Periodic Reporting for period 3 - UniMab (Immune-privileged, immortal, myogenic stem cells for gene therapy of Muscular Dystrophy.)
Periodo di rendicontazione: 2024-01-01 al 2025-06-30
Duchenne muscular dystrophy (DMD) is a devastating incurable disease, affecting thousands with heavy burden on the health systems. This project combines the development of a safe, “immune-privileged cell” with genetic engineering to correct many dystrophin gene mutations for an efficacious and cost affordable therapy.
The applicant pioneered systemic intra-arterial transplantation of mesoangioblasts (blood vessel-derived progenitors) that proved safe in DMD patients and is being implemented for efficacy. However, this personalised approach would prove prohibitively expensive for healthcare systems, as pricing of successful gene therapies is showing. We made the striking observation that human mesoangioblasts can be indefinitely expanded with a novel culture medium, even after genetic manipulation and cloning.
In this project cells have been first genome edited to delete endogenous HLA (β2-microglubin and class II CTA) to make them invisible to the immune system while inserting the tolerogenic HLA-E, normally expressed by the placenta to prevent rejection of the foetus whose paternal antigens are "stranger" for the mother.
Selected clones will be engineered to express a small nuclear RNA (snRNA) that causes skipping of a given exon of the dystrophin gene. Due to the syncytial nature of muscle fibres, the snRNA also enters and corrects the genetic defect in neighbouring, dystrophic nuclei, thus amplifying of one log the therapeutic effect. Five different cell lines would correct the mutation in 60% of DMD patients.
The cell lines will be transplanted in humanized DMD mice and assessed for the ability to escape immune surveillance and to differentiate in dystrophin expressing myofibers, establishing pre-clinical safety and efficacy for an off the shelf, affordable product. The applicant has unique expertise to successfully complete this project, whose strategy may be expanded to other recessive monogenic diseases, for a ground breaking impact in regenerative medicine.
The applicant pioneered systemic intra-arterial transplantation of mesoangioblasts (blood vessel-derived progenitors) that proved safe in DMD patients and is being implemented for efficacy. However, this personalised approach would prove prohibitively expensive for healthcare systems, as pricing of successful gene therapies is showing. We made the striking observation that human mesoangioblasts can be indefinitely expanded with a novel culture medium, even after genetic manipulation and cloning.
In this project cells have been first genome edited to delete endogenous HLA (β2-microglubin and class II CTA) to make them invisible to the immune system while inserting the tolerogenic HLA-E, normally expressed by the placenta to prevent rejection of the foetus whose paternal antigens are "stranger" for the mother.
Selected clones will be engineered to express a small nuclear RNA (snRNA) that causes skipping of a given exon of the dystrophin gene. Due to the syncytial nature of muscle fibres, the snRNA also enters and corrects the genetic defect in neighbouring, dystrophic nuclei, thus amplifying of one log the therapeutic effect. Five different cell lines would correct the mutation in 60% of DMD patients.
The cell lines will be transplanted in humanized DMD mice and assessed for the ability to escape immune surveillance and to differentiate in dystrophin expressing myofibers, establishing pre-clinical safety and efficacy for an off the shelf, affordable product. The applicant has unique expertise to successfully complete this project, whose strategy may be expanded to other recessive monogenic diseases, for a ground breaking impact in regenerative medicine.
During the first 30 months of the project we have achieved important results that allow us to predict the successful completion before the end of the funding period. The results can be summarised as followed:
1. Definition of a novel tissue culture medium that allows indefinite proliferation of mesoangioblasts.
2. Successful deletion of major histocompatibility antigens (HLA) from human mesoangioblasts.
3. Development of a novel lentiviral vector, expressing additional genes that reduce the immune response against mesoangioblasts.
4. Development of conditions to analyse immunogenicity of original and edited cells in mixed lymphocyte reactions.
5. Jackson Lab is producing for us immune-deficient, dystrophic mice that will allow to test the immune privilege of edited mesoangioblasts after transplantation, documented by survival and production of dystrophin.
Of notice, this is the first time that complete elimination of HLA is achieved in a human adult stem/progenitor cells that can be extensively expanded in culture and eventually banked.
1. Definition of a novel tissue culture medium that allows indefinite proliferation of mesoangioblasts.
2. Successful deletion of major histocompatibility antigens (HLA) from human mesoangioblasts.
3. Development of a novel lentiviral vector, expressing additional genes that reduce the immune response against mesoangioblasts.
4. Development of conditions to analyse immunogenicity of original and edited cells in mixed lymphocyte reactions.
5. Jackson Lab is producing for us immune-deficient, dystrophic mice that will allow to test the immune privilege of edited mesoangioblasts after transplantation, documented by survival and production of dystrophin.
Of notice, this is the first time that complete elimination of HLA is achieved in a human adult stem/progenitor cells that can be extensively expanded in culture and eventually banked.
Similar strategies have already been successful with embryonic and reprogrammed stem cells, the only one that until now was possible to proliferate extensively in culture. Unfortunately these cells develop a number of mutations and chromosome alterations that so far have prevented their use in the clinics.
The possibility of indefinitely proliferate mesoangioblasts allows now to prepare immune privileged adult stem cells that show chromosome stability and differentiation potency even after extensive in vitro proliferation.
Thus we can now use these cells in pre- clinical models to test their safety and efficacy in restoring dystrophin production and muscle function. This will set the stage for a novel clinical trial in Duchenne patients affected by a mutation of exon 51 of the dystrophin gene with "intent to cure". Most importantly this approach will be extended to other mutation of the dystrophin gene and then to other forms of muscular dystrophy.
Finally the same approach could be extended to very or extremely rare genetic diseases of skeletal and smooth muscles, while maintaining sustainable costs.
The possibility of indefinitely proliferate mesoangioblasts allows now to prepare immune privileged adult stem cells that show chromosome stability and differentiation potency even after extensive in vitro proliferation.
Thus we can now use these cells in pre- clinical models to test their safety and efficacy in restoring dystrophin production and muscle function. This will set the stage for a novel clinical trial in Duchenne patients affected by a mutation of exon 51 of the dystrophin gene with "intent to cure". Most importantly this approach will be extended to other mutation of the dystrophin gene and then to other forms of muscular dystrophy.
Finally the same approach could be extended to very or extremely rare genetic diseases of skeletal and smooth muscles, while maintaining sustainable costs.