Combined Stem Cell and Gene Therapy Cures Fanconi’s Anaemia

Approach cures human genetic disease in vitro Sunday, 31 May 2009 A study led by researchers at the Salk Institute for Biological Studies, has catapulted the field of regenerative medicine significantly forward, proving in principle that a human genetic disease can be cured using a combination of gene therapy and induced pluripotent stem (iPS) cell technology. The study, published in the May 31, 2009 early online edition of Nature, is a major milestone on the path from the laboratory to the clinic. "It's been ten years since human stem cells were first cultured in a Petri dish," says the study's leader Juan-Carlos Izpisúa Belmonte, Ph.D., a professor in the Gene Expression Laboratory and director of the Center of Regenerative Medicine in Barcelona (CMRB), Spain. "The hope in the field has always been that we'll be able to correct a disease genetically and then make iPS cells that differentiate into the type of tissue where the disease is manifested and bring it to clinic." Although several studies have demonstrated the efficacy of the approach in mice, its feasibility in humans had not been established. The Salk study offers the first proof that this technology can work in human cells. Belmonte's team, working with Salk colleague Inder Verma, Ph.D., a professor in the Laboratory of Genetics, and colleagues at the CMRB, and the CIEMAT in Madrid, Spain, decided to focus on Fanconi anaemia (FA), a genetic disorder responsible for a series of haematological abnormalities that impair the body's ability to fight infection, deliver oxygen, and clot blood. Caused by mutations in one of 13 Fanconi anaemia (FA) genes, the disease often leads to bone marrow failure, leukaemia, and other cancers. Even after receiving bone marrow transplants to correct the haematological problems, patients remain at high risk of developing cancer and other serious health conditions.

---

Shown in green are genetically-corrected fibroblasts from Fanconi anaemia patients are reprogrammed to generate induced pluripotent stem cells, which, in turn, can be differentiated into disease-free hematopoietic progenitors, capable of producing blood cells in vitro. Credit: Courtesy of Dr. Juan-Carlos Belmonte, Salk Institute for Biological Studies.

---

After taking hair or skin cells from patients with Fanconi anaemia, the investigators corrected the defective gene in the patients' cells using gene therapy techniques pioneered in Verma's laboratory. They then successfully reprogrammed the repaired cells into induced pluripotent stem (iPS) cells using a combination of transcription factors, Oct4, Sox2, Klf4 and c-Myc. The resulting FA-iPS cells were indistinguishable from human embryonic stem cells and iPS cells generated from healthy donors. Since bone marrow failure as a result of the progressive decline in the numbers of functional hematopoietic stem cells is the most prominent feature of Fanconi anaemia, the researchers then tested whether patient-specific iPS cells could be used as a source for transplantable hematopoietic stem cells. They found that FA-iPS cells readily differentiated into hematopoietic progenitor cells primed to differentiate into healthy blood cells. "We haven't cured a human being, but we have cured a cell," Belmonte explains. "In theory we could transplant it into a human and cure the disease." Although hurdles still loom before that theory can become practice — in particular, preventing the reprogrammed cells from inducing tumours — in coming months Belmonte and Verma will be exploring ways to overcome that and other obstacles. In April 2009, they received a $6.6 million from the California Institute Regenerative Medicine (CIRM) to pursue research aimed at translating basic science into clinical cures. "If we can demonstrate that a combined iPS–gene therapy approach works in humans, then there is no limit to what we can do," says Verma. About the Salk Institute for Biological Studies: The Salk Institute for Biological Studies is one of the world's preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused both on discovery and on mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer's, diabetes, and cardiovascular disorders by studying neuroscience, genetics, cell and plant biology, and related disciplines. Faculty achievements have been recognized with numerous honours, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent non-profit organization and architectural landmark. Reference: Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells Ángel Raya, Ignasi Rodríguez-Pizà, Guillermo Guenechea, Rita Vassena, Susana Navarro, María José Barrero, Antonella Consiglio, Maria Castellà, Paula Río, Eduard Sleep, Federico González, Gustavo Tiscornia, Elena Garreta, Trond Aasen, Anna Veiga, Inder M. Verma, Jordi Surrallés, Juan Bueren & Juan Carlos Izpisúa Belmonte Nature advance online publication 31 May 2009, doi:10.1038/nature08129 ......... ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://cellnews-blog.blogspot.com/ and http://www.geocities.com/giantfideli/index.html

---

Adult Bone Marrow Stem Cells Injected Into Skeletal Muscle Can Repair Heart Tissue

Adult Bone Marrow Stem Cells Injected Into Skeletal Muscle Can Repair Heart Tissue Friday, 29 May 2009 University at Buffalo researchers have demonstrated for the first time that injecting adult bone marrow stem cells into skeletal muscle can repair cardiac tissue, reversing heart failure. Using an animal model, the researchers showed that this non-invasive procedure increased myocytes, or heart cells, by two-fold and reduced cardiac tissue injury by 60 percent. The therapy also improved function of the left ventricle, the primary pumping chamber of the heart, by 40 percent and reduced fibrosis, the hardening of the heart lining that impairs its ability to contract, by up to 50 percent. "This work demonstrates a novel non-invasive mesenchymal stem cell (MSC) therapeutic regimen for heart failure based on an intramuscular delivery route," said Techung Lee, Ph.D., UB associate professor of biochemistry and senior author on the paper. Mesenchymal stem cells are found in the bone marrow and can differentiate into a variety of cell types. "Injecting MSCs or factors released by MSCs improved ventricular function, promoted myocardial regeneration, lessened apoptosis (cell death) and fibrotic remodelling, recruited bone marrow progenitor cells and induced myocardial expression of multiple growth factor genes," Lee said. "These findings highlight the critical 'cross-talks' between the injected MSCs and host tissues, culminating in effective cardiac repair for the failing heart." The heart disease death rate has dropped significantly in the last three decades due to better treatments, resulting in large numbers of people living with heart failure. This advance has lead to another health hurdle: The only therapy available to reverse the decline in cardiac function is heart transplantation, and donor hearts are very scarce. Clinical trials of myocardial stem cell therapy traditionally have relied on surgery — infusing the stem cells directly into the heart or injecting them into the myocardium, the heart muscle — invasive methods that can result in harmful scar tissue, arrhythmia, calcification or small vessel blockages. "In our research with a swine model of heart failure," said Lee, "we've found that only 1-to-2 percent of MSCs infused into the myocardium grafted into the heart, and there was no evidence that they differentiated into heart muscle cells. In addition, diseased tissue is not a healthy environment for cell growth.” "For these reasons, and because patients with heart failure are not good surgical risks, it made sense to explore a non-invasive cell delivery approach," said Lee. An important feature of MSCs is their ability to produce a plethora of tissue healing effects, known as "trophic factors," which can be harnessed for stem cell therapy for heart failure. Lee noted that the multiple trophic factors produced by MSCs have been shown in the literature to be capable of reducing tissue injury, inhibiting fibrosis, promoting angiogenesis, stimulating recruitment and proliferation of tissue stem cells, and reducing inflammatory oxidative stress, a common cause of cardiovascular disease and heart failure. "Since skeletal muscle is the most abundant tissue in the body and can withstand repeated injection of large number of stem cells, we thought it would be a good method to deliver MSCs," Lee said. "We hypothesized that MSCs, via secretion of these functionally synergistic trophic factors, would be able to rescue the failing heart even when delivered away from the myocardium.” "This study proves our hypothesis," said Lee. "We've demonstrated that injecting MSCs, or trophic factors released by MSCs, into skeletal muscle improved ventricular function, promoted regeneration of heart tissue, decreased cell death and improved other factors that cause heart failure.” "This non-invasive stem cell administration regimen, if validated clinically, is expected to facilitate future stem cell therapy for heart failure." Lee said the next step is to use genetic and pharmacological engineering to make the stem cells more active, so good therapeutic effects can be achieved with fewer cells. "That is our goal. It would reduce the cost of stem cell therapy and make it more affordable for patients in the future." Reference: Heart Failure Therapy Mediated by the Trophic Activities of Bone Marrow Mesenchymal Stem Cells: A Non-invasive Therapeutic Regimen Arsalan Shabbir, David Zisa, Gen Suzuki, and Techung Lee Am J Physiol Heart Circ Physiol (April 24, 2009). doi:10.1152/ajpheart.00186.2009 ......... ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://cellnews-blog.blogspot.com/ and http://www.geocities.com/giantfideli/index.html

---

‘Floppy Baby’ Syndrome – Heart Protein Saves Muscle

New study shows heart muscle protein can replace its missing skeletal muscle counterpart to give mice with myopathy a long and active life Tuesday, 26 May 2009 In a world first, West Australian scientists have cured mice of a devastating muscle disease that causes a Floppy Baby Syndrome – a breakthrough that could ultimately help thousands of families across the globe. A heart muscle protein can replace its missing skeletal muscle counterpart to give mice with myopathy a long and active life. The research, published online today in the Journal of Cell Biology, reveals how a team at the Western Australian Institute for Medical Research (WAIMR) has restored muscle function in mice with one type of Floppy Baby Syndrome – a congenital myopathy disorder that causes babies to be born without the ability to properly use their muscles. The currently incurable genetic diseases render most of the affected children severely paralysed and take the lives of the majority of these children before the age of one. The contraction machinery protein, actin, exists in different forms in the adult heart and skeletal muscles. The heart form, ACTC, is also the dominant form in skeletal muscle of the foetus. However, during development, the skeletal form, ACTA1, increases in production and by birth has taken over. It is not clear why the switch occurs, or why it does not occur in the heart, but it happens in every higher vertebrate and, for that reason, has been considered vitally important. Mutations to the ACTA1 gene cause a rare but serious myopathy. Most patients die within the first year of life and some are born almost completely paralyzed. Mice lacking ACTA1 die nine days after birth. Dr Kristen Nowak, lead author on the publication, wondered if ACTC could compensate for a lack of ACTA1. The two proteins differ only slightly but, like the developmental switch in production, this difference is conserved across species. Many researchers therefore assumed such compensation would never work.

---

Muscle cell architecture looks normal in transgenic mice that lack ACTA1 but express human ACTC. Credit: Nowak, K.J., et al. 2009. J. Cell Biol. doi:10.1083/jcb.200812132.

---

But it did. Nowak and colleagues crossed Acta1 mutant mice with transgenic mice that express human ACTC at high levels in skeletal muscle cells. The resulting mice did not die at nine days. In fact, almost all of them (93.5%) survived more than three months, and some more than two years. The mice's locomotor performance was comparable with wild type, as was their overall muscle strength (though individual muscle fibres were slightly weaker), and their endurance was actually higher — they ran faster and for longer. This begs the question, Why do we even have ACTA1? Besides pondering that, Nowak and colleagues are also working out how to boost endogenous ACTC as a possible therapy for ACTA1-lacking patients. Dr Kristen Nowak said the team was extremely encouraged that it had been able to cure a group of mice born with the condition. "The mice with Floppy Baby Syndrome were only expected to live for about nine days, but we managed to cure them so they were born with normal muscle function, allowing them to live naturally and very actively into old age," she said. "This is an important step towards one day hopefully being able to better the lives of human patients – mice who were cured of the disease lived more than two years, which is very old age for a mouse." Dr Nowak said the team was able to cure the mice with the recessive form of the genetic condition by replacing missing skeletal muscle actin – a protein integral in allowing muscles to contract – with similar actin found in the heart. "Earlier in our search to tackle these diseases, we discovered a number of children who, despite having no skeletal muscle actin in their skeletal muscle due to their genetic mutation, were not totally paralysed at birth," she said. "On closer inspection, we found it was because heart actin – another form of the protein – was abnormally "switched on" in their skeletal muscles.” "We had already begun investigating whether we could use heart actin to treat skeletal muscle actin disease, so that discovery spurred us on, and we've now proved it can be done – we can use heart actin to overcome the absence of skeletal muscle actin in mice." Heart actin is found in cardiac muscle and, during foetal development, it also works in skeletal muscles in the body, but by birth, heart actin has almost completely disappeared within skeletal muscle. Using genetic techniques, the WAIMR research team has reactivated the heart actin after birth in place of skeletal muscle actin, reversing the effects of the congenital myopathy. Head of the WAIMR research group Professor Nigel Laing said the team's next step was to apply their findings to human patients. "We are now screening more than a thousand already-approved medications looking for one that might increase heart actin in skeletal muscles, which could potentially offer a treatment for many patients," he said. "Current therapies only target the effects of these conditions, not the condition itself – we hope our approach could lead to a much greater improvement for a range of muscle diseases." This discovery is the latest for the team, which has been investigating debilitating muscle diseases for more than 20 years. The first major breakthrough for actin disease was in 1999, when the team identified that defects in the skeletal muscle actin gene, ACTA1 – responsible for producing skeletal muscle actin, cause multiple muscle diseases. Since then, the team has classified and named a new muscle disease 'Laing Myopathy' – named after Professor Nigel Laing – and helped implement worldwide screening for families at risk of genetic muscle disease. WAIMR Director Professor Peter Klinken said he was thrilled WAIMR was playing such an integral part in helping tackle devastating muscle diseases. "The persistence and determination shown by Professor Laing and his team over many, many years is nothing short of inspiring," he said. "They've asked some big questions in their quest to find a cure for this Floppy Baby Syndrome and have worked tirelessly to find the answers to those questions in the hope of helping families across the world.” "Research institutes like ours exist to help people live healthier lives and I am delighted at the important discoveries we are making in this field." About Floppy Baby Syndrome: The skeletal muscle actin mutations which cause congenital myopathies can be classified into five individual diseases which affect thousands of families worldwide. Children with recessive muscle actin diseases have no skeletal muscle actin because of mutations in the skeletal muscle actin gene which "knock out" the gene function. In Australia, dozens of families are affected by congenital myopathies, which bring high emotional costs and personal suffering, as well as financial and community burdens. This research was funded by the National Health and Medical Research Council, WAIMR and a number of patient support groups including the Association Française contre les Myopathies (French Muscular Dystrophy Association) and the US Muscular Dystrophy Association. The research project centred at the WAIMR laboratory was a collaborative effort with groups at the Medical Research Council and the University of Oxford in the United Kingdom, Cincinnati Children's Hospital Medical Center as well as the Centre for Microscopy, Characterisation and Analysis at the University of Western Australia and Perth-based Proteomics International, which have also assisted the team's work. Reference: Rescue of skeletal muscle alfa-actin–null mice by cardiac (fetal) alfa-actin Kristen J. Nowak, Gianina Ravenscroft, Connie Jackaman, Aleksandra Filipovska, Stefan M. Davies, Esther M. Lim, Sarah E. Squire, Allyson C. Potter, Elizabeth Baker, Sophie Clément, Caroline A. Sewry, Victoria Fabian, Kelly Crawford, James L. Lessard, Lisa M. Griffiths, John M. Papadimitriou, Yun Shen, Grant Morahan, Anthony J. Bakker, Kay E. Davies, and Nigel G. Laing J. Cell Biol., May 25, 2009: jcb.200812132v1 ......... ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at http://cellnews-blog.blogspot.com/ and http://www.geocities.com/giantfideli/index.html

---