Method for modifying genes
AssigneeOxford University Innovation Limited
InventorsTudor A. FULGA, Yale S. MICHAELS, Thomas A. MILNE
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Abstract
The present invention relates to method of modulating the level of expression of an endogenous gene in a cell, the method comprising inserting a heterologous microRNA (miRNA) response element (MRE) into the 3′-untranslated region (3′-UTR) of the gene. The binding of endogenous miRNAs to the MRE results in or leads to a repression of the level of expression of the gene. The invention also relates to cells and transgenic animals whose endogenous genes comprise heterologous MRE in their 3′-UTRs.
Claims26
A method for modifying an endogenous gene, the method comprising the step of:
- (i) inserting a heterologous MRE into the 3′-UTR of the gene.
A method of modulating the level of expression of a gene in a cell, wherein the gene is expressed or is capable of being expressed in the cell, wherein the cell is one which expresses a miRNA or is capable of expressing the miRNA, the method comprising the step:
- (i) inserting a heterologous MRE into the 3′-UTR of the gene, wherein the heterologous MRE is one which is capable of being bound by the miRNA with a first affinity, and wherein the binding of the miRNA to the MRE results in or leads to a reduction of the level of expression of the gene.
A cell, wherein one or more of the cell's endogenous genes comprise a heterologous MRE in the gene's 3′-UTR.
A transgenic non-human animal, wherein one or more of the animal's endogenous genes comprise a heterologous MRE in the gene's 3′-UTR.
A plurality of nucleic acid molecules, each nucleic acid molecule independently comprising:
- (a) a gene comprising a different MRE in its 3′-UTR, wherein the nucleotide sequences of the genes are the same apart from the nucleotide sequences of the MREs,
- wherein the nucleotide sequences of the MREs have all independently been derived from the nucleotide sequence of a common MRE to produce modified MREs,
- wherein the common MRE is capable of being bound by a miRNA with a first affinity, and wherein the modified MREs are capable of being bound by the miRNA with a plurality of different affinities.
A method for inhibiting the production of a disease-promoting activator by a cell, wherein the cell comprises a first gene which encodes an agent which initiates the disease, wherein the cell also comprises a second gene which encodes an activator which promotes proliferation of the disease, the method comprising the steps of:
- (a) modifying the nucleotide sequence of the 3′-UTR of the second gene to introduce a MRE for a miRNA which is differentially upregulated in cells which comprise the first gene,
- wherein the binding of the miRNA to the MRE represses the production of the activator, and thereby limiting proliferation of the disease.
A method for prophylactically inhibiting the production of a disease-promoting activator by a cell, wherein the cell is at risk of comprising a first gene which encodes an agent which promotes the disease, wherein the cell also comprises a second gene which encodes an activator which promotes proliferation of the disease, the method comprising the steps of:
- (a) modifying the nucleotide sequence of the 3′-UTR of the second gene to introduce a MRE for a miRNA which is differentially upregulated in cells which comprise the first gene,
- wherein, in cells which comprise the first gene, the binding of the miRNA to the MRE represses the production of the activator, and thereby limiting proliferation of the disease.
A cell whose genome comprises an endogenous gene encoding an activator wherein the activator is one which is capable of promoting the proliferation of the cell, and wherein the nucleotide sequence of the gene encoding the activator comprises a heterologous MRE in the 3′-UTR.
A cell whose genome comprises an endogenous gene which is capable of causing a disease, and wherein the nucleotide sequence of the gene comprises a heterologous MRE in the 3′-UTR.
A process for producing a non-human transgenic animal, the process comprising the steps:
- (i) inserting a heterologous MRE into the 3′-UTR of a gene in the genome of a totipotent or pluripotent cell;
- and
- (ii) culturing the cell under conditions such that the cell develops into a transgenic animal;
- wherein the gene encodes an agent which promotes proliferation of a disease.
A cell which expresses an immune checkpoint polypeptide, wherein the nucleotide sequence of the gene encoding the immune checkpoint polypeptide comprises a heterologous MRE in the 3′-UTR, preferably wherein the cell is a T-cell or a T-cell derived from a stem cell and wherein the immune checkpoint polypeptide is PD-1.
A method of producing a modified cell, the method comprising the step of:
- (a) in the genome of a cell, inserting the nucleotide sequence of a heterologous MRE into the 3′-UTR of a gene which encodes an immune checkpoint polypeptide,
- preferably wherein the cell is a T-cell, or a T-cell derived from a stem cell and wherein the immune checkpoint polypeptide is PD-1.
A method of producing a plurality of modified cells, the method comprising the step of:
- (a) in the genomes of a plurality of cells, inserting the nucleotide sequences of heterologous MREs in the 3′-UTRs of genes which encode a common immune checkpoint polypeptide,
- thereby producing a plurality of cells each of which comprises a different heterologous MRE.
A method of treating cancer cells in a subject, wherein the cancer cells have the potential of being detected by the immune system, the method comprising the steps of:
- (a) in the genome of a T-cell which has been obtained from the subject, inserting the nucleotide sequence of a heterologous MRE in the 3′-UTR of a gene which encodes an immune checkpoint polypeptide thus producing a modified T-cell, wherein the heterologous MRE is one which is capable of being bound by a miRNA which is differentially upregulated in activated T-cells;
- and
- (b) introducing the modified T-cell into the subject;
- wherein the modified T-cell, when activated by contact with the cancer cells, is not negatively regulated by the cancer cells, and wherein the modified T-cell promotes the destruction or rejection of the cancer cells.
Dependencies are inferred from the claim text.