Tag Archives: gene therapy

Research Roundup: Ebola vaccine hope for apes, gene therapy for dogs, and research into stroke

Welcome to the second of our Research Roundups. These Friday posts aim to inform our readers about the many stories that relate to animal research each week. Do you have an animal research story we should include in next week’s roundup? You can send it to us via our Facebook page or through the contact form on the website.

  • The first orally administered vaccine for Ebola developed for the conservation of wild apes, has completed its first and final biomedical research trial for the foreseeable future. The study, published in the journal Scientific Reports, shows that the vaccine was effective and did not induce health complications or lead to signs of stress in the apes. Lead investigator, Peter Walsh statedIn an ideal world, there would be no need for captive chimpanzees. But this is not an ideal world. It is a world where diseases such as Ebola, along with rampant commercial poaching and habitat loss, are major contributors to rapidly declining wild ape populations.Oral vaccines offer a real opportunity to slow this decline. The major ethical debt we owe is not to a few captive animals, but to the survival of an entire species we are destroying in the wild: our closest relatives.

One of the captive chimpanzees in the research trial receiving the oral Ebola vaccination. Credit: Matthias Schnell, Thomas Jefferson University.

  • A new compound, P7C3-A20, has been shown to prevent brain cell death and to promote new cell growth in a rat model of ischemic stroke. Nearly 87% of all strokes are ischemic strokes. Strokes kill 130,000 Americans yearly, with someone in the USA having a stroke every 40 seconds and with a death occurring every 4 minutes.
  • A new gene therapy, which aims to treat the fatal muscle-wasting disease, myotubular myopathy or MTM, has shown considerable success in dogs. Like humans, dogs naturally get this disease as a result of a genetic defect which tends to lead to breathing difficulty and early death. One year after a single gene therapy treatment, the dogs with the condition were indistinguishable from the control group. This offers huge promise for future human therapies for MTM. Results were published in Molecular Therapy.

Image from Science Daily

  • A team of scientists have prevented and alleviated two autoimmune diseases, multiple sclerosis (MS) and type 1 diabetes, in early stage mouse models. Autoimmune diseases affect an estimated 23 million Americans, and this research using mice highlights the importance of animal research in alleviating these debilitating diseases.
  • A new study finds that Lactobacillus, a common bacteria found in yogurt, may be used to alleviate symptoms associated with depression in mice – providing hope for the 7% of the population that experience a major depressive episode at least once in their lifetime. The study was published in Scientific Reports.

Image by Understanding Animal Research.

  • Canadian animal rights group, Last Chance for Animals, has alleged mistreatment of animals at the Contract Research Organisation, ITR Laboratories. The footage was included in a CTV W5 news report. In response to the infiltration video, ITR Labs released a statement saying they had parted ways with a number of technicians seen inappropriately handling animals in the footage. The Canadian Council of Animal Care also released a statement explaining that an inspection of the ITR facilities was now being organized.

Clinical trial success for Cystic Fibrosis gene therapy: built on animal research

This morning the Cystic Fibrosis Gene Therapy Consortium (GTC) announced the results of clinical trial in 140 patients with cystic fibrosis, which demonstrate the potential for gene therapy to slow – and potentially halt – the decline of lung function in people with the disorder. It is a success that is built on 25 years of research, in which studies in animals have played a crucial role.

Cystic fibrosis is one of the most commonly inherited diseases, affecting about one in every four thousand children born in the USA, and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene produces a channel that allows the transport of chloride ions across membranes in the body, and the many mutations identified in cystic fibrosis sufferers either reduce the activity of the channel or eliminate it entirely. This defect in chloride ion transport leads to defects in several major organs including the lungs, digestive system, pancreas, and liver. While the severity of the disease and the number of organs affected varies considerably, cystic fibrosis patients often ultimately require lung transplant s, and too many still die early in their 20’s and 30’s as the disease progresses.

In a paper published in Lancet Respiratory Medicine today (1), the GTG members led by Professor Eric Alton of Imperial College London compared monthly delivery to the airway of a non-viral plasmid vector containing the CFTR gene in the liposome complex pGM160/GL67A using a nebuliser with a placebo group who received saline solution via the nebuliser. They reported stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group after a year. This is the first time that gene therapy has been shown to safely stabilise the disease, and while the difference between the treated and control group was modest, and the therapy is not yet ready to go into clinical use, it provides a sound bases for further development and improvement.


The Chief Executive of the Cystic Fibrosis Trust, which is one of the main funders of the GTC, has welcomed the results, saying:

Further clinical studies are needed before we can say that gene therapy is a viable clinical treatment. But this is an encouraging development which demonstrates proof of concept.

“We continue to support the GTC’s ground-breaking work as well as research in other areas of transformational activity as part of our mission to fight for a life unlimited by cystic fibrosis.”

So how did animal research pave the way for this trial?

Following the identification of the CFTR gene in 1989 scientists sought to create animal models of cystic fibrosis with which to study the disease, and since the early 1990’s more than a dozen mouse models of cystic fibrosis have been created. In some of these the CFTR gene has been “knocked out”, in other words completely removed, but in others the mutations found in human cystic fibrosis that result in a defective channel have been introduced. These mouse models show many of the defects seen in human cystic fibrosis patients and over the past few years have yielded important new information about cystic fibrosis, and in 1993 Professor Alton and colleagues demonstrated that it is possible to deliver a working copy of the CFTR gene using liposomes to the lungs of CFTR knockout mice and correct some of the deficiencies observed.

To get a working copy of the CFTR gene to the lungs of cystic fibrosis patients Professor Alton and colleagues needed three things:

• A DNA vector containing the working CFTR gene that is safe and  can express sufficient amounts of the CFTR channel protein in the lungs to correct the disease

• A lipid-like carrier that can form a fatty sphere around the DNA vector to so that it can cross the lipid membrane of cells in the lung, as “naked” DNA will not do this efficiently.

• A nebuliser device that produces an aerosol of the gene transfer agent so that it can be inhaled into the lungs of the patient.

Several early attempts to use gene therapy using viral vectors to deliver the working copy of the CFTR gene to patients failed because the immune response rapidly neutralised the adenoviral vector (see this post for more information on challenges using adenoviral vectors), and while attempts to use non-viral vectors were more promising, it was found that they caused a mild inflammation in most patients, which would make then unsuitable for long term use. As reported in a paper published in 2008 the GTC members developed and assessed in mice a series of non-viral DNA vectors, repeatedly modifying them and testing their ability to both drive CFTR gene expression in the lungs and avoid inducing inflammation. They finally hit on a vector – named pGM169 – which fulfilled both key criteria.

Earlier the consortium had undertaken a study to determine which carrier molecule to use in their non-viral gene transfer agent (GTA). To do this they assessed 3 GTA’s, each consisting of a lipid like molecule that could form a sphere around the non-viral DNA vector; either the 25 kDa-branched polyethyleneimine (PEI), the cationic liposome GL67A, or as a compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. Because there are significant differences in airway physiology between mouse and human they carried out this study in sheep, whose lung physiology more closely matches that of humans. The study identified the cationic liposome GL67A as the most promising candidate, resulting in robust expression of the CFTR transgene in the sheep lungs.

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

Studies in sheep play a key role in the development of gene therapy for cystic fibrosis

It now remained to bring the DNA vector and carrier together. In a 2013 publication the consortium reported that repeated aerosol doses of pGM169/GL67A to sheep over a 32 week period were safe and induced expression of the CFTR transgene in the sheep lungs, although the level of expression varied between individuals (this variation was also observed in human CF patients in the clinical trial reported today). A final study, this time in mice, assessed the suitability of the Trudell AeroEclipse II nebuliser as a device to create stable pGM169/GL67A aerosols, finding that it did so in a reproducible fashion. When aerosolized to the mouse lung, the new pGM169/GL67A formulation was capable of directing persistent CFTR transgene expression for at least 2 months, with minimal inflammation. These studies provided the evidence to support the gene delivery system and dosage strategy used in the clinical trial reported today.

The trial results announced today are an important accomplishment, but they mark a beginning rather than the end for Cystic Fibrosis gene therapy. It will be necessary to improve the efficiency of the therapy before it can enter widespread clinical use. Animal research will certainly play an important part in this work, notably the observation that the efficiency of CFTR gene delivery using this strategy was varied between individuals in both sheep and humans indicates that sheep are a good model in which to assess changes to improve the consistency and effectiveness of the gene therapy.

If you would like to know more about this cystic fibrosis gene therapy clinical trial you can watch two videos recorded at a meeting for cystic fibrosis patients at ICL on the  Cystic Fibrosis Trust website.

Paul Browne

1) Alton E.W.F.W. et al. “Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial” Lancet Respiratory Medicine Published online July 3, 2015

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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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

Successful gene therapy for hemophilia A in dogs – humans next!

On Wednesday we were saddened learned that double Nobel laureate Fred Sanger had died,  so it was fitting that yesterday also saw the announcement of an important scientific advance that owes everything to the molecular biology revolution he helped to launch – one that may improve the lives of many thousands of people with Hemophilia A.

Hemophilia can affect dogs, and research on dogs with haemophilia has helped develop therapies for the disease. Image: Understanding Animal Research.

Hemophilia can affect dogs, and research on dogs with haemophilia has helped develop therapies for the disease. Image: Understanding Animal Research.

Hemophilia A is caused by a deficiency in the production of coagulation factor VIII, which leads to an increased risk of bleeding, and is due to defects in the gene located on the X-chromosome that lead to either insufficient production of factor VIII or production of defective factor VIII. Patients with severe Hemophilia A require frequent intravenous injections of recombinant factor VIII to prevent serious bleeding.  The BBC reported on Wednesday that a team of scientists based in the US and France have developed a gene therapy that successfully treated hemophilia in 2 dogs, and continued to prevent serious bleeds more than 2 years following treatment. Their therapeutic strategy involved isolating bone marrow hematopoietic stem cells (stem cells that give rise to all types of blood cell – including red blood cells, white blood cells and the platelets that are crucial to clotting) and transforming them with a lentiviral vector containing a gene encoding factor VIII under the control of a promoter that had previously been shown to drive expression of the drives the expression the target gene in the platelets of mice and dogs.  The transformed hematopoietic stem cells were then infused back into the same dog from which they had been isolated. Writing in the Nature Communications paper (1) reporting this study lead author Dr David Wilcox of the Medical College of Wisconsin and his colleagues discuss why dogs were the ideal subjects for preclinical evaluation of this therapy (for a great article on the crucial role of dogs in haemophilia research see this article from the magazine HEMAWARE).

 A canine model for haemophilia A exists, which results from a genetic mutation causing a large inversion of the FVIII gene (that resembles a molecular genetic defect found in about 40% of humans with the severe haemophilia A)4. Likewise, canine haemophilia A is essentially identical to the human disease in its clinical presentation characterized by severe-intermittent episodes of joint bleeding and haemorrhage. Protein replacement therapy is the most common treatment of severe bleeding episodes for haemophilia A but it has been confounded by the formation of inhibitory antibodies to transfused human FVIII in 30% of patients5,6. Similarly, 100% of dogs utilized from the Chapel Hill colony for this study develop inhibitory antibodies after being transfused with human FVIII (ref. 7), albeit severe bleeding is successfully treated with canine FVIII supplements. Thus, canine haemophilia A appears to be an ideal system to determine whether platelets can be used successfully to deliver human FVIII to the site of a vascular injury as a feasible approach to improve haemostasis within a ‘large-animal’ model of haemophilia A with the ability to form inhibitory antibodies to human FVIII.”

The above quotation also refers to an advantage of the technique they used over previous gene therapy methods developed to treat hemophilia A, one that the BBC article surprisingly didn’t pick up on. The BBC article mentions that an advantage that targeting expression of factor VIII to the platelets over previous studies where factor VIII was expressed in the liver of dogs with hemophilia A is that it would be suitable for patients who have damaged livers, but this is not the main advantage. The production of inhibitory antibodies against recombinant factor VIII by the patient is a problem that reduces the effectiveness of current therapies in about a third of people with hemophilia A, and this was also a problem in gene therapy techniques previously tested in clinical trials where factor VIII is secreted into the bloodstream from tissues such as the liver. Dr Wilcox and colleagues had the idea that by targeting expressing factor VIII specifically to platelets it would not be exposed to and blocked by inhibitory antibodies.

Laboratory Mice are the most common species used in research

GM mice are aiding the development of innovative therapies for many diseases, and haemophilia A is no exception.

The test this theory they turned to a genetically modified mouse model of hemophilia A, which had already proven very useful in earlier stages of the development of gene therapy to treating hemophilia A. In a study undertaken in GM mice (2) which had been immunized so that they produced inhibitory antibodies against human factor VIII, Dr Wilcox and colleagues at the Medical college of Wisconsin demonstrated that the their lentiviral vector that directed factor VIII expression specifically to the platelets resulted in the expression of therapeutic levels of factor VIII associated with platelets in the blood, even 6 months after treatment.

What’s more, they showed that this was possible using a nonmyeloablative conditioning regime before infusing the transformed hematopoietic stem cells. Conditioning regimes reduce the immune response to a transplant (and in cancers such as leukemia also eradicate the cancerous cells, using drugs such as the nitrogen mustards that we discussed earlier this week) but the myeloablative conditioning regimes that are very effective in treating leukemia carry significant risks, for example from infections following the procedure. Nonmyeloablative conditioning that does not completely destroy the patient’s reduces the risk of infection and transplant related death, and is thus more appropriate for conditions that are not immediately life threatening. This study paved the way for the evaluation of platelet specific factor VIII therapy in dogs that was reported on Wednesday. It is noteworthy that in the dogs, which were also treated using a nonmyeloablative pre-transplant conditioning regimen,  no inhibitory antibodies were detected against human factor VIII (unlike with previous gene therapy techniques), indicating that when associated with platelets it is sequestered from the immune system.

Almost 2 years ago we reported on the success of a small clinical trial of gene therapy in the treatment of hemophilia B following studies in mice and monkeys. We hope that with the development of a gene therapy technique that requires a milder conditioning regime and can avoid inhibitory antibodies this success will soon be repeated in hemophilia A.

Paul Browne

1)      Du LM, Nurden P, Nurden AT, Nichols TC, Bellinger DA, Jensen ES, Haberichter SL, Merricks E, Raymer RA, Fang J, Koukouritaki SB, Jacobi PM, Hawkins TB, Cornetta K, Shi Q, Wilcox DA. “Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A.” Nat Commun. 2013 Nov 19;4:2773. doi: 10.1038/ncomms3773. Pubmed 24253479

2)      Kuether EL, Schroeder JA, Fahs SA, Cooley BC, Chen Y, Montgomery RR, Wilcox DA, Shi Q. “Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity.” J Thromb Haemost. 2012 Aug;10(8):1570-80. doi: 10.1111/j.1538-7836.2012.04791.x.PubMed 22632092. PMC3419807

Trial of gene therapy in heart failure launches following success in rats and pigs.

Heart failure is a deadly condition that affects about two out of every hundred adults in the USA, and occurs when the heart is unable to provide sufficient pump action to maintain blood flow to meet the needs of the body. Among the more common causes are heart attacks and hypertension, but less frequently it can also be caused by viral infections or autoimmune diseases.

While the therapies available for heart failure have improved a lot in recent years thanks to the development of drugs such as Ivabradine, heart failure is still a major cause of death and disability, particularly among the over 65’s. As you might expect scientists around the world are developing several innovative approaches to treating heart failure – the British Heart Foundation’s “Mending Broken Hearts” appeal is an excellent example of the concerted effort now underway – and we have highlighted on this blog and our Facebook page  techniques ranging from electrical stimulation of the vagus nerve to collagen patches that stimulate tissue repair.

To those animal research has added another: Gene Therapy!

Image courtesy of Imperial College London

Image courtesy of Imperial College London

Yesterday the BBC reported the recent launch in the UK of a Clinical trial of gene therapy for heart failure, and Professor Peter Weissberg of the British Heart Foundation, who funded much of the basic and applied research leading up to this trial, noted the promise that this approach holds:

Whilst drugs can offer some relief, there is currently no way of restoring function to the heart for those suffering with heart failure. This early clinical study is the culmination of years of BHF funded laboratory research and offers real promise.

“Gene therapy is one of the new frontiers in heart science and is a great example of the cutting edge technologies that the BHF is using to fight heart failure. Gene therapy aims to improve the function of weak heart muscle cells, whereas our Mending Broken Hearts Appeal is aimed at finding ways to replace dead heart muscle cells after a heart attack. Both approaches are novel and both offer great potential for the future.””

This trial, which is being run by researchers at Imperial College London and the Royal Brompton Hospital, is part of a multinational multicentre trial of 200 patients – CUPID-2b – which seeks to assess whether injection into heart tissue of a adeno-associated virus 1-based gene therapy vector driving expression of the enzyme SERCA2a can repair damaged heart tissue and improve cardiac function. The reasoning behind this is that as a calcium transport protein SERCA2a plays a key role in maintaining the correct balance of calcium ions in heart muscle cells, and studies in both human heart failure patients and animal models of heart failure the amount of SERCA2a is lower than normal. A combination of studies in human heart muscle tissue and animal models of heart failure over several years demonstrated that this decrease is associated with calcium overload, an abnormal heart rhythm and tissue damage, suggesting that increasing the amount of SERCA2a in the injured heart tissue may reverse the damage.

In 2010 paper was published reporting on the first clinical trial of this therapy1 (available to read for free), whose primary goal was to assess the safety of the technique, and it noted that studies in animal models of heart failure provided vital evidence underpinning the decision to move it into clinical trials.

In preclinical HF models in rodents,(20) pigs,(18) and sheep,(21) increasing the level of SERCA2a using recombinant AAV vectors was well tolerated and restoration of SERCA2a levels resulted in significant improvement in cardiac function and energetics, even when the underlying pathophysiology or insult (eg, mitral valve rupture or pacing induced heart failure) was not corrected. Based on these findings, this first-in-human Phase 1/2 Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial(4) aims to restore levels of this key enzyme in HF patients via gene transfer of the SERCA2a cDNA by delivering a recombinant AAV (AAV1/SERCA2a) via percutaneous intra-coronary infusion.”

These studies, first in short- term studies in rats published in 2007 and subsequently longer duration studies in pigs and sheep published in 2008, indicated that this therapy was safe, could restore SERCA2a to normal levels, promoted heart muscle repair and improved heart function.

It’s just one more example of how animal research is contribution to the exciting field of gene therapy, and to advances in treating heart failure.

Paul Browne

1)      Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg B, Borow K, Dittrich H, Zsebo KM, Hajjar RJ; Calcium Up-Regulation by Percutaneous Administration of Gene Therapy In Cardiac Disease (CUPID) Trial Investigators. “Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID Trial), a first-in-human phase 1/2 clinical trial.” J Card Fail. 2009 Apr;15(3):171-81. doi: 10.1016/j.cardfail.2009.01.013. PMCID: PMC2752875

Italian Gene Therapy Success – Thanks to Animal Research

Italian scientists announced yesterday that they had successfully treated the deadly genetic disorders metachromatic leukodystrophy and Wiskott-Aldrich syndrome in a small clinical trial, marking and another major landmark in the developing field of gene therapy. As you might expect the breakthrough has received widespread media coverage, including CBS, the BBC and the Italian edition of the Huffington Post. While these reports do a reasonable job of explaining the significance of these two small trials, they do miss out one important fact, that this advance rests on many years of basic and applied scientific research, much of which required animal studies.

Many of the reports highlight the fact that this trial – led by  Dr Luigi Naldini, Dr Alessandra Biffi and Dr Alessandro Aiuti of the San Raffaele Telethon Institute for Gene Therapy – involved the use of parts of HIV virus to create a vector that can then insert function versions of the genes that are missing or defective into bone marrow stem cells of the patients, before transplanting the corrected cells back into the patients. This utilisation of a deadly virus to create a therapy that saves lives still takes people by surprise, but really it should not as this is not the first time. Last year we discussed on this blog how such a lentiviral vector had been used to successfully treat acute lymphoblastic leukemia while back in 2009 we saw how a similar gene therapy vector was used to treat the neurological disorder cerebral X-linked adrenoleukodystrophy (X-ALD). Animal research made crucial contributions to these two therapies, and it wasn’t surprising to find that it played an equally crucial role in developing gene therapy for metachromatic leukodystrophy and Wiskott-Aldrich syndrome.

In the paper published online in Science (1) reporting on the outcome of the trial of gene therapy in 3 boys with Wiskott-Aldrich Syndrome (WAS) , the Telethon team write that:

We developed a SIN lentiviral vector coding for human WASP* under the control of a 1.6 kb reconstituted WAS gene promoter (LV-w1.6W) (3). The use of this endogenous promoter ensures that the transgene is expressed in a physiological manner (4), restoring WASP expression and function in human and murine WAS cells (3, 30–34). Its moderate enhancer activity combined with the SIN LTR design reduces the risk of insertional mutagenesis (35), as shown by in vitro transformation assays (36) and preclinical in vivo studies in WASP-deficient mice (34, 37). These data provided the rationale for a phase I/II clinical trial in which LV-w1.6W was used as a gene therapy vector for treatment of patients with WAS.

*WASP, a protein that regulates the cytoskeleton and mutated in the syndrome

An open-access paper published earlier this year in the journal Molecular Therapy (2) describes the role of studies undertaken in immunodeficient mice to develop and evaluate the lentiviral vector to treat Wiskott-Aldrich Syndrome in more detail.

In the Science paper (3) reporting the results of the trial of gene therapy in 3 boys with metachromatic leukodystrophy, the Telethon team highlight the key contribution of studies in mouse models of metachromatic leukodystrophy (MLD) in evaluating the ability of hematopoietic stem cells (HSCs) transformd using the lentiviral vector containing the functional arylsulfatase A  gene – deficient in MLD – to treat the disease:

In a mouse model of MLD, we have demonstrated that disease manifestations can be prevented and corrected by lentiviral vector (LV)–based HSC-GT but not by HSCT (8, 9, 16). This is consistent with the observation that HSCT fails to provide consistent benefits in MLD patients (3, 5–7). LV–based HSC-GT induced extensive and supra-physiological expression of the functional ARSA gene throughout the HSC progeny, which in turn mediated widespread cross-correction of CNS and PNS resident cells (8, 9).

This was only the last in a long series of animal studies that led to the clinical trial, an open-access review of progress being made in the development of gene therapy for a range of leukodystrophies published by Dr Biffi in 2011 highlights not only this important preclinical work, but also the basic and translational studies undertaken using a variety of different vector types in mice and monkeys that provided the data that allowed scientists to develop an effective therapy.

At a time when science and medicine in Italy is under attack from charlatans who are promoting dubious stem cell therapies, and scientific activists are campaigning against laws that threaten the very future of medical research in Italy, this weeks good news from the San Raffaele Telethon Institute for Gene Therapy is a reminder that there are many excellent scientists in Italy who are conducting medical research at the highest level, and that their work depends on animal research. We hope that this breakthrough – and there can be no doubt that this is a breakthrough – heralds a better future for science in Italy.

Speaking of Research

1) Aiuti A. et al. “Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome” Science. Published online 11 July 2013, DOI: 10.1126/science.1233151

2) Scaramuzza S. et al. “Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott-Aldrich syndrome.” Mol Ther. 2013 Jan;21(1):175-84. doi: 10.1038/mt.2012.23.

3) Biffi A. et al. “Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy” Science. Published online 11 July 2013, DOI: 10.1126/science.1233158

4) Biffi A, Aubourg P, Cartier N. “Gene therapy for leukodystrophies.” Hum Mol Genet. 2011 Apr 15;20(R1):R42-53. doi: 10.1093/hmg/ddr142.