Tag Archives: mouse

Animal research successes spur growth in science…but PeTA can only complain

What do multiple myeloma, influenza, advanced breast cancer, atrial fibrillation, thyroid cancer, ear infection, advanced ovarian cancer and obesity all have in common? One commonality is obvious – they cause suffering, sickness and sometimes death in people around the world. Another commonality is less obvious – these are each conditions that are now being treated with new drugs just approved by the U.S. Food and Drug Administration (FDA) in the past three months alone. That’s right… in the period from Thanksgiving 2014 until now, new drugs that treat each of these conditions have become available, and these agents will be used to treat the illnesses that may affect millions of Americans. Eventually, they will likely have enormous worldwide impacts on these diseases. That’s something to be thankful for.

While some are thankful that the scientific progress is successfully tackling human suffering and disease, others cast doubt on the way that progress is achieved. In a newly published analysis entitled “Trends in animal use at US research facilities” [1], employees of People for the Ethical Treatment of Animals (PeTA) – a self-avowed animal rights organization – report that, amongst the largest research universities in the United States, the number of animals involved in research has grown by over 70% during the past 15 years. In their publication, the authors express alarm over the growing use of animals not covered by the Animal Welfare Act (AWA), mostly mice and fish, in biomedical research, without making any mention of the impact of this research growth.

This growth in animal research in the US is directly linked to an accelerating pace of scientific study and its benefits. A brief visit to the FDA’s “New Drugs at FDA page” makes it quickly apparent that the rate of approval of new medications is astounding. Where is this progress coming from? At least in part, it’s coming from the scientific discoveries that are pouring out of the research laboratories located in colleges and universities, institutes and pharmaceutical and biotechnology companies around the globe. A good example is the innovative BiTE antibody Blincyto (blinatumomab) which was approved for use in treating B-cell acute lymphoblastic leukemia in December 2014 (clinical evaluation against other cancers is ongoing); as we discussed in a blog post in 2008, animal research – particularly studies in mice – played a key role in its development and early evaluation.

Thanks to the researchers that occupy laboratories around the world, scientific discoveries are coming faster than ever, and all of us benefit. It’s not just that there is more research being done – it’s that the impact of the science is better than ever thanks to more advanced technologies, accumulating knowledge of how the body works and more advanced animals models, including ones that mimic human disease processes in increasingly sophisticated ways that promote new discoveries and new opportunities to develop novel drugs.

Why is the scale of animal research growing in the US? The answer is clear: scientific progress is cumulative. One discovery often enables multiple other lines of work. The discovery of the structure of DNA, for example, enabled thousands of efforts to find the genetic causes of disease. Because of this, successes build on successes and research grows.

What is the consequence of the growth in animal research? The answer is: new treatments, new cures, less sickness and longer, healthier lives.

In their paper, the PeTA employees fail to mention any of the following accomplishments, allow of which resulted from the growing scientific research efforts around the world:

But this isn’t the end. To these existing accomplishments, add the work that was started in the past 15 years and will yet unfold in the forthcoming decade AND the overwhelming progress in basic/fundamental research that will lead to new treatments and cures throughout the first half of the 21st century, and you have the recipe for a growing animal research infrastructure in this country.

As recent statistics from the UK indicate, the increase in the use of mice and fish in research is driven almost entirely by the increasing number of studies that involve the use of genetically-modified (GM) animals. In other words, the increase is driven by scientific and technological advances that had a profound impact on biomedical research over the past 15 years, rather than any desire to avoid using species regulated by the AWA (while mice and fish studied in Universities are not covered by the AWA, research involving them is regulated in multiple ways, including through the federal Office of Laboratory Animal Welfare which issues the PHS Guide for the Care and Use of Laboratory Animals).

“Recent statistics from the UK indicate, the increase in the use of mice and fish in research is driven almost entirely by the increasing number of studies that involve the use of genetically-modified (GM) animals.”

Growing study of GM animals has occurred because these models are enormously useful. To take just one example, the National Institute of Child Health and Development recently published an online article entitled “It’s in the DNA: Animal Models Offer Clues to Human Development”, discussing the role of animal models in helping to understand human development and developmental disorders. But this is far from the only example, studies in GM mice are key to many of the state-of-the-art emerging fields in biomedical research. These range from the very new areas of optogenetics – which uses light to control activation of individual cells – and gene editing techniques such as CRISPR that have the potential to cure genetic disorders, to new therapies such as cancer immunotherapy and treatments for rare genetic disorders such as progeria and Pompe disease which are being used to successfully treat patients for whom effective therapies were previously unavailable.

The rise in the numbers of zebra fish is also driven by their value as research models. As vertebrates they share over 84% of the genes that cause disease when defective in humans, while their rapid reproduction and transparent eggs make them ideal subjects for genetic and developmental studies. It’s not surprising that they are both an increasingly popular species in basic biomedical research, and in the preclinical evaluation of potential new therapies and of the environmental safety of chemicals.

In recent years zebra fish have become an increasingly popular species in biomedical research.

What the statistics presented by PeTA in their article don’t tell you is that, while the number of experiments and studies have increased, animal research increasingly involves Refined techniques that produce minimized harm to the subjects and Reduced numbers of animals per study. And of course, animal research directly led to the ability to Replace animals in some types of studies, altogether. The efficacy and efficiency of animal research is advancing, and individual discoveries are, on average, being made with fewer animals. That is a fact missed entirely by the PeTA article.

Furthermore, within the concept of refinement is the idea that researchers should use animals that will suffer less in a laboratory setting wherever possible [2]. So replacing a small number of “higher” mammals with a high number of “lower” animals is consistent with the 3Rs principles of animal welfare. PeTA neglect to mention that USDA statistics show a 40% fall in the use of AWA-covered species over the last 15 years, and it is likely that a small proportion of the rise in use of non-AWA covered species is due to technological advances that have allowed non-AWA species (e.g. GM mice) to replace AWA species (e.g. monkeys) in some studies, for example to develop new treatments for HIV/AIDS, in line with the principle of Refinement we have outlined.

Number of animals used annually for research in the US

“PeTA neglect to mention that USDA statistics show a 40% fall in the use of AWA-covered species over the last 15 years”

Through the implementation of these 3Rs, scientists ensure that they engage in socially-responsible and ethical work. What the authors of the PeTA study should do is to explain how achieving their end goal of a virtual end to animal research, which will reverse the trend of accelerating discovery and medical progress upon which it depends, is ethical or defensible.

  1. Goodman, J., Chandna A., and Roe K. 2015. Trends in animal use at US research facilities in: J Med Ethics. 0:1-3
  2. Richmond, J., 2014. Refinement Alternatives: Minimizing Pain and Distress in Allen, D. and Waters M. ed. In Vivo Toxicity Testing” in: Reducing, Refining and Replacing the Use of Animals in Toxicity Testing. Cambridge: RSC. pp. 133

David Jentsch

Primate research and twenty years of stem cell firsts

This guest post is by Jordana Lenon, B.S., B.A., Senior Editor, Wisconsin National Primate Research Center and University of Wisconsin-Madison Stem Cell and Regenerative Medicine Center. The research will also be featured this evening in a public talk at UW-Madison’s Wednesday Nite at the Lab. WN@tL: “Twenty Years of Stem Cell Milestones at the UW.”  Details and link are below. Update 1/8/15:  Dr. William Murphy’s talk  can now be viewed at:  http://www.biotech.wisc.edu/webcams?lecture=20150107_1900

As we enter 2015, the 20th anniversary of the first successful isolation and culture of primate pluripotent stem cells in the world, it’s time to look back and see how far we’ve come. Thanks to a young reproductive biologist who came from the University of Pennsylvania’s VMD/PhD program to the Wisconsin National Primate Research Center at the University of Wisconsin-Madison in 1991, and to those whose research his groundbreaking discoveries informed, the fields of cell biology and regenerative medicine will never be the same.

stem cell colonies

Pluripotent stem cells are right now being used around the world to grow different types of cells—heart muscle cells, brain cells, pancreatic cells, liver cells, retinal cells, blood cells, bone cells, immune cells and much more.

Cultures of these cells are right now being used to test new drugs for toxicity and effectiveness.

More and more of these powerful cells are right now moving out of the lab and into preclinical (animal) trials and early human clinical trials to treat disease. The results are being published in peer-reviewed scientific journal articles on stem cell transplant, injection and infusion, reprogramming, immunology, virology and tissue engineering.

Pluripotent stem cells and their derivatives are right now being studied to learn more about reproduction and development, birth defects, and the genetic origins of disease.

Embryonic, induced pluripotent, tissue specific (adult), and other types of stem cells and genetically reprogrammed cells are all being used by researchers due to the open and collaborative environment of scientific and medical enterprises in the U.S. and around the world.

All of this is happening right now because of discoveries made 20 years ago by researchers at the Wisconsin National Primate Research Center.

Here is a brief timeline of stem cell breakthroughs by WNPRC scientists:

  • 1995-James Thomson becomes the first to successfully isolate and culture rhesus monkey embyronic stem cells (ES cells) at the Wisconsin Regional Primate Research Center (PNAS)
  • 1996-Thomson repeats this feat with common marmoset ES cells (Biol Reprod).
  • 1998-Thomson publishes the neural differentiation of rhesus ES cells (APMIS).
  • 1998-Thomson’s famous breakthrough growing human ES (hES) cells is published in Science. (This research occurred off campus, with private funding.)

Many subsequent stem cell “firsts” were accomplished by scientists who conducted lengthy training with James Thomson or Ted Golos, reproduction and development scientists at the Wisconsin National Primate Research Center. These highlights include the following accomplishments by Primate Center researchers:

  • 2003-WNPRC Post-doctoral trainee Thomas Zwaka achieves homologous recombination with hES cells. A method for recombining segments of DNA within stem cells, the technique makes it possible to manipulate any part of the human genome to study gene function and mimic human disease in the laboratory dish (Nature Biotechnology).
  • 2004-WNPRC Post-doctoral trainee Behzad Gerami-Naini develops an hES model that mimics the formation of the placenta, giving researchers a new window on early development (Endocrinology).
  • 2005- WNPRC scientist Igor Slukvin and post-doc Maxim Vodyanik become the first to culture lymphocytes and dendritic cells from human ES cells (Blood, J Immunol).
  • 2005-WiCell’s Ren-He Xu, who completed his post-doctoral research at the WNPRC, grows hES cells in the absence of mouse-derived feeder cells (Nature Methods).
  • 2006-WiCell’s Tenneille Ludwig, a graduate student/post-doc/assistant scientist through the Primate Center with Barry Bavister, then James Thomson, formulates a media that supports hES cells without the need for contaminating animal products (Nature Biotechnology). Co-authoring the work is another former Primate Center post-doc, Mark Levenstein.
  • 2007-Junying Yu, WNPRC and Genome Center, in Jamie Thomson’s lab, grows induced pluripotent stem cells, or iPS cells. (Science). These are genetically reprogrammed mature cells that act like embryonic stem cells, but without the need to destroy the embryo.

Researchers at all of the National Primate Research Centers continue to make advances in this remarkable field of research and medicine. A few more milestones include the following:

  • 2007- Shoukhrat Mitalipov at the Oregon National Primate Research Center successfully converted adult rhesus monkey skin cells to embryonic stem cells using somatic cell nuclear transfer (Nature)
  • 2012- Shoukhrat Mitalipov at the Oregon National Primate Research Center generation chimeric rhesus monkeys using embryonic cells (Cell)
  • 2012-Alice Tarantal at the California NPRC successfully transplants human embryonic stem cells differentiated toward kidney lineages into fetal rhesus macaques.
  • 2013-Qiang Shi at the Texas Biomedical Research Institute and Gerald Shatten at the University of Pittsburgh – and previously with the Oregon National Primate Research Center and Wisconsin National Primate Research Center – genetically programs baboon embryonic stem cells to restore a severely damaged artery.
  • 2013-Shoukhrat Mitalipov at the Oregon National Primate Research Center produces human embryonic stem cells through therapeutic cloning, or somatic cell nuclear transfer (Cell)

NPRC Stem Cell Timeline 01.06.15

Before all of this happened, we must note that non-primate mammalian embryonic stem cells were first successfully isolated and cultured in 1981, by Martin Evans and Matthew Kaufman at the University of Cambridge, England. That breakthrough occurred almost 35 years ago. Jamie Thomson studied mouse embryonic stem cells in Pennsylvania before working on primate cells.

Even before that, in 1961, Ernest McCulloch and James Till at the Ontario Cancer Institute in Canada discovered the first adult stem cells, also called somatic stem cells or tissue-specific stem cells, in human bone marrow. That was 55 years ago.

So first it was human stem cells, then mouse, then monkey, then back to humans again. Science speaks back and forth. It reaches into the past, makes promises in the present, and comes to fruition in the future.

In every early talk I saw Jamie Thomson give about his seminal stem cell discoveries in the late 1990s and early 2000s – to staff, scientists, to the public, to Congress, to the news media – he would explain why he came to UW-Madison in the early 1990s to try to advance embryonic stem cell research. In large part, he said, it was because we had a National Primate Research Center here at UW-Madison, and also that we had leading experts in transplant and surgery at our medical school. After he joined the WNPRC as a staff pathologist and set up his lab, first he used rhesus and then marmoset embryos before expanding to cultures using human IVF patient-donated embryos off campus with private funding from Geron Corporation in Menlo Park, California.

Human And Mouse EmbryoIn these early talks, Jamie included images (see above) showing how very differently the mouse blastocyst (a days-old embryo, before implantation stage) is structured from the nonhuman primate and human primate blastocysts concerning germ layer organization and early development (ectoderm, mesoderm and endoderm). He also was able to show for the first time how differently stem cells derived from these early embryos grow in culture. In contrast to the mouse ES cells, the monkey cells, especially those of the rhesus monkey, grow in culture almost identically to human cells.

At the time, Thomson predicted that more scientists would study human ES cells in their labs over monkey ES cells, if human ES cells could become more standardized and available. Yet he emphasized that the NPRCs and nonhuman primate models would continue to play a critical role in this research, especially when it would advance to the point when animal models would be needed for preclinical research before attempting to transplant cells and tissues grown from ES cells. Both predictions have come true.

Jamie closed his talks, and still does, with this quotation:

“In the long run, the greatest legacy for human ES cells may be not as a source of tissue for transplantation medicine, but as a basic research tool to understand the human body.”

This simply and elegantly reminds us how basic research works: Many medical advances another 20 years from now will have an important link to the discoveries of today, which have their underpinnings in that early research in Jamie Thomson’s lab 20 years ago. It will become easy to forget where it all started, when many diseases of today, if not completely cured, will become so preventable, treatable and manageable that those diagnosed with them will spend more time living their lives than thinking about how to survive another day.

Just as I did not have to worry about polio, and my children did not have to worry about chicken pox, my grandchildren will hopefully see a world where leukemia, blindness, diabetes and mental illness do not have the disabling effects or claim as many young lives as they do today.

***

_______________________________________________________

WN@tL “Twenty Years of Stem Cell Milestones at the UW”

http://www.uwalumni.com/event/wntl-twenty-years-of-stem-cell-milestones-at-the-uw/

January 7 – 7:00PM – 8:15PM CT
Location: UW Biotechnology Center 425 Henry Mall, Room 1111, Madison, WI 53706
Cost: Free

Speaker: William L. Murphy, Stem Cell and Regenerative Medicine Centerwnatl_williammurphy

Don’t miss this fascinating talk covering stem cell milestones at the UW. Professor Murphy will talk about the work of his team at the Stem Cell and Regenerative Medicine Center, where they are creating biological materials that could radically change how doctors treat a wide range of diseases.

Bio: Murphy is the Harvey D. Spangler Professor of Engineering and a co-director of the Stem Cell and Regenerative Medicine Center. His work includes developing biomaterials for stem cell research. Specifically, Murphy uses biomaterials to define stem cell microenvironments and develop new approaches for drug delivery and gene therapy. His lab also uses bio-inspired approaches to address a variety of regenerative medicine challenges, including stem-cell differentiation, tissue regeneration and controlled drug delivery. Murphy has published more than 100 scientific manuscripts and filed more than 20 patent applications.

Stem cell therapy allows blind to see again, thanks to animal research

A team of scientists led by stem cell pioneer Professor Robert Lanza has reported today in the Lancet (1) the first evidence for the long-term safety of  retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) in patients who took part in a trial undertaken in four centres in the US. substantial improvements in vision were also recorded in almost half the treated patients, compared to no improvement in untreated patients.

This is the first time that clinical benefits have been demonstrated in the medium to long term in patients with any disese treated with hESC-derived cells, and is a major milestone in the development of the field of regenerative medicine. It’s an achievement that is due to many years of animal research.

Image:UCL/PA

Image:UCL/PA

The trial focused on 18 patients with two different types of macular degeneration,  Stargardt’s macular dystrophy and nine with dry atrophic age-related macular degeneration, that are common causes of blindness in adults and children and for which no effective treatments are currently available.

Nine patients with Stargardt’s macular dystrophy and nine with dry atrophic age-related macular degeneration received injections of 50,000 to 150,000 RPE cells behind the retina of their worst-affected eye. Robert Lanza, adjunct Professor at the Institute for Regenerative Medicine, Wake Forest University School of Medicine and Chief Scientific Officer at Advanced Cell Technology who funded the trial, describes the results:

The vision of most patients improved after transplantation of the cells. Overall, the vision of the patients improved by about three lines on the standard visual acuity chart, whereas the untreated fellow eyes did not show similar improvements in visual acuity. The patients also reported notable improvements in their general and peripheral vision, as well as in near and distance activities”

Professor Steven Shwartz, who led the team at the Jules Stein Eye Institute that took part in this trial, noted how important this result is to both the patients in this trial and the field of hESC-derived stem cell medicine.

Our results suggest the safety and promise of hESCs to alter progressive vision loss in people with degenerative diseases and mark an exciting step towards using hESC-derived stem cells as a safe source of cells for the treatment of various medical disorders requiring tissue repair or replacement,

You can listen to interviews with Steven Schwartz and several of the participants in this clinical trial in an NPR broadcast here.

In 2011 we discussed the launch of trials of these hESC-derived RPE cells, including some of those whose results are reported today,  at Moorfields Eye Hospital in London and the Jules Stein Eye Institute at UCLA. A paper published in the Journal Stem Cells in 2009 showed how studies in rodent models retinal degerneration paved the way for these trials by demonstrating that RPE cells derived from hESCs were safe and could restore vision:

Assessments of safety and efficacy are crucial before human ESC (hESC) therapies can move into the clinic. Two important early potential hESC applications are the use of retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration and Stargardt disease, an untreatable form of macular dystrophy that leads to early-onset blindness. Here we show long-term functional rescue using hESC-derived RPE in both the RCS rat and Elov14 mouse, which are animal models of retinal degeneration and Stargardt, respectively. Good Manufacturing Practice-compliant hESC-RPE survived subretinal transplantation in RCS rats for prolonged periods (>220 days). The cells sustained visual function and photoreceptor integrity in a dose-dependent fashion without teratoma formation or untoward pathological reactions. Near-normal functional measurements were recorded at >60 days survival in RCS rats. To further address safety concerns, a Good Laboratory Practice-compliant study was carried out in the NIH III immune-deficient mouse model. Long-term data (spanning the life of the animals) showed no gross or microscopic evidence of teratoma/tumor formation after subretinal hESC-RPE transplantation. These results suggest that hESCs could serve as a potentially safe and inexhaustible source of RPE for the efficacious treatment of a range of retinal degenerative diseases.”

This work – and earlier studies of RPE cells derived from ESCs – built on decades of basic stem cell research, starting with the pioneering work of Gail Martin, Matthew Kaufman and Martin Evans in mice, and the subsequent derivation of ESCs in macaques and then humans by James Thompson and colleagues at the university of Wisconsin- Madison.

Laboratory Mice are the most common species used in research

The humble mouse has played a key role in the development of stem cell medicine.

Today’s announcement is a major milestone in regenerative medicine, and one that id justifiably being celebrated, but we should also remember the many years of careful research that has led up to this moment. As with many medical advances much of the early research on embryonic stem cells was undertaken without any immediate clinical application in mind, but it nevertheless created the knowledge that is now driving an important emerging field of medicine. This is a lesson we need to remember when we donate to charities, when we discuss the importance of research with others, and most of all when we go to the ballot box!

Paul Browne

1) Schwartz SD et al. “Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies” Lancet published onlin3 15 October 2014. Link.

2) Lu B et al. “Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration.”
Stem Cells. 2009 Sep;27(9):2126-35. doi: 10.1002/stem.149.

How to help girls with Rett syndrome, and strike a blow against extremism!

Today we have a guest post by Dr Nicoletta Landsberger, Associate Professor at the University of Insubria and Principle Investigator at the San Raffaele Rett Research Center. The San Raffaele Rett Research Center is supported by the Pro Rett Ricerce (proRett), a small but energetic Italian patient organization that funds research in Italy and abroad to find a cure for the neurodevelopmental disorder Rett syndrome, which affects about 1 in 10,000 girls. 

A fortnight ago Dr Landsberger was forced to cancel a fundraising event – which included a raffle – for proRett due to the threat of disruption from animal rights extremists. Our friends in Pro-Test Italia wrote an open letter to Italian prime minister Matteo Renzi about this attack on medical progress, and bought 200 tickets for the raffle (worth 400 Euros).

Regular readers of this blog will be well aware of the recent increase in animal rights extremism in Italy, but the campaign against a charity that seeks to find effective therapies for a disease that devastates many thousands young lives around the world marks a new low. We need to support our friends in Italy, to support the children who suffer from Rett syndrome, and to send a strong message to animal rights extremists that their intimidation and bullying will not be tolerated. We are not asking you to march in the streets, or to sign a petition, or even to write a letter, we are asking you to do something a lot simpler; we are asking you to make a donation to proRett.

Please take a few minutes to give proRett what you can via their PayPal account, even a small donation will help (The PayPal account is in Italian, but essentially identical to the English language version. United States is Stati Unita in Italian, and United Kingdom is Regno Unito. If you are unsure of anything just use Google Translate).

Imagine Anna, a wonderful eight months girl sitting in her high chair and turning the pages of a book while watching it. Imagine Anna’s mother showing you other pictures of her daughter, smiling to her siblings or grasping objects. Everything seems normal, but then, few months later, the pictures are different. Anna is not smiling anymore, the expression of her face is different, the brightness has disappeared and in many pictures Anna has protruding jaws. Anna’s mother tells me “this is when I realized that something was changing…. At that time Anna’s progress stopped, the ability to hold the book and turn its pages was lost, overcome by continuous stereotyped hand-wringing movements. Rett syndrome and its regression phase were taking Anna away, locking her in her body for good”.

Anna is now 16, she is wheel chair bound, unable to talk and to play; like most girls affected by Rett syndrome she suffers from seizures, hypotonia, constipation, scoliosis, osteopenia, and breathing irregularities. Like most girls affected (over 90%) by typical Rett syndrome she carries a mutation in the X-linked MECP2 gene.

Today, almost 30 years after Rett syndrome was internationally recognized as a unique disorder mainly affecting girls, we know that it is a rare genetic disease, and that because of its prevalence (roughly 1:10.000 born girls) can be considered one of the most frequent causes of intellectual disability in females worldwide.

Rett syndrome is a pediatric neurological disorder with a delayed onset of symptoms and has to be clinically diagnosed relying on specific criteria. Girls affected by typical Rett Syndrome are born apparently healthy after a normal pregnancy and uneventful delivery and appear to develop normally usually throughout the first 6-18 months of life. Then their neurological development appears to arrest and, as the syndrome progresses, a regression phase occurs that leads to a documented loss of early acquired developmental skills, such as purposeful hand use, learned single words/babble and motor skills. During the regression phase, patients develop gait abnormalities and almost continuous stereotypic hand wringing, washing, clapping, and mouthing movements that constitute the hallmark of the disease. Many other severe clinical features are associated with typical Rett syndrome, including breathing abnormalities, seizures, hypotonia and weak posture, scoliosis, weight loss, bruxism, underdeveloped feet, severe constipation and cardiac abnormalities. Rett syndrome patients often live into adulthood, even though a slight increase in the mortality rate is observed, which is often caused by sudden deaths, probably triggered by breathing dysfunctions and cardiac alterations. There are no effective therapies available to slow or stop the disease, only treatments to help manage symptoms.

Genetic analyses show that most cases are caused by a mutation in the X-linked MECP2 gene, and many different missense mutations and deletions have been identified within the MECP2 gene of girls with Rett syndrome that prevent the protein from functioning correctly. The formal genetic proof of the involvement of the MECP2 gene in Rett syndrome is further provided by a number of diverse mouse models carrying different MECP2 alterations, which display the same symptoms observed in human patients (for more information see this recent open-access review by David Katz and colleagues) . These animals that fully recapitulate the disease have permitted us to demonstrate that the neurons have a constellation of minor defects, but that no degeneration is occurring, and that our brain need MECP2 at all times. Whenever the gene gets inactivated the disease appears.

Genetically modified mice have made crucial contributions to our understanding of Rett syndrome. Image courtesy of Understanding Animal Research.

Genetically modified mice have made crucial contributions to our understanding of Rett syndrome. Image courtesy of Understanding Animal Research.

Rett syndrome is mainly a neuronal disease, and obviously the amount of research we can do with the girls’ brains is limited. Because of this a range of mouse models of the disease have been instrumental for the study of the pathology. Furthermore, the same mice have permitted scientists to find the first molecular pathways that appear altered in the disease leading to test some therapeutic molecules in mice. Translational research leads to a clinical trial; and this is the case here, for example a clinical trial of IGF1 therapy is currently under way. Importantly, in 2007, Professor Adrian Bird and colleagues at the University of Edinburgh demonstrated in a mouse model that it is in principle possible to reverse Rett syndrome, and that MECP2-related disorders can be treated even at late stages of disease progression. However, the functional role(s) of MECP2 and their relevance to different aspects of development and neurological function are not fully understood, and different mutations in the MECP2 have varying effects on these roles, which any treatments will have to account for. Research indicates that too much MECP2 expression can be damaging, so scientists will need to find a way to express just the right amount of MECP2, in just the areas it is required. The clinical community has decided that no drug can be given to Rett syndrome girls without having first been tested in two different laboratories and on at least two diverse mice models of the disease. Nevertheless, this research is very promising, and not just for those with Rett syndrome and their families, as the insights gained through developing therapies for Rett syndrome are likely to be applicable to therapeutic strategies for a wide range of neurodevelopmental disorders. Studies in mouse models of Rett syndrome have a crucial role to play in this ongoing work.

proRETT is an association founded in 2004 by parents of children born with Rett syndrome, who began their activity by raising funds for the US based Rett Syndrome Research Foundation (now the International Rett Syndrome Foundation). proRett now supports the work of top Rett researchers in Italy, the UK and USA. I am a professor of molecular biology who has worked on MECP2 since I was a post-doctoral fellow in the team of the late Dr Alan P Wolffe at the National Institute of Child Health and Human Development.

In 2005 I met with proRETT to launch a collaboration in order to accelerate the scientific interest in the disease in Italy and abroad, and over the next few years   we worked together to organize two international scientific meetings (e.g. the European Working Group on Rett Syndrome) and attracted the interest of several Italian researcher to the disease. In 2010 proRETT felt the necessity to support more research in Italy and decided to open a laboratory – the San Raffaele Rett Research Center  – at the prestigious San Raffaele Scientific Institute in Milan. The laboratory, which I lead, employs 2 post-doctoral scientists, 3 PhD students and an undergraduate student. Further a second laboratory employing 8 scientists, supervised by myself and Danish researcher Dr. Charlotte Kilstrup-Nielsen, and fully dedicated to Rett syndrome is located at the University of Insubria in Busto Arsizio. As I outlined earlier, our research, as well as that of many other laboratories in the world, is interested in defining the molecular pathways that get deregulated because of a dysfunctional MECP2.  We are also examining the role of the gene during early development and outside of the brain itself. Eventually we hope to develop some novel protocols of gene therapy that can reverse Rett syndrome.

The Rett syndrome research team at the University of Insubria in Busto Arsizio

The Rett syndrome research team at the University of Insubria in Busto Arsizio

Because one of the two labs supported by proRETT is in Busto Arsizio and in Busto Arsizio there is a strong female volleyball team – Unendo Yamamay – almost one year ago we decided to organize a match of the Yamamay team dedicated to proRETT. The idea was for a female team to support research on a disease that affects girls, with both volleyball and research in the same town. The team were keen to help and the event was scheduled to be held on Saturday 15th March 2014. That evening we would have been the guests of Yamamay, and we were going to hold a raffle to raise money for research.

Unfortunately, once the event was announced last month, the trouble started. It began when the Busto Arsizio branch of the large Italian animal rights group the Lega Anti Vivisesione published decontextualised images of dead mice (seems familiar – SR)not belonging to my lab on their facebook page and claimed that our activities were unscientific  in order to stir up anger amongst their supporters against our lab (you can read more details about this in Italian here). They then tried to start a boycott of Unendo Yamamay and started a mass  e-mailing campaign, writing on social networks and to the proRETT and Unendo Yamamay. At the end of this nightmare, and because the local police headquarters was not confident about keeping the event safe from disruption by violent animal rights extremists, we had to give up. The match went ahead but proRETT were no longer guests, with Unendo Yamamay issuing a statement expressing their extreme regret at the events leading to the cancellation that had “caused serious harm to persons engaged daily in medical research against this terrible disease”.

Organizers had hoped to sell 6 thousand tickets for the lottery in aid of Rett syndrome research

Organizers had hoped to sell 6 thousand tickets for the lottery in aid of Rett syndrome research

The cancellation was felt as a tragedy by the parents, who, obviously, felt themselves even more alone than before. Because of that we decided to hold the raffle in our university in Busto Arsizio on Friday evening the in order to raise some money for proRETT, where we were joined by some parents and girls with Rett syndrome, as well as several journalists, and the president of Pro-Test Italia, who chose to show solidarity by attending. In the end we raised almost 6,000 euros from the raffle, less than we had initially hoped, but enough to show us and the parents of girls with Rett syndrome that there are still good people who are prepared to stand up for vital research.

We need to make sure this never happens in Italy again. This fight goes beyond Rett girls but is in the name of the progress of biomedical science in Italy and in the world; it is in the name of a future with less suffering. We would like the parents of Rett girls  and researchers dedicated to curing this disease to not feel alone, so we ask you to join good people in Italy and across the world to show your support for our girls, and your contempt for animal rights extremism, by making a small donation to proRETT.

Thank you.

Nicoletta Landsberger

To learn more about the role of animal research in advancing human and veterinary medicine, and the threat posed to this progress by the animal rights lobby, follow us on Facebook or Twitter.

From clinic to mouse to clinic: New HIV gene therapy shows promise!

Yesterday a team of University of Pennsylvania researchers – led by Dr Pablo Tebas, Professor Carl June, and Dr Bruce Levine – announced the successful conclusion of a clinical trial to evaluate the safety of a new gene therapy technique for treating HIV. It is a result that may eventually allow millions of HIV positive people to control the infection without having to take daily medication.

Two technicians in Penn Medicine's Clinical Cell and Vaccine Production Facility hold up a bag of modified T cells. Image: Penn Medicine

Two technicians in Penn Medicine’s Clinical Cell and Vaccine Production Facility hold up a bag of modified T cells. Image: Penn Medicine

Their study, published in the New England Journal of Medicine, involved taking a sample of T-cells from 12 patients and then using an adenoviral vector to introduce into these cells an enzyme known as a zinc-finger nuclease (ZFN) that has been targeted to the CCR5 receptor gene so that it introduces a mutation called CCR5-delta-32.  They then expanded the number of T-cells in vitro until they had billions of the transformed T-cells ready for transplant back into the patients.

Most HIV strains need to bind to CCR5 to infect T-cells, and the CCR5-delta-32 mutation prevents this binding and subsequent infection, as was dramatically demonstrated in the case of the “Berlin patient”, so the Pennsylvania team are hoping that their method will enable long-term control of HIV infection in patients, so that they may no longer need to take anti-retroviral medication.

An important part of the development of this therapy was its evaluation in vivo in an animal model of HIV infection. To do this they turned to mice rather than the more usual SIV/macaque model, as the sequence of the CCR5 gene at the site targeted by ZFN in macaques is not conserved with humans and would require the design and assembly of a distinct ZFN binding set for testing in SIV infection. Mice don’t normally become infected with HIV, but by using NOG mice that have been genetically modified so that their own immune system do not develop and then transplanting human immune cells into the mice they were able to produce mice with “humanized” immune systems that could be used to evaluate the ability of their ZFN modified T-cells to block HIV infection. In a paper published in the journal Nature Biotechnology in 2008, the team led by Carl June reported that the transformed human T-cells could successfully engraft and proliferate when transplanted into the NOG mice, and protect against subsequent HIV infection.

To our knowledge, genome editing that is sufficiently robust to support therapy in an animal model has not been shown previously. The ZFN-guided genomic editing was highly specific and well tolerated, as revealed by examination of the stability, growth and engraftment characteristics of the genome-modified sub-population even in the absence of selection…We also observed a threefold enrichment of the ZFN-modified primary human CD4+ T cells and protection from viremia in a NOG mouse model of active HIV-1 infection. As predicted for a genetically determined trait, the ZFN-modified cells demonstrated stable and heritable resistance in progeny cells to HIV-1 infection both in vitro and in vivo. These results demonstrate that ZFN-mediated genome editing can be used to reproduce a CCR5 null genotype in primary human cells.”

Following this they also undertook more extensive regulatory studies in mice to demonstrate that there were no toxicities associated with the ZFIN transformation of the T-cells.

While the clinical trial announced yesterday focused on the safety of the technique, the authors also reported that HIV RNA became undetectable in one of four patients who could be evaluated, and that the blood level of HIV DNA decreased in most patients, which bodes well for future trials when larger quantities of ZFN-modified cells will be transplanted.

This is not the first time that the pioneering work of Bruce Levine and Carl June has caught our attention, they are the same researchers who have hit the headlines with an innovative “Chimeric Antibody Receptor” gene therapy for leukemia that is part of the cancer immunotherapy revolution now underway. Their latest breakthrough is another indication of how gene therapy is becoming an important part of 21st century medicine.

Paul Browne

To learn more about the role of animal research in advancing human and veterinary medicine, and the threat posed to this progress by the animal rights lobby, follow us on Facebook at: https://www.facebook.com/SpeakingofResearch

Visionary Science: Gene therapy saves sight thanks to animal research

Yesterday the BBC News and Guardian Newspaper reported that a team led by surgeon Professor Robert Maclaren at the Oxford Eye Hospital had succeeded in using gene therapy to halt the decline in vision in six patients with the progressive eye disorder choroideremia.

All six patients were taking part in a clinical trial, and what was especially exciting was the sustained improvement in vision in the two patients whose vision had deteriorated the most. This is great news for the patients themselves, and as the technique is likely to be applicable to many different genetic eye disorders it is also good news for many millions of people who may benefit in future. It is also an excellent example of how years of research in mice, dogs and monkeys can lead to an important clinical advance.

Choroideremia is caused by a defect in the CHM gene, which encodes the Rab escort protein 1 (REP1), and lack of this protein leads to gradual degeneration of the retinal epithelium layer  (RPE) and rod photoreceptor cells in the eye, causing a progressive decline in vision that usually starts with night blindness and loss of peripheral vision, and eventually leads to total blindness.

To halt this decline Professor Maclean’s team used a vector  based on a modified adeno-associated virus serotype 2 (AAV2) which could express the healthy CHM gene in the eye and produce REP1.  Why did they choose AAV2 out of all the potential virus vectors available? The Lancet paper reporting on this trial cites a key study published in the Journal of Molecular Medicine in  2013* by Professor Maclaren and colleagues, which describes the development and evaluation of the vector used in the trial. In their introduction and discussion they discuss the rational for choosing the AAV2 vector:

With a functional fovea, safety with regard to avoiding a vector-related inflammatory reaction is of paramount importance. Two recent clinical trials had demonstrated that serotype 2 adeno-associated viral (AAV2) vectors have no long-term retinal toxicity when administered at the dose range 1010–1011 genome particles [12, 13]. Importantly, in addition to transducing the RPE, AAV2 is also known to target rod photoreceptors efficiently in the non-human primate [14], providing the ideal tropism for a CHM gene therapy strategy.

… Although one might argue that other serotypes such as AAV8 may be more efficient in targeting photoreceptors, AAV2 with the CBA promoter remains the gold standard for retinal transduction as evidenced by the sustained vision in Briard dogs treated with AAV2 vector over a decade ago [35].

12. Cideciyan AV, Aleman TS, Boye SL, Schwartz SB, Kaushal S, Roman AJ, Pang JJ, Sumaroka A, Windsor EA, Wilson JM, et al. Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc Natl Acad Sci U S A. 2008;105:15112–15117. doi: 10.1073/pnas.0807027105.  13. Jacobson SG, Cideciyan AV, Ratnakaram R, Heon E, Schwartz SB, Roman AJ, Peden MC, Aleman TS, Boye SL, Sumaroka A, et al. Gene therapy for Leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years. Arch Ophthalmol. 2011;130:9–24. doi: 10.1001/archophthalmol.2011.298.  35. Bennicelli J, Wright JF, Komaromy A, Jacobs JB, Hauck B, Zelenaia O, Mingozzi F, Hui D, Chung D, Rex TS, et al. Reversal of blindness in animal models of Leber congenital amaurosis using optimized AAV2-mediated gene transfer. Mol Ther. 2008;16:458–465. doi: 10.1038/sj.mt.6300389.

So which two clinical trials are they referring to? Well, as you can see from the references they are referring to the successful trials of gene therapy for Leber Congenital Amaurosis (LCA)whose development we discussed on this blog back in 2009. As McLaren and colleagues point out, the sustained expression of RPE65 and long-term recovery of vision in the Briard dog model of LCA was a key factor in their decision.  The observation that AAV2 could be used to drive gene expression in rod photoreceptors was also important, as Maclaren and colleagues had previously generated a genetically modified mouse model of Choroideremia by knocking out CHM expression in the eye, and established that in Choroideremia the degeneration of rod photoreceptors is independent of the degeneration of the RPE, so it is crucial that the vector can drive healthy gene expressed in both the rods and RPE.

To develop the vector Maclaren and colleagues first compared the efficiency of 3 different promoters (promoters are sections of DNA that promote gene expression) -AAV2/2-EFS, AAV2/5-EFS and AAV2/2-CBA  – in driving expression of the CHM gene when added in vitro in a variety of dog and human fibroblast (connective tissue cell)  lines in an AAV2 vector, and then when injected in vivo in the retinas of healthy mice. These studies demonstrated that the most efficient AAV2 vector – named AAV2/2-CBA-REP1 – could drive expression of high levels of REP1 in both the RPE and rod photoreceptors of mice. After identifying the most effective AAV2 vector for expressing REP1  they assessed whether it was capable of expressing REP1 in isolated human retina’s obtained post-mortem from human donors, which it did. They then evaluated whether there as any toxicity associated with expressing REP1 in vivo in the retina of healthy mice, finding that AAV2/2-CBA-REP1 was non-toxic even when injected into the retina at high doses, and that it did not adversely affect vision.

Following these studies the question remained; would injection of AAV2/2-CBA-REP1 stop deterioration of vision in choroideremia?

To address this Maclaren and colleagues turned again to the genetically modified mouse model of choroideremia thay they had created earlier. Injection of the vector into the retinas of these CHM mice:

Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham injected eyes confirming that AAV2/2-CBA-REP1 is a promising  vector suitable for choroideremia gene therapy in human clinical trials.”

In other words the therapy worked in the mouse model of choroideremia, paving the way for the successful clinical trial reported this week.

This new therapy is another example of the importance of animal studies to the development of new clinical techniques and therapies, but also highlights the fact that medical science is a long game, with basic and applied research conducted more than a decade, even two decades,  ago being crucial to this week’s exciting announcement. This is something policy makers would do well to remember!

Paul Browne

* While this paper was published in 2013, the work it reports was completed several years earlier, before the clinical trial was launched in 2011.

1) Tanya Tolmachova, Oleg E. Tolmachov, Alun R. Barnard, Samantha R. de Silva, Daniel M. Lipinski, Nathan J. Walker, Robert E. MacLaren,corresponding author and Miguel C. Seabra “Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo”  J Mol Med (Berl). 2013 July; 91(7): 825–837. PMCID: PMC3695676

Animal research brings hope to the girl whose skin never heals

On Friday the BBC broadcast a moving report about a young girl named Sohana Collins, who suffers from the painful and life threatening genetic disorder epidermolysis bullosa (EB), caused by mutations in the type VII collagen gene (Col7a1).  The report also included an interview with Prof John McGrath, Professor of Molecular dermatology at Kings College London, who is leading a clinical trial – EBSTEM – of mesenchymal stem cell therapy for EB that Sohana is part of, who spoke about the potential for this therapy to help people with EB.

Sohana Collins, who is participating in the EBSTEM trial. Image: BBC News

Sohana Collins, who is participating in the EBSTEM trial. Image: BBC News

Type VII collagen (col7) is a key component of the basement membrane of the skin, a layer of protein structures that acts as a kind of cement that binds the outermost layer of the skin – the epidermis – to the underlying dermal layer, and lack of clo7 leads to the two layers to move independently of each other. This shearing movement at the dermal-epidermal junction has  the result that even the slightest injury can lead to blisters and sores, and people with EB have a very high risk of developing skin cancers. The EBSTEM trial seeks to determine if infused mesenchymal stem cells from healthy donors can migrate to the skin and produce col7, restoring the basement membrane and relieving the symptoms of EB. The clinical trial registration document for EBSTEM notes that evidence from both animal studies and (subsequent) small clinical studies indicates that mesenchymal stem cells have the potential to treat this condition.

So where does animal research fit in to this work? Well, as a 2012 review (1) by Prof. McGrath points out, genetically modified mouse models of EB have both provided key information on the role of col7 and how its absence leads to the lesions seen in EB, and also provide a system in which novel therapies can be evaluated.

A number of model systems have been developed to examine the pathomechanistic consequences of mutations in heritable skin diseases, and many of these systems are also being utilized for development of molecular therapies. Particularly valuable towards understanding of disease mechanisms has been the development of transgenic animal models which recapitulate the clinical features noted in patients; these genetically modified animals have played a major role in advancing our understanding of the disease mechanisms in different forms of EB (Bruckner-Tuderman et al., 2010; Natsuga et al, 2010). Besides providing direct evidence for the structural role of many of the basement membrane zone adhesion molecules, the development of transgenic mice with EB phenotypes has provided novel information on the complex secondary effects mediated by signaling pathways and other systems that modify the EB phenotypes. In addition to transgenic animals, EB phenotypes have been observed in a number of animal species, both domestic and wild, as a result of naturally occurring mutations (Jiang and Uitto, 2005; Bruckner-Tuderman et al., 2010). In many cases, the suitability of these animal models of human disease for preclinical testing of gene-, protein-, and cell-based molecular therapies has been documented.”

A key early study was that of Professor John Wagner and colleagues at the University of Minnesota, who in 2008 reported that intravenous injection of wild-type bone marrow-derived cells could migrate to the skin lesions, produce the missing col7 protein, prevent blister formation, and extend survival in a genetically modified mouse model of EB  (2), providing the first evidence that stem cell therapy might benefit people with EB.  This study led Prof. Wagner and a team of researchers – including Prof. McGrath – to undertake a clinical trial of bone marrow transplantation in 6 EB patients, using standard chemoablative pre-conditioning procedures prior to transplant (which as we discussed in a recent post is quite a harsh procedure). The results were promising, new type col7 was noted in the basement membrane at the dermal-epidermal junction and clinical improvement was sustained for at least 1 year after bone marrow transplantation. However, two of the six children who completed the treatment died of complications of the procedure, that the risks of this kind of standard bone marrow transplant are too great in EB patients.

Subsequently another study in col7 deficient mice led by Dr Vitali Alexeev at Thomas Jefferson University indicated that when mesenchymal stem cells (a particular population of multipotent cells present in the bone marrow and other tissues that are being investigated as potential therapies for diseases such as multiple sclerosis) were transplanted into the skin they secreted col7, which was distributed throughout the treated area and formed connections with another collagen molecule – col4 – that necessary to restore the basement membrane (3). This study also demonstrated that the mesenchymal stem cells home in on areas of damage at the dermal-epidermal junction. This study – combined with the earlier observation that bone marrow derived stem cells were injected intravenously in the GM mouse model of EB they ameliorated their condition – provided good evidence that intravenous injection of mesenchymal stem cells may be a viable treatment for EB, and supporting decision to launch the EBSTEM trial.

A futher advantage of using mesenchymal stem cells is that while the EBSTEM trial is using bone-marrow derived mesenchymal stem cells, mesenchymal stem cells can potentially be obtained more easily from several other tissues, including fat tissue, which may provide a more abundant source of cells for transplant in the future. A drawback with intravenously injecting mesenchymal stem cells, compared to bone marrow transplantation, is that the benefits are less long lasting, and  the procedure will need to be repeated every few months (the optimum frequency required will be determined in later trials, but based on previous experience with MSCs it is likely to be about once every 6 months).

We wish Sohana and the other participants in this trial, and Professor McGrath and his colleagues, the very best of luck. While this new therapy is not a cure for EB, we hope that it will prove a major step towards that goal.

Paul Browne

1)      Uitto J, Christiano AM, McLean WH, McGrath JA. “Novel molecular therapies for heritable skin disorders.” J Invest Dermatol. 2012 Mar;132(3 Pt 2):820-8. doi: 10.1038/jid.2011.389. PMID: 22158553 PMCID: PMC3572786

2)      Tolar J, Ishida-Yamamoto A, Riddle M, McElmurry RT, Osborn M, Xia L, Lund T, Slattery C, Uitto J, Christiano AM, Wagner JE, Blazar BR. “Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells” Blood. 2009 Jan 29;113(5):1167-74. doi: 10.1182/blood-2008-06-161299. PMID: 18955559 PMCID: PMC2635082

3)      Alexeev V, Uitto J, Igoucheva O. “Gene expression signatures of mouse bone marrow-derived mesenchymal stem cells in the cutaneous environment and therapeutic implications for blistering skin disorder.” Cytotherapy. 2011 Jan;13(1):30-45. doi: 10.3109/14653249.2010.518609. PMID: 20854215