Methods and compositions for preparing CD4-deficient car t cells and Anti-CD4 car t cells and uses thereof

Abstract

To provide a method of killing or inhibiting a population of CD4+ cells.SOLUTION: There are provided methods for 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).SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 820,387, filed March 19, 2019, entitled "METHODS AND COMPOSITIONS FOR GENERATING CD4 DEFICIENT CAR T CELLS AND ANTI-CD4 CAR T CELLS," which is expressly incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT NIH Contract / Grant Number: RO1 AI118500 "Generating T cells with optimized anti-HIV chimeric antigen receptors and disruption of CCR5 as a strategy to target HIV-infected cells"

[0003] Sequence Listing Reference This application has been filed with an electronic Sequence Listing, which has been provided as an approximately 4kb file under filename SCRI206WOSEQLIST, created on March 13, 2020. The information set forth in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0004] Some aspects of the methods and compositions provided herein relate to disrupting at least one CD4 gene in a cell, such as a CD4+ T cell. In some embodiments, the disruption involves the use of a CRISPR guide polynucleotide. Some embodiments also involve the generation and use of cells with at least one disrupted CD4 gene and a chimeric antigen receptor (CAR). Additionally, some aspects of the methods and compositions provided herein relate to CARs, such as anti-CD4 CARs and anti-CD19 CARs, and their use to treat diseases, such as HIV, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL). [Background technology]

[0005] CD4+ T cells play a central role in the functioning of the immune system. In the immune system, CD4+ T cells can assist B cells in producing antibodies, induce the enhancement of the bactericidal activity of macrophages, recruit neutrophils, eosinophils, and basophils to the site of infection or inflammation, and control a series of immune responses, such as regulating the immune system, by producing cytokines and chemokines. CD4 expression can also be found on other types of cells. CD4 also plays a crucial role in the performance of some pathogens, for example, it is essential for HIV infection of human cells. CD4 cells can also be infected by other pathogens, such as HTLV and EBV. There are also diseases caused by CD4+ T cells. There are also various CD4+ malignancies. Furthermore, CD4+ T cells are thought to play a role in some autoimmune and alloimmune diseases. Thus, there is a need to develop novel cell therapies that involve CD4 destruction and / or target CD4 T cells. Summary of the Invention [Means for solving the problem]

[0006] Some of the embodiments of the methods and compositions provided herein include the following aspects.

[0007] Embodiment 1: An isolated nucleic acid for CD4 gene disruption, the nucleic acid comprising a CRISPR (clustered regularly interspaced short palindromic repeats) guide polynucleotide capable of hybridizing to the CD4 gene or configured to hybridize to the CD4 gene.

[0008] Embodiment 2: The isolated nucleic acid of embodiment 1, wherein said guide polynucleotide is capable of hybridizing to the sense strand of the CD4 gene or is configured to hybridize to the sense strand of the CD4 gene.

[0009] Embodiment 3: The isolated nucleic acid according to embodiment 1, wherein said guide polynucleotide is capable of hybridizing to the antisense strand of the CD4 gene or is configured to hybridize to the antisense strand of the CD4 gene.

[0010] Embodiment 4: The isolated nucleic acid according to any one of embodiments 1 to 3, wherein said guide polynucleotide is capable of hybridizing to the first exon of the CD4 gene or is configured to hybridize to the first exon of the CD4 gene.

[0011] Embodiment 5: The isolated nucleic acid according to any one of embodiments 1 to 3, wherein said guide polynucleotide is capable of hybridizing to the third exon of the CD4 gene or is configured to hybridize to the third exon of the CD4 gene.

[0012] Embodiment 6: The isolated nucleic acid according to any one of embodiments 1 to 5, wherein said guide polynucleotide targets a protospacer adjacent motif (PAM).

[0013] Embodiment 7: The isolated nucleic acid of embodiment 6, wherein the PAM comprises an NGG motif.

[0014] Aspect 8: A nucleic acid according to any one of aspects 1 to 7, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8 or a complementary strand thereof.

[0015] Embodiment 9: A nucleic acid according to any one of embodiments 1 to 8, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:6 or its complementary strand.

[0016] Embodiment 10: A nucleic acid according to any one of embodiments 1 to 9, comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8, or a complementary strand thereof.

[0017] Embodiment 11: A nucleic acid according to any one of embodiments 1 to 10, comprising the nucleotide sequence shown in SEQ ID NO: 6 or a complementary strand thereof.

[0018] Embodiment 12: The nucleic acid according to any one of embodiments 1 to 11, wherein the guide polynucleotide comprises RNA.

[0019] Embodiment 13: A ribonucleoprotein (RNP) comprising a nucleic acid according to any one of embodiments 1 to 12.

[0020] Embodiment 14: An RNP according to embodiment 13, comprising a Cas9 protein.

[0021] Embodiment 15: A method for producing a genetically modified cell, the method comprising the step of introducing an RNP according to embodiment 13 or 14 into the cell.

[0022] Embodiment 16: The method of embodiment 15, further comprising introducing into said cell a second polynucleotide encoding a Cas9 protein.

[0023] Embodiment 17: The method of embodiment 16, wherein the step of introducing the RNP into the cell comprises electroporation or lipofection.

[0024] Embodiment 18: A method for producing a genetically modified cell, comprising the steps of: introducing into a cell a first polynucleotide encoding the nucleic acid according to any one of aspects 1 to 12; and introducing into the cell a second polynucleotide encoding a Cas9 protein; The method includes:

[0025] Embodiment 19: The method according to embodiment 18, wherein the step of introducing the first polynucleotide and / or the second polynucleotide into the cell comprises electroporation or lipofection.

[0026] Embodiment 20: The method according to any one of embodiments 15 to 19, further comprising the step of introducing into said cell a third polynucleotide encoding a chimeric antigen receptor (CAR).

[0027] Embodiment 21: The method of embodiment 20, wherein said CAR specifically binds to CD4 protein.

[0028] Embodiment 22: The method of embodiment 20, wherein the CAR specifically binds to CD19 protein.

[0029] Embodiment 23: The method of embodiment 20, wherein the CAR specifically binds to an HIV protein.

[0030] Embodiment 24: The method of embodiment 20, wherein said CAR comprises an antigen-binding domain derived from an HIV-neutralizing antibody.

[0031] Embodiment 25: The method of embodiment 24, wherein said HIV neutralizing antibody is selected from PGT128, PG9 and PGT145.

[0032] Embodiment 26: The method of any one of embodiments 20 to 25, wherein said CAR is expressed from an inducible promoter.

[0033] Embodiment 27: The method according to any one of embodiments 15 to 26, further comprising the step of introducing into said cell a fourth polynucleotide encoding a suicide gene system.

[0034] Embodiment 28: The method according to embodiment 27, wherein said suicide gene system is a herpes simplex virus thymidine kinase (HSVTK) / ganciclovir (GCV) suicide gene system or an inducible caspase suicide gene system.

[0035] Embodiment 29: The method according to any one of embodiments 15 to 28, wherein the cell is a mammalian cell.

[0036] Embodiment 30: The method according to any one of embodiments 15 to 29, wherein the cell is a human cell.

[0037] Embodiment 31: The method according to any one of embodiments 15 to 30, wherein the cells express CD4.

[0038] Embodiment 32: The method according to any one of embodiments 15 to 31, wherein the cell is a lymphocyte.

[0039] Embodiment 33: The method according to any one of embodiments 15 to 32, wherein the cell is a T cell.

[0040] Embodiment 34: The method according to any one of embodiments 15 to 33, wherein said cell is a primary cell.

[0041] Embodiment 35: The method according to any one of embodiments 15 to 34, wherein said cells are cells isolated from peripheral blood mononuclear cells.

[0042] Embodiment 36: The method according to any one of embodiments 15 to 35, wherein the cells express CD3 or CD8.

[0043] Embodiment 37: The method according to any one of embodiments 15 to 36, wherein said cell is a CD4+ cell, a natural killer cell or a cell derived from a natural killer T cell.

[0044] Embodiment 38: The method according to any one of embodiments 15 to 37, wherein the cell is an ex vivo cell.

[0045] Embodiment 39: A genetically modified cell produced by a method according to any one of embodiments 15 to 38.

[0046] Embodiment 40: A genetically modified cell comprising at least one CD4 gene disrupted by a CRISPR (clustered regularly interspaced short palindromic repeats) guide polynucleotide.

[0047] Embodiment 41 The genetically modified cell according to embodiment 40, wherein said at least one CD4 gene is disrupted in the first exon.

[0048] Embodiment 42: The genetically modified cell according to embodiment 40, wherein said at least one CD4 gene is disrupted in the third exon.

[0049] Aspect 43: The genetically modified cell according to any one of aspects 40 to 42, wherein the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8 or a complementary strand thereof.

[0050] Embodiment 44: The genetically modified cell according to any one of embodiments 40 to 43, wherein the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6 or its complementary strand.

[0051] Aspect 45: The genetically modified cell according to any one of aspects 40 to 44, wherein the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8 or a complementary strand thereof.

[0052] Embodiment 46: The genetically modified cell according to any one of embodiments 40 to 45, wherein the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 6 or a complementary strand thereof.

[0053] Embodiment 47: A genetically modified cell according to any one of embodiments 40 to 46, further comprising a chimeric antigen receptor (CAR).

[0054] Embodiment 48: The genetically modified cell of embodiment 47, wherein the CAR specifically binds to CD4 protein.

[0055] Embodiment 49: The genetically modified cell of embodiment 47, wherein the CAR specifically binds to CD19 protein.

[0056] Embodiment 50: The genetically modified cell of embodiment 47, wherein the CAR specifically binds to an HIV protein.

[0057] Embodiment 51: The genetically modified cell of embodiment 50, wherein the CAR comprises an antigen-binding domain derived from an HIV-neutralizing antibody.

[0058] Embodiment 52: The genetically modified cell according to embodiment 51, wherein said HIV neutralizing antibody is selected from PGT128, PG9 and PGT145.

[0059] Embodiment 53: A genetically modified cell according to any one of embodiments 40 to 52, wherein the CAR is expressed from an inducible promoter.

[0060] Embodiment 54: A genetically modified cell according to any one of embodiments 40 to 53, further comprising a polynucleotide encoding a suicide gene system.

[0061] Embodiment 55: The genetically modified cell according to embodiment 54, wherein said suicide gene system is a herpes simplex virus thymidine kinase (HSVTK) / ganciclovir (GCV) suicide gene system or an inducible caspase suicide gene system.

[0062] Embodiment 56: A genetically modified cell according to any one of embodiments 40 to 55, which is a mammalian cell.

[0063] Embodiment 57: A genetically modified cell according to any one of embodiments 40 to 56, which is a human cell.

[0064] Embodiment 58: A genetically modified cell according to any one of embodiments 40 to 57, which is a lymphocyte.

[0065] Embodiment 59: A genetically modified cell according to any one of embodiments 40 to 58, which is a T cell.

[0066] Embodiment 60: A genetically modified cell according to any one of embodiments 40 to 59, which is a primary cell.

[0067] Embodiment 61: A genetically modified cell according to any one of embodiments 40 to 60, which is a cell isolated from a peripheral blood mononuclear cell.

[0068] Embodiment 62: A genetically modified cell according to any one of embodiments 40 to 61, which expresses CD3 or CD8.

[0069] Embodiment 63: A genetically modified cell according to any one of embodiments 40 to 62, which is a cell derived from a CD4+ cell, a natural killer cell or a natural killer T cell.

[0070] Embodiment 64: A pharmaceutical composition comprising a genetically modified cell according to any one of embodiments 40 to 63 and a pharma- ceutically acceptable excipient.

[0071] Embodiment 65: A genetically modified cell population having enhanced resistance to HIV infection, comprising a genetically modified cell according to any one of embodiments 40 to 63, the cell population having enhanced resistance to HIV infection compared to a cell in which at least one CD4 gene is not disrupted.

[0072] Embodiment 66: A method for killing or suppressing a CD4+ cell population, the method comprising the step of contacting a CD4+ cell population with a genetically modified cell according to any one of embodiments 40 to 63.

[0073] Embodiment 67: The method of embodiment 66, wherein said CD4+ cell population comprises HIV.

[0074] Embodiment 68: The method of embodiment 66, wherein said CD4+ cell population comprises acute myeloid leukemia (AML) cells or acute lymphocytic leukemia (ALL) cells.

[0075] Embodiment 69: A method for reducing HIV-infected CD4+ cells, comprising the step of contacting a population of CD4+ cells with a genetically modified cell according to any one of embodiments 40 to 63.

[0076] Embodiment 70: The method according to any one of embodiments 66 to 69, wherein said population of CD4+ cells and said genetically modified cells are derived from the same subject.

[0077] Embodiment 71: The method according to any one of embodiments 66 to 70, wherein said CD4+ cell population is an ex vivo cell population.

[0078] Embodiment 72: The method according to any one of embodiments 66 to 70, wherein said CD4+ cell population is an in vivo cell population.

[0079] Embodiment 73: A method of treating, suppressing or palliating HIV infection, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) in a subject, comprising: 64. A method comprising the step of administering to a subject in need of treatment, inhibition or amelioration of HIV infection, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), a cell population comprising the genetically modified cell according to any one of aspects 40 to 63.

[0080] Embodiment 74: The method of embodiment 72, wherein the genetically modified cells are autologous cells of the subject.

[0081] Embodiment 75: The method of embodiment 73 or 74, wherein the genetically modified cell comprises a chimeric antigen receptor (CAR).

[0082] Embodiment 76: The method of embodiment 75, wherein the CAR comprises an anti-CD4 CAR.

[0083] Embodiment 77: The method of embodiment 75, wherein said CAR specifically binds to CD19 protein.

[0084] Embodiment 78: The method of embodiment 75, wherein the CAR specifically binds to an HIV protein.

[0085] Embodiment 79: The method of embodiment 75, wherein the CAR comprises an antigen-binding domain derived from an HIV-neutralizing antibody.

[0086] Embodiment 80: The method of embodiment 79, wherein said HIV neutralizing antibody is selected from PGT128, PG9 and PGT145.

[0087] Embodiment 81: The method according to any one of embodiments 73 to 80, further comprising the step of inducing expression of said CAR in said cell.

[0088] Embodiment 82: The method according to any one of embodiments 73 to 80, further comprising a step of suppressing or reducing expression of said CAR in said cell.

[0089] Embodiment 83: The method of any one of embodiments 73 to 82, wherein expression of the CAR is suppressed or reduced following administration of the cell population to the subject.

[0090] Embodiment 84: The method of any one of embodiments 73 to 82, wherein the inhibition or reduction of expression of the CAR is confirmed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the cell population to the subject.

[0091] Embodiment 85: The method of any one of embodiments 73 to 83, wherein the suppression or reduction in expression of the CAR is confirmed 1 week, 2 weeks, 3 weeks, or 4 weeks after administration of the cell population to the subject.

[0092] Embodiment 86: The method according to any one of embodiments 73 to 85, wherein the subject is a mammal.

[0093] Embodiment 87: The method according to any one of embodiments 73 to 86, wherein the subject is a human.

[0094] Embodiment 88: A cell population comprising a genetically modified cell according to any one of embodiments 40 to 63 for use in a pharmaceutical preparation for the treatment of HIV, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), or for use in the treatment of HIV, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).

[0095] Embodiment 89: Use of a cell population comprising a genetically modified cell according to any one of embodiments 40 to 63 in the preparation of a medicament for the treatment of HIV, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). [Brief description of the drawings]

[0096] [Figure 1] FIG. 1 shows a schematic diagram showing the exons and introns of the CD4 gene and the locations of six CRISPR guide polynucleotides (G1, G2, G3, G4, G5, G6, G7 and G8) in exon 1 and exon 3 of the CD4 gene.

[0097] [Diagram 2]Figure 2A shows flow cytometry data plotting side scatter area (SSC-A) on the Y axis against CD4+ cell lines stained for CD4 on the X axis. Shown are untreated cells (mock), cells treated with Cas9 only, or cells treated with Cas9 and CRISPR guide polynucleotides (G1, G2, G3, G4, G5, or G6). Figure 2B shows flow cytometry data plotting CD4 on the Y axis against CD4+ cell lines stained for CD8 on the X axis (bottom). Shown are untreated cells (mock), or cells treated with Cas9 and CRISPR guide polynucleotides (G6, G7, or G8).

[0098] [Diagram 3] Flow cytometry data for CD4 and CD8 staining of human primary T cells is shown for untreated cells (mock), cells treated with Cas9 alone, cells treated with CRISPR guide polynucleotide 6 alone, or cells treated with Cas9 and CRISPR guide polynucleotides (G3 or G6).

[0099] [Figure 4] Figure 4A shows a comparison of the CD4 genomic sequences from 17 untreated control clones with the consensus sequence shown at the bottom of the figure. The sequence of G6 is shown below the sequence in the figure. Figure 4B shows a comparison of the CD4 genomic sequences from 20 clones treated with Cas9 and G6 CRISPR guide polynucleotide with the G6 insert sequence shown in the figure.

[0100] [Diagram 5]FIG. 5A shows flow cytometry data of CD4 and CD8 staining of untreated primary CD3+ cells as a control. FIG. 5B shows flow cytometry data of CD4 and CD8 staining of primary CD3+ cells transduced with lentivirus containing polynucleotides encoding anti-CD4 CAR only as a control. FIG. 5C shows flow cytometry data of CD4 and CD8 staining of primary CD3+ cells treated with G6 CRISPR guide polynucleotide only as a control. FIG. 5D shows flow cytometry data of CD4 and CD8 staining of primary CD3+ cells treated with Cas9 and G6 CRISPR guide polynucleotide. FIG. 5E shows flow cytometry data of CD4 and CD8 staining of primary CD3+ cells treated with Cas9 and G6 CRISPR guide polynucleotide and transduced with lentivirus containing polynucleotides encoding anti-CD4 CAR.

[0101] [Figure 6] FIG. 6A shows flow cytometry data of untreated primary CD3+ cells as a control. FIG. 6B shows flow cytometry data of primary CD3+ cells transduced with a lentiviral vector containing a polynucleotide encoding only an anti-CD4 CAR expressing blue fluorescent protein as a control. FIG. 6C shows flow cytometry data of primary CD3+ cells treated with only a G6 CRISPR guide polynucleotide as a control. FIG. 6D shows flow cytometry data of primary CD3+ cells treated with Cas9 and G6 CRISPR guide polynucleotide. FIG. 6E shows flow cytometry data of primary CD3+ cells treated with Cas9 and G6 CRISPR guide polynucleotide and transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD4 CAR expressing blue fluorescent protein. The plot shown in FIG. 6 shows side scatter area (SSC-A) on the Y axis against blue fluorescent protein (BFP) on the X axis.

[0102] [Figure 7] Figure 7A shows flow cytometry data from CD4 and CD8 staining of primary CD3+ cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD4 CAR only, backgated to analyze cells positive for BFP (expressed from the CAR). Figure 7B shows flow cytometry data from CD4 and CD8 staining of primary CD3+ cells treated with Cas9 and G6 CRISPR guide polynucleotides and transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD4 CAR, backgated to analyze cells positive for BFP (expressed from the CAR).

[0103] [Figure 8] Functional data shown in flow cytometry data when T cells or CAR T cells were mixed with target cells. Target cells were untransduced K562 cells or K562 cells transduced with a lentiviral vector containing a polynucleotide encoding CD19. Effector cells were untransduced T cells, T cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD19 CAR, or T cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD19 CAR and transfected with a G6 CRISPR guide polynucleotide and Cas9. The top panel of FIG. 8 shows gating based on lymphocyte morphology (as determined by side scatter plotted against forward scatter). The bottom panel of FIG. 8 shows gating of live cells from lymphocyte morphology gated cells.

[0104] [Figure 9]Functional data shown in flow cytometry data when T cells or CAR T cells were mixed with target cells. Target cells were untransduced K562 cells or K562 cells transduced with a lentiviral vector containing a polynucleotide encoding CD19. Effector cells were untransduced T cells, T cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD19 CAR, or T cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD19 CAR and transfected with a G6 CRISPR guide polynucleotide and Cas9. The top row of Figure 9 shows the expression of CAR in live lymphocytes identified in Figure 8, measured by expression of BFP encoded downstream of this CAR. The second, third, and fourth rows of Figure 9 show the expression of intracellular cytokines in CAR-expressing cells identified in the first plot, measured by flow cytometry. The second row of Figure 9 shows the expression of intracellular TNF-α, assessed with a PE-labeled anti-TNF-α antibody. The third row of Figure 9 shows intracellular IL-2 expression assessed with anti-IL-2 antibody labeled with PE-Cy7, and the fourth row of Figure 9 shows IFN-γ expression assessed with anti-IFN-γ antibody labeled with APC.

[0105] [Figure 10] Flow cytometry data for TNF-α expression in CD19-expressing K562 target cells when mixed with effector cells. Effector cells were T cells containing an anti-CD19 CAR (left panel) or CD4-depleted T cells containing an anti-CD19 CAR (right panel). SSC-A was measured by staining with PE-A (top panel) or CD4-PerCPCy5.5-A and CD8-Alexa Fluor 700A (bottom panel). CD4 depletion identified an increased CD4- and CD8-double negative population in live TNF-α-expressing CAR+ (BFP+) lymphocytes. This population is shown in the circled area in the bottom right panel.

[0106] [Figure 11] Flow cytometry data for IL-2 expression in CD19-expressing K562 target cells when mixed with effector cells. Effector cells were T cells containing an anti-CD19 CAR (left panel) or CD4-depleted T cells containing an anti-CD19 CAR (right panel). SSC-A was measured by staining with PE-Cy7-A (top panel) or CD4-PerCPCy5.5-A and CD8-Alexa Fluor 700A (bottom panel). CD4 depletion identified an increased CD4- and CD8-double negative population in live IL-2-expressing CAR+ (BFP+) lymphocytes. This population is shown in the circled area in the bottom right panel.

[0107] [Figure 12] Flow cytometry data for IFN-γ expression in CD19-expressing K562 target cells when mixed with effector cells. Effector cells were T cells containing an anti-CD19 CAR (left panel) or CD4-depleted T cells containing an anti-CD19 CAR (right panel). SSC-A was measured by staining with APC-A (top panel) or CD4-PerCPCy5.5-A and CD8-Alexa Fluor 700A (bottom panel). CD4 depletion identified an increased CD4- and CD8-double negative population of live CAR+ (BFP+) lymphocytes expressing IFN-γ. This population is shown in the circled area in the bottom right panel.

[0108] [Figure 13] Flow cytometry data showing mixing of CD19-expressing K562 target cells with CD4-depleted T cells containing anti-CD19 CAR as effector cells, followed by gating on lymphocytes, live cells, and CAR+ cells and staining for TNF-α, IL-2, and IFN-γ expression. The figures on the left show cytokine expression in CD4+ CAR T cells. The figures on the right show cytokine expression in CAR T cells genetically engineered to not express CD4.

[0109] [Figure 14] Flow cytometry data showing cell morphology (side scatter on the Y-axis plotted against forward scatter on the X-axis) for various combinations of target and effector cells. Target cells were a CD4+ cell line (A3.01 cells) transduced with a lentiviral vector containing a polynucleotide encoding green fluorescent protein (GFP). Effector cells were untransduced T cells, T cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD4 CAR, or T cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD4 CAR and transfected with a polynucleotide encoding a G6 CRISPR guide targeting CD4 and Cas9. Target and effector cells were mixed in a 2:1 ratio and incubated for 22 hours.

[0110] [Figure 15] Flow cytometry data showing the fate of GFP-expressing CD4+ target cells under various conditions are shown. Target cells were a CD4+ cell line (A3.01 cells) transduced with a lentiviral vector containing a polynucleotide encoding green fluorescent protein (GFP). Effector cells were untransduced T cells, T cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD4 CAR, or T cells transduced with a lentiviral vector containing a polynucleotide encoding an anti-CD4 CAR and transfected with a polynucleotide encoding a G6 CRISPR guide targeting CD4 and Cas9. Target and effector cells were mixed at a 2:1 ratio and incubated for 22 hours. The bottom right panel shows that mixing with anti-CD4 CAR-expressing cells eliminated the majority of GFP-expressing CD4+ cells.

[0111] [Figure 16]Figure 1 shows a time course of HIV p24 capsid concentration monitored over a 5-day period in viral cultures in the presence of various effector cells, including control cells (mock), CD4-disrupted cells, cells containing anti-CD4 CAR, and CD4-disrupted cells containing anti-CD4 CAR. Both anti-CD4 CRISPR-treated effector cells and anti-CD4 CAR-expressing cells reduce HIV proteins, but the combination of these treatments reduces HIV more effectively than either method alone.

[0112] [Figure 17] Flow cytometry data is shown for HIV-infected peripheral blood mononuclear cells (PBMCs) as target cells mixed with control cells (mock) or CD4-depleted cells. Upon CD4 depletion, a 65% decrease in the percentage of HIV+ (p24+) cells was observed (compare the two panels on the bottom right, showing a decrease from 5.12% to 1.78% infected cells).

[0113] [Figure 18] Flow cytometry data are shown when HIV-infected PBMCs were mixed with GFP+ control cells (mock) or CD4-depleted GFP+ cells, showing that upon CD4 depletion, more GFP-positive cells survive.

[0114] [Figure 19] Flow cytometry data for control T cells (mock), AML target cells (MOLM13 cells) containing the mCherry marker, and T cells containing the anti-CD4 CAR are shown. Measurements were performed by flow cytometry and side scatter (Y-axis) was plotted against Texas cherry red (X-axis). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0115] Some embodiments provided herein relate to methods and compositions for disrupting at least one CD4 gene in a cell, such as a CD4+ T cell. In some such embodiments, the disruption includes the use of a CRISPR guide polynucleotide. Some embodiments include the generation of a cell with at least one disrupted CD4 gene and a chimeric antigen receptor (CAR) and its use in medicine. Some embodiments of the methods and compositions provided herein relate to a cell, such as a T cell, with a CAR (such as an anti-CD4 CAR or an anti-CD19 CAR), and further to the use of the cell with the CAR in medicine, for treating disorders such as, for example, HIV, acute myeloid leukemia (AML), CD4+ acute lymphoblastic leukemia (ALL). In some embodiments, disrupting at least one CD4 gene in a cell, such as a CD4+ T cell, results in a cell with enhanced resistance to HIV infection compared to a cell in which at least one CD4 gene is not disrupted. Some embodiments include the use of an anti-CD19 CAR to treat, inhibit or alleviate disorders such as acute lymphoblastic leukemia (ALL) and / or HIV. In some such embodiments, expression of CD4 may be problematic when using allogeneic T cells. As used herein, "disrupted" includes knocking out the function of a gene such that the gene product is not thereafter expressed and / or such that the gene product produced from the disrupted gene has no activity.

[0116] Some embodiments provided herein include highly efficient disruption of the CD4 gene in cells, such as T cells, by using specific CRISPR guide polynucleotides. In some embodiments, the genetically modified T cells with disrupted CD4 gene may further include an anti-CD4 CAR. The genetically modified cells can be directed to other cells expressing CD4. Some embodiments include efficient elimination of CD4+ cell populations by such genetically modified cells. Some embodiments include reducing HIV infection in HIV-infected cell populations and / or increasing cell viability in HIV-infected cell populations. Some embodiments include targeting and killing AML cells. Some embodiments include targeting and killing ALL cells.

[0117] The function of CD4+ T cells is important as an aspect of cell-based therapies such as CD4+ CAR T cells. However, expression of CD4 on CAR T cells can be problematic, for example, 1) in HIV-infected patients where HIV can use CD4 to infect CAR T cells, 2) when attempting to produce an anti-CD4 CAR T cell product that is expected to kill CD4+ anti-CD4 CAR T cells, or 3) when attempting to produce a therapeutic cell product using non-autologous cells.

[0118] In previous reports, it was unknown whether CD4+ T cells genetically modified to not express CD4 would continue to function similarly to CD4+ T cells. CD4 is a co-receptor of the T cell receptor (TCR) and interacts with antigen-presenting cells in cooperation with the TCR. Therefore, CD4+ T cells genetically modified to not express CD4 are expected to lose TCR-related functions. However, as described herein, CD4+ CAR T cells genetically modified to disrupt the CD4 gene were unexpectedly found to retain CD4+ functions, such as producing cytokines in the presence of cells expressing the target antigen and killing cells expressing the target antigen.

[0119] CD4+ CAR T cells are critical to the efficacy of current CAR T cell therapies. Combining CD4 destruction with the use of anti-CD4 CARs allows for the production of anti-CD4 CAR T cell preparations that contain CD4- / CD8- cells that behave similarly to CD4+ cells. Anti-CD4 CAR T cells may be beneficial in a variety of settings, for example in HIV infection as a way to target and eradicate the reservoir of CD4+ HIV-infected cells.

[0120] Typically, effective treatments using CAR T cells include the use of CD4+CAR T cells. On the other hand, anti-CD4 CAR T cells are expected to target and kill CD4+T cells. As shown herein, anti-CD4 CAR-containing CD4+T cells that retain CD4+T cell function even after genetic modification to not express CD4 may be useful in certain treatment methods. In some such embodiments, it may be useful to limit the effect of CAR T cells containing anti-CD4 CARs so that the treated subject can retain a CD4+T cell population after treatment. Thus, there is great clinical interest in the temporary elimination of CD4+T cells in a subject. For example, the temporary elimination of CD4+T cells in a subject may eliminate HIV-infected CD4 T cells in the subject, thereby facilitating the treatment, mitigation, or suppression of HIV. Additionally, the temporary elimination of CD4+T cells in a subject may be applicable to a wide range of treatments, including the treatment of autoimmune diseases in which autoreactive CD4 T cells may be involved or dominate, the suppression of cross-reactivity in transplant patients, and the treatment, suppression, or mitigation of CD4+ malignancies. The embodiments provided herein include a method for producing anti-CD4 CAR T cells. By depleting CD4, anti-CD4 CAR T cells can be produced that do not express CD4, which can protect them from targeted killing by anti-CD4 CAR T cells. Surprisingly, the embodiment of the method of the present invention for depleting the CD4 gene in T cells was found to be efficient. By transfecting cells with a ribonucleoprotein containing a CRISPR guide RNA targeting the CD4 gene just before transducing the anti-CD4 CAR, a substantially homogenous CD4-negative CAR T cell population was generated, including CD8+ CAR T cells and CD8- CAR T cells. Furthermore, the CD4-depleted CAR T cells were found to have similar functions to CD4+ CAR T cells, as measured by cytokine expression in a mixed target and effector cell assay.In some embodiments, after treatment in a subject, it may be desirable to suppress or reduce expression of the anti-CD4 CAR in the genetically modified cells so as not to eliminate all CD4+ cells in the subject's body. Some such embodiments may include regulating expression of the anti-CD4 CAR in the genetically modified cells to eliminate CD4+ cells for a short period of time, which may include, for example, expressing the CAR from an inducible promoter or expressing the CAR in a pharmacologically regulatable manner, or incorporating a suicide system into the genetically modified cells to avoid eliminating CD4+ cells for a long period of time.

[0121] In some embodiments, the CD4 gene-disrupted genetically engineered T cells can also contain CARs targeting human epitopes other than CD4 (such as CD19) or pathogen-derived epitopes (such as HIV Env). CAR T cells engineered not to express CD4 may be an attractive option for various clinical applications for several reasons. More specifically, CD4+ CAR T cells engineered not to express CD4 are no longer reactive to antigens recognized by the native T cell receptor, which may have the advantage of enhancing the potency of CAR-specific responses. In addition, when using allogeneic CAR T cells, CD4 disruption may minimize CD4-mediated GVHD. Furthermore, when considering the use of CAR T cells in HIV-infected individuals, CD4 disruption may provide efficient protection of CAR T cells from HIV infection.

[0122] Acute myeloid leukemia (AML) is a challenging malignancy with a high mortality rate. A limitation in the treatment of AML is the difficulty in identifying specific epitopes on AML that can be safely targeted without significantly ablating the hematopoietic system or causing off-target toxicity to other tissues. To date, efforts have been made to develop therapies, mainly focusing on epitopes such as CD33 and CD123, but it is unclear whether these therapies are safe or effective. A subset of AML cells expresses CD4. CD4 expression is particularly increased in AML with rearrangements of the KMT2A (MLL) gene at 11q23. Furthermore, the survival rate of AML with high CD4 expression is significantly lower than that of AML with normal to low CD4 expression.

[0123] As described herein, the killing ability of anti-CD4 CAR T cells against AML cells was tested in a killing assay. PBMCs were stimulated, transduced with anti-CD4 CAR constructs, and expanded for about 3 weeks. In the examples disclosed herein, anti-CD4 CAR T cells targeted and effectively killed AML cells.

[0124] Certain aspects of the methods and compositions provided herein are described in U.S. Pat. No. 10,273,280 and U.S. Patent Application Publication No. 2020 / 0000937, both of which are expressly incorporated by reference in their entireties.

[0125] Definition of Terms As used herein, "nucleic acid", "nucleic acid molecule" or "polynucleotide" refers to molecules such as, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), chemical synthesis, in vitro transcription, ligation, cleavage, endonuclease action or exonuclease action. Nucleic acid molecules may be composed of natural nucleotide monomers (such as DNA or RNA), or analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications at the sugar moiety and / or at the pyrimidine or purine base moiety. Modifications at the sugar moiety include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines or azide groups, and the sugar moiety may be etherified or esterified. Additionally, the entire sugar moiety may be replaced with conformationally or electronically similar structures, such as, for example, azasugars and / or carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or similar bonds. Linkages similar to phosphodiester bonds include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and phosphoroamidate bonds. "Nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded.

[0126] As used herein, the term "coding strand" has its common and ordinary meaning in the context of this specification, including, but not limited to, a DNA strand having the same base sequence as the RNA transcript produced, except that thymines are replaced by uracils. The coding strand contains codons and the non-coding strand contains anticodons.

[0127] As used herein, "encode" refers to the property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, mRNA, etc., serving as a template for the synthesis of another macromolecule, such as a given amino acid sequence. Thus, a particular gene encodes a protein when its corresponding mRNA is transcribed and translated to produce the protein in a cell or other biological system.

[0128] As used herein, the term "regulatory element" has its general and ordinary meaning in the context of the present specification, and includes, for example, a segment of a nucleic acid molecule capable of increasing or decreasing the expression of a particular gene in an organism, including, but not limited to, a segment of a nucleic acid molecule capable of affecting the transcription and / or translation of an operably linked transcribable DNA molecule. Regulatory elements, such as promoters (e.g., MND promoters), leaders, introns, transcription termination regions, and the like, are DNA molecules that have gene regulatory activity and play an essential role in the overall expression of genes in living cells. Regulatory elements include, but are not limited to, CAAT boxes, CCAAT boxes, Pribnow boxes, TATA boxes, SECIS elements, mRNA polyadenylation signals, A boxes, Z boxes, C boxes, E boxes, G boxes, hormone response elements such as insulin gene regulatory sequences, DNA binding regions, activation regions, and / or enhancer regions.

[0129] Some embodiments provided herein include a CRISPR (clustered regularly interspaced short palindromic repeats) / CAS system. As used herein, the term "CRISPR / CAS system" refers to a gene editing system in which expression of one or two guide RNAs complementary to the gene to be knocked out or expressed is required for the CRISPR / CAS system to function. Cas9 is an RNA-guided DNA endonuclease enzyme that can form a complex by associating with one or two guide RNAs and cleave a sequence complementary to the target portion of the guide RNA through this complex. In some embodiments, the CRISPR / CAS system includes a target gene (such as CD4), a guide RNA, and a Cas9 endonuclease or a derivative or fragment thereof as basic components. In some embodiments, one aspect of gene editing using CRISPR / Cas9 is a system that can efficiently deliver guide RNA to a wide variety of cells. In some embodiments, this delivery may include guide RNA as a nucleic acid produced in vitro, for example, guide RNA produced by in vitro transcription or chemical synthesis. In some embodiments, the nucleic acid encoding the guide RNA is rendered nuclease resistant by incorporation of modified bases, such as 2'O-methyl bases. In some embodiments, the guide RNA can be incorporated into a ribonucleoprotein (RNP) complex.

[0130] Some embodiments provided herein include a CRISPR / Cpf1 system. The CRISPR / Cpf1 system uses Cpf1, a single-stranded RNA-guided endonuclease of the class 2 CRISPR Cas9 system. Class 2 effectors rely on a single effector protein (such as Cas9), but Cpf1 is involved in robust DNA interference through a mechanism different from Cas9. Cpf1 is a CRISPR-associated DNA nuclease that utilizes two components to program RNA. In some embodiments of the compositions provided herein, the composition further includes two or more sequences obtained from a viral genome. In some embodiments, the two or more sequences obtained from a viral genome encode a promoter, a protein, a gene cassette for expressing a foreign gene, a coagulation factor, an antibody or a portion thereof (e.g., a binding moiety or scFv), a CAS9 enzyme, a Cas9-VP64 enzyme, a nuclease, a protein with a defective function in a particular disease, a recombination template for modifying a genomic site by homologous recombination repair, or a cell surface protein. In some embodiments, the nuclease includes Cpf1. Further examples of CRISPR systems for use in the embodiments provided herein are disclosed in U.S. Patent Publication Nos. 2019 / 0071717, 2019 / 0071657, and 2019 / 0062735, all of which are incorporated by reference in their entireties.

[0131] In some embodiments, the use of chemically modified CRISPR guide polynucleotides (such as RNA) is envisioned. Chemically modified guide RNAs have been used to perform CRISPR-Cas genome editing in human primary cells (Hendel, A. et al., Nat Biotechnol. 2015 Sep; 33(9):985-9). The chemical modification of the guide RNA may include modifications that confer nuclease resistance. The nuclease may be an endonuclease, an exonuclease, or both. Some examples of chemical modifications include, but are not limited to, 2'-fluoro, 2'O-methyl, phosphorothioate dithiol 3'-3' end linkages, 2-amino-dA, 5-methyl-dC, C-5 propynyl-C, C-5 propynyl-U, and morpholino. The present invention is not limited to these examples, and other chemical modifications, alternatives and variations of these exemplary chemical modifications are also included in the present invention.

[0132] As used herein, "chimeric antigen receptor (CAR)" refers to a genetically engineered protein receptor that can confer specificity to effector immune cells, such as T cells. The use of CAR-expressing T cells can promote proliferation and activation in vivo, but the effects of CAR-expressing T cells are not limited thereto. CARs can also be designed to give T cells the tropism to target cells expressing a specific cell surface antigen, and T cells with such target tropism can activate lymphocytes (such as T cells) by recognizing the target. The specificity of a monoclonal antibody or its binding fragment or scFv can be transferred to T cells by transferring the coding sequence of the CAR using a vector. When CARs are used to treat a subject in need of treatment, a technique called adoptive cell transfer is used. In this technique, T cells are taken from the subject to be treated, and the resulting T cells are genetically engineered to express a CAR specific for the antigen. The T cells, now capable of recognizing and targeting the antigen due to the expression of the CAR, are then reintroduced into the patient. In some embodiments, lymphocytes expressing CARs are described that can be delivered to a subject for targeting of specific cells. In some embodiments, CAR-expressing lymphocytes can exhibit bispecificity by expressing two CARs. In some embodiments, CAR-expressing lymphocytes can exhibit bispecificity by expressing a CAR and a specific T cell receptor (TcR). TcRs are molecules present on the surface of T lymphocytes, or T cells, and can recognize antigens. In some embodiments, CAR-expressing lymphocytes can exhibit bispecificity by expressing a bispecific CAR. The structure of a CAR may comprise a fusion protein of a single chain variable fragment (scFv) from a monoclonal antibody linked to a transmembrane domain and an intracellular signaling domain. CARs often comprise an extracellular scFv linked to an intracellular CD3 zeta domain (first generation CARs).In addition, a costimulatory domain (e.g., CD28 and / or 4-1BB) can be further linked to the scFv, thereby enhancing the therapeutic efficacy of the CAR in a subject in need of treatment (second generation CAR). T cells expressing a CAR molecule can recognize and kill target cells expressing the specific antigen targeted by the CAR.

[0133] In some embodiments, the promoter used in the present invention may be an inducible promoter or a constitutive promoter. Inducible promoters include, but are not limited to, tamoxifen-inducible promoters, tetracycline-inducible promoters, and doxycycline-inducible promoters (e.g., tre promoters). Constitutive promoters include, for example, SV40, CMV, UBC, EF1α, PGK, and CAGG. In some embodiments, the promoter is a tamoxifen-inducible promoter, a tetracycline-inducible promoter, or a doxycycline-inducible promoter (e.g., tre promoter). In some of the embodiments provided herein, the expression of the protein is induced by tamoxifen and / or its metabolites. Metabolites of tamoxifen include active metabolites such as 4-hydroxytamoxifen (afimoxifen) and N-desmethyl-4-hydroxytamoxifen (endoxifen), which can exert 30 to 100 times the affinity of tamoxifen for the estrogen receptor. In some embodiments, the metabolite of tamoxifen is 4-hydroxytamoxifen (afimoxifen) and / or N-desmethyl-4-hydroxytamoxifen (endoxifen). In some embodiments, an inducible promoter is designed and / or engineered to have low basal activity, high levels of expression, and / or the ability to be switched on and off for short periods of time. In another embodiment, the CAR can be engineered to allow expression to be regulated by using pharmacological agents independent of promoter function, including but not limited to the systems described herein using rapamycin, rapamycin analogs, or HCV NS3 protease inhibitors.

[0134] As used herein, "suicide gene therapy", "suicide gene" and "suicide gene system" may refer to a method of destroying cells by apoptosis, which requires a suicide gene that causes cells to undergo apoptosis and commit suicide. By utilizing suicide gene therapy, the safety of genetically modified immune cells can be improved and adverse events that may occur due to the injection of genetically modified immune cells can be managed. There are several known methods for carrying out suicide gene therapy. In some embodiments, genetically modified cells can be further modified with a suicide gene ex vivo. The suicide gene may be, but is not limited to, a gene encoding a factor that can convert a non-toxic prodrug into a toxic compound at the cellular level. Such a prodrug can be administered to a subject, and the prodrug can be used to selectively eliminate genetically modified cells modified with a suicide gene. A suicide system using the herpes simplex thymidine kinase (Hsv-tk) / ganciclovir (GCV) suicide system has also been reported (Casucci et al. 2011, Journal of Cancer 2011, 2; which is expressly incorporated herein in its entirety by reference). In some embodiments, the suicide gene system is the herpes simplex virus thymidine kinase (HSVTK) / ganciclovir (GCV) suicide gene system or the inducible caspase suicide gene system.

[0135] As used herein, "subject" or "patient" refers to any organism to which the embodiments described herein may be used or administered, for example, for experimental, diagnostic, prophylactic and / or therapeutic purposes. Subjects or patients include, for example, animals. In some embodiments, the subject is a mouse, a rat, a rabbit, a non-human primate, and a human. In some embodiments, the subject is a cow, a sheep, a pig, a horse, a dog, a cat, a primate, or a human.

[0136] As used herein, the terms "treatment" and "treating" may refer, depending on the context, to both therapeutic and prophylactic treatments, which aim to prevent or alleviate (alleviate) a targeted pathological condition, to prevent a pathological condition, to pursue or obtain a beneficial outcome, or to reduce the likelihood that an individual will develop a pathological condition even if the treatment ultimately fails. Subjects in need of treatment or therapy include those already suffering from a pathological condition, those prone to developing a pathological condition, or those in whom a pathological condition needs to be prevented.

[0137] Specific Polynucleotides Some embodiments of the methods and compositions provided herein include disrupting the CD4 gene in a cell. For example, the CD4 gene can be disrupted by introducing an insertion, deletion, or mutation into the CD4 gene. In some embodiments of the methods and compositions provided herein, one or both alleles of the CD4 gene can be disrupted in a cell. For example, one or both alleles of the CD4 gene can be disrupted in the diploid genome of a cell by using the methods and compositions provided herein. In some embodiments, disrupting the CD4 gene can suppress the expression of a functional CD4 protein in a cell. For example, disrupting the CD4 gene can prevent the expression of the CD4 protein, can reduce the expression of the CD4 protein, or can express a non-functional CD4 protein variant or fragment thereof. In some embodiments, a non-functional CD4 protein variant may not bind to a ligand or receptor that specifically binds to a functional CD4 protein, or may not efficiently bind to a ligand or receptor that specifically binds to a functional CD4 protein. In some embodiments, the non-functional CD4 protein variant or fragment thereof may not bind to the ligand binding domain of the chimeric antigen receptor or may not bind efficiently to the ligand binding domain of the chimeric antigen receptor. In some embodiments, disrupting the CD4 gene may include using an isolated nucleic acid to disrupt at least one CD4 gene in a cell. Some of the isolated nucleic acids include a CRISPR (clustered regularly interspaced short palindromic repeats) guide polynucleotide that can hybridize to the CD4 gene or is configured to hybridize to the CD4 gene. In some embodiments, the guide polynucleotide comprises RNA.

[0138] In some embodiments, the guide polynucleotide is capable of hybridizing to the sense strand of the CD4 gene or is configured to hybridize to the sense strand of the CD4 gene. In some embodiments, the guide polynucleotide is capable of hybridizing to the antisense strand of the CD4 gene or is configured to hybridize to the antisense strand of the CD4 gene. In some embodiments, the guide polynucleotide is capable of hybridizing to a portion of the CD4 gene or is configured to hybridize to a portion of the CD4 gene, thereby disrupting the expression of the CD4 gene and disrupting regulatory elements of the CD4 gene, such as promoters, exons, splice sites, introns, etc. In some embodiments, the guide polynucleotide is capable of hybridizing to an exon of the CD4 gene or is configured to hybridize to an exon of the CD4 gene, including the first exon, the second exon, the third exon, the fourth exon, the fifth exon, the sixth exon, the seventh exon, the eighth exon, or the ninth exon of the CD4 gene. In some embodiments, the guide polynucleotide is capable of hybridizing to an exon of the CD4 gene or is configured to hybridize to the first exon of the CD4 gene. In some embodiments, the guide polynucleotide is capable of hybridizing to an exon of the CD4 gene or is configured to hybridize to the third exon of the CD4 gene.

[0139] In some embodiments, the guide polynucleotide may comprise a nucleotide sequence having at least a specific identity (%) to the nucleotide sequence set forth in any one of SEQ ID NOs: 1-8 or its complementary strand. In some embodiments, the guide polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-8 or its complementary strand, or a sequence identity within a range of any two of the above percentages. In some embodiments, the guide polynucleotide comprises a nucleotide sequence having at least a specific identity (%) to the nucleotide sequence set forth in SEQ ID NO: 6 or its complementary strand. In some embodiments, the guide polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6 or its complementary strand, or a range of sequence identity between any two of the percentages. In some embodiments, the guide polynucleotide may comprise a nucleotide sequence set forth in any of SEQ ID NOs: 1-8 or its complementary strand. In some embodiments, the guide polynucleotide may comprise a nucleotide sequence set forth in SEQ ID NO: 6 or its complementary strand.

[0140] The specific method for producing the cells Some of the embodiments of the methods and compositions provided herein include the method of producing genetically modified cells.Some of these embodiments include the use of gene targeting systems to specifically modify the CD4 locus in cells.Some embodiments include the use of CRISPR (clustered regularly interspaced short palindromic repeats) systems, such as the CRISPR / Cas9 system, with guide polynucleotides.

[0141] In some embodiments, the genetically modified cell can be generated by introducing a CRISPR guide polynucleotide and a Cas9 protein into a target cell. In some embodiments, one or more vectors encoding a CRISPR guide polynucleotide and / or one or more vectors encoding a Cas9 protein can be introduced into the target cell. In some embodiments, the CRISPR guide polynucleotide and / or the Cas9 protein can be directly introduced into the target cell. For example, a ribonucleoprotein (RNP) containing a CRISPR guide polynucleotide and / or a Cas9 protein can be prepared and introduced into the target cell. Methods for introducing the vector or RNP into the cell can include lipofection or electroporation.

[0142] In some embodiments, the guide polynucleotide may comprise a guide polynucleotide disclosed herein. In some embodiments, the guide polynucleotide may comprise a nucleotide sequence having at least a certain identity (%) with the nucleotide sequence shown in any of SEQ ID NOs: 1-8 or its complementary strand. In some embodiments, the identity with the nucleotide sequence shown in any of SEQ ID NOs: 1-8 or its complementary strand may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a percentage between any two of these percentages. In some embodiments, the guide polynucleotide may comprise a nucleotide sequence having at least a certain identity (%) with the nucleotide sequence shown in SEQ ID NO: 6 or its complementary strand. In some embodiments, the identity with the nucleotide sequence shown in SEQ ID NO: 6 or its complementary strand may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a percentage between any two of these percentages. In some embodiments, the guide polynucleotide may comprise a nucleotide sequence as set forth in any one of SEQ ID NOs: 1 to 8, or a complementary strand thereof. In some embodiments, the guide polynucleotide may comprise a nucleotide sequence as set forth in SEQ ID NO: 6, or a complementary strand thereof.

[0143] Some of the embodiments of the methods and compositions provided herein include a method of producing a genetically modified cell comprising a CAR. Some of such embodiments may include a step of introducing a polynucleotide encoding a CAR into a cell. In some embodiments, the CAR comprises an antigen binding domain that specifically binds to CD4 protein or CD19 protein. Examples of anti-CD4 CARs or anti-CD19 CARs are disclosed in U.S. Patent Publication No. 9,951,118, U.S. Patent Publication No. 2018 / 0371052, U.S. Patent Publication No. 2017 / 0107286, and U.S. Patent Publication No. 2019 / 0062430 (all of which are expressly incorporated herein by reference in their entirety).

[0144] In some embodiments, the polynucleotide encoding the CAR comprises an inducible promoter such that expression of the CAR can be induced in a cell or pharmacologically regulated in a cell.

[0145] Some embodiments of the methods and compositions provided herein include methods of making a genetically modified cell, further comprising introducing into the cell a polynucleotide encoding a suicide gene system, in some embodiments, the suicide gene system is the herpes simplex virus thymidine kinase (HSVTK) / ganciclovir (GCV) suicide gene system or an inducible caspase suicide gene system.

[0146] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cell that expressed CD4 protein prior to disruption of the CD4 gene. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a cell isolated from peripheral blood mononuclear cells. In some embodiments, the cell is a CD3 expressing cell. In some embodiments, the cell is a CD8 expressing cell. In some embodiments, the cell is an in vivo cell. In some embodiments, the cell is an ex vivo cell.

[0147] Some of the embodiments of the methods and compositions provided herein include cells produced by the methods disclosed herein.

[0148] Specific cells Some of the embodiments of the methods and compositions provided herein include genetically modified cells that can be used in medicine. In some embodiments, the genetically modified cells include at least one disrupted CD4 gene. In some of such embodiments, the CD4 gene is disrupted by the CRISPR guide polynucleotides provided herein.

[0149] In some embodiments, the at least one CD4 gene is disrupted in the first exon. In some embodiments, the at least one CD4 gene is disrupted in the third exon. In some embodiments, the at least one CD4 gene is disrupted by a CRISPR guide polynucleotide comprising a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8 or its complementary strand. In some embodiments, the at least one CD4 gene is disrupted by a CRISPR guide polynucleotide comprising a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 6 or its complementary strand. In some embodiments, the at least one CD4 gene is disrupted by a CRISPR guide polynucleotide comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8 or its complementary strand. In some embodiments, the at least one CD4 gene is disrupted by a CRISPR guide polynucleotide comprising a nucleotide sequence shown in SEQ ID NO: 6 or its complementary strand.

[0150] Some of the embodiments of the methods and compositions provided herein include recombinant cells comprising a CAR, which can be used in medicine. In some embodiments, the CAR comprises an antigen-binding domain that specifically binds to CD4 protein. Examples of anti-CD4 CARs are disclosed in US Patent Publication No. 2018 / 0371052, which is expressly incorporated herein by reference in its entirety. In some embodiments, the polynucleotide encoding the CAR comprises an inducible promoter so that expression of the CAR can be induced in the cell or pharmacologically regulated in the cell.

[0151] In some embodiments, the CAR comprises an anti-CD19 CAR. In some embodiments, the CAR comprises an anti-HIV CAR. In some of such embodiments, the anti-HIV CAR may comprise an scFv derived from an anti-HIV neutralizing antibody. Examples of such antibodies include PGT128, PG9 and PGT145. Examples of methods and compositions that may be included in the embodiments provided herein are disclosed in U.S. Patent Publication No. 2017 / 0044240, which is expressly incorporated herein by reference in its entirety. Further examples of CARs that may be included in the embodiments provided herein are disclosed in U.S. Patent Publication No. 2018 / 0009891, U.S. Patent Publication No. 2017 / 0015746, U.S. Patent Publication No. 9,951,118, U.S. Patent Publication No. 2018 / 0371052, U.S. Patent Publication No. 2017 / 0107286, U.S. Patent Publication No. 2019 / 0062430, and WO2017 / 027291, which are expressly incorporated by reference in their entireties.

[0152] Some embodiments of the methods and compositions provided herein include genetically engineered cells that contain a suicide gene system. In some embodiments, the suicide gene system is the herpes simplex virus thymidine kinase (HSVTK) / ganciclovir (GCV) suicide gene system or the inducible caspase suicide gene system.

[0153] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cell that expressed CD4 protein prior to disruption of the CD4 gene. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a cell isolated from peripheral blood mononuclear cells. In some embodiments, the cell is a CD3 expressing cell. In some embodiments, the cell is a CD8 expressing cell. In some embodiments, the cell is a cell derived from a CD4+ cell, a natural killer cell, or a natural killer T cell. In some embodiments, the cell is an in vivo cell. In some embodiments, the cell is an ex vivo cell.

[0154] Some embodiments of the methods and compositions provided herein include pharmaceutical compositions comprising the genetically modified cells disclosed herein and a pharma- ceutically acceptable excipient.

[0155] Specific methods for killing or inhibiting cells Some embodiments of the methods and compositions provided herein include a method of killing or suppressing a CD4+ cell population comprising contacting a CD4+ cell population with a genetically modified cell disclosed herein. In some such embodiments, the CD4+ cell population comprises a virus. In some embodiments, the virus is HIV. In some embodiments, the CD4+ cell population and the genetically modified cell are from the same subject. In some embodiments, the CD4+ cell population and the genetically modified cell are from different subjects. In some embodiments, the population of CD4+ cells and / or the genetically modified cells is an ex vivo cell population. In some embodiments, the population of CD4+ cells and / or the genetically modified cells is an in vivo cell population.

[0156] Some embodiments of the methods and compositions provided herein include methods of reducing, inhibiting, or ameliorating HIV replication in a population of CD4+ cells infected with HIV. Some such embodiments include contacting a population of CD4+ cells with a genetically modified cell disclosed herein. In some embodiments, the population of CD4+ cells and the genetically modified cells are from the same subject. In some embodiments, the population of CD4+ cells and / or the genetically modified cells are from different subjects. In some embodiments, the population of CD4+ cells and / or the genetically modified cells is an ex vivo cell population. In some embodiments, the population of CD4+ cells and / or the genetically modified cells is an in vivo cell population. In some embodiments, the amount of HIV replication in a population of HIV-infected CD4+ cells contacted with the genetically modified cells is reduced by at least 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 75%, 90%, 99%, 100%, or a percentage within a range including any two of these percentages, as compared to the amount of HIV replication in a population of HIV-infected CD4+ cells not contacted with the genetically modified cells.

[0157] Some embodiments of the methods and compositions provided herein include methods of improving the viability of a population of CD4+ cells infected with HIV. Some such embodiments include contacting a population of CD4+ cells with a genetically modified cell disclosed herein. In some embodiments, the population of CD4+ cells and the genetically modified cell are from the same subject. In some embodiments, the population of CD4+ cells and the genetically modified cell are from different subjects. In some embodiments, the population of CD4+ cells and / or the genetically modified cell is an ex vivo cell population. In some embodiments, the population of CD4+ cells and / or the genetically modified cell is an in vivo cell population. In some embodiments, the viability of a population of HIV-infected CD4+ cells contacted with a genetically modified cell is improved by at least 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 75%, 90%, 99%, 100%, or a percentage between any two of these percentages, compared to the viability of a population of HIV-infected CD4+ cells not contacted with the genetically modified cell.

[0158] Some embodiments of the methods and compositions provided herein include methods of treating, suppressing or ameliorating HIV infection in a subject having HIV infection. Some such embodiments include administering to a subject in need of treatment, suppression or amelioration of HIV infection a cell population comprising the genetically modified cells disclosed herein. In some embodiments, the genetically modified cells are autologous cells obtained from the subject. In some embodiments, the genetically modified cells are not autologous cells obtained from the subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a subject selected to receive therapy for HIV infection. In some embodiments, the selection of the subject is performed by clinical and / or diagnostic evaluation. Such diagnostic or clinical evaluation may be performed by assessing the presence and / or amount of HIV infection in the subject, such as by analyzing the presence or absence of HIV virus, HIV protein or HIV nucleic acid in the subject.

[0159] In some embodiments, the genetically modified cell comprises a CAR. In some embodiments, the CAR comprises an anti-CD4 CAR and / or an anti-CD19 CAR.

[0160] Some embodiments further comprise inducing the expression of the CAR in the cell. For example, the polynucleotide encoding the CAR may comprise an inducible promoter, or may be engineered to allow regulation of the inducible promoter with a pharmacological agent. In some such embodiments, the expression of the CAR in the cell comprising the polynucleotide encoding the CAR can be induced or suppressed by administering an inducer to a subject.

[0161] Some embodiments further include suppressing or reducing the expression of the CAR in the cell. In some such embodiments, administration of an inducer for inducing the expression of the CAR in the cell comprising the polynucleotide encoding the CAR can be stopped or its dosage can be reduced. In some embodiments, an inhibitor for reducing the expression of the CAR in the cell can be administered to the subject. In some embodiments, after administering the cell population to the subject, the expression of the CAR is suppressed or reduced. In some embodiments, the expression of the CAR is suppressed or reduced for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or a period between any two of these periods, after administering the cell population to the subject. In some embodiments, the expression of the CAR is suppressed or reduced for at least 1 week, 2 weeks, 3 weeks, or 4 weeks, or a period between any two of these periods, after administering the cell population to the subject.

[0162] Some embodiments further include a method of treatment with additional therapy. For example, HIV management may include the use of antiretroviral drugs to control HIV infection. The types of drugs used in the treatment, treatment or management of HIV include, but are not limited to, entry or fusion inhibitors (e.g., maraviroc or enfuvirtide), nucleoside reverse transcriptase inhibitors (e.g., zidovudine, abacavir, lamivudine, emtricitabine or tenofovir), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine, efavirenz, etravirine or rilpivirine), integrase inhibitors (e.g., elvitegravir or dolutegravir) and / or protease inhibitors (e.g., lopinavir, indinavir, nelfinavir, amprenavir, ritonavir, darunavir or atazanavir). Combination therapy using any of these classes of drugs in combination with one or more of the CAR-containing T cells described herein can also be used in the treatment and management of HIV.

[0163] Specific Kit Some of the embodiments of the methods and compositions provided herein include kits for generating genetically modified cells. Some of such kits may include an isolated polynucleotide for disruption of the CD4 gene in a cell. In some embodiments, the isolated polynucleotide includes a CRISPR guide polynucleotide comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence set forth in any of SEQ ID NOs: 1-8 or its complementary strand. In some embodiments, the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6 or its complementary strand. In some embodiments, the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1-8 or its complementary strand. In some embodiments, the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO:6 or its complement. Some embodiments may further comprise a polynucleotide encoding a Cas9 protein. Some embodiments may further comprise a Cas9 protein. EXAMPLES

[0164] Example 1 - Destruction of CD4 in T cells using CRISPR-Cas9 This example relates to disrupting the CD4 gene in cells using Cas9 and CRISPR guide polynucleotides. Six CRISPR guides were designed to target the CD4 gene. Figure 1 shows the location that each CRISPR guide targets, and Table 1 shows the sequence of each CRISPR guide. [Table 1]

[0165] Each guide was used to disrupt the CD4 gene in A3.01 cells (a human T cell line) that highly express CD4. Each guide was introduced into the cells by electroporation as a ribonucleoprotein (RNP). The relative efficacy of each guide was assessed by measuring its ability to reduce the expression of CD4 on the cell surface by flow cytometry. Data were acquired 7 days after transfection of Cas9 and guide. As controls, non-modified cells not stained for CD4 (no treatment with CRISPR / Cas9), non-modified cells stained for CD4 (no treatment with CRISPR / Cas9), or cells treated with Cas9 alone without a guide and stained for CD4 were used (Figure 2A, top row). In the absence of CRISPR / Cas9, approximately 96% of the cells were CD4+. In the presence of various CRISPR / Cas9 guides targeting CD4, CD4 expression was reduced from approximately 94 to 64% in cells treated with the various guides (Figure 2A, middle and bottom rows). Based on theory, it was unclear which guide would perform best. Surprisingly, some of the guides predicted to disrupt CD4 expression were unable to do so efficiently. In fact, both guide 3 and guide 6 were significantly more active in disrupting the CD4 gene than the other guides. Guide 6 performed best, reducing CD4 expression by 64%.

[0166] Guide 3 and Guide 6 were tested for their ability to deplete CD4 on primary lymphocytes. The relative efficacy of the two guides was assessed by measuring their ability to reduce the expression of CD4 on the cell surface by flow cytometry.

[0167] In addition, two other guides were tested. Figure 2B shows flow cytometry data plotting CD4 on the Y axis against CD4+ cell lines stained for CD8 on the X axis (bottom). The figure shows untreated cells (mock) or cells treated with Cas9 and CRISPR guide polynucleotides (G6, G7, or G8). G6 performed better than G7 and G8. The percentage of CD4+ cells was lowest in G6-treated cells compared to G7-treated and G8-treated cells. Also, the percentage of CD4-CD8- double negative cells was higher in G6-treated cells compared to G7-treated and G8-treated cells.

[0168] CD3+ cells were isolated from primary human peripheral blood mononuclear cells (PBMCs). Guides were introduced as RNPs using electroporation. Data were obtained 11 days after transfection of Cas9 and guides, and cells were stained for CD4 and CD8. Controls included cells that were neither transfected nor stained; mock-transfected cells stained for CD4 and CD8; cells transfected with Cas9 without guides and stained for CD4 and CD8; or cells transfected with guide 6 without Cas9 and stained for CD4 and CD8 (Figure 3). Test cells were treated with guide 3 or guide 6 and CRISPR / Cas9 (Figure 3). The percentage of CD4+ cells was about 15.6% in mock-transfected cells, compared to about 0.99% in guide 6-transfected cells. Thus, the use of guide 6 reduced cell surface CD4 expression by about 94%. Additionally, the percentage of CD4+ cells in mock-transfected cells was approximately 15.6%, whereas that in cells transfected with Guide 3 was 15.0%. Thus, the use of Guide 3 reduced cell surface CD4 expression by approximately 4%. Guide 6 performed better than Guide 3 in primary cells.

[0169] To further confirm the disruption of CD4 at the gene level, we performed colony sequencing of CD4 in mock-transfected (Figure 4A) or guide6-transfected primary cells (Figure 4B). We sequenced the CD4 gene region from 17 untreated control clones and 20 clones treated with RNP containing G6 and Cas9. The consensus sequence is shown at the bottom of the top and bottom panels of Figure 4A and Figure 4B, and the location of the 20-base pair guide6 CRISPR site is shown below the consensus sequence. All 20 clones treated with RNP containing G6 and Cas9 had insertions, deletions, or mutations in the guide6 CRISPR site.

[0170] Example 2 - Generation of anti-CD4 CAR T cells and their activity This example concerns the activity of primary CD3+ T cells containing a disrupted CD4 gene and an anti-CD4 CAR to eliminate CD4+ target cell populations. Primary CD3+ T cells were isolated, transfected with an anti-CD4 CRISPR RNP (guide6) and transduced with an anti-CD4 chimeric antigen receptor (CAR) containing a blue fluorescent protein (BFP) reporter. This anti-CD4 CAR contained an scFv having the amino acid sequence shown in SEQ ID NO: 9 (DIVMTQSPSSLAVSVGEKVTMICKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYRTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLQESGPELVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAREKDNYATGAWFAYWGQGTTVTVSS).

[0171] The cells were expanded in vitro for one week and CD4 and CD8 staining and CAR (BFP) expression were assessed by flow cytometry.

[0172] As shown in Figure 5B, transduction of anti-CD4 CAR resulted in efficient depletion of CD4 positive cells (the percentage of CD4+ cells decreased from 27.1% to 0.24%). Also, as shown in Figure 5D, transfection of anti-CD4 CRISPR / Cas9 RNP resulted in depletion of CD4 positive cells (the percentage of CD4+ cells decreased from 27.1% to 8.17%). Furthermore, as shown in Figure 5E, the combination of transfection of anti-CD4 CRISPR / Cas9 RNP and transduction of anti-CD4 CAR resulted in significant depletion of CD4+ cells (decreased from 27.1% to 0.01%) and a double-negative cell population that was both CD4 and CD8 negative (increased from 0.63% to 20.7%). Thus, compared to controls, non-CD4-disrupted anti-CD4 CAR T cells were able to eliminate almost all CD4+ cells, CD4 gene-disrupted cells without anti-CD4 CARs were used, and CD4 cell numbers were significantly reduced. On the other hand, when anti-CD4 CRISPR-treated and anti-CD4 CAR-transduced cells were used, a significant elimination of CD4+ T cells was observed, with a clear emergence of a double-negative (CD4-negative and CD8-negative) CAR(BFP)+ cell population.

[0173] Cells were treated with anti-CD4 CRISPR / Cas9 RNP (guide 6) and transduced with anti-CD4 CAR containing blue fluorescent protein (BFP). The expression of BFP in these cells was evaluated by flow cytometry. As shown in Figure 6B and Figure 6E, the expression of BFP was essentially restricted to only cells transduced with CAR containing BFP. Also, as shown in Figure 7A and Figure 7B, CAR (BFP+) live cells were gated and then stained for CD4 (Y axis) and CD8 (X axis). Because the expression of CAR was not very high (17.8%), back-gating on BFP+ cells further demonstrated that CD4+ cells were depleted (0.029%) and a double-negative cell population (27.7%) appeared, especially in anti-CD4 CRISPR-treated anti-CD4 CAR T cells.

[0174] Example 3 - Functional analysis of CD4-disrupted CAR T cells This example concerns the activity of primary CD3+ T cells containing a disrupted CD4 gene and an anti-CD19 CAR, as measured by intracellular cytokine expression. It is predicted that disruption of CD4 may result in loss of function of CD4+ lymphocytes. With this in mind, we investigated the function of CAR T cells genetically engineered to not express CD4.

[0175] To measure CAR T cell function, expression of the appropriate cytokines (TNF-α, IL-2, and IFN-γ) in CAR T cells was assessed upon contact with cells expressing the target antigen. Non-CD4-depleted and CD4-depleted anti-CD19 CAR T cells were generated and mixed with target cells (K562 cells or K562 cells genetically modified to express CD19) at a 2:1 effector to target cell ratio. BFP was expressed from the anti-CD19 CAR construct. After 6 hours, intracellular cytokine expression in each cell was assessed by flow cytometry. Figure 8 shows flow cytometry data when non-CD4-depleted or CD4-depleted anti-CD19 CAR T cells were mixed with CD19-expressing or non-CD19-expressing target cells. Each cell was assessed for its lymphocyte morphology (FIG. 8, top panel) and for viable / dead cell staining (FIG. 8, bottom panel).

[0176] Cytokine (TNF-α, IL-2, and IFN-γ) expression was evaluated in BFP+CAR T cells. Anti-CD19 CAR T cells expressed cytokines only in the presence of target cells expressing CD19 (Figure 9). Negative controls did not produce significant amounts of cytokines. Wild-type and CD4-depleted CAR T cells produced similar amounts of cytokines. Thus, CAR T cells had similar functions, as assessed by comparing the percentage of cells expressing cytokines, regardless of whether CD4 was depleted. Unexpectedly, for TNF-α and IL-2, the percentage of cells expressing cytokines was higher in CD4-depleted CAR T cells, suggesting that CD4 depletion in CAR T cells may have a unique advantage. This was theoretically unexpected.

[0177] By back-gating the cytokine-producing CAR T cells, it was found that even among the CD4-depleted CAR T cells, double-negative cells (CD4-negative and CD8-negative) were producing cytokines. Therefore, the phenotype of the TNFα-producing CAR T cells was examined. As shown in FIG. 10, when CD4 was not destroyed, the majority of the TNFα-producing cells were CD4+. After CD4 destruction, the majority of the TNFα-producing cells were CD4-negative and CD8-negative double-negative cells. The phenotype of the IL-2-producing CAR T cells was also examined. As shown in FIG. 11, when CD4 was not destroyed, the majority of the IL-2-producing cells were CD4+, but after CD4 destruction, the majority of the IL-2-producing cells were CD4-negative and CD8-negative double-negative cells. Furthermore, the phenotype of the IFN-γ-producing CAR T cells was also examined. As shown in FIG. 12, when CD4 was not destroyed, the majority of the IFN-γ-producing cells were CD4+, but after CD4 destruction, the majority of the IFNγ-producing cells were CD4-negative and CD8-negative double-negative cells.

[0178] Cytokine production was compared between CD4-positive and CD4-negative anti-CD19 CAR T cells in CD4-depleted single cell samples. As shown in Figure 13, CD4 could not be depleted with 100% efficiency, resulting in a mixture of CD4+ and CD4-CAR T cells. CD4+ CAR T cells that were not CD4-depleted produced similar amounts of cytokines as CD4-depleted CAR T cells.

[0179] The cell morphology of anti-CD4 CAR T cells when mixed with a GFP+CD4+ cell line (A3.01 cells) was compared between CD4-depleted and non-CD4-depleted cells as assessed by flow cytometry. Mock T cells, CD4-depleted CAR T cells, or non-CD4-depleted CAR T cells were mixed with GFP-expressing A3.01 cells at a 2:1 ratio and incubated for 22 hours. As shown in Figure 14, CD4-depleted CAR T cells were mixed with a CD4+ cell line (A3.01) and forward and side scatter measurements showed that they had similar cell morphology.

[0180] The activity of CD4-disrupted CAR T cells was evaluated in terms of their ability to kill target cells. As shown in Figure 15, CD4-disrupted and non-CD4-disrupted anti-CD4 CAR T cells were equally effective in eliminating GFP+CD4+ cell lines, as shown by the reduction in GFP / FITC+ cells in the two bottom right panels compared to the bottom left panels. Thus, CD4-disrupted CAR T cells were functional and killed target cells as efficiently as non-CD4-disrupted CD4+ CAR T cells.

[0181] Example 4 - CD4 disruption protects T cells and CAR T cells from HIV infection This example relates to the activity of T cells in which the CD4 gene was destroyed by treatment with Cas9 and G6, or T cells containing anti-CD19 CAR and in which the CD4 gene was destroyed, when co-cultured with HIV-infected cells. Each T cell was mixed with allogeneic cells infected with HIV at a ratio of 2:1.

[0182] A variety of T cells were generated, including control T cells (mock), CD4-disrupted T cells, T cells containing anti-CD4 CAR, and CD4-disrupted T cells containing anti-CD4 CAR. These generated T cells were mixed with HIV-infected allogeneic cells at a 2:1 ratio. HIV protein production was monitored in the cultures over a 5-day period as measured by HIV capsid (p24) ELISA. As shown in Figure 16, HIV replicated in HIV-infected cells mixed with mock effector T cells. However, in the presence of non-CAR, CD4-disrupted T cells, less HIV protein was produced, indicating that CD4 disruption reduces HIV replication. Non-CD4-disrupted anti-CD4 CAR T cells reduced HIV protein by more than 50%, demonstrating for the first time that anti-CD4 CAR T cell therapy can suppress HIV replication. CD4-disrupted anti-CD4 CAR T cells produced less virus than non-CAR CD4-disrupted T cells or non-CD4-disrupted anti-CD4 CAR T cells, demonstrating the benefit of combining these strategies.

[0183] CD4-disrupted and untreated mock cells were labeled with GFP using a GFP-expressing lentiviral vector and mixed with allogeneic peripheral blood mononuclear cells (PBMCs) infected with HIV at a 2:1 ratio. After 2–3 days, the percentage of HIV-infected cells was measured by intracellular staining of HIV capsid (p24) and assessed by flow cytometry. Without CD4 disruption, approximately 5.1% of all cells were infected with HIV (Figure 17, bottom, center panel). In contrast, when T cells were exposed to anti-CD4 CRISPR, only 1.8% of cells were HIV positive (Figure 17, bottom, right panel). The percentage of HIV-infected cells was reduced by 65%, indicating that CD4 disruption reduces HIV infection in T cells.

[0184] CD4-depleted cells and untreated mock cells were labeled with GFP using a GFP-expressing lentiviral vector and mixed with allogeneic PBMCs infected with HIV at a 2:1 ratio. After 2 days, GFP expression in each cell was evaluated by flow cytometry. As shown in the left panel of Figure 18, when CD4 was not depleted, almost all GFP+ cells disappeared, presumably due to HIV infection. On the other hand, as shown in the right panel of Figure 18, when CD4 was depleted, more GFP+ cells survived, presumably protecting GFP+ cells from HIV infection. More specifically, the percentage of GFP cells that survived HIV infection increased significantly from 0.31% to 3.11% after CD4 depletion, a 10-fold increase in survival rate. Thus, an increase in survival rate of genetically modified cells was observed in HIV infection.

[0185] Example 5 - Anti-CD4 CAR T cells targeting AML cells MOLM-13 cells are an AML cell line. MOLM-13 cells were labeled by transduction with the mCherry marker. MOLM-13 cells (target cells) were mixed with T cells containing anti-CD4 CAR (effector cells) or control T cells without anti-CD4 CAR (mock cells) at a 1:1 ratio and incubated for 21 hours. The cell populations were then analyzed by flow cytometry.

[0186] As shown in FIG. 19, measurements were performed by flow cytometry, and side scatter (Y axis) was plotted against Texas cherry red (X axis). Shown are mock T cells alone (top left plot), AML cells (MOLM-13 cells) alone (top center plot), and anti-CD4 CAR T cells alone (top right plot). AML (MOLM-13) target cells were transduced with mCherry. 98% of the target cells were mCherry positive (top center plot). The bottom plot of FIG. 19 shows the results of mock T cells or CAR T cells mixed with AML cells. The mCherry signal was reduced by 86% in the target and effector cell population compared to the target and mock cell population, indicating that anti-CD4 CAR T cells effectively killed AML cells.

[0187] Example 6 - CD4-anti-CD4 CAR T cells targeting AML cells CD4- T cells are generated using Cas9 and G6 as described above and transduced with a polynucleotide encoding an anti-CD4 CAR. MOLM-13 cells (target cells) are labeled by transduction with the mCherry marker. MOLM-13 cells are mixed in a 1:1 ratio with (1) CD4+ T cells containing an anti-CD4 CAR (effector CD4+ cells), (2) CD4- T cells containing an anti-CD4 CAR (effector CD4- cells), (3) CD4+ control T cells without an anti-CD4 CAR (mock CD4+ cells), or (4) CD4- control T cells without an anti-CD4 CAR (mock CD4- cells) and incubated for 21 hours. The incubated cell populations are analyzed by flow cytometry. The mixed population of target and effector CD4- cells results in increased levels of AML cell killing compared to the mixed population of target and effector CD4+ cells.

[0188] As used herein, the term "comprising" is synonymous with the terms "including," "containing," or "characterized by" and is open-ended and inclusive and does not exclude additional elements or steps not described herein.

[0189] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention may be modified, as may the manufacturing methods and equipment. Such modifications will be readily apparent to those of skill in the art in light of this disclosure or practice of the invention disclosed herein. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but will encompass all modifications and other aspects that fall within the true scope and spirit of the invention.

[0190] All references cited herein, including but not limited to published patent applications, unpublished patent applications, patents, and journal articles, are incorporated herein by reference in their entirety and made a part of this specification. In the event that any reference, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or take precedence over such conflicting matter.

[0191] The present invention includes the following inventions. [1] A method for killing or suppressing a CD4+ cell population, comprising contacting the CD4+ cell population with a genetically modified cell that contains at least one CD4 gene disrupted by a CRISPR (clustered regularly interspaced short palindromic repeats) guide polynucleotide. [2] The method according to [1], wherein the CD4+ cell population contains HIV. [3] The method according to [1], wherein the CD4+ cell population comprises acute myeloid leukemia (AML) cells or acute lymphocytic leukemia (ALL) cells. [4] The method according to [1], wherein the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8 or a complementary strand thereof. [5] The method according to [1], wherein the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:6 or a complementary strand thereof. [6] The method according to [1], wherein the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8 or a complementary strand thereof. [7] The method according to [1], wherein the at least one CD4 gene is disrupted with a CRISPR guide polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:6 or a complementary strand thereof. [8] The method according to [1], wherein the genetically modified cell contains a chimeric antigen receptor (CAR). [9] The method according to [8], wherein the CAR specifically binds to CD4 protein.

[10] The method according to [8], wherein the CAR specifically binds to CD19 protein.

[11] The method according to [8], wherein the CAR specifically binds to an HIV protein.

[12] The method described in [8], wherein the CAR comprises an antigen-binding domain derived from an HIV neutralizing antibody.

[13] The method according to

[12] , wherein the HIV neutralizing antibody is selected from PGT128, PG9 and PGT145.

[14] The method of [8], wherein the CAR is expressed from an inducible promoter.

[15] The method according to [1], wherein the genetically modified cell contains a polynucleotide encoding a suicide gene system.

[16] The method according to

[15] , wherein the suicide gene system is a herpes simplex virus thymidine kinase (HSVTK) / ganciclovir (GCV) suicide gene system or an inducible caspase suicide gene system.

[17] The method according to [1], wherein the genetically modified cells are mammalian cells.

[18] The method according to [1], wherein the genetically modified cells are human cells.

[19] The method according to [1], wherein the genetically modified cells are lymphocytes.

[20] The method according to [1], wherein the genetically modified cell is a T cell.

[21] The method according to [1], wherein the genetically modified cells are primary cells.

[22] The method according to [1], wherein the genetically modified cells are cells isolated from peripheral blood mononuclear cells.

[23] The method according to [1], wherein the genetically modified cells express CD3 or CD8.

[24] The method according to [1], wherein the genetically modified cells comprise cells derived from CD4+ cells, natural killer cells or natural killer T cells.

[25] An isolated nucleic acid for disrupting the CD4 gene, the nucleic acid comprising a CRISPR (clustered regularly interspaced short palindromic repeats) guide polynucleotide capable of hybridizing to the CD4 gene or configured to hybridize to the CD4 gene.

[26] The isolated nucleic acid of

[25] , wherein the guide polynucleotide is capable of hybridizing to the first exon or the third exon of the CD4 gene, or is configured to hybridize to the first exon or the third exon of the CD4 gene.

[27] The isolated nucleic acid of

[25] or

[26] , wherein the guide polynucleotides target adjacent identical protospacer adjacent motifs (PAMs).

[28] The isolated nucleic acid described in

[27] , wherein the PAM includes an NGG motif.

[29] A nucleic acid according to any one of

[25] to

[28] , comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 8 or a complementary strand thereof.

[30] The nucleic acid according to any one of

[25] to

[28] , comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence shown in SEQ ID NO: 6 or its complementary strand.

[31] The nucleic acid according to any one of

[25] to

[28] , comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8 or a complementary strand thereof.

[32] The nucleic acid according to any one of

[25] to

[28] , comprising the nucleotide sequence shown in SEQ ID NO: 6 or a complementary strand thereof.

[33] The nucleic acid according to any one of

[25] to

[32] , wherein the guide polynucleotide comprises RNA.

[34] A ribonucleoprotein (RNP) comprising the nucleic acid according to any one of

[25] to

[33] .

[35] The RNP described in

[34] , comprising a Cas9 protein.

[36] A method for producing a genetically modified cell, the method comprising the step of introducing a ribonucleoprotein (RNP) according to

[34] or

[35] into a cell.

[37] The method of

[36] , further comprising the step of introducing into the cell a second polynucleotide encoding a Cas9 protein.

[38] A method for producing a genetically modified cell, comprising the steps of: introducing into a cell a first polynucleotide encoding the nucleic acid described in any one of

[25] to

[33] ; and introducing into the cell a second polynucleotide encoding a Cas9 protein.

[39] The method according to any one of

[36] to

[38] , further comprising the step of introducing into the cell a third polynucleotide encoding a chimeric antigen receptor (CAR).

[40] The method according to

[39] , wherein the CAR specifically binds to CD4 protein.

[41] The method according to

[39] or

[40] , wherein the CAR specifically binds to CD19 protein.

[42] The method according to any one of

[39] to

[41] , wherein the CAR specifically binds to an HIV protein.

[43] The method according to

[42] , wherein the CAR comprises an antigen-binding domain derived from an HIV-neutralizing antibody.

[44] The method according to

[43] , wherein the HIV neutralizing antibody is selected from PGT128, PG9 and PGT145.

[45] The method according to any one of

[39] to

[44] , wherein the CAR is expressed from an inducible promoter.

[46] The method according to any one of

[38] to

[45] , further comprising the step of introducing into the cell a fourth polynucleotide encoding a suicide gene system.

[47] The method according to

[46] , wherein the suicide gene system is a herpes simplex virus thymidine kinase (HSVTK) / ganciclovir (GCV) suicide gene system or an inducible caspase suicide gene system.

[48] ​​The method according to any one of

[38] to

[47] , wherein the cell is a mammalian cell.

[49] The method according to any one of

[38] to

[48] , wherein the cells are human cells.

[50] The method according to any one of

[38] to

[49] , wherein the cells express CD4.

[51] The method according to any one of

[38] to

[50] , wherein the cell is a lymphocyte.

[52] The method according to any one of

[38] to

[51] , wherein the cell is a T cell.

[53] The method according to any one of

[38] to

[52] , wherein the cells are primary cells.

[54] The method according to any one of

[38] to

[53] , wherein the cells are cells isolated from peripheral blood mononuclear cells.

[55] The method according to any one of

[38] to

[54] , wherein the cells express CD3 or CD8.

[56] The method according to any one of

[38] to

[55] , wherein the cell is a cell derived from a CD4+ cell, a natural killer cell, or a natural killer T cell.

[57] The method according to any one of

[38] to

[56] , wherein the cells are ex vivo cells.

[58] A genetically modified cell produced by the method according to any one of

[38] to

[57] .

[59] A genetically modified cell having enhanced resistance to HIV infection, comprising the genetically modified cell described in

[58] , and having enhanced resistance to HIV infection compared to a cell in which at least one CD4 gene is not disrupted.

[60] A pharmaceutical composition comprising the genetically modified cell according to

[58] or

[59] and a pharma- ceutical acceptable excipient.

[61] A method for killing or suppressing a CD4+ cell population, comprising the step of contacting a CD4+ cell population with a genetically modified cell described in

[58] or

[59] or a pharmaceutical composition described in

[60] .

[62] The method according to

[61] , wherein the CD4+ cell population contains HIV.

[63] The method of

[61] or

[62] , wherein the CD4+ cell population comprises acute myeloid leukemia (AML) cells or acute lymphoblastic leukemia (ALL) cells.

[64] A method for reducing or suppressing HIV replication in an HIV-infected CD4+ cell population in a subject, preferably a human, comprising the step of contacting an HIV-infected CD4+ cell population in vivo or in vitro, preferably in a subject, with a genetically modified cell according to

[58] or

[59] , wherein the subject may be a subject selected for treatment of HIV infection, for example, the subject is selected by clinical and / or diagnostic evaluation, such as by assessment of the presence and / or amount of HIV infection in the subject, e.g., analysis of the presence or absence of HIV virus, HIV protein or HIV nucleic acid in the subject.

[65] The method according to any one of

[61] to

[64] , wherein the CD4+ cell population and the genetically modified cells are derived from the same subject.

[66] The method according to any one of

[61] to

[65] , wherein the CD4+ cell population is an ex vivo cell population.

[67] The method according to any one of

[61] to

[65] , wherein the CD4+ cell population is an in vivo cell population.

[68] A method for treating, suppressing or alleviating HIV infection in a subject having HIV infection, treating, suppressing or alleviating acute myeloid leukemia (AML) in a subject having AML, or treating, suppressing or alleviating acute lymphoblastic leukemia (ALL) in a subject having ALL, comprising administering to the subject a cell population comprising the genetically modified cell of

[58] or

[59] , wherein the subject may be a subject selected for treatment of HIV infection, AML and / or ALL, for example, the subject is a subject selected by clinical and / or diagnostic evaluation, such as by evaluation of the presence or absence and / or amount of HIV infection or AML in the subject, such as analysis of the presence or absence of HIV virus, HIV protein or HIV nucleic acid and / or AML markers identified by blood tests or prognostic sequencing.

[69] The method according to

[68] , wherein the genetically modified cells are autologous cells of the subject.

[70] The method according to any one of

[61] to

[69] , wherein the genetically modified cell contains a CAR, and the method further comprises a step of inducing expression of the CAR in the cell.

[71] The method according to any one of

[61] to

[70] , wherein the genetically modified cell contains a CAR, and the method further comprises a step of suppressing or reducing expression of CAR in the cell.

[72] The method of

[69] or

[70] , wherein expression of the CAR is suppressed or reduced after administration of the cell population to the subject.

[73] The method according to any one of

[69] to

[72] , wherein inhibition or reduction of expression of the CAR is confirmed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days after administration of the cell population to the subject.

[74] The method according to any one of

[69] to

[73] , wherein inhibition or reduction of expression of the CAR is confirmed 1 week, 2 weeks, 3 weeks or 4 weeks after administration of the cell population to the subject.

[75] The method according to any one of

[61] to

[74] , wherein the subject is a mammal.

[76] The method according to any one of

[61] to

[75] , wherein the subject is a human.

[77] Use of a cell population comprising the genetically modified cells according to

[58] or

[59] in a pharmaceutical agent for the treatment, suppression or palliative treatment of HIV, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).

[78] Use of a cell population comprising the genetically modified cells according to

[58] or

[59] in the preparation of a medicinal product for the treatment, suppression or palliative treatment of HIV, acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).

Claims

[Claim 1] 1. A method for killing or suppressing a CD4+ cell population, comprising contacting the CD4+ cell population with a genetically modified cell that contains at least one CD4 gene disrupted by a CRISPR (clustered regularly interspaced short palindromic repeats) guide polynucleotide.

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