Projects / Programmes
Designed safety switch for cell-based immunological therapy
| Code |
Science |
Field |
Subfield |
| 3.01.00 |
Medical sciences |
Microbiology and immunology |
|
| Code |
Science |
Field |
| B000 |
Biomedical sciences |
|
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
Cell-based immunotherapy, cancer immunotherapy, chimeric antigen receptor (CAR), stem cells, CRISPR/Cas9, essential genes, split proteins
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
34529 |
PhD Vida Forstnerič |
Biotechnology |
Head |
2018 - 2020 |
40 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
21,007 |
Abstract
Ce
Cell-based therapy is emerging as a new pillar of human therapy, with successful applications of CAR T-cell based immunotherapy of cancer and stem cell based therapies. Unlike small molecules, therapeutic cells have the capacity to sense conditions in the tissue, produce a therapeutic response, proliferate, engraft and survive extended periods in patients. As cells react to a certain tissue microenvironment and physiological milieu, their plasticity may allow them to skew away from their primary therapeutic purpose, potentially leading to harmful side-affects, including Graft-versus host disease, chronic or hyper-acute toxicity or malignancy. Introduction of a safety switch allowing reliable elimination of therapeutic cells is essential for safer and wider use of cell based therapies. Safety switches published to date are predominantly based on the inducible activation of a suicide gene, such as thymidine kinase or caspase-9. A major drawback of these systems is their sensitivity to loss-of-function mutations, which would allow circumvention of the safety mechanism and positive selection of mutated cells. We propose to design a universal cell therapy safety mechanism applicable to CAR-T cell therapy and other types of cell therapies allowing rapid and complete eradication of therapeutic cells after the completion of therapy or in case of the onset of adverse effects. The main princple of the project is to introduce a survival signal that can not be bypassed by point mutations. Cell survival will be based on the chemicaly-induced reconstitution of a split essential gene replacing the knocked-out version of the essential gene. Using the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, we will inactivate a selected endogenous essential gene and substitute its function by an inducible split complementary protein designed based on the essential gene with known tertiary stucture. The split protein will dimerize only in the presence of a small chemical regulator, therefore preventing cell survival upon removal of the exogenous signal. The proposed mechanism could be used for multiple types of cell therapy, such as CAR T-cell therapy, transgenic TCR T-cell therapy and stem cell therapy.
Significance for science
In a therapeutic setting, cells can act as part cell and part device, moving to specific sites in the body, integrating inputs and executing complex funtions in response. These attributes can be successfully harnessed in the treatement of cancer, infections, autoimmunity, neurodegenerative diseases and tissue repair and regeneration. However, the complexity of cells and the challenge of precisely predicting and controlling their actions in vivo presents daunting scientific, regulatory, cultural and economic obstacles for the establishment of cell-based therapy as a more widespread pharmaceutical platform. Currently, stem cell therapy and T-cell immunotherapy remain based on autologous, ex vivo engineered cells, or cells from allogeneic donors, which can greatly limit use of therapy, especially in immunocompromised individuals, heavily treated patients and infants where sufficient propagation of autologues cells is not always possible. Further prospectives are aimed towards an ultimate goal of engineering universal, off-the-shelf therapeutic cells to overcome patient compatibility issues and increase accessibility of such therapies. With the tools and knowledge avaliable to scientists today, possibilities arise to generate and assemble in new ways a tooklit of molecular parts allowing rational design of and precise and rigurous control over therapeutic cells in vivo.
Our research proposal harnesses diverse and modular approaches of synthetic biology for engineering mammalian cells for use in cell-based immunotherapy, aimed to increasing safety. Current safety mechanisms include inducible suicide gene strategies, all of which are able to, by a single point mutation, shed or inactivate the control circuit as a mechanism of escape. Such cells would be prone to positive selection in vivo and would further represent a safety concern, thus not ultimately resolving the issue. The originality of our design is in that it bypasses this crucial issue since it is based on the conditional reconstitution of an essential function rather than on activation of a toxic molecule, which can be easily bypassed by point mutations. In our design, any single point mutation, causing inactivation of the safety mechanism, could not present a safety issue, since the specific cell in which the mutation occurs would be selected against, and not propagated, as is the case in current suicide gene strategies. Our device is designed to represent a sure-fire universal survival switch for cells which would allow for rapid termination of therapy if toxicity where to occur at any stage or upon completion of therapy to prevent further unwanted activation of therapeutic cells.
Significance for the country
In a therapeutic setting, cells can act as part cell and part device, moving to specific sites in the body, integrating inputs and executing complex funtions in response. These attributes can be successfully harnessed in the treatement of cancer, infections, autoimmunity, neurodegenerative diseases and tissue repair and regeneration. However, the complexity of cells and the challenge of precisely predicting and controlling their actions in vivo presents daunting scientific, regulatory, cultural and economic obstacles for the establishment of cell-based therapy as a more widespread pharmaceutical platform. Currently, stem cell therapy and T-cell immunotherapy remain based on autologous, ex vivo engineered cells, or cells from allogeneic donors, which can greatly limit use of therapy, especially in immunocompromised individuals, heavily treated patients and infants where sufficient propagation of autologues cells is not always possible. Further prospectives are aimed towards an ultimate goal of engineering universal, off-the-shelf therapeutic cells to overcome patient compatibility issues and increase accessibility of such therapies. With the tools and knowledge avaliable to scientists today, possibilities arise to generate and assemble in new ways a tooklit of molecular parts allowing rational design of and precise and rigurous control over therapeutic cells in vivo.
Our research proposal harnesses diverse and modular approaches of synthetic biology for engineering mammalian cells for use in cell-based immunotherapy, aimed to increasing safety. Current safety mechanisms include inducible suicide gene strategies, all of which are able to, by a single point mutation, shed or inactivate the control circuit as a mechanism of escape. Such cells would be prone to positive selection in vivo and would further represent a safety concern, thus not ultimately resolving the issue. The originality of our design is in that it bypasses this crucial issue since it is based on the conditional reconstitution of an essential function rather than on activation of a toxic molecule, which can be easily bypassed by point mutations. In our design, any single point mutation, causing inactivation of the safety mechanism, could not present a safety issue, since the specific cell in which the mutation occurs would be selected against, and not propagated, as is the case in current suicide gene strategies. Our device is designed to represent a sure-fire universal survival switch for cells which would allow for rapid termination of therapy if toxicity where to occur at any stage or upon completion of therapy to prevent further unwanted activation of therapeutic cells.
Most important scientific results
Final report
Most important socioeconomically and culturally relevant results
Final report
