Methods and compositions for engineering CD4-deficient CAR T cells and anti-CD4 CAR T cells and uses thereof
Some aspects of the methods and compositions provided herein relate to the disruption of at least one CD4 gene in a cell, such as a CD4+ T cell. In some embodiments, the disruption comprises use of a CRISPR guide polynucleotide. Some embodiments also include the preparation and use of a cell having at least one disrupted CD4 gene and a chimeric antigen receptor (CAR). Some aspects of the methods and compositions provided herein relate to CARs, such as an anti-CD4 CAR or an anti-CD19 CAR, and use to treat disorders including HIV, acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL).
1 . A genetically modified cell comprising an inactivated endogenous CD4 gene, wherein the genetically modified cell:
comprises a chimeric antigen receptor (CAR), wherein the CAR specifically binds a target antigen; and
wherein the genetically modified cell is prepared by introducing into a source cell a guide RNA (gRNA), which hybridizes to a first exon or a third exon of a CD4 gene, wherein the source cell is a CD3+ cell; and
wherein the genetically modified cell comprises an activity to produce a cytokine against a target cell comprising the target antigen, wherein a level of the cytokine produced by the genetically modified cell is the same or greater than a level of the cytokine produced by a cell comprising the CAR and which lacks an inactivated endogenous CD4 gene.
2 . A pharmaceutical composition comprising the genetically modified cell of claim 1 , and a pharmaceutically acceptable excipient.
3 . A method of killing or inhibiting a population of CD4+ cells, comprising contacting the population of CD4+ cells with the genetically modified cell of claim 1 .
4 . A method of reducing or inhibiting HIV replication in a population of CD4+ cells comprising an HIV-infected cell, the method comprising contacting the population of CD4+ cells with the genetically modified cell of claim 1 .
5 . A method of treating, inhibiting, or ameliorating a disorder in a subject, wherein the disorder is selected from an HIV infection, an acute myeloid leukemia (AML) or an acute lymphocytic leukemia (ALL), comprising administering to the subject a population of cells comprising the genetically modified cell of claim 1 .
6 . The genetically modified cell of claim 1 , wherein the source cell is a CD4+ T cell.
7 . The genetically modified cell of claim 1 , wherein the target antigen is CD4, CD19, or an HIV protein.
8 . The genetically modified cell of claim 1 , wherein the target antigen is CD4.
9 . The genetically modified cell of claim 1 , wherein the genetically modified cell comprises a cytotoxic activity against a target cell comprising the target antigen.
10 . The genetically modified cell of claim 1 , wherein the cytokine is selected from TNF-alpha, IL-2 or IFN gamma.
11 . The genetically modified cell of claim 1 , wherein the source cell is a CD4+ T cell and the target antigen is CD4.
12 . A system comprising:
the genetically modified cell of claim 1 ; and
a target cell comprising the target antigen.
13 . The system of claim 12 , wherein the target cell is selected from an HIV-infected cell, an acute myeloid leukemia (AML) cell, or an acute lymphocytic leukemia (ALL) cell.
14 . A CD3+ cell comprising an inactivated endogenous CD4 gene, the cell comprising:
a guide RNA (gRNA), which hybridizes to a first exon or a third exon of a CD4 gene,
a Cas 9 nuclease or polynucleotide encoding the Cas 9 nuclease, and
a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR specifically binds a target antigen; and
wherein the cell comprises an activity to produce a cytokine against a target cell comprising the target antigen, wherein a level of the cytokine produced by the cell is the same or greater than a level of the cytokine produced by a cell comprising the CAR and which lacks an inactivated endogenous CD4 gene.
15 . The cell of claim 14 , wherein the gRNA comprises a sequence having at least 95% sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOS: 01-08 or complement thereof.
16 . The cell of claim 14 , wherein the gRNA comprises the sequence set forth in SEQ ID NO:06 or a complement thereof.
17 . The cell of claim 14 , wherein the cell is derived from a CD4+ T cell.
18 . The cell of claim 14 , wherein the target antigen is CD4, CD19, or an HIV protein.
19 . The cell of claim 14 , wherein the target antigen is CD4.
20 . The cell of claim 14 , wherein the gRNA comprises the sequence set forth in SEQ ID NO:08 or a complement thereof.