Chimeric antigen receptor compositions and methods of use

Abstract

Provided herein are polynucleotides, vectors, compositions and methods for preparing and using genetically modified immune cells that express a chimeric antigen receptor (CAR) and a CD70 short hairpin RNA (shRNA), as well as method of using such modified immune cells for treating diseases or disorders, such as cancer or autoimmune disorders.

Description

Attorney Docket No.: 000218-0156-WO1CHIMERIC ANTIGEN RECEPTOR COMPOSITIONS AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 720,121, filed November 13, 2024, the contents of which are herein incorporated by reference in their entireties.FIELD

[0002] The disclosure is directed to molecular biology, and more specifically, to compositions and methods for preparing and using genetically modified immune cells that express a chimeric antigen receptor (CAR) and a CD70 shRNA.INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is herein incorporated by reference in its entirety. Said XML copy, created on November 12, 2025, is named “000218-0156-W01-SL.xml” and is 101,384 bytes in size.BACKGROUND

[0004] The discovery of cell-based therapies capable of recognizing and binding to a specific target protein with high affinity and avidity has been a focus of the biopharmaceutical industry. There remains a need for more efficacious cell-based therapies that demonstrate enhanced in vivo activity, persistence and extended treatment duration compared to traditional cell-based therapies.SUMMARY

[0005] In one aspect, provided herein is a polynucleotide comprising: (a) a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and (b) a nucleic acid sequence encoding a CD70 shRNA, wherein the CD70 shRNA comprises the sequence set forth in SEQ ID NO: 49.

[0006] In another aspect, provided herein is a transposon comprising: (a) a nucleic acid sequence encoding a CAR; and (b) a nucleic acid encoding an CD70 shRNA, wherein the CD70 shRNA comprises the sequence set forth in SEQ ID NO: 49.

[0007] In some embodiments, the CAR comprises (a) an ectodomain comprising an antigen recognition region that binds an antigen; (b) a transmembrane domain, and (c) anAttorney Docket No.: 000218-0156-WO1endodomain comprising at least one costimulatory domain. In some embodiments, the ectodomain of (a) further comprises a signal peptide and / or a hinge between the antigen recognition region and the transmembrane domain. In some embodiments, the signal peptide and / or transmembrane domain comprises a sequence encoding a human CD2, CD36, CD3s, CD3y, CD3< CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide and / or transmembrane domain. In some embodiments, the endodomain comprises a human CD3(^ endodomain. In some embodiments, the at least one costimulatory domain comprises a human 4-1BB, CD28, CD3(^, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In some embodiments, the hinge comprises a sequence derived from a human CD8a, IgG4, and / or CD4 sequence.

[0008] In some embodiments, the CAR targets an oncogenic gene product comprising CD70, CD7, BCMA, CD 19, CD20, MUC1, PSMA or a combination thereof.

[0009] In some embodiments, the transposon further comprises a nucleic acid encoding an inducible caspase polypeptide, a nucleic acid encoding a chimeric stimulatory receptor, a nucleic acid encoding a selection gene, a nucleic acid encoding a therapeutic agent, or a combination thereof. In some embodiments, the selection gene comprises a dihydrofolate reductase (DHFR) resistance gene. In some embodiments, the transposon is a piggyBac transposon.

[0010] In some embodiments, the transposon comprises one or more promoters. In some embodiments, the one or more promoters is an elongation factor 1 -alpha (EFla) promoter, a U6 promoter or both an EFla promoter and a U6 promoter. In some embodiments, the promoter is an EFla promoter operably linked to the CAR and the CD70 shRNA.

[0011] In another aspect, provided herein is a vector comprising a polynucleotide or a transposon described herein.

[0012] In another aspect, provided herein is a cell comprising a CAR and a CD70 shRNA described herein or a transposon described herein.

[0013] In another aspect, provided herein is a population of cells comprising a CAR and a CD70 shRNA described herein. In some embodiments, the cells are immune cells. In some embodiments, the cells are T-cells.

[0014] In some embodiments, a portion of the immune cells comprises a genetic modification, which reduces or inhibits expression of a T-cell receptor or a major histocompatibility complex (MHC). In some embodiments, the genetic modification is an insertion of a sequence encoding a P-2 microglobulin (P2M) and wherein the genetic modification reduces or inhibits expression of an MHC I. In some embodiments, the genetic modification is anAttorney Docket No.: 000218-0156-WO1insertion of a sequence encoding an a chain (TCRa), a P chain (TCRP), or a combination thereof and wherein the genetic modification reduces or inhibits expression of a TCR.

[0015] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of cells express the CD70 shRNA.

[0016] In another aspect, provided herein is a composition comprising a cell or a population of cells described herein. In another aspect, provided herein is a pharmaceutical composition comprising a composition described herein and a pharmaceutically acceptable carrier.

[0017] In another aspect, provided herein is a method of treating a disease or disorder in a subject in need thereof comprising administering a therapeutically effective amount of a composition of or a pharmaceutical composition described herein. In some embodiments, the disease or disorder is one or more of Acute myeloid Leukemia (AML), T cell lymphoma, Non-Hodgkin lymphoma (NHL), Renal cell carcinoma (RCC), or an autoimmune disease or disorder. In some embodiments, the autoimmune disorder is selected from: autoimmune neutropenia, Guillain-Barre syndrome, epilepsy, autoimmune encephalitis, Isaacs' syndrome, nevus syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, or membranous nephropathy.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGs. 1A and IB show a schematic of transposon cassettes with two different embedded shRNA designs FIG. 1 A shows a canonical shRNA design under a separate U6 promoter from the CAR. FIG. IB shows the shRNA and CAR as a multi-cistronic transgene design under expression control of a single promoter. The single promotor design demonstrated optimal shRNA expression, less residual CD70 surface expression and optimal potency. The canonical design with the U6 promoter resulted in cellular toxicity likely due to overexpression of shRNA (data not shown).

[0019] FIGs. 2A-2C show in vivo analysis of shRNAs for depletion of CD70 in an anti-CD70 CAR-T. shRNA #776 (SEQ ID NO: 49) provided the greatest potency compared to the other shRNAs and outperformed both WT and CC-edited CD70 CAR-T cells in vivo.Attorney Docket No.: 000218-0156-WO1

[0020] FIG. 3 shows the potency of anti-CD70 CAR-T cells in vivo against the Nomo-1 AML xenograft cell line. NSG mice were inoculated with Nomo-1 tumor cells and then treated with anti-CD70 CAR-T cells. Tumor burden was monitored by bioluminescent imaging (BLI).

[0021] FIG. 4A shows CD70 expression for Anti-CD70 CAR-T cells expressing a tool VH binder targeting CD70 with either no disruption of CD70 expression (WT) or with depletion of CD70 expression via Cas-CLOVER gene editing (KO) or via an anti-CD70 miRNA-embedded shRNA (shRNA) incorporated in the transgene. CD70 expression on final product CAR-T cells was measured by flow cytometry with a fluorescence-minus-one (FMO) staining control. shRNA-mediated depletion of CD70 results in a CAR-T product with less residual CD70 surface expression.

[0022] FIG 4B. shows potency of anti-CD70 CAR-T cells in vivo against the Nomo-1 AML xenograft cell line, which endogenously expresses CD70. NSG mice were inoculated with Nomo-1 tumor cells and then treated with anti-CD70 CAR-T cells. Tumor burden was monitored by bioluminescent imaging (BLI) and compared to control mice that did not receive CAR-T treatment (PBS). shRNA-mediated depletion of CD70 results in a CAR-T product with greater potency, outperforming both WT and CC-edited CD70 CAR-T cells in vivo.

[0023] All documents cited herein, including any cross referenced or related patent or application are hereby incorporated herein by reference in their entireties for all purposes, unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.DETAILED DESCRIPTIONCD70 shRNA

[0024] CD70 expression on T-cells can lead to T-cell fratricide which can diminish the potency of a CAR-T product. In one aspect, provided herein are shRNA agents targeting a CD70 nucleic acid sequence (CD70 shRNA) that can be incorporated into a CAR-T product.Attorney Docket No.: 000218-0156-WO1In certain embodiments, the CD70 nucleic acid sequence is the mRNA having Accession No. NM_001252.5 (SEQ ID NO: 50). In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid sequence fully complementary along its length to a portion of CD70. In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid sequence fully complementary along its length to the region of nucleotides 776 to 797 of SEQ ID NO: 50 or at least a 16, at least a 17, at least a 18, and least a 19, at least a 20, at least a 21 nucleobase portion thereof. In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid portion of the sequence comprising the sequence TACACTTTTTCTCTTGAACTTA (SEQ ID NO: 48). In certain embodiments, the antisense nucleic acid portion of the CD70 shRNA has at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 nucleotides of SEQ ID NO: 48. In certain embodiments, the CD70 shRNA comprises or consists of the nucleic acid sequence tcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgcaagttcaagagaaaaagtgtatagtgaagcc acagatgtatacactttttctcttgaacttatgcctactgcctcggacttcaaggggctagaattcga (shRNA 776; SEQ ID NO: 49).

[0025] In certain embodiments, the CD70 shRNA comprises the nucleic acid sequence TAAGTTCAAGAGAAAAAGTGTA (SEQ ID NO: 59). In certain embodiments, the CD70 shRNA comprises the nucleic acid sequence CTGACAGGTTGAAGCAAGTAGA (SEQ ID NO: 60). In certain embodiments, the CD70 shRNA comprises the nucleic acid sequence TCGTGATGGCATCTACATGGTA (SEQ ID NO: 61). In certain embodiments, the CD70 shRNA comprises the nucleic acid sequence TGCTGCTGATTAGGGTTTTTTA (SEQ ID NO: 62). In certain embodiments, the CD70 shRNA comprises the nucleic acid sequence CAAGAGAAAAAGTGTACACACA (SEQ ID NO: 63).

[0026] A person of skill in the art will appreciate that the antisense sequences disclosed herein may be RNA or DNA. Thus, in some embodiments, the thymidine in SEQ ID NOs 49-52 and 59-63 may be replaced with uracil without affecting the sequence’s ability to bind the target.

[0027] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or at least about 99% identical to SEQ ID NO: 49.

[0028] In certain embodiments, the shRNA comprises a nucleotide sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 94%, about 96%, about 98%, or about 99% identical to SEQ ID NO: 49.Attorney Docket No.: 000218-0156-WO1

[0029] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about a 100 nucleobase (nb), at least about a 125 nb, at least about a 130 nb, at least about a 135 nb, at least about a 140 nb, at least about a 145 nb, at least about a 150 nb, at least about a 152 nb, at least about a 154 nb, at least about a 156 nb portion of SEQ ID NO: 49.

[0030] In certain embodiments, the shRNA comprises a nucleotide sequence that is about a 100 nucleobase (nb), about a 125 nb, about a 130 nb, about a 135 nb, about a 140 nb, about a 145 nb, about a 150 nb, about a 152 nb, about a 154 nb, about a 156 nb portion of SEQ ID NO: 49.

[0031] In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid sequence fully complementary along its length to the region of nucleotides 22 to 43 of SEQ ID NO: 50 or at least a 16, at least a 17, at least a 18, and least a 19, at least a 20, at least a 21 nucleobase portion thereof. In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid portion of the sequence comprising the sequence TCTACTTGCTTCAACCTGTCAG (SEQ ID NO: 51). In certain embodiments, the antisense nucleic acid portion of the CD70 shRNA has at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 nucleotides of SEQ ID NO: 51. In certain embodiments, the CD70 shRNA comprises or consists of the nucleic acid sequence tcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgatgacaggttgaagcaagtagatagtgaagcc acagatgtatctacttgcttcaacctgtcagtgcctactgcctcggacttcaaggggctagaattcga (shRNA 22; SEQ ID NO: 52).

[0032] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96% and least about 98%, at least about 99% identical to SEQ ID NO: 52.

[0033] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about a 100 nucleobase (nb), at least about al25 nb, at least about a 130 nb, at least about a 135 nb, at least about a 140 nb, at least about a 145 nb, at least about a 150 nb, at least about a 152 nb, at least about a 154 nb, at least about a 156 nb portion of SEQ ID NO: 52.

[0034] In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid sequence fully complementary along its length to the region of nucleotides 445 to 466 of SEQ ID NO: 50 or at least a 16, at least a 17, at least a 18, and least a 19, at least a 20, at least a 21 nucleobase portion thereof. In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid portion of the sequence comprising the sequenceAttorney Docket No.: 000218-0156-WO1TACCATGTAGATGCCATCACGA (SEQ ID NO: 53). In certain embodiments, the antisense nucleic acid portion of the CD70 shRNA has at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 nucleotides of SEQ ID NO: 53. In certain embodiments, the CD70 shRNA comprises or consists of the nucleic acid sequence tcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgccgtgatggcatctacatggtatagtgaagcc acagatgtataccatgtagatgccatcacgatgcctactgcctcggacttcaaggggctagaattcga (shRNA 448; SEQ ID NO: 54).

[0035] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96% and least about 98%, at least about 99% identical to SEQ ID NO: 54.

[0036] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about a 100 nucleobase (nb), at least about 125 nb, at least about 130 nb, at least about 135 nb, at least about 140 nb, at least about 145 nb, at least about 150 nb, at least about 152 nb, at least about 154 nb, at least about 156 nb portion of SEQ ID NO: 54.

[0037] In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid sequence fully complementary along its length to the region of nucleotides 735 to 756 of SEQ ID NO: 50 or at least a 16, at least a 17, at least a 18, and least a 19, at least a 20, at least a 21 nucleobase portion thereof. In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid portion of the sequence comprising the sequence TAAAAAACCCTAATCAGCAGCA (SEQ ID NO: 55). In certain embodiments, the antisense nucleic acid portion of the CD70 shRNA has at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 nucleotides of SEQ ID NO: 55. In certain embodiments, the CD70 shRNA comprises or consists of the nucleic acid sequence tcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgcgctgctgattagggttttttatagtgaagccac agatgtataaaaaaccctaatcagcagcatgcctactgcctcggacttcaaggggctagaattcga (shRNA 735; SEQ ID NO: 56).

[0038] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96% and least about 98%, at least about 99% identical to SEQ ID NO: 56.Attorney Docket No.: 000218-0156-WO1

[0039] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about a 100 nucleobase (nb), at least about 125 nb, at least about 130 nb, at least about 135 nb, at least about 140 nb, at least about 145 nb, at least about 150 nb, at least about 152 nb, at least about 154 nb, at least about 156 nb portion of SEQ ID NO: 56.

[0040] In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid sequence fully complementary along its length to the region of nucleotides 782 to 803 of SEQ ID NO: 50 or at least a 16, at least a 17, at least a 18, and least a 19, at least a 20, at least a 21 nucleobase portion thereof. In certain embodiments, the CD70 shRNA comprises an antisense nucleic acid portion of the sequence comprising the sequence TGTGTGTACACTTTTTCTCTTG (SEQ ID NO: 57). In certain embodiments, the antisense nucleic acid portion of the CD70 shRNA has at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 nucleotides of SEQ ID NO: 57. In certain embodiments, the CD70 shRNA comprises or consists of the nucleic acid sequence tcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgaaagagaaaaagtgtacacacatagtgaagc cacagatgtatgtgtgtacactttttctcttgtgcctactgcctcggacttcaaggggctagaattcga (shRNA 782; SEQ ID NO: 58).

[0041] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 96% and least about 98%, at least about 99% identical to SEQ ID NO: 58.

[0042] In certain embodiments, the shRNA comprises a nucleotide sequence that is at least about a 100 nucleobase (nb), at least about 125 nb, at least about 130 nb, at least about 135 nb, at least about 140 nb, at least about 145 nb, at least about 150 nb, at least about 152 nb, at least about 154 nb, at least about 156 nb portion of SEQ ID NO: 58.Chimeric Antigen Receptors

[0043] In one aspect, provided herein are chimeric antigen receptors (CARs) and cells comprising the same. In certain embodiments of the disclosure, the chimeric antigen receptor comprises (a) an ectodomain comprising a ligand recognition region; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.

[0044] In some aspects, a CAR disclosed herein comprises: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatoryAttorney Docket No.: 000218-0156-WO1domain. As used throughout the disclosure, a CAR comprising a VH as the antigen recognition region can be referred to as a VCAR.

[0045] In some aspects, a CAR disclosed herein comprises a transmembrane domain. In certain embodiments, the transmembrane domain may comprise a sequence encoding a human CD2, CD38, CD3s, CD3y, CD3< CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In certain embodiments, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain.

[0046] The CD8a transmembrane domain may comprise the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 3). In some embodiments, the CD8a transmembrane domain comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 3). The CD8a transmembrane domain may be encoded by the nucleic acid comprising the sequence atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 4).

[0047] In some embodiments, a CAR provided herein comprises an endodomain comprising one or more co-stimulation domains. In certain embodiments, the endodomain may comprise a human CD3(^ endodomain.

[0048] The CD3(^ costimulatory domain may comprise the amino acid sequence:RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 5). In some embodiments, the CD3(^ costimulatory domain comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 5).

[0049] The CD3(^ costimulatory domain may be encoded by a nucleic acid comprising the sequence: cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 6).Attorney Docket No.: 000218-0156-WO1

[0050] In certain embodiments, the at least one costimulatory domain may comprise a human 4-1BB intracellular domain, a TACI intracellular domain, a CD28 intracellular domain, a CD40 intracellular domain, an ICOS intracellular domain, a MyD88 intracellular domain, or an OX-40 intracellular domain, or any combination thereof. In certain embodiments, the endodomain comprises a CD28 co-stimulatory domain and a 4- IBB costimulatory domain. In certain embodiments, the endodomain comprises a CD3(^ co-stimulatory domain and a 4-1BB costimulatory domain. In certain embodiments, the endodomain comprises a CD3(^ costimulatory domain and a TACI costimulatory domain.

[0051] The 4-1BB costimulatory domain may comprise the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7). In some embodiments, the 4- IBB costimulatory domain comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7). The 4-1BB costimulatory domain may be encoded by a nucleic acid comprising the sequence aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 8). The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain. The CD28 costimulatory domain comprises an amino acid sequence comprising: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 11).

[0052] In certain embodiments, the CD28 costimulatory domain is encoded by a nucleic acid sequence comprising: agaagcaagcggagccggctgctgcacagcgactacatgaacatgacccctagacggcccggacctaccagaaagcactaccag ccttacgctcctcctagagacttcgccgcctaccggtcc (SEQ ID NO: 12).

[0053] The TACI costimulatory domain may comprise the amino acid sequence KKRGDPCSCQPRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQES AVTPGTPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGA (SEQ ID NO: 13). In some embodiments, the TACI costimulatory domain comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence KKRGDPCSCQPRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQES AVTPGTPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGA (SEQ ID NO: 13).

[0054] In certain embodiments, the TACI costimulatory domain is encoded by a nucleic acid comprising the sequenceaaaaagcggggcgatccttgctcctgccagccaagatcaagacccagacagtctcctgccaagtcctctcaggaccacgccatggaaAttorney Docket No.: 000218-0156-WO1gctggcagccctgtgtctacatctcccgagccagtggaaacatgctccttttgtttccctgagtgtcgggccccaactcaagaaagcgc agtgacaccaggcactccagatccaacctgcgctggccgatggggatgccatacaagaacaaccgtgctccaaccatgtccacacat tcccgatagcggactgggaattgtgtgtgtccccgcacaagaaggtggcccaggggcc (SEQ ID NO: 14). The TACI costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

[0055] In some aspects, a CAR disclosed herein comprises a hinge region between the transmembrane domain and the ectodomain. In certain embodiments, the hinge region may comprise a sequence derived from a human CD8a, IgG4, and / or CD4 sequence. In certain embodiments of the VCARs of the disclosure, the hinge region may comprise a sequence derived from a human CD8a sequence. The hinge region may comprise the amino acid sequenceTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9). In some embodiments, the hinge region comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9). The human CD8a hinge region may be encoded by a nucleic acid comprising the sequence actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 10).

[0056] In some aspects, a CAR provided herein further comprises a signal peptide. In certain embodiments, the signal peptide may comprise a sequence encoding a human CD2 signal peptide, a human CD36 signal peptide, a human CD3s signal peptide, a human CD3y signal peptide, a human CD3(^ signal peptide, a human CD4 signal peptide, a human CD8a signal peptide, a human CD19 signal peptide, a human CD28 signal peptide, a human 4-1BB signal peptide or a human GM-CSFR signal peptide. In certain embodiments of the VCARs of the disclosure, the signal peptide may comprise a human CD8a signal peptide. The human CD8a signal peptide may comprise the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 1). In some embodiments, the signal peptide comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 1). The human CD8a signal peptide may be encoded by a nucleic acid comprising the sequence atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 2).

[0057] VCARs of the disclosure may bind an antigen with an affinity of a KD of less than or equal to 10'9M, less than or equal to 10'10M, less than or equal to 10'11M, less than or equalAttorney Docket No.: 000218-0156-WO1to 10'12M, less than or equal to 10'13M, less than or equal to 10'14M, and less than or equal to 10’15M. The KD may be determined by surface plasmon resonance.

[0058] In certain embodiments, a CAR as disclosed comprises an ectodomain comprising an antigen recognition region that binds to a specific antigen. In certain embodiments, a CAR as disclosed herein comprises an ectodomain comprising an antigen recognition region that binds to CD70, CD7, BCMA, CD 19, CD20, MUC1, PSMA or a combination thereof. In certain embodiments of the VCARs of the disclosure, the VCAR comprises a single domain antibody, a VHH (also known as a nanobody), a VH, or a combination thereof. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a recombinant sequence. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a chimeric sequence. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a human sequence. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a humanized sequence.

[0059] In certain embodiments, an antigen recognition region may comprise two VHs to produce a bispecific or tandem VCAR. The two VHs present in a tandem VCAR may bind to the same antigen or to different antigens.

[0060] In certain embodiments of the VCARs of the disclosure, the VCAR comprises a single domain antibody. In some embodiments, the single domain antibody is a VHH or a VH antibody. In some embodiments, the VH antibody is a UniDab antibody. In some embodiments, VH antibody is not a fragment of a naturally occurring monoclonal antibody.

[0061] In certain embodiments, the VH comprises a human or a humanized sequence. In certain embodiments, the VH comprises a non-naturally occurring sequence. In certain embodiments, the VH is not naturally occurring. In certain embodiments, the VH comprises a recombinant or chimeric sequence. In certain embodiments, the VH is produced by an in vitro procedure of affinity selection and recombination.Cells and Modified Cells

[0062] In another aspect, provided herein are cells modified to comprise the CARs (e.g., VCAR or dual CARs) provided herein. Cells and modified cells of the disclosure can be mammalian cells. In some embodiments, the cells and modified cells are human cells. In some embodiments, the cell is not a germ cell. In some embodiments, the cell is not a human germ cell.

[0063] In some embodiments, cells and modified cells of the disclosure can be somatic cells. In some embodiments, cells and modified cells of the disclosure can be differentiated cells. InAttorney Docket No.: 000218-0156-WO1some embodiments, cells and modified cells of the disclosure can be autologous cells or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment following administration to a subject. Allogeneic cells may be any type of cell. Allogeneic cells can be stem cells or can be derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0064] Cells and modified cells of the disclosure can be immune cells. The immune cells of the disclosure can be iPSCs, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T-cell), stem memory T cells (TSCM cells), central memory T cells (TCM), stem cell-like T cells, B lymphocytes (B-cells), antigen presenting cells (APCs), cytokine induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes or osteoclasts.

[0065] In some embodiments, the cells of the disclosure are immune precursor cells. The immune precursor cells can be any cells that can differentiate into one or more types of immune cells. The immune precursor cells can be multipotent stem cells that can self-renew and develop into immune cells. The immune precursor cells can be hematopoietic stem cells (HSCs) or descendants thereof. The immune precursor cells can be precursor cells that can develop into immune cells. Non-limiting examples of HSC descendent cells include multipotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocyte cells (T-cells), B lymphocyte cells (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes and macrophages.

[0066] In some embodiments, the immune precursor cells are hematopoietic progenitor cells (HPCs). Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent and umbilical cord blood.

[0067] HSCs can be isolated or derived from a primary or cultured stem cell. HSCs can be isolated or derived from, for example, an embryonic stem cell, a multipotent stem cell, a pluripotent stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).

[0068] HSCs produced by the disclosed methods can retain features of “primitive” stem cells that, while isolated or derived from an adult stem cell and while committed to a single lineage, share characteristics of embryonic stem cells. For example, the “primitive” HSCs produced by the disclosed methods may retain their “sternness” following division and may not differentiate. Without wishing to be bound by theory, as an adoptive cell therapy, the “primitive” HSCs produced by the disclosed methods are believed to not only replenish theirAttorney Docket No.: 000218-0156-WO1numbers but also expand in vivo. “Primitive” HSCs produced by the disclosed methods can be therapeutically effective when administered as a single dose.

[0069] Primitive HSCs can be CD34+. Primitive HSCs can be CD34+ and CD38-. Primitive HSCs can be CD34+, CD38- and CD90+. Primitive HSCs can be CD34+, CD38-, CD90+ and CD45RA-. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[0070] Primitive HSCs, HSCs, and / or HSC descendent cells can be modified according to the disclosed methods to express an exogenous sequence (e.g., a chimeric antigen receptor or therapeutic protein). Modified primitive HSCs, modified HSCs, and / or modified HSC descendent cells can be forward differentiated to produce a modified immune cell including, but not limited to, a modified T cell, a modified natural killer cell and / or a modified B-cell.

[0071] The modified immune or immune precursor cells can be NK cells. The NK cells can be cytotoxic lymphocytes that differentiate from lymphoid progenitor cells. Modified NK cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. In some aspects, non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0072] The modified immune or immune precursor cells can be B cells. In some aspects, B cells are a type of lymphocyte that expresses B cell receptors on the cell surface. In some aspects, B cell receptors bind to specific antigens. In some embodiments, modified B cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0073] Modified T cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Without wishing to be bound by theory, unlike traditional biologies and chemotherapeutics, the disclosed modified-T cells may retain the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments. To achieve this, in some embodiments, modified-T cells not only drive an initial response, but also persist in the patient as a stable population of viable memory T cells to prevent potential relapses. Alternatively, in some aspects, when it is not desired, the modified T cells do not persist in the patient.

[0074] In some embodiments, the modified T cell further comprise an inducible safety switch that may be activated by administering to the subject a ligand which results in the death of the modified T cell. In some embodiments, an inducible proapoptotic polypeptide is operably linked to a ligand binding region that may be optimized to bind a chemical inducer of dimerization. In some aspects, when the ligand binding region specifically binds theAttorney Docket No.: 000218-0156-WO1induction agent, proapoptotic target molecules can become cross-linked, and, consequently, activated to selectively induce apoptosis in a cell containing an inducible proapoptotic polypeptide including, but not limited to, inducible caspase polypeptides including inducible caspase 9 (iCas9) polypeptides. In some embodiments, the inducible caspase 9 polypeptides may comprise a truncated caspase 9 polypeptide encoded by a truncated or modified amino acid and / or nucleic acid sequence encoding the truncated caspase 9 polypeptide.

[0075] In some embodiments, the inducible proapoptotic polypeptide can comprise (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible safety switches such as iCas9 are further described in international patent application publication No.WO 2018 / 068022, which is incorporated herein by reference in its entirety for examples of inducible safety switches that may be used in the cells described herein. An illustrative amino acid sequence of an iCas9 safety switch is set forth in SEQ ID NO: 46. In some embodiments, the iCas9 safety switch is encoded by the nucleic acid sequence set forth in SEQ ID NO: 43.

[0076] Intensive efforts have been focused on the development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as of a modified-T cell product containing early memory T cells, especially stem cell memory (TSCM) or stem cell-like T cells. Without wishing to be bound by theory, stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (TCM) T cells or TCM like cells, effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and longterm modified-T cell engraftment. A linear pathway of differentiation may be responsible for generating these cells: Naive T cells (TN) > TSCM > TCM > TEM > TE > TTE, whereby TN is the parent precursor cell that directly gives rise to TSCM, which then, in turn, directly gives rise to TCM, etc. Compositions of T cells of the disclosure can comprise one or more of each parental T cell subset with TSCM cells being the most abundant (e.g., TSCM > TCM > TEM > TE > TTE).

[0077] The immune cell precursor can be differentiated into or is capable of differentiating into an early memory T cell, a stem cell like T-cell, a Naive T cells (TN), a TSCM, a TCM, a TEM, a TE, or a TTE. The immune cell precursor can be a primitive HSC, an HSC, or a HSC descendent cell of the disclosure. The immune cell can be an early memory T cell, a stem cell like T-cell, a Naive T cells (TN), a TSCM, a TCM, a TEM, a TE, or a TTE.Attorney Docket No.: 000218-0156-WO1

[0078] The methods of the disclosure can be used to modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of a plurality of modified T cells in the population expresses one or more cell-surface marker(s) of an early memory T cell. In some embodiments, at least 20% of cells in the population express one or more cell-surface marker(s) of an early memory T cell. In some embodiments, at least 25% of cells in the population express one or more cell-surface marker(s) of an early memory T cell. In some embodiments, at least 50% of cells in the population express one or more cellsurface marker(s) of an early memory T cell. In some embodiments, at least 75% of cells in the population express one or more cell-surface marker(s) of an early memory T cell.Examples of cell surface markers of early memory T cells include CD95, CD45RA, CCR7, CD62L, CD28, CD27, CDlla, CD58, CD122 (IL-2RP), CXCR3, and CD127.

[0079] In some embodiments, the population of modified early memory T cells comprises a plurality of modified stem cell-like T cells. In some embodiments, the population of modified early memory T cells comprises a plurality of modified stem cell memory T cells (TSCM cells). In some embodiments, the population of modified early memory T cells comprises a plurality of modified TCM cells.

[0080] In some aspects, the methods of the disclosure can be used to modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cellsurface marker(s) of a stem cell-like T cell. In some embodiments, the population of modified stem cell-like T cells comprises a plurality of modified TSCM cells. In some embodiments, the population of modified stem cell-like T cells comprises a plurality of modified TCM cells.

[0081] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM) or a TscM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L.

[0082] The cell-surface markers of a TSCM or a TscM-like cell can also comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp. The cell-Attorney Docket No.: 000218-0156-WO1surface markers of a TSCM or a TscM-like cell can also comprise one or more of CD45RA, CD95, IL-2R , CCR7, and CD62L.

[0083] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of a central memory T cell (TCM) or a TcM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L. The cell-surface markers of a TCM or a TcM-like cell can also comprise one or more of CD45RO, CD95, IL-2R0, CCR7, and CD62L.

[0084] The methods of the disclosure can be used to modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of a naive T cell (TN). The cell-surface markers of a TN cell can comprise one or more of CD45RA, CCR7 and CD62L.

[0085] The methods of the disclosure can be used to modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of an effector T-cell (modified TEFF). The cell-surface markers of a modified TEFF can comprise one or more of CD45RA, CD95, and IL-2Rp.

[0086] The methods of the disclosure can modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells of the population expresses one or more cell-surface marker(s) of a stem cell-like T cell, a stem memory T cell (TSCM) or a central memory T cell (TCM).

[0087] The modified cells of the population may comprise a transgene. The transgene may be, for example, a gene encoding a CAR. The populations of cells disclosed herein may be characterized by their expression of certain cell surface markers, for example, CD34, CD90, CD38, CD45RA and CD49f.

[0088] In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the populationAttorney Docket No.: 000218-0156-WO1comprise a transgene, and at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express CD34 (i.e., the cells have the cell-surface marker phenotype CD34+). In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 70% to about 99%, about 75% to about 95% or about 85% to about 95% of the population of modified cells express CD34 (i.e., the cells have the cellsurface marker phenotype CD34+). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express both the protein encoded by the transgene and CD34. In some embodiments, the transgene encodes a CAR.

[0089] In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and wherein at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express CD34 and do not CD38 (i.e., the cells have the cell-surface marker phenotype CD34+ and CD38-). In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 45% to about 90%, about 50% to about 80% or about 65% to about 75% of the population of modified cells express CD34 and do not express CD38 (i.e., the cells have the cell-surface marker phenotype CD34+ and CD38-). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express both the protein encoded by the transgene and CD34, but do not express CD38. In some embodiments, the transgene encodes a CAR.Attorney Docket No.: 000218-0156-WO1

[0090] In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express CD34 and CD90 and do not express CD38 ( / .< ., the cells have the cell-surface marker phenotype CD34+, CD38- and CD90+). In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified cells express CD34 and CD90 and do not express CD38 (i.e., the cells have the cellsurface marker phenotype CD34+, CD38- and CD90+). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express the protein encoded by the transgene, CD34 and CD90, but do not express CD38. In some embodiments, the transgene encodes a CAR.

[0091] In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express CD34 and CD90 and do not express CD38 or CD45RA (i.e., the cells have the cell-Attorney Docket No.: 000218-0156-WO1surface marker phenotype CD34+, CD38-, CD90+, and CD45RA-). In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified cells express CD34 and CD90 and do not express CD38 or CD45RA (i.e., the cells have the cell-surface marker phenotype CD34+, CD38-, CD90+, and CD45RA-). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express the protein encoded by the transgene, CD34 and CD90 but do not express CD38 or CD45RA. In some embodiments, the transgene encodes a CAR.

[0092] In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express CD34, CD90 and CD49f and do not express CD38 or CD45RA (i.e., the cells have the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+). In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 0.02% to about 30%, about 0.02% to about 2%, about 0.04% to about 2% or about 0.04% to about 1% of the population of modified cells express CD34, CD90 and CD49f and do not express CD38 or CD45RA (i.e., the cells have the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+). In some embodiments, at least 70%, at least 75%, at leastAttorney Docket No.: 000218-0156-WO180%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express the protein encoded by the transgene, CD34, CD90 and CD49f, but does not express CD38 or CD45RA. In some embodiments, the transgene encodes a CAR.

[0093] In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells CD34 and CD90 and do not express CD45RA ( / .< ., the cells have the cell-surface marker phenotype CD34+, CD90+ and CD45RA-). In some embodiments, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 0.2% to about 5%, about 0.2% to about 3% or about 0.4% to about 3% of the population of modified cells express CD34 and CD90 and do not express CD45RA (i.e., the cells have the cell-surface marker phenotype CD34+, CD90+ and CD45RA-). In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express both the protein encoded by the transgene, CD34 and CD90, but do not express CD45RA. In some embodiments, the transgene encodes a CAR.

[0094] Compositions and methods of producing and / or expanding the immune cells or immune precursor cells (e.g., the disclosed CAR-T-cells) and buffers for maintaining or enhancing a level of cell viability and / or a stem-like phenotype of the immune cells or immune precursor cells (e.g., the disclosed CAR-T-cells) are disclosed elsewhere herein andAttorney Docket No.: 000218-0156-WO1are disclosed in more detail in U.S. Patent No. 10,329,543 and PCT Publication No.WO 2019 / 173636, each of which is incorporated herein by reference in its entirety.Methods of Introducing Nucleic Acids into Cells

[0095] In some embodiments, a CAR cell can be produced by introducing a nucleic acid encoding the CAR into the cell. In some embodiments, a transgene and / or genomic editing construct are also introduced into the cell.

[0096] In some aspects, introducing a nucleic acid sequence, transgene and / or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise one or more of topical delivery, adsorption, absorption, electroporation, spinfection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetofection or by nanoparticle-mediated delivery. In some aspects, introducing a nucleic acid sequence, transgene and / or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In some aspects, introducing a nucleic acid sequence, transgene and / or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ by mechanical transfection can comprise cell squeezing, cell bombardment, or gene gun techniques. In some aspects, introducing a nucleic acid sequence, transgene and / or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ by nanoparticle-mediated transfection can comprise liposomal delivery, delivery by micelles, and delivery by polymerosomes.

[0097] Gene editing tools can also be delivered to cells using one or more poly(histidine)-based micelles. Poly(histidine) (e.g., poly(L-histidine)), is a pH-sensitive polymer due to the imidazole ring providing an electron lone pair on the unsaturated nitrogen. That is, poly(histidine) may have amphoteric properties through protonation-deprotonation. In particular, at certain pHs, poly(histidine)-containing triblock copolymers may assemble into a micelle with positively charged poly(histidine) units on the surface, thereby enabling complexing with the negatively-charged gene editing molecule(s). Without wishing to be bound by theory, using these nanoparticles to bind and release proteins and / or nucleic acids in a pH-dependent manner may provide an efficient and selective mechanism to perform a desired gene modification. Without wishing to be bound by theory, this micelle-based delivery system provides substantial flexibility with respect to the charged materials, as well as a large payload capacity, and targeted release of the nanoparticle payload. In one example, site-specific cleavage of the double stranded DNA is enabled by delivery of a nuclease using the poly(histidine)-based micelles. Without wishing to be bound by a particular theory, it isAttorney Docket No.: 000218-0156-WO1believed that in the micelles that are formed by the various triblock copolymers, the hydrophobic blocks aggregate to form a core, leaving the hydrophilic blocks and poly(histidine) blocks on the ends to form one or more surrounding layer.

[0098] In an aspect, the disclosure provides triblock copolymers made of a hydrophilic block, a hydrophobic block, and a charged block. In some aspects, the hydrophilic block may be poly(ethylene oxide) (PEO), and the charged block may be poly(L-histidine). An example triblock copolymer that can be used is a PEO-b-PLA-b-PHIS, with variable numbers of repeating units in each block varying by design.

[0099] Diblock copolymers that can be used as intermediates for making triblock copolymers can have hydrophilic biocompatible polyethylene oxide) (PEO), which is chemically synonymous with PEG, coupled to various hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(ortho esters), poly(peptides), poly(phosphazenes) and poly(saccharides), including but not limited by poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(s-caprolactone) (PCL), and poly (trimethylene carbonate) (PTMC). In some embodiments, polymeric micelles comprised of 100% PEGylated surfaces possess improved in vitro chemical stability, augmented in vivo bioavailability, and prolonged blood circulatory half-lives.

[0100] Polymeric vesicles, polymersomes and poly(Histidine)-based micelles, including those that comprise triblock copolymers, and methods of making the same, are described in further detail in U.S. Patent Nos. 7,217,427; 7,868,512; 6,835,394; 8,808,748; 10,456,452; U.S. Publication Nos. 2014 / 0363496; 2017 / 0000743; and 2019 / 0255191; and PCT Publication No. WO 2019 / 126589, each of which is incorporated herein by reference in its entirety.

[0101] Introducing a nucleic acid sequence, transgene and / or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise a non-viral vector. The non-viral vector can comprise a nucleic acid encoding a CAR. The non-viral vector can comprise plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmids, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). The non-viral vector can comprise a transposon as described herein, such as a piggyBac transposon.

[0102] Introducing a nucleic acid sequence, transgene and / or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise a viral vector. The viral vector can be a non-integrating non-chromosomal vector. Non-limiting examples of non-integrating non-chromosomal vectors include adeno-associated virus (AAV), adenovirus, and herpesAttorney Docket No.: 000218-0156-WO1viruses. The viral vector can be an integrating chromosomal vector. Non-limiting examples of integrating chromosomal vectors include adeno-associated vectors (AAV), Lentiviruses, and gamma-retroviruses.

[0103] Introducing a nucleic acid sequence, transgene and / or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise a combination of vectors. Non-limiting examples of vector combinations include viral and non-viral vectors, a plurality of non-viral vectors, or a plurality of viral vectors. Non-limiting examples of vector combinations include a combination of a DNA-derived and an RNA-derived vector, a combination of an RNA and a reverse transcriptase, a combination of a transposon and a transposase, a combination of a non-viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.

[0104] Genome modification can comprise introducing a nucleic acid sequence, transgene and / or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ to stably integrate a nucleic acid sequence, transiently integrate a nucleic acid sequence, produce sitespecific integration of a nucleic acid sequence, or produce a biased integration of a nucleic acid sequence. The nucleic acid sequence can encode a CAR.

[0105] The nucleic acid sequence or transgene can be about 1 kb to about 15 kb in size. The nucleic acid sequence or transgene can be at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 11 kb, at least 12 kb, at least 13 kb, at least 14 kb, at least 15 kb in size. The nucleic acid sequence or transgene can be about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb or about 15 kb in size.

[0106] In some embodiments, another means for introducing a nucleic acid encoding a CAR includes using a transposon system. The present disclosure provides a transposon comprising a nucleic acid encoding a CAR. In a preferred aspect, the transposon is a plasmid DNA transposon comprising a nucleotide sequence encoding the CAR (e.g., VCAR) as disclosed herein flanked by two cis -regulatory insulator elements. In some embodiments, the present disclosure also provides a composition comprising a transposon. In a preferred aspect, the composition comprising the transposon can further comprise a plasmid comprising a nucleotide sequence encoding a transposase. The nucleotide sequence encoding the transposase may be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.Attorney Docket No.: 000218-0156-WO1

[0107] A transposon of the present disclosure can be a piggyBac™ (PB) transposon. In some aspects when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase a piggyBac-like (PBL) transposase or a Super piggyBac™ (SPB) transposase. In some embodiments, the sequence encoding the SPB transposase is an mRNA sequence.

[0108] Non-limiting examples of PB transposons and PB, PBL and SPB transposases are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No.8,399,643 and PCT Publication No. WO 2010 / 099296, each of which is incorporated herein by reference in its entirety for examples of transposon systems that may be used in the methods described herein.

[0109] In some embodiments, the PB, PBL and SPB transposases recognize transposonspecific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and inserts the contents between the ITRs at the sequence 5’-TTAT-3’ within a chromosomal site (a TTAT target sequence) or at the sequence 5’-TTAA-3’ within a chromosomal site (a TTAA target sequence). The target sequence of the PB or PBL transposon can comprise or consist of 5’-CTAA-3’, 5’-TTAG-3’, 5’-ATAA-3’, 5’-TCAA-3’, 5’AGTT-3’, 5 ’-ATTA-3’,5’-GTTA-3’, 5’-TTGA-3’, 5’-TTTA-3’, 5’-TTAC-3’, 5’-ACTA-3’, 5’-AGGG-3’,5 ’-CT AG-3’, 5’-TGAA-3’, 5’-AGGT-3’, 5’-ATCA-3’, 5’-CTCC-3’, 5 ’-T AAA-3’,5’-TCTC-3’, 5’TGAA-3’, 5’-AAAT-3’, 5’-AATC-3’, 5’-ACAA-3’, 5’-ACAT-3’,5’-ACTC-3’, 5’-AGTG-3’, 5 ’-AT AG-3’, 5 ’-C AAA-3’, 5’-CACA-3’, 5 ’-C ATA-3’,5’-CCAG-3’, 5’-CCCA-3’, 5’-CGTA-3’, 5’-GTCC-3’, 5’-TAAG-3’, 5’-TCTA-3’,5’-TGAG-3’, 5’-TGTT-3’, 5’-TTCA-3’5’-TTCT-3’ and 5’-TTTT-3’. In some aspects, thePB or PBL transposon system has no payload limit for the genes of interest that can be included between the ITRs.

[0110] Exemplary amino acid sequence for one or more PB, PBL and SPB transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810 and U.S. Patent No.8,399,643, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the compositions and methods described herein. Illustrative sequences of transposases and transposons that may be used in methods described herein are also shown in Table 1 below.

[0111] Table 1: Illustrative Transposon and Transposase SequencesAttorney Docket No.: 000218-0156-WO1Attorney Docket No.: 000218-0156-WO1Attorney Docket No.: 000218-0156-WO1<<<< <<<<<Attorney Docket No.: 000218-0156-WO1Attorney Docket No.: 000218-0156-WO1Attorney Docket No.: 000218-0156-WO1<<<<<<Attorney Docket No.: 000218-0156-WO1

[0112] In some embodiments, the PB transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15:MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTS SGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEI YAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIR PTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSK YGIKILMMCDSGYKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCD NWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVS YKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSR KTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRK RLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCK KCKKVICREHNIDMCQSCF (SEQ ID NO: 15).

[0113] In some embodiments, the PB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the PB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15.

[0114] The PB or PBL transposase can also comprise or consist of the amino acid sequence of SEQ ID NO: 15 with amino acid substitution at positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 15. The transposase can comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 15 comprising one, two, three or all of the following amino acid substitutions: BOV, G165S, M282V, N538K.

[0115] In some embodiments, the SPB transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16:MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTS SGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEI YAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIR PTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKY GIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDN WFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSY KPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRK TNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKR LEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKK CKKVICREHNIDMCQSCF (SEQ ID NO: 16).

[0116] In some embodiments, the SPB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 with one, two, three, four or five conservative aminoAttorney Docket No.: 000218-0156-WO1acid substitutions. In some embodiments, the SPB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16.

[0117] In certain aspects wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the transposases can further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 15 or SEQ ID NO: 16. Such mutations are described in more detail in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of mutations that may be introduced into the transposases disclosed herein.

[0118] The PB, PBL or SPB transposases can be isolated or derived from an insect, vertebrate, crustacean or urochordate as described in more detail in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods and compositions disclosed herein. In preferred aspects, the PB, PBL or SPB transposases can be isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0119] In some aspects, a hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred aspect, a hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No.6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and WO 2019 / 17363, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods and compositions disclosed herein. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077, which is incorporated herein by reference in its entirety for examples of mutations that may be introduced into the transposases disclosed herein.

[0120] In some aspects, the PB or PBL transposase is integration deficient. In some aspects, an integration deficient PB or PBL transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wildtype transposase. Examples of integration deficient PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods andAttorney Docket No.: 000218-0156-WO1compositions disclosed herein. A list of integration deficient amino acid substitutions is disclosed in US patent No. 10,041,077, which is incorporated herein by reference in its entirety for examples of mutations that may be introduced into the transposases disclosed herein.

[0121] In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods and compositions disclosed herein.

[0122] A transposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example as disclosed in U.S. Patent No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase. In some embodiments, the Sleeping Beauty transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the Sleeping Beauty transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the Sleeping Beauty transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, a hyperactive Sleeping Beauty (SB100X) transposase comprises or consists of an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the sequence set forth in SEQ ID NO: 18. In some embodiments, the SB100X transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the SB100X transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18.

[0123] A transposon of the present disclosure can be a Helraiser transposon. An exemplary Helraiser transposon includes Helibatl. In some embodiments, the Helibatl transposon comprises or consists of a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, the Helibatl transposon comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 19. In some aspects, when the transposon is a Helraiser transposon (e.g., a HelibatlAttorney Docket No.: 000218-0156-WO1transposon), the transposase is a Helitron transposase (for example, as disclosed in WO 2019 / 173636). In some embodiments, the Helitron transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the Helitron transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the Helitron transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20.

[0124] A transposon of the present disclosure can be a Tol2 transposon. A Tol2 transposon may include inverted repeats, and subterminal sequences. In some embodiments, the Tol2 transposon comprises or consists of a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, the Tol2 transposon comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 21. In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in WO 2019 / 173636). In some embodiments, the Tol2 transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the Tol2 transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the Tol2 transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0125] A transposon of the present disclosure can be a TcBuster transposon. In some aspects, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (for example, as disclosed in WO 2019 / 173636). The TcBuster transposase can comprise or consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence. In some embodiments, a TcBuster transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the TcBuster transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the TcBuster transposase comprises or consists of theAttorney Docket No.: 000218-0156-WO1amino acid sequence set forth in SEQ ID NO: 23. The polynucleotide encoding a TcBuster transposase can comprise or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence. In some embodiments, a TcBuster transposase is encoded by a polynucleotide comprising or consisting of an nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 24. In some embodiments, the TcBuster transposase is encoded by the nucleic acid sequence set forth in SEQ ID NO: 24.

[0126] In some aspects, a mutant TcBuster transposase comprises one or more sequence variations when compared to a wild type TcBuster transposase as described in more detail in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety for examples of mutations that may be introduced into the transposases disclosed herein.

[0127] The transposon can be a nanotransposon. A nanotransposon can comprise, consist essentially of, or consist of (a) a sequence encoding a transposon insert, comprising a sequence encoding a first inverted terminal repeat (ITR), a sequence encoding a second inverted terminal repeat (ITR), and an intra-ITR sequence; (b) a sequence encoding a backbone, wherein the sequence encoding the backbone comprises a sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints, and a sequence encoding a selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints, and (c) an inter-ITR sequence. In some aspects, the inter-ITR sequence of (c) comprises the sequence of (b). In some aspects, the intra-ITR sequence of (a) comprises the sequence of (b).

[0128] The sequence encoding the backbone can comprise between 1 and 600 nucleotides, inclusive of the endpoints. In some aspects, the sequence encoding the backbone consists of between 1 and 50 nucleotides, between 50 and 100 nucleotides, between 100 and 150 nucleotides, between 150 and 200 nucleotides, between 200 and 250 nucleotides, between 250 and 300 nucleotides, between 300 and 350 nucleotides, between 350 and 400 nucleotides, between 400 and 450 nucleotides, between 450 and 500 nucleotides, between 500 and 550 nucleotides, between 550 and 600 nucleotides, each range inclusive of the endpoints.

[0129] The inter-ITR sequence can comprise between 1 and 1000 nucleotides, inclusive of the endpoints. In some aspects, the inter-ITR sequence consists of between 1 and 50 nucleotides, between 50 and 100 nucleotides, between 100 and 150 nucleotides, between 150 and 200Attorney Docket No.: 000218-0156-WO1nucleotides, between 200 and 250 nucleotides, between 250 and 300 nucleotides, between 300 and 350 nucleotides, between 350 and 400 nucleotides, between 400 and 450 nucleotides, between 450 and 500 nucleotides, between 500 and 550 nucleotides, between 550 and 600 nucleotides, between 600 and 650 nucleotides, between 650 and 700 nucleotides, between 700 and 750 nucleotides, between 750 and 800 nucleotides, between 800 and 850 nucleotides, between 850 and 900 nucleotides, between 900 and 950 nucleotides, or between 950 and 1000 nucleotides, each range inclusive of the endpoints.

[0130] The nanotransposon can be a short nanotransposon (SNT), wherein the inter-ITR sequence comprises between 1 and 200 nucleotides, inclusive of the endpoints. The inter-ITR sequence can consist of between 1 and 10 nucleotides, between 10 and 20 nucleotides, between 20 and 30 nucleotides, between 30 and 40 nucleotides, between 40 and 50 nucleotides, between 50 and 60 nucleotides, between 60 and 70 nucleotides, between 70 and 80 nucleotides, between 80 and 90 nucleotides, or between 90 and 100 nucleotides, each range inclusive of the endpoints.

[0131] The selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints, can comprise a sequence encoding a sucrose-selectable marker. The sequence encoding a sucrose-selectable marker can comprise a sequence encoding an RNA-OUT sequence. The sequence encoding an RNA-OUT sequence can comprise or consist of 137 base pairs (bp). The selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints, can comprise a sequence encoding a fluorescent marker. The selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints, can comprise a sequence encoding a cell surface marker.

[0132] The sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints, can comprise a sequence encoding a mini origin of replication. In some aspects, the sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints, comprises a sequence encoding an R6K origin of replication. The R6K origin of replication can comprise an R6K gamma origin of replication. The R6K origin of replication can comprise an R6K mini origin of replication. The R6K origin of replication can comprise an R6K gamma mini origin of replication. The R6K gamma mini origin of replication can comprise or consist of 281 base pairs (bp).

[0133] In some aspects of the nanotransposon, the sequence encoding the backbone does not comprise a recombination site, an excision site, and / or a ligation site. In some aspects, neither the nanotransposon nor the sequence encoding the backbone comprises a product of a recombination site, an excision site, and / or a ligation site. In some aspects, neither theAttorney Docket No.: 000218-0156-WO1nanotransposon nor the sequence encoding the backbone is derived from a recombination site, an excision site, and / or a ligation site.

[0134] In some aspects of the nanotransposon, a recombination site comprises a sequence resulting from a recombination event. In some aspects, a recombination site comprises a sequence that is a product of a recombination event. In some aspects, the recombination event comprises an activity of a recombinase (e.g., a recombinase site).

[0135] In some aspects of the nanotransposon, the sequence encoding the backbone does not further comprise a sequence encoding foreign DNA.

[0136] In some aspects of the nanotransposon, the inter-ITR sequence does not comprise a recombination site, an excision site, a ligation site or a combination thereof. In some aspects, the inter-ITR sequence does not comprise a product of a recombination event, an excision event, a ligation event or a combination thereof. In some aspects, the inter-ITR sequence is not derived from a recombination event, an excision event, a ligation event or a combination thereof. In some aspects, the inter-ITR sequence comprises a sequence encoding foreign DNA. In some aspects, the intra-ITR sequence comprises at least one sequence encoding an insulator and a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell. The mammalian cell can be a human cell. In some aspects, the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell and a second sequence encoding an insulator. In some aspects, the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, a polyadenosine (poly A) sequence and a second sequence encoding an insulator. In some aspects, the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, at least one exogenous sequence, a polyadenosine (poly A) sequence and a second sequence encoding an insulator.

[0137] In certain aspects, a transposon cassette comprises a nucleic acid sequence encoding a CAR and a CD70 shRNA as illustrated in FIGS. 1 A and IB. In certain aspects, as shown in FIG. IB, the transposon cassette comprises a left end ITR, an EFla promoter operably associated with the nucleic acids encoding: an iCas9 safety switch, a CAR, a DHFR mutein selectable marker, a CD70 shRNA; and a right end ITR. In certain aspects, a transposon cassette is constructed via Gibson assembly. Without wishing to be bound by a particular theory, the overexpression of shRNA mediated by U6 promoter (FIG. 1 A) may result in cellular toxicity (data not shown). Expression from a multi -ci stronic transgene under controlAttorney Docket No.: 000218-0156-WO1of the single promoter (e.g., the EFla promoter in Fig. IB) may reduce overexpression of the shRNA and reduce cellular toxicity.

[0138] Nanotransposons are described in more detail in International Patent Application Publication No. WO 2020 / 132396, which is incorporated herein by reference in its entirety for examples for nanotransposons that may be used in the methods and compositions described herein.

[0139] Furthermore, vectors may be used to introduce nucleic acids into cells. A vector of the present disclosure can be a viral vector or a recombinant vector. Viral vectors can comprise a sequence isolated or derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof. The viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses comprise two or more inverted terminal repeat (ITR) sequences located in cis next to a sequence encoding an scFv or a CAR of the disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03.

[0140] A vector of the present disclosure can be a nanoparticle. Non-limiting examples of nanoparticle vectors include nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof ), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymersomes), micelles, lipids (e.g., liposomes), organic molecules (e.g., carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g., calcium phosphate or gold) or any combination thereof. A nanoparticle vector can be passively or actively transported across a cell membrane.Attorney Docket No.: 000218-0156-WO1Gene Editing Compositions and Methods for Targeted Genome Editing at Selected Locus

[0141] Genome modification can comprise introducing a nucleic acid sequence, transgene and / or a genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ to stably integrate a nucleic acid sequence. The stable chromosomal integration can be a random integration, a site-specific integration, or a biased integration. The site-specific integration can be non-assisted or assisted. In some embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In some embodiments, the site-directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. In some embodiments, the transgene with homologous nucleotide extensions enables genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end-joining. The site-specific integration can occur at a safe harbor site. Genomic safe harbor sites are sites in the genome that are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements are expressed (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome, which can pose a risk to the host organism. Non-limiting examples of potential genomic safe harbors include intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

[0142] Alternatively, the site-specific transgene integration can occur at a site that disrupts expression of a target gene. Disruption of target gene expression can occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. Non-limiting examples of target genes targeted by site-specific integration include TRAC, TRAB, PDI, any gene encoding an immunosuppressive protein, and genes encoding proteins involved in allo-rej ection.

[0143] Further alternatively, the site-specific transgene integration can occur at a site that results in enhanced expression of a target gene. Enhancement of target gene expression can occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0144] Enzymes can be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. Enzymes can create single-strand breaks or double-strandAttorney Docket No.: 000218-0156-WO1breaks. Non-limiting examples of break-inducing enzymes include transposases, integrases, endonucleases, CRISPR / Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVER™, and CPF1. Break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, or as a nucleoprotein complex with a guide RNA (gRNA). Non-limiting examples of break-inducing enzymes are described in International Patent Application Publications No. WO 2016 / 205554, No.WO 2019 / 126578, and No. WO 2018 / 064681, each of which is incorporated by reference in its entirety for examples of gene editing compositions that may be used in the methods disclosed herein. Exemplary mutant Cas-CLOVER break-inducing enzymes of the disclosure are also described below.

[0145] The site-specific transgene integration can be controlled by a vector-mediated integration site bias.

[0146] The site-specific transgene integration site can be a non-stable chromosomal insertion. The integrated transgene can be become silenced, removed, excised, or further modified. The genome modification can be a non-stable integration of a transgene. The non-stable integration can be a transient non-chromosomal integration, a semi-stable non chromosomal integration, a semi -persistent non-chromosomal insertion, or a non-stable chromosomal insertion. The transient non-chromosomal insertion can be epi-chromosomal or cytoplasmic. In one aspect, the transient non-chromosomal insertion of a transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.

[0147] The genome modification can be a semi-stable or persistent non-chromosomal integration of a transgene. A DNA vector encodes a scaffold / matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of a non-viral vector allowing for autonomous replication in the nucleus of dividing cells.

[0148] The genome modification can be a non-stable chromosomal integration of a transgene. The integrated transgene can become silenced, removed, excised, or further modified.

[0149] The transgene can comprise a sequence encoding for a therapeutic agent. The therapeutic agent can be a protein or an RNA that provides a therapeutic benefit when administered to a cell or a subject. The therapeutic agent can be a therapeutic protein or a therapeutic RNA. The therapeutic agent can be human beta-globin (HBB), T87Q human betaglobin (HBB T87Q), BAF chromatin remodeling complex subunit (BCL11 A) shRNA, insulin like growth factor 2 binding protein 1 (IGF2BP1), interleukin 2 receptor gamma (IL2RG), alpha galactosidase A (GLA), alpha-L-idurondase (IDUA), iduronate 2-sulfatase (IDS), cystinosin lysosomal cysteine transporter (CTNS). The transgene can comprise aAttorney Docket No.: 000218-0156-WO1nucleic acid sequence encoding Factor VIII or Factor IX. The transgene can comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

[0150] The transgene can comprise a nucleic acid sequence encoding a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical. In one aspect, the transgene can comprise a sequence for a CAR. In one aspect, the transgene comprising a CAR that specifically binds to CD70, CD7, BCMA, CD 19 CD20, MUC1, PSMA or a combination thereof. The transgene can comprise a sequence encoding for an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. The transgene can be integrated into the genome of the HSC. The integration can be stable or transient.

[0151] In gene editing systems that involve inserting new or existing nucleotides / nucleic acids, insertion tools e.g., DNA template vectors, transposable elements (transposons or retrotransposons) must be delivered to the cell in addition to the cutting enzyme (e.g., a nuclease, recombinase, integrase or transposase). Examples of such insertion tools for a recombinase may include a DNA vector. Other gene editing systems require the delivery of an integrase along with an insertion vector, a transposase along with a transposon / retrotransposon, etc. An example recombinase that may be used as a cutting enzyme is the CRE recombinase. Non-limiting examples of integrases that may be used in insertion tools include viral based enzymes taken from any of a number of viruses including AAV, gamma retrovirus, and lentivirus. Examples transposons / retrotransposons that may be used in insertion tools are described in more detail herein.

[0152] The present disclosure provides methods of targeted genome editing comprising introducing a gene editing composition and / or a cell comprising the gene editing composition. The gene editing composition can comprise a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. The sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof can comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or the nuclease domain thereof can comprise one or more of a CRISPR / Cas protein,Attorney Docket No.: 000218-0156-WO1a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease.dCas9-Clo051 (Cas-CLOVER) fusion proteins

[0153] Examples of fusion proteins that may be used for the gene editing methods disclosed herein include fusion proteins comprising a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise a Clo051 nuclease or a nuclease domain thereof. The gene editing composition can further comprise a guide sequence. The guide sequence may comprise an RNA sequence. Illustrative sequences for gene editing system components are also shown in Table 2 below.

[0154] The disclosure provides compositions comprising a small, Cas9 (Cas9) operatively linked to an effector. The disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, Cas9 (Cas9). A small Cas9 construct of the disclosure can further comprise an effector comprising a type IIS endonuclease. In some embodiments, the fusion protein comprises a Staphylococcus aureus Cas9 with an active catalytic site comprising the amino acid sequence of SEQ ID NO: 25.

[0155] The disclosure provides compositions comprising an inactivated, small, Cas9 (dSaCas9) operatively linked to an effector. The disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, inactivated Cas9 (dSaCas9). A small, inactivated Cas9 (dSaCas9) construct of the disclosure can further comprise an effector comprising a type IIS endonuclease. In some embodiments, the fusion protein comprises a dSaCas9 comprising the amino acid sequence of SEQ ID NO: 26, which includes a D10A and a N580A mutation to inactivate the catalytic site.

[0156] The disclosure provides compositions comprising an inactivated Cas9 (dCas9) operatively linked to an effector. The disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9). An inactivated Cas9 (dCas9) construct of the disclosure can further comprise an effector comprising a type IIS endonuclease.

[0157] The dCas9 can be isolated or derived from Streptococcus pyogenes. The dCas9 can comprise the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28. The dCas9 can also comprise amino acid substitutions at amino acid positions 10 and 840 of SEQ ID NO: 27 orAttorney Docket No.: 000218-0156-WO128, which inactivate the catalytic site. In some aspects, these substitutions are D10A and H840A.

[0158] An illustrative Clo051 nuclease domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 29. In some aspects, the Clo051 nuclease domain comprises at least one amino acid substitution. In some aspects, the amino acid substitution is in the alpha-helix-loop domain of the Clo051 nuclease. In some aspects, the amino acid substitution is at position 35, 37, 60, 98, 100 or 146 of SEQ ID NO: 29.

[0159] An illustrative dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 30. The illustrative dCas9-Clo051 fusion protein can be encoded by a polynucleotide which comprises, consists essentially of, or consists of, the nucleic acid sequence of SEQ ID NO: 31. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0160] An illustrative dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 32. The illustrative dCas9-Clo051 fusion protein can be encoded by a polynucleotide which comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 33. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0161] A dCas9-Clo051 fusion (Cas-CLOVER) fusion protein of the disclosure may further comprise at least one nuclear localization sequence (NLS). In some embodiments, the dCas9-Clo051 fusion protein of the disclosure comprises at least two nuclear localization sequences. In some embodiments, the NLS is on the N-terminal end of the dCas9-Clo051 fusion protein (NLS-dCas9-Clo051). In some embodiments, the NLS is on the C-terminal end of the dCas9-Clo051 fusion protein (dCas9-Clo051-NLS). In some embodiments, the NLS is on the N-terminal end and at the C-terminal end of the dCas9-Clo051 fusion protein (“NLS-dCas9-Clo051-NLS” or “wildtype Cas-CLOVER”).

[0162] The NLS-dCas9-Clo051-NLS (“wildtype Cas-CLOVER”) fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 30.

[0163] NLS-dCas9-Clo051-NLS amino acid sequence (NLS amino acid sequence is bolded and underlined):MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELL VNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVR ENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAV NVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDKKYSIGLAIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTR RKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQAttorney Docket No.: 000218-0156-WO1TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYI DGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK PAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQK AQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKM KNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQK GNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDI<VLSAYNI<HRDI<PIREQAENIIHLFTLTNLGAPAAFI<YFDTTIDRI<RYTS TKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVSS (SEQ ID NO: 30).

[0164] In some embodiments, the wildtype Cas-CLOVER fusion protein comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the wildtype Cas- CLOVER fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 30 with one, two, three, four or five conservative amino acid substitutions.

[0165] The nucleic acid encoding the NLS-dCas9-Clo051-NLS (“wildtype Cas-CLOVER”) fusion protein can be DNA or RNA. In some embodiments, a dCas9-Clo051 fusion protein comprising two NLS regions is encoded by a DNA sequence comprising, consisting essentially of, or consisting of SEQ ID NO: 34.

[0166] DNA sequence encoding the NLS-dCas9-Clo051-NLS (sequence encoding the NLS is bolded and underlined): atggcaccaaagaagaaaagaaaagtggagggcatcaagtcaaacatcagcctgctgaaagacgaactgcggggacagattagt cacatcagtcacgagtacctgtcactgattgatctggccttcgacagcaagcagaatagactgtttgagatgaaagtgctggaactgctg gtcaacgagtatggcttcaagggcagacatctgggcgggtctaggaaacctgacggcatcgtgtacagtaccacactggaagacaac ttcggaatcattgtcgataccaaggcttattccgagggctactctctgccaattagtcaggcagatgagatggaaaggtacgtgcgcga aaactcaaatagggacgaggaagtcaaccccaataagtggtgggagaatttcagcgaggaagtgaagaaatactacttcgtctttatct caggcagcttcaaagggaagtttgaggaacagctgcggagactgtccatgactaccggggtgaacggatctgctgtcaacgtggtca atctgctgctgggcgcagaaaagatcaggtccggggagatgacaattgaggaactggaacgcgccatgttcaacaattctgagtttat cctgaagtatggaggcgggggaagcgataagaaatactccatcggactggccattggcaccaattccgtgggctgggctgtcatcac agacgagtacaaggtgccaagcaagaagttcaaggtcctggggaacaccgatcgccacagtatcaagaaaaatctgattggagccct gctgttcgactcaggcgagactgctgaagcaacccgactgaagcggactgctaggcgccgatatacccggagaaaaaatcggatctAttorney Docket No.: 000218-0156-WO1gctacctgcaggaaattttcagcaacgagatggccaaggtggacgatagtttctttcaccgcctggaggaatcattcctggtggaggaa gataagaaacacgagcggcatcccatctttggcaacattgtggacgaagtcgcttatcacgagaagtaccctactatctatcatctgagg aagaaactggtggactccaccgataaggcagacctgcgcctgatctatctggccctggctcacatgatcaagttccgggggcattttct gatcgagggagatctgaaccctgacaattctgatgtggacaagctgttcatccagctggtccagacatacaatcagctgtttgaggaaa acccaattaatgcctcaggcgtggacgcaaaggccatcctgagcgccagactgtccaaatctaggcgcctggaaaacctgatcgctc agctgccaggagagaagaaaaacggcctgtttgggaatctgattgcactgtccctgggcctgacacccaacttcaagtctaattttgatc tggccgaggacgctaagctgcagctgtccaaagacacttatgacgatgacctggataacctgctggctcagatcggcgatcagtacg cagacctgttcctggccgctaagaatctgagtgacgccatcctgctgtcagatattctgcgcgtgaacacagagattactaaggcccca ctgagtgcttcaatgatcaaaagatatgacgagcaccatcaggatctgaccctgctgaaggctctggtgaggcagcagctgcccgag aaatacaaggaaatcttctttgatcagagcaagaatggatacgccggctatattgacggcggggcttcccaggaggagttctacaagtt catcaagcccattctggaaaagatggacggcaccgaggaactgctggtgaagctgaatcgggaggacctgctgagaaaacagagg acatttgataacggaagcatccctcaccagattcatctgggcgaactgcacgccatcctgcgacggcaggaggacttctacccatttct gaaggataaccgcgagaaaatcgaaaagatcctgaccttcagaatcccctactatgtggggcctctggcacggggaaatagtagattt gcctggatgacaagaaagtcagaggaaactatcaccccctggaacttcgaggaagtggtcgataaaggcgctagcgcacagtccttc attgaaaggatgacaaattttgacaagaacctgccaaatgagaaggtgctgcccaaacacagcctgctgtacgaatatttcacagtgtat aacgagctgactaaagtgaagtacgtcaccgaagggatgcgcaagcccgcattcctgtccggagagcagaagaaagccatcgtgg acctgctgtttaagacaaatcggaaagtgactgtcaaacagctgaaggaagactatttcaagaaaattgagtgtttcgattcagtggaaat cagcggcgtcgaggacaggtttaacgcctccctggggacctaccacgatctgctgaagatcatcaaggataaggacttcctggacaa cgaggaaaatgaggacatcctggaggacattgtgctgacactgactctgtttgaggatcgcgaaatgatcgaggaacgactgaagact tatgcccatctgttcgatgacaaagtgatgaagcagctgaaaagaaggcgctacaccggatggggacgcctgagccgaaaactgatc aatgggattagagacaagcagagcggaaaaactatcctggactttctgaagtccgatggcttcgccaacaggaacttcatgcagctga ttcacgatgactctctgaccttcaaggaggacatccagaaagcacaggtgtctggccagggggacagtctgcacgagcatatcgcaa acctggccggcagccccgccatcaagaaagggattctgcagaccgtgaaggtggtggacgaactggtcaaggtcatgggacgaca caaacctgagaacatcgtgattgagatggcccgcgaaaatcagacaactcagaagggccagaaaaacagtcgagaacggatgaag agaatcgaggaaggcatcaaggagctggggtcacagatcctgaaggagcatcctgtggaaaacactcagctgcagaatgagaaact gtatctgtactatctgcagaatggacgggatatgtacgtggaccaggagctggatattaacagactgagtgattatgacgtggatgccat cgtccctcagagcttcctgaaggatgactccattgacaacaaggtgctgaccaggtccgacaagaaccgcggcaaatcagataatgt gccaagcgaggaagtggtcaagaaaatgaagaactactggaggcagctgctgaatgccaagctgatcacacagcggaaatttgata acctgactaaggcagaaagaggaggcctgtctgagctggacaaggccggcttcatcaagcggcagctggtggagacaagacagat cactaagcacgtcgctcagattctggatagcagaatgaacacaaagtacgatgaaaacgacaagctgatcagggaggtgaaagtcat tactctgaaatccaagctggtgtctgactttagaaaggatttccagttttataaagtcagggagatcaacaactaccaccatgctcatgac gcatacctgaacgcagtggtcgggaccgccctgattaagaaataccccaagctggagtccgagttcgtgtacggagactataaagtgt acgatgtccggaagatgatcgccaaatctgagcaggaaattggcaaggccaccgctaagtatttcttttacagtaacatcatgaatttcttt aagaccgaaatcacactggcaaatggggagatcagaaaaaggcctctgattgagaccaacggggagacaggagaaatcgtgtggg acaagggaagggattttgctaccgtgcgcaaagtcctgtccatgccccaagtgaatattgtcaagaaaactgaagtgcagaccgggg gattctctaaggagagtattctgcctaagcgaaactctgataaactgatcgcccggaagaaagactgggaccccaagaagtatggcgg gttcgactctccaacagtggcttacagtgtcctggtggtcgcaaaggtggaaaaggggaagtccaagaaactgaagtctgtcaaagag ctgctgggaatcactattatggaacgcagctccttcgagaagaatcctatcgattttctggaagccaagggctataaagaggtgaagaa agacctgatcattaagctgccaaaatactcactgtttgagctggaaaacggacgaaagcgaatgctggcaagcgccggagaactgca gaagggcaatgagctggccctgccctccaaatacgtgaacttcctgtatctggctagccactacgagaaactgaaggggtcccctgag gataacgaacagaagcagctgtttgtggagcagcacaaacattatctggacgagatcattgaacagatttcagagttcagcaagagag tgatcctggctgacgcaaatctggataaagtcctgagcgcatacaacaagcaccgagacaaaccaatccgggagcaggccgaaaat atcattcatctgttcaccctgacaaacctgggcgcccctgcagccttcaagtattttgacaccacaatcgatcggaagagatacacttcta ccaaagaggtgctggatgctaccctgatccaccagagtattaccggcctgtatgagacacgcatcgacctgtcacagctgggaggcg atgggagccccaagaaaaagcggaaggtgtctagttaatga (SEQ ID NO: 34).gRNAs

[0167] In some aspects, the Cas-CLOVER fusion proteins described above are used in conjunction with a guide sequence. A guide sequence in the context of a Cas-Clover systemAttorney Docket No.: 000218-0156-WO1or a CRISPR-Cas9 system can be any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. The guide sequence may form a duplex with a target sequence. The duplex may be a DNA duplex, an RNA duplex, or a RNA / DNA duplex. The terms “guide molecule” and “guide RNA” and “single guide RNA” are used interchangeably herein to refer to RNA-based molecules that are capable of forming a complex with a Cas-Clover or a CRISPR-Cas protein and comprises a guide sequence. The guide molecule or guide RNA may encompass RNA-based molecules having one or more chemically modifications (e.g., by chemical linking two ribonucleotides or by replacement of one or more ribonucleotides with one or more deoxyribonucleotides), as described herein.

[0168] The guide RNA can comprise a sequence complementary to a target sequence within a genomic DNA sequence. The target sequence within a genomic DNA sequence can be a target sequence within a safe harbor site of a genomic DNA sequence. Exemplary target sequences include but are not limited to HBB, TRAC, B2M, TCRb, GAPDH or SOX17.

[0169] The guide RNA can comprise a sequence complementary to at least one target sequence on a transposon, plasmid or vector. In some aspects, the complementary sequence to the guide RNA on the transposon, plasmid or vector is located within the transgene for targeted nucleic acid insertion. In some aspects, the complementary sequence to the guide RNA on the transposon, plasmid or vector is located within the transgene for targeted nucleic acid insertion. In some aspects, the complementary sequence on the transposon, plasmid or vector facilitates binding of a gRNA which is bound to an effector molecule, thereby tethering all components.

[0170] The term “target region”, “target sequence” or “protospacer” as used interchangeably herein refers to the region of the target gene to which the Cas-Clover system or the CRISPR / Cas9-based system targets. The Cas-Clover or the CRISPR / Cas9-based system may include more than one gRNA, wherein the gRNAs target different DNA sequences. The target DNA sequences may be overlapping. The Cas-Clover system may include at least two gRNAs, wherein the gRNAs target different DNA sequences. The target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer. Different Type II systems have differing PAM requirements. For example, the S. pyogenes Type II system uses an “NGG” sequence, where “N” can be any nucleotide.

[0171] The guide RNA or the guide RNA of a Cas-Clover protein or a CRISPR-Cas protein may comprise a tracr-mate sequence (encompassing a “direct repeat” in the context of anAttorney Docket No.: 000218-0156-WO1endogenous CRISPR system) and a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system). In some embodiments, the Cas-Clover or the CRISPR-Cas system or complex as described herein does not comprise and / or does not rely on the presence of a tracr sequence. In certain embodiments, the guide molecule may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.

[0172] In certain embodiments, the guide sequence or spacer length of the guide molecules is 15 to 50 nucleotides in length. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides in length. In certain embodiments, the spacer length is from 15 to 17 nucleotides in length, from 17 to 20 nucleotides in length, from 20 to 24 nucleotides in length, from 23 to 25 nucleotides in length, from 24 to 27 nucleotides in length, from 27-30 nucleotides in length, from 30-35 nucleotides in length, or greater than 35 nucleotides in length.

[0173] In some embodiments, the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length.

[0174] In some embodiments, the sequence of the guide molecule (direct repeat and / or spacer) is selected to reduce the degree secondary structure within the guide molecule. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide RNA participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

[0175] As described above, the Cas-Clover system and the CRISPR / Cas9 system utilizes targeting gRNA and a shuttling gRNA that provides the targeting of the Cas-Clover system and the CRISPR / Cas9-based system. The gRNA may be a fusion of two noncoding RNAs: a crRNA and a tracrRNA. The sgRNA may target any desired DNA sequence by exchangingAttorney Docket No.: 000218-0156-WO1the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics the naturally occurring crRNA: tracrRNA duplex involved in the Type II Effector system. This duplex, which may include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide for the Cas9 to cleave the target nucleic acid.

[0176] In some embodiment, the gRNA targets a region upstream of the target gene (e.g., B2M gene locus), e.g., between 0-1000 bp upstream of a target gene. In some embodiments, the gRNA targets a region between 0-50 bp, 0-100 bp, 0-150 bp, 0-200 bp, 0-250 bp, 0-300 bp, 0-350 bp, 0-400 bp, 0-450 bp, 0-500 bp, 0-550 bp, 0-600 bp, 0-650 bp, 0-700 bp, 0-750 bp, 0-800 bp, 0-850 bp, 0-900 bp, 0-950 bp or 0-1000 bp upstream of the transcription start site of the target gene. In some embodiments, the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp or about 1500 bp upstream of the target gene.

[0177] In some embodiments, the gRNA targets a region downstream of a target gene (e.g., B2M gene locus), e.g., between 0-1000 bp downstream of a target gene. In some embodiments, the gRNA targets a region between 0-50 bp, 0-100 bp, 0-150 bp, 0-200 bp, 0-250 bp, 0-300 bp, 0-350 bp, 0-400 bp, 0-450 bp, 0-500 bp, 0-550 bp, 0-600 bp, 0-650 bp, 0-700 bp, 0-750 bp, 0-800 bp, 0-850 bp, 0-900 bp, 0-950 bp or 0-1000 bp downstream of the target gene. In some embodiments, the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp or about 1500 bp downstream of the target gene.

[0178] gRNA can be divided into a target binding region and a Cas9 binding region. The target binding region hybridizes with a target region in a target gene. Methods for designing such target binding regions are known in the art, see, e.g., Doench et al., Nat Biotechnol. (2014) 32: 1262-7; and Doench et al., Nat Biotechnol. (2016) 34: 184-91, incorporated by reference herein in their entirety. Design tools are available at, e.g., Feng Zhang lab's target Finder, Michael Boutros lab's Target Finder (E-CRISP), RGEN Tools (Cas-OF Finder), CasFinder, and CRISPR Optimal Target Finder. In certain embodiments, the target binding region can be between about 15 and about 50 nucleotides in length (about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length). In certain embodiments, the target binding region can be between about 19 and about 21 nucleotides in length. In oneAttorney Docket No.: 000218-0156-WO1embodiment, the target binding region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[0179] In one embodiment, the target binding region is complementary, e.g., completely complementary, to the target region in the target gene. In one embodiment, the target binding region is substantially complementary to the target region in the target gene. In one embodiment, the target binding region comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are not complementary to the target region in the target gene. As used throughout the disclosure, the term “substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0180] Exemplary sgRNAs of the disclosure include but are not limited to sequences for targeting HBB, B2M, TRAC or GAPDH gene locus. Exemplary sgRNAs of the disclosure comprise, consist essentially of or consists of the sequences as shown in Table 3.

[0181] In certain aspects, the cells of the disclosure are modified cells. The cells of the disclosure can be modified by the methods of targeted genome editing described herein or known in the art.

[0182] Modulation” or “regulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. A reduction of a level of expression or activity of a gene is when at least 50%, at least 75%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the expression of the gene in a cell or the functional activity of the gene in a cell is reduced when compared to the naturally occurring wild-type counterpart of the cell.

[0183] In certain aspects, the cells of the disclosure comprise a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M). In certain aspects, the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I). In certain aspects, the cells of the disclosure comprise a modification of an endogenous sequence encoding a T-cell Receptor (TCR). In certain aspects, the modification reduces or eliminates a level of expression or activity of the TCR. In certain embodiments, the TCR is TCRb. In certain aspects, the cells are modified using a Cas-Clover system and sgRNA in Table 2.Attorney Docket No.: 000218-0156-WO1Table 2: Illustrative Sequences for Gene Editing<<< <<<<< <<<<<<<<Attorney Docket No.: 000218-0156-WO1<<<<< <<<<<<<<<<Attorney Docket No.: 000218-0156-WO1Attorney Docket No.: 000218-0156-WO1<<<<Attorney Docket No.: 000218-0156-WO1Attorney Docket No.: 000218-0156-WO1<<<<Attorney Docket No.: 000218-0156-WO1Attorney Docket No.: 000218-0156-WO1Attorney Docket No.: 000218-0156-WO1

[0184] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in PCT Application Numbers PCT / US2024 / 016659, PCT / US2016 / 037922, PCT / US2018 / 066941, PCT / US2017 / 054799, U.S. Patent Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185 and U.S. Patent No.Attorney Docket No.: 000218-0156-WO110,415,024, each of which is incorporated herein by reference in its entirety. Exemplary gene editing compositions including mutant Cas-CLOVER and methods of using these compositions for gene editing are described herein.Methods of Expressing a CAR

[0185] The disclosure provides methods of expressing a CAR or dual CAR on the surface of a cell. In some aspects, the method comprises (a) obtaining a cell population; (b) contacting the cell population with a composition comprising a CAR or dual CAR, or with a sequence encoding the CAR or dual CAR, under conditions sufficient to transfer the CAR or dual CAR across a cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the sequence encoding the CAR or dual CAR; and (d) expanding and / or selecting at least one cell from the modified cell population that express the CAR or dual CAR on the cell surface.

[0186] In some aspects, the cell population can comprise leukocytes, including, for example, CD4+ and / or CD8+ leukocytes. The cell population can comprise CD4+ and CD8+ leukocytes in an optimized ratio. In some embodiments, the optimized ratio of CD4+ to CD8+ leukocytes does not naturally occur in vivo. The cell population can also comprise a tumor cell.

[0187] In some aspects, the conditions sufficient to transfer the CAR or the sequence encoding the CAR, transposon, or vector across a cell membrane of at least one cell in the cell population comprises at least one of an application of one or more pulses of electricity at a specified voltage, a buffer, and one or more supplemental factor(s). In some aspects, the conditions suitable for integration of the sequence encoding the CAR comprise at least one of a buffer and one or more supplemental factor(s).

[0188] The buffer can comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucleofection buffer or any combination thereof. The one or more supplemental factor(s) can comprise (a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof; (b) a salt, a mineral, a metabolite or any combination thereof; (c) a cell medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and (e) a reagent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, the chemokine, the interleukin or any combination thereof can comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11,Attorney Docket No.: 000218-0156-WO1IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-lFl, IL-1 beta / IL-lF2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F Heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L or any combination thereof. The salt, the mineral, the metabolite or any combination thereof can comprise HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS / FCS, Human serum, serumsubstitute, antibiotics, pH adjusters, Earle’s Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgCh, Na2HPC>4, NAH2PO4, Sodium lactobionate, Mannitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NC>3)2, Tris / HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell medium can comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium or any combination thereof. The inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof comprise inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspasel, Pro-ILIB, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3p (GSK-3 P) (e.g. TWS119), or any combination thereof. Examples of such inhibitors can include Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK or any combination thereof. The reagent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without a NLS sequence, a TREX1 enzyme or any combination thereof.

[0189] The expansion and selection steps can occur concurrently or sequentially. The expansion can occur prior to selection. The expansion can occur following selection, and, optionally, a further (i.e. second) selection can occur following expansion. Concurrent expansion and selection can be simultaneous. The expansion and / or selection steps can proceed for a period of 10 to 14 days, inclusive of the endpoints.Attorney Docket No.: 000218-0156-WO1

[0190] The expansion can comprise contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell through the CAR, thereby generating an expanded cell population. The antigen can be presented on the surface of a substrate. The substrate can have any form, including, but not limited to a surface, a well, a bead or a plurality thereof, and a matrix. The substrate can further comprise a paramagnetic or magnetic component. The antigen can be presented on the surface of a substrate, wherein the substrate is a magnetic bead, and wherein a magnet can be used to remove or separate the magnetic beads from the modified and expanded cell population. The antigen can be presented on the surface of a cell or an artificial antigen presenting cell. Artificial antigen presenting cells can include, but are not limited to, tumor cells and stem cells.

[0191] In some aspects, wherein the transposon or vector comprises a selection gene, the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying a cell expressing the selection gene as surviving the selection and identifying a cell failing to express the selection gene as failing to survive the selection step.

[0192] The disclosure provides a composition comprising the modified, expanded and selected cell population of the methods described herein.

[0193] A more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in PCT Publication No. WO 2020 / 051374, which is incorporated herein by reference in its entirety for examples of methods that may be used to modify the cells disclosed herein.Methods of Use

[0194] The disclosure also provides the use of a disclosed composition or pharmaceutical composition for the treatment of a disease or disorder in a subject, e.g., cancer or an autoimmune disorder, as known in the art or as described herein, using the disclosed compositions and pharmaceutical compositions, e.g., administering to the subject a therapeutic effective amount of the composition or pharmaceutical composition comprising cells expressing a CAR or dual CAR). In one aspect, the subject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[0195] The disclosure provides a method for modulating or treating cancer in a cell, tissue, organ, animal or subject. Non-limiting examples of a cancer include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia, chronicAttorney Docket No.: 000218-0156-WO1myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin’s lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome / hypercalcemia of malignancy, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer related bone resorption, cancer related bone pain, and the like.

[0196] In certain aspects, provided herein are methods of treating multiple myeloma (MM). MM is a hematological malignancy characterized by the uncontrolled proliferation of monoclonal plasma cells in the bone marrow. Despite advancements in therapeutic strategies, including the use of immunomodulatory agents, proteasome inhibitors, and monoclonal antibodies, relapsed and refractory multiple myeloma (RRMM) remains a significant clinical challenge.

[0197] In certain aspects, the CAR cells of the present disclosure are modified to recombinantly express dihydrofolate reductase (DHFR), which advantageously renders the CAR cells resistant to methotrexate (MTX). The MTX resistant CAR cells (“MTX-CAR cells”) may be used in methods of treating a subject in need thereof in combination with subsequent MTX administration to eliminate activated T-cells and NK cells targeting the MTX-CAR cells thereby increasing the in vivo persistence and efficacy of the MTX-CAR cells in the subject. Modified cells can be formulated for storage at any temperature including room temperature and body temperature. Modified cells can be formulated for cryopreservation and subsequent thawing. Modified cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. Modified cells can be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and / or CAR expression level to ensure a minimal level of cell function and CAR expression. Modified cells can be formulated in a pharmaceutically acceptable carrier at a prescribed density with one or more reagents to inhibit further expansion and / or prevent cell death. The CAR may comprise a wildtype form of the DHFR gene or a mutein thereof. An illustrative amino acid sequence of a DHFR mutein is set forth in SEQ ID NOs: 44 and 47.Attorney Docket No.: 000218-0156-WO1

[0198] The methods of the present disclosure comprise administering an effective amount of MTX to a subject at a predetermined time post administration of the MTX CAR cells. As used herein as it relates to the methods herein, “an effective dose” of MTX is a dose that is sufficient to eliminate the subject’s activated T-cells and NK cells targeting the previously administered MTX-CAR cells but spares the MTX-CAR cells. An exemplary effective amount of MTX is an MTX serum level in a subject of about 200 nM, which equates to serum levels calculated for low dose MTX therapies for autoimmune disorder patients. As disclosed herein, the MTX-CAR cells of the present disclosure are resistant to MTX levels well in excess of 200 nM.

[0199] The methods can optionally further comprise co-administration or combination therapy for treating such cancer, wherein the administering of any composition or pharmaceutical composition disclosed herein, further comprises administering, before concurrently, and / or after, at least one chemotherapeutic agent (e.g., an alkylating agent, a mitotic inhibitor, a radiopharmaceutical).

[0200] In some aspects, the subject does not develop graft vs. host (GvH) and / or host vs. graft (HvG) following administration. In one aspect, the administration is systemic. Systemic administration can be any means known in the art and described in detail herein. Preferably, systemic administration is by an intravenous injection or an intravenous infusion. In one aspect, the administration is local. Local administration can be any means known in the art and described in detail herein. Preferably, local administration is by intra-tumoral injection or infusion, intraspinal injection or infusion, intracerebroventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.

[0201] In some aspects, the therapeutically effective dose of the MTX-CAR cells is a single dose. In some aspects, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 doses that are manufactured simultaneously. In some aspects, where the composition is autologous cells or allogeneic cells, the dose is an amount sufficient for the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0202] In one example, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising a VCAR that specifically binds to an antigen on a tumor cell. In aspects where the composition comprises a modified cell or cell population, the cell or cell population may be autologous or allogeneic.

[0203] In some aspects of the methods of treatment described herein, the treatment can be modified or terminated. Specifically, in aspects where the composition used for treatmentAttorney Docket No.: 000218-0156-WO1comprises an inducible proapoptotic polypeptide (iCASp9 or iC9), apoptosis may be selectively induced in the cell by contacting the cell with an induction agent. A treatment may be modified or terminated in response to, for example, a sign of recovery or a sign of decreasing disease severity / progression, a sign of disease remission / cessation, and / or the occurrence of an adverse event. In some aspects, the method comprises the step of administering an inhibitor of the induction agent to inhibit modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (for example, when a sign or symptom of the disease reappear or increase in severity and / or an adverse event is resolved).

[0204] In certain aspects, provided are methods of increasing in vivo persistence of MTX-CAR cells in a subject comprising administering to the subject a therapeutically effective amount of MTX-CAR cells; and administering at a predetermined time post administration of the MTX-CAR cells an effective amount of MTX sufficient to eliminate (reduce by at least 25%) activated T-cells and NK cells targeting the MTX-CAR cells leading to an increased in vivo persistence of the MTX-CAR cells. In certain embodiments, the MTX-CAR cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product. In certain embodiments, the oncogenic gene product is CD70, CD7, BCMA, CD 19, CD20, MUC1, PSMA or a combination thereof. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX-CAR-T cells).

[0205] In certain aspects, provided are methods of increasing in vivo persistence of MTX-CAR-T -T cells in a subject comprising administering to the subject a therapeutically effective amount of MTX-CAR-T cells, wherein the MTX-CAR-T cells comprise a heterologous nucleic acid encoding dihydrofolate reductase (DHFR); and, administering at a predetermined time post administration of the MTX-CAR-T cells, an effective amount of MTX sufficient to eliminate (reduce by X) activated T-cells and NK cells targeting the MTX-CAR-T cells leading to an increased in vivo persistence of the MTX-CAR cells. In certain embodiments, the MTX-CAR-T cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product. In certain embodiments, the oncogenic gene product is CD70, CD7, BCMA, CD19, CD20, MUC1, PSMA or a combination thereof. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX-CAR-T cells).

[0206] In certain aspects, provided are methods of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of MTX-CAR-T cells; and administering at a predetermined time post administration of the MTX-CAR-T cells an effective amount of MTX sufficient to eliminate (reduce by X) activated T-cells and NK cells targeting the CAR cells leading to an increased in vivo persistence of the MTX-Attorney Docket No.: 000218-0156-WO1CAR-T cells and increased treatment efficacy of the MTX-CAR-T cells compared to no MTX administration. In certain embodiments, the MTX-CAR-T cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product. In certain embodiments, the oncogenic gene product is CD70, CD7, BCMA, CD 19, CD20, MUC1, PSMA or a combination thereof. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX-CAR-T cells).

[0207] In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 10 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 15 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 20 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 30 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 40 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 50 minutes.

[0208] In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 1 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 2 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 3 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 4 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 5 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 6 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 7 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 8 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 9 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 10 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 11 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 12 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 13 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 14 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 15 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 16 hr. In certain embodiments, the predetermined time post administration ofAttorney Docket No.: 000218-0156-WO1the MTX-CAR cells is about 17 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 18 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 19 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 20 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 21 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 22 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 23 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 24 hr.

[0209] In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 2 days. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 3 days. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 4 days. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 5 days. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 6 days.

[0210] In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 1 week. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 2 weeks. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 3 weeks. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 1 month.

[0211] In certain embodiments, the effective amount of MTX at a predetermined time post administration of the MTX-CAR cells is a single dose. In certain embodiments, the single dose is administered on Day 3 post-administration of the MTX-CAR cells. In certain embodiments, the single dose is administered on Day 6 post-administration of the MTX-CAR cells. In certain embodiments, the effective amount of MTX at a predetermined time post administration of the MTX-CAR cells is a multi-dose regiment. In certain embodiments, two doses are administered on Days 3 and 6 post-administration of the MTX-CAR cells. In certain embodiments, three doses are administered on Days 1, 3, and 6 post-administration of the MTX-CAR cells. In certain embodiments, four doses are administered on Days 1, 3, 6 and 11 post-administration of the MTX-CAR cells. In certain embodiments, eight doses are administered on Days 1, 3, 6, 11, 14, 18, 22 & 26 post-administration of the MTX-CAR cells.

[0212] In certain aspects, provided are methods of treating cancer in a subject in need thereof as described above further comprising administering at a predetermined time an effectiveAttorney Docket No.: 000218-0156-WO1dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR cells. In certain aspects, the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR cells is one day (Day -1) prior to administration of the MTX-CAR cells. In certain aspects, the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR cells is two days (Day -2) prior to administration of the MTX-CAR cells. In certain aspects, the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR cells is three days (Day -3) prior to administration of the MTX-CAR cells. In certain aspects, the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR cells is four days (Day -4) prior to administration of the MTX-CAR cells. In certain aspects, the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR cells is five days (Day -5) prior to administration of the MTX-CAR cells.Nucleic Acids and Vectors

[0213] In another aspect, provided herein are nucleic acids and vectors comprising the nucleic acids provided herein. The term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid. A vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

[0214] The isolated nucleic acid compositions of this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some aspects, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra, or Sambrook, supra).

[0215] Methods of amplification of RNA or DNA are well known in the art and can be used according to the disclosure without undue experimentation, based on the teaching and guidance presented herein.Attorney Docket No.: 000218-0156-WO1

[0216] Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Pat. Nos.4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 to Innis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 to Gyllensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, et al; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediated amplification that uses anti-sense RNA to the target sequence as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with the tradename NASBA), the entire contents of which references are incorporated herein by reference. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0217] For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the disclosure and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.

[0218] The isolated nucleic acids of the disclosure can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra . Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.Recombinant Expression Cassettes

[0219] The disclosure further provides recombinant expression cassettes comprising a nucleic acid or polynucleotide of the disclosure. A nucleic acid or polynucleotide sequence of the disclosure can be used to construct a recombinant expression cassette that can be introducedAttorney Docket No.: 000218-0156-WO1into at least one desired host cell. A recombinant expression cassette may comprise a polynucleotide of the disclosure operably linked to transcriptional initiation regulatory sequences that can direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the disclosure.

[0220] In some aspects, isolated nucleic acid or polynucleotide sequences that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in the intron) of a non-heterologous form of a polynucleotide of the disclosure so as to up or down regulate expression of a polynucleotide of the disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and / or substitution.Expression Vectors and Host Cells

[0221] The disclosure also provides to vectors that include isolated nucleic acid or polynucleotide molecules of the disclosure, host cells that are genetically engineered with the recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra, Ausubel, et al., supra, each entirely incorporated herein by reference.

[0222] The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[0223] The DNA insert may be operatively linked to an appropriate promoter. The expression constructs may further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs can include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.

[0224] Expression vectors may include at least one selectable marker. Such markers can include, e.g., ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos.Attorney Docket No.: 000218-0156-WO15,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokaryotes (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the above-described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0225] Expression vectors may include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure can comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably the selectable cell surface marker distinguishes those cells modified by a composition or method of the disclosure from those cells that are not modified by a composition or method of the disclosure. Such cell surface markers can include, e.g., but are not limited to, “cluster of designation” or “classification determinant” proteins (often abbreviated as “CD”) such as a truncated or full-length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers may further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8): 1277-87).

[0226] Expression vectors can include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCE, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0227] Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the disclosure.Alternatively, nucleic acids of the disclosure can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding a protein scaffold of the disclosure. Such methods are well known in the art, e.g., as described in U.S. Pat. Nos.5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.Attorney Docket No.: 000218-0156-WO1

[0228] Illustrative of cell cultures useful for the production of proteins disclosed herein are bacterial, yeast, and mammalian cells as known in the art. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, and include the COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Agl4, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va.(www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2 / 0-Agl4 cells (ATCC Accession Number CRL-1851). In a preferred aspect, the recombinant cell is a P3X63Ab8.653 or an SP2 / 0-Agl4 cell.

[0229] Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (EFla Promoter) (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer, and / or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al., supra, Sambrook, et al., supra. Other cells useful for production of nucleic acids or proteins of the present disclosure are known and / or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources. An illustrative sequence encoding the EFla promoter is set forth in SEQ ID NO: 42.

[0230] When eukaryotic host cells are employed, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.

[0231] In certain aspects, provided herein are nucleic acids or polynucleotides comprising in the 5’ to 3’ direction: a nucleic acid sequence encoding iCas9 safety switch; a nucleic acidAttorney Docket No.: 000218-0156-WO1sequence encoding a chimeric antigen receptor (CAR) as disclosed herein; and a nucleic acid encoding a selectable marker.

[0232] In certain aspects, provided herein are nucleic acid or polynucleotides comprising in the 5’ to 3’ direction: a promoter; a nucleic acid sequence encoding iCas9 safety switch; a nucleic acid sequence encoding one or more chimeric antigen receptor (CAR) as disclosed herein; and a nucleic acid encoding a selectable marker.

[0233] In certain aspects, the nucleic acid sequence encoding iCas9 safety switch further comprises a 3’ 2A sequence. In certain aspects, the nucleic acid sequence encoding a chimeric antigen receptor (CAR) further comprises a 3’ 2A sequence.

[0234] In certain aspects, the promoter is the EFla promoter. In certain embodiments, the EFla promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 42. In certain aspects, the iCas9 safety switch comprises the nucleic acid sequence set forth in SEQ ID NO: 43. In certain aspects, the iCas9 safety switch comprises the amino acid sequence set forth in SEQ ID NO: 46. In certain aspects, the selectable marker is a recombinant dihydrofolate reductase mutein gene (mDHFR). In certain aspects, the mDHFR comprises the nucleic acid sequence set forth in SEQ ID NO: 44. In certain aspects, the mDHFR comprises the amino acid sequence set forth in SEQ ID NO: 47. In certain aspects, the one or more CAR targets a gene product comprising CD70, CD7, BCMA, CD 19, CD20, MUC1, PSMA or a combination thereof.

[0235] In certain aspects, provided are transposons comprising any of the polynucleotides of the present disclosure. In one embodiment, the transposon is a piggyBac transposon. In one embodiment, the piggyBac left end ITR of the piggyBac transposon comprises the nucleic acid sequence set forth in SEQ ID NO: 41. In one embodiment, the piggyBac right end ITR of the piggyBac transposon comprises the nucleic acid sequence set forth in SEQ ID NO: 45.

[0236] In certain aspects, provided are vectors comprising any of the polynucleotides of the present disclosure. In one embodiment, the vector comprises any of the transposons of the present disclosure.

[0237] In certain aspects, provided are cells comprising the polynucleotides, transposons or vectors of the present disclosure. In one embodiment, the cell expresses an iCas9 safety switch, one or more functional CAR as disclosed herein, and a selectable marker.

[0238] In certain aspects, provided are compositions comprising a T-cell population isolated from at least one healthy human donor; wherein the T-cell population expresses one or more chimeric antigen receptor as disclosed herein, and a selectable marker.Attorney Docket No.: 000218-0156-WO1

[0239] In certain aspects, provided are compositions comprising a T-cell population isolated from a patient having a disease or disorder; wherein the T-cell population expresses one or more chimeric antigen receptor as disclosed herein and mDHFR.

[0240] Illustrative sequences for the proteins that may be used in the constructs and methods described herein are set forth in Table 3.Table 3: Illustrative Nucleic Acid and Related Protein for use in the Polynucleotides, Transposons, Vectors and Cells of the Present DisclosureAttorney Docket No.: 000218-0156-WO1<<<<Definitions

[0241] As used throughout the disclosure, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0242] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more standard deviations. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.Attorney Docket No.: 000218-0156-WO1

[0243] The disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various aspects, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the disclosure or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0244] The disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout the disclosure, the term "fragment" refers to a portion of the DNA sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a DNA sequence comprising coding sequences may encode protein fragments that retain biological activity of the native protein and hence DNA recognition or binding activity to a target DNA sequence as herein described. Alternatively, fragments of a DNA sequence that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a DNA sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the disclosure.

[0245] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.Attorney Docket No.: 000218-0156-WO1

[0246] “Gene expression” refers to the conversion of the information contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, micro-RNA, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

[0247] The term “operatively linked” or its equivalents (e.g., “linked operatively”) means two or more molecules are positioned with respect to each other such that they are capable of interacting to affect a function attributable to one or both molecules or a combination thereof. As used throughout the disclosure, the term "operably linked" refers to the expression of a gene that is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between a promoter and a gene can be accommodated without loss of promoter function.

[0248] Non-covalently linked components and methods of making and using non-covalently linked components, are disclosed. The various components may take a variety of different forms as described herein. For example, non-covalently linked (i.e., operatively linked) proteins may be used to allow temporary interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate enables a functional association only or primarily under circumstances where such association is needed for the desired activity. The linkage may be of duration sufficient to allow the desired effect.

[0249] In some aspects of the present disclosure, a method for directing proteins to a specific locus in a genome of an organism is disclosed. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are capable of operatively linking via a non-covalent linkage.

[0250] A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.Attorney Docket No.: 000218-0156-WO1

[0251] Nucleic acids of the disclosure may be single- or double-stranded. Nucleic acids of the disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. Nucleic acids of the disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. Nucleic acids of the disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof. Nucleic acids of the disclosure may contain combinations of deoxyribo- and ribo-nucleotides. Nucleic acids of the disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids of the disclosure may be synthesized to comprise non-natural amino acid modifications. Nucleic acids of the disclosure may be obtained by chemical synthesis methods or by recombinant methods.

[0252] Nucleic acids of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Nucleic acids of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic acids of the disclosure may contain modified, artificial, or synthetic nucleotides that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring.

[0253] Given the redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotide sequences are contemplated herein.

[0254] As used throughout the disclosure, the term "promoter" refers to a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter,Attorney Docket No.: 000218-0156-WO1SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 Alpha promoter, CAG promoter, or SV40 late promoter and the CMV IE promoter.

[0255] A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In an aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference.

[0256] Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0257] As used herein, “conservative” amino acid substitutions may be defined as set out in Tables A, B, or C below. In some aspects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 5.Attorney Docket No.: 000218-0156-WO1Table 5: Conservative Amino Acid Substitutions I

[0258] Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in Table 6.Table 6: Conservative Amino Acid Substitutions II

[0259] Alternately, exemplary conservative substitutions are set out in Table 7.Attorney Docket No.: 000218-0156-WO1Table 7: Conservative Amino Acid Substitutions III

[0260] It should be understood that the polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution. As used throughout the disclosure, “sequence identity” may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). The terms "identical" or "identity" when used in the context of twoAttorney Docket No.: 000218-0156-WO1or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0261] As used throughout the disclosure, the term "endogenous" refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.

[0262] As used throughout the disclosure, the term "exogenous" refers to nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non-naturally occurring genome location.

[0263] The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. By "introducing" is intended presenting to the cell the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell. The methods of the disclosure do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.Attorney Docket No.: 000218-0156-WO1EXAMPLES

[0264] The Examples in this section are provided for illustration and are not intended to limit the invention.Example 1: shRNA-mediated depletion of CD70

[0265] Anti-CD70 CAR-T cells expressing a tool VH binder targeting CD70 were generated either without disruption of the CD70 gene (WT), with Cas-CLOVER mediated gene editing at the CD70 locus (KO) or with various miRNA-embedded shRNA sequences included in the transgene. See FIG. IB for configuration of shRNA 22, 448, 735, 776, and 782. For shRNA 22Front (22F), the configuration in FIG. IB was used except that the shRNA was placed at the front of the transgene, immediately next to the EFla promoter and upstream of iC9. CD70 expression on final product CAR-T cells was assessed by flow cytometry at the end of production with comparison to a flourescence-minus-one (FMO) control. shRNA 776 demonstrated the lowest level of CD70 expression as compared to the other shRNAs tested (FIGs. 2A, 2B and 2C)Example 2: In Vivo Analysis of shRNAs for depletion of CD70 in an Anti-CD70 CAR-T

[0266] The potency of anti-CD70 CAR-T cells produced as described in Example 1 was assessed in vivo against the Nomo-1 AML xenograft cell line, which endogenously expresses CD70. NSG mice were inoculated with Nomo-1 tumor cells and then treated with anti-CD70 CAR-T cells. Tumor burden was monitored by bioluminescent imaging (BLI) and compared to control mice that did not receive CAR-T treatment (PBS) (FIG.3). shRNA #776 (SEQ ID NO: 49) provided the greatest potency compared to the other shRNA and outperformed both WT and CC-edited CD70 CAR-T cells in vivo.Example 3: Robust In Vivo Tumor Control by Anti-CD70 CAR-T cells with shRNA depletion of CD70 by shRNA 776

[0267] Anti-CD70 CAR-T cells, expressing a tool VH binder targeting CD70, were produced either with no disruption of CD70 expression (WT) or with depletion of CD70 expression via Cas-CLOVER gene editing (CC) or via an anti-CD70 miRNA-embedded shRNA (shRNA) incorporated in the transgene under a common promoter (see FIG. IB). CD70 expression on final product CAR-T cells was measured by flow cytometry with a fluorescence-minus-one (FMO) staining control (FIG. 4A). shRNA-mediated depletion of CD70 results in lessAttorney Docket No.: 000218-0156-WO1residual CD70 surface expression and less CD70 related fratricide (as evidenced by the potency demonstrated in Example 3).

[0268] The potency of anti-CD70 CAR-T cells produced as described in Example 1 was assessed in vivo against the Nomo-1 AML xenograft cell line, which endogenously expresses CD70. NSG mice were inoculated with Nomo-1 tumor cells and then treated with anti-CD70 CAR-T cells. Tumor burden was monitored by bioluminescent imaging (BLI) and compared to control mice that did not receive CAR-T treatment (PBS) (FIG. 4B). shRNA-mediated depletion of CD70 with shRNA776 (SEQ ID NO: 49) results in a CAR-T product with greater potency, outperforming both WT and CC-edited CD70 CAR-T cells in vivo.

Claims

Attorney Docket No.: 000218-0156-WO1CLAIMSWe claim:

1. A polynucleotide comprising:(a) a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and (b) a nucleic acid sequence encoding a CD70 shRNA, wherein the CD70 shRNA comprises the sequence set forth in SEQ ID NO: 49.

2. A transposon comprising:(a) a nucleic acid sequence encoding a CAR; and(b) a nucleic acid encoding an CD70 shRNA, wherein the CD70 shRNA comprises the sequence set forth in SEQ ID NO: 49.

3. The polynucleotide of claim 1 or the transposon of claim 2, wherein the CAR comprises (a) an ectodomain comprising an antigen recognition region that binds an antigen; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.

4. The polynucleotide or transposon of claim 3, wherein the ectodomain of (a) further comprises a signal peptide and / or a hinge between the antigen recognition region and the transmembrane domain.

5. The polynucleotide or transposon of claim 3, wherein the signal peptide and / or transmembrane domain comprises a sequence encoding a human CD2, CD36, CD3s, CD3y, CD3(^, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide and / or transmembrane domain.

6. The polynucleotide or transposon of claim 3, wherein the endodomain comprises a human CD3(^ endodomain.

7. The polynucleotide or transposon of claim 3, wherein the at least one costimulatory domain comprises a human 4-1BB, CD28, CD3(^, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.

8. The polynucleotide or transposon of claim 3, wherein the CAR targets an oncogenic gene product comprising CD70, CD7, BCMA, CD 19, CD20, MUC1, PSMA or a combination thereof.

9. The polynucleotide or transposon of claim 3, wherein the hinge comprises a sequence derived from a human CD8a, IgG4, and / or CD4 sequence.Attorney Docket No.: 000218-0156-WO110. The transposon of any one of claims 2-9, wherein the transposon further comprises a nucleic acid encoding an inducible caspase polypeptide, a nucleic acid encoding a chimeric stimulatory receptor, a nucleic acid encoding a selection gene, a nucleic acid encoding a therapeutic agent, or a combination thereof.

11. The transposon of claim 10, wherein the selection gene comprises a dihydrofolate reductase (DHFR) resistance gene.

12. The transposon of any one of claims 2-11, wherein the transposon is a piggyBac transposon.

13. The transposon of any one of claims 2-12, wherein the transposon comprises one or more promoters.

14. The transposon of claim 13, wherein the one or more promoters is an elongation factor 1 -alpha (EFla) promoter, a U6 promoter or both an EFla promoter and a U6 promoter.

15. The transposon of claim 14, wherein the promoter is an EFla promoter operably linked to the CAR and the CD70 shRNA.

16. A vector comprising the polynucleotide of any one of claims 1, or 3-9.

17. A vector comprising the transposon of any one of claims 2-15.

18. A cell comprising the polynucleotide of any one of claims 1, or 3-9.

19. A cell comprising the transposon of any one of claims 2-8.

20. A population of cells comprising the polynucleotide of any one of claims 1, or 3-9.

21. The population of cells of claim 20, wherein the cells are immune cells.

22. The population of cells of claim 20 or 21, wherein cells are T-cells.

23. The population of cells of claims 21 or 22, wherein a portion of the immune cells comprises a genetic modification which reduces or inhibits expression of a T-cell receptor or a major histocompatibility complex (MHC).

24. The population of cells of claim 23, wherein the genetic modification is an insertion of a sequence encoding a P-2 microglobulin (P2M) and wherein the genetic modification reduces or inhibits expression of an MHC I.Attorney Docket No.: 000218-0156-WO125. The population of cells of claim 23, wherein the genetic modification is an insertion of a sequence encoding an a chain (TCRa), a P chain (TCRP), or a combination thereof and wherein the genetic modification reduces or inhibits expression of a TCR.

26. The population of cells of any one of claims 20-25, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of cells express the CD70 shRNA.

27. A composition comprising the cell of claim 18 or 19.

28. A composition comprising the population of cells of any one of claims 20-26.

29. A pharmaceutical composition comprising the composition of claim 27 or 28 and a pharmaceutically acceptable carrier.

30. A method of treating a disease or disorder in a subject in need thereof comprising administering a therapeutically effective amount of the composition of claim 27 or 28 or the pharmaceutical composition of claim 29.

31. The method of claim 30, wherein the disease or disorder is one or more of Acute myeloid Leukemia (AML), T cell lymphoma, Non-Hodgkin lymphoma (NHL), Renal cell carcinoma (RCC), or an autoimmune disease or disorder.

32. The method of claim 31, wherein the autoimmune disorder is selected from: autoimmune neutropenia, Guillain-Barre syndrome, epilepsy, autoimmune encephalitis, Isaacs' syndrome, nevus syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, or membranous nephropathy.

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