PROBLEM

Muscular dystrophies are a heterogeneous group of inherited disorders presenting a large clinical variability regarding age of onset, patterns of skeletal muscle involvement, heart damage, rate of progression and mode of inheritance. Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD), the most severe case, are facing several problems and no therapy is available for this disease as for all muscular dystrophies.

BACKGROUND

Cell therapy is one promising approach to correct genetic diseases by contributing to tissue regeneration; stem cells can be isolated from a healthy donor or, when possible from the same patient. In the first case cells will be transplanted under a regime of immune suppression while in the second case, cells will have to be genetically corrected before transplantation in the same patient from which they were derived. The recent identification of different types of multi-potent stem cells, some of which are suitable for protocols of cell therapy, has disclosed new perspectives in the treatment of genetic diseases.

HYPOTHESIS

Our previous work indicated that CD133+ stem cells, a recently identified population of progenitor cells, produce functional improvement upon intra-arterial injection in a mouse model of muscular dystrophy. Thus it could be possible to focus upon this type of stem cell for autologous transplantation in DMD animal models.

RESEARCH

Recently, transplantation of engineered dystrophic canine muscle-derived CD133+ cells has given promising results in Golden Retriever Muscular Dystrophy (GRMD) dogs, the most reliable animal model that shows a form of dystrophy very similar to DMD (and even more severe in most cases).

We isolated CD133+ stem cells from muscle biopsies of GRMD dogs, we then expanded them characterized the CD133+ cells by FACS analysis.

The issue was then to use a specific-designed lentiviral vector capable of eliminating the mRNA segment from exon 6 to 8 in canine dystrophin gene. We tested different concentration of lentiviral vectors and  verified the restoration of dystrophin transcript in culture by RT-PCR.

Two dystrophic dogs were treated with serial intrarterial injections of autologous transduced cells.

The transplanted animals were analysed at different times; most of the biopsies in all muscles were morphologically less affected than those of untreated dogs.

Functional improvement of treated dogs was also assessed by a 15 min timed running test and by a combined clinical grading score. The untreated littermates became slower over the treatment time, whereas two treated dogs ran faster after treatment.

OBSERVATION

Because of these results, we plan a pilot clinical trial, based on intra-muscular and intra-arterial transplantation of autologous engineered muscle derived CD133+ cells. Efficacy and possible adverse effects will be evaluated to test whether this approach may represent a first step towards an efficacious therapy for muscular dystrophy.