Final Report Summary - MYOAMP (Amplification of human myogenic stem cells in clinical conditions)
In Europe over 30 000 young people, a number equivalent to the population of Monaco, are affected by Duchenne muscular dystrophy (DMD), either from genetic transmission of through new mutations (1/3 of the cases). Among the muscular dystrophies, the DMD has one of the most severe prognosis and finding a treatment to at least improve the quality of life of the affected children is a dramatic issue for the families.
It is a fact that the families are often desperate to see the last results obtained by researchers turned into more clinical trials to at least bring some improvement to their children's health. On the other hand, it is also true that despite this urgent need, families are also aware that designing a therapy is a difficult exercise which involves a lot of assessment and agreements from the regulatory authorities in which safety is the major keyword. Many clinical trials using muscle progenitor cells have been developed in the past for DMD with very limited success.
A solution may be found in combinations of new therapeutic approaches. From this perspective, MYOAMP intended to speed up the assessment of the potential of a new cell therapy approach and to set up the conditions for future clinical trials which could not be set-up within its budget and time limits.
By gathering the expertise of the partners, MYOAMP generated guidelines originating from concerted efforts and validated by both clinicians and European Small and medium-sized enterprises (SMEs) to facilitate the initiation of clinical trials involving stem cell therapy.
Furthermore, the participation of clinicians and SMEs ensured that the guidelines generated by MYOAMP are complying with both clinical and economical conditions. The constant exchange with patients' associations allowed a rapid transfer of information to the European population concerned directly by MYOAMP efforts. Finally, the synergy established with other consortia, such as TREAT-NMD, a consortium, or ICE, an international consortium, maximised the outcomes of MYOAMP concerted efforts, and ensured that these efforts will be translated into clinical trials, some of them already being set up, and a collaboration between MYOAMP partners and these consortia, initiated within the frame of MYOAMP, is still ongoing.
MYOAMP novelty: coupling exon-skipping to cell therapy
The recent emergence of new therapeutic venues, based upon post-transcriptional genetic corrections called 'exon-skipping', or genetic surgery, has raised new hope for the DMD community. Using viral transfer approaches it has given very promising results but unfortunately cannot reach every muscle of the body and several injections would trigger an immune response to the vector. Autologous cell therapy may be used as a complement or alternative if the cell type used fulfilled the criteria of being an efficient vector and bringing a functional benefit to the diseased muscle.
Autologous muscle progenitors cannot be used since these are already defective in dystrophic muscle, while stem cells from other origins are ideal candidates, as long as their myogenic and proliferative potentials are ensured. In this perspective, several cell types like mesoangioblasts, which have already been used in mouse and dog models of muscular dystrophy, and AC133 cells have a therapeutic potential for skeletal muscle, as demonstrated in the mouse within MYOAMP. An example of the result of implantation of AC133 cells into regenerating muscles of immunodeficient mice is shown below to illustrate the myogenic potential of these cells.
Paving the way to clinical trials
Very little was known about how the conditions required to amplify these stem cells isolated from humans can be set up in Good manufacturing practices (GMP) conditions, which is an essential step required before any clinical trial.
Many groups have used animal models to investigate the possibilities of using autologous cell therapy for muscular dystrophies, but these data are dispersed, not always comparable and information regarding human cells is sparse. Moreover, little attention has been focussed on the transfer of this knowledge towards applications for therapeutic trials.
The feasibility of autologous myoblast transfer therapy has already been demonstrated for cardiac repair, even if in cardiac therapy, injected cells were mainly used to counteract the development of fibrosis in patients devoid of any defect in skeletal muscle. The fact that pre-clinical trials developed in mouse models of muscular dystrophies have been successful as compared to clinical trials, which used mostly allogenic cells and resulted in very limited clinical benefit for the patients, illustrated the urgent need for pre-clinical studies using human cells.
MYOAMP addressed the question of the amplification of myogenic stem cells used in autologous cell therapy and their safety. The cells transduced with a lentiviral construct allowing exon-skipping have been selected through functional tests and further amplified. The safety and ethics have been defined through two specific work packages at all steps of the amplification of the transduced cells. In vitro and in vivo approaches aimed at improving our understanding of the regulation of proliferation, while the culture conditions and effect of cytokines have been assessed, and telomere length measurements (reflecting the mitotic clock status of the cell) have been defined and are being adapted for SMEs.
The number of cells to be injected at each implantation, which is purely empirical in many clinical trials, has been tested and optimised in a model of implantation of human cells in immuno-deficient mice, in order to define the maximum number of cells to be finally amplified in GMP conditions.
The stability of the parameters initially examined in non-GMP conditions, have been checked through amplification in various conditions, to allow the definition of both guidelines for GMP production and key-parameters to be followed during the GMP amplification.
Therefore, MYOAMP succeded in defining conditions to isolate and amplify human myogenic stem cells to be used in future clinical trials in GMP conditions. Fuethermore, the constant collaboration between academic, Research and development (R&D) and SME partners ensured that these conditions comply with SME requirements, and an extensive market and patent analysis has been carried out within MYOAMP.
European added value
The main outcome of MYOAMP is the definition of protocols to obtain amplified human stem cells with an optimised efficiency for clinical trials.
In addition to basic knowledge on the amplification mechanisms, MYOAMP provided guidelines to obtain these cells in a reproducible and safe manner that can be directly transferred to clinical applications, and adressed technical, ethical and safety issues in a GMP environment.
To ensure a maximum success, MYOAMP partners have worked from the beginning in close contact with several patient associations including the Duchenne Parent Projects in France and United Kingdom, the Association Monegasque Contre les Myopathies, and the Association Francaise contre les Myopathies. The end-users had thus the possibility to be the first to know about the progress made by the participants of MYOAMP. This coordination between patients association, scientists and clinicians was a key-effort to bring the expected results to the next step, convince the national health institutions of the necessity to support the clinical trials, and define the conditions for these trials. An international concerted effort, named ICE, was set up as a result of MYOAMP concerted efforts, and one of MYOAMP partners is setting up a clinical trial in Italy using the results generated by the MYOAMP consortium.
It is a fact that the families are often desperate to see the last results obtained by researchers turned into more clinical trials to at least bring some improvement to their children's health. On the other hand, it is also true that despite this urgent need, families are also aware that designing a therapy is a difficult exercise which involves a lot of assessment and agreements from the regulatory authorities in which safety is the major keyword. Many clinical trials using muscle progenitor cells have been developed in the past for DMD with very limited success.
A solution may be found in combinations of new therapeutic approaches. From this perspective, MYOAMP intended to speed up the assessment of the potential of a new cell therapy approach and to set up the conditions for future clinical trials which could not be set-up within its budget and time limits.
By gathering the expertise of the partners, MYOAMP generated guidelines originating from concerted efforts and validated by both clinicians and European Small and medium-sized enterprises (SMEs) to facilitate the initiation of clinical trials involving stem cell therapy.
Furthermore, the participation of clinicians and SMEs ensured that the guidelines generated by MYOAMP are complying with both clinical and economical conditions. The constant exchange with patients' associations allowed a rapid transfer of information to the European population concerned directly by MYOAMP efforts. Finally, the synergy established with other consortia, such as TREAT-NMD, a consortium, or ICE, an international consortium, maximised the outcomes of MYOAMP concerted efforts, and ensured that these efforts will be translated into clinical trials, some of them already being set up, and a collaboration between MYOAMP partners and these consortia, initiated within the frame of MYOAMP, is still ongoing.
MYOAMP novelty: coupling exon-skipping to cell therapy
The recent emergence of new therapeutic venues, based upon post-transcriptional genetic corrections called 'exon-skipping', or genetic surgery, has raised new hope for the DMD community. Using viral transfer approaches it has given very promising results but unfortunately cannot reach every muscle of the body and several injections would trigger an immune response to the vector. Autologous cell therapy may be used as a complement or alternative if the cell type used fulfilled the criteria of being an efficient vector and bringing a functional benefit to the diseased muscle.
Autologous muscle progenitors cannot be used since these are already defective in dystrophic muscle, while stem cells from other origins are ideal candidates, as long as their myogenic and proliferative potentials are ensured. In this perspective, several cell types like mesoangioblasts, which have already been used in mouse and dog models of muscular dystrophy, and AC133 cells have a therapeutic potential for skeletal muscle, as demonstrated in the mouse within MYOAMP. An example of the result of implantation of AC133 cells into regenerating muscles of immunodeficient mice is shown below to illustrate the myogenic potential of these cells.
Paving the way to clinical trials
Very little was known about how the conditions required to amplify these stem cells isolated from humans can be set up in Good manufacturing practices (GMP) conditions, which is an essential step required before any clinical trial.
Many groups have used animal models to investigate the possibilities of using autologous cell therapy for muscular dystrophies, but these data are dispersed, not always comparable and information regarding human cells is sparse. Moreover, little attention has been focussed on the transfer of this knowledge towards applications for therapeutic trials.
The feasibility of autologous myoblast transfer therapy has already been demonstrated for cardiac repair, even if in cardiac therapy, injected cells were mainly used to counteract the development of fibrosis in patients devoid of any defect in skeletal muscle. The fact that pre-clinical trials developed in mouse models of muscular dystrophies have been successful as compared to clinical trials, which used mostly allogenic cells and resulted in very limited clinical benefit for the patients, illustrated the urgent need for pre-clinical studies using human cells.
MYOAMP addressed the question of the amplification of myogenic stem cells used in autologous cell therapy and their safety. The cells transduced with a lentiviral construct allowing exon-skipping have been selected through functional tests and further amplified. The safety and ethics have been defined through two specific work packages at all steps of the amplification of the transduced cells. In vitro and in vivo approaches aimed at improving our understanding of the regulation of proliferation, while the culture conditions and effect of cytokines have been assessed, and telomere length measurements (reflecting the mitotic clock status of the cell) have been defined and are being adapted for SMEs.
The number of cells to be injected at each implantation, which is purely empirical in many clinical trials, has been tested and optimised in a model of implantation of human cells in immuno-deficient mice, in order to define the maximum number of cells to be finally amplified in GMP conditions.
The stability of the parameters initially examined in non-GMP conditions, have been checked through amplification in various conditions, to allow the definition of both guidelines for GMP production and key-parameters to be followed during the GMP amplification.
Therefore, MYOAMP succeded in defining conditions to isolate and amplify human myogenic stem cells to be used in future clinical trials in GMP conditions. Fuethermore, the constant collaboration between academic, Research and development (R&D) and SME partners ensured that these conditions comply with SME requirements, and an extensive market and patent analysis has been carried out within MYOAMP.
European added value
The main outcome of MYOAMP is the definition of protocols to obtain amplified human stem cells with an optimised efficiency for clinical trials.
In addition to basic knowledge on the amplification mechanisms, MYOAMP provided guidelines to obtain these cells in a reproducible and safe manner that can be directly transferred to clinical applications, and adressed technical, ethical and safety issues in a GMP environment.
To ensure a maximum success, MYOAMP partners have worked from the beginning in close contact with several patient associations including the Duchenne Parent Projects in France and United Kingdom, the Association Monegasque Contre les Myopathies, and the Association Francaise contre les Myopathies. The end-users had thus the possibility to be the first to know about the progress made by the participants of MYOAMP. This coordination between patients association, scientists and clinicians was a key-effort to bring the expected results to the next step, convince the national health institutions of the necessity to support the clinical trials, and define the conditions for these trials. An international concerted effort, named ICE, was set up as a result of MYOAMP concerted efforts, and one of MYOAMP partners is setting up a clinical trial in Italy using the results generated by the MYOAMP consortium.
