A team of researchers in France, led by Dr. Ana Buj-Bello (Genethon/Inserm) and teams at the University of Washington and Harvard Medical School in the United States, achieved a new step towards the treatment of myotubular myopathy by gene therapy. The researchers demonstrated the efficacy of administration of a therapeutic vector by a single intravenous injection and identified the dose that restores long-term muscular strength in a large animal model of the disease.

This work, published today in Molecular Therapy, has been achieved thanks to donations from the French Telethon and the support of the Myotubular Trust.
Myotubular myopathy is an X-linked genetic disease affecting 1 in 50,000 newborn boys. It is caused by mutations in the MTM1 gene encoding myotubularin, a protein involved in the functioning of muscle cells. The severe form of the disease leads to hypotonia, generalized muscle weakness and death in early infancy due to breathing difficulties. Dogs naturally affected by this myopathy also have a reduced life expectancy. To date, there is no effective treatment for this rare disease.

In the present study, Genethon’s team developed and manufactured an adeno-associated virus (AAV) vector able to deliver a normal copy of the MTM1 gene in the entire musculature. The AAV product was administrated by a simple intravenous injection in ten week-old dogs manifesting the first symptoms of the disease – instead of the locoregional route of administration used in previous studies (Science Translational Medicine, January 2014).

The treatment restored whole-body muscle strength and function, and prolonged the life of affected dogs. Treated dogs were indistinguishable from normal animals 9 months after product injection.

Dr. Ana Buj-Bello, Genethon/Inserm said: « We report a dose-finding study and show the therapeutic benefit of the vector by a single intravenous injection. It is a clinically-relevant route of administration and represents a step towards a clinical trial in patients”.

© Fabrice Hyber, pour Organoïde/Institut Pasteur

French researchers have identified a marker that makes it possible to differentiate “dormant” HIV-infected cells from healthy cells. This discovery will make it possible to isolate and analyze reservoir cells which, by silently hosting the virus, are responsible for its persistence even among patients receiving antiviral treatment, whose viral load is undetectable. It offers new therapeutic strategies for targeting infected cells. This research is part of the ANRS strategic program “Réservoirs du VIH”. It is the result of a collaboration between the CNRS, Montpellier University, Inserm, the Institut Pasteur, the Henri-Mondor AP-HP hospital in Créteil, the Gui de Chauliac hospital (CHU de Montpellier) and the VRI (Vaccine Research Institute), and is published in the journal Nature on March 15, 2017. A patent owned by the CNRS has been filed for the diagnostic and therapeutic use of the identified marker.

Since 1996, there has been consensus among the scientific community that a cure for HIV will involve targeting “reservoir cells” that host the virus in the organisms of patients undergoing triple therapy. HIV can remain hidden in these reservoirs, in latent form, for several decades, eluding the immune system’s response and antiviral treatments, without any viral protein being expressed. But if treatment ceases, the virus massively proliferates and the disease progresses again. Patients must therefore receive treatment for life. To envisage eliminating this dormant virus, a first stage consists in distinguishing the HIV-infected reservoir cells from their healthy counterpart cells, which resemble them to a very large degree. This is what has been achieved by a team of researchers, who have identified a marker of reservoir cells: a protein present only on the surface of infected cells.

Hypothesizing that HIV might leave a mark on the surface of its host cell, researchers from the Institut de génétique humaine (CNRS/Montpellier University) first worked in vitro on an infection model developed in their laboratory. After comparing infected cells and healthy cells,[1] they noticed one particular protein, coded by a gene among the hundred of those expressed in a specific way by infected cells. Present only on the surface of the infected cells, the CD32a protein thus met, in vitro, the criteria of a reservoir cell marker. This was then confirmed by experiments on clinical samples. By studying blood samples from 12 patients living with HIV and receiving treatment,[2] the researchers isolated the cells expressing the marker and observed that almost all were HIV carriers. In vitro, the activation of these cells induced a production of viruses capable of reinfecting healthy cells whereas their elimination entailed a significant delay in viral production.

In the fight against HIV, this discovery paves the way to a better fundamental understanding of viral reservoirs, which it will now be possible to isolate more easily and analyze directly. In the longer term, it should lead to therapeutic strategies aiming to eliminate the latent virus from the organism and make remission—at least temporary—possible in the absence of antiviral treatments.

This research received support from the ANRS, MSD Avenir, the European Commission, the Fondation Bettencourt Schuelle, the Fondation pour la recherche médicale and the Vaccine Research Institute (VRI).

[1] The cells studied are T CD4 lymphocytes, whose numbers are progressively reduced upon infection by HIV. The number of these cells is thus used by doctors to monitor the progression of the disease and the efficacy of treatments.

[2] Patients monitored by the Clinical Immunology and Infectious Disease Department at the Henri-Mondor AP-HP hospital in Créteil and by the Infectious and Tropical Disease Department at Gui de Chauliac hospital (CHU de Montpellier).

On 13 June last, the Inserm Ethics Committee assembled over a hundred individuals at its annual seminar. All those present had the benefit of an ethical perspective on many problems posed by biomedical research. One of the questions addressed was that of CRISPR-Cas9 technology. The Ethics Committee has devoted a specific opinion to it, while the NIH has just obtained a first green light for a human cancer immunotherapy trial.

The Inserm Ethics Committee (CEI) may receive referrals or carry out investigations on its own initiative to reflect on ethical questions raised by medical science- and health-related research carried out within the Institute. At the end of its reflection, it issues an opinion in the form of notes that may evolve as new contributions are added. In 2015, the CEO of Inserm requested the Ethics Committee to specifically examine questions related to the development of CRISPR technology, and particularly:

1- What are the questions raised by the technology as such?

2- Does the rapidity of its development raise particular problems?

3- Does the simplicity of its use call for regulation of its implementation in the laboratory?

Given the technical advantages of the method, and its very rapid dissemination, the question now is to evaluate where, when and how its use might pose an ethical problem. It seemed immediately important to distinguish three areas associated with different issues:

1/ application of the technology to humans, which essentially raises the question of germ line modifications;

2/ application to animals, particularly “pest” species, which raises the question of potential horizontal gene transfer and the emergence of irreversible damage to biodiversity;

3/ risks of damage to the environment.

Recommendations of the Inserm Ethics Committee

The Committee immediately proposes that Inserm adopt the following principles:

1- To encourage research aimed at evaluating the efficacy and safety of CRISPR technology and other recently published genome editing technologies, in experimental models that can allow case-by-case determination of the benefit/risk balance of a therapeutic application, including any applications that involve germ cells and the embryo. This information is essential to the future determination of what might be authorised for human use in terms of therapeutic approaches.

2- The potentially adverse effects of gene drive systems must be evaluated before any use outside of a laboratory, observing the containment rules already in force for other genetic modifications. Evaluations must be made over long periods, given the transmissible nature of the driver gene. Reversibility measurements should be provided for in the event of escape or adverse effects. Such analyses and the design of multiple scenarios require the constitution of pluridisciplinary teams.

3- To comply with the prohibition of any modification of the germ line nuclear genome for reproductive purposes in the human species, and not support any application to modify the legal conditions until uncertainties about the risks have been clearly evaluated, and until a broad consultation involving multiple partners from civil society has ruled on this scenario.

4- To participate in any national or international initiative dealing with questions of freedom of research and medical ethics, including initiatives with emerging countries that will also be affected by the development of genome editing technologies.

5- Finally, to draw attention to the more philosophical question which contrasts the plasticity of life with the idea of a human nature founded on the only biological constant. Awareness needs to be increased regarding the utopia and dystopias that can be generated by some therapeutic promises.

Research efforts associating scientists from the CNRS, UVSQ and INSERM within the Laboratoire END-ICAP[1], working in collaboration with a team from the University of Bern, has demonstrated the therapeutic potential of a new class of synthetic oligonucleotides[2] in the treatment of Duchenne muscular dystrophy (DMD) using RNA “surgery”. Tested in the mouse, this new generation of molecules proved to be clinically superior to those currently under evaluation in DMD patients, notably for restoring cardio-respiratory and central nervous system function. These findings were published on 2 February 2015 in Nature Medicine.

Study of Duchenne Muscular Dystrophy-  Necrotic lesions (cellular death) of muscular fibers made up of myofribils. Gomori trichrome stain. ©Inserm/Fardeau, Michel

Neuromuscular diseases include several hundred conditions, mainly of genetic origin, which are defined by a defect in muscle control or the destruction of muscle tissue. Taken together, they affect tens of thousands of people in France, and represent a major public health challenge. The most emblematic of these conditions, Duchenne Muscular Dystrophy (DMD) is caused by mutations that affect the gene coding for dystrophin, a protein essential for the correct functioning of muscle cells. This particularly severe and disabling disease does not yet benefit from any satisfactory treatment.

RNA “surgery” is an approach that has been developed in order to correct certain genetic abnormalities. This therapy is based on using small sequences of antisense oligonucleotides (AON)[3] that can bind to — and specifically act on — messenger RNA, and allow the synthesis of a missing protein. Several studies are underway to synthesize different types of AONs designed to act on dystrophin production. Despite the encouraging results of some clinical trials, existing AONs have limitations: their level of toxicity is sometimes high and they cannot act at the cardiac level or cross the blood-brain barrier. Designing a therapy that would be effective simultaneously for all skeletal muscles, the heart and the central nervous system, remains a challenge.

The authors of this work have developed new nucleotides for AON synthesis: tricyclo-DNA (tcDNA). These AON-tcDNA, synthetic analogs of DNA, hybridize with target RNA and cause the excision of a fragment of RNA[4]. By acting on that part of the gene that carries an error, they allow the synthesis of a truncated — but stable and functional — dystrophin. The monitoring of DMD mice treated with these AON-tcDNA has shown that these analogs perform better than their previous equivalents. Administered intravenously, the AON-tcDNA are efficiently distributed throughout the skeletal muscle, and also reach the cardiac tissue and central nervous system, which was not the case for their predecessors. In addition, the restoration of dystrophin production is more effective than with previous AONs. After a twelve-week treatment, the mice displayed highly significant improvements, not only in muscle function but also in cardio-respiratory function, which are the principal targets in patients suffering from this neuromuscular disease.

The scientists also demonstrated a correction of emotional responses, which are naturally exacerbated in DMD subjects and may cause learning disabilities and cognitive defects. This part of the study, performed in collaboration with a team from the Institut des Neurosciences Paris Saclay (CNRS/Université Paris-Sud), showed that dystrophin is crucial to the proper functioning of certain neurons and that the behavioral disorders observed in the context of a dystrophin deficit were at least partially reversible in adult mice with DMD.

As well as these promising results, AON-tcDNA are characterized by a lengthy residence time within tissues, which in the future will allow treatments to be administered at longer intervals. Another advantage is that they are not degraded but gradually excreted by the body, thus making the treatment reversible and limiting its toxicity. The toxicological analyses required are still ongoing but their initial results suggest that these new AONs are well tolerated at high doses in mice.

The mechanisms responsible for the efficacy of these third-generation AONs are still poorly understood but several of their properties may be involved, including notably their strong affinity for RNA and their ability to form “nanoparticle” aggregates in a spontaneous manner.

The chemistry of tcDNA thus opens numerous perspectives for their application in various genetic diseases. Above all, this is a new step towards a systemic drug therapy[5] for Duchenne muscular dystrophy.