Cell mediated gene therapy for Duchenne muscular dystrophy: trans-correction of resident nuclei to amplify dystrophin expression

This research projects aims at optimising a treatment that was recently tested in patients affected by Duchenne muscular dystrophy, with the idea to extend it to other rare genetic diseases of the muscle, so that one strategy may in the future be adapted for more diseases.
Muscular dystrophies are caused by mutations in a single gene that leads to the impaired function and death of muscles in the human body. This in turn causes disability of variable severity that, in the worse cases, are devastating; they compromise quality of life and lead to a premature death. Although individual muscular dystrophies are rare or extremely rare, together they affect many thousands of people and represent a major challenge for National Health Services, in charge of providing palliative therapies and medical assistance, often over decades.
In order to "cure" genetic diseases it is necessary to replace or repair the defective gene and this can be achieved by using viral vectors (viruses that have been modified in the laboratory to deliver a correct version of the gene into cells) or stem cells (cells which can be used to generate any other type of cell in the body). To make sure they're accepted by the body, stem cells can be derived from a related donor (e.g. a sibling) or from the patient themselves, after having been "repaired" in the lab. These therapies have been successful so far for several genetic diseases affecting the blood, the skin, and the cornea. This has been possible because it is relatively easy to remove the diseased tissue and replace it. This approach is not possible for diseases affecting the heart, the liver or the brain. In these cases, healthy cells will have to correct the genetic defect and somehow help the function of the resident diseased cells.
The innovative aspect of our treatment is based on the fact that stem cells which are injected into the arteries of patients end up distributed uniformly throughout the body. Some of these stem cells can then cross out of the blood vessel, move into the surrounding tissue of the body and eventually repair it. However, the process is not yet efficient enough to result in a noticeable improvement in patients. In order to achieve this, we are studying in detail each step of the transplantation procedure. In this specific project, we are developing a strategy by which, a single, genetically corrected cell will also correct neighbouring nuclei inside a newly regenerated muscle fibre that is composed of very many nuclei. This will amplify the therapeutic effect. The successful completion of the research programme will rapidly lead to a new clinical trial, that may be then extended to more rare genetic diseases of muscle, increasing the benefit for patients.

In order to "cure" genetic diseases it is necessary to replace or repair the defective gene: this can be achieved with viral vectors, small molecules that correct the genetic defect, or stem/progenitor cells that are incorporated into the tissue. All these strategies are in pre-clinical or clinical experimentation for Duchenne Muscular Dystrophy, due to mutations in dystrophin gene. The absence of the protein leads to muscle damage during contraction and in time to wheelchair dependence, cardiac or respiratory failure and premature death. The innovative aspect of our experimental strategy is based upon intra-arterial delivery of mesoangioblasts (vessel-associated myogenic progenitors) that distribute uniformly throughout the body districts downstream of the injected artery. Some of these stem cells cross out of the blood vessel, move into the surrounding tissue of the body and eventually repair it. However, the process is not yet efficient enough and work is ongoing to implement these steps. Here we focus on enhancing genetic correction by using a lentivector that encodes a small nuclear RNA (U7) engineered to skip exon 51 of the dystrophin gene. As the snRNA assembles with proteins in the cytoplasm, it then enters all neighbouring nuclei thus, amplifying the therapeutic effect aim. This is tested by co-culturing in vitro one DMD genetically corrected cell with an excess (10, 30) of DMD cells and measuring the amount of dystrophin produced in these hybrid myotubes. After exploring the underlying molecular mechanisms in vivo experiments are conducted with human cells in immune deficient mice and with dystrophic, genetically corrected rat mesoangioblasts in newly developed DMD rat carrying a mutation in exon 52 of the dystrophin gene. Functional test on corrected rats will test the effect on motility. The successful completion of the research programme will rapidly lead to a new clinical trial, on young DMD patients with an implement protocol and "intent to cure".

The first beneficiaries of this programme will be patients affected by genetic, recessive, muscular dystrophies. In the future also patients affected by other rare genetic diseases of the mesoderm, may benefit from this therapeutic approach, if successful. It is important to stress that many recessive genetic diseases of the mesoderm are so rare that they will unlikely become the target of a specific research programme aimed at finding a possible therapy. The important added value of this project is a "one serves all" strategy, though of course adjustments will have to be made for each specific disease. However having available a common platform will dramatically cut time and costs for these patients that currently receive only palliative therapies and together present a major burden for the National Health System.
Patient associations may also benefit from this work and eventually decide to be involved in co-sponsoring further programmes and developments, as is already happening in the case of muscular dystrophy.
Beside Academic beneficiaries, students, training medical doctors, nurses and medical personnel in general may be involved and benefit for the future clinical translation of this therapeutic strategy. Moreover, although the programme is developed for rare disease that usually elicit a modest interest in companies, the possibility of applying intra-arterial delivery to MSC therapies that currently employ intra-venous injection, may significantly improve their efficacy and attract their interest, as most of these trials are sponsored by SME.
If successful this project should elicit the interest of policy-makers in the National Health Systems, both nationally and internationally for the possible adoption of new medical therapies, that, although associated with a very high cost, would lead, if successful to a dramatic reduction of the current costs related to long-term palliative treatments, medical, surgical and rehabilitation assistance. Last and most important, recovery of patient to at least partially normal and productive life will bear an additional advantage both in ethical and economic terms.
Finally, though its communication plans, this project may also impact on the general public and young students by popularising prespectives and problems of regenerative medicine to a wide audience.