BioMAP analysis
View Patent ↗The involvement of an expression product in a cell in a pathway is determined by genetically modifying the cell, incubating the cell with predetermined factors in induce a physiological state and measuring parameters affected by the pathway. Changes in the levels of the parameters as a result of the presence of the expressed product indicate that the expression product is involved with the pathway.
1. A method for characterization of a genetic agent according to its mechanism of action on cellular signaling pathways, the method comprising:
expressing said genetic agent in a cell, wherein said cell is present in a cell culture assay combination, wherein said cell culture assay combination comprises cells and at least two factors sufficient to provide a physiological state of interest involving at least two pathways;
recording changes in at least two different cellular parameter readouts whose levels vary as a result of introduction of said genetic agent;
deriving a biomap dataset from said parameter readouts wherein said biomap comprises data normalized to be a ratio of test to control data on the same cell type under control conditions in the absence of said genetic agent, and said parameters are optimized so that the set of data in the biomap is sufficiently informative that it can discriminate the mechanism of action of said agent; and
analyzing said biomap by a multiparameter pattern recognition algorithm to quantify relatedness of said biomap to reference biomaps that include known genetic agents that target specific pathways, wherein the presence or absence of relatedness to said reference biomaps provides a characterization of said genetic agent mechanism of action.
2. The method according to claim 1 , wherein said genetic agent is introduced into said cell by transfection.
3. The method according to claim 1 , wherein at least four parameters are measured.
4. The method according to claim 1 , wherein said genetic agent is expressed in a panel of cell culture assay combinations; a biomap dataset is derived from each cell culture assay combination in said panel; and each biomap dataset in compared to a reference biomap dataset.
5. The method according to claim 4 , wherein said assay combinations in said panel vary in the factors or cells that are present.
6. The method according to claim 1 , wherein said cells are endothelial cells.
7. The method according to claim 6 , wherein said endothelial cells are primary cells.
8. The method according to claim 7 , wherein said cells are primary HUVEC cells.
9. The method according to claim 6 , wherein one of said at least two pathways is a pathway associated with inflammation.
10. The method according to claim 9 , wherein said parameters include at least two of ICAM-1, VCAM-1, Mig, IL-8, and HLA-DR.
11. The method according to claim 1 , wherein said cell is a leukocyte, cancer cell, or mast cell.
12. The method according to claim 1 , wherein said genetic agent is constitutively active.
13. The method according to claim 1 , wherein said genetic agent encodes an antisense nucleic acid.
14. A method for determining whether a genetic agent influences at least one pathway in a cell in a cell culture, the method comprising:
expressing said genetic agent in a genetically modified cell to overproduce the product of said genetic agent as compared to a cell in the basal state, wherein said cell is present in a cell culture assay combination, wherein said cell culture assay combination comprises at least two factors sufficient to provide a physiological state of interest involving at least two pathways in said cell in said cell culture, said pathways being indicated by levels of selected cellular parameters;
incubating said genetically modified cell in said culture for sufficient time for expression of said genetic agent and growth of said cell;
incubating said basal state cell in said culture for a time analogous to said incubating of said genetically modified cell; and
recording changes in at least two different said cellular parameter readouts for said genetically modified cell and said basal level cell whose levels respond to said pathways as a result of introduction of said genetic agent;
deriving a biomap dataset from said changes in parameter readouts wherein said biomap comprises data normalized to be a ratio of test to control data on the same cell type under control conditions in the absence of said genetic agent, and said parameters are optimized so that the set of data in the biomap is sufficiently informative that it can discriminate the pathway influence of said agent; and
analyzing said biomap by a multiparameter pattern recognition algorithm to quantify relatedness of said biomap to reference biomaps that include known genetic agents that target specific pathways, wherein the presence or absence of relatedness to said reference biomaps provides a characterization of said genetic agent in influencing said at least one pathway.
15. The method according to claim 14 , wherein said genetically modified cell and said basal level cell are endothelial cells.
16. The method according to claim 14 , wherein said parameters include at least two of ICAM-1, VCAM-1, Mig, IL-8, and HLA-DR.
17. The method according to claim 14 , wherein said at least one pathway includes at least one of NF□B and Jak/STAT.
18. A method for determining whether a genetic agent influences at least one pathway in a cell in a cell culture, wherein said pathway includes at least one of NF□B and Jak/STAT, the method comprising:
expressing said genetic agent in a genetically modified cell to overproduce the product of said genetic agent as compared to a cell in the basal state, wherein said cell is present in a cell culture assay combination, wherein said cell culture assay combination comprises at least two factors sufficient to provide a physiological state of interest involving at least two pathways in said cell in said cell culture, wherein said factors include at least one of IL-1, IFN-gamma, and TNF-alpha, said pathways being indicated by levels of selected cellular parameters;
incubating said genetically modified cell in said culture for sufficient time for expression of said genetic agent and growth of said cell;
incubating said basal level cell in said culture for a time analogous to said incubating of said genetically modified cell;
recording changes in at least two different said cellular parameter readouts for said genetically modified cell and said basal level cell whose levels respond to said pathways as a result of introduction of said genetic agent;
deriving a biomap dataset from said changes in parameter readouts wherein said biomap comprises data normalized to be a ratio of test to control data on the same cell type under control conditions in the absence of said genetic agent, and said parameters are optimized so that the set of data in the biomap is sufficiently informative that it can discriminate the pathway influence of said agent; and
analyzing said biomap by a multiparameter pattern recognition algorithm to quantify relatedness of said biomap to reference biomaps that include known genetic agents that target specific pathways, wherein the presence or absence of relatedness to said reference biomaps provides a characterization of said genetic agent in influencing said at least one pathway.
19. The method according to claim 18 , wherein said at least one pathway is involved with the inflammatory state.
20. The method according to claim 18 , wherein said parameters include at least two of ICAM-1, VCAM-1, Mig, IL-8, and HLA-DR.
21. The method according to claim 18 , wherein said changes in said levels are compared to at least one BioMAP of levels of parameters for a selected state.