration. The findings were presented by Abeona Therapeutics’ a leading clinical-stage biopharmaceutical company focused on developing novel gene therapies for life-threatening rare diseases, and the scientific and clinical collaborators at Stanford University School of Medicine, a center of excellence for the treatment of patients with epidermolysis bullosa.

The company also unveiled updated clinical data from the ongoing Phase 1/2 clinical trial for the EB-101 gene therapy program for patients with, along with supportive natural history data for 128 patients with the fatal skin disease.

 “Last week at the SID conference, our EB-101 team of clinical investigators and scientific collaborators presented data from the ongoing Phase 1/2 gene therapy clinical trial and a supportive natural history study of patients with RDEB that highlight the unprecedented wound healing and durable collagen C7 expression of four patients through two years’ post-treatment, including one patient that has continued to see EB-101 treated wounds remain healed three years post-treatment. The relevance of these benefits is highlighted when compared to non-treated control wounds evaluated from the 128-patient natural history study, which showed that RBEB patients suffer chronic and recurrent wounds that do not heal on their own and persist for several years,” said Timothy J. Miller, Ph.D., President and CEO of Abeona Therapeutics.

In the Phase 1/2 trial, EB-101 was administered to non-healing chronic wounds [mean length of time wounds were unhealed (unclosed) was 8.5 years prior to the gene therapy administration] on each subject and assessed for wound healing at predefined time points over years. The primary endpoint of the clinical trial is to assess safety and evaluate wound closure after EB-101 administration compared to control untreated wounds. Secondary endpoints include expression of full-length collagen C7 and restoration of anchoring fibrils at three and six months’ post-administration.

As reflected at the conference by Stanford collaborators, wounds were evaluated at three, six, 12, 24 and 36 months for appearance, durability, and resistance to blistering**:

Wound healing >50%:  defined as >50% closure after EB-101 administration was observed in:
– 100% (36/36 treated wounds, n=6 subjects) at 3 months;
– 89% (32/36 treated wounds, n=6 subjects) at 6 months;
– 83% (20/24 treated wounds, n=4 subjects) at 12 months,
– 88% (21/24 treated wounds, n=4 subjects) at 24 months,
– 100% (6/6 treated wounds, n=1 subject) at 36 months post-administration.

Wound healing >75%: defined as >75% closure after EB-101 administration was observed in:
– 83% (30/36 treated wounds, n=6 subjects) at 3 months;
– 61% (22/36 treated wounds, n=6 subjects) at 6 months;
– 50% (12/24 treated wounds, n=4 subjects) at 12 months;
– 71% (17/24 treated wounds, n=4 subjects) at 24 months;
– 83% (5/6 treated wounds, n=1 subject) at 36 months post-administration.

Collagen VII (C7) expression observed: C7 and morphologically normal NC2 reactive anchoring fibrils – the “zipper” that holds skin onto the underlying tissue and the primary deficit in RDEB patients – have been observed in EB-101 treatments up to two years post administration.

Natural History Study

Data from a supportive natural history study of 1,436 wounds of 128 patients with RDEB, established by Stanford and EBCare Registry, were also presented at the conference. The natural history study characterized both chronic non-healing wounds, defined as an area that does not heal ≥12 weeks, and recurrent wounds, defined as an area that partially heals but then easily re-blisters. Results presented were characterized as 1041 recurrent wounds and 395 chronic open wounds.

Notably, in the natural history study, 13 RDEB patients with a total of 15 chronic wounds were treated with an allograft product, including Apligraf® and Dermagraft®***. Of these wounds treated with allografts, only 7% (1/15 treated wounds) remained healed after 12 weeks, and 0% (0/15 treated wounds) remained healed after 24 weeks. This is a meaningful finding of the natural history study, as there are no approved therapies for RDEB patients that demonstrate significant wound closure after two months post-application.

Investigators at Stanford University are enrolling patients for the ongoing Phase 2 portion of the Phase 1/2 clinical trial (NCT01263379). The EB-101 program has been granted orphan drug designation from the European Medicines Agency (EMA).

Image credit: Samantha Okazaki / Today

Thorough Genotyping and Repurposed Drugs Key to Treating Small-Cell Lung Cancer, says Cancer Expert

Small cell lung cancer (SCLC) is an aggressive disease characterized by quick growth and spread. While there has been a gradual decrease in incidence of SCLC in recent years, likely reflecting the decreased prevalence of tobacco use, little progress has been made in treating SCLC due to its complex pathogenesis.

The majority of patients, including those with limited-stage disease and those who initially respond to chemo- and radiation- therapy (two traditional pillars of cancer therapy), become resistant to treatment resulting in a very small percentage (approximately 6%) who survive 5 years after being diagnosed.

Smoking is the main risk factor for SCLC, with only 2-3% of patients categorized as never-smokers.

Identifying Therapeutic Targets in Small-Cell Lung Cancer

From the molecular point of view, SCLC is characterized by a multitude of alterations, owing to the fact that cells are exposed to a myriad of carcinogens contained in cigarette smoke, which bind and mutate DNA. These alterations affect numerous genes and pathways, but among these there are few obvious therapeutic targets. This means that the driver genes responsible for most SCLC development and progression have yet to be identified with any certainty.

However, new high-throughput technologies, which allow comprehensive gene profiling, have revealed promising findings. For example, 20% of SCLC patient tumors bear alterations in the MYC gene family. This discovery has helped to identify a subset of patients sensitive to an oncogenic kinase downstream in the MYC pathway, allowing for better designed, biomarker-driven clinical trials for these, often repurposed, therapeutic agents.

Similarly, PARP1 and Notch have been found overexpressed in SCLC. In order to target PARP1, an enzyme which, when it malfunctions, leads to replication of damaged DNA, researchers are currently evaluating the efficacy of PARP inhibitors for treatment of SCLC. And, to investigate targeting of the Notch signaling pathway, which influences the cellular life-cycle, the FDA is in the process of approving Tarextumab, a selective Notch inhibitor, in the treatment of SCLC.

Another issue with SCLC tumors is that they are mostly characterized by the loss of two crucial oncosuppressor genes, named RB, RB2\p130 i and TP53, which are less actionable pharmaceutically because it is much more difficult to restore a loss of function rather than block an oncogenic gain of function. Although challenging, researchers are nonetheless trying to develop strategies in this direction.

Repurposing Existing Drugs

Also important to the progress of SCLC therapies, more effective drug identification and testing, through the use of powerful mouse models of the human disease, put researchers in a good position to tackle this cancer type and attempt better defined targeted approaches.

Recent immunotherapy approaches have emerged as a significant new pillar in cancer therapy and are being assessed in numerous clinical trials for a multitude of tumors, including SCLC. In particular, two new agents, nivolumab and ipilimumab, have recently been developed to treat other forms of cancer, such as unresectable or metastatic malignant melanoma, advanced non-small-cell lung cancer (NSCLC), and advanced renal-cell carcinoma. These agents have also been tested for applications in SCLC. Nivolumab and ipilimumab are constituted by monoclonal antibodies functioning through direct inhibition of CTLA4 and PD1, respectively, which are key negative regulators of the antitumoral immune function. Bristol-Myers Squibb (BMS) was able to obtain the National Comprehensive Cancer Network indication for use of nivolumab and nivolumab plus ipilimumab in patients with SCLC who progressed after one or more previous regimens. The indication was achieved upon the publication on Lancet Oncology by Scott Antonia and colleagues, who reported the efficacy of nivolumab monotherapy and nivolumab plus ipilimumab, achieving antitumour activity with durable responses and manageable safety profiles in previously treated SCLC patients, enrolled in the CheckMate-032 clinical trial.

Data was also presented at the World Lung Cancer Congress on the immunotherapy drug pembrolizumab, another therapeutic antibody against PD1, already approved for other diseases, which showed good efficacy.

One additional drug in this category is rovalptizumab teserine, a first-in-class antibody-drug conjugate comprised of a humanized monoclonal antibody against DLL3 and a toxin. DLL3 is a Notch ligand found to be expressed on 80% of SCLC. There is a 3rd line trial which is biomarker driven, meaning that they test for DLL3 expression and patients are eligible if they have “high” DLL3.

 

Doctors Treat Deadly Cancerous Disorders with Gene-Guided, Targeted Therapy

Genomic testing of biopsies from patients with deadly, treatment-resistant cancerous blood syndromes called histiocytoses allowed doctors to identify genes fueling the ailments and use targeted molecular drugs to successfully treat them.

Researchers from the Cincinnati Children’s Cancer and Blood Diseases Institute report their data in Journal of Clinical Investigation Insight (JCI Insight). They recommend the regular use of comprehensive genomic profiling at diagnosis to positively impact clinical care, as well as rigorous clinical trials to verify and extend the diagnostic and treatment conclusions in their study.

Histiocytoses are a group of disorders in which abnormal accumulations of white blood cells form tumors on vital organs, leading to systemic organ damage or death. About half of the patients can be treated successfully with chemotherapy, but others are treatment resistant.

Study authors conducted genomic profiling of biopsies from 72 child and adult patients with a variety of treatment-resistant histiocytoses, including the most common one in children, Langerhans cell histiocytosis (LCH), according to the lead investigator, Ashish Kumar, MD, PhD.

Twenty-six patients with treatment-resistant disease had gene mutations involving either BRAF or MAP2K1 that directly activate the MAP-kinase cancer pathway. Researchers determined such patients would benefit from the targeted molecular therapies dabrafenib or trametinib, which block the MAP kinase pathway. The approved cancer drugs were prescribed off label to the histiocytosis patients.

“In the last year, three patients we treated were infants with disease that was resistant to several rounds of intense chemotherapy. In the past, these children either would have suffered serious complications including death or would have had to endure more intensive treatments and the ensuing toxicities, including the risk of death,” Kumar said. “All three are thriving now on one oral medication that put their disease into remission.”

Potentially Reversible Changes in Gene Control ‘Prime’ Pancreatic Cancer Cells to Spread

Epigenetic changes, not DNA mutations, drive some metastasis

multicenter team of researchers reports that a full genomic analysis of tumor samples from a small number of people who died of pancreatic cancer suggests that chemical changes to DNA that do not affect the DNA sequence itself yet control how it operates confer survival advantages on subsets of pancreatic cancer cells. Those advantages, the researchers say, let such cancer cells thrive in organs like the liver and lungs, which receive a sugar-rich blood supply.

In a summary of the study, published online in the journal Nature Genetics on Jan. 16, the
research teams also report evidence that an experimental drug — not approved for human use — can reverse these “epigenetic” changes to block tumor formation in lab-grown pancreatic cancer cells. These findings may lead to more effective treatment strategies against metastatic pancreatic cancer, which is universally lethal.

“What we found astonished us,” says study leader Andrew Feinberg, M.D., Bloomberg Distinguished Professor of Epigenetics at The Johns Hopkins University and a Johns Hopkins Kimmel Cancer Center member. “Changes in genes’ regulation — not in the DNA sequence of genes themselves — were the driving force behind successful metastases in our experiments, and, as far as we know, this is the first genomewide experimental evidence for this phenomenon.”

Metastasis, or cancer spread by the formation of tumors at new sites, is generally what makes cancers deadly because surgery and other treatments are unlikely to find and destroy every cancer cell. That is particularly true, Feinberg says, for pancreatic cancer, which usually goes undetected until after it has spread. Because it is so deadly and because the number of new cases is increasing, it is predicted to be the second leading cause of cancer deaths in the western world by 2020, trailing only lung cancer, he adds. In 2016, the disease was predicted to strike an estimated 53,070 Americans and kill 41,780, according to the National Cancer Institute.

To better understand the formation of metastases in pancreatic cancer, Christine Iacobuzio-Donahue, M.D., Ph.D., professor of pathology at Memorial Sloan Kettering Cancer Center, collected tumor samples from eight patients with the most common form of pancreatic cancer (pancreatic ductal adenocarcinoma) immediately after their deaths.

Samples were taken from the original, or primary, tumor in the pancreas and from any detectable nearby and distant metastatic tumors. The tumors’ genomes were then analyzed for genetic mutations, or alterations in their DNA, by her team and that of Bert Vogelstein, M.D., professor of oncology at the Johns Hopkins University School of Medicine and the co-director of the Ludwig Center at the Johns Hopkins Kimmel Cancer Center. In a companion paper, they report finding no genetic mutations directly linked to or likely responsible for the success of the metastases.

Working on the same tumor samples, Feinberg’s team looked for other kinds of changes — specifically in the so-called epigenome, the array of reversible chemical and structural changes to DNA and the proteins around which it is wrapped. Epigenetic changes do not alter the information encoded in the DNA sequence itself but determine whether and to what extent specific genes are used by cells.

The research groups then examined the landscape of the pancreatic cancer epigenome using a combination of stains on patient tissues, direct examination of the proteins that wrap DNA and whole-genome sequencing of the detected epigenetic changes to map precisely where they were located.

Feinberg says no major changes were seen in the tumors of patients whose cancer spread only locally. But tumors from patients with distant metastases to the lung and liver showed massive epigenetic changes that mapped to large, blocklike segments of the genome, both in the distant metastases themselves and in the section, or “subclone,” of the primary tumors they came from.

One explanation for that, says Iacobuzio-Donahue, is that “Distant metastases have to travel long distances along the ‘highways’ of the blood vessels, land in a good spot and colonize, while local metastases just pinch off the primary tumor and go a short distance on ‘familiar side roads,’ so they are usually more similar to the primary tumor.”

Beyond pancreatic cancer, the blocklike segments where the epigenetic changes were located could be universal across other cancers. “Although we haven’t tested this idea yet, we know that similar epigenetic regions are important in other types of cancer, such as colon cancer, so it’s likely that these large-scale epigenetic changes are occurring in them too,” says Feinberg.

Because epigenetic changes affect gene activity, the research team categorized the affected genes by function to see if the changes might have specific consequences among different subclones from the same patient. This analysis, done on separate samples from the same patient, revealed that many of the affected genes confer advantages to cancer cells by, for example, enhancing cell migration or resistance to chemotherapy.

The teams next looked for what might control the epigenetic changes. Aware that cancers rewire their metabolism in ways that could change the epigenome and that distant metastases in pancreatic cancer naturally spread to organs fed by a sugar-rich blood supply, the researchers wondered if the tumor cells had altered the way they use the basic form of sugar, glucose.

In biochemical tests, they learned that distant metastatic tumors consumed excessive amounts of glucose when compared to local metastases. They also found that distant metastases and their precursors processed glucose through a growth-promoting series of metabolic reactions called the pentose phosphate pathway, which burns (or oxidizes) glucose-derived molecules into building blocks for tumors. Particularly important was an enzyme called 6-phosphogluconate dehydrogenase (PGD).

Those results may answer another poorly understood question about metastatic cancer biology, the researchers report. Oliver McDonald, M.D., Ph.D., assistant professor of pathology, microbiology and immunology at Vanderbilt University and co-first author of the study, says: “In pancreatic cancer, the fact that it may take years for a primary tumor to develop, while metastases can progress very quickly, is somewhat of an enigma. The changes we found in glucose utilization could be the answer.”

To see if PGD and the pentose phosphate pathway were tied to the epigenetic changes the researchers had detected in distant metastases, they treated tumor cells from different sites in a single patient with the drug 6-aminonicotinamide (6AN), which is known to inhibit PGD but is not used in humans because of its severe side effects. The drug had no effect on the epigenetic state of DNA taken from the local metastasis but reversed the epigenetic changes seen in cells from the distant metastasis.

Significantly, treatment with 6AN specifically decreased the activity of genes with malignant, cancer-spreading functions, like cell cycle control and DNA repair. But most importantly, the researchers say, in three laboratory tests of tumor growth, the drug strongly blocked tumor formation in distant metastases and their precursors.

The investigators caution that application of their findings to the treatment of pancreatic cancer must await further understanding of the complicated genetics and biology of metastases. “We still don’t know how the pentose phosphate pathway gets turned on, for example,” says McDonald, “but we’re working on figuring it out.”

Feinberg adds that the groups are also trying to learn how activation of the pentose phosphate pathway leads to the massive epigenetic changes seen. “Hopefully, these investigations will help develop new drugs to treat this aggressive cancer and others,” he says.

Other authors of the report include Xin Li, Rakel Tryggvadottir and Tal Salz of the Johns Hopkins University School of Medicine; Anna Word, Sonoko Natsume and Kimberly Stauffer of Vanderbilt University Medical Center; Tyler Saunders, Alvin Makohon-Moore and Yi Zhong of Memorial Sloan Kettering Cancer Center; Samantha Mentch, Marc Warmoes and Jason Locasale of Duke University School of Medicine; Alessandro Carrer and Kathryn Wellen of the University of Pennsylvania Perelman School of Medicine; and Hao Wu of Emory University.

Gut Bacteria May Hold Key to Treating Autoimmune Disease

Defects in the body’s regulatory T cells (T reg cells) cause inflammation and autoimmune disease by altering the type of bacteria living in the gut, researchers from The University of Texas Health Science Center at Houston have discovered. The study, “Resetting microbiota by Lactobacillus reuteri inhibits T reg deficiency–induced autoimmunity via adenosine A2A receptors,” which will be published online December 19 in The Journal of Experimental Medicine, suggests that replacing the missing gut bacteria, or restoring a key metabolite called inosine, could help treat children with a rare and often fatal autoimmune disease called IPEX syndrome.

T reg cells suppress the immune system and prevent it from attacking the body’s own tissues by mistake. Defects in T reg cells therefore lead to various types of autoimmune disease. Mutations in the transcription factor Foxp3, for example, disrupt T reg function and cause IPEX syndrome. This inherited autoimmune disorder is characterized by a variety of inflammatory conditions including eczema, type I diabetes, and severe enteropathy. Without a stem cell transplant from a suitable donor, IPEX syndrome patients usually die before the age of two.

Autoimmune diseases can also be caused by changes in the gut microbiome, the population of bacteria that reside within the gastrointestinal tract. In the study, the team led by Yuying Liu and J. Marc Rhoads at The University of Texas Health Science Center at Houston McGovern Medical School find that mice carrying a mutant version of the Foxp3 gene show changes in their gut microbiome at around the same time that they develop autoimmune symptoms. In particular, the mice have lower levels of bacteria from the genus Lactobacillus. The researchers discovered that by feeding the mice with Lactobacillus reuteri, they could “reset” the gut bacterial community and reduce the levels of inflammation, significantly extending the animals’ survival.

Bacteria can secrete metabolic molecules that have large effects on their hosts. The levels of a metabolite called inosine were reduced in mice lacking Foxp3 but were restored to normal after resetting the gut microbiome with L. reuteri. The researchers found that, by binding to cell surface proteins called adenosine A2A receptors, inosine inhibits the production of Th1 and Th2 cells. These pro-inflammatory T cell types are elevated in Foxp3-deficient mice, but their numbers are diminished by treatment with either L. reuteri or inosine itself, reducing inflammation and extending the animals’ life span.

“Our findings suggest that probiotic L. reuteri, inosine, or other A2A receptor agonists could be used therapeutically to control T cell–mediated autoimmunity,” says Yuying Liu.

Conflict of interest statement: Some of the authors of this study, including Yuying Liu and J. Marc Rhoads, have a patent application pending on use of inosine and A2A agonists in IPEX syndrome.

After One Dose of Gene Therapy, Hemophilia B Patients Maintain Near-Normal Levels of Clotting Factor

At ASH Meeting, CHOP Hematologist Leads Clinical Trial in Which All Subjects Safely Maintain Factor IX Expression that Curtails Disabling Bleeding

Researchers are reporting the highest and most sustained levels to date of an essential blood-clotting factor IX in patients with the inherited bleeding disorder hemophilia B. After receiving a single dose of an experimental gene therapy in a clinical trial, patients with hemophilia produced near-normal levels of clotting factor IX, allowing them to stop clotting factor infusions and to pursue normal activities of daily life without disabling bleeding episodes.

Lindsey A. George, MD, a hematologist at Children’s Hospital of Philadelphia (CHOP)is the lead investigator of the phase 1/2 clinical trial sponsored by Spark Therapeutics, Inc. and Pfizer, Inc. The American Society of Hematology (ASH) today highlighted updated findings from that trial in a press conference during its annual meeting in San Diego. George will present those study results tomorrow at an ASH plenary scientific session.

Katherine High, MD, a senior author of the study and Spark Therapeutics’s president and chief scientific officer, described the updated interim trial data at today’s press conference. The clinical trial of nine adult hemophilia B patients, aged 18 to 52 years, used a single dose of a gene therapy product engineered to enter patients’ liver cells and direct the production of the blood clotting factor that they lack.

George notes, “Our goal in this trial was to evaluate the safety of the gene therapy product and secondarily, to determine if we could achieve levels of factor IX that could decrease bleeding events in patients.” She added, “These patients have a severe or moderate level of hemophilia, with baseline clotting factor level less than or equal to 2 percent of levels in healthy people. In current treatment, patients with hemophilia give themselves intravenous doses of factor IX up to a couple times a week. While generally effective, factor levels fluctuate, and patients may suffer painful, disabling joint bleeds when their clotting factor levels drop. Such a regimen requires significant planning of daily activities.”

In the current trial, said George, the patients maintained factor levels of approximately 30 percent, enough to lift them out of the severe category. “At these new levels, hemophilia patients do not typically need to self-treat with factor to avoid bleeding events,” she said, adding, “This represents a potential dramatic improvement in their quality of life and a shift in the way we think about treating hemophilia.” A factor level of 30 percent is near-normal, she added, and patients would be expected to experience bleeding only in the event of major trauma or surgery.

One subject self-infused two days after receiving the gene therapy vector. Beyond this, no patients had any bleeding events or required factor for any reason. With significant reduction in bleeding events and factor use, six of the first seven patients reported increased physical activity and all reported improved quality of life. Two additional patients received the gene therapy product too recently to determine quality-of-life measures.

Previous hemophilia gene therapy trials have been frustrated by an immune response to the gene therapy product that limited the success of the therapy. In the current trial, two patients experienced an immune response to the gene therapy that did not result in safety concerns, and were treated with steroids. The patients are still undergoing treatment but have maintained factor IX activity without bleeding.

George reported that she is cautiously optimistic, acknowledging that this trial is a small study, with a short follow-up period as yet. However, as the researchers continue to monitor patients in the current trial, next steps will be to discuss with the U.S. Food and Drug Administration the outlines of a larger, phase 3 clinical trial. No gene therapies for any genetic diseases have yet been approved for clinical use in the U.S.

Formerly a research leader at CHOP, High pursued groundbreaking preclinical investigations in hemophilia B gene therapy and provided scientific expertise to previous gene therapy trials in hemophilia and other genetic disorders at CHOP before moving to Spark Therapeutics, which was spun off from CHOP in 2013. CHOP maintains a financial interest in the company.

Successfully Treating Genetically Determined Autoimmune Enteritis

Using targeted immunotherapy, doctors have succeeded in curing a type of autoimmune enteritis caused by a recently discovered genetic mutation. This report comes from researchers at the Department of Biomedicine of the University of Basel and University Hospital Basel. Their results raise new possibilities for the management of diarrhea, which is often a side effect of melanoma treatment.

Immunodeficiencies can arise due to gene mutations in immune system proteins. As such mutations rarely occur, these immunodeficiencies often go unrecognized or are detected too late for effective treatment. Currently, there are more than 300 different known genetically determined immunodeficiencies, with new examples being described almost every week.

Prof. Mike Recher’s research group at the Department of Biomedicine of the University of Basel and University Hospital Basel recently discovered a genetic immunodeficiency associated with serious, chronic autoimmune enteritis in an adult patient. Happily, according to the researchers’ report in the Journal of Allergy and Clinical Immunology, they were able not only to describe the new mutation, but also to successfully treat the patient with targeted therapy.

Autoimmune reaction caused by mutation

The patient had a rare mutation in the CTLA-4 protein found on the surface of T-cells. Normally, this protein prevents immune cells from attacking an patient’s own body. However, as it was not functioning adequately due to the mutation, T-cells attacked the patient’s own intestinal cells, causing chronic inflammation. This resulted in the patient suffering from severe diarrhea and weight loss.

These unusual symptoms led the cantonal hospital of Graubünden to refer the patient to the special clinic for immunodeficiency at the University Hospital Basel. Initial immunological investigations suggested a genetically determined dysregulation of the immune system. The new CTLA-4 gene mutation was discovered following subsequent analysis of the entire genome at the University Hospital Zurich. Further investigations showed that the mutation causes reduced CTLA-4 function, which led to increased infiltration of the intestinal mucosa by T-cells and therefore to chronic diarrhea.

Treatment with therapeutic antibodies

Working in close cooperation with University Hospital Basel’s gastroenterology department, the doctors opted for a therapy that uses a new drug from the monoclonal antibody group to prevent the T-cells from penetrating the intestinal mucosa. This drug (vedolizumab) blocks a specific adhesion molecule on the surface of the T-cell and thereby inhibits immune cells from binding themselves to receptors present in the intestine, preventing the T-cells from penetrating the blood vessels in the intestinal tissue. This treatment produced the desired outcome: after three months, the patient’s chronic diarrhea had stopped completely.

Preventing diarrhea in melanoma patients

In some diseases, however, CTLA-4 inhibition can be used therapeutically, as in the treatment of skin cancer (melanoma). The drug Ipilimumab works similarly to the CTLA-4 mutation, meaning that immune system T-cells are no longer properly inhibited and can more efficiently attack the malignant skin cancer cells. One of the side-effects of this therapy is autoimmune intestinal inflammation – analogous to the inflammation that occurs in patients with the CTLA-4 gene mutation. It is possible that melanoma patients, who suffer severe diarrhea due to the inhibition of their CTLA-4 function, will benefit from this new insight, which opens up new therapeutic possibilities for Vedolizumab.

Cooperation between regional hospitals, basic research and university medical departments

This case demonstrates the importance of precise diagnosis of the molecular causes of an illness in enabling targeted, personalized treatment. “In order to expand our knowledge in these areas, doctors in clinics and regional hospitals must be on the alert for unusual disease phenotypes and refer such patients to specialized university hospital clinics for further evaluation,” says study author Mike Recher. “We also need clinical university centers that are closely linked to research laboratories.”

Researching Proinsulin Misfolding to Understand Diabetes

  According to the World Health Organization, 422 million adults across the globe have diabetes. In fact, the number of adults with the disease continues to grow each year.

To help the growing patient population, researchers at the University of Michigan are going down to the molecular level. Here, they’re trying to determine what makes cells in the diabetic pancreas less efficient in generating insulin molecules.

Diabetes occurs when the body’s pancreas does not produce enough insulin to keep blood sugar levels under control.

“Ten years ago, we found that when insulin is being made in the pancreatic beta cells, a certain subfraction of new synthesized insulin molecules, called proinsulin, cannot fold properly,” says Ming Liu, Ph.D., research associate professor of internal medicine at U-M and co-investigator on a new study on the topic.

“This problem is known as proinsulin misfolding, and several different groups around the world have now come up with similar observations. We also found that in animals in which production of misfolded proinsulin molecules reaches 30 percent of total proinsulin, that is enough for these animals to develop diabetes from pancreatic beta cell failure.”

A four-member team of U-M faculty are currently zeroing in on misfolded proinsulin.

“When you are born, you receive two copies of the gene encoding proinsulin, one from your mom and one from your dad,” says Billy Tsai, Ph.D., co-investigator and professor of cell and developmental biology at U-M. “There is a special kind of diabetes, called Mutant Ins-gene Induced Diabetes of Youth (MIDY), in which the patients with diabetes have a mutation in one of the copies so that as much as half of all of their proinsulin may be misfolded.”

Tsai explains that in the pancreatic beta cell, proinsulin first is targeted, or delivered, to the endoplasmic reticulum (ER) compartment, in order to begin the process of making insulin. When proteins are made in the ER, if things go right, they acquire their natural folded three-dimensional shape that is needed in order to function as they should.

If they don’t acquire and retain that proper shape, then the cell recognizes them as being defective protein molecules and works to destroy them so that they don’t wreak havoc within the cell.

“In the MIDY disease, having that one mutated gene making proinsulin is bad news,” Tsai says. “The cell has to figure out a way to recognize the bad protein molecules that come from the mutant gene and destroy them. And if it doesn’t, it turns out that misfolded proinsulin can have a “dominant-interfering” effect on the normal bystander proinsulin molecules that are made from the other, good gene.”

He adds that the normal proinsulin would ordinarily be made into insulin and that would help to lower blood sugar. But, when the misfolded proinsulin physically attaches itself to the normal bystander proinsulin, that blocks the ability of beta cells to make the normal proinsulin into insulin.

Liu and Tsai are joined in the study by U-M colleagues Peter Arvan, M.D., Ph.D., professor and chief of the Division of Metabolism, Endocrinology & Diabetes, and Ling Qi, Ph.D., professor of molecular and integrative physiology.

The team explains that the pancreatic cells have a way of rectifying the protein misfolding problems. The major way is by recognizing misfolded or damaged proteins and ejecting them from the ER to the cell’s proteosome, a major cellular garbage disposal that has the responsibility of chopping up proteins targeted for destruction. That process is called Endoplasmic Reticulum Associated Degradation (ERAD).

The idea is that if misfolded proinsulin is chopped up and degraded, then the remaining normal proinsulin can move through the beta cell and be successfully converted into biologically active insulin, to lower blood sugar.

The team is now researching if there is a way to stimulate the degradative pathway in order to get rid of more of the mutant protein.

“We think we can rectify this diabetic disease by manipulating the ERAD pathway so we can restore normal insulin secretion,” says Tsai.

“We’re trying to show proof of principle that if we manipulate the cells to have increased ability to degrade misfolded proinsulin, we can increase the amount of normal insulin that can be made and secreted. The hope is this would then help in the development of drugs that would stimulate ERAD to generate the same beneficial effect.”

The team explains this type of research has not been reported before in the diabetes field.

“There have been extensive studies on proteins undergoing ERAD,” says Qi. “Researchers know that protein misfolding is important for certain diseases, but we’re now focusing in on diabetes.”

Arvan agrees, “To understand protein misfolding diseases, we have to know more about protein folding. This is an exciting step in the field of diabetes research.”

The team has just received a four-year, multi-investigator grant from the National Institutes of Health, from which important new answers are expected.

Gene therapy for blistering skin disease appears to enhance healing in clinical trial

Grafting sheets of a patient’s genetically corrected skin to open wounds caused by the blistering skin disease epidermolysis bullosa appears to be well-tolerated and improves wound healing, according to a phase-1 clinical trial conducted by researchers at the Stanford University School of Medicine.

The results mark the first time that skin-based gene therapy has been demonstrated to be safe and effective in patients.

The findings will be published Nov. 1 in JAMA. Associate professors of dermatology Peter Marinkovich, MD, and Jean Tang, MD, PhD, share senior authorship of the study. Senior scientist Zurab Siprashvili, PhD, is the lead author.

For the study, four adult patients with recessive dystrophic epidermolysis bullosa, an excruciatingly painful genetic skin disease, received the skin grafts.

“Our phase-1 trial shows the treatment appears safe, and we were fortunate to see some good clinical outcomes,” said Tang. “In some cases, wounds that had not healed for five years were successfully healed with the gene therapy. This is a huge improvement in the quality of life for these people.”

People with epidermolysis bullosa lack the ability to properly produce a protein called type-7 collagen that is needed to anchor the upper and lower layers of the skin together. As a result, the layers slide across one another upon the slightest friction, creating blisters and large open wounds. The most severe cases are fatal in infancy. Other patients with recessive dystrophic EB can live into their teens or early adulthood with supportive care. Often these patients die from squamous cell carcinoma that develops as a result of constant inflammation in response to ongoing wounding.

The Stanford researchers showed that it was possible to restore functional type-7 collagen protein expression in patient skin grafts to stop blistering and allow wounds to heal. They also found that the protein continued to be expressed and that wound healing was improved during a year of follow up.

Looking to build upon results

The researchers seek to build upon these promising early results in a new trial that will include patients ages 13 and older.

“Moving into the pediatric population may allow us to intervene before serious chronic wounds and scars appear,” said Marinkovich, who directs the Stanford Blistering Disease Clinic. Repeated rounds of wounding and scarring on the fingers and palms, for example, often lead to fusion of the skin and the formation of what’s known as a “mitten hand.”

Siprashvili used a virus to deliver a corrected version of the type-7 collagen gene into batches of each patient’s skin cells that had been harvested and grown in the laboratory. He coaxed these genetically corrected cells to form sheets of skin about the size of an iPhone 5. The sheets were then surgically grafted onto the patient’s chronic or new wounds in six locations.

The researchers tracked the status of the grafts at one-, three- and six-month intervals for at least a year, checking to see if they stayed in place and caused wound closure. They also looked for any evidence of an immune reaction to the grafts, and whether the grafts continued to make the corrected type-7 collagen protein.

All 24 grafts were well-tolerated, the researchers found. Furthermore, they could detect expression of the type-7 collagen protein in the correct location of the skin in nine out of 10 tissue biopsies at three months. After 12 months, they were able to detect the collagen protein in five out of 12 biopsies.

Wound healing

Similar results were seen with wound healing. After three months, 21 of the 24 grafts were intact. This number dropped to 12 out of 24 after one year.

“Even a small improvement in wound healing is a huge benefit to the overall health of these patients,” said Tang. “For example, it may reduce the likelihood of developing squamous cell carcinoma that often kills these patients in young adulthood.”

Coupling grafts with hand surgery to break up scarred, fused tissue could help patients maintain the use of their hands, Marinkovich said.

Tang, Marinkovich and their colleagues will continue to monitor the patients in the phase-1 trial throughout their lifetimes to assess any long-term effects of the grafts.

The completion of the phase-1 trial and the potential to improve upon these outcomes is due to a concerted, long-term effort at Stanford to find ways to help young patients with this devastating disease.

The researchers are now starting a phase-2 clinical trial and are looking for new patients. For more information, send an email to tangy@stanford.edu or mpm@stanford.edu.

“This trial represents the culmination of two decades of dedicated clinical and basic science research at Stanford that began with the arrival of the former dean of the School of Medicine, Eugene Bauer, who set up the multidisciplinary EB Center at Stanford,” said Tang. “We have been working for a long time to get to this potential therapy into patients. We had to discover the genes and proteins involved and the responsible mutations. We then had to learn to deliver the corrected gene and grow those cells into sheets suitable for grafting.”

“We could not have reached this point without the support of the EB patients and their families,” said Marinkovich. “Since the time of my research training in the laboratory of Robert Burgeson, PhD, who discovered type-7 collagen, I’ve been deeply motivated to contribute to the EB community, and it is very satisfying to be able to finally see this molecular therapy come to fruition.”

The work is an example of Stanford Medicine’s focus on precision health, the goal of which is to anticipate and prevent disease in the healthy and precisely diagnose and treat disease in the ill.

New Treatment Leaves Liver Cancer Cells In Limbo

Scientists have shown that a mutation in a gene called Arid1b can cause liver cancer. The gene normally protects against cancer by limiting cell growth, but when mutated it allows cells to grow uncontrollably. The researchers have shown that two existing drugs can halt this growth in human cells. This points to a new approach to treating liver cancer.

These early results could be translated into a treatment relatively quickly, says Jesus Gil of the MRC Clinical Sciences Centre (CSC), based at Imperial College London, and who led the study. This is because the drugs are already used to treat other types of cancer. Gemcitabine is used on bladder, pancreas and ovary cancer, whilst DON has been tested in clinical trials. Both are known to be safe in people so will not require the usual toxicity testing.

According to MacMillian Cancer Support, 4200 people in the UK are diagnosed with liver cancer annually. “Liver cancer is a deadly disease,” says Luca Tordella, a postdoctoral researcher at the CSC who played a key role in the study. He says the new treatment will be most effective in people who have a mutation in the Arid1b gene. “It’s important to better classify patients into groups, according to their genes. One advance of personalised medicine is to understand which drug will work best on you, and which on me.”

The CSC team began by looking for mutations in the DNA of 100 people with liver cancer. They focused in on genes known to regulate a cellular state called senescence. This is a built-in safety mechanism that helps to protect the cell against cancer. Senescence is triggered when a cell grows abnormally fast and, once induced, it acts to keep the cell in limbo, such that it is alive but can no longer grow or divide. Bypassing senescence is a hallmark of many types of cancer.

It has previously been suggested that the Arid1b gene plays a role in liver cancer. The CSC team are the first to show that it does so by disrupting senescence. Working with researchers at the University of Tübingen, Germany, they mutated Arid1b in mice and human cells, and this stopped the cells from entering senescence. When Arid1b was mutated in mice that also had a mutation in the gene Ras, the mice developed liver cancer.

The researchers also identified exactly how Arid1b stimulates senescence. In healthy cells, Arid1b is part of a bigger complex (called SWI/SNF) that regulates the activity of hundreds of genes. One of these genes produces an enzyme that breaks down the building blocks of DNA. Without these blocks, the cell cannot continue to grow and divide so enters senescence. But if Arid1b is mutated, the blocks cannot be degraded and the cell continues to grow, bypassing senescence and potentially growing out of control.

Gil and Tordella have shown that it’s possible to induce senescence by treating human cells, which have an Arid1b mutation, with either gemcitabine or DON. Both drugs inhibit the synthesis of these blocks, called nucleotides, and thereby induce senescence and stop cancer in its tracks.

“Around 20% of patients with liver cancer have mutations on the genes encoding for components of the SWI/SNF complex. What we suggest is if we treat these people with drugs that target the degradation of nucleotides, they will respond,” says Gil. “We plan to continue to research this in the lab to develop treatments to target liver cancer.”

Asthma Research Unexpectedly Yields New Treatment Approach For Inherited Enzyme Disease

Experiments designed to reveal how a protein protects the lungs from asthma-related damage suggest a new way to treat a rare disease marked by the inability of cells to break down fats, according to a report in EBioMedicine published online Oct. 25.

The study results address Gaucher’s disease, which is caused by a genetic glitch in cell structures called lysosomes that process fats and remove cellular waste. Found mostly in Jews of Eastern and Central European origin, the condition may come with joint pain, blood disorders, enlarged spleens and livers, memory loss, and lung damage.

At a cellular level, Gaucher’s disease is associated with abnormally low production of the protein progranulin, as well as with the misplaced buildup of the enzyme beta-glucocerebrosidase, or GBA, outside lysosomes, instead of inside where it is needed.

Led by researchers from NYU Langone Medical Center, the new study found that a manufactured version of progranulin reversed most effects of Gaucher’s disease in mouse and human cell studies, including GBA accumulation.

“Our results suggest a new way to treat Gaucher’s disease that corrects abnormal enzyme delivery by progranulin to lysosomes, as opposed to current treatment strategies that temporarily replenish lysosomal GBA stores, which are then steadily consumed,” says senior study investigator Chuanju Liu, PhD, a professor in the Departments of Orthopaedic Surgery and Cell Biology at NYU Langone. The research team and NYU Langone hold a patent on related, potential therapies.

Among the study’s other key findings was that progranulin must bind to other molecules to transport the enzyme to lysosomes, specifically the protective “heat shock” protein 70. If unshielded, cellular GBA molecules fold up and stick together outside lysosomes.

Researchers also found that adding synthetic progranulin, or Pcgin, to blood cells obtained from patients with Gaucher’s, led to a 40 percent reduction in GBA clumping within a week. Pcgin was used because it is chemically more stable than progranulin and poses no risk of uncontrolled tumor-like cell growth in test animals, say the authors.

“Our new experiments are the first to explain why reduced progranulin is a key characteristic of Gaucher’s, and why the mice engineered to lack the protein serve as such a good model to test new therapies,” says lead study investigator Jian Jinlong, MD, PhD, an associate research scientist at NYU Langone.

Along with their role in brain disorders, progranulin shortages had been tied by previous research to cell swelling in asthmatic lungs. In the current set of experiments in progranulin-deficient mice, adding Pcgin reduced lung-tissue swelling by as much as 60 percent, an effect seen with current GBA-replacement treatments.

According to Liu, further research is needed to determine the precise mechanism by which progranulin reduces cell swelling, a process that would likely yield even more drug targets for Gaucher’s disease.

Experts estimate that as many as one in 50,000 Americans has some form of Gaucher’s, while one in 500 Jews of Ashkenazi descent has the disease.

Genome Engineering Paves The Way For Sickle Cell Cure

A team of physicians and laboratory scientists has taken a key step toward a cure for sickle cell disease, using CRISPR-Cas9 gene editing to fix the mutated gene responsible for the disease in stem cells from the blood of affected patients.

For the first time, they have corrected the mutation in a proportion of stem cells that is high enough to produce a substantial benefit in sickle cell patients.

The researchers from the University of California, Berkeley, UCSF Benioff Children’s Hospital Oakland Research Institute (CHORI) and the University of Utah School of Medicine hope to re-infuse patients with the edited stem cells and alleviate symptoms of the disease, which primarily afflicts those of African descent and leads to anemia, painful blood blockages and early death.

“We’re very excited about the promise of this technology,” said Jacob Corn, a senior author on the study and scientific director of the Innovative Genomics Initiative at UC Berkeley. “There is still a lot of work to be done before this approach might be used in the clinic, but we’re hopeful that it will pave the way for new kinds of treatment for patients with sickle cell disease.”

In tests in mice, the genetically engineered stem cells stuck around for at least four months after transplantation, an important benchmark to ensure that any potential therapy would be lasting.

“This is an important advance because for the first time we show a level of correction in stem cells that should be sufficient for a clinical benefit in persons with sickle cell anemia,” said co-author Mark Walters, a pediatric hematologist and oncologist and director of UCSF Benioff Children’s Hospital Oakland’s Blood and Marrow Transplantation Program.

The results will be reported in the Oct. 12 issue of the online journal Science Translational Medicine.

Sickle cell disease is a recessive genetic disorder caused by a single mutation in both copies of a gene coding for beta-globin, a protein that forms part of the oxygen-carrying molecule hemoglobin. This homozygous defect causes hemoglobin molecules to stick together, deforming red blood cells into a characteristic “sickle” shape. These misshapen cells get stuck in blood vessels, causing blockages, anemia, pain, organ failure and significantly shortened lifespan. Sickle cell disease is particularly prevalent in African Americans and the sub-Saharan African population, affecting hundreds of thousands of people worldwide.

The goal of the multi-institutional team is to develop genome engineering-based methods for correcting the disease-causing mutation in each patient’s own stem cells to ensure that new red blood cells are healthy.

The team used CRISPR-Cas9 to correct the disease-causing mutation in hematopoietic stem cells – precursor cells that mature into red blood cells – isolated from whole blood of sickle cell patients. The corrected cells produced healthy hemoglobin, which mutated cells do not make at all.

Future pre-clinical work will require additional optimization, large-scale mouse studies and rigorous safety analysis, the researchers emphasize. Corn and his lab have joined with Walters, an expert in developing curative treatments such as bone marrow transplant and gene therapy for sickle cell disease, to initiate an early-phase clinical trial to test this new treatment within the next five years.

Notably, research groups might be able to apply the approach described in this study to develop treatments for other blood diseases such as β-thalassemia, severe combined immunodeficiency (SCID), chronic granulomatous disease, rare disorders like Wiskott-Aldrich syndrome and Fanconi anemia, and even HIV infection.

“Sickle cell disease is just one of many blood disorders caused by a single mutation in the genome,” Corn said. “It’s very possible that other researchers and clinicians could use this type of gene editing to explore ways to cure a large number of diseases.”

“There is a clear path for developing therapies for certain diseases,” said co-senior author Dana Carroll of the University of Utah, who co-developed one of the first genome editing techniques over a decade ago. “It’s very gratifying to see gene editing technology being brought to practical applications.”

The work is the fruit of the Innovative Genomics Initiative, a joint effort between UC Berkeley and UCSF that aims to correct DNA mutations that underlie human disease using CRISPR-Cas9, a pioneering technology co-developed by scientists at UC Berkeley that has made genome editing easier and more efficient than ever before.

Smallest-Reported Artificial Virus Could Help Advance Gene Therapy

Gene therapy is a kind of experimental treatment that is designed to fix faulty genetic material and help a patient fight off or recover from a disease. Now scientists have engineered the smallest-reported virus-like shell that can self-assemble. It could someday carry potentially therapeutic DNA or RNA and transfer it to human cells. The report appears in the Journal of the American Chemical Society.

The story of gene therapy is fraught with much hype and high-profile failures. But, hype and failures aside, it remains a promising route to treat a range of ailments, from rare genetic diseases to common conditions such as diabetes. Clinical trials to test various gene therapy treatments are underway. One possible approach is to copy the way viruses behave. When they infect people, viruses inject their genetic material into human cells. Artificial viruses have been engineered to mimic this step, but they tend to clump or are not uniform in size, which can hinder their effectiveness. Max Ryadnov and colleagues wanted to address these issues.

Rather than using full proteins, the researchers used short peptide sequences designed to assemble into tiny gene carriers, which are smaller than previously reported synthetic viruses and even naturally occurring viruses. Lab testing showed that their artificial viral shells were uniform in size and didn’t clump. The particles could encase DNA or RNA and transfer the genetic material to human cells without harm. Depending on the introduced material, the recipient cells then either expressed a new protein or stopped expressing their own protein.