Tag Archives: mouse

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

A new drug to treat type II diabetes: Thank the…Gila monster?

Earlier this week Lyxumia (generic name Lixisenatide), a new drug that helps to control type II diabetes, was launched in the UK. In addition to being an effective and saft therapy for type II diabeted, including in some patients that do not respond to current first-line therapies, Lyxumia is relatively inexpensive when compared to current therapies for type II diabetes, which will help to save the health services money that can be invested in other therapies.

Lyxumia belongs to a new class of drugs known as the glucagon-like peptide 1 receptor agonists that work by increasing the secretion of insulin in response to consumption of food, and is administered by a once daily injection.That animal research played a key role in the development of the glucagon-like peptide 1 (GLP-1) receptor agonists for treating diabetes should not be a surprise, but when I took a quick look at the paper (1) reporting the preclinical development of Lyxumia (them called ZP10A)  I got a surprise.

The low half-life of native GLP-1 (90-120 s) (Deacon et al., 1995; Egan et al., 2003) has led to extensive research to find new compounds with pharmakokinetic properties suitable for development of a drug candidate. Exendin-4 was first isolated from the salivary gland of the Gila monster (Heloderma suspectum), and characterization showed that the peptide was structurally related to, but distinct from GLP-1 with a sequence homology of only 52%. Further characterization of exendin-4 showed that the peptide is a potent agonist for the mammalian GLP-1 receptor  with a longer in vivo half-life and prolonged duration of action compared with GLP-1 (Raufman et al., 1992; Young et al., 1999). Recent studies have shown that administration of exendin-4 induces pancreatic endocrine differentiation, islet proliferation and an increase in β-cell mass (Edvell and Lindström, 1999; Xu et al., 1999), indicating that exendin-4 may exert insulinotropic effects on the β-cells (Greig et al., 1999; Parkes et al., 2001).

Yes, you read it correctly, the development of effective GLP-1 receptor agonists started with a discovery made by a scientist studying venom peptides found in the the saliva of a large lizard!

The Gila monster - an unlikely ally in the fight against diabetes. Image courtesy of Jeff Servoss

The Gila monster – an unlikely ally in the fight against diabetes. Image courtesy of Jeff Servoss

This should actually not come as so much of a surprise, venom is an incredibly rich source of bioactive molecules, and scientists around the world are studying the venom of a bewildering array of animals in order to identify everything from better painkillers to therapies for Parkinson’s disease. Recently EU recognized the value of such research by setting up the VENOMICS project to provide tools and resources to the scientists engaged in it.

Lyxumia itself was created as a synthetic analogue of exendin-4, and following   in the db/db mouse model of diabetes the team at Zealand Pharma concluded that:

[T]hese studies demonstrate that ZP10A is an effective antidiabetic compound that effectively improves FBG and glucose tolerance, resulting in a long-term improvement of total glucose control. Furthermore, the sustained effect on glucose metabolism, and pancreatic expression of insulin even after discontinuation of ZP10A treatment indicates that ZP10A preserves β-cell function in diabetic db/db mice. Therefore, it is concluded that ZP10A is not only a promising candidate for the treatment of human type 2 diabetes but also it has the potential to prevent the progression of the disease.

On the basis of these very promising results ZP10A underwent further preclinical evaluation in collaboration with Sanofi-Aventis before entering into successful clinical trials.

The availability of a new and cost effective therapy to help people to manage type-2 diabetes is very welcome, but the story of the development of the Glucagon-like peptide 1 receptor agonists reminds us that new therapies can lurk in the most unlikely – and indeed most unpleasant – places!

Paul Browne

1) Thorkildsen C, Neve S, Larsen BD, Meier E, Petersen JS. “Glucagon-like peptide 1 receptor agonist ZP10A increases insulin mRNA expression and prevents diabetic progression in db/db mice.” J Pharmacol Exp Ther. 2003 Nov;307(2):490-6. Epub 2003 Sep 15.

Treating Progeria; How GM mice give hope to some very special children

Something big is going on right now in the world of research.

Something very specific for some very special children with a very rare disease. It may not be widely known by name but I am sure you have seen these children. The disease is called Progeria. From the Progeria Research Foundation’s website, we learn:

Hutchinson-Gilford Progeria Syndrome “Progeria” or “HGPS” is a rare, fatal genetic condition characterized by an appearance of accelerated aging in children*.  Its name is derived from Greek and means “prematurely old.”  While there are different forms of Progeria, the classic type is Hutchinson-Gilford Progeria Syndrome, which was named after the doctors who first described it in England: in 1886 by Dr. Jonathan Hutchinson, and in 1897 by Dr. Hastings Gilford.

Progeria affects approximately 1 in 4 – 8 million newborns.  There are an estimated 200-250 children living with Progeria worldwide at any one time.  It affects both sexes equally and all races.  Since The Progeria Research Foundation was created in 1999, we have discovered children with Progeria living in over 40 countries.”

Most of us will have come across a picture of one of these children in the papers, on TV, or on the internet. We remember them because they look different from other kids their age. If you ever get the privilege to chat with them, you will find that are some of the wisest people you will ever meet. To speak with them is truly inspiring because of their personalities and outlook on life. It is also heart wrenching because we know most will never reach their twenties.

About eight years ago I was working as a veterinary technician in a research facility. During that time a new investigator moved his lab into our facility, and we received his colony of mice a few weeks before he arrived. After we had cared for the mice for a few days, we started to see some very strange things. The weanlings were sometimes very small, and occasionally they were also thin. It was strange to see mice that were so young but  looked like such old men. The reason was simple, these mice had been genetically modified to carry the same defective Lamin A gene that is responsible for Hutchinson-Gilford progeria syndrome in children. The ‘sick’ mice we saw were actually mice with Progeria!”

GM mice aided the development of a therapy for Progeria

GM mice aided the development of a therapy for Progeria

Several years later Dr. Stephen G. Young and colleagues at UCLA published a study that detailed what they found within this small population of mice (1). Once a GM model of mice had been developed, cells from these mice were studied (2). When a farnesyltransferase inhibitor  was used in vitro on these cells, it showed this drug was a possible treatment for this terrible disease. Once this was learned, they went on to the next step which was to test farnesyltransferase inhibitor in vitro on cells from actual Progeria patients (3). When these studies looked very promising, confirming that the process occurring in the mouse and human cells were very similar, the GM mice were once again indispensable for the first in vivo study to determine if farnesyltransferase inhibitors could improve the health of mice with Progeria (1). This is the part that cannot be replicated by any calculations, test tube chemicals or computer programs. Without in vivo studies, it is impossible to know what a treatment will do in a living creature. The mice that were born with Progeria were given a farnesyltransferase inhibitor. Would they get better or would they stay the same? Once the study was complete, all results were compared and this therapy looked very promising indeed!

Professor Young gave a talk on his progeria research to the Congressional Medical Research Caucus in 2009, in which he discusses his group’s GM mouse studies in much more detail, and you can watch the video here.

From there, a drug needed to be developed that could be evaluated in children with Progeria. This is a process that can often take many years, but fortunately some farnesyltransferase inhibitors designed as cancer treatments looked promising (see more about it here). lonafarnib was selected for clinical trials in progeria because it had already been assessed in pediatric cancer clinical trials where it had a demonstrated an acceptable safety profile. This is how decades of drug development happened in less than 10 years.

Researchers were able to move many steps ahead, much closer to the Progeria clinical trials that were needed. Remember, the one thing these children do not have is time. They grow old and die, sometimes as young as seven, and very rarely live past twenty. Most die in their teens. If a completely new drug had been needed, nearly every child alive with the disease that day would have passed away by the time it was ready for a clinical trial.

I think it is very important to explain briefly genetic disease and the role GM play in finding treatments and cures. Francis Collins is a well known and oft cited geneticist and physician, and currently Director of the National Institutes of Health, who gave a TED talk in April 2012 about this very topic.  Dr. Collins has long been interested in Progeria, he led the team that first identified defects in the Lamin A gene as a cause of Hutchinson-Gilford progeria syndrome in 2003, and later in 2008 published a study that examine the effect of farnesyltransferase inhibitors on cardiac defects in a mouse model of Progeria (cardiac defects are the most common cause of death in children with Progeria).


At the most basic, a genetic disease is caused when there is a faulty gene somewhere in the genetic code. While the *reason* the gene is broken may be a mystery, there are roughly 4,000 genetic diseases that scientists at least know what gene is causing the problem, which is the case for Progeria. Scientists know what is causing the problem, but how do you fix it? Dr. Collins has a vision of accelerating the transition from the bench to the bedside, and the example of progeria shows that one of best tools for finding the treatments and cures is Genetically Modified mice. Our GM mice.

In the case with Progeria, researchers were able to create the same disease in mice that was found in humans, effectively mirroring the disease. By doing this, they are able to study not just the disease itself, but study treatments on a live organism with the disease. With GM mice, researchers are able to find treatments and cures at an unprecedented pace. As Dr. Mark Kieran, who led the first clinical trial of  lonafarnib to treat progeria (4), said:

PRF (Progeria Research Foundation)provides a model for disease research organizations, and is a good example of successful translational research, moving from gene discovery to clinical treatment at an unprecedented pace,”

There are over 4,000 genetic diseases known to us right now, yet only 250 of them have treatments. If we can find help for these people so quickly, why are there so few cures? One reason is that in many cases there are still no mouse model available to study. In our case of Progeria, a mouse model of the disease was developed which sped up research by years or even decades. Without GM mice, this treatment would not be available now. Progeria clinical trials moved very quickly compared to most treatments and it was announced in September of 2012. Finally, these children had a treatment! While this is not a cure, it is a huge step forward. With early diagnosis and treatment, these children have a much better chance at a normal life!

Because of the extremely rare occurrence of this disease, these children can be hard to find, especially in less developed countries where they may have never seen this disease before. In 2009, the Progeria Research Foundation  (PRF)launched the “Find the Other 150” campaign. As of September 2012, they were aware of 96 of the estimated 200-250 children living with Progeria. If you are aware of any of these children, please visit www.FindTheOther150.org to find information on how to participate in future studies.

I have spent nearly a decade in this field now. I will always remember those mice and those children. To see a treatment developed and to even have played a small part it helping it happen is humbling. Will I make headlines? No. Will my name ever be in a published paper? Probably not. Will I make millions off any of the discoveries I participate it? Never. I went into this field knowing full well I will never get rich or retire early and wealthy. That is not why I am here.  I choose to do what I do because of people out there like these Progeria kids. I do this for them, and all the millions of cancer patients out there like my late husband. I do this so we can find a cure.

And to know I had even a tiny part in making that cure happen, that, is priceless.

Pamela Bass

1)  Yang SH, Meta M, Qiao X, Frost D, Bauch J, Coffinier C, Majumdar S, Bergo MO, Young SG, Fong LG.”A farnesyltransferase inhibitor improves disease phenotypes in mice with a Hutchinson-Gilford progeria syndrome mutation.” J Clin Invest. 2006 Aug;116(8):2115-21

2)  Yang SH, Bergo MO, Toth JI, Qiao X, Hu Y, Sandoval S, Meta M, Bendale P, Gelb MH, Young SG, Fong LG.”Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation.” Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10291-6. Epub 2005 Jul 12.

3) Toth JI, Yang SH, Qiao X, Beigneux AP, Gelb MH, Moulson CL, Miner JH, Young SG, Fong LG. “Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes.” Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12873-8. Epub 2005 Aug 29.

4) Gordon LB, Kleinman ME, Miller DT, Neuberg DS, Giobbie-Hurder A, Gerhard-Herman M, Smoot LB, Gordon CM, Cleveland R, Snyder BD, Fligor B, Bishop WR, Statkevich P, Regen A, Sonis A, Riley S, Ploski C, Correia A, Quinn N, Ullrich NJ, Nazarian A, Liang MG, Huh SY, Schwartzman A, Kieran MW. “Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson-Gilford progeria syndrome.” Proc Natl Acad Sci U S A. 2012 Oct 9;109(41):16666-71. doi: 10.1073/pnas.1202529109. Epub 2012 Sep 24.