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

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences


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.

Technical Summary

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".

Planned Impact

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.
Title La trama della vita (The frame of life). Marsilio editor. 
Description Living forever is a long-lasting dream, present in many novels, paintings and musical pieces. Who knows whether this dream may one day come true? Regenerative medicine, the topic of the book, promises not only to cure terrible and so far incurable diseases, but also to repair or replace our cells and tissues so that, in theory, we may live "forever young and healthy". Do we really want this? Moreover, for few spectacular successes, there have been many more failures. Nevertheless, the expectations raised in patients have been exploited by commercially driven "stem cell clinics" that offer to cure any disease with stem cells in exchange for much money. These and other topics such as the impact on society and health economics are discussed in this book. 
Type Of Art Creative Writing 
Year Produced 2018 
Impact The book, currently in Italian, explains to a lay audience promises and problems of regenerative medicine and its impact on society. The book was sold out and is currently a finalist for the Galieo prize for scientific outreach. 
Description Regenerative Medicine: Scientific Advances and Regulatory Framework The Federation of European Academies of Medicine
Geographic Reach Europe 
Policy Influence Type Contribution to a national consultation/review
URL https://www.feam.eu/?post_type=events&p=1764
Description A universal donor mesoangioblast for the therapy of muscular dystrophy and other recessive genetic diseases of the mesoderm.
Amount £178,000 (GBP)
Funding ID 20150256 
Organisation University of Manchester 
Sector Academic/University
Country United Kingdom
Start 04/2019 
End 03/2020
Description Immortal, universal donor cells for the ex vivo gene therapy of muscular dystrophy: development of a pre-clinical mouse model.
Amount £61,937 (GBP)
Funding ID MR/S015116/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2019 
End 05/2020
Description Recreating the ideal niche: environmental control of cell identity in regenerating and diseased muscles
Amount € 3,482,746 (EUR)
Funding ID EC H2020-MSCA-ITN-2019-860034 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 04/2020 
End 03/2024
Title A clinical trial on chip: a simple, cheap and reliable method to predict efficacy of a new therapy. 
Description We observed that co-cultures of myotubes and motoneurones have been used since decades and promote mytoube maturation in vitro via both electrical and chemical signals. We recently used E11 mouse embryonic motonerones to innervate cultures of adult human myotubes in a ECM gel. To our surprise we observed that within 24 hours from the establishment of synaptic connections, cultures were rhythmically and vigorously twitching, something that should not occur with a fully mature synapsis, where contraction only occurs upon motoneuron stimulation. This creates a stressful situation that leads to death and detachment of all DMD myotubes within the next 24h while healthy myotubes continue to twitch for another 3-4 days. Remarkably, DMD myotubes, genetically corrected with a small nuclear RNA (U7) engineered to skip exon 51 of the dystrophin gene, behaved as healthy myotubes, thus showing that this method reveals phenotypic conversion that revert the dystrophic phenotype. We want to develop and standardise this method, test a number of pilot therapies (e.g. steroids) to offer this tool to the scientific community. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact In these years many clinical trials have been carried out for Duchenne Muscular Dystrophy. Unfortunately they are very expensive, time consuming and so far have not provided a clear indication of efficacy, though many showed promise that they may lead to efficacy but, again after much time and money. This tool aims at developing a clinical trial on chip to offer a simple, reliable and cheap tool to predict the effect of a given therapeutic strategy. 
Description Functional tests on motor activity of transplanted DMD rats 
Organisation Manchester Metropolitan University
Country United Kingdom 
Sector Academic/University 
PI Contribution We will transplant DMD rats with wt or genetically corrected DMD cells and will subject animals to physiological analysis perofrmed by our collaborator.
Collaborator Contribution Prof. Hans Daegan, and expert muscle physiologist will collaborate on the project by testing motility on transplanted DMD rats on treadmills and rotarod, and also measuiring force of contraction and restistance to fatigue in isolated transplanted muscles.
Impact We expect to accurately measure the physiological response of DMD rats to cell transplantation.
Start Year 2019
Description In vivo trasplantation of mesoangioblasts in steroid treated dystrophic mice 
Organisation University of Bari
Department Department of Biosciences, Biotechnology and Pharmacological Sciences
Country Italy 
Sector Academic/University 
PI Contribution We have completed experiments on the binding of mesoangioblasts on endothelium in the presence or absence of inflammation. We now need to complete experiments in vivo on dystrophic mice subjected to different anti-inflammatory regimes. To this end we will send cells to our collaborators and they will transplant and study the mice.
Collaborator Contribution Prof. Anna Maria De Luca, University of Bari, is an internationally recognised molecular pharmacologist with a long expertise in muscular dystrophy. Ornella Cappellari, senior post-doc has a part time appointemt in Bari and in Manchester. She has been trained i microsurgery technicques in the laboratory of Prof. Dominic Wells at the Royal Veterinary College.
Impact The collaboration has just started. It will result in ajoint publication.
Start Year 2019
Title Mesoangioblast-based gene therapy for Duchenne Muscular Dystrophy: a phase I/IIa study/DMD04 
Description Mesoangioblast-mediated exon 51 skipping for genetic correction of dystrophin, based upon a single injection in individual skeletal muscles of five non-ambulant patients affected by Duchenne Muscular Dystrophy: a non randomized, open-label, phase I/IIa study. We are testing safety of genetically corrected autologous mesoangioblasts by intramuscular injection in a foot muscle (Extensor digitorum brevis) of 5 non-ambulant DMD patients. Mesoangioblats were corrected with a lentivral vector expressing the U7 small nuclear RNA (snRNA) engineered to skip exon 51 of the dystrophin gene. The snRNA diffuses to neighbouring resident nuclei of the dystrophin muscle fibre thus amplifying the therapeutic effect. In case of dystrophin production = 10% of a healthy muscle, cells will also be injected in the thumb muscle, whose increased force of contraction would ameliorate the quality of patients' life. The Wellcome Trust HICF funded this first in man clinical trial that is expected to start in Manchester in 2020. 
Type Therapeutic Intervention - Cellular and gene therapies
Current Stage Of Development Early clinical assessment
Year Development Stage Completed 2020
Development Status Under active development/distribution
Impact If successful, this trial will pave the way to systemic distribution of autologous genetically corrected mesoangioblasts in young patients. This would be a subsequent trial with intent to cure. 
Description Annual Meeting of the Manchester Network of Regenerative Medicine. Manchester 26 Febriary 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact This is an annual meeting of the Manchester Network of Regenerative Medicine. Manchester. In this meeting there are long and short talks (I delivered a long talk while my post-doc Francesco Galli a short one) on regenerative medicine. The meeting is attended by researchers, clinicians and students.
Year(s) Of Engagement Activity 2019
Description Conference on Congenital Muscular Dystrophy type 1A 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact This day was a gathering for all experts in the field willing to share their knowledge and gain new insights.
Year(s) Of Engagement Activity 2019
URL https://mdc1a.com/conference/
Description ERC-EIC workshop on Cell and Gene Therapy 29.06.2021 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact The workshop took place online on 29 June 2021 14h00-18h00 CET, with around 150 participants, including ERC and EIC grant holders, representatives from industry, venture capitalists, and innovation leaders. This workshop was intended to bring science and innovation together, with the aim of better assessing areas of innovation potential in specific scientific domains, as well as the bottlenecks in moving from knowledge to innovation.
Year(s) Of Engagement Activity 2021
Description Genoa Festival of Science 24.10-04.11.2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact In Genoa, the 17th edition of the Science Festival from October 24 until November 4th, 2019. As in the previous years, the festival combines hundreds of different initiatives and events, designed to fulfill and stimulate the interest of visitors of all ages and all levels of knowledge. A special attention is paid to the latest developments in science and technologies and to researchers coming from emerging countries.

Exhibitions, workshops, shows and conferences have as a goal the promotion and popularization of science among people of all ages.
Researchers, schools and families gather at Genoa Science Festival to experience and understand science through a series of interactive activities. The goal of all these interdisciplinary proposals is to overcome the traditional opposition between scientific and humanistic culture.
Year(s) Of Engagement Activity 2019
URL https://www.italybyevents.com/en/events/liguria/science-festival-genoa/
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact This is an annual international meeting of Duchenne Parent Project (Italy) dedicated to children with DMD and their parents. In these meetings leading scientists and clinicians present the results of ongoing research work and clinical trials.
Year(s) Of Engagement Activity 2019