Genetically engineered t cells with regnase-1 and/or tgfbrii disruption have improved functionality and persistence

Abstract

A population of genetically engineered T cells, comprising a disrupted Reg1 gene and/or a disrupted TGFBRII gene. Such genetically engineered T cells may comprise further genetic modifications, for example, a disrupted CD70 gene. The population of genetically engineered T cells exhibit one or more of (a) improved cell growth activity; (b) enhanced persistence; and (c) reduced T cell exhaustion, (d) enhanced cytotoxicity activity, (e) resistant to inhibitory effects induced by TGF-b, and (f) resistant to inhibitory effects by fibroblasts and/or inhibitory factors secreted thereby, as compared to non-engineered T cell counterparts.

What is claimed is: 
     
         1 . A population of genetically engineered T cells, comprising:
 (i) a disrupted Regnase-1 (Reg1) gene; and/or   (ii) a disrupted Transforming Growth Factor Beta Receptor II (TGFBRII) gene.   
     
     
         2 . The population of genetically engineered T cells of  claim 1 , which comprises both (i) and (ii). 
     
     
         3 . The population of genetically engineered T cells of  claim 1 , wherein the population of genetically engineered T cells, as compared to non-engineered T cell counterparts, has one or more of the following features:
 (a) improved cell growth activity;   (b) enhanced persistence;   (c) reduced T cell exhaustion;   (d) resistant to inhibitory effects induced by TGF-β;   (e) enhanced cell killing capacity; and   (f) resistant to inhibitory effects by fibroblasts and/or inhibitory factors secreted thereby.   
     
     
         4 . The population of genetically engineered T cells of  claim 1 , wherein the T cells are further engineered to express a chimeric antigen receptor (CAR). 
     
     
         5 . The population of genetically engineered T cells of  claim 1 , wherein the disrupted Reg1 gene is genetically edited in exon 2 and/or exon 4. 
     
     
         6 . The population of genetically engineered T cells of  claim 1 , wherein the disrupted TGFBRII gene is genetically edited in exon 1, exon 2, exon 3, exon 4, or exon 5, optionally wherein the disrupted TGFBRII gene is genetically edited in exon 4 or exon 5. 
     
     
         7 . The population of genetically engineered T cells of  claim 1 , wherein the disrupted Reg1 gene, the disrupted TGFBRII gene, or both are genetically edited by a CRISPR/Cas-mediated gene editing system. 
     
     
         8 . The population of genetically engineered T cells of  claim 7 , wherein the CRISPR/Cas-mediated gene editing comprises a guide RNA (gRNA) targeting a site in the Reg1 gene that comprises a nucleotide sequence listed in Table 22, which optionally is selected from the group consisting of SEQ ID NO: SEQ ID NO: 320, 322, 323, and 327. 
     
     
         9 . The population of genetically engineered T cells of  claim 8 , wherein the gRNA targeting the Reg1 gene comprises a nucleotide sequence listed in Table 22, which optionally is selected from the group consisting of SEQ ID NO: 24, 32, 36, or 52. 
     
     
         10 . The population of genetically engineered T cells of  claim 9 , wherein the disrupted Reg1 gene comprises a nucleotide sequence selected from those listed in Table 31, 33, 34, or 38. 
     
     
         11 . The population of genetically engineered T cells of  claim 7 , wherein the CRISPR/Cas-mediated gene editing system comprises a guide RNA (gRNA) targeting a site in the TGFBRII gene that comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 269, 275, 281, 287, 293, 299, 305, 311, and 317. 
     
     
         12 . The population of genetically engineered T cells of  claim 11 , wherein the gRNA targeting the TGFBRII gene comprises a spacer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 266, 272, 278, 284, 290, 296, 302, 308, and 314. 
     
     
         13 . The population of genetically engineered T cells of  claim 7 , wherein the gRNA further comprises a scaffold sequence. 
     
     
         14 . The population of genetically engineered T cells of  claim 13 , wherein:
 the gRNA targeting the Reg1 gene comprises the nucleotide sequence of any of SEQ ID NO: 22, 30, 34, or 50; and/or   the gRNA targeting the TGFBRII gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 270, 300, 306, or 312.   
     
     
         15 . The population of genetically engineered T cells of  claim 1 , which further comprises:
 (iii) a disrupted T cell receptor alpha chain constant region (TRAC) gene,   (iv) a disrupted beta-2-microglobulin (β2M) gene,   (v) a disrupted CD70 gene, or   (vi) a combination of any of (iii)-(v).   
     
     
         16 . The population of genetically engineered T cells of  claim 15 , wherein the T cells comprise a disrupted T cell receptor alpha chain constant region (TRAC) gene. 
     
     
         17 . The population of genetically engineered T cells of  claim 15 , wherein the T cells comprise a disrupted beta-2-microglobulin (β2M) gene. 
     
     
         18 . The population of genetically engineered T cells of  claim 15 , wherein the T cells comprise a disrupted CD70 gene. 
     
     
         19 . The population of genetically engineered T cells of  claim 15 , wherein the disrupted TRAC gene, the disrupted β2M gene, and/or the disrupted CD70 gene is genetically edited by one or more CRISPR/Cas-mediated gene editing system 
     
     
         20 . The population of genetically engineered T cells of  claim 4 , wherein the T cells comprise a nucleic acid encoding the CAR, and wherein the nucleic acid is inserted in the genome of the T cells. 
     
     
         21 . The population of genetically engineered T cells of  claim 20 , wherein the nucleic acid encoding the CAR is inserted in the disrupted Reg1 gene, the disrupted TGFBRII gene, the disrupted TRAC gene, the disrupted β2M, or the disrupted CD70 gene. 
     
     
         22 . The population of genetically engineered T cells of  claim 21 , wherein the nucleic acid encoding the CAR is inserted in the disrupted TRAC gene, and optionally wherein the nucleic acid encoding the CAR replaces the deleted fragment comprising SEQ ID NO: 69 in the TRAC gene. 
     
     
         23 . The population of genetically engineered T cells of  claim 15 , wherein the disrupted Reg1 gene comprises a nucleotide sequence listed in Table 31, 33, 34, or 38, the disrupted TRAC gene comprises a nucleotide sequence listed in Table 24; the disrupted β2M comprises a nucleotide sequence listed in Table 25 and/or the disrupted CD70 gene comprises a nucleotide sequence listed in Table 26. 
     
     
         24 . The population of genetically engineered T cells of  claim 4 , wherein the CAR comprises an extracellular antigen binding domain specific to a tumor antigen, a co-stimulatory signaling domain of 4-1BB or CD28, and a cytoplasmic signaling domain of CD3ζ. 
     
     
         25 . The population of genetically engineered T cells of  claim 24 , wherein the tumor antigen is CD19, BCMA, CD70, CD33, or PTK7. 
     
     
         26 . The population of genetically engineered T cells of  claim 24 , wherein the CAR binds CD19 (anti-CD19 CAR) and wherein the extracellular antigen binding domain in the anti-CD19 CAR is a single chain variable fragment (scFv) that binds CD19 (anti-CD19 scFv). 
     
     
         27 . The population of genetically engineered T cells of  claim 24 , wherein the anti-CD19 scFv comprises (i) a heavy chain variable region (V H ) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 124; and (ii) a light chain variable region (V L ) that comprises the same light chain CDRs as those in SEQ ID NO: 125; optionally wherein the V H  comprises the amino acid sequence of SEQ ID NO: 124 and the V L  comprises the amino acid sequence of SEQ ID NO: 125. 
     
     
         28 . The population of genetically engineered T cells of  claim 27 , wherein the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 120. 
     
     
         29 . The population of genetically engineered T cells of  claim 28 , wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO:353. 
     
     
         30 . The population of genetically engineered T cells of  claim 24 , wherein the CAR binds CD70 (anti-CD70 CAR) and wherein extracellular antigen binding domain in the anti-CD70 CAR is a single chain variable fragment (scFv) that binds CD70 (anti-CD70 scFv). 
     
     
         31 . The population of genetically engineered T cells of  claim 30 , wherein the anti-CD70 scFv comprises (i) a heavy chain variable region (V H ) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 143; and (ii) a light chain variable region (V L ) that comprises the same light chain CDRs as those in SEQ ID NO: 144; optionally wherein the V H  comprises the amino acid sequence of SEQ ID NO: 143 and the V L  comprises the amino acid sequence of SEQ ID NO: 144. 
     
     
         32 . The population of genetically engineered T cells of  claim 31 , wherein the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 140 or 142. 
     
     
         33 . The population of genetically engineered T cells of  claim 32 , wherein the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO:354. 
     
     
         34 . The population of genetically engineered T cells of  claim 24 , wherein the CAR binds BCMA (anti-BCMA CAR) and wherein the extracellular antigen binding domain in the anti-BCMA CAR is a single chain variable fragment (scFv) that binds BCMA (anti-BCMA CAR). 
     
     
         35 . The population of genetically engineered T cells of  claim 34 , wherein the anti-BCMA scFv comprises (i) a heavy chain variable region (V H ) that comprises the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 149; and (ii) a light chain variable region (V L ) that comprises the same light chain CDRs as those in SEQ ID NO: 150; optionally wherein the V H  comprises the amino acid sequence of SEQ ID NO: 149 and the V L  comprises the amino acid sequence of SEQ ID NO: 150. 
     
     
         36 . The population of genetically engineered T cells of  claim 35 , wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 148. 
     
     
         37 . The population of genetically engineered T cells of  claim 36 , wherein the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 146 or SEQ ID NO:355. 
     
     
         38 . The population of genetically engineered T cells of  claim 1 , wherein the genetically engineered T cells are derived from primary T cells of one or more human donors. 
     
     
         39 . The population of genetically engineered T cells of  claim 1 , wherein the genetically engineered T cells show cytokine-dependent growth. 
     
     
         40 . A method for preparing the population of genetically engineered T cells of  claim 1 , the method comprising:
 (a) providing a plurality of cells, which are T cells or precursor cells thereof;   (b) genetically editing the Reg1 gene and/or the TGFBRII gene; and   (c) producing the population of genetically engineered T cells having disrupted Reg1 gene and/or the TGFBRII gene.   
     
     
         41 . The method of  claim 40 , wherein step (b) comprises genetically editing both the Reg1 gene and the TGFBRII gene. 
     
     
         42 . The method of  claim 40 , wherein step (b) is performed by one or more CRISPR/Cas-mediated gene editing systems. 
     
     
         43 . The method of  claim 40 , wherein step (b) is performed by delivering to the plurality of cells an RNA-guided nuclease and a gRNA targeting the Reg1 gene. 
     
     
         44 . The method of  claim 43 , wherein the gRNA targeting the Reg1 gene is specific to an exon of the Reg1 gene selected from the group consisting of exon 2 and exon 4. 
     
     
         45 . The method of  claim 44 , wherein the gRNA targeting the Reg1 gene comprises a spacer having a nucleotide sequence of SEQ ID NO: 24, 32, 36, or 52. 
     
     
         46 . The method of  claim 40 , wherein step (b) is performed by delivering to the plurality of cells an RNA-guided nuclease and a gRNA targeting the TGFBRII gene. 
     
     
         47 . The method of  claim 46 , wherein the gRNA targeting the TGFBRII gene is specific to an exon of the TGFBRII gene selected from the group consisting of exon 1, exon 2, exon 3, exon 4, and exon 5, preferably wherein the gRNA targeting the TGFBRII gene is specific to exon 4 or exon 5. 
     
     
         48 . The method of  claim 47 , wherein the gRNA targeting the TGFBRII gene comprises a spacer having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 272, 300, 308, and 314. 
     
     
         49 . The method of  claim 40 , wherein the gRNA targeting the Reg1 gene and the gRNA targeting the TGFBRII gene further comprises a scaffold sequence. 
     
     
         50 . The method of  claim 49 , wherein the gRNA targeting the Reg1 gene comprises a nucleotide sequence of SEQ ID NO: 22, 30, 34, or 50; and/or wherein the gRNA targeting the TGFBRII gene comprises a nucleotide sequence of SEQ ID NOs: 270, 300, 306, or 312. 
     
     
         51 . The method of  claim 40 , wherein the plurality of T cells in step (a) comprises one or more of the following genetic modifications:
 (i) engineered to express a chimeric antigen receptor (CAR);   (ii) has a disrupted T cell receptor alpha chain constant region (TRAC) gene;   (iii) has a disrupted β2M gene; and   (iv) has a disrupted CD70 gene.   
     
     
         52 . The method of  claim 40 , wherein the method further comprises:
 (i) delivering to the T cells a nucleic acid encoding a chimeric antigen receptor (CAR);   (ii) genetically editing a TRAC gene to disrupt its expression;   (iii) genetically editing a β2M gene to disrupt its expression;   (iv) genetically editing a CD70 gene to disrupt its expression; or   (v) a combination thereof.   
     
     
         53 . The method of  claim 52 , wherein one or more of (i)-(iv) are performed by one or more CRISPR/Cas-mediated gene editing system comprising one or more RNA-guided nucleases and one or more gRNAs targeting the TRAC gene, the β2M gene, and/or the CD70 gene. 
     
     
         54 . The method of  claim 53 , wherein the gRNA targeting the TRAC gene comprises the nucleotide sequence of SEQ ID NO: 59. 
     
     
         55 . The method of  claim 53 , wherein the gRNA targeting the β2M gene comprises the nucleotide sequence of SEQ ID NO: 63. 
     
     
         56 . The method of  claim 53 , wherein the gRNA targeting the CD70 gene comprises the nucleotide sequence of SEQ ID NO: 55. 
     
     
         57 . The method of  claim 56 , wherein the method comprises delivering to the T cells one or more ribonucleoprotein particles (RNP), comprising the RNA-guided nuclease, one or more of the gRNAs, and the nucleic acid encoding the CAR. 
     
     
         58 . The method of  claim 50 , wherein the RNA-guided nuclease is a Cas9 nuclease, which optionally is a  S. pyogenes  Cas9 nuclease. 
     
     
         59 . The method of  claim 50 , wherein the nucleic acid encoding the CAR is in an AAV vector. 
     
     
         60 . The method of  claim 50 , wherein the nucleic acid encoding the CAR comprises a left homology arm and a right homology arm flanking the nucleotide sequence encoding the CAR; and wherein the left homology arm and the right homology arm are homologous to a genomic locus in the T cells, allowing for insertion of the nucleic acid into the genomic locus. 
     
     
         61 . The method of  claim 60 , wherein the genomic locus is in the Reg1 gene, the TGFBRII gene, the TRAC gene, the β2M gene, or the CD70 gene; optionally wherein the genomic locus is the TRAC gene. 
     
     
         62 . The method of  claim 50 , wherein the method comprising disrupting the TRAC gene by a CRISPR/Cas-mediated gene editing system comprising a gRNA comprising nucleotide sequence of SEQ ID NO: 47 and the nucleic acid encoding the CAR is inserted at the site targeted by the gRNA. 
     
     
         63 . The method of  claim 40 , wherein the method comprising delivering to the T cells a nucleic acid encoding a CAR, which is specific to CD70, and genetically editing the CD70 gene to disrupt its expression. 
     
     
         64 . The method of  claim 40 , wherein the T cells of step (a) are derived from primary T cells of one or more human donors. 
     
     
         65 . A population of genetically engineered T cells, which is prepared by a method of  claim 40 . 
     
     
         66 . A method for eliminating undesired cells in a subject, the method comprising administering to a subject in need thereof a population of genetically engineered T cells set forth in  claim 4 . 
     
     
         67 . The method of  claim 66 , wherein the undesired cells are cancer cells, which optionally are hematopoietic cancer cells or solid tumor cells. 
     
     
         68 . The method of  claim 66 , wherein the undesired cells are CD19 + , BCMA + , CD70 + , CD33 + , or PTK7 + . 
     
     
         69 . A guide RNA (gRNA) targeting a Reg1 gene, comprising a nucleotide sequence specific to a fragment in exon 1, exon2, exon3, or exon4, optionally in exon 2 or exon 4 of the Reg1 gene. 
     
     
         70 . The gRNA of  claim 69 , wherein the gRNA comprises a spacer listed in Table 22, which optionally is selected from the group consisting of SEQ ID NO: 24, 32, 36, and 52. 
     
     
         71 . The gRNA of  claim 69 , wherein the gRNA further comprises a scaffold sequence. 
     
     
         72 . The gRNA of  claim 69 , wherein the gRNA comprises one or more modified nucleotides. 
     
     
         73 . The gRNA of  claim 72 , wherein the gRNA comprises one or more 2′-O-methyl phosphorothioate residues at the 5′ and/or 3′ terminus of the gRNA. 
     
     
         74 . The gRNA of  claim 73 , which comprises the nucleotide sequence listed in Table 22, which optionally is any of SEQ ID NOs: 22, 23, 30, 31, 34, 35, 50, or 51. 
     
     
         75 . A guide RNA (gRNA) targeting a TGFBRII gene, comprising a nucleotide sequence specific to a fragment in exon 1, exon 2, exon 3, exon 4, or exon5 of the TGFBRII gene, preferably wherein the gRNA comprises a nucleotide sequence specific to exon 4 or exon 5 of the TGFBRII gene. 
     
     
         76 . The gRNA of  claim 75 , wherein the gRNA comprises a spacer listed in Table 32, which optionally has the nucleotide sequence selected from the group consisting of SEQ ID NOs: 272, 302, 308, and 314. 
     
     
         77 . The gRNA of  claim 76 , wherein the gRNA further comprises a scaffold sequence. 
     
     
         78 . The gRNA of  claim 75 , wherein the gRNA comprises one or more modified nucleotides. 
     
     
         79 . The gRNA of  claim 78 , wherein the gRNA comprises one or more 2′-O-methyl phosphorothioate residues at the 5′ and/or 3′ terminus of the gRNA. 
     
     
         80 . The gRNA of  claim 79 , which comprises the nucleotide sequence listed in Table 39, optionally wherein the gRNA comprises the nucleotide sequence of any one of SEQ ID NO: SEQ ID NOs: 270, 271, 300, 301, 306, 307, 312, and 313.