Author Archives: Blue Sky Science

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.

Understanding addiction: NIDA article highlights contribution of animal research

Professor David Jentsch is a highly respected UCLA neuroscientist who specialises in the study of addiction, one of the most widespread and serious medical problems in our society today. Sadly, by devoting his career to finding out how to better treat a condition that ruins – and all too often ends – many millions of lives in the USA and around the world every year, David has found himself, his colleagues, and his friends and neighbors under attack from animal rights extremists whose tactics have ranged from harassment, stalking and intimidation, to arson and violence.

Did this extremist campaign persuade David to abandon his research?

No chance!

In 2009 David responded to the extremist campaign against him and his colleagues by helping to found Pro-Test for Science to campaign for science and against animal rights extremism at UCLA, and has been a key contributor to Speaking of Research, writing articles on the role of animal studies in the development of new therapies for addiction, what his studies on rodents and vervet monkeys involve, and how addiction research can help us to understand obesity.

Vervet monkeys involved in David Jentsch's research program live in outdoor social groups to ensure optimal welfare

Vervet monkeys involved in David Jentsch’s research program live in outdoor social groups to ensure optimal welfare

This week the NIH’s National institute on Drug Abuse (NIDA) has published an excellent article on David’s ongoing research entitled  “Methamphetamine Alters Brain Structures, Impairs Mental Flexibility”, which highlights the importance of non-human primate research in identifying how addiction alters the brain and why some individuals are more prone to develop damaging methamphetamine dependency than others. You can read the article in full here.

Human chronic methamphetamine users have been shown to differ from nonusers in the same ways that the post-exposure monkeys differed from their pre-exposure selves. The researchers’ use of monkeys as study subjects enabled them to address a question that human studies cannot: Did the drug cause those differences, or were they present before the individuals initiated use of the drug? The study results strongly suggest that the drug is significantly, if not wholly, responsible”

This knowledge of how drug use disrupts brain function will be crucial to development effective clinical interventions for methamphetamine addiction, and the huge scale and devastating impact of methamphetamine use makes it clear that such interventions are desperately needed, as David highlights in the article’s conclusion.

Methamphetamine dependence is currently a problem with no good medical treatments, when you say a disease like methamphetamine dependence is costly, it’s not just costing money, but lives, productivity, happiness, and joy. Its impact bleeds through families and society.”

At a time when animal rights activists in many countries are pushing to ban addiction research involving animals, the NIDA article on the work of David and his colleagues shows why this work is so valuable, and just what would be lost if animal rights extremists are allowed to have their way.

Speaking of Research

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

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Statement on postponement of Pro-Test for Science rally

Dear colleagues, students, friends and supporters,

We want to thank each and every person that put aside their valuable personal time when they committed to attending this weekend’s Pro-Test rally in Westwood. Your agreement to participate is a testament to your commitment to scientific research and to the scientists who have been targeted at UCLA.

Our real goal was to positively change the climate for researchers at work and at home, where protesters are conducting their campaigns of terror. Counter demonstrating was but one way that this can be accomplished, and indeed, we believe a multi-faceted approach is required. Your commitment to this demonstration has evoked a renewed motivation in the University to work with us to create new strategies to bring under control the activities of animal rights extremists who insist on conducting campaigns of harassment, intimidation and threats against scientists and their families. At the recommendation of the University and to give these strategies an opportunity to develop and take effect, Pro-Test for Science has decided to defer the event planned for this Saturday.

We want to thank our supporters, and those who may object to aspects of the work but still hold that moral disputes ought to be resolved in the court of public opinion by civil debate. We will continue to express our expert views to the public so that society can take informed decisions in matters of basic, medical research and public health.

Pro-Test for Science


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/

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

Cancer Immunotherapy: A breakthrough made through animal research

The prestigious journal Science has published its top 10 Breakthroughs of the Year 2013, and top of the list is a development that promises to have a huge impact on the lives of millions of people in the coming decades – Cancer Immunotherapy.

The article focuses on three particular therapies that have recently shown great
promise in clinical trials – chimeric antigen receptors, anti-CTLA4 therapy, and anti-PD1 therapy – all of which highlight the fact that his is a field
where animal research is making an absolutely critical contribution.

Regular readers will remember that we discussed how studies in mouse xenograft models of acute lymphoblastic leukaemia (ALL) contributed to the development of chimeric antigen receptor (CAR) therapy that has now shown very promising results in clinical trials against ALL and Chronic Lymphocytic Leukemia, and as Science reports is now being evaluated against many other cancers.

The Science news article on cancer immunotherapy notes that a mouse study published in Science provided key evidence that antibodies that target the protein CTLA-4 – a receptor that acts to suppress the activate the T cells of the immune system – can increase the effectiveness of the immune system in eliminating tumor cells.

Similarly – as discussed in this open access review – the development of anti-PD1 immunotherapy started when was found that PD-1 knockout mice developed autoimmmune disorders, indicating that PD-1 played a role in regulating the immune response. Subsequent preclinical studies in a variety of mouse cancer models demonstrated that administration of antibodies against PD-1 greatly increased the ability of the immune system to attack the tumors, even well established and metastatic tumors.

Laboratory Mice are the most common species used in research

Cancer Immunotherapy – adding even more accomplishments to an already impressive CV!

The examples of CAR, Anti-CTLA4 and anti-PD1 therapies highlight how the field of cancer immunotherapy is maturing, but it is a field which has already delivered some important therapies.  For, example back in 2009 Emma Stokes wrote an article for this blog on the discovery and development of Rituximab, a chimeric antibody therapy that has revolutionized the treatment of B-cell cancers such as Non-Hodgkin’s lymphoma. This work has not stood still either, last week the BBC reported on the successful trial of a new chimeric antibody therapy named GA101 in patients with chronic lymphocytic leukaemia (CLL) and other B-cell conditions. GA101 targets the same protein – CD20 – as Rituximab, but was designed to induce a more powerful anti-cancer activity with fewer adverse effects. The abstract of the 2010 paper reporting on the preclinical research leading to the development of GA101 highlights the role played by studies in mouse models of cancer and in monkeys.

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell–depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.”

Of course there are another 9 breakthroughs on Science’s list, and it’s notable that several others involve animal research. One of these is CRISPR, a technique that allows scientists to modify the genes of organisms in vivo or cells in vitro with unprecedented precision, and more recently showed potential in mouse studies as a therapy for genetic disorders. Another is CLARITY, a technique that renders brain tissue transparent so that it can be studied in more detail than has previously been possible, and which joins a range of new techniques that are part of a revolution in neuroscience. Of course there was also the news of the first human stem cells created through cloning by Professor Mitalipov at Oregon Health and Science University, a pioneering scientist whose work we have discussed on several occasions.

The choice of cancer immunotherapy, and indeed of this list as a whole, is a reminder at the end of what has been a very difficult year for science in several countries across the world of the extraordinary progress that is being made, and why it is vital to support the scientists who make it happen. As we bid farewell to 2013 and greet 2014 we can only guess at what new discoveries and breakthroughs the year will bring, but we also know that now – perhaps more than any time in recent history – we need to join together across the world to stand up for science!

Paul Browne

Italian science rallies for animal research at the Mario Negri Institute.

On Saturday 30th November around 400 researchers, scientists and students met at the Mario Negri Research Institute in Milan, Italy, to take part in the “Io Sto Con La Ricerca” (I’m With Research) Convention, organized by Silvio Garattini, Director of the Institute. This Convention aimed to emphasize the importance of biomedical research to human health and the role of animal research within it.

The event was organized in response to a concurrent animal rights march in the center of the city, which activists from the Animal Amnesty group had planned to end next to the Mario Negri Institute. The protesters were not only rallying against animal experimentation, but specifically against the Institute’s Director Garattini, who they condemn as a cruel “vivisector”. Just days before the rally, Garattini has received anonymous death threats from activists, which resulted in the authorities banning the animal rights march from ending at the Mario Negri Institute, and prompted the Mayor of Milan to issue a public statement to condemn the intimidation and offer support to the scientists who were meeting at the Mario Negri Institute.

The convention about to get underway. Image from Pro-Test Italia

The convention about to get underway. Image from Pro-Test Italia

Many important Italian scientists and politicians participated in the conference, making speeches about the importance of animal research for science and human health and also offering solidarity with Silvio Garattini. Speakers included Gianluca Vago, Rector of the University of Milan; Francesco Brancati, President of UNAMSI, an association for medical and health information; Emilia de Biasi, President of the Senate Health Commission; Cristina Tajani, Research Authority of the City of Milan. It was also fantastic to see Paola Zaratin, the Director of Scientific Research for the Italian Multiple Sclerosis Society (AISM), one of the largest medical research charities in Italy. Other participants included the directors of every Research Institute of Milan, and Agnese Collino, a new member of Pro-Test Italia’s scientific committee, whose speech covered the misinformation surrounding animal research – often incorrectly described as a useless, outdated and cruel research method by animal rights activists – and the way animal rights activists misrepresent and distort images and quotations to advance their cause.

Dr Agnese Collino addresses the problem of disinformation in the animal research debate. Image from Pro-Test Italia

Dr Agnese Collino addresses the problem of disinformation in the animal research debate. Image from Pro-Test Italia

The Convention went without a hitch of any kind, in a very calm and positive atmosphere, with people listening carefully and with interest to the speeches. Many journalists attended the event and wrote articles comparing it with the animal rights rally. Most described both views without taking a side, a slight but important change in the way they used to report news about this topic.  Some did even better, for example the national newspaper La Republica published a series of images from the convention and the Milan daily newspaper Il Giornale published a great article entitled “The researchers rebel: We don’t torture animals”, while the popular and prestigious Milanese newspaper Corriere della Sera published an editorial in support of Silvio Garattini and the Mario Negri Institute. Director Silvio Garattini declared the event a great success, and thanked participants for helping to ensure that the public heard the viewpoint of researchers.

We hope that this event, together with other events that Pro-Test Italia is involved in this week, could make people aware of what research actually is and how important it is for our lives, with the purpose of defeating ignorance and misinformation about this topic. Disinformation has caused increasing trouble for researchers, such as the adoption, by the Italian Parliament, of a new law, that places greater restrictions on Italian research that exists across the rest of Europe.

Andrea Tosini, Pro-Test Italia

Update: Yesterday the journal Science has reported that more than 13,000 people, the great majority  Italian scientists, have signed a petition asking the EU commission to take action to save animal research - and indeed medical research in general – in Italy from damaging laws passed by the Italian parliament. It’s another sign of just how seriously the Italian scientific community is taking this threat to their countries scientific future.

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