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US 2022/0228168 A1Published application
Affinity-based multiplexing for live-cell monitoring of complex cell populations
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology
InventorsJacob Borrajo, Paul Blainey, Mohamad Najia, Hong Anh Anna Le, FuNien Tsai
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Abstract
Compositions and methods for inducing and isolating virus-like particles (VLPs), and for allowing real-time assessment of VLP-captured analytes obtained from targeted living mammalian cells, are provided.
Claims25
A mammalian cell comprising a virus like particle (VLP) producing protein and an epitope-tagged viral surface protein.
A composition selected from the group consisting of:
- A mammalian cell comprising an epitope-tagged virus like particle (VLP) producing protein;
- and
- A mammalian cell comprising a virus like particle (VLP) producing protein and a viral surface protein of a virus differing from the virus of the virus like particle (VLP) producing protein.
(canceled)
A method selected from the group consisting of:
- A method for obtaining an expression profile of a living cell, the method comprising:
- (a) providing a living cell;
- (b) introducing a nucleic acid sequence encoding for a VLP producing protein to the living cell, wherein introduction of the nucleic acid sequence encoding for a VLP producing protein is sufficient to induce budding of VLPs from the living cell;
- (c) isolating VLPs produced by the living cell via binding of a VLP protein;
- and
- (d) performing RNA sequencing upon the isolated VLPs, thereby obtaining expression profile information for the isolated VLPs, wherein the expression profile information for the isolated VLPs reflects the expression profile of the living cell,
- thereby obtaining an expression profile of the living cell;
- A method for obtaining an expression profile of a living cell, the method comprising:
- (a) providing a living cell;
- (b) introducing a first nucleic acid sequence encoding for a VLP producing protein and a second nucleic acid encoding for an epitope-tagged viral surface protein to the living cell, wherein introduction of the first nucleic acid sequence encoding for a VLP producing protein is sufficient to induce budding of VLPs from the living cell;
- (c) isolating VLPs produced by the living cell via binding of the epitope-tagged viral surface protein;
- and
- (d) performing RNA sequencing upon the isolated VLPs, thereby obtaining expression profile information for the isolated VLPs, wherein the expression profile information for the isolated VLPs reflects the expression profile of the living cell,
- thereby obtaining an expression profile of the living cell;
- A method for obtaining a first analyte profile for a first population of living cells and a second analyte profile for a second population of living cells, the method comprising:
- (a) providing a first population of living cells;
- (b) introducing a first nucleic acid sequence encoding for a VLP producing protein and a second nucleic acid encoding for a first epitope-tagged viral surface protein to the first population of living cells, wherein introduction of the first nucleic acid sequence encoding for a VLP producing protein is sufficient to induce budding of VLPs from the first population of living cells;
- (c) providing a second population of living cells;
- (d) introducing the first nucleic acid sequence encoding for a VLP producing protein and a second nucleic acid sequence encoding for a second epitope-tagged viral surface protein to the second population of living cells, wherein introduction of the first nucleic acid sequence comprising a nucleic acid sequence encoding for a VLP producing protein is sufficient to induce budding of VLPs from the second population of living cells;
- (e) isolating VLPs produced by the first population of living cells via binding of the first epitope-tagged viral surface protein;
- (f) obtaining a first analyte profile from the isolated VLPs of the first population of living cells;
- (g) isolating VLPs produced by the second population of living cells via binding of the second epitope-tagged viral surface protein;
- and
- (h) obtaining a second analyte profile from the isolated VLPs of the second population of living cells,
- thereby obtaining a first analyte profile for a first population of living cells and a second analyte profile for a second population of living cells;
- A method for obtaining a first analyte profile for a first population of living cells and a second analyte profile for a second population of living cells, the method comprising:
- (a) providing a first population of living cells;
- (b) introducing a first nucleic acid sequence encoding for a VLP producing protein to the first population of living cells, wherein introduction of the first nucleic acid sequence encoding for a VLP producing protein is sufficient to induce budding of VLPs from the first population of living cells;
- (c) providing a second population of living cells;
- (d) introducing a second nucleic acid sequence encoding for a VLP producing protein to the second population of living cells, wherein introduction of the second nucleic acid sequence encoding for a VLP producing protein is sufficient to induce budding of VLPs from the second population of living cells;
- (e) isolating VLPs produced by the first population of living cells via binding of a first VLP protein;
- (f) obtaining a first analyte profile from the isolated VLPs of the first population of living cells;
- (g) isolating VLPs produced by the second population of living cells via binding of a second VLP protein;
- and
- (h) obtaining a second analyte profile from the isolated VLPs of the second population of living cells,
- thereby obtaining a first analyte profile for a first population of living cells and a second analyte profile for a second population of living cells;
- A method for assessing a test compound for efficacy and/or toxicity in living cells, the method comprising:
- (a) providing a population of living cells;
- (b) introducing a nucleic acid sequence encoding for a VLP producing protein to the living cells, wherein introduction of the nucleic acid sequence encoding for the VLP producing protein is sufficient to induce budding of VLPs from the living cells;
- (c) contacting the living cells with a test compound;
- (d) isolating VLPs produced by the living cells via binding of a VLP protein;
- and
- (e) obtaining analyte profile information from the isolated VLPs, wherein the analyte profile information indicates the efficacy and/or toxicity of the test compound,
- thereby assessing a test compound for efficacy and/or toxicity in living cells;
- and
- A method for assessing a test compound for efficacy and/or toxicity in living cells, the method comprising:
- (a) providing a population of living cells;
- (b) introducing a first nucleic acid sequence encoding for a VLP producing protein and a second nucleic acid sequence encoding for an epitope-tagged viral surface protein to the living cells, wherein introduction of the nucleic acid sequence encoding for a VLP producing protein is sufficient to induce budding of VLPs from the living cells;
- (c) contacting the living cells with a test compound;
- (d) isolating VLPs produced by the living cells via binding of the epitope-tagged viral surface protein;
- and
- (e) obtaining analyte profile information from the isolated VLPs, wherein the analyte profile information indicates the efficacy and/or toxicity of the test compound,
- thereby assessing a test compound for efficacy and/or toxicity in living cells.
50 more elements
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Dependencies are inferred from the claim text.