In vivo LPX_FLIP delivery for single cell sequencing analysis
New treatments to fight immunopathologies originating from enhanced immune reactivity against host self-antigens in the course of transplantation (graft vs host disease, GvHD) and autoimmunity are urgently needed. Therapeutic approaches based on either systemic or local immunosuppression, which eliminate or suppress polyclonal, autoreactive T-cell specificities, compromise the entire systemic immunity with many side effects, sometimes even life threatening. One of the most promising, clinically relevant new strategies is mRNA-based lipoplexes (LPX-RNA) to generate tolerogenic myeloid cells. Lipoplexes are lipid carriers that can be used as a delivery system. These are easy to induce and personalized to the patients’ needs. This therapeutic strategy will open new avenue to treat autoimmune diseases and GvHD, providing a therapeutic treatment that will reduce cost, time and administration care.
LPX-RNA complex injection in mice have been performed in University of Verona with successful results without any side effects (Sasso M.S. et al. Biomaterials. 2016). Their preliminary in vitro data demonstrate that transfection switches the molecular program of monocytes towards an immunosuppressive phenotype. Preliminary in vivo experiments show that the complexes are preferentially taken up by myeloid compartment in the spleen compared to lymphocytes. Similar results have been reported in literature (Sasso M.S. Biomaterials. 2016; Sahin U. Nature. 2016).
The purpose of this study is to replicate the animal experiment here at NTNU where cells from injected mice can be sequenced using single cell sequencing. This will allow us to identify the molecular effects of the transfected RNA and evaluate the activation of the MDSC-like transcriptional program. Simple and controlled experimental systems, like cell cultures is therefore not suitable to observe the overall effect on a living subject. Mice (n=12, C57BL/6N) as the best model will be injected with LPX-RNA and euthanized after 18 hours, allowing collection of spleen and bone marrow. The harvested cells will then be sequenced. Using single cell sequencing also allows us to overcome the high variability of in vivo transfection and thus minimizing the number of mice. Because the transfection protocol has been previously optimized using cell lines, the number of mice used is limited further. As the only procedure the mice will go through is an injection (under anesthesia) that is neither toxic, immunogenic or with other side effects. The mice will thus only experience a mild and passing discomfort.
LPX-RNA complex injection in mice have been performed in University of Verona with successful results without any side effects (Sasso M.S. et al. Biomaterials. 2016). Their preliminary in vitro data demonstrate that transfection switches the molecular program of monocytes towards an immunosuppressive phenotype. Preliminary in vivo experiments show that the complexes are preferentially taken up by myeloid compartment in the spleen compared to lymphocytes. Similar results have been reported in literature (Sasso M.S. Biomaterials. 2016; Sahin U. Nature. 2016).
The purpose of this study is to replicate the animal experiment here at NTNU where cells from injected mice can be sequenced using single cell sequencing. This will allow us to identify the molecular effects of the transfected RNA and evaluate the activation of the MDSC-like transcriptional program. Simple and controlled experimental systems, like cell cultures is therefore not suitable to observe the overall effect on a living subject. Mice (n=12, C57BL/6N) as the best model will be injected with LPX-RNA and euthanized after 18 hours, allowing collection of spleen and bone marrow. The harvested cells will then be sequenced. Using single cell sequencing also allows us to overcome the high variability of in vivo transfection and thus minimizing the number of mice. Because the transfection protocol has been previously optimized using cell lines, the number of mice used is limited further. As the only procedure the mice will go through is an injection (under anesthesia) that is neither toxic, immunogenic or with other side effects. The mice will thus only experience a mild and passing discomfort.