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US 2024/0026352 A1Published application
Targeted gene regulation of human immune cells with crispr-cas systems
OwnerCharles A. Gersbach, Sean McCutcheoninventors
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Abstract
Disclosed herein are CRISPR/Cas systems comprising a fusion protein and at least one gRNA targeting a gene or a regulatory element thereof in a cell such as an immune cell, and vector compositions encoding the same. The systems and compositions may be used in methods of modulating expression of a gene in a cell such as an immune cell, as well as in methods of treating a disease such as cancer, autoimmune diseases, or viral infections.
Claims127
A CRISPR/Cas system comprising:
- a fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein, and wherein the second polypeptide domain has an activity selected from transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, histone methylase activity, DNA methylase activity, histone demethylase activity, and/or DNA demethylase activity;
- and
- at least one guide RNA (gRNA) targeting a gene or a regulatory element thereof in an immune cell.
A CRISPR/Cas system comprising:
- a fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein, and wherein the second polypeptide domain has an activity selected from transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, histone methylase activity, DNA methylase activity, histone demethylase activity, and/or DNA demethylase activity;
- and
- at least one guide RNA (gRNA) targeting a gene selected from B2M, TIGIT, CD2, EGFR, and IL2RA, or a regulatory element thereof in a cell.
A vector composition comprising:
- a polynucleotide sequence encoding a fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein, and wherein the second polypeptide domain has an activity selected from transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, histone methylase activity, DNA methylase activity, histone demethylase activity, or DNA demethylase activity;
- and
- a polynucleotide sequence encoding at least one guide RNA (gRNA) targeting a gene or a regulatory element thereof in an immune cell.
A vector composition comprising:
- a polynucleotide sequence encoding a fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein, and wherein the second polypeptide domain has an activity selected from transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, histone methylase activity, DNA methylase activity, histone demethylase activity, or DNA demethylase activity;
- and
- a polynucleotide sequence encoding at least one guide RNA (gRNA) targeting a gene selected from B2M, TIGIT, CD2, EGFR, and IL2RA, or a regulatory element thereof in a cell.
The vector composition of claim 58, wherein the gRNA comprises a polynucleotide sequence selected from SEQ ID NOs:
- 58-70 or 102-120, a variant thereof, or a fragment thereof, or is encoded by or targets a polynucleotide sequence selected from SEQ ID NOs:
- 45-57 or 83-101.
The vector composition of claim 58 or 57, wherein the gRNA targets B2M or a regulatory element thereof.
A method of screening for one or more putative gene regulatory elements in a genome that modulate a gene target or a phenotype of an immune cell, the method comprising:
- (a) contacting a plurality of modified target immune cells with a library of gRNAs, each gRNA targeting a gene regulatory element in an immune cell, thereby generating a pool of test immune cells,
- (b) selecting a population of test immune cells having a modulated gene or phenotype;
- (c) quantifying the frequency of the gRNAs within the population of selected immune cells, wherein the gRNAs that target gene regulatory elements that modulate the phenotype are overrepresented or underrepresented in the selected immune cells;
- and
- (d) identifying and characterizing the gRNAs within the population of selected immune cells thereby identifying the gene regulatory elements that modulate the phenotype,
- wherein the modified target immune cell comprises a fusion protein, the fusion protein comprising a first polypeptide domain comprising a Cas protein and a second polypeptide domain having an activity selected from transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, histone methylase activity, DNA methylase activity, histone demethylase activity, or DNA demethylase activity.
3 more elements
A method of screening a library of gRNAs for modulation of gene expression in a cell, the method comprising:
- (a) generating a library of vectors with a library of gRNAs, each gRNA targeting a target gene or a regulatory element thereof in a cell, the library of vectors comprising:
- a polynucleotide sequence encoding a fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein, and wherein the second polypeptide domain has an activity selected from transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, histone methylase activity, DNA methylase activity, histone demethylase activity, or DNA demethylase activity;
- a polynucleotide sequence encoding a reporter protein operably linked to the polynucleotide sequence encoding the fusion protein;
- and
- a polynucleotide sequence encoding one of the gRNAs;
- (b) transducing a plurality of cells with the library of gRNAs;
- (c) culturing the transduced cells;
- (d) sorting the cultured cells based on the growth of the cells or on the level of expression of the gene or the reporter protein;
- and
- (e) sequencing the gRNA from each cell sorted in step (d).
7 more elements
A method of screening a library of gRNAs for modulation of gene expression in a cell, the method comprising:
- (a) generating a library of vectors with a library of gRNAs, each gRNA targeting a target gene or a regulatory element thereof in a cell, the library of vectors comprising:
- a polynucleotide sequence encoding a fusion protein, wherein the fusion protein comprises two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein, and wherein the second polypeptide domain has an activity selected from transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, histone methylase activity, DNA methylase activity, histone demethylase activity, or DNA demethylase activity;
- and
- a polynucleotide sequence encoding one of the gRNAs;
- (b) transducing a plurality of cells with the library of gRNAs;
- (c) culturing the transduced cells;
- (d) capturing the gRNA from the transduced cells;
- and
- (e) sequencing the gRNA from each transduced cell captured in step (d).
6 more elements
Dependencies are inferred from the claim text.