Engineering human artificial chromosomes containing the dystrophin locus for autologous cell therapy of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy (1/3500 males). It affects skeletal and cardiac muscles, leading to progressive loss of movements, confinement to a wheelchair and finally to total paralysis and assisted ventilation. Death occurs usually in the third decade of life because of cardiac and/or respiratory failure. Improved medical assistance has increased the lifespan of patients but still there is no efficacious therapy and steroids are the only palliative treatment available. Among several novel strategies that are entering clinical experimentation, our group focused on cell therapy, based upon intra-arterial infusion of mesoangioblasts (MABs), stem/progenitor cells associated to blood vessels that showed efficacy in dystrophic mice and dogs. Although a phase I/II clinical trial based upon allogeneic transplantation of mesoangioblasts is currently running, gene therapy of autologous cells would be preferable (no immune suppression and no need of HLA-matched donor) but the large size of the dystrophin gene (2.4Mb) hampers the use of viral vectors. Our group pioneered the use of human artificial chromosomes (HACs) containing the whole dystrophin locus for muscular dystrophy (DYS-HAC), showing efficacy of this strategy in dystrophic mouse cells. Based on the above, we now plan to develop a definitive strategy that would allow translation to human cells and boosting the therapeutic effect.
First, we will insert into the DYS-HAC an "immortalizing cassette" containing genes that allow bypassing senescence (telomerase and Bm1) and a suicide gene as a safety device. Preliminary results confirm that this cassette works in human cells. The cassette is floxed and thus can be excised from the HAC before cells are infused into patients.The DYS-HAC will also contain an inducible MyoD (i.e linked to the Estrogen Receptor: MyoD-ER), a myogenic master gene that will allow induction of muscle differentiation (which is variable among different patients).
The optimal number of copies of dystrophin to include will be determined to achieve the optimal level of expression for a single genetically corrected nucleus to compensate for the muscle fibre resident nuclei that cannot synthesize it. All the subsequent generations of HACs will be transferred in human dystrophic mesoangioblasts that will be then challenged for their ability to repair dystrophic muscle and ameliorate the disease.
The PI is a leader in the field of cell therapy; his expertise, and that of his colleagues, will guarantee a high probability of success for this ambitious but realistic project, whose results may lead to rapid clinical translation and, at the same time, pave the way for other monogenic diseases, characterized by mutations of a very large gene.

The aim of this project is to develop a novel cell therapy for Duchenne Muscular Dystrophy (DMD) based upon transplantation of autologous mesoangioblasts (MABs; vessel associated progenitors) engineered with a human artificial chromosome (HAC) vector containing the entire 2.4 megabases of the human dystrophin locus (DYS-HAC). Specifically we will insert into the DYS-HAC:
a) Floxed immortalizing sequences (human telomerase reverse transcriptase (hTERT), Bmi-1 and the suicide gene Herpex Simplex Virus Thymidine Kinase (HSV-TK). These will allow us to extend, reversibly, the lifespan of primary human MABs, excision of the floxed cassette by a non-integrating lentiviral vector expressing the Cre recombinase and negative selection of cells where the cassette has not been excised;
b) An inducible MyoD (MyoD-ER) where the myogenic factor is linked to the Estrogen Receptor, to induce myogenesis at will, since mesoangioblasts vary in their myogenic potency among different patients.
c) Two or more full-length human dystrophin cDNAs to compensate for gene dosage insufficiency deriving from fusion of relatively few genetically corrected cells in a fiber containing a large majority of genetically defective nuclei. Transfer of different HACs, each with a different selection marker, will allow us to determine the number of copies required for optimal protein production.
Subsequent generations of HACs, progressively incorporating the above mentioned functions, will be built and transferred into MABs from DMD patients. Proliferation, differentiation, transformation/tumorigenicity of cells will be tested in vitro and in vivo (scid/mdx dystrophic and immune-deficient mice) before and after excision of floxed immortalizing sequences (by a non-integrating lentiviral vector). Engraftment, dystrophin production, amelioration of the pathology and functional recovery will be tested in transplanted mice. This project will set the stage for a definitive cell therapy of DMD.

Although cardiovascular and neurological diseases remain the most widespread and disabling in the Western world, musculoskeletal impairments are among the most frequent causes of disability, encompassing more than 150 diseases and affecting every age and socioeconomic group. The classic example is muscular dystrophy, which can appear from infancy to adulthood and affects over 300,000 European citizens. This group of diseases alone poses serious social and economic problems for each national health system, considering the cost for repeated hospitalizations, home assistance, physiotherapy, surgery, medical devices for a devastating disease that takes now decades from diagnosis to death with a progressively deterioration of life quality.
Our project aims to demonstrate safety and efficacy for rapid clinical translation of this cell therapy strategy for Duchenne muscular dystrophy, though restricted to patients in a paediatric age and whose mutation cannot be treated by the simpler exon-skipping strategy. We aim to achieve a significant stabilization/amelioration of clinical symptoms. Importantly, even a partial effect would produce an enormous benefit for patients.
Together with patients, their families would benefit of the success of this project, not only in terms of emotional impact but also in practical terms, due to a reduction of the daily heavy burden related to continuous assistance to patients who are non ambulant and, later, totally unable to move.
The National Health Service would also indirectly benefit, not only for the implementation of available therapies, but also in economic terms. Although cell therapies are expensive and patient-tailored, they would still be cheaper that the total costs for the NHS, related to diagnosis, medical care, surgery, physiotherapy, drugs, medical devices that are required for decades.