Chimeric antigen receptor (CAR) T cells and uses thereof

Abstract

The present disclosure provides plasmids, nucleic acids, or constructs for use with the CRISPR / CAS9 system to genetically engineer T cells. In some aspects, disclosed herein are methods of using such engineered T cells to treat cancer.

Description

[Technical Field]

[0001] Related Applications This PCT application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 431,354, "Chimeric Antigen Receptor (CAR) T Cells and Uses Thereof," filed December 9, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference The Sequence Listing submitted on December 11, 2023 as an .XML file titled "10935-020WO1_ST26.xml," created on December 7, 2023, with a file size of 115,746 bytes, is incorporated herein by reference pursuant to 37 C.FR § 1.52(e)(5).

[0003] The present disclosure relates to engineered T cells, compositions, and methods of their use. [Background technology]

[0004] T cells have potent antitumor activity and have been successfully used in several clinical trials. Modifying T cells with chimeric antigen receptors (CARs) can improve targeting and increase specificity. Generating CAR-T cells using rentoviral transduction has several limitations, including random integration of the transgene, which can lead to unintended consequences such as oncogene activation, gene silencing, or adversely affecting the antitumor efficacy of CAR-T. New methods and vectors are needed to engineer T cells. Summary of the Invention

[0005] Methods and compounds for delivering a CRISPR / CAS9 gene editing system to T cells are disclosed.

[0006] In one aspect, disclosed herein are plasmids, nucleic acids, and / or constructs for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (e.g., an scFv targeted to a receptor on a target cell (e.g., CD33), a transmembrane domain (e.g., an NKG2D transmembrane domain, a CD4 transmembrane domain, a CAR polypeptide ... , CD8 transmembrane domain, CD28 transmembrane domain, and / or CD3ξ transmembrane domain), a costimulatory domain (e.g., a CAR comprising a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination of a 2B4 domain, a CD28 costimulatory domain, and / or a 4-1BB costimulatory domain), and a CD3ξ signaling domain), and a right homology arm, wherein the left and right homology arms are each 1000 bp or less in length (e.g., 30 bp, 300 bp, 600 bp in length). In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain and / or a 4-1BB costimulatory domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain.

[0007] Also disclosed herein are T cells comprising a plasmid, nucleotide acid, and / or construct for a CRISPR / Cas9 integration system for use in any of the preceding embodiments, wherein the left and right homologous arms are the same length or different lengths. In some embodiments, the homologous arms specifically hybridize to the adeno-associated virus integration site 1 (AAVS1) on human chromosome 19.

[0008] In some embodiments, disclosed herein is a T cell comprising a plasmid, nucleic acid, and / or construct for a CRISPR / Cas9 integrated system for use in any of the preceding aspects, wherein the plasmid, nucleic acid, or construct further comprises a murine leukemia virus-derived (MND) promoter.

[0009] In some embodiments, the plasmid, nucleic acid, or construct of any preceding aspect is contained within and / or delivered into a T-cell adeno-associated virus (AAV) vector (e.g., an AAV vector comprising an AAV6 serotype) that comprises the plasmid, nucleic acid, or construct of any preceding aspect. In some embodiments, the AAV vector further comprises a plasmid, nucleic acid, or construct encoding a crRNA, a tracer RNA (trcrRNA), and a Cas endonuclease. The AAV vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

[0010] Also disclosed herein are methods for treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and / or myelodysplastic syndrome (MDS), etc.), comprising administering to a subject having cancer the modified cells of any of the preceding aspects.

[0011] In one aspect, disclosed herein is a method of generating chimeric antigen receptor (CAR) T cells, comprising: a) obtaining an AAV vector comprising a plasmid, nucleic acid, or construct comprising a ribonucleoprotein (RNP) complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA, a transgene (e.g., a chimeric antigen receptor against a tumor antigen), wherein the transgene is flanked by homology arms, and the homology arms are 1000 bp or less in length; and b) introducing the transgene and the RNP complex into a T cell, wherein the transgene (e.g., a chimeric antigen receptor against a tumor antigen) is introduced into the T cell via infection with an adenovirus-associated virus (AAV), and the RNP complex hybridizes to a target sequence in the genomic DNA of the T cell, and DNA repair enzymes in the T cell insert the transgene into the host genome at the target sequence (e.g., by homology-directed repair), thereby generating a CAR T cell. In some embodiments, the RNP complex can be introduced into cells by electroporation, hi some embodiments, the RNP complex can be introduced into cells by viral delivery with the same or a different AAV (i.e., reinfection).

[0012] In one aspect, disclosed herein is a method of genetically modifying T cells (including, but not limited to, primary or expanded cells), comprising: a) obtaining an AAV vector comprising a plasmid, nucleic acid, or construct comprising a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA, a chimeric antigen receptor (CAR) polynucleotide sequence, wherein the polynucleotide sequence is flanked by homology arms, and the homology arms are 1000 bp or less in length; and b) introducing the polynucleotide sequence and the RNP complex into a T cell, wherein the polynucleotide sequence is introduced into the cell via infection with AAV, the RNP complex hybridizes to a target sequence in the genomic DNA of the T cell, and DNA repair enzymes in the T cell insert the transgene into the host genome at the target sequence in the genomic DNA of the T cell, thereby generating a modified T cell.

[0013] In some embodiments, a method of genetically modifying a T cell of any preceding aspect disclosed herein, wherein the T cell is infected with an AAV disclosed herein at a multiplicity of infection (MOI) of about 5-500K.

[0014] Also disclosed herein are methods of genetically modifying T cells of any of the preceding aspects, wherein primary cells are incubated for approximately 4 to 10 days in the presence of IL-2, IL-15, and / or IL-7, either before or after infection, or before or after electroporation, and irradiated feeder cells, plasma membrane particles, or exosomes. In some embodiments, disclosed herein are methods of genetically modifying T cells of any of the preceding aspects, further comprising expanding the primary T cells for approximately 4 to 10 days in the presence of irradiated feeder cells, plasma membrane particles, or exosomes prior to infection, wherein the irradiated feeder cells, plasma membrane particles, or exosomes express membrane-bound 4-1BBL, membrane-bound IL-21, membrane-bound IL-15, or any combination thereof. Also disclosed herein are methods of genetically modifying T cells of any of the preceding aspects, further comprising expanding the modified T cells on irradiated feeder cells, plasma membrane particles, or exosomes after infection, wherein the irradiated feeder cells, plasma membrane particles, or exosomes express membrane-bound 4-1BBL, membrane-bound IL-21, or membrane-bound IL-15, or any combination thereof.

[0015] In some aspects, disclosed herein are methods for treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and / or myelodysplastic syndromes (MDS), etc.), comprising administering to the subject a therapeutically effective amount of T cells, wherein the T cells comprise a plasmid, nucleic acid, and / or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises, in order, a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and a right homology arm, wherein the left homology arm and the right homology arm are each 1000 bp or less (e.g., 600 bp) in length.

[0016] In some embodiments, disclosed herein are plasmids, nucleic acids, or constructs for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, the plasmids, nucleic acids, or constructs comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, wherein the polynucleotide sequence is flanked by a protospacer adjacent motif (PAM) and a sequence encoding a crisprRNA (crRNA), or is flanked by two PAMs and a sequence encoding a crRNA. In some aspects, the disclosed plasmids, nucleic acids, or constructs can be used in any of the methods for treating, reducing, lowering, inhibiting, ameliorating, or preventing cancer and / or metastasis of the preceding aspects, or in any of the methods for generating CAR T cells and / or genetically modifying T cells of the preceding aspects.

[0017] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows CD33CAR-T cells generated by the disclosed technology. [Figure 2] Figure 2 shows the distribution of events per chromosome from the dGH assay in sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23. [Figure 3] Figure 3 shows the distribution of event rates by cell number in sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23 from the dGH assay. [Figure 4] Figure 4 shows a summary of inversion and sister chromatid exchange (SCE) events in sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23 from the dGH assay. [Figure 5]Figure 5 shows an overview of the size differences counted per chromosome in sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23 from the dGH assay. [Figure 6] Figure 6 shows an exemplary karyogram. SCE events are present on chromosome 1p (Chr1p), 5q (Chr5q), Chr7q, Chr8q, Chr9q, Chr10qx2, Chr14q, ChrXp, and ChrXq. Size differences are observed between homologs on Chr2. There is a small inversion on Chr8p, and Chr16q has a full-arm deletion. [Figure 7] Figure 7 shows an exemplary karyogram. SCE events are present in Chr2p, Chr4q, Chr7q, and Chr12q. Size differences are observed between the Chr2, Chr5, and Chr11 homologs. There is a recurrent small inversion in Chr8p. Chr2 has a disrupted chromatid. [Figure 8] Figure 8 shows next-generation sequencing (NGS) coverage (gray) of the entire vector. Black arrows indicate the positions of primers. The vector map is shown at the bottom. The Y-axis is limited to 100X. [Figure 9] Figure 9 shows TLA sequence coverage of the entire human genome using primer set 2. Chromosomes are shown on the y-axis and chromosomal locations on the x-axis. Identified integration sites are circled. [Figure 10] Figure 10 shows TLA sequence coverage (gray) at the vector integration locus, chr4:15,737,375-15,822,498. The upper arrow in Set 1 indicates the location of the breakpoint sequence. The lower arrow in Set 1 indicates the location of primer Set 1. The bars in Set 2 indicate the location of the homology arms. The Y axis is limited to 2500X. [Figure 11] FIG. 11 shows a schematic of genetic modification of NK cells. [Figure 12]Figures 12A and 12B show the successful generation of NK cells expressing CD33CAR using a combination of Cas9 / RNP and AAV6. Figure 12A shows representative flow cytometry showing the expression levels of CD33CAR in NK cells 7 days after Cas9 / RNP electroporation targeted for transduction with AAVS1 and AAV6 (MOI=7.5x10). Figure 12B shows that the expression levels of CD33CAR in NK cells after transduction and electroporation were stable for 7 and 14 days (n=3). [Figure 13] Figure 13 shows the successful generation of CD38KOCD33CAR-expressing NK cells using a combination of Cas9 / RNP and AAV6. Representative flow cytometry shows the expression levels of CD33CAR in NK cells 21 days after Cas9 / RNP electroporation and AAV6 transduction (MOI=7.5x10) (n=1). [Figure 14] Figures 14A, 14B, 14C, 14D, 14E, and 14F show that NK cells expressing CD33CAR exhibit efficient cytotoxicity against CD33+ tumors. Figures 34A and 34D show that CD33CAR NK cells exhibit significantly greater degranulation than wild-type NK cells when cocultured with Kasumi-1 (**adjusted P value = 0.004, *adjusted P value = 0.01 for HL60). Figures 34B and 34C show that expressing CD33CAR on NK cells enhances the antitumor activity of NK cells against Kasumi-1, as shown by representative cytotoxicity assays performed at different effector:target (E:T) ratios and in three donors (****adjusted P value < 0.0001). Figures 34E and 34F show that this enhanced cytotoxic activity was observed only with CD33CAR-Gen2 NK cells against HL-60. [Figure 15] Figure 15 shows successful generation of CD38KO NK cells from ex vivo expanded PB-NK cells using Cas9 / RNP. CD38 expression on NK cells before and after CD38 depletion with Cas9 / RNP (n5), representative fluorescence-activated cell sorter (FACS) analysis of purified CD38KO NK cells. [Figure 16]Figures 16A, 16B, 16C, and 16D show favorable metabolic reprogramming of CD38KO NK cells. Figure 36A shows a heatmap of DEGs in significantly altered pathways (cholesterol biosynthesis and OXPHOS) determined by IPA based on normalized RNA-seq data of paired CD38WT and CD38KO NK cells (n = 6). Figure 36B shows a principal component analysis (PCA) of DEGs, demonstrating the consistent effect of CD38 deletion for each donor despite extensive donor variation. Figure 36C shows summary data from metabolic analysis of paired CD38WT and CD38KO NK cells (n = 3, mean ± SD). Figure 36D shows a graphical analysis of basal OCR, ECAR, OCR / ECAR, and spare respiratory capacity (SRC) from Figure 36C. All experiments were performed five times. FCCP, carbonylcyanium-4-(trifluoromethoxy)phenylhydrazone, ROT / AA, rotenone and antimycin A. [Figure 17] FIG. 17 shows the relative expression of mutant genes identified in CD38KO NK cells in wild-type (WT) expanded human NK cells. [Figure 18] FIG. 18 shows the relative expression of mutated genes identified in CD38KOCD33CAR NK cells in WT-expanded human NK cells. [Figure 19] Figure 19 shows an OCI-AML-3 xenograft model demonstrating survival of mice administered CAR-NK cells compared to unmodified NK cells. [Figure 20] Figure 20 shows the CD33CAR construct. Sig-leucel utilizes a second-generation anti-CD33CAR. The extracellular binding domain is an anti-CD33 scFv based on the heavy and light chain sequences derived from the human monoclonal antibody HuM195 (lintuzumab) and connected with a Whitlow linker. The hinge, stalk, and transmembrane domains are derived from human CD8α. The intracellular signaling domain consists of the CD3ζ activation domain and the 4-1BB costimulatory domain. [Figure 21] FIG. 21 shows the AAV6 vector sequence map. [Figure 22]FIG. 22 shows a schematic of clinical CD38KOCD33CARNK therapy. [Figure 23] FIG. 23 shows a flow diagram outlining the production and testing of CD38KOCD33CARNK. [Figure 24] Figures 24A and 24B show CD33CAR-expressing NK cells with improved effector function compared to wild-type (WT) expanded peripheral blood NK cells. Figure 6A shows CD38 expression on AML cells. AML cells cocultured with WT-NK or CD33CAR-NK cells caused AML cell death, as shown by cell viability assessment and SPADE plots (color-coded pRb expression indicates viable cells). Green arrows indicate viable AML cells, and red arrows indicate dead / dying AML cells. CD33CAR-NK cells were shown to kill more AML cells, and surviving AML cells exhibited decreased surface expression of CD33 and increased expression of CD38. Figure 6B shows Bioplex results demonstrating higher secretion of IFN-γ and TNF-α from CD33CAR-NK cells cocultured with AML cells. [Figure 25] FIG. 25 shows a schematic diagram of a directed genomic hybridization (dGH) assay using targeted probes. [Figure 26] Figure 26 shows cells lacking transgene signal. In the bottom panel, the telomere signal layer is removed. An overlay of telomere and centromeric bracketed probes can be seen as transgene probes, as seen in the top panel. Sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23. [Figure 27] Figure 27 shows a cell with an inversion / SCE event in the target region of the centromeric probe (circled). Bracketed probe signals for telomeres and centromeric regions can appear as transgene probes. Sample KromaTiD-WT-ctrl / 8AM-dGH-8 / 1 / 23. [Figure 28]Figure 28 shows a cell with an inversion / SCE event in the target region of the telomere probe (circled). The right panel is enlarged and the centromeric layer has been removed to better visualize the telomere probe signal pattern. An overlay of telomeric and centromeric bracketed probe signals can appear as a transgene probe, as seen in the left panel. [Figure 29] Figure 29 shows a cell with a transgene inserted into both copies of CD38 (circled). The right panel shows the transgene insertion signal layer. An overlay of the telomeric and centromeric bracketed probe signals can appear as a transgene probe in the merged image. Sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23. [Figure 30] Figure 30 shows a cell with a transgene inserted into both copies of CD38 (circled). In the right panel, the telomere probe signal layer is removed. The overlay of telomere and centromere bracketed probe signals can appear as transgene probe signals in the merged image. Sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23. [Figure 31] Figure 31 shows a cell with one transgene insertion on-target in CD38 (boxed) and one off-target (boxed). In the bottom panel, the telomere probe signal is removed. An overlay of the telomere and centromere bracketed probe signals can appear as transgene probe signals in the composite image. Sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23. [Figure 32] Figure 32 shows the design of CD33CAR-Gen2 and CD33CAR-Gen4v2. [Figure 33] Figure 33 shows the anti-AML activity of CD33CAR-NK. [Figure 34] FIG. 34 shows that CD33C ANK cells have enhanced anti-AML activity. [Figure 35]FIG. 35 is a schematic diagram showing the generation of CD38KO / CD33-CAR NK cells resistant to fratricide. [Figure 36] Figure 36 shows CD38KO / CD33-CAR NK cells resistant to fratricide targeted to residual AML. DETAILED DESCRIPTION OF THE INVENTION

[0019] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best currently known embodiment. In light of this, those skilled in the art will recognize and appreciate that many variations can be made to the various embodiments of the invention described herein and still obtain the beneficial results of the disclosure. It will also be apparent that some desirable advantages of the disclosure can be obtained by selecting some features of the disclosure without utilizing other features. Thus, those skilled in the art will recognize that many modifications and adaptations to the disclosure are possible and desirable in certain circumstances and are a part of the disclosure. Therefore, the following description is illustrative of the principles of the disclosure, but not limiting.

[0020] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the drawings and examples, but this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0021] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "comprises" and variations thereof are used synonymously with the term "includes" and variations thereof and are open, non-limiting terms. Although the terms "comprises" and "includes" have been used herein to describe various implementations, the terms "consisting essentially of" and "consisting of" can be used in place of "comprises" and "includes" to provide more specific implementations, and are also disclosed. As used in this disclosure and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0022] For a complete understanding of terms used in this specification, the following definitions are provided.

[0023] The terms "about" and "approximately" are defined as "approximate" as understood by one of ordinary skill in the art. In one non-limiting embodiment, these terms are defined to within 10%. In another non-limiting embodiment, these terms are defined to within 5%. In yet another non-limiting embodiment, these terms are defined to within 1%.

[0024] Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It will be further understood that the endpoints of each range are significant both in relation to the other endpoint, and independently of the other endpoint. Additionally, certain values ​​are disclosed herein, and it is understood that each value is also disclosed "about" that particular value in addition to the value itself. For example, if a value of "10" is disclosed, "about 10" is also disclosed. As will be appreciated by those of ordinary skill in the art, when a value is disclosed "less than or equal to," it is understood that "greater than or equal to" and possible ranges between those values ​​are also disclosed. For example, if the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point of "10" and a specific data point of 15 are disclosed, it is understood that values ​​between 10 and 15, as well as values ​​greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and equal to 10, greater than 15, less than 15, less than or equal to 15, and equal to 15, are considered to be disclosed. It is also understood that each unit between two specified units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0025] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarterial, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "concurrent administration," "administration in combination," "simultaneous administration," or "administered simultaneously" means that compounds are administered at the same time in time or essentially immediately after each other. In the latter case, the two compounds are administered close enough in time that the observed results are indistinguishable from those achieved when the compounds are administered at the same time in time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, through an entry into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to the area of ​​the administration point or an area immediately adjacent thereto, but does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent is readily detectable in the local vicinity of the administration point, but is undetectable or detectable in negligible amounts in distant parts of the subject's body. Administration includes self-administration and administration by another.

[0026] "Biocompatible" generally refers to a material and any metabolic or breakdown products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects in the subject.

[0027] A "control" is a substitute subject or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0028] The terms "complementary" or "substantially complementary" refer to hybridization or base pairing between nucleotides or nucleic acids, or the formation of a duplex, such as between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site of a single-stranded nucleic acid. Complementary nucleotides are typically A and T / U, or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, when optimally aligned and compared, pair with at least about 80%, usually at least about 90%-95%, and more preferably about 98%-100% of the nucleotides of the other strand, with appropriate nucleotide insertions or deletions. Alternatively, substantial complementarity exists when an RNA or DNA strand hybridizes to its complement under selective hybridization conditions. Typically, selective hybridization occurs when there is about 65% or greater complementarity, or about 75% or greater, or about 90% or greater complementarity over a range of at least 14-25 nucleotides. See Kanehisa (1984) Nucl. Acids Res. Acids Res.

[0029] As used herein, the term "comprises" and variations thereof are used synonymously with the term "includes" and variations thereof and are open and non-limiting terms. Although the terms "comprises" and "includes" are used herein to describe various implementations, the terms "consisting essentially of" and "consisting of" can be used instead of "comprises" and "includes" to provide more specific implementations, and are also disclosed.

[0030] A "composition" refers to any substance that has a beneficial biological effect. Beneficial biological effects include therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, vectors, polynucleotides, cells, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "composition" is used, or when a particular composition is specifically identified, it should be understood that this term includes the composition itself as well as pharmaceutically acceptable, pharmacologically active vectors, polynucleotides, salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0031] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. The DNA polynucleotide may encode an RNA that is translated into a protein (mRNA) (thus, DNA and mRNA together encode a protein), or the DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, microRNA (miRNA), "non-coding" RNA (ncRNA), guide RNA, etc.).

[0032] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0033] "Fragments" can include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not associated with other sequences, so long as the activity of the fragment is not significantly altered or impaired compared to the unaltered peptide or protein. These modifications can provide additional properties, such as removing or adding amino acids capable of disulfide bonding, increasing biolongevity, or altering secretion characteristics. In any case, the fragment must possess a biologically active property, such as modulating transcription of a target gene.

[0034] The term "gene" or "gene sequence" refers to a coding sequence or a regulatory sequence, or fragments thereof. A gene can include any combination of coding sequences, regulatory sequences, or fragments thereof. Thus, a "gene" referred to herein can be all or a portion of a naturally occurring gene. The polynucleotide sequences referred to herein are used synonymously with the term "gene" and can include any coding, non-coding, or regulatory sequence, fragments thereof, and combinations thereof. The terms "gene" or "gene sequence" include, for example, regulatory sequences that are upstream of the coding sequence (e.g., a ribosome binding site).

[0035] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences are used in BLAST or BLASTM with the default parameters described below. "Substantially identical" refers to two or more sequences or subsequences that contain a specified percentage of identical amino acid residues or nucleotides (i.e., about 60% identity, preferably 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 higher identity over a specified region when compared and aligned for closest correspondence over a comparison window or designated region). Such sequences are then said to be "substantially identical." This definition also refers to or can be applied to the complement of a test sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As explained below, preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) nucleotide sequence identity is defined as the percentage of amino acids in a candidate sequence that match the nucleotides of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including the algorithm needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0036] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test and reference sequences are input into computer, and subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage for test sequence compared with reference sequence based on program parameters.

[0037] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschule et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschule et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is terminated when the cumulative alignment score drops by a quantity X from its maximum achieved value, when the cumulative score falls below zero due to the accumulation of one or more negatively scoring residue alignments, or when either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. For example, the BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as default a word length of 3, an expectation (E) of 10, alignment (B) of 50, expectation (E) of 10, M=5, N=-4 in the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), and a comparison of both strands.

[0038] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0039] As used herein, the terms "naturally occurring" or "unmodified" or "wild-type" as applied to a nucleic acid, polypeptide, cell, or organism refer to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a source in nature and is present in an organism (including a virus) that has not been intentionally modified by man in the laboratory is wild-type (and naturally occurring).

[0040] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, or composition by a statistically significant amount. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, so long as the increase is statistically significant.

[0041] "Reduction" can refer to any change that results in a lower amount of a symptom, disease, composition, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of a gene product containing the substance is less than the output of the gene product without the substance. A reduction can also be, for example, a change in the symptoms of a disorder, such that the symptoms are less than those previously observed. A reduction can be any individual, median, or average decrease in a statistically significant amount of a condition, symptom, activity, or composition. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, as long as the reduction is statistically significant.

[0042] As used herein, the term "nucleic acid" refers to a polymer composed of nucleotides, such as deoxyribonucleotides (DNA) or ribonucleotides (RNA). As used herein, the terms "ribonucleic acid" and "RNA" refer to a polymer composed of ribonucleotides. As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to a polymer composed of deoxyribonucleotides.

[0043] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0044] As used herein, the term "operably linked" can refer to the placement of regulatory sequences useful for expression of a nucleic acid coding sequence in a nucleic acid molecule in an appropriate position relative to the coding sequence to effect expression of the coding sequence. This same definition can also apply to the placement of coding sequences and / or transcriptional control elements (e.g., promoters, enhancers, termination elements) and / or selectable markers in an expression vector. The term "operably linked" can also refer to the placement of polypeptide segments within a single polypeptide chain, where the individual polypeptide segments can be, without limitation, proteins, fragments thereof, linking peptides, and / or signal peptides. The term "operably linked" can refer to the direct fusion of different individual polypeptides within a single polypeptide or fragment thereof, whether there are no intervening amino acids between the different segments or when the individual polypeptides are connected to each other via one or more intervening amino acids.

[0045] "Primers" are a subset of probes that can support some type of enzymatic manipulation and can hybridize to a target nucleic acid so that enzymatic manipulation can occur. Primers can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with enzymatic manipulation.

[0046] A "probe" is typically a molecule that can interact with a target nucleic acid in a sequence-specific manner, for example, through hybridization. Nucleic acid hybridization is well understood in the art and is discussed herein. Typically, probes can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.

[0047] A "protein coding sequence," or a sequence encoding a specific protein or polypeptide, is a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into mRNA (in the case of DNA) and translated into a polypeptide in vitro or in vivo (in the case of mRNA). The boundaries of the coding sequence are determined by a start codon at the 5'-terminus (N-terminus) and a translation stop nonsense codon at the 3'-terminus (C-terminus). Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence is typically located 3' to the coding sequence.

[0048] The term "polynucleotide" refers to a single- or double-stranded polymer composed of nucleotide monomers.

[0049] The term "polypeptide" refers to a compound consisting of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

[0050] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0051] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operatively linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, direct expression of the gene product in a tissue-specific manner.

[0052] A "pharmaceutically acceptable" ingredient can refer to an ingredient that is not biologically or otherwise undesirable, i.e., an ingredient that can be incorporated into the pharmaceutical formulations of the present invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation in which it is contained. When used in reference to human administration, the term generally means that the ingredient has met the necessary standards of toxicology and manufacturing testing or that it is included in the inactive ingredient guide prepared by the U.S. Food and Drug Administration.

[0053] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally refers to a carrier or excipient useful in preparing a safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations, and materials further described herein.

[0054] "Pharmacologically active" (or simply "active") can refer to derivatives or analogs (e.g., salts, esters, amides, complexes, metabolites, isomers, fragments, etc.) that have the same type of pharmacological activity as the parent compound, and to approximately the same extent, in a "pharmacologically active" derivative or analog.

[0055] An "effective amount" of a drug refers to an amount of the drug sufficient to provide a desired effect. The amount of a drug that is "effective" will vary from subject to subject, depending on many factors, such as the subject's age and general condition, the specific drug, and so forth. Therefore, it is not always possible to specify a quantified "effective amount." However, an appropriate "effective amount" for any subject may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug may refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to achieve a therapeutic effect may vary according to factors such as the subject's age, sex, and weight. Dosage regimens can be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0056] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., the prevention of a disorder or other undesirable physiological condition (e.g., cancer). These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0057] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a drug) refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is control of cancer. In some embodiments, the desired therapeutic result is control of metastasis. In some embodiments, the desired therapeutic result is reduction in tumor size. In some embodiments, the desired therapeutic result is prevention and / or treatment of recurrence. The therapeutically effective amount of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term can also refer to an amount of therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition being treated, the subject's tolerance, the drug and / or drug formulation being administered (e.g., potency of the therapeutic agent, concentration of the drug in the formulation, etc.), and various other factors understood by those of skill in the art. In some cases, the desired biological or medical response is achieved after multiple administrations of the composition over a period of days, weeks, or years.

[0058] As used herein, a "transgene" refers to exogenous genetic material (e.g., one or more polynucleotides) that has been or can be artificially provided to a cell. The term can be used to refer to a "recombinant" polynucleotide encoding any of the polypeptides disclosed herein that are the subject of this disclosure. The term "recombinant" refers to a sequence (e.g., a polynucleotide or polypeptide sequence) that is not present in the cell in which the sequence is artificially provided, or a sequence that is joined to other polynucleotides in an arrangement that is not present in the cell in which the sequence is artificially provided. The term "artificial" refers to non-natural occurrence in a host cell and is understood to include manipulation by humans, machines, exogenous agents (e.g., enzymes, viruses, etc.), other non-natural manipulation, or a combination thereof. A transgene can include, but is not limited to, a gene operatively linked to a promoter (e.g., an open reading frame). When a transgene is artificially provided to a cell, the transgene can be integrated into a chromosome of the host cell, present extrachromosomally, or any combination thereof.

[0059] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also discussed in the sentence in which they are relied upon and are individually and specifically incorporated by reference herein for the material contained therein.

[0060] Plasmids, nucleic acids, and / or constructs for genetically modifying cells and methods thereof Disclosed herein are engineered T cells comprising a plasmid, nucleic acid, and / or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a left homology arm, a chimeric antigen receptor (CAR) polypeptide (e.g., an scFv targeted to a receptor (e.g., CD33) on a target cell), a transmembrane domain (e.g., an NKG2D transmembrane domain, a CD4 transmembrane domain), a CAR ... In some embodiments, the CAR comprises, in order, a CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3ξ transmembrane domain), a costimulatory domain (e.g., a CAR comprising a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination of a 2B4 domain, a CD28 costimulatory domain, and / or a 4-1BB costimulatory domain), and a CD3ξ signaling domain), and a right homology arm, wherein the left and right homology arms are each 1000 bp or less in length (e.g., about 30 bp, about 300 bp, about 600 bp in length). In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain and / or a 4-1BB costimulatory domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain.

[0061] Generally, a "CRISPR system" or "CRISPR integrated system" refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated "Cas" genes. In some embodiments, one or more elements of the CRISPR system are derived from a Type I, Type II, or Type III CRISPR system. CRISPR systems are known in the art. See, e.g., U.S. Patent No. 8,697,359, incorporated herein by reference in its entirety.

[0062] An endonuclease / RNP (e.g., Cas9 / RNP) consists of a three-component recombinase endonuclease protein (e.g., Cas9 endonuclease) complexed with a CRISPR locus. The endonuclease complexed with a CRISPR locus can be referred to as a CRISPR / Cas guide RNA. The CRISPR locus contains a synthetic single guide RNA (gRNA) that consists of an RNA that can hybridize to a complementary repeat RNA (crRNA) and a trans-complementary repeat RNA (tracrRNA) complexed with a target sequence. Thus, the CRISPR / Cas guide RNA hybridizes to a target sequence within the genomic DNA of a cell. In some cases, the Class 2 CRISPR / Cas endonuclease is a Type II CRISPR / Cas endonuclease. In some cases, the Class 2 CRISPR / Cas endonuclease is a Cas9 polypeptide, and the corresponding CRISPR / Cas guide RNA is a Cas9 guide RNA. These Cas9 / RNPs can cleave genomic targets with higher efficiency compared to approaches that rely on foreign DNA because they are delivered as a functional complex. In addition, the rapid clearance of Cas9 / RNPs from cells can reduce off-target effects such as the induction of apoptosis.

[0063] To generate the RNP complex, the crRNA and tracrRNA can be mixed in a 1:1, 2:1, or 1:2 ratio at a concentration of about 50 μM to about 500 μM (e.g., 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 225 μM, 250 μM, 275 μM, 300 μM, 325 μM, 350 μM, 375 μM, 400 μM, 425 μM, 450 μM, 475 μM, or 500 μM), preferably between 100 μM and about 300 μM, most preferably about 200 μM, at 95°C for about 5 minutes to form the crRNA:tracrRNA complex (i.e., guide RNA). The crRNA:tracrRNA complex can be mixed with a Cas endonuclease (e.g., Cas9) at a final dilution of about 20 μM to about 50 μM (e.g., 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, or 50 μM).

[0064] Upon binding to a target sequence in a target cell, the CRISPR locus can modify the genome by introducing one or more base pair insertions or deletions, insertion of a heterologous DNA fragment (e.g., a donor polynucleotide), deletion of an endogenous DNA fragment, inversion or translocation of an endogenous DNA fragment, or a combination thereof, into the target DNA. Thus, the disclosed method can be used to generate knockouts or knock-ins when combined with DNA for homologous recombination. Herein, it is shown that adeno-associated virus (AAV)-mediated transfer of Cas9 / RNPs is a relatively efficient method that overcomes previous limitations of genetic modification in cells (such as T cells, B cells, macrophages, NK cells, NKT cells, fibroblasts, osteoblasts, hepatocytes, neurons, epithelial cells, and muscle cells).

[0065] The CRISPR / Cas9 system has recently been shown to facilitate high-precision genome editing using adeno-associated virus (AAV) vectors that serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of approximately 4.5 kilobases limits the size of the donor, including the homologous arms. Therefore, in certain embodiments, it is recommended that any transcript and any transgene exceeding 100 bp have homologous arms with each arm being at least 800 bp long; many systems use asymmetric arms of 800 bp and 1000 bp for a total of 1800 bp. Therefore, AAV vectors cannot carry transgenes larger than approximately 2.5 kb. In one aspect, disclosed herein is an AAV CRISPR / CAS9 nucleotide delivery system comprising a donor assembly plasmid with homology arms of 30 bp to 1000 bp, and the donor assembly plasmid may be 30 bp, 50 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, 210 bp, 220 bp, 230 bp, 240 bp, 250 bp, 260 bp, 270 bp, 280 bp, 290 bp, 300 bp, 310 bp, 320 bp, 330 bp, 340 bp, 350 bp, 360 bp, 370 bp, 380 bp, 390 bp, 400 bp, 410 bp, 420 bp, 430 bp, 440 bp, 450 bp, 460 bp, 470 bp, 480 bp, 490 bp , 500bp, 510bp, 520bp, 530bp, 540bp, 550bp, 560bp, 570bp, 580bp, 590bp, 600bp, 610bp, 620bp, 63 0bp, 640bp, 650bp, 660bp, 670bp, 680bp, 690bp, 700bp, 710bp, 720bp, 730bp, 740bp, 750bp, 760bp , 770bp, 780bp, 790bp, 800bp, 810bp, 820bp, 830bp, 840bp, 850bp, 860bp, 870bp, 880bp, 890bp, 900bp, 910bp, 920bp, 930bp, 940bp, 950bp, 960bp, 970bp, 980bp, 990bp, or 1000bp.For example, the homology arms can be symmetric 30 bp homology arms, symmetric 300 bp homology arms, symmetric 500 bp homology arms, symmetric 600 bp homology arms, symmetric 800 bp homology arms, symmetric 1000 bp homology arms, or asymmetric 800 bp homology arms including an 800 bp left homology arm (LHA) and a 1000 bp right homology arm (RHA) for homologous recombination (HR), or no homology arms at all for non-homologous end joining using a homology-independent targeted integration (HITI) plasmid. In some examples, the plasmids with or without homology arms are those disclosed in International Publication No. WO 2020 / 198675, which is incorporated herein by reference in its entirety. In some embodiments, the plasmids include a clinically approved splice acceptor (SA) (SEQ ID NO: 10) and a clinically approved polyadenylation terminator (PA) (e.g., BGH polyA terminator SEQ ID NO: 11). It is understood and contemplated herein that the homology arms can be symmetric (same length on both sides) or asymmetric (different lengths on both sides), to accommodate different transgene lengths.That is, the length of the homologous arm can have any combination of the left homologous arm (LHA) length and the right homologous arm (RHA) length, and can be LHA 30 bp (SEQ ID NO: 2) and RHA 30 bp (SEQ ID NO: 1), LHA 30 bp and RHA 100 bp, LHA 30 bp and RHA 300 bp (SEQ ID NO: 3), LHA 30 bp and RHA 500 bp (SEQ ID NO: 5), LHA 30 bp and RHA 800 bp (SEQ ID NO: 7), LHA 30 bp and RHA 1000 bp, LHA100bp and RHA30bp, LHA100bp and RHA100bp, LHA100bp and RHA300bp, LHA100bp and RHA500bp, LHA100bp and RHA800bp, LHA100bp and RHA1000bp, LHA300bp (SEQ ID NO: 4) and RHA30bp, LHA300bp and RHA100bp, LHA300bp and RHA300bp, LHA300bp and RHA500bp, LHA300bp and RHA800bp, LHA300bp and RHA1000bp, LHA500bp (SEQ ID NO: 6) and RHA30bp, LHA500bp and RHA100bp, LHA500bp and RHA300bp, LHA500bp and RHA500bp, LHA500bp and RHA800bp, LHA500bp and RHA1000bp, LHA800bp (SEQ ID NO: 8), RHA30bp, LHA800bp and RHA100bp, LHA800bp and RHA300bp, LHA800bp and RHA500bp, LHA800bp and RHA800bp, LHA800bp and RHA1000bp, LHA1000bp and RHA30bp, LHA1000bp and RHA100bp, LHA1000bp and RHA300bp, LHA1000bp and RHA500bp, LHA1000bp and RHA800bp, LHA1000bp and RHA1000bp.

[0066] There are several methods for delivering DNA templates, including viral and non-viral methods. In non-viral approaches, single-stranded or double-stranded DNA templates are typically electroporated with Cas9 / RNP, but this approach is less efficient than viral transduction. For viral gene delivery, adeno-associated viruses (AAVs), including AAV6, have been safely used in clinical trials and are useful as vectors for sensitive primary immune cells, including T cells.

[0067] Transcripts delivered via AAV vectors can be packaged as linear single-stranded (ss) DNA (ssAAV) or linear self-complementary (sc) DNA (scAAV) approximately 4.7 kb in length. The advantage of scAAV vectors is that they contain mutated inverted terminal repeats (ITRs) required for replication, helping to avoid the rate-limiting step of double-strand generation compared to ssDNA vectors. Due to the limited packaging capacity of scAAV, HAs of 30 bp, 300 bp, 500 bp, and 800-1000 bp have been designed for the right and left sides of the Cas9 target site, providing researchers with the ability to find optimal HA lengths and select HA lengths based on transgene size. Furthermore, due to the limited packaging capacity compared to ssAAV, scAAV may not be suitable for large transgenes, such as chimeric antigen receptors (CARs) targeting CD33. Therefore, both ssAAV and scAAV have been designed and tested based on transgene size, offering a wide range of options for gene insertion in primary T cells.

[0068] The efficiency of recombination has been shown to improve with increasing HA length. Therefore, in the case of the ssAAV backbone, the longest possible lengths of the left and right homology arms (HA) were used for mCherry (e.g., 800-1000 bp of HA) and CD33CAR-T (e.g., 600 bp of HA). Because homology arm design is a time-consuming procedure requiring multiple optimizations, the CRISPaint approach was also explored, a homology-independent method for gene insertion or tagging. In this method, a DNA template encoding a gene of interest is provided with the same Cas9 target site containing a crRNA and a sequence encoding a PAM sequence (also referred to here as PAMg, e.g., SEQ ID NO: 9). Introduction of the Cas9 complex simultaneously cleaves the template DNA and genomic DNA. As a result, the CRISPaint template is presented as a linear double-stranded DNA that can be integrated via non-homologous repair mechanisms. Thus, in one aspect, disclosed herein are plasmids, nucleic acids, and / or constructs for delivering a donor transgene to a cell and integrating that transgene (e.g., a CAR) into the cell in combination with CRISPR / Cas9. Thus, disclosed herein are plasmids, nucleic acids, and / or constructs for use in the CRISPR / Cas9 integration system of any preceding aspect, wherein the left and right homologous arms are the same length or different lengths.

[0069] In some aspects, the homology arms specifically hybridize to adeno-associated virus integration site 1 (AAVS1) on human chromosome 19. In some embodiments, the LHA is 600 bp in length. In some embodiments, the LHA comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 31, or a fragment thereof. In some embodiments, the RHA is 600 bp in length. In some embodiments, the RHA comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 32, or a fragment thereof.

[0070] The plasmids, nucleic acids, or constructs disclosed herein comprise a polynucleotide sequence encoding a chimeric antigen receptor CAR polypeptide. As used herein, "chimeric antigen receptor" or "CAR" refers to a chimeric receptor that targets a cancer antigen and attracts cells expressing the receptor to cancer cells expressing the target antigen. Typically, a CAR comprises a molecule that recognizes a peptide derived from a tumor antigen presented by an MHC molecule, or an antibody or fragment thereof (e.g., Fab', scFv, Fv, etc.) expressed on the surface of the CAR cell that targets the cancer antigen. The receptor is fused to a signaling domain (e.g., a CD3ζ domain, an NKG2C, or an NKp44 domain, etc.) via a linker. The tumor antigen target is a protein produced by tumor cells that elicits an immune response. The choice of antigen-binding domain will depend on the particular type of cancer to be treated. Tumor antigens are known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CALX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos Related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUL, RU2, SSX2, AKAP-4, LCK, OY-TESl, PAX5, SART3, CLL-1, fucosyl-GM1, GloboH, MN-CAIX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, polysialic acid, PLAC1, RUL, RU2(AS), intestinal carboxylesterase, lewisY, sLE, LY6K, muthsp70-2, M-CSF, MY CN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutants, Ras mutants, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPVE6,E7,Sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART1, XAGE1, ELF2M, ERG (TMPRSS2ETS fusion gene), NA17, neutrophil elastase, sarcoma metastasis breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, These include CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGF II, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TNAg, Tie2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. Non-limiting examples of tumor antigens include: differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal rearrangements such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, IL13Ra2, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA15-3 / CA27.29 / BCAA, CA195, CA242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM,These include HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilum C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0071] The CAR polypeptide can also comprise a transmembrane domain (e.g., an NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3ξ transmembrane domain, etc.) and a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination of a 2B4 domain, a CD28 costimulatory domain, and / or a 4-1BB costimulatory domain). In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain and / or a 4-1BB costimulatory domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain. For example, in some embodiments, the CAR polypeptide comprises an IgG4 hinge domain, a CD4 transmembrane domain, a CD28 costimulatory domain, a CD3 zeta polypeptide, and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell, including, but not limited to, a cancer cell expressing a target antigen (e.g., CD33). In some embodiments, the CAR polypeptide comprises an IgG4 hinge domain, an NKG2D transmembrane domain, a 2B4 costimulatory domain, a CD3 zeta polypeptide, and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell, including, but not limited to, a cancer cell expressing a target antigen (e.g., CD33). In some embodiments, the CAR polypeptide is as shown in Figure 6B. In some embodiments, a polynucleotide encoding a CAR polypeptide described herein comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO:22, SEQ ID NO:23, or a fragment thereof.

[0072] In some embodiments, a polynucleotide encoding an scFv described herein comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 18, or a fragment thereof. In some embodiments, a polynucleotide encoding an scFv described herein comprises SEQ ID NO: 18, or a fragment thereof.

[0073] In some embodiments, a polynucleotide encoding an IgG4 hinge described herein comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 19, or a fragment thereof. In some embodiments, a polynucleotide encoding an IgG4 hinge described herein comprises SEQ ID NO: 19 or a fragment thereof.

[0074] In some embodiments, a polynucleotide encoding a CD28 costimulatory domain described herein comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 20, or a fragment thereof. In some embodiments, a polynucleotide encoding a CD28 costimulatory domain described herein comprises SEQ ID NO: 20, or a fragment thereof.

[0075] In some embodiments, a polynucleotide encoding CD3 zeta (CD3z or CD3ζ) described herein comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO:21, SEQ ID NO:28, or a fragment thereof. In some embodiments, a polynucleotide encoding CD3 zeta described herein comprises SEQ ID NO:21, SEQ ID NO:28, or a fragment thereof.

[0076] In some embodiments, a polynucleotide encoding an NKG2D transmembrane domain described herein comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 24, or a fragment thereof. In some embodiments, a polynucleotide encoding an NKG2D transmembrane domain comprises SEQ ID NO: 24, or a fragment thereof.

[0077] In some embodiments, a polynucleotide encoding a 2B4 domain described herein comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 26, or a fragment thereof. In some embodiments, a polynucleotide encoding a 2B4 domain comprises SEQ ID NO: 26, or a fragment thereof.

[0078] In some embodiments, the polynucleotide encoding the anti-CD33 scFv comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 29, or a fragment thereof. In some embodiments, the polynucleotide encoding the anti-CD33 scFv comprises SEQ ID NO: 29, or a fragment thereof.

[0079] In some embodiments, the MND promoter described herein comprises a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 30, or a fragment thereof. In some embodiments, the MND promoter comprises SEQ ID NO: 30.

[0080] In some embodiments, the expression vectors described herein comprise one or more linker sequences, wherein the linker sequences comprise a sequence at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 25 or a fragment thereof. In some embodiments, the expression vectors described herein comprise one or more linker sequences, wherein the linker sequences comprise SEQ ID NO: 25 or a fragment thereof.

[0081] Thus, in some embodiments, the T cells disclosed herein comprise a plasmid, nucleic acid, or construct comprising a polynucleotide sequence encoding a CAR polypeptide, wherein the CAR polypeptide comprises a transmembrane domain (e.g., an NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3ξ transmembrane domain), a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination of the 2B4 domain, the CD28 costimulatory domain, and / or the 4-1BB costimulatory domain), CD3 zeta, and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell (e.g., a cancer cell expressing CD33). In some embodiments, the CAR polypeptide specifically binds to CD33.

[0082] The plasmids, nucleic acids, and / or constructs disclosed herein can be integrated into the genome of transduced T cells via HITI, CRISPaint, or other non-homologous end joining (NHEJ) techniques, thereby offering the advantage of higher integration efficiency. In some examples, NHEJ plasmids, nucleic acids, and / or constructs are those disclosed in International Publication No. WO 2020 / 198675, the entire contents of which are incorporated herein by reference. To aid in identifying cleavage sites for transgene removal upon integration, the plasmids, nucleic acids, and / or constructs contain one or more PAMg sequences (i.e., sequences encoding a protospacer adjacent sequence (PAM) and a crRNA (i.e., gRNA)) (SEQ ID NO: 9) to target donor transgene integration. In some instances, in an NHEJ DNA template (e.g., a CRISPaint DNA template), a single (PAMg) or double (PAMgPAMg) Cas9 target sequence is integrated around a transgene (e.g., a polynucleotide encoding a CAR, such as the CD33CAR disclosed herein), but within the ITRs. Thus, Cas9 can simultaneously cleave the gDNA and the CRISPaint DNA template, allowing integration at the genomic DSB.

[0083] Thus, in some aspects, disclosed herein are T cells comprising a plasmid, nucleic acid, or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, wherein the polynucleotide sequence is flanked by one protospacer adjacent motif (PAM) and one polynucleotide sequence encoding a crisprRNA (crRNA) or by two polynucleotide sequences encoding two PAMs and a crRNA. In some aspects, disclosed herein are T cells comprising a plasmid, nucleic acid, or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integration system, wherein the plasmid, nucleic acid, or construct comprises, in order, a polynucleotide sequence encoding a protospacer adjacent motif (PAM) sequence and a crRNA, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and a polynucleotide sequence encoding a PAM sequence and a crRNA.

[0084] Furthermore, despite the advantages of using single-stranded (SS) plasmids, nucleic acids, and / or constructs for inserting larger transgenes, SS plasmids, nucleic acids, and / or constructs may require more time to fold and function as double-stranded DNA within cells prior to integration, which increases DNA sensing mechanisms and cytotoxicity in some cells (e.g., T cells, B cells, macrophages, NK cells, fibroblasts, osteoblasts, hepatocytes, neurons, epithelial cells, muscle cells, etc.). This issue is overcome by using self-complementary (SC) (double-stranded) structures to shorten the exposure time to exogenous DNA within cells.

[0085] It is understood and contemplated herein that crispr RNA (crRNA) is used to target Cas9 nuclease activity to the target site and cleave the donor plasmid, allowing the donor transgene to recombine into the host DNA. In some cases, the crRNA is combined with a tracrRNA to form a guide RNA (gRNA). The disclosed plasmids, nucleic acids, and / or constructs use AAV integration, targeting intron 1 of the protein phosphatase 1 regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, designated AAVS1, as the transgene integration site. This locus is a "safe harbor gene," allowing stable, long-term transgene expression in many cell types. Because disruption of PPP1R12C is not associated with any known disease, the AAVS1 locus is often considered a safe harbor for transgene targeting. Because the AAVS1 site is used as the target location, the CRSPR RNA (crRNA) must target the DNA. Guide RNAs disclosed herein include GGGGCCACTAGGGACAGGAT (SEQ ID NO: 17) or any 10-nucleotide sense or antisense contiguous fragment thereof. Thus, in some examples, the PAM+ sequence encoding the crRNA includes SEQ ID NO: 9. While AAVS1 is used herein for exemplary purposes, it is understood and contemplated herein that other "safe harbor genes" can be used with equivalent results and can be substituted for AAVS1 if more appropriate given the particular cell type or transgene being transfected. Examples of other safe harbor genes include, but are not limited to, CC chemokine receptor type 5 (CCR5), the ROSA26 locus, and TRAC.

[0086] As an example, the plasmids, nucleic acids, or constructs disclosed herein further comprise a murine leukemia virus (MND) promoter.

[0087] As noted above, the use of AAV as a vector to deliver the disclosed CRISPR / Cas9 plasmids and any donor genes is limited to a maximum of approximately 4.5 kb. One way to increase the allowable transgene size is to create additional room by replacing the Cas from another bacterial source (e.g., the commonly used Streptococcus pyogenes Cas9 (SpCas9) with a synthetic Cas9), which is understood and contemplated herein. Cas substitutions can also be used to increase target specificity, thereby reducing the need for gRNAs. Thus, for example, the Cas can be derived from Staphylococcus aureus (SaCas9), Acidaminococcus spp. (AsCpf1), Lachnospira casebacterium (LbCpf1), Neisseria meningitidis (NmCas9), Streptococcus thermophilus (StCas9), Campylobacterjejuni (CjCas9), enhanced SpCas9 (eSpCas9), SpCas9-HF1, Fokl-fused dCas9, multiplying Cas9 (xCas9), and / or catalytically inactive Cas9 (dCas9).

[0088] The term "Cas protein" or "Cas" refers to a polypeptide encoded by a Cas (CRISPR-associated) gene. Cas proteins include proteins encoded by genes in the cas locus, including adaptive and interference molecules. Interference molecules of bacterial adaptive immune complexes include endonucleases. Cas endonucleases described herein contain one or more nuclease domains.

[0089] It is understood and contemplated herein that the use of a particular Cas9 allows for the PAM sequence to be varied to screen targets using the Cas9 endonuclease (or alternatives). Suitable PAM sequences for use herein include NGG (SpCas9PAM), NNGRRT (SaCas9PAM), NNNNGATT (NmCas9PAM), NNNNRYAC (CjCas9PAM), NNAGAAW (St), TTTV (LbCpflPAM and AsCpflPAM), TYCV (LbCpflPAM mutant and AsCpflPAM mutant), where N is any nucleotide, V is A, C, or G, Y is C or T, W is A or T, and R is A or G.

[0090] In one aspect, disclosed herein is a method of genetically modifying T cells, the method comprising: a) obtaining a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA (gRNA) specific for a target DNA sequence in the T cell, and a plasmid, nucleic acid, or construct comprising a transgene (e.g., a chimeric antigen receptor for a tumor antigen), wherein the transgene is flanked by homology arms; and b) introducing the transgene and the RNP complex into the T cell, wherein the transgene is introduced into the T cell via infection with an adeno-associated virus (AAV), and the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell. In one aspect, the method can further comprise introducing the RNP complex into the T cell via electroporation (e.g., when modifying T cells). In one aspect, the method can further comprise re-infecting the target cell (e.g., T cell) with a second AAV virus comprising the RNP complex. In one embodiment, if the transgene is small enough, the same AAV can contain both the transgene and the RNP complex, hi a further embodiment, the transgene and the RNP complex can be encoded on the same plasmid, nucleic acid, or construct.

[0091] In one aspect, disclosed herein is a method of genetically modifying T cells, comprising: a) obtaining a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA and an AAV vector comprising a plasmid, nucleic acid, or construct comprising a transgene (e.g., a chimeric antigen receptor for a tumor antigen), wherein the transgene is flanked by a PAM and a crRNA or flanked by sequences encoding two PAMs and two crRNAs; and b) introducing the transgene and RNP complex into T cells, wherein the transgene is introduced into the T cells via infection with AAV, the ribonucleoprotein (RNP) complex hybridizes to a target sequence within the genomic DNA of the T cells, and DNA repair enzymes in the T cells insert the transgene into the host genome at the target sequence (e.g., by non-homologous end joining), thereby generating a modified T cell. In one embodiment, the method can further comprise introducing the RNP complex into the T cell via electroporation (e.g., in the case of modifying a T cell). In one embodiment, the method can further comprise re-infecting the target cell (e.g., a T cell) with a second AAV virus comprising the RNP complex. In one embodiment, if the transgene is sufficiently small, the same AAV can comprise both the transgene and the RNP complex. In a further embodiment, the transgene and the RNP complex can be encoded on the same plasmid, nucleic acid, or construct.

[0092] In some embodiments, the AAVs described herein can be used as vectors to deliver the disclosed prime editing plasmids and any donor transgenes described herein (e.g., polynucleotides encoding CARs). Prime editing is a "search and replace" genome editing technique that mediates targeted insertions, deletions, base-interchanges, and combinations thereof in human cells without the need for DSBs or donor DNA templates. Prime editing uses a fusion protein containing a catalytically deficient Cas9 endonuclease, an engineered reverse transcriptase, an RNA-programmable nicks, and / or a prime editing guide RNA (pegRNA) to directly copy genetic information from the pegRNA extension into a target genomic locus. Methods for designing and using prime editing techniques are known in the art. For examples, see Anzalone, AV, Randolph, PB, Davis, JR, et al. Search and replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019), the contents of which are incorporated herein by reference.

[0093] In one aspect, the T cells are primary T cells from a donor source, which may be from, for example, an allogeneic or autologous donor source for adoptive transfer therapy (i.e., the ultimate recipient of the modified cells), a T cell line, or an expanded T cell source obtained from a primary T cell source or a T cell line.

[0094] Prior to transduction of T cells, T cells can be incubated in a medium suitable for T cell proliferation. It is understood and contemplated herein that the culture conditions can include the addition of cytokines, antibodies, and / or feeder cells. Thus, in one aspect, disclosed herein is a method of genetically modifying T cells, further comprising incubating the T cells for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to transduction of the T cells in a medium that supports T cell proliferation, wherein the medium further comprises cytokines, antibodies, and / or feeder cells. For example, the medium can comprise IL-2, IL-7, IL-12, IL-15, IL-18, and / or IL-21. In one aspect, the medium can also comprise an anti-CD3 antibody. In one aspect, the feeder cells can be purified from feeder cells that stimulate the T cells. For example, T cell-stimulating feeder cells for use in the claimed invention disclosed herein can be irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or non-irradiated autologous or allogeneic PBMCs, RPMI8866, HFWT, K562, K562 cells transduced with membrane-bound IL-15 and 41BBL, or IL-21, or any combination thereof, or EBV-LCL. In some embodiments, the feeder cells are provided in combination with a solution of IL-21, IL-15, and / or 41BBL. The feeder cells can be seeded into the cell culture at a ratio of 1:2, 1:1, or 2:1. It is understood and contemplated herein that the culture period can be 1 to 14 days (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) after AAV infection, preferably 3 to 7 days, and most preferably 4 to 6 days. For example, the medium can include IL-2, IL-7, IL-12, IL-15, IL-18, and / or IL-21.

[0095] It is understood and contemplated herein that incubation conditions for primary cells and expanded cells may vary. In one aspect, culture of primary cells prior to AAV infection includes medium, cytokines (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, and / or IL-21), and / or anti-CD3 antibodies for less than 5 days (e.g., 1, 2, 3, or 4 days). For expanded T cells, culture can be performed in the presence of feeder cells (e.g., at a 1:1 ratio) in addition to or instead of cytokines (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, and / or IL-21) and / or anti-CD3 antibodies. Culture of expanded T cells can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to transduction. Thus, in one aspect, disclosed herein is a method of genetically modifying T cells, the method comprising culturing primary T cells for 4 days in the presence of IL-2, IL-7, or IL-15 prior to infection and / or electroporation with an AAV vector (if the RNP complex is introduced by electroporation), or culturing expanded cells for 4, 5, 6, or 7 days in the presence of irradiated feeder cells prior to infection and / or electroporation with AAV if the RNP complex is introduced by electroporation.

[0096] After transduction of T cells (e.g., via AAV infection or electroporation), the modified T cells can now be expanded in medium containing feeder cells that stimulate the modified T cells. Thus, the modified T cells retain their viability and proliferation potential and can be expanded after AAV infection and / or electroporation using irradiated feeder cells (if the RNP complex is introduced via electroporation). For example, T cell-stimulating feeder cells for use in the claimed inventions disclosed herein can be irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or non-irradiated autologous or PBMCs, RPMI8866, HFWT, K562, K562 cells transduced with membrane-bound IL-15 and 41BBL, or IL-21, or any combination thereof, or EBV-LCL. In some embodiments, the T cell feeder cells are provided in combination with a solution of IL-21, IL-15, and / or 41BBL. Feeder cells can be seeded into cultures of T cells at a ratio of 1:2, 1:1, or 2:1. It is understood and contemplated herein that the culture period can be 1 to 14 days (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) after infection and / or electroporation, preferably 3 to 7 days, and most preferably 4 to 6 days. In some embodiments, the medium for culturing the modified T cells can further comprise cytokines such as, for example, IL-2, IL-7, IL-12, IL-15, IL-18, and / or IL-21.

[0097] It is understood, and contemplated herein, that in one aspect, one purpose of the disclosed methods of genetically modifying cells is to generate modified cells. Accordingly, disclosed herein are modified T cells made by the disclosed methods. Accordingly, in one aspect, disclosed herein are modified T cells (including, but not limited to, CAR T cells) comprising any of the plasmids, nucleic acids, constructs, or vectors disclosed herein. For example, disclosed herein are anti-CD33 CAR T cells, which can also comprise an scFv targeting CD33, a transmembrane domain (e.g., an NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3ξ transmembrane domain, etc.), and a costimulatory domain (e.g., a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination of a 2B4 domain, a CD28 costimulatory domain, and / or a 4-1BB costimulatory domain). In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain and / or a 4-1BB costimulatory domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain.

[0098] In one aspect, disclosed herein is a method of generating a chimeric antigen receptor (CAR) T cell, comprising: a) obtaining a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA and an AAV vector comprising a plasmid, nucleic acid, or construct comprising a transgene (e.g., a chimeric antigen receptor for a tumor antigen), wherein the transgene is flanked by a PAM and a crRNA or by two PAMs and a crRNA; and b) introducing the RNP complex into a T cell, wherein the transgene is introduced into the T cell via infection of a target cell with an adeno-associated virus (AAV), wherein the ribonucleoprotein (RNP) complex hybridizes to a target sequence within the genomic DNA of the T cell, and wherein DNA repair enzymes in the T cell insert the transgene into the host genome at the target sequence (e.g., by non-homologous end joining), thereby generating a modified T cell. In one embodiment, the method can further include introducing the RNP complex into a T cell via electroporation. In one embodiment, the method can further include re-infecting the target cell (e.g., a T cell) with a second AAV virus containing the RNP complex. In one embodiment, if the transgene is sufficiently small, the same AAV can contain both the transgene and the RNP complex. In a further embodiment, the transgene and the RNP complex can be encoded on the same plasmid, nucleic acid, or construct.

[0099] In some aspects, disclosed herein are methods of genetically modifying T cells, comprising: a) obtaining an AAV vector comprising a plasmid, nucleic acid, or construct comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA, wherein the polynucleotide sequence is flanked by homology arms, and the homology arms are 800 bp or less in length; and b) introducing the polynucleotide sequence and the RNP complex into a T cell, wherein the polynucleotide sequence is introduced into the T cell via infection of the T cell with AAV, the RNP complex hybridizes to a target sequence in the genomic DNA of the T cell, and DNA repair enzymes in the T cell insert the transgene into the host genome at the target sequence in the genomic DNA of the T cell, thereby generating a modified T cell.

[0100] In one aspect, the modified T cells used in the disclosed immunotherapy methods and generated by the disclosed modification methods are primary T cells from a donor source, which can be, for example, from an allogeneic or autologous donor source for adoptive transfer therapy (i.e., the ultimate recipient of the modified T cells), a T cell line, or a source of expanded cells obtained from a primary T cell source or a T cell line. While primary T cells can be used, it is understood and contemplated herein that the disclosed modifications of T cells can occur ex vivo or in vitro.

[0101] As used herein, T cells refer to primary T cells or expanded T cells. Primary T cells can be cultured for about 4 to 10 days in the presence of IL-2, IL-7, and / or IL-15 prior to infection with an AAV vector. In one example, primary cells are expanded for about 4 to 10 days in the presence of irradiated feeder cells, plasma membrane particles, or exosomes prior to infection. In some embodiments, the irradiated feeder cells, plasma membrane particles, or exosomes express membrane-bound 4-1BBL, membrane-bound IL-21, or membrane-bound IL-15, or any combination thereof.

[0102] After transduction of T cells, the engineered T cells can be expanded and stimulated before administering the engineered T cells to a subject. For example, disclosed herein are methods for adaptively transplanting T cells into a subject in need thereof, wherein the T cells are expanded with irradiated feeder cells, plasma membrane (PM) particles, or exosomes (EX) expressing membrane-bound IL-21 (mbIL-21) prior to administration to the subject (PM particles and EX-exosomes expressing mbIL-21 are referred to as PM21 particles and EX21 exosomes, respectively). In some embodiments, expansion can further include irradiated feeder cells, plasma membrane (PM) particles, or exosomes expressing membrane-bound IL-15 (mbIL-15) and / or membrane-bound 4-1BBL (mb4-1BBL). It is understood and contemplated herein that in some embodiments, stimulation and expansion of engineered T cells occurs in vivo following or simultaneously with administration of the engineered cells to the subject. Thus, methods of immunotherapy are disclosed herein that involve administering IL-21 or mbIL-21-containing PM particles, exosomes containing mbIL-21, and / or irradiated mbIL-21-expressing feeder cells to transfer T cells into a subject, followed by expansion of T cells in the subject. In some embodiments, the expansion further comprises administration of PM particles, exosomes, and / or irradiated feeder cells expressing IL-15 and / or 4-1BBL, or membrane-bound IL-15 and / or 4-1BBL.

[0103] In some embodiments, the methods disclosed herein comprise infecting T cells with AAV at an MOI range of about 1 to about 1000 KMOI (e.g., about 5 to 500 KMOI). For example, the methods disclosed herein comprise infecting T cells with AAV at an MOI of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500.

[0104] A. Hybridization / Selective Hybridization The term hybridization typically refers to a sequence-driven interaction between at least two nucleic acid molecules, such as a primer or probe and a gene. A sequence-driven interaction refers to an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide-specific manner. For example, G interacting with C, or A interacting with T, is a sequence-driven interaction. Typically, a sequence-driven interaction occurs on the Watson-Crick or Hoogsteen face of a nucleotide. The hybridization of two nucleic acids is affected by several conditions and parameters known to those skilled in the art. For example, the salt concentration, pH, and temperature of the reaction all affect whether two nucleic acid molecules hybridize.

[0105] Parameters for selective hybridization between two nucleic acid molecules are known to those of skill in the art. For example, in some embodiments, conditions for selective hybridization can be defined as stringent hybridization conditions. For example, the stringency of hybridization is controlled by both the temperature and salt concentration of either or both of the hybridization and washing steps. For example, hybridization conditions to achieve selective hybridization can involve hybridization in a high ionic strength solution (6X SSC or 6X SSPE) at a temperature approximately 12-25°C below Tm (the melting temperature at which half of the molecules dissociate from their hybridization partner), followed by washing at a temperature and salt concentration combination selected so that the wash temperature is approximately 5-20°C below Tm. Temperature and salt conditions are easily determined empirically in preliminary experiments in which a sample of reference DNA immobilized on a filter is hybridized to a target nucleic acid, followed by washing under conditions of different stringency. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. Stringency can be achieved using these conditions as described above or as known in the art. Preferred stringent hybridization conditions for DNA:DNA hybridizations are approximately 68°C (in aqueous solution) in 6X SSC or 6X SSPE, followed by a wash at 68°C. If desired, the stringency of the hybridization and wash can be reduced as the desired degree of complementarity decreases, and depending on the GC or AT richness of any region where variability is sought. If desired, the stringency of the hybridization and wash can be increased as the desired degree of complementarity increases, and depending on the GC or AT richness of any region where high complementarity is desired, all as known in the art.

[0106] Another way to define selective hybridization is by looking at the amount (percentage) of one nucleic acid bound to the other nucleic acid. For example, in some embodiments, selective hybridization conditions are when at least about 60, 65, 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, 100% of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in, for example, 10-, 100-, or 1000-fold excess. This type of assay is useful when both the limiting and non-limiting primers are in, for example, their k d or only one of the nucleic acid molecules is 10-fold, 100-fold, or 1000-fold less than their k d The reaction can be carried out under conditions exceeding 100°C.

[0107] Another way to define selective hybridization is by examining the percentage of primers that are enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments, selective hybridization conditions are those where at least about 60, 65, 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, 100% of the primers are enzymatically manipulated under conditions that promote enzymatic manipulation. For example, if the enzyme manipulation is DNA extension, selective hybridization conditions are when at least about 60, 65, 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, 100% of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or those described in the art appropriate for the enzyme performing the manipulation.

[0108] It is understood that, just as with homology, various methods for determining the level of hybridization between two nucleic acid molecules are disclosed herein. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise specified, meeting the parameters of any method will be sufficient. For example, if 80% hybridization is required, as long as hybridization occurs within the required parameters in any one of these methods, it will be considered disclosed herein.

[0109] One of skill in the art will understand that if a composition or method, either collectively or individually, meets any one of these criteria for determining hybridization, it is a composition or method disclosed herein.

[0110] B. Nucleic acid The various molecules disclosed herein are based on nucleic acids. The disclosed nucleic acids are composed of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. For example, when a vector is expressed in a cell, it is understood that the expressed mRNA is typically composed of A, C, G, and U. Similarly, for example, when an antisense molecule is introduced into a cell or cellular environment, for example, through exogenous delivery, it is understood that it is advantageous for the antisense molecule to be composed of nucleotide analogs, which reduces degradation of the antisense molecule in the cellular environment.

[0111] a) Nucleotides and related molecules Nucleotides are molecules containing a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate and sugar moieties to form an internucleoside linkage. The base moieties of nucleotides are adenine-9-yl (A), cytosin-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. Non-limiting examples of nucleotides would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are a wide variety of these types of molecules available in the art and available herein.

[0112] A nucleotide analog is a nucleotide that contains some type of modification in either the base, sugar, or phosphate moiety. Modifications to nucleotides are well known in the art and may include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications in the sugar or phosphate moieties. There are a wide variety of these types of molecules available in the art and available herein.

[0113] Nucleotide substitutes are molecules that have similar functional properties as nucleotides but do not contain a phosphate moiety, such as peptide nucleic acids (PNAs). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes can adopt a double helix type structure when interacting with an appropriate target nucleic acid. There are a wide variety of these types of molecules available in the art and available herein.

[0114] Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs, for example, to enhance cellular uptake. The conjugates can be chemically linked to the nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are a wide variety of these types of molecules available in the art and available herein.

[0115] A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, N1, and C6 positions of a purine-based nucleotide, nucleotide analog, or nucleotide substitute, and the C2, N3, and C4 positions of a pyrimidine-based nucleotide, nucleotide analog, or nucleotide substitute.

[0116] A Hoogsteen interaction is an interaction that occurs on the Hoogsteen face of a nucleotide or nucleotide analog that is exposed in the major groove of duplex DNA. The Hoogsteen face includes the reactive groups (NH or O) at the N7 and C6 positions of purine nucleotides.

[0117] b) Array There are various sequences related to protein molecules involved in the signaling pathways disclosed herein, such as CD33, 4-1BB, NKG2D, or 2B4, all of which are encoded by or are nucleic acids. Sequences of human and other analogs of these genes, as well as alleles, splice variants, and other types of variants of these genes, are available in various protein and gene databases, including Genbank. Those skilled in the art will understand how to analyze sequence discrepancies and differences and how to adjust compositions and methods related to a particular sequence to other related sequences. Primers and / or probes can be designed for any given sequence given the information disclosed herein and known in the art.

[0118] c) Primers and probes Compositions comprising primers and probes are disclosed and can interact with the disclosed nucleic acids, such as CD33, disclosed herein. In certain embodiments, primers are used to support DNA amplification reactions. Typically, primers may be extended in a sequence-specific manner. Extension of a primer in a sequence-specific manner includes any method in which the sequence and / or composition of the nucleic acid molecule to which the primer hybridizes or otherwise associates induces or influences the composition or sequence of the product produced by primer extension. Thus, extension of a primer in a sequence-specific manner includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence-specific manner are preferred. In certain embodiments, primers are used in DNA amplification reactions, such as PCR or direct sequencing. It should be understood that in certain embodiments, primers can also be extended using non-enzymatic techniques, e.g., nucleotides or oligonucleotides used to extend the primer are modified so that they chemically react to extend the primer in a sequence-specific manner. Typically, the disclosed primers hybridize to the disclosed nucleic acids or regions of the nucleic acids, or they hybridize to the complement of the nucleic acid or the complement of a region of the nucleic acid.

[0119] In certain embodiments, the size of the primer or probe for interacting with a nucleic acid can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification, or simple hybridization of the probe or primer. Exemplary primers or probes comprise at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 1 8, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0120] In other embodiments, the primers or probes are 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, It can be up to 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0121] Primers for the CD33 gene can typically be used to generate an amplified DNA product containing a region of the CD33 gene or the full-length gene, generally such that the size of the product can be accurately determined to within 3 nucleotides, or within 2 nucleotides, or within 1 nucleotide.

[0122] In certain embodiments, the product comprises at least 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, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0123] In other embodiments, the product is 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, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides in length.

[0124] C. Delivery of Compositions to Cells Several compositions and methods exist that can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can be broadly divided into two categories: viral-based delivery systems and non-viral-based delivery systems. For example, nucleic acids can be delivered through several direct delivery systems, such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, and cosmids, or via the transfer of genetic material into cells or carriers, such as cationic liposomes. Suitable means for transfection, including viral vectors, chemical transfectants, or physical and mechanical methods such as electroporation and direct diffusion of DNA, are described, for example, in Wolff, J.A., et al., Science, 247, 1465-1468, (1990), and Wolff, J.A. Nature, 352, 815-818, (1991). Such methods are well known in the art and can be readily adapted for use with the compositions and methods described herein. In certain cases, the methods will be modified to work specifically with large DNA molecules. Furthermore, these methods can be used to target specific diseases and cell populations by using the targeting properties of the carrier.

[0125] a) Nucleic Acid-Based Delivery Systems A transfer vector can be any nucleotide construct used to deliver genes to cells (e.g., a plasmid) or used as part of a general strategy for delivering genes, such as a recombinant retrovirus or adenovirus (Rametal. Cancer Res. 53:83-88, (1993)). In some examples, the plasmids described herein can be DNA templates or nucleotide constructs that include the polynucleotide sequences provided herein.

[0126] As used herein, a plasmid, nucleic acid, or construct, or viral vector, is an agent that transports the disclosed nucleic acid without being degraded by cells and contains a promoter that drives gene expression in the delivered cells. Viral vectors include, for example, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, AIDS virus, neurotrophic virus, Sindbis virus, and other RNA viruses, including those with an HIV backbone. Also preferred are viral families that share the properties of these viruses and are suitable for use as vectors. Retroviruses include murine moloney leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as vectors. Retroviral vectors are commonly used vectors because they can carry larger genetic payloads, i.e., transgenes or marker genes, than other viral vectors. However, they are less useful in non-proliferating cells. Adenoviral vectors are relatively stable, easy to handle, highly concentrated, can be administered in aerosol formulations, and are capable of gene transfer into non-dividing cells. Poxvirus vectors are large, have several sites for gene insertion, are thermostable, and can be stored at room temperature. A preferred embodiment is a viral vector designed to suppress the immune response of the host organism caused by viral antigens. Preferred vectors of this type carry the coding region for interleukin 8 or 10.

[0127] Viral vectors can have a higher transfection capacity than chemical or physical methods for introducing genes into cells. Viral vectors typically contain nonstructural early genes, structural late genes, RNA polymerase III transcripts, inverted terminal repeats necessary for replication and encapsidation, and a promoter controlling the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more early genes removed, and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA. This type of construct can carry up to approximately 8 kb of foreign genetic material. The necessary functions of the removed early genes are usually supplied by cell lines engineered to express the gene products of the early genes in trans.

[0128] (1) Adeno-associated virus vector Another type of viral vector is based on adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV-type vectors can transport approximately 4-5 kb, and wild-type AAV is known to stably integrate into chromosome 19 (e.g., AAV integration site 1 (AAVS1)). Vectors with this site-specific integration property are preferred. The AAV used can be derived from any AAV serotype, including, but not limited to, AAC1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and recombinant (rAAV) AAVs such as AAV-Rh74, and / or synthetic AAVs (e.g., AAV-DJ, Anc80). AAV serotypes can be selected based on cell or tissue tropism. AAV vectors used in the disclosed compositions and methods can be single-stranded (SS) or self-complementary (SC).

[0129] In another type of AAV virus, the AAV has a pair of inverted terminal repeats (ITRs) flanking at least one cassette having a promoter that directs cell-specific expression operably linked to a heterologous gene, heterologous in this context refers to any nucleotide sequence or gene that is not native to AAV or B19 parvovirus.

[0130] Typically, the AAV and B19 coding regions are deleted, resulting in a safe, non-cytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration but not cytotoxicity, and the promoter drives cell-specific expression.

[0131] Thus, the disclosed vectors provide DNA molecules that can be integrated into mammalian chromosomes without substantial toxicity.

[0132] The inserted genes in viruses and retroviruses usually contain promoters and / or enhancers that help control the expression of the desired gene product. A promoter is generally a sequence of DNA that functions when in a relatively fixed location with respect to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors and may contain upstream elements and response elements.

[0133] It is understood that the packaging capacity of AAV is limited, and this is taken into consideration herein. One way to overcome the loading capacity of AAV vectors is to use two vectors, where the transgene is split between two plasmids and the two portions of the transgene are joined into a single full-length transgene using a 3' splice donor and a 5' splice acceptor. Alternatively, the two transgenes can be engineered to have significant overlap, and the two segments are joined into a full-length transcript by homologous recombination.

[0134] D. Expression Systems The nucleic acid delivered to a cell typically contains an expression control system. For example, inserted genes in viral and retroviral systems usually contain promoters and / or enhancers that help control the expression of the desired gene product. A promoter is generally a sequence of DNA that functions when it is in a relatively fixed position with respect to the transcription start site. A promoter contains core elements required for the basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0135] a) Viral promoters and enhancers Preferred promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, including the genomes of viruses such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and, most preferably, cytomegalovirus, or heterologous mammalian promoters such as the beta-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication (Fierce et al., Nature, 273:113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, P. J. Tal., Gene 18:355-360 (1982)). Of course, promoters from host cells or related species are also useful herein.

[0136] Enhancers generally refer to sequences of DNA that function at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78:993 (1981)) or 3' (Lusky, M. L. et al., Mol. Cell Biol. 3:1108 (1983)). Furthermore, enhancers can be located within introns (Banerji, J. Lett. et al., Cell 33:729 (1983)) and within the coding sequence itself (Osborne, T. F. et al., Mol. Cell Biol. 4:1293 (1984)). They are usually 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters may also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of gene expression. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), but typically, for gene expression, enhancers from eukaryotic cell viruses will be used for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0137] The promoter and / or enhancer can be specifically activated either by light or by specific chemical events that trigger their function. The system can be regulated by agents such as tetracycline and dexamethasone. There are also ways to enhance gene expression of viral vectors by exposure to radiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0138] In certain embodiments, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs, the promoter and / or enhancer region is active in all eukaryotic cell types, even if it is only expressed in certain types of cells at certain times. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, the cytomegalovirus (full-length promoter), and the LTR of retroviral vectors.

[0139] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types, such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0140] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells) may also contain sequences necessary for transcription termination, which can affect mRNA expression. These regions are transcribed as polyadenylation segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated region also includes a transcription termination site. The transcription unit preferably also contains a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcription unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. A homologous polyadenylation signal is preferably used in transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of approximately 400 bases. The transcription unit also preferably contains other standard sequences that, alone or in combination with the above sequences, improve expression or stability from the construct.

[0141] b) Marker Viral vectors can contain nucleic acid sequences encoding marker products. This marker product is used to determine whether the gene has been delivered to a cell and is being expressed once delivered. Preferred marker genes are the E. coli lacZ gene (encoding β-galactosidase) and green fluorescent protein.

[0142] In some embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into mammalian host cells, the transformed mammalian host cells can survive when placed under selective pressure. There are two widely used categories of selection regimes. The first category is based on cellular metabolism and the use of mutant cell lines that lack the ability to grow independently of a supplemented medium. Two examples include CHODHFR- cells and mouse LTK- cells. These cells lack the ability to grow without added nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes required for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented medium. An alternative to supplementing the medium is to alter the growth requirements of cells lacking the respective genes by introducing an intact DHFR or TK gene. Individual cells not transformed with the DHFR or TK genes will not be able to survive in unsupplemented medium.

[0143] The second category is dominant selection, which refers to selection schemes that can be used with any cell type and do not require the use of mutant cell lines. These schemes typically use drugs to stop host cell growth. Those cells carrying the novel gene will express a protein that conveys drug resistance and survive selection. Examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid (Mulligan, R.C. and Berg, P., Science 209:1422 (1980)), or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5:410-413 (1985)). Three examples use bacterial genes under eukaryotic control to convey resistance to the appropriate drugs, G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0144] E. Peptides a) Protein variants Protein variants and derivatives are well understood by those skilled in the art and can include amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional, or deletional variants. Insertions include amino- and / or carboxyl-terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions will usually be smaller than those of amino- or carboxyl-terminal fusions, e.g., on the order of one to four residues. Derivatives of immunogenic fusion proteins, such as those described in the Examples, are generated by fusing a polypeptide of sufficient size to confer immunogenicity to a target sequence by in vitro crosslinking or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about two to six residues are deleted at any one site within the protein molecule. These variants are typically prepared by site-directed mutagenesis of nucleotides within the DNA encoding the protein, thereby producing DNA encoding the variant, followed by expression of the DNA in recombinant cell culture. Well-known techniques for making substitution mutations at predetermined sites in DNA with a known sequence include M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues but can occur at several different locations at once; insertions are usually on the order of about 1 to 10 amino acid residues, and deletions range from about 1 to 30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., deletion of two residues or insertion of two residues. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at the final construct. Mutations should not shift the sequence out of reading frame and preferably do not create complementary regions that could produce secondary mRNA structure. Substitution variants are those in which at least one residue has been removed and a different residue has been inserted in its place. Such substitutions are generally made in accordance with Tables 5 and 6 below, and are referred to as conservative substitutions.

[0145] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 6, i.e., by selecting residues that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions generally expected to result in the greatest changes in protein properties would be those resulting in (a) a hydrophilic residue, e.g., seryl or threonyl, being substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline being substituted for (or by) any other residue; (c) a residue with an electropositive side chain, e.g., lysyl, arginyl, or histidyl, being substituted for (or by) an electronegative residue, e.g., glutamyl or aspar; or (d) a residue with a bulky side chain, e.g., phenylalanine, being substituted for a residue without a side chain, e.g., glycine in this case; or (e) increasing the number of sites for sulfation and / or glycosylation.

[0146] For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue with another, or replacing one polar residue with another. Substitutions include, for example, combinations of glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine, and the like. Such conservatively substituted variations of each explicitly disclosed sequence are included in the mosaic polypeptides provided herein.

[0147] Substitutional or deletional mutagenesis can be used to insert sites for N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletion of cysteines or other labile residues may also be desirable. Deletion or substitution of potential proteolysis sites, e.g., Arg, is accomplished, for example, by deleting one of the basic residues or substituting one with a glutaminyl or histidyl residue.

[0148] Certain post-translational derivatizations are the result of the action of recombinant host cells on expressed polypeptides. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the O-amino groups of lysine, arginine, and histidine side chains (Tecreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86

[1983] ), acetylation of the N-terminal amine, and, in some cases, amidation of the C-terminal carboxyl.

[0149] It should be understood that one way of defining variants and derivatives of the proteins disclosed herein is by defining them in terms of homology / identity to a particular known sequence. Specifically disclosed are variants of these and other proteins disclosed herein that have at least 70%, 75%, 80%, 85%, 90%, or 95% homology to the set forth sequence. Those skilled in the art can readily understand how to determine the homology of two proteins. For example, homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0150] Other methods for calculating homology can be performed using published algorithms. Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0151] For nucleic acids, the same type of homology can be obtained, for example, by the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989.

[0152] It is understood that the descriptions of conservative variation and homology can be combined together in any combination, such as in an embodiment where a variant has at least 70% homology to a particular sequence, where the variant is a conservative variation.

[0153] Because this specification discusses various proteins and protein sequences, it is understood that the nucleic acids capable of encoding those protein sequences are also disclosed. This would include all degenerate sequences related to a particular protein sequence, i.e., all nucleic acids having a sequence that encodes a particular protein sequence, as well as all nucleic acids containing degenerate nucleic acids that encode disclosed variants and derivatives of a protein sequence. Thus, while each specific nucleic acid sequence may not be described herein, it is understood that each and every sequence is actually disclosed and described herein through the disclosed protein sequences. It is also understood that, although there is no amino acid sequence that represents the specific DNA sequence encoding the protein in an organism in which a particular variant of a disclosed protein is disclosed herein, known nucleic acid sequences encoding that protein are also known, disclosed, and described herein.

[0154] It is understood that there are numerous amino acid and peptide analogs that can be incorporated into the disclosed compositions. For example, there are numerous D-amino acids or amino acids with different functional substituents than those shown in Tables 5 and 6. Opposite stereoisomers of naturally occurring peptides, as well as stereoisomers of peptide analogs, are disclosed. These amino acids can be readily incorporated into a polypeptide chain by charging a tRNA molecule with the amino acid of choice and engineering a genetic construct that inserts the analog amino acid into the peptide chain in a site-specific manner, e.g., using an amber codon.

[0155] Molecules can be produced that resemble peptides but are not joined via natural peptide bonds. For example, amino acid or amino acid analog linkages can include CH2NH--, --CH2S--, --CH2--CH2--, --CH=CH-- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CHH2SO-- (these and others are described in Spatola, A. F., In Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends PharmSci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185(1979)(--CH2NH--,CH2CH2--);Spatolaetal.Life Sci 38:1243-1249(1986)(--CHH2--S);Hann J.Chem.Soc Perkin Trans.I307-314(1982)(--CH--CH--,cis and trans);Almquist et al.J.Med.Chem.23:1392-1398(1980)(--COCH2--);Jennings-White et al.Tetrahedron Lett 23:2533(1982)(--COCH2--);Szelke et al.European Appln. EP 45665 CA (1982):97:39405 (1982) (--CH(OH)CH--); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH--); and Hruby Life Sci 31:189-199 (1982) (--CH--S--), each of which is incorporated herein by reference. A particularly preferred non-peptide bond is --CHNH--.It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

[0156] Amino acid analogs and analogs, as well as peptide analogs, often have more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broader spectrum of biological activity), reduced antigenicity, etc.

[0157] Because D-amino acids are not recognized by peptidases and the like, D-amino acids can be used to generate more stable peptides. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used to generate more stable peptides. Cysteine ​​residues can be used to cyclize or link two or more peptides together. This can be beneficial for constraining peptides into a particular conformation.

[0158] F. Pharmaceutical Carriers / Pharmaceutical Product Delivery As mentioned above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject together with a nucleic acid or vector, without causing any undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition with which it comes into contact. The carrier will necessarily be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as will be well known to those skilled in the art.

[0159] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, topically (including topical intranasal administration or administration by inhalant), etc. As used herein, "topical intranasal administration" means delivery of a composition to the nose and nasal passages via one or both nostrils and can include delivery by a spray or droplet mechanism, or by aerosolization of the nucleic acid or vector. Administration of a composition by inhalant can be through the nose or mouth via delivery by a spray or droplet mechanism. Delivery can also be directly to any region of the respiratory system (e.g., lungs) via intubation. The exact amount of composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration, etc. Thus, it is not possible to specify an exact amount for every composition. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0160] Parenteral administration of compositions, when used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution in liquid prior to injection, or as emulsions. A recent revision of parenteral administration approaches involves the use of slow or sustained release to maintain a constant dosage. See, for example, U.S. Patent No. 3,610,795, incorporated herein by reference.

[0161] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992), and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). Generally, receptors are involved in pathways of either constitutive or ligand-induced endocytosis. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and then either recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0162] G. Methods of Treating Cancer The plasmids, nucleic acids, constructs, vectors, and modified T cells disclosed herein can be used to treat, inhibit, reduce, diminish, alleviate, and / or prevent any disease in which uncontrolled cell proliferation occurs, such as cancer. Recent advances have been made in cancer immunotherapy. Genetically modified chimeric antigen receptor (CAR) T cells are an excellent example of engineered immune cells that have been successfully deployed in cancer immunotherapy. It is understood and contemplated herein that the disclosed plasmids, nucleic acids, constructs, and methods can be used to generate cancer-targeting CAR-T cells.

[0163] Accordingly, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and / or myelodysplastic syndrome (MDS), etc.), the methods comprising administering to a subject having cancer an engineered T cell as disclosed herein. For example, disclosed herein are methods for treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and / or myelodysplastic syndromes (MDS)), comprising administering to the subject a therapeutically effective amount of modified T cells, wherein the modified T cells comprise a plasmid, nucleic acid, and / or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises, in order, a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (e.g., a CAR that targets CD33), and a right homology arm, wherein the left homology arm and the right homology arm are each 1000 bp or less (e.g., 600 bp) in length.

[0164] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, a reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to native or control levels.

[0165] "Reduce" or other forms of this term, such as "reducing" or "reduction," refers to a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value; in other words, it is relative, although it is understood that reference to a standard or relative value is not necessarily required. For example, "reducing tumor growth" means reducing the rate of tumor growth compared to a standard or control.

[0166] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, stabilize or slow the development or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduction and therefore does not require a comparison to a control. As used herein, something may be reduced but not prevented, although something that is reduced may be prevented. Similarly, something may be prevented but not reduced, although something that is prevented may be reduced. It is understood that where reduction or prevention is used, the use of other words is expressly disclosed unless specifically specified otherwise.

[0167] The term "treatment" refers to the medical management of a patient with the intent to cure, alleviate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, condition, or disorder; preventative treatment, i.e., treatment directed at minimizing or partially or completely inhibiting the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed at ameliorating the associated disease, condition, or disorder.

[0168] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, e.g., a mammal. In one aspect, a subject can be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. A subject can also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, a subject can be a human or a veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0169] As noted above, the plasmids, nucleic acids, constructs, vectors, and modified T cells disclosed herein can be used to treat, suppress, reduce, decrease, ameliorate, and / or prevent cancer. A representative, but non-limiting list of cancers that can be treated using the disclosed compositions is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, acute lymphocytic leukemia (ALL), hairy cell leukemia (HCL), myelodysplastic syndromes (MDS), myeloid leukemia (including, but not limited to, acute myeloid leukemia (AML) and chronic myeloid leukemia (CML)), bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer, including small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, cancer of the mouth, pharynx, larynx, and squamous cell carcinoma of the lung, cervical cancer, carcinoma), breast cancer, as well as epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostate cancer, or pancreatic cancer.

[0170] In one aspect, disclosed herein is a method for adoptively transferring engineered T cells into a subject in need thereof, the method comprising: a) obtaining the T cells to be engineered; and b) obtaining a ribonucleoprotein (RNP) complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA and an AAV vector comprising a plasmid, nucleic acid, or construct comprising a transgene (e.g., a chimeric antigen receptor for a tumor antigen), the transgene being flanked by homology arms and linked to the homology arms. The method includes obtaining a modified T cell, wherein the transgene is less than 1000 bp; c) introducing the transgene and RNP complex into a T cell, wherein the transgene is introduced into the T cell via infection with an adeno-associated virus (AAV), the RNP complex hybridizes to a target sequence within the T cell's genomic DNA, and DNA repair enzymes in the T cell insert the transgene into the host genome (e.g., by homologous repair) at the target sequence within the target cell's genomic DNA, thereby generating a modified T cell; and d) transferring the modified T cell to a subject. In one embodiment, the transgene can be contained on the same plasmid, nucleic acid, or construct as the Cas9 endonuclease, or encoded on a second plasmid, nucleic acid, or construct within the same or a different AAV vector. In one embodiment, the target cell can be transduced using electroporation with the RNP complex prior to or simultaneously with infection of the cell with the AAV containing the transgene.

[0171] In one aspect, the modified T cells used in the disclosed immunotherapy methods can be primary cells from a donor source (e.g., an allogeneic donor source for adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified cells), a T cell line, or a primary T cell source or an expanded T cell source obtained from a cell line. While primary T cells can be used, it is understood and contemplated herein that the disclosed modifications of T cells can occur ex vivo or in vitro.

[0172] Also disclosed herein is a plasmid, nucleic acid, or construct comprising, in order, a left homologous arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and a right homologous arm, wherein the left and right homologous arms are each 1000 bp or less in length.

[0173] In another aspect, disclosed herein is a plasmid, nucleic acid, or construct, AAV vector, or modified T cell for use as a medicament. Disclosed herein is the use of a plasmid, nucleic acid, or construct, AAV vector, or modified T cell disclosed herein for the manufacture of a medicament.

[0174] Also disclosed herein is a plasmid, nucleic acid, or construct, AAV vector, or modified cell disclosed herein for use in cancer treatment. Also disclosed herein is the use of a plasmid, nucleic acid, or construct, AAV vector, or modified cell disclosed herein for the manufacture of a medicament for cancer treatment.

[0175] Also disclosed herein are chimeric antigen receptor (CAR) T cells generated using the methods of generating CAR T cells disclosed herein for use in treating cancer. Also disclosed herein is the use of chimeric antigen receptor (CAR) T cells generated using the methods of generating CAR T cells disclosed herein for the manufacture of a medicament for treating cancer.

[0176] Although several embodiments of the present disclosure have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0177] By way of non-limiting example, examples of specific embodiments of the present disclosure are provided below. [Example]

[0178] The following examples are intended to illustrate compositions, devices, methods, and results in accordance with the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that would be apparent to one skilled in the art.

[0179] Example 1: KromaTiD dGH screen whole genome analysis report - Sample KromaTiD-CD33CARNK / 8AM-dGH / 8 / 1 / 23. Assay: Standard dGHSCREENAS-0002.1 (50 cells)

[0180] Metaphase and karyotype qualification: Samples were prepared and qualified for dGH analysis before performing the assay.

[0181] The dGH screen is designed for samples with a globally normal karyotype and is not qualified for analysis of highly rearranged genomes.

[0182] A spread resolution of 350+ (G-band equivalent) is selected for the analysis.

[0183] Assay Description: The Five-Color Whole Genome Assay (5CWG or dGHSCREEN) is a combination assay of dGH paints for all 24 human chromosomes.

[0184] The assay includes five color panels composed of unique arrays of high-density (HD) dGH chromosome paints, such that chromosomes painted with the same color can be distinguished by size, shape, and centromere position.

[0185] Results include chromosome-specific attribution of inter- and intra-chromosomal structural events, including inversions, translocations, aneuploidies (gains and losses), insertions, centromeric abnormalities, and complex events across samples.

[0186] Prior to analysis, images of metaphase spreads drawn with the dGH screen are qualified, processed, and sorted into chemokaryograms for rapid and consistent assay reading.

[0187] Cell-by-cell event evaluation is performed to exploit population-level analysis of events from random to clonal.

[0188] Please refer to Tables 2 to 6 and Figures 2 to 7. Example 2: Transgene analysis and integration site sequencing of one sample of human cells containing the 33_CD33CARV4(LHCD8-41)CD38_ssAAV-BackBoneKan vector.

[0189] One sample of transgenic human cells carrying the 33_CD33CARV4(LHCD8-41)CD38_ssAAV-BackBoneKan vector sequence was analyzed. Table 7 shows the summary of the integrated sequencing.

[0190] The analysis showed that: 1) Vector integrity: a. Determining the presence of sequence variants and their allele frequencies. b. Determining the presence of inter-vector breakpoints, indicative of ligation of multiple vector copies and / or structural rearrangements within a single vector sequence. 2) Identification of the vector integration site and breakpoint sequences between the vector and the genome. 3) Assessment of the presence and abundance of off-target integration sites. 4) Assessment of the presence of structural variations surrounding the vector integration site.

[0191] The generated data show that the vector successfully integrated at the target site in a proportion of cells. Additionally, three sequence and seven structural mutations were detected within the vector. Finally, seven random integration events were detected at the sgRNA cleavage site. Targeted locus amplification (TLA), sequencing, and data mapping

[0192] Viable, frozen human primary NK cells were used and processed according to the Cergentis TLA protocol. TLA was performed with two independent primer sets specific for the vector sequence and the genome (Table 8). Next-generation sequencing (NGS) reads were aligned to the vector sequence and the host genome. The human hg38 genome was used as the host reference genome sequence.

[0193] Result CD38 KO / CD33CARV4NK FIG. 8 shows NGS coverage across the vector sequence using primer sets 1 and 2.

[0194] Coverage is observed throughout the vector sequence, Vector:1-6,346, indicating that the vector is integrated into the backbone. Low coverage in the backbone indicates integration in a subset of cells. Coverage is also observed in the ITRs, indicating that in addition to integration through the homology arms, ITR-based integration also occurred in this sample. Coverage in the ITR regions and backbone also indicates the presence of episomal (non-integrated) copies of the vector in this sample.

[0195] The boundaries of the homologous arms at positions 145 and 3,666 allow for quantification of alleles containing correctly integrated TGs within the data for Primer Set 2. At position 145, 34% of reads represent successful targeting events, compared to 38% at position 3,666.

[0196] Sequence and structural variations were called in the covered regions.

[0197] Sequence Variations. The sequence variations detected are shown in Table 9. All variations located in homologous arms are considered to be genomic variations detected due to incomplete integration of this homologous arm in a subset of samples.

[0198] Vector polymerization and structural variations. The identified intervector breakpoint sites are shown in Table 10. A total of seven structural variations were identified. Complete reads were also found at all breakpoint locations, indicating polymerization of (partial) vector sequences. Using TLA, it is not possible to determine the exact order of (partial) copies or to confirm the presence of at least one complete copy. Furthermore, all breakpoint sequences are considered to represent polymerization of ectopic (non-integrated) copies.

[0199] Breakpoint sites 1 and 6, 2 and 7, and 4 and 10 represent the same breakpoint sequence but are not reported twice due to homology within the vector. Therefore, breakpoint sequences may have arisen from both designated vector locations. Due to the heterogeneous nature of the samples, these fusions are expected to be present in a subset of samples.

[0200] Example 3: Relapsed / Refractory CD33 + Siglenkabtageneunileucel (Sig-leucel) for the treatment of acute myeloid leukemia overview Sig-leucel is composed of universal donor peripheral blood natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) targeting CD33. CD33CARNK cells are generated by a combination of techniques including feeder cell expansion of NK cells, CRISPR / Cas9 gene editing, and adenovirus-associated virus (AAV) gene delivery. Cas9 / RNP electroporation initiates disruption of the CD38 locus in combination with AAV-mediated CAR delivery with homologous arms for site-specific gene insertion. The sig-leucel second-generation CAR protein consists of an extracellular portion harboring a humanized anti-CD33 single-chain antibody fragment (scFv) and an intracellular portion containing cell signaling (CD3-ζ) and costimulatory (4-1BB) domains. Based on current knowledge, this gene editing combination will not be used to generate clinical products with NK cells. The purpose of sig-leucel is to target CD33. +Treatment of patients with relapsed / refractory acute myeloid leukemia (AML).

[0201] background Disease background - relapsed / refractory AML Despite advances in our understanding of AML biology, survival rates for patients with relapsed or refractory disease remain stagnant. With current standard relapse regimens, complete response rates (CR rates) remain at 50–70%, even with the addition of anti-CD33 antibody therapy. For patients with a morphologic response to reinduction and consolidation with hematopoietic stem cell transplantation (HSCT), the 2-year overall survival (OS) rate is less than 50%. The current accepted standard of care for relapsed AML is chemotherapy with cytarabine (Ara-C) and fludarabine. The importance of high-dose cytarabine in both initial and salvage therapy for the treatment of AML is well established.

[0202] Fludarabine is widely used to lymphocytose patients before lymphocyte infusion, and fludarabine-containing regimens, usually combined with cytarabine and antirubicin, are used for reinduction of newly diagnosed refractory or relapsed AML. Gandhi et al. showed that fludarabine enhances the increased intracellular retention of Ara-CTP, the active metabolite of cytarabine, in AML blasts. This led to the development of the highly active FLAG (fludarabine, cytarabine, G-CSF) regimen for AML (Estey E, Plunkett W, Gandhi V, et al.: Fludarabine and arabinosylcytosine therapy of refractory and relapsed acute myelogenous leukemia. Leuk Lymphoma 9:343-50, 1993).

[0203] According to a study by the International Berlin-Frankfurt-Munster Study Group, pediatric patients with first relapsed or primary resistant AML who received FLAG chemotherapy had a CR rate of 59% after two courses of treatment (Kaspers GJ, Zimmermann M, Reinhardt D, et al: Improved outcome in pediatric relapsed acute myeloid leukemia: results of a randomized trial on liposomal daunorubicin by the International BFM Study Group. J Clin Oncol 31:599-607, 2013).

[0204] The median absolute percentage of leukemic blasts on day 28 was 4%. A retrospective analysis by the Japanese Pediatric Leukemia / Lymphoma Study Group, which studied outcomes for relapsed pediatric acute myeloid leukemia (AML), showed a 65% CR after FLAG-based reinduction (Moritake H, Tanaka S, Miyamura T, et al: The outcomes of relapsed acute myeloid leukemia in children: Results from the Japanese Pediatric Leukemia / Lymphoma Study Group AML-05R study. Pediatr Blood Cancer: e28736, 2020).

[0205] Despite most patients undergoing transplantation, the 5-year overall survival rate was 36.1%. In a recent COG study of children with relapsed / refractory AML, patients were treated with liposomal daunorubicin / cytarabine (CPX-351) followed by FLAG (Cooper TM, Absalon MJ, Alonzo TA, et al: Phase I / II Study of CPX-351 Followed by Fludarabine, Cytarabine, and Granulocyte-Colony Stimulating Factor for Children With Relapsed Acute Myeloid Leukemia: A Report From the Children's Oncology Group. J Clin Oncol 38:2170-2177, 2020).

[0206] The best responses in patients with initial refractory disease or first relapse included 54% complete remission (CR) with partial platelet recovery, 14% CR, and 13.5% CR with incomplete blood test recovery. The relapse studies reported the CR rate after first relapse. Patients with second or subsequent relapses show increasingly poor response rates to conventional chemotherapy regimens, with a 25% CR rate after the third treatment attempt and a 17% CR rate after four to six treatment attempts. Similar to acute lymphoblastic leukemia, the depth of response before HSCT is an important prognostic marker in AML. These results refer to the morphologic CR rate, and strategies to improve pre-HSCT MRD-negative response rates are important for improving outcomes in this high-risk patient population.

[0207] NK cell therapy for AML Natural killer (NK) cells are cytotoxic lymphocytes that play a key role in recognizing malignant and virus-infected cells, bridging the gap between innate and adaptive immune responses. In hematological malignancies, innate NK cells exhibit qualitative and quantitative dysfunction, and poor NK cell function at diagnosis predicts a poor prognosis. At the time of AML diagnosis, NK cell phenotypes can be stratified into highly functional and dysfunctional groups with distinct transcriptional alterations in pathways involved in cytotoxicity, intracellular signaling, and metabolism. Patients with dysfunctional NK cell profiles at diagnosis were at increased risk of relapse. Furthermore, patients with a "less mature" NK cell profile had reduced overall and relapse-free survival.

[0208] NK cell activation and cytotoxicity are independent of tumor antigens and are instead regulated by the balance of activating and inhibitory NK receptor signaling. Activating receptors recognize ligands on the surface of cancer or virus-infected cells that represent danger, while inhibitory receptors are responsible for self-recognition. NK receptor classes include natural cytotoxicity receptors (NCRs), C-type lectin receptors, and killer cell immunoglobulin-like receptors (KIRs). The inhibitory effect of class I HLA on KIR stimulation may limit the clinical efficacy of autologous NK cells. Clinical evidence demonstrating the benefit of alloreactive NK cells in AML comes from studies of the graft-versus-leukemia (GVL) effect in allogeneic stem cell transplantation. Stem cell transplantation 22 Early NK cell recovery after transplant and increased NK cell numbers in the graft are associated with improved transplant outcomes in leukemia.

[0209] Further evidence for NK cell-mediated GVL has been provided in the setting of HLA-mismatched HSCT. Ruggeri et al. observed that AML patients undergoing haploidentical HSCT had a reduced relapse rate when there were HLA differences between the donor and recipient in the GVL direction in a NK cell-deficient ligand model (Ruggeri L, Capanni M, Urbani E, et al: Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295:2097-100, 2002).

[0210] This concept is called "ligand-ligand mismatch," and similar studies have confirmed the importance of NK alloreactivity in AML patients undergoing HSCT. Similarly, patients who underwent HLA-mismatched transplants with donor-recipient pairs that mismatched KIR genes experienced reduced relapse and improved survival. These clinical observations are also supported by mouse models in which allogeneic NK cells demonstrated superior antitumor activity and survival compared with autologous NK cells.

[0211] Supported by this early clinical evidence, adoptive NK cell therapy to enhance the GVL effect was investigated. The earliest trials were performed with NK cells isolated from the leukocyte compartment of healthy donors using immunomagnetic cell selection and overnight IL-2 activation. Using this approach, Miller et al. demonstrated the ability to induce up to 2 x 10 7 It has been shown that infusion of haploidentical NK cells / kg can induce remission of poor-prognosis AML without graft-versus-host disease (GVHD) (Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al: Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105:3051-7, 2005).

[0212] In a similar study, Rubnitz et al. reported on the safety of KIR-mismatched NK cell infusion as a post-remission boost therapy for children with AML, with no relapses reported in 10 treated patients (Rubnitz JE, Inaba H, Ribeiro RC, et al: NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia. J Clin Oncol 28:955-9, 2010).

[0213] Other studies using NK cells obtained by this approach have also shown that NK cell infusion is safe and not associated with severe infusion reactions, graft-versus-host disease (GVHD), or graft rejection in the setting of allogeneic sarcoma transplantation (HSCT) in patients with lymphoma and myeloma. However, response rates in these studies have been variable (OS ranging from 29% to 73%), and the dose of NK cells generated by this approach is typically ≤10 7 / kg single dose.

[0214] A genetically modified K562 cell line used as irradiated feeder cells has been developed, allowing for the generation of large numbers of clinical-grade NK cells from normal donor, patient, umbilical cord blood, and embryonic / pluripotent stem cells. Peripheral blood NK cells have been infused and expanded using this approach in Phase I / II trials for hematological malignancies and pediatric solid tumors. In these trials, up to 3 × 10 8 More than 300 infusions of expanded and activated NK cells at a dose of 100 cells / kg have been administered to over 100 patients, showing evidence of early efficacy without dose-limiting toxicity.K562-derived feeder cells were genetically modified to express membrane-bound IL-21 and 4-1BBL.

[0215] In a phase I / II study (NCT01904136), 25 patients with myeloid malignancies (AML, MDS, CML) underwent stem cell transplantation (SCT) from a semi-synonymous donor and received three infusions (days -2, 7, and 28) of NK cells expanded from the donor's peripheral blood. 4 ~10 8 Thirteen patients treated during the Phase I dose-escalation trial were reported, with only one patient experiencing a relapse, and relapse-free survival and viral reactivation rates were unexpectedly low. No patients developed GvHD greater than grade II, which was lower than the historical incidence of this regimen without NK cells. The lowest dose level, 10 4 One infection-related graft failure was observed in a patient treated at 1 / kg. No dose-limiting toxicities (DLTs) were observed in the phase I and phase II expansion studies, and non-DLT adverse events were minimal, including transient fever, rash, and tachycardia. Updated phase II results showed promising results, with a 2-year relapse rate of 4% compared to 38% (p=0.014) in a case-matched control cohort from the CIBMTR database.

[0216] Twenty-two patients with myeloid leukemia undergoing matched allogeneic hematopoietic stem cell transplantation received NK cells expanded from umbilical cord blood (N=10) or peripheral blood of related matched (N=9) or partially matched (N=3) donors for 10 6 ~10 8 No dose-limiting toxicities were observed, and non-DLT adverse events were minimal, including fever, rash, and tachycardia.

[0217] In completed phase I / II studies (NCT01787474 in the US and NCT02809092 in Brazil), 28 adult patients with relapsed / refractory AML were treated with reinduction chemotherapy (fludarabine / cytarabine + / - GCSF) followed by six infusions (three times weekly for two weeks) of expanded haploidentical NK cells for 10 days. 6 ~10 7The drug was administered at a dose of 0.05 mg / kg. In the first 13 patients reported, 78.6% responded, with 50% achieving a complete response. The first subject treated in this trial began treatment with 91% blasts in his bone marrow. Twenty days after completing the NK cell infusion, his bone marrow contained 5% blasts by flow cytometry. No further treatment was administered, and another bone marrow was obtained 40 days after completing treatment, showing only 0.6% blasts. 100 days after treatment, blasts had further decreased to 0.07%, suggesting an ongoing immunological anti-leukemia response elicited by NK cells.

[0218] In the clinical trials mentioned above, subjects received patient-specific NK cell products (autologous or related allogeneic donor). As with autologous CAR T cells, these studies experienced high costs, delays, and subject withdrawals due to the manufacturing required after enrollment.

[0219] To solve this problem, an approach to define optimal "universal" donors was developed, partnering with Be-the-Match Biotherapies (BTMB) to identify these donors and collect peripheral blood mononuclear cells (MNC(A)) by apheresis. Using a stepwise algorithm, ideal alloreactive donors were selected from individuals with KIR-B genotype, HLA-approved groups C1, C2, and Bw4, and cytomegalovirus (CMV)+ serostatus. The rationale for the universal donor selection criteria is as follows: 1) Killer cell immunoglobulin-like receptors (KIRs) are genetically highly polymorphic and are inherited independently of HLA. KIR genes can be classified into inhibitory (KIRA haplotype) or activating (KIRB haplotype) haplotypes based on gene content. Individuals with the KIR-B genotype have a higher number of activated NK cell receptors, which results in enhanced alloreactivity and antitumor function. 2) NK cells are licensed (enhanced in their attack potential) when they express inhibitory killer immunoglobulin receptors (KIRs) for self-HLA class I molecules. This allows NK cells to recognize "self" and spare self cells from attack. Therefore, targets lacking self-HLA class I molecules are more likely to be recognized by licensed NK cells. The inhibitory KIR genes known to be involved in NK alloreactions are: (i) 2DL1, which binds to HLA-C group 2 alleles; (ii) 2DL2 and 2DL3, which bind to HLA-C group 1 alleles; and (iii) 3DL1, which binds to HLA-BBw4 alleles. According to the missing ligand model, for each NK cell expressing an inhibitory KIR gene, an alloreactivity will occur only if the corresponding ligand is absent from the receptor but present in the donor; for example, any donor with a group C1 allele will exhibit an alloreactivity to any individual lacking the group C1 allele. Thus, a donor with HLA from the C1, C2, and Bw4 families is predicted by this model to exhibit an alloreactivity to any receptor lacking C1, C2, or Bw4. 3) While inhibitory KIRs prevent alloreactivity, activating KIRs (aKIRs) recognize activating ligands that promote NK cell cytolysis. Inheritance of activating KIRs is widely variable, with an individual potentially having 0 to 7 aKIRs. Data from patients undergoing stem cell transplantation indicate that patients receiving allogeneic transplants from donors with more activating KIRs have better outcomes than those receiving allogeneic transplants from donors with fewer activating KIRs. 4) Finally, NKG2C expression is induced in patients with CMV infection and correlates with an adaptive NK cell phenotype and improved leukemia-free relapse survival.

[0220] Therefore, the optimal universal donor was defined as one with an HLA genotype consisting of C1, C2, and Bw4 alleles, a KIR genotype with inhibitory KIRs binding C1, C2, and Bw4 (resulting in maximal licensing), a high proportion of activating KIRs, and exposure to CMV with high NKG2C expression.

[0221] Considering data on Caucasian donors, the C1 / C2 / Bw4 allele is present in 32% of the population. Of the 23 KIR genotypes that represent 80% of the population, 25.3% fulfill all of these conditions. Approximately 90% of adults are thought to be exposed to CMV. Therefore, an "ideal" NK cell donor can be identified in approximately 1 in 16 healthy individuals. In collaboration with BTMB, an apheresis product was identified and collected from 10 different donors as starting material for NK cells.

[0222] Six patients were enrolled in a phase 1 study (NCT04220684) investigating the safety of universal donor NK cells in combination with chemotherapy for the treatment of primary refractory or relapsed AML or MDS in adults. Universal donor NK cells were produced by an expansion technique utilizing irradiated CSTX002 feeder cells as described above. All six patients received 1x10 7 Each patient received 1000 NK cells / kg, with a total of six planned NK cell infusions over two weeks (days 0, 2, 4, 7, 9, and 11). Five patients tolerated all six NK cell infusions administered within two weeks. No transfusion-related reactions, neurotoxicity, or graft-versus-host disease were observed. One patient developed symptoms concerning for cytokine release syndrome (CRS) associated with streptococcal bacteremia after the first infusion of NK cells, and subsequent infusions were therefore withheld. Because definitive attribution to bacteremia was not possible, attribution to NK cells was deemed probable. CRS-related symptoms resolved with the administration of steroids.

[0223] These initial trials demonstrate the safety and initial clinical efficacy of multiple administrations of allogeneic expanded NK cells for AML. However, treatment of extensive relapsed disease may require more potent NK cell products because leukemic blasts exploit mechanisms that promote NK cell dysfunction, including altered expression of activating receptors and tumor downregulation of NK receptor ligands. Improving NK cell function and tumor antigen recognition is critical for the success of cell therapy in AML.

[0224] CD33 as a target in AML The safety of CAR-directed cell therapy products depends primarily on the CAR target molecule. An ideal CAR target is a tumor-associated antigen that is highly expressed on tumor cells and not expressed on other normal tissues in the body. CD33 is a salivary acid-binding immunoglobulin-related lectin (siglec) present on the surface of hematopoietic cells. CD33 is expressed on bone marrow-derived cells and leukemic blasts, but importantly, it is not present on pluripotent hematopoietic stem cells or non-hematopoietic cells. CD33 is expressed in more than 80% of AML patients. Given the high expression of CD33 on AML blasts and leukemic progenitor cells, there is a long precedent for exploiting this target in immunotherapy approaches.

[0225] Gemtuzumab-ozogamicin (GO) is a humanized anti-CD33 monoclonal antibody conjugated to the DNA-binding cytotoxic drug calicheamicin, approved by the FDA for use in pediatric and adult CD33-positive acute myeloid leukemia (AML). Upon binding to CD33, GO is internalized, releasing calicheamicin, which induces DNA double-strand breaks and results in cell death. When combined with chemotherapy, GO is effective in reducing minimal residual disease in patients with relapsed and refractory AML. Commonly cited toxicities include myelosuppression related to off-target tumors and sinus venosus obstruction (SOS) due to calicheamicin hepatotoxicity. In the largest pediatric GO study to date, 1,022 pediatric patients with newly diagnosed AML were enrolled in the Children's Oncology Group trial AAML0531 and randomly assigned to receive standard induction chemotherapy or standard induction chemotherapy combined with GO. Although the addition of GO did not affect the remission rate after induction, disease-free survival was improved in patients receiving GO, associated with a reduced risk of relapse (HR, 0.73; 95% CI, 0.58–0.91; P = .006; 3-year RR: 32.8% ± 4.6% vs. 41.3% ± 4.9%), consistent with adult data. Grade 3–5 adverse events in this study were similar between study arms, including the incidence of hematologic toxicities and time to neutrophil recovery. However, a post-hoc analysis noted a higher rate of prolonged neutrophil recovery (>59 days) in GO patients during INT2 (12.0% vs. 6.3%, P = .01), which may have contributed to the increased treatment-related mortality in the low-risk group. The clinical efficacy of GO may be limited by pharmacokinetics and resistance mechanisms, including overexpression of drug efflux pumps in AML cells. However, the acceptable safety profile and lack of off-target toxicity seen with GO support further exploration of CD33 as an immunotherapy target.

[0226] Lintuzumab (HuM195) is a humanized anti-CD33 monoclonal antibody with high binding affinity and cytotoxic activity against CD33-positive cells. Although there were initial reports of clinical efficacy in adults with AML, large phase III randomized studies using lintuzumab in combination with chemotherapy in adults with relapsed AML failed to demonstrate significant improvements in response rates or survival in patients receiving the antibody. Importantly, all of these studies noted only infusion-related adverse events with the addition of lintuzumab to chemotherapy, further supporting the safety of CD33 as a therapeutic target. Due to concerns that antibody therapy alone was insufficient for disease control in leukemia, lintuzumab development shifted to radiolabeled HuM195 to enhance its efficacy.

[0227] Following the success of CD19 CAR T-cell therapy, there has been a recent movement to develop CAR T cells for AML. Ten adults with relapsed / refractory AML were enrolled in a phase I trial using home-grown CD33 CAR T cells. Only three patients were able to receive CAR T-cell infusions; four patients had CD33 CAR T-cell products that did not meet release criteria, two had rapidly progressing AML before apheresis, and one patient died before receiving cells. Two patients developed cytokine release syndrome, and one patient exhibited neurotoxicity associated with CAR T-cell infusion. There were no dose-limiting toxicities. None of the patients had a clinical response to treatment, and all three died of progressive disease. This study highlights the feasibility issues of using autologous T cells in patients with relapsed / refractory leukemia; the majority of patients enrolled in this trial (7 of 10) were unable to receive the study treatment due to challenges in collecting and manufacturing autologous T cells from heavily pretreated patients with the immunosuppressive environment of active leukemia. A Phase I / II study of autologous CD33-CAR-T cells in pediatric patients with relapsed / refractory AML is currently underway (NCT03971799). This study experienced numerous subject withdrawals, manufacturing issues, and treatment delays. These studies highlight the need for "off-the-shelf" cell therapies to improve the speed and efficacy of harvested cell therapy in this patient population.

[0228] CAR NK cells Historically, genetic modification of NK cells has been unsuccessful due to their resistance to viral transduction. In contrast to T cells, the innate function of NK cells as a first antiviral defense makes them relatively resistant to traditional viral transduction-based genetic modification approaches. Alternative NK cell sources and novel genetic engineering techniques have enabled successful genetic modification of NK cells using non-viral methods. In a phase I / II clinical trial utilizing CAR-NK cells, 11 patients with CLL or NHL received a single injection of coldblood-derived, off-the-shelf CD19CAR-NK cells. The CAR-NK cells were equipped with CD19CAR, IL-15, and caspase suicide genes. The CAR-NK cells were well tolerated, with no dose-limiting toxicities, no reports of cytokine release syndrome or neurotoxicity, and a 73% response rate. CAR-NK cells expanded in vivo and remained detectable for at least one year after infusion. Similar to data reported in CAR-T cell trials, patients who responded to treatment had higher peak CAR-NK cell proliferation than non-responders. There are many other CAR-NK cell targets in development for hematologic malignancies, including CD33, CD123, CD20, CD19 / 20, and BCMA.

[0229] CD38 knockout Daratumumab, an FDA-approved monoclonal antibody against CD38, has transformed the treatment landscape for multiple myeloma, with an overall response rate of over 80% when combined with chemotherapy. Preclinical and clinical reports also suggest a role for targeting CD38 in other hematologic malignancies. NK cells have high levels of CD38 on their surface and are depleted in patients treated with daratumumab as a result of NK-on-NK ADCC, a process called fratricide. CD38-negative or low-CD38 NK cells are resistant to daratumumab-induced fratricide and, compared with CD38-positive NK cells, demonstrate enhanced tumor cytotoxicity when combined with daratumumab. To overcome NK cell-on-NK killing caused by daratumumab, CRISPR / Cas9 has been used to generate CD38-knockout NK cells (CD38 KO These CD38KO NK cells are resistant to daratumumab-induced fratricide, have a favorable metabolic profile, and improve ADCC against CD38-expressing multiple myeloma. CD38 is expressed on AML blasts, and daratumumab significantly reduces tumor burden in AML mouse models. This study demonstrated a subpopulation of AML blasts that highly express CD38 but weakly express CD33.

[0230] This disclosure provides site-directed insertion of a CD33CAR into the CD38 locus. The objectives of this two-part gene editing are threefold: first, to maintain CD33 antigen-specific targeting by the CAR while avoiding random transgene insertion and subsequent insertional mutagenesis seen with viral transduction techniques; second, to improve the metabolic fitness of NK cells; and third, to enhance therapeutic efficacy and prevent NK cell fratricide by targeting these CD38 CARs. KO This opens the door to clinical trials combining CD33C ANK cells with anti-CD38 monoclonal antibodies.

[0231] The present disclosure provides a method for the treatment of relapsed / refractory AML patients with CD38 KOCD33 CAR NK cells also offer safety benefits. As mentioned above, CD33 is highly expressed on AML blasts and leukemia progenitor cells, and this target has a long history of demonstrating its safety in cancer immunotherapy. There are advantages to using NK cells over autologous T cells in CAR-engineered cell therapy. While T cells and NK cells have similar therapeutic functions, CAR NK cells have the additional ability to recognize tumors via their innate NK cell receptors, potentially preventing relapse due to antigen escape. Furthermore, allogeneic HLA-mismatched NK cells can be safely administered without causing GVHD, highlighting the ability to manufacture universal donor or "off-the-shelf" CAR NK cells, avoiding the cost and time constraints associated with CAR T-cell therapy manufacturing. Using a standardized healthy donor cell bank as a cell source also avoids the challenges associated with collecting and manufacturing autologous cell therapy products from heavily pretreated, active leukemia patients. Finally, NK cells are safe, with cytokine release syndrome and neurotoxicity minimal in previous NK cell studies.

[0232] Unlike T cells, NK cells are much more difficult to efficiently transduce with rentoviral vectors. To solve this problem, NK cells were engineered using CRISPR gene editing delivered as Cas9 / ribonucleoprotein (Cas9 / RNP) via electroporation to introduce a double-strand break (DSB) at the CD38 locus, followed by AAV6 transduction for delivery of CAR DNA. The CAR is placed between the ITRs of the AAV backbone and surrounded by 600 bp of homology arms, which target the CAR to the DSB at the CD38 locus. This method is suitable for clinical use in the development of CD38 KO CD33C A is used to generate NK cells. In addition to improving transduction efficiency, this approach also improves safety by limiting transgene copy number, reducing insertional mutagenesis, and improving gene transcription uniformity.

[0233] Here, CD38 KOCD33CAR NK cells were successfully generated using the described methodology, and in vitro data demonstrated successful depletion of CD38 and expression of CD33CAR, resulting in greater anti-AML activity than unmodified NK cells.

[0234] Research into the effects and effectiveness of Meganium Generation of CD33C ANK cells CD33CAR NK cells were generated by targeted insertion of the CD33CAR construct into the AAVS1 locus on human chromosome 19. AAVS1 is a well-validated "safe harbor" for DNA transgene integration. This approach reliably generated CARNK cells with a transduction efficiency of over 60% in peripheral blood NK cells (Figures 12A and 12B).

[0235] The same approach was used to deliver CAR DNA to the CD38 locus. Instead of targeting AAVS1, homologous arms were designed for the CD38 locus on chromosome 4, allowing for simultaneous knockout of CD38 and knock-in of CD33CAR to this locus (13). To generate CD33CAR NK cells, Cas9 / RNP targeting the CD38 locus was electroporated, followed by AAV6 transduction of the CD33CAR construct containing homologous arms in the CD38 targeting region.

[0236] Antitumor effect of CD33C ANK cells CD33CAR-NK cells were shown to be highly effective against several AML cell lines and AML patient samples, as assessed by NK cell degranulation (Figures 14A and 14B) and three separate cytotoxicity assays: a calcein 4-hour cytotoxicity assay (Figures 14C, 14D, 14E), a real-time cell analysis (RTCA, xCELLigence) cytotoxicity assay (Figure 14F), and CyTOF analysis (Figure 5A). CyTOF analysis demonstrated upregulation of CD38 in surviving AML cells after culture with CD33CAR NK cells (Figure 24A). Furthermore, CAR-expressing NK cells exhibit significantly higher cytokine secretion compared to wild-type (WT) NK cells (Figure 24B).

[0237] CD38 knockout NK cells CD38 is a transmembrane glycoprotein that plays a key role in cellular metabolism. Targeting CD38 in human peripheral blood NK cells using Cas9 / RNP not only improves antibody-dependent cytotoxicity of NK cells in combination with dartomumab, but also improves the metabolic fitness of NK cells. Metabolic fitness plays an important role in NK cell function within the tumor microenvironment. RNA-seq was performed to compare WT and CD38 KO performed on NK cells and CD38 KO Significant changes in pathways involved in cholesterol biosynthesis (P=0.00001) and oxidative phosphorylation (P=0.00001) were observed in NK cells (Figure 16A). Analysis of genes in these pathways revealed that CD38 KO We identified a small but significant increase in the expression of mitochondrial genes specifically related to ATP synthesis, NAD recycling, and electron transport in NK cells. KO Examination of NK cell metabolism revealed that CD38 KO NK cells are CD38 WT The OCR:ECAR ratio was significantly higher in CD38-deficient NK cells compared to NK cells (Figures 16C and 16D). These results indicate that CD38 deletion induces NK cells to preferentially use OXPHOS to achieve their bioenergetic needs. Importantly, CD38 KONK cells are CD38 WT They also had higher spare respiratory capacity and mitochondrial respiratory capacity compared to NK cells (Figure 16D).

[0238] CD38 KO Anti-AML activity of CD33C ANK cells CD38 generated from healthy donors using the method shown in Figure 33 KO CD33C ANK cells were co-cultured with the AML cell line Kasumi-1 for 4 hours. To assess the antitumor effect, AML cells were labeled with calcein-AM and expressed CD38 at different effector-to-target (E:T) ratios. KO CD33CARNK(v4) cells were added. Cytotoxicity of labeled tumor cells was quantified by measuring the fluorescence of calcein released into the supernatant. KO Data from CD33CARNK(v4) cells demonstrate the improved anti-AML activity of these new cells, as shown in FIG.

[0239] Genomic safety studies conducted As described above, this example demonstrates the safe production of expanded NK cells derived from an allogeneic, specifically universal donor source. In particular, the approach of RNP electroporation and ex vivo targeted gene insertion using AAV vectors was utilized here because it requires less formal guidance and has fewer precedents for construction. This approach may improve the safety profile of genetically engineered products.

[0240] Electroporation of pre-complexed Cas9 / gRNA(RNP) allows for tighter control of genome exposure to editing enzymes compared to vector-based expression, without the potential for sustained expression.

[0241] Site-specific insertion alleviates the random and unknown nature of insertion sites common in retroviral and lentiviral gene insertion.

[0242] Applying this methodology to a universal donor platform will allow for the full testing and release of off-the-shelf products compared to home-grown products.

[0243] With this in mind, the following genomic assay was developed.

[0244] 1. Evaluation of CD33CAR Integration Random integration of genetic material into the host genome is associated with poor outcomes in patients undergoing genetically engineered T cells and gene therapy. The Cas9 / RNP site-specific CAR insertion approach achieved highly efficient CAR expression while overcoming safety concerns associated with random integration of CAR into the human genome via lentivirus.

[0245] First, in CD33CAR NK cells, CD33CAR was inserted into the AAVS1 locus, a human safe harbor locus. Using targeted locus amplification (TLA), this gene editing approach demonstrated low off-target CAR integration. The vector integrated as intended at human chromosome chr19:55,115,754-55,115,767, with the expected integration occurring in intron 1 of PPP1R12C.

[0246] Here, CD38 KO Similar TLA analysis was performed on CD33CARNK cells. Similar to the data from the safe harbor AAVS1 site, only a single integration hotspot was identified at the intended CD38 locus on chromosome 4.

[0247] Overall, the targeted locus amplification analysis indicates that this approach results in highly targeted insertion of CD33CAR at the intended site.

[0248] 2. Identifying Off-Target Effects of CD38-Specific CRISPR Using Churchill Churchill is a bioinformatics approach developed and utilized by the Clinical Genomics Institute at Nationwide Children's Hospital to streamline whole genome sequence analysis. Churchill fully automates the analytical process required to align raw sequence data, undergoing the complex and computationally intensive process of alignment, post-alignment processing, local realignment, recalibration, and variant discovery, with the goal of identifying variants of clinical relevance. This approach is directed at CD38 KO NK cells and CD38 KO Whole genome sequence data of CD33CAR NK cells was analyzed and used to compare with unmodified expanded NK cells from the same donor.

[0249] Whole-genome sequencing (WGS) was performed and used to identify off-target effects of Cas9 / RNP targeted to CD38. Next-generation sequencing data were processed through Churchill, and reads were aligned to the GRCh37 reference genome using BWAMEM (v0.7.15). Mutations were called using the Mutect2 tool in the Genetic Analysis Toolkit (GATKv4.0.5.1, Broad Institute) and annotated using SnpEff (v4.3). Knockout-specific single-nucleotide polymorphisms and insertion-deletion mutations (indels) were detected compared to wild-type (WT) NK cells. Because repair of DNA breaks generated by Cas9 / RNP varies between cells and is close to regions of guide RNA homology, clustered events were not filtered out as is typically done in somatic genome analysis. Therefore, a Mutect2 filter was applied to identify CD38. KO Those occurring at any frequency in cells but absent in WTNK cells were included, and only those that passed the applied filters or clustered events, nonsynonymous mutations, and those within coding regions were included.

[0250] 26 genes with single nucleotide polymorphisms and indels were identified in CD38 KOWe found that the mutations were unique to NK cells and had potential moderate or high impact. In total, 18 genes had mutations classified as moderate impact (missense and non-frameshift) by SnpEff, and seven genes (including CD38) had mutations classified as high impact (start-loss, stop-gain, and frameshift) (Table 11). By RNA-seq, only four of the off-target genes with high-impact mutations (CC2D1B, DENND4B, KMT2C, and WDR89) were expressed at meaningful levels in NK cells (Figure 17).

[0251] The same approach was applied to CD38 KO When applied to CD33CAR NK cells, we found 13 genes (including CD38) with high-impact mutations compared to matched expanded but unmodified NK cells from the same donor (Table 12). Seven were expressed at detectable levels in expanded NK cells by RNAseq (Figure 18), but none of the CD38 genes described above were expressed at high-impact mutations (Table 12). KO These WGS data demonstrate the ability to detect mutational events with high sensitivity and do not identify recurrent off-target effects caused by CD38-targeted Cas9 / RNP, with or without subsequent insertion of CD33CAR.

[0252] clinical research Generation of CD38 knockout CD33C A NK cells Considering the advantages of targeting CD38 in NK cells and the low off-target effects when using Cas9 / RNP, NK cells were generated with a CD33CAR gene inserted into the CD38 locus. A similar approach was used to insert a CAR (Cas9 / RNP AAV6) by engineering homologous arms of the CD38 targeting site, achieving simultaneous knockout of CD38 and knock-in of CD33CAR into this locus (Figure 11). KO AAV6 vectors were utilized to generate CD33CARNK cells. Flow cytometry was used to determine CAR transduction and CD38 knockout efficiency (Figure 13).

[0253] Preclinical research in vitro research CD38 KO To assess the function of CD33C ANK cells in vitro, their effect on CD33+ tumor cell lines was measured. KO CD33C ANK cells were co-cultured with AML cell lines and patient samples, and supernatants were collected after 4 and 48 hours, and cytokine production was measured by Luminex multiplex cytokine analysis. To assess antitumor efficacy, AML cells were labeled with calcein-AM and expressed CD38 at different effector-to-target (E:T) ratios. KO CD33C-A NK cells are added. Cytotoxicity of the labeled tumor cells is quantified by measuring the fluorescence of calcein released into the supernatant. Real-time efficacy is measured using the xCELLigence RTCAMP instrument (ACEA Biosciences). The assay is performed using wild-type NK cells and CD38 KO This was carried out to compare CD33CARNK cells and further using CD33CAR-NK cells inserted into the AAVS1 locus to assess the impact of CD38 deletion.

[0254] in vivo studies. CD38 KO To further evaluate the efficacy of CD33CARNK, in vivo studies will be performed using a xenograft mouse model. Briefly, NSG mice will be inoculated with 1x10 CD33CARNK cells via tail vein injection. 6 Animals in the treatment groups were inoculated with luciferase-transduced MOLM-13, MV-4-11, or OCI-AML3 AML cells. Animals in the treatment groups were inoculated with 10 mAb by tail vein injection on days 7 and 14 after tumor injection. 7 CD38 KO Mice receive CD33C A NK cells. Low doses of IL-2 are administered along with the NK cells, with the dose in mice being 4x10 compared to the human study. 8This is similar to NK cells / kg, but in the absence of endogenous homeostatic cytokines. Two control groups of animals receive either unmodified expanded NK cells or no NK cells. To track the antitumor response, weekly bioluminescence imaging and peripheral blood samples for flow cytometry evaluation of leukemia cells are performed. Survival curves are generated. All animal experiments are performed in accordance with protocols approved by the Nationwide Children's Hospital Institutional Animal Care and Use Committee (IACUC).

[0255] CD38 KO Safety evaluation of CD33C ANK cells. CD33 is a documented target for antibody and CAR T cell therapies with established safety in humans. There are no established models for testing the safety of targeting human CD33 in mouse models, nor are there methods or materials for generating equivalent mouse anti-CD33-expanded CAR T cell NK cells for immunocompatible mouse models. As noted above, human safety data will be established for expanded universal donor NK cells. Preclinical and product release safety testing will focus on generating additional genomic safety data.

[0256] Assessment of on-target and off-target integration frequency. The Churchill assay described above was performed for each CD38 KO This will be performed on CD33C ANK cell products to validate CD38 on-target alterations and identify off-target Cas9 / RNP effects. KO Whole genome sequencing data from CD33C ANK cells will be compared to unmodified expanded NK cells from the same donor. Product release for clinical use requires that at least 50% of CD38 reads be identified as mutated. Remaining mutations will be cataloged and accessed as baseline product information if patient safety studies are required.

[0257] TLA is used to broadly identify CAR integration sites. This data is orthogonal to the dGH Insight assay, but because it is not a fully qualified assay using CAR probes, it is reported over at least three validation runs to gather baseline data on key integration sites.

[0258] dGH Insight: A targeted fluorescence-based genomic hybridization assay will be developed in collaboration with KromaTiD. This assay will identify site-specific insertion of CAR at the CD38 locus using a CAR-specific probe and two CD38 locus-specific probes flanking the targeted insertion site. G-banding results will be analyzed for each CD38 KO CD33CAR NK cell products are reported from 200 metaphases. Release criteria are ≥90% CAR insertions at the intended CD38 locus.

[0259] Classic G-banding: To assess overall genomic integrity, each CD38 KO G-banding results from 20 metaphases of the CD33C ANK cell product are reported (performed by KromaTiD). G-banding results do not support release criteria but are for informational purposes only. (See Figures 2-10 and 26-32.)

[0260] dGH screening: To further assess genomic integrity, the results of dGH screening were compared with each CD38 KO Data from 50 metaphases of the CD33C ANK cell product (performed by KromaTiD) are reported. Data reports summarize results regarding on-target and off-target integrations and structural mutations related to the CD38 locus. Results from dGH screening do not support the release criteria but complement the G-banding data and are for informational purposes only. (See Figures 2-10 and 26-32.)

[0261] Evaluation of residual AAV6 vector. The AAV6 virus will be used only to provide a template for in vitro insertion of the CAR gene. The vector copy number used for transduction is low (10 4 ~10 5 Considering the MOI, dilution during culture, the expected 2,000-fold expansion of NK cells over two weeks, and the final wash step, the final AAV infectious particles were at a biologically meaningful level (a typical gene therapy dose is 10 13 ~10 14 Nonetheless, to demonstrate safety, the presence of vector copy numbers is expected to be so low that capsids are undetectable.

[0262] A digital droplet PCR assay for AAV2 ITR was developed and validated as an indicator of viral genome content used in AAV6 pseudotyped vector release testing. Here, viral genome copy number is reported to quantify residual virus in validation runs and clinical products.

[0263] Pharmaceuticals and Manufacturing Product Description The proposed drug product consists of NK cells derived from healthy donors selected based on optimal universal donor criteria, expanded in vitro on CSTX-002 feeder cells in IL-2-containing medium, and genetically modified by Cas9 / RNP electroporation and AAV6 transduction to express the CD38 KO CD33C A results in NK cells.

[0264] viral vectors Product name: CD33CARV4(LHCD8-41)CD38_ssAAV AAV serotype: Serotype#AAV6

[0265] DNA encoding the homology arm (HA) of the CD33CAR and CD38 loci is cloned between the ITRs of the AAV2 backbone, as follows: CD38 right HA, BGHpA, MND promoter, CD33CAR, BGHpA, and CD38 left HA. See Figure 21.

[0266] Vector generation and purification The AAV viral vector CD33CARV4(LHCD8-41)CD38_ssAAV is produced by Andelyn Biosciences at their clinical manufacturing facility located at 575 Children's Crossroads, Columbus, Ohio.

[0267] Viral vectors were produced at Andelyn Biosciences by cotranscription of three DNA plasmids into human embryonic kidney (HEK) 293 cells: the vector plasmid, the AAV helper plasmid, and the Ad helper plasmid. Plasmids used to produce viral vectors were produced by Andelyn Biosciences and Aldevron. The plasmids are listed and described in the production section.

[0268] The manufacturer distributes the vector product into single-use vials and stores them until use in cell manufacturing. The vectors are supplied frozen (≤-60°C) and transported to the Abigail Wexner Research Institute Cell-Based Therapeutics Core Facility (CBT) at Nationwide Children's Hospital (AWRI-NCH) where they are stored at ≤-60°C until use in manufacturing.

[0269] The sequence of the AAV viral vector CD33CARV4 (LHCD8-41) CD38_ssAAV is shown in SEQ ID NO: 50. The AAV2-ITRddPCR assay was used to measure the physical titer.

[0270] Research batches of the vector were manufactured by Andelyn Biosciences using the same final plasmid as the final clinical vector. This material was used for process development and some early preclinical studies. A single batch of GMP-like vector product was manufactured under a toxicology process plan, but in the Andelyn GMP cleanroom, and underwent extensive clinical-grade testing.

[0271] Plasmid generation The AAV viral vector CD33CARV4(LHCD8-41)CD38_ssAAV was produced using the following plasmids: Vector Plasmid CD33CARV4(LHCD8-41)CD38_ssAAVBackBoneKan AAV helper plasmid pNLRepcap6-Kan Ad helper plasmid pHelpKanV4

[0272] The vector plasmid CD33CARV4(LHCD8-41)CD38_ssAAVBackBoneKan is a toxicology-grade plasmid manufactured in Andelyn Biosciences' GMP facility. The vector plasmid contains a kanamycin resistance gene.

[0273] The AAV helper plasmid pNLRepcap6-Kan is a research HD grade plasmid manufactured by Aldevron. The helper plasmid contains a kanamycin resistance gene.

[0274] The Ad helper plasmid pHelp-KanV4 is a GMP-S grade plasmid manufactured by Aldevron, and the Ad helper plasmid contains a kanamycin resistance gene.

[0275] To confirm the identity and purity of each plasmid, sequencing was also performed using NextGenPacBio sequencing. The CoA of the vector plasmid contains PacBio sequencing results, while the CoA of the AAV helper plasmid and the CoA of the Ad helper plasmid contain only Sanger sequencing results. The PacBio sequencing results for these two plasmids after release are shown below.

[0276] NextGenPacBio sequencing covered the Ad helper plasmid (pHelpKanV4) at an average read depth of 152,383x. Sequence analysis revealed four positions that differed from the expected plasmid map, all representing single-nucleotide insertions with frequencies ranging from 10.38% to 16.27%. The mutations were located in the following regions relative to sequencing from the PsiI restriction site used for linearization prior to sequencing: 47:T insertion into the poly(T)(9) region of the E4 gene 834:A insertion into the poly(A)(13) region of the E4 gene 2641: G insertion towards the end of the VA gene 5933: Insertion of A into ColE1 origin site

[0277] These variants may affect AAV replication efficiency during vector generation but should not affect the production or structure of the AAV viral vector or its transgene. Nine other variants were present in the sequence at frequencies between 0.1 and 0.21%, with the remaining variants occurring at frequencies <0.01%, which is within the statistical error range of this deep sequencing approach.

[0278] NextGenPacBio sequencing covered the AAV helper plasmid pNLRepcap6-Kan at an average read depth of 247,055x. Sequence analysis revealed one position that differed from the expected plasmid map, a single-nucleotide insertion at a frequency of 11.79%. The variant was located in the following region relative to sequencing from the AsiSI restriction site used for linearization prior to sequencing: 830: Insertion of A at the site of the pUC origin of replication.

[0279] This mutation affects plasmid replication but does not affect the production or structure of the AAV viral vector or its transgene.

[0280] Genome editing components CRISPR gene editing of NK cells is delivered via electroporation as a Cas9 / ribonucleoprotein (Cas9 / RNP) complex to introduce a double-strand break prior to AAV6 transduction. The Cas9 protein and a single guide RNA (sgRNA) are complexed together to form a ribonucleoprotein (RNP) complex. The sgRNA targets exon 1 of the CD38 gene.

[0281] Non-clinical sgRNA and Cas9 reagents were used to generate cells for preclinical studies. Non-clinical grade (GMP-like) sgRNA and research-grade Cas9 protein will be used for preclinical studies. Manufacture of the CARNK cell test product required under this IND for clinical use will use GMP-grade sgRNA and CTSTrueCutCas9 protein, which will be complexed together prior to electroporation. GMPCD38-sgRNA will be manufactured by Synthego. GMPCTSTrueCutCas9 protein will be manufactured by ThermoFisherScientific.

[0282] The sgRNA is resuspended in PBS and combined with the ready-to-use suspended Cas9 protein to form the ribonucleoprotein, Cas9 / RNP complex. USP or GMP-grade PBS is used. The mixture is incubated at room temperature and can be used within 20 minutes of completing the incubation, or it can be stored on ice until use.

[0283] The Cas9 / RNP complex is added to NK cells suspended in the TheraPEAKP3 Primary Cell Nucleofector Solution Set (Lonza), manufactured according to applicable GMP standards and intended to support GMP manufacturing. Electroporation is performed using the 4D-Nucleofector System (Lonza).

[0284] All gene editing procedures will be performed at the Nationwide Children's Hospital (NCH) Abigail Wexner Research Institute (AWRI-NCH) Cell-Based Therapy Core Facility (CBT) and will operate under applicable GMP guidelines.

[0285] Universal donor NK cells Donors undergo the infectious disease testing and screening required for HCT / P donors, which is performed at BTMB facilities in accordance with 21 CFR Part 1271 and in accordance with the FDA guidance document "Determining Donor Eligibility for Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT / Ps)" and any issued supplemental guidance documents. Donors are tested for KIR and HLA genotype, infectious disease markers, NK cell proliferation, and NKG2C content.

[0286] Donors who meet all selection criteria undergo MNC(A) collection at a BTMB-accredited collection center. The MNC(A) product is then transported to a CBT core facility for additional processing. The apheresis collection is washed to remove platelets, and magnetic immunodepletion of CD3+ cells is performed on the apheresis product using the Miltenyi Clini MACS. CD3-depleted MNCs are stored in cryobags in the vapor phase of liquid nitrogen until thawed and used for NK cell manufacturing.

[0287] This serves as the cell source material used in the manufacture of pharmaceutical products.

[0288] CD38 KO CD33CARNK cell manufacturing All CD38 KOCD33C ANK cell products are manufactured at the Abigail Wexner Research Institute at Nationwide Children's Hospital (AWRI-NCH) Cell-Based Therapy Core Facility (CBT). The CBT Core Facility is part of the NCH Cell Manufacturing Facility, a controlled-access ISO Class 7 / 8 facility used to manufacture biologics under cGMP conditions. The CBT Core Facility includes multiple dedicated laboratories used for research and development, cell manufacturing, quality control, and temperature-controlled storage.

[0289] CD38 KO The CD33CAR NK cell product is produced from CD3+-depleted MNCs (A) and expanded by recurrent stimulation with irradiated feeder cells expressing membrane-bound IL-21 and 4-1BBL (clone CSTX002). After one week of expansion, the NK cells are genetically modified, rested for two days, and then expanded in culture for an additional 14 days. The product is cryopreserved at various cell concentrations and released / distributed for infusion into individual patients at specified doses.

[0290] cell culture On day 0 of NK expansion, CD3-depleted MNCs are thawed and washed, then expanded by recurrent weekly stimulation with irradiated CSTX002 feeder cells (IFCs). Briefly, IFCs are added at an IFC-to-effective TNC ratio of approximately 1:2 (also known as Stimulation 1 or Stim1) in cell culture medium containing 5% immune cell serum replacement and 100 IU / mL recombinant human IL-2. Additional fresh IL-2 is added at 100 IU / mL every 1–3 days. On day 7, expanded NK cells are aliquoted and stored in cryobags in the vapor phase of liquid nitrogen until thawed for the remaining manufacturing steps.

[0291] On day 7, NK cells were thawed and genetically modified using CRISPR / Cas9 in combination with AAV6. CD33CAR was inserted into the CD38 locus via site-specific insertion using CRISPR / Cas9. First, CD38 was targeted with gRNA via electroporation of precomplexed Cas9 / RNP into proliferating NK cells. Briefly, proliferating NK cells were harvested, washed, and resuspended in 20 μl of TheraPEAKP3 primary cell 4D-nucleofactor solution. Five μl of precomplexed Cas9 / RNP targeting CD38 was added to the cell suspension. Cas9 / RNP was electroporated into NK cells using the 4D-nucleofactor system. After electroporation, cells were rested for 30 minutes in medium containing IL-2. Cells were then counted, and AAV6 was added at a multiplicity of infection (MOI) of 75,000.

[0292] After transfection, cells were rested in culture for 2 days, then stimulated with irradiated feeder cells at a 1:1 IFC to viable TNC ratio (also called Stim2) and cultured for an additional 7 days in the presence of 100 IU / mL IL-2.

[0293] The cells are transferred to the Xuri Cell Expansion System for further expansion, restimulated with irradiated feeder cells at a 1:1 IFC to viable TNC ratio (also called Stim3) and cultured in the presence of IL-2 for an additional 7 days.

[0294] Harvesting and cryopreservation On day 23, cells are harvested and cryopreserved in aliquots. KO The CD33C ANK cell product is cryopreserved in a medium containing DMSO, human serum albumin (HSA), and Plasmalyte A. The product is released / distributed for infusion at doses prescribed to individual patients.

[0295] Product distribution, thawing, and infusion will be performed in accordance with effector cell therapy protocols and / or institutional clinical standard operating procedures. The product will be infused immediately after thawing without any additional manipulations such as washing or culturing.

[0296] Flow diagram outlining product testing For an overview of the manufacturing process and testing, please refer to the manufacturing flow chart (Figure 23).

[0297] important material

[0298] CD38 KO Critical ingredients that affect the quality, safety, potency, and purity of the CD33C A NK cell product are listed in Table 13. Excipients present in the final product are listed in bold italics. CD38 KO CD33C A NK cell release criteria and additional testing Testing will be conducted according to the existing CBT core facility standard operating procedures (SOPs). See the flow diagram (Figure 23) for an overview of the process and testing. CD38 KO CD33CAR NK cells will be administered at four dose levels: a. Dose level 1: 1x10 7 CAR-NK cells / kg (±20%) Dose level 2: 3x10 7 CAR-NK cells / kg (±20%) Dose Level 3: 1x10 8 CAR-NK cells / kg (±20%) b. Dose level 4: 1x10 8 Two doses of CAR-NK cells / kg (±20%), 1 week apart Safety testing using TLA, Churchill, dGH screen, custom dGH probe, and G-banding.

[0299] Targeted locus amplification: CD38 KO Genome-wide mapping of CD33CAR integration will be performed using TLA technology (Cergentis B.V.). KOFor each validation run, genomic DNA was isolated from NK cells expressing CD33CAR, cross-linked, fragmented, and re-guided using a kit provided by Cergentis, and then submitted to Cergentis for sequencing.

[0300] Next-generation sequencing (NGS) data processed through Churchill. High-fidelity Cas9 has been shown to have low off-target editing because it degrades quickly after electroporation. To study off-target effects in CRISPR-modified NK cells, CD38 KO WGS was performed on each single batch of CD33CAR NK cells. Next-generation sequencing data was processed through Churchill, and reads were aligned to the GRCh37 reference genome using BWAMEM (v0.7.15). Mutations were called using the Mutect2 tool in the Genetic Analysis Toolkit (GATK v4.0.5.1, Broad Institute) and annotated using SnpEff (v4.3). Knockout-specific single-nucleotide polymorphisms and insertion-deletion mutations (indels) were identified by comparison with expanded NK cells from the same donor that were not electroporated or transduced. Because repair of Cas9 / RNP-generated DNA breaks varies between cells and is close to regions of guide RNA homology, clustered events are not filtered out as is typically done in somatic genome analysis. Mutect2 filters were applied to identify CD38 KO These include events occurring at any frequency in CD33CAR NK cells but absent in non-electroporated or non-transduced expanded NK cells from the same donor, and only those that passed all applied filters, clustered events, non-synonymous mutations, and those in coding regions.

[0301] dGH Screen: a. Final CD38 KOSamples of CD33C ANK cells are prepared using the recommended KromaTiD dGH cell preparation kit to preserve metaphase spreads for hybridization. Samples are sent to KromaTiD for hybridization with genome-wide fluorescent probes. High-resolution karyotyping is performed by image analysis and the following is reported (Figures 6 and 7). i. Reciprocal, balanced, allelic translocations ii. Orientation events such as inversions and sister chromatid exchanges iii. Chromosome gain and loss events including sister chromatid fusion, double centrioles / acentric, fragmentation / chromothripsis, polyploidy and aneuploidy.

[0302] Custom dGH Insight. Final CD38 KO CD33CAR NK cell samples are prepared and submitted to KromaTiD for dGH screening, hybridized with a two-color probe for the CD38 knock-in site and CD33CAR. The analysis reports the total number and distribution of cells with CAR insertions and the percentage inserted at the CD38 locus.

[0303] G-banding. Final CD38 KO CD33C ANK cell samples will be prepared for standard G-banding analysis and submitted to KromaTiD. Standard metaphase karyotype analysis will be performed and reported.

[0304] Safety testing of residual AAV6 Based on the MOI of the initial transduction event, the wash steps, and the media dilution steps, the MOI of residual AAV6 was 10. 8 In products containing NK cells / mL, 2.5x10 5 At the highest dose level, this is considered to be less than 10 viral particles / mL. 14 This shows that the dose of virions / kg administered is nearly 1 billion times lower than the dose of virions / kg administered.

[0305] Clinical Trial Overview Current treatment options for relapsed and / or refractory AML are limited, and the overall survival rate for this patient population is low. The best chance for durable remission in this high-risk group is allogeneic hematopoietic stem cell transplantation (HSCT), and patients must achieve a complete response before HSCT for optimal success. Given the critical role of NK alloreactivity in mediating anti-leukemic effects in AML, current translational efforts in AML are directed toward adoptive immunotherapy using functionally activated NK cells. While the safety of adoptive transfer of ex vivo-expanded haploidentical NK cells has been demonstrated in patients with AML and other cancers, NK cell therapy in this context has yet to be realized. One of the major obstacles in adoptive NK cell immunotherapy is obtaining sufficient cell numbers and making them readily available for infusion into leukemia patients. Here, we demonstrate that large quantities of NK cells can be expanded in vitro, generating a bank of universal donor-derived NK cells for "off-the-shelf" NK cell therapy. Phase I trials utilizing this universal donor NK cell bank in adult and pediatric patients with relapsed / refractory AML are NCT04220684 and NCT05503134.

[0306] The purpose of this study is to confirm the safety and evaluate the efficacy of universal donor-derived CD38 knockout CD33C A RNK cells in pediatric patients with relapsed / refractory acute myeloid leukemia (AML). This study will examine the safety and efficacy of universal donor-derived CD38 knockout CD33C A RNK cells administered by intravenous infusion to relapsed / refractory AML patients. KO This was a non-randomized, open-label, phase I study of CD33CARNK cells followed by dose-escalation of induction chemotherapy (fludarabine / cytarabine).

[0307] See Figure 22 for study design and Table 15 for protocol summary.

[0308] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0309] TIFF2026504616000001.tif87170TIFF2026504616000002.tif11170TIFF2026504616000003.tif55170TIFF202650461600000 4.tif30170TIFF2026504616000005.tif31170TIFF2026504616000006.tif93170TIFF2026504616000007.tif30170TIFF202650 4616000008.tif53170TIFF2026504616000009.tif45170TIFF2026504616000010.tif49170TIFF2026504616000011.tif178170 TIFF2026504616000012.tif35170TIFF2026504616000013.tif188170TIFF2026504616000014.tif153170TIFF20265046160000 15.tif69170TIFF2026504616000016.tif194170TIFF2026504616000017.tif192170TIFF2026504616000018.tif225170TIFF20 26504616000019.tif45170TIFF2026504616000020.tif174170TIFF2026504616000021.tif68170TIFF2026504616000022.tif1 46170TIFF2026504616000023.tif216170TIFF2026504616000024.tif119170TIFF2026504616000025.tif103170TIFF20265046 16000026.tif214170TIFF2026504616000027.tif87170TIFF2026504616000028.tif114170TIFF2026504616000029.tif109170

[0310] array 1. SEQ ID NO: 1 - 30 bp right homology arm (RHA) gattggtgacagaaaaagccccatccttagg 2. Array No. 2 - 30 bp left homologous arm (LHA) ttatctgtcccctccaccccacagtggggc 3. Array No. 3 - 300 bp RHA gattggtgacagaaaagccccatccttaggcctcctccttcctagtctcctgatattgggtctaacccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctccttgccagaacctctaaggtttgcttacgatggagccagagaggatcctgggagggagagcttggcagggggtgggagggaagggggggatgcgtgacctgcccggttctcagtggccaccctgcgctaccctctcccagaacctgagctgctctgacgcggctgtc 4. Array No. 4 - 300 bp LHA gttctcctgtggattcgggtcacctctcactcctttcatttgggcagctcccctaccccccttacctctctagtctgtgctagctcttccagccccctgtcatggcatcttccaggggtccgagagctcagctagtcttcttcctccaacccgggcccctatgtccacttcaggacagcatgtttgctgcctccagggatcctgtgtccccgagctgggaccaccttatattcccagggccggttaatgtggctctggttctgggtacttttatctgtcccctccaccccacagtggggc 5. Array No. 5 - 500 bp RHA gattggtgacagaaaagcccatccttaggcctcctccttcttctctctgattgggtctaaccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctccttgccagaacctctaaggtttgcttacgatggagccagagaggatcctgggagggagagcttggcaggggtgggagggagggggggatgcgtgacctgccccggttctcagtggc caccctgcgctaccctctcccagaacctgagctgctctgacgcggctgtctggtgcgtttcactgatcctggtgctgcagcttccttacacttcccaagaggagaagcagtttggaaaaacaaaatcagaataagttggtcctgagttctaactttggctcttcacctttctagtcccaatttatattgttcctccgtgcgtcagttttacctgtgagataaggccagtagccagccccgtcctggcag 6. sequence number 6-500bpLHA tcccttttcctctcttctggggcctgtgccatctctcgtttcttaggatggccttctccgacggatgtctcccttgcgtcccgcctccccttcttgtaggcctgcatcatcaccgtttttctggacaaccccaaagtaccccgtctccctggctttagccacctctccatcctcttgctttctttgcctggacaccccgttctcctgtggattcgggtcacctctcactcctttcatttgggcagctc ccctaccccccttacctctctagtctgtgctagctcttccagccccctgtcatggcatcttccaggggtccgagagctcagctagtcttcttcctccaacccgggcccctatgtccacttcaggacagcatgtttgctgcctccagggatcctgtccccgagctgggaccaccttatattcccagggccggttaatgtggctctggttctgggtacttttatctgtcccctccaccccacagtgggc 7. sequendi no. 7-800bpRHA gattggtgacagaaaagccccatccttaggcctcctccttcctagtctcctgatattgggtctaacccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctccttgccagaacctctaaggtttgcttacgatggagccagagaggatcctgggagggagagcttggcagggggtgggagggaagggggggatgcgtgacctgcccggttctcagtggccaccctgcgctaccctctcccagaacctgagctgctctgacgcggctgtctggtgcgtttcactgatcctggtgctgcagcttccttacacttcccaagaggagaagcagtttggaaaaacaaaatcagaataagttggtcctgagttctaactttggctcttcacctttctagtccccaatttatattgttcctccgtgcgtcagttttacctgtgagataaggccagtagccagccccgtcctggcagggctgtggtgaggaggggggtgtccgtgtggaaaactccctttgtgagaatggtgcgtcctaggtgttcaccaggtcgtggccgcctctactccctttctctttctccatccttctttccttaaagagtccccagtgctatctgggacatattcctccgcccagagcagggtcccgcttccctaaggccctgctctgggcttctgggtttgagtccttggcaagcccaggagaggcgctcaggcttccctgtcccccttcctcgtccaccatctcatgcccctggctctcctgccccttccctacaggggttcctggctctgctcttcagactgagccccgttcccctgcatccccgttcccctgcatcccccttcccctgcatcccccagaggccccaggccacctacttggcctggaccccacgagaggccaccccagccctgtctaccaggctgccttttgggtggattctcctccaactgtggggtgactgcttgg。 8. SEQ ID NO: 8 - 800 bp LHA Tgctttctctgacctgcattctctcccctgggcctgtgccgctttctgtctgcagcttgtggcctgggtcacctctacggctggcccagatccttccctgccgcctccttcaggttccgtcttcctccactccctcttccccttgctctctgctgtgttgctgcccaaggatgctctttccggagcacttccttctcggcgctgcaccacgtgatgtcctctgagcggatcctccccgtgtctgggtcctctccgggcatctctcctccctcacccaaccccatgccgtcttcactcgctgggttcccttttccttctccttctggggcctgtgccatctctcgtttcttaggatggccttctccgacggatgtctcccttgcgtcccgcctccccttcttgtaggcctgcatcatcaccgtttttctggacaaccccaaagtaccccgtctccctggctttagccacctctccatcctcttgctttctttgcctggacaccccgttctcctgtggattcgggtcacctctcactcctttcatttgggcagctcccctaccccccttacctctctagtctgtgctagctcttccagccccctgtcatggcatcttccaggggtccgagagctcagctagtcttcttcctccaacccgggcccctatgtccacttcaggacagcatgtttgctgcctccagggatcctgtgtccccgagctgggaccaccttatattcccagggccggttaatgtggctctggttctgggtacttttatctgtcccctccaccccacagtggggc 9. SEQ ID NO: 9 - PAM g (sequence encoding PAM + crRNA) ccaatcctgtccctagtggcccc 10. SEQ ID NO: 10 - splice acceptor atcgatcgcaggcgcaatcttcgcatttcttttttccag 11. SEQ ID NO: 11 - BGH polyA terminator cctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattc 12. SEQ ID NO: 12 - mCherry gtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaa 13. SEQ ID NO: 13 - 30 bp plasmid carrying the mCherry transgene ttatctgtcccctccaccccacagtggggccactagggacagcgatcgggtacatcgatcgcaggcgcaatcttcgcatttcttttttccaggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaacgcggccgccctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattcgattggtgacagaaaagccccatccttagg 14. SEQ ID NO:14 - 300 bp plasmid carrying the mCherry transgene 15. SEQ ID NO:15 - 500 bp plasmid carrying the mCherry transgene 16. SEQ ID NO:16 - 800 bp plasmid carrying the mCherry transgene 17. SEQ ID NO: 17 - crRNA GGGGCCACTAGGGACAGGAT 18. SEQ ID NO: 18 - scFv atgctgctgctggtgacctctctgctgctgtgcgagctgccacacccagccttcctgctgatcccagacatccagatgacacagagccccagctccctgagcgcctccgtgggcgacagagtgaccatcacatgtagggcctctgagagcgtggataactatggcatcagcttcatgaattggtttcagcagaagcctggcggcgccccaaagctgctgatctacgcagccagcatgcagggctccggcgtgccctctcggttctccggctctggcagcggcaccgacttcaccctgacaatctctagcctgcagccagacgatttcgccacatactattgccagcagagcaaggaggtgccctggacctttggccagggcacaaaggtggagatcaagggctccacctctggcagcggcaagcctggcagcggagagggctccacaaagggacaggtgcagctggtgcagtccggagccgaggtgaagaagccaggctcctctgtgaaggtgtcttgtaaggccagcggctataccttcacagactacaacatgcactgggtgcgccaggcaccaggacagggcctggagtggatcggctacatctatccttacaacggcggcaccggctataatcagaagtttaagtccaaggccaccatcacagccgatgagtctaccaatacagcctacatggagctgagcagcctgcggtccgaggacacagccgtgtactattgcgcccggggcagacccgctatggactattggggccagggcaccctggtgacagtgtctag 19. SEQ ID NO: 19 - IgG4-Hinge gagagcaagtacggaccaccttgcccaccatgtcctgcaccagagttcctgggaggaccttccgtgttcctgtttcctccaaagccaaaggacaccctgatgatcagccggacccagaggtgacatgcgtggtggtggacgtgagccaggaggaccccgaggtgcagttc aactggtacgtggatggcgtggaggtgcacaatgccaagaccaagccaagagaggagcagtttaactccacctatagggtggtgtctgtgctgacagtgctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtgtccaataagggcctgccttctcttatcg agaagaccatctctaaggcaaagggacagccaagggagccacaggtgtatacactgcccctagccaggaggagatgaccaagaaccaggtgtccctgacatgtctggtgaagggcttttacccttctgacatcgccgtggagtgggagagcaatggccagccagagaacaa ttataagaccacaccacccgtgctggactctgatggcagcttctttctgtacagccgcctgaccgtggataagtcccggtggcaggagggcaacgtgttctcctgctctgtgatgcacgaggccctgcacaatcactacacacagaagagcctgtccctgtctctgggcaag 20. sequence number 20-CD28 atgttttgggtgctggtggtgggaggcgtgctggcctgttatccctgctggtgaccgtggccttcatcatcttttggtgcgctccaagcggagccggggcggacactctgactacatgaacatgaccccacggagacccggacctacaaggaagcactatcagccctacgcccctccacggacttcgcagcatatcgcagc 21. sequence number 21-CD3z cgggtgaagtttagcagatccgccgatgcaccagcatatcagcaggcagaatcagctgtacaacgagctgaatctgggcaggcgcgaggatacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagcccaggcgcaagaaccctcaggagg gcctgtataatgagctgcagaaggacaagatggccgaggcctacagcgagatcggcatgaagggagagcggagaaggggcaagggacacgatggcctgtatcagggcctgtccaccgccacaaaggacacctacgatgcactgcacatgcaggccctgccacctcggtga 22. SEQ ID NO: 22-CD33CAR-Gen2-ssAAV 23. SEQ ID NO: 23-CD33CAR-Gen4v2 24. SEQ ID NO: 24 - NKG2D transmembrane domain agcaacctgttcgtggcctcctggatcgccgtgatgatcatctttcgcatcggcatggccgtggccatcttctgctgtttctttttcccatcc 25. SEQ ID NO:25 - Linker ggaggctctggaggaggctccggc 26. SEQ ID NO: 26-2B4 tggcggagaaagcggaaggagaagcagagcgagacctcccctaaggagtttctgacaatctatgaggacgtgaaggatctgaagaccaggcgcaatcacgagcaggagcagaccttcccaggaggaggctctacaatctacagcatgatccagtcccagagcagcgccccaaccagccag gagccagcctatacactgtactctctgatccagcctagccggaagtctggcagccgcaagcggaaccactccccatctttcaattctaccatctatgaagtgatcggcaagagccagcctaaggcccagaacccagccagactgtccaggaaggagctggagaattttgacgtgtactct 27. SEQ ID NO:27 - Linker ggaggcagcggaggaggctctggc 28. SEQ ID NO: 28-CD3z cgcgtgaagttcagccggtccgccgatgccccagcctataagcagggccagaaccagctgtacaacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggcggggccgggaccccgagatgggaggcaagccccggagaaagaaccctcaggagggcctgtataatgagctgcagaaggacaagatggccgaggcctactccgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtatcagggcctgagcaccgccacaaaggacacctacgatgccctgcacatgcaggccctgcctccacggtgatga 29. SEQ ID NO: 29 - anti - CD33 scFv atgctgctgctggtgacctccctgctgctgtgcgagctgccacaccctgcctttctgctgatcccagacatccagatgacacagagccccagctccctgtctgccagcgtgggcgacagagtgaccatcacatgtagggcctccgagtctgtggataactatggcatcagctttatgaattggttccagcagaagccaggaggcgcccctaagctgctgatctacgcagcctccatgcagggctctggcgtgcccagccgctttagcggctccggctctggcaccgatttcaccctgacaatctctagcctgcagccagacgattttgccacatactattgccagcagtccaaggaggtgccctggaccttcggccagggcacaaaggtggagatcaagggcagcacctccggctctggcaagcctggctccggagagggctctacaaagggacaggtgcagctggtgcagagcggagccgaggtgaagaagccaggctcctctgtgaaggtgagctgtaaggcctccggctatacctttacagactacaacatgcactgggtgagacaggcaccaggacagggcctggagtggatcggctacatctatccttacaacggcggcaccggctataatcagaagttcaagagcaaggccaccatcacagccgatgagtccaccaatacagcctacatggagctgagcagcctgaggagcgaggacacagccgtgtactattgcgccagaggcaggcctgctatggactattggggccagggcaccctggtgacagtgtctagc 30 - SEQ ID NO: 30 - MND promoter atcgatcacgagactagcctcgagaagcttgatatcgaattccacggggttggacgcgtcttaattaaggatccaaggtcaggaacagagaaacaggagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagttggaacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctagaggatcgatcccccgggctgcaggaattcaagcgagaagacaagggcagaaagcacc 31. SEQ ID No. 31 - 600 bp, LHA, AAVS1 (gen4v2 and gen2) gctgcaccacgtgatgtcctctgagcggatcctccccgtgtctgggtcctctccgggcatctctcctccctcacccaaccccatgccgtcttcactcgctgggttcccttttccttctccttctggggcctgtgccatctctcgtttcttaggatggccttctccgacggatgtctcccttgcgtcccgcctccccttcttgtaggcctgcatcatcaccgtttttctggacaaccccaaagtaccccgtctccctggctttagccacctctccatcctcttgctttctttgcctggacaccccgttctcctgtggattcgggtcacctctcactcctttcatttgggcagctcccctaccccccttacctctctagtctgtgctagctcttccagccccctgtcatggcatcttccaggggtccgagagctcagctagtcttcttcctccaacccgggcccctatgtccacttcaggacagcatgtttgctgcctccagggatcctgtgtccccgagctgggaccaccttatattcccagggccggttaatgtggctctggttctgggtacttttatctgtcccctccaccccacagtggggc 32. SEQ ID No. 32 - 600 bp, RHA, AAVS1 (gen4v2 and gen2) gattggtgacagaaaagccccatccttaggcctcctccttcctagtctcctgggtctaaccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctccttgccagaacctctaa ggtttgcttacgatggagccagagaggatcctgggagggagagcttggcaggggtgggagggaaggggggatgcgtgacctgcccggttctcagtggccaccctgcgctaccctctcccagaacctgagctgctctgacgggctgtc tggtgcgtttcactgatcctggtgctgcagcttccttacacttcccaagaggagaagcagtttggaaaaacaaaatcagaataagttggtcctgagttctaactttggctcttcacctttctagtcccaattttatattgttcctccgtg cgtcagttttacctgtgagataaggccagtagccagccccgtcctggcagggctgtggtgaggaggggggtgtccgtgtggaaaactccctttgtgagaatggtgcgtcctaggtgttcaccaggtcgtggccgcctctactccctttct 33. sequence number 33-gRNA TTCTCCTGTGGATTCGGGTCAC 34. sequence number 34-gRNA CTCTCTGGCTCCATCGTAAGCA 35. sequence number 35-gRNA TCCTGGGCAAACAGCATAA 36. sequence number 36-gRNA GAGCTGCAGAAGGACAAGAT 37. sequence number 37-gRNA CTCTGTGTCATCTGGATGTCTG 38. sequence number 38-gRNA CTTTGAGCTCTACTGGCTTCTG 39. sequence number 39-gRNA GCGAGTGAAGACGGCATG 40. SEQ ID NO: 40 - gRNA GTCTGTGCTAGCTCTTCCAG 41. SEQ ID NO: 41 - gRNA GCGATGTCAGAAGGGTAAA 42. SEQ ID NO: 42 - gRNA GGCGGACACTCTGACTACAT 43. SEQ ID NO: 43 - Promoter GGCATGGGGTTGGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCAGACACGGGGAGGATCCGCTCAGAGGACATCACGTGGTGCAGCGGCGCGCGGCCGCAGAAAGGGAGTAGAGGCGGCCACGACCTGGTGAACACCTAGGACGCACCATTCTCACAAAGGGAGTTTTCCACACGGACACCCCCCTCTCACCACAGCCCTGCCAGGACGGGCTGGCTACTGGCCTTATCTC 44. SEQ ID NO: 44 - Promoter GCGAGTGAAGACGGCATGGGGTTGGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCAGACACGGGGAGGATCCGCTCAGAGGACATCACGTGGTGCAGCGGCGCCCGGCCGCAGGAAGGG AGTAGAGGCGGCCACGACCTGGTGAACACCTAGGACGCACCATTCTCACAAAGGGAGTTTTCCACACGGACACCCCCCTCCTCACCACAGCCCTGCCAGGACGGGGCTGGCTACTGGCCTTA 45. SEQ ID NO: 45 - Promoter GCGAGTGAAGACGGCATGGGGTTGGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCAGACACGGGGAGGATCCGCTCAGAGGACATCACGTGGTGCAGCGGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCAGAAAGGGAGTAGAGGCGGCCACGACCTGGTGAACACCTAGGACGCACCATTCTCACAAAGGGAGTTTTCCACACGGA 46. Sequence number 46 - Promoter GCGAGTGAAGACGGCATGGGGTTGGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCAGACACGGGGAGGATCCGCTCAGAGGACATCACGTGGTGCAGCGGCCGCAGAAAGGGAGTAGAGGCGGCCACGACCTGGTGAACACCTAGGACGCACCATTCTCACAAAGGGAGTTTTCCACACGGACACCCCCCTCCTCACCACAGCCCTGCCAGGACGGGGCTGGCTACTGGCCTT 47. Sequence number 47 - PAMgPAMgmCherry structure CCAATCCTGTCCCTAGTGGCCCCCACTAGGGACAGCGATCGGGTACATCGATCGCAGGCGCAATCTTCGCATTTCTTTTTTCCAGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAACGCGGCCGCCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCCCAATCCTGTCCCTAGTGGCCCC 48 - PAM gRNA mCherry Structure CCAATCCTGTCCCTAGTGGCCCCCACTAGGGACAGCGATCGGGTACATCGATCGCAGGCGCAATCTTCGCATTTCTTTTTTCCAGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAACGCGGCCGCCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC 49. SEQ ID NO: 49 - CD33CARV4 (LHCD8-41) CD38ssAAV-backbone MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGGAPKLLIYAASMQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK GSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS 50. SEQ ID NO: 50 - CD33CARV4 (LHCD8-41) CD38ssAAV-backbone 51. SEQ ID NO: 51 - Off-target reference sequence CCCCGCCTGGAGCCCTATGGCCAACTGCGAGTTCAGCCCGGTGTCCGGGGACAAA 52. SEQ ID NO: 52 - Off-target alteration sequence CCTTGGAGTCGCAGCTAGCCTCTGTGAGG 53. SEQ ID NO: 53 - Off-target alteration sequence TTTATTAGTAG 54. SEQ ID NO: 54 - Off-target alteration sequence AAACTCTAGG 55. SEQ ID NO: 55 - Off-target alteration sequence ACCAACGAACTGTAAGGGCT 56. SEQ ID NO: 56 - Off-target alteration sequence GCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC 57. SEQ ID NO: 57 - Off-target alteration sequence GATCCACTTTTATAAATATTAAAGTGGATCTGTATACAT 58. SEQ ID NO: 58 - Non-target reference sequence AAGAGTGACAACCTGGAACAGAGAGAGACC 59. SEQ ID NO: 59 - IgG Hinge CD4 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 60. Sequence number 60-41BB-L KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 61. SEQ ID NO: 61-CD3z RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. 1. An engineered T cell comprising a plasmid, nucleic acid, and / or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises, in order, a left homologous arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and a right homologous arm, wherein the left homologous arm and the right homologous arm are each 1000 bp or less in length.

2. 2. The modified T cell of claim 1, wherein the CAR polypeptide comprises a transmembrane domain, a costimulatory domain, a CD3ζ signaling domain, and a single-chain variable fragment (scFv) that specifically binds to a receptor on a target cell.

3. The modified T cell of claim 2 , wherein the receptor comprises CD33.

4. The modified T cell of claim 2, wherein the scFV that specifically binds to CD33 comprises a sequence that is at least 90% identical to SEQ ID NO: 29 or a fragment thereof.

5. The modified T cell of claim 2, wherein the scFv that specifically binds to CD33 comprises SEQ ID NO: 29 or a fragment thereof.

6. 6. The modified T cell of any one of claims 2-5, wherein the transmembrane domain of the CAR polypeptide comprises a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

7. 6. The modified T cell of any one of claims 2-5, wherein the costimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination thereof.

8. The modified T cell of any one of claims 1 to 7, further comprising a polyadenylation signal between the transgene and the right homology arm.

9. The modified T cell of any one of claims 1 to 8, wherein the left homologous arm and the right homologous arm are the same length.

10. 10. The modified T cell of claim 9, wherein the homology arms are each 30 bp in length.

11. 10. The modified T cell of claim 9, wherein the homology arms are each 300 bp in length.

12. 10. The modified T cell of claim 9, wherein the homology arms are each 600 bp in length.

13. 10. The modified T cell of claim 9, wherein the homology arms are each 1000 bp in length.

14. The modified T cell of any one of claims 1 to 8, wherein the left homologous arm and the right homologous arm are of different lengths.

15. The modified T cell of any one of claims 1 to 14, wherein the homologous arms specifically hybridize to the adeno-associated virus integration site 1 (AAVS1) of human chromosome 19.

16. 16. The modified T cell of any one of claims 1 to 15, further comprising a murine leukemia virus-derived (MND) promoter.

17. 17. The modified T cell of any one of claims 1 to 16, wherein the plasmid, the nucleic acid, or the construct is contained within an adeno-associated virus (AAV) vector.

18. The modified T cell of claim 17, wherein the AAV serotype comprises AAV6.

19. 19. The modified T cell of claim 17 or 18, wherein the vector further comprises a plasmid, nucleic acid, or construct encoding a crRNA, a tracer RNA (trcrRNA), and a CAS endonuclease.

20. 20. The modified T cell of any one of claims 17 to 19, wherein the vector is a single-stranded AAV (ssAAV).

21. The modified T cell of any one of claims 17 to 19, wherein the vector is a self-complementary AAV (scAAV).

22. 22. The modified T cell of any one of claims 17 to 21, wherein the vector comprises a sequence at least 90% identical to SEQ ID NO: 22 or SEQ ID NO: 23, or a fragment thereof.

23. 23. The modified T cell of any one of claims 1 to 22, wherein the T cell is expanded in the presence of irradiated feeder cells, plasma membrane particles, or exosomes that express membrane-bound IL-21, membrane-bound 4-1BBL, and / or membrane-bound IL-15, or any combination thereof.

24. 24. A method of treating cancer in a subject, comprising administering to the subject the modified T cell of any one of claims 1-23.

25. 1. A method of treating cancer in a subject, comprising administering to the subject engineered T cells comprising a plasmid, nucleic acid, and / or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises, in order, a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and a right homology arm, wherein the left homology arm and the right homology arm are each 1000 bp or less in length.

26. 26. The method of claim 24 or 25, wherein the cancer comprises leukemia.

27. 1. A method for genetically modifying T cells, comprising: a) obtaining a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA and an AAV vector comprising a plasmid, nucleic acid, or construct comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, wherein the polynucleotide sequence is flanked by homologous arms, and the homologous arms are 800 bp or less in length; and b) introducing the polynucleotide sequence encoding the CAR polypeptide and the RNP complex into the cell, wherein the polynucleotide sequence encoding the CAR polypeptide is introduced into the T cell via infection of the cell with the AAV, the RNP complex hybridizes to a target sequence within the genomic DNA of the cell, and DNA repair enzymes in the T cell insert the polynucleotide sequence encoding the CAR polypeptide into the host genome at the target sequence within the genomic DNA of the cell, thereby generating a modified T cell.

28. 28. The method of claim 27, wherein the T cells are primary T cells or expanded T cells.

29. 29. The method of claim 28, wherein the primary T cells are incubated in the presence of IL-2, IL-7, and / or IL-15 for about 4 to 10 days prior to infection.

30. 30. The method of claim 28 or 29, wherein the primary T cells are expanded in the presence of irradiated feeder cells, plasma membrane particles, or exosomes for about 4 to 10 days prior to infection.

31. 31. The method of claim 30, wherein the irradiated feeder cells, the plasma membrane particles, or the exosomes express membrane-bound 4-1BBL, membrane-bound IL-21, or membrane-bound IL-15, or any combination thereof.

32. 31. The method of any one of claims 27 to 30, further comprising expanding the modified T cells on irradiated feeder cells, plasma membrane particles, or exosomes after infection, wherein the irradiated feeder cells, plasma membrane particles, or exosomes express membrane-bound 4-1BBL, membrane-bound IL-21, membrane-bound IL-15, or any combination thereof.

33. 32. The method of any one of claims 27-31, further comprising expanding the modified T cells with IL-2, IL-7, and / or IL-15 after infection.

34. 33. The method of any one of claims 27 to 32, wherein the T cells are infected with the AAV at a multiplicity of infection (MOI) of about 5 to 500,000.

35. The method of any one of claims 27 to 33, wherein the RNP complex is introduced into the T cells via electroporation.

36. 35. The method of any one of claims 27 to 34, wherein the RNP complex is introduced into the T cell via transfection, and the RNP complex is encoded on the same or a different AAV.

37. 36. The method of any one of claims 27-35, wherein the CAR polypeptide comprises a transmembrane domain, a costimulatory domain, a CD3ζ signaling domain, and a single-chain variable fragment (scFv) that specifically binds to a receptor on a target cell.

38. 38. The method of claim 37, wherein the receptor comprises CD33.

39. 38. The method of claim 37, wherein the scFV that specifically binds to CD33 comprises a sequence that is at least 90% identical to SEQ ID NO: 29 or a fragment thereof.

40. 38. The method of claim 37, wherein the scFv that specifically binds to CD33 comprises SEQ ID NO: 29 or a fragment thereof.

41. 41. The method of any one of claims 27-40, wherein the transmembrane domain of the CAR polypeptide comprises a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

42. 42. The method of any one of claims 27-41, wherein the costimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination thereof.

43. 43. The method of any one of claims 27 to 42, wherein the left homologous arm and the right homologous arm are the same length.

44. 44. The method of claim 43, wherein the homology arms are each 600 bp in length.

45. The method of any one of claims 27 to 42, wherein the left homologous arm and the right homologous arm are of different lengths.

46. 46. ​​The method of any one of claims 27 to 45, wherein the homologous arms specifically hybridize to the adeno-associated virus integration site 1 (AAVS1) of human chromosome 19.

47. 47. The method of any one of claims 27 to 46, wherein the plasmid, the nucleic acid, or the construct further comprises a murine leukemia virus-derived (MND) promoter.

48. The method of any one of claims 27 to 47, wherein the AAV serotype comprises AAV6.

49. 49. The method of any one of claims 27 to 48, wherein the vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

50. 50. The method of any one of claims 27 to 49, wherein the vector comprises a sequence at least 90% identical to SEQ ID NO: 22 or SEQ ID NO: 23 or a fragment thereof.

51. 1. A method for generating chimeric antigen receptor (CAR) T cells, comprising: a) obtaining a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA and an AAV vector comprising a plasmid, nucleic acid, or construct comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, wherein the polynucleotide sequence is flanked by homologous arms, and the homologous arms are 1000 bp or less in length; and b) introducing the polynucleotide sequence encoding the CAR polypeptide and the RNP complex into a T cell, wherein the polynucleotide sequence encoding the CAR polypeptide is introduced into the T cell via infection of the T cell with the AAV, the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell, and a DNA repair enzyme in the T cell inserts the polynucleotide sequence encoding the CAR polypeptide into a host genome at the target sequence within the genomic DNA of the T cell, thereby generating a CAR T cell.

52. 52. The method of claim 51, wherein the T cells are primary T cells or expanded T cells.

53. 53. The method of claim 52, wherein the primary T cells are incubated in the presence of IL-2, IL-7, and / or IL-15 for about 4 to 10 days prior to infection.

54. 54. The method of claim 52 or 53, wherein the primary T cells are expanded in the presence of irradiated feeder cells, plasma membrane particles, or exosomes for about 4 to 10 days prior to infection.

55. 55. The method of claim 54, wherein the irradiated feeder cells, the plasma membrane particles, or the exosomes express membrane-bound 4-1BBL, membrane-bound IL-21, or membrane-bound IL-15, or any combination thereof.

56. 56. The method of any one of claims 51 to 55, further comprising expanding the CAR T cells with irradiated feeder cells, cell membrane particles, or exosomes after infection, wherein the irradiated feeder cells, cell membrane particles, or exosomes express membrane-bound 4-1BBL, membrane-bound IL-21, or membrane-bound IL-15, or any combination thereof.

57. 57. The method of any one of claims 51-56, further comprising expanding the CAR T cells with IL-2, IL-7, and / or IL-15 after infection.

58. 58. The method of any one of claims 51-57, wherein the T cells are infected with the AAV at an MOI of about 5-500K.

59. 59. The method of any one of claims 51 to 58, wherein the RNP complex is introduced into the T cells via electroporation.

60. 60. The method of any one of claims 51 to 59, wherein the RNP complex is introduced into the T cell via transfection, and the RNP complex is encoded on the same or a different AAV.

61. 61. The method of any one of claims 51-60, wherein the CAR polypeptide comprises a transmembrane domain, a costimulatory domain, a CD3ζ signaling domain, and a single-chain variable fragment (scFv) that specifically binds to a receptor on a target cell.

62. 62. The method of claim 61, wherein the receptor comprises CD33.

63. 62. The method of claim 61, wherein the scFV that specifically binds to CD33 comprises a sequence that is at least 90% identical to SEQ ID NO: 29 or a fragment thereof.

64. 62. The method of claim 61, wherein the scFv that specifically binds to CD33 comprises SEQ ID NO: 29 or a fragment thereof.

65. 65. The method of any one of claims 51-64, wherein the transmembrane domain of the CAR polypeptide comprises a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

66. 66. The method of any one of claims 51-65, wherein the costimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination thereof.

67. 67. The method of any one of claims 51 to 66, wherein the left homologous arm and the right homologous arm are the same length.

68. 68. The method of claim 67, wherein the homology arms are each 600 bp in length.

69. The method of any one of claims 51 to 66, wherein the left homologous arm and the right homologous arm are of different lengths.

70. 70. The method of any one of claims 51 to 69, wherein the homologous arms specifically hybridize to the adeno-associated virus integration site 1 (AAVS1) of human chromosome 19.

71. 71. The method of any one of claims 51 to 70, wherein the plasmid, the nucleic acid, or the construct further comprises a murine leukemia virus-derived (MND) promoter.

72. The method of any one of claims 51 to 71, wherein the AAV serotype comprises AAV6.

73. 73. The method of any one of claims 51 to 72, wherein the vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

74. 74. The method of any one of claims 51 to 73, wherein the vector comprises a sequence at least 90% identical to SEQ ID NO: 22 or SEQ ID NO: 23 or a fragment thereof.

75. 75. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of CAR T cells generated using the method of any one of claims 51-74.

76. 1. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of T cells, wherein the T cells comprise a plasmid, nucleic acid, and / or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises, in order, a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and a right homology arm, wherein the left homology arm and the right homology arm are each 1000 bp or less in length.

77. 77. The method of Claim 75 or 76, wherein the CAR polypeptide comprises a transmembrane domain, a costimulatory domain, a CD3ζ signaling domain, and a single chain variable fragment (scFv) that specifically binds to a receptor on a target cell.

78. 78. The method of claim 77, wherein the receptor comprises CD33.

79. 78. The method of claim 77, wherein the scFV that specifically binds to CD33 comprises a sequence that is at least 90% identical to SEQ ID NO: 29 or a fragment thereof.

80. 78. The method of claim 77, wherein the scFv that specifically binds to CD33 comprises SEQ ID NO: 29 or a fragment thereof.

81. 81. The method of any one of claims 75-80, wherein the transmembrane domain of the CAR polypeptide comprises a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

82. 82. The method of any one of claims 75-81, wherein the costimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination thereof.

83. 83. The method of any one of claims 75 to 82, further comprising a polyadenylation signal between the transgene and the right homologous arm.

84. 84. The method of any one of claims 75 to 83, wherein the left homologous arm and the right homologous arm are the same length.

85. 85. The method of claim 84, wherein the homology arms are each 30 bp in length.

86. 85. The method of claim 84, wherein the homology arms are each 300 bp in length.

87. 85. The method of claim 84, wherein the homology arms are each 600 bp in length.

88. 85. The method of claim 84, wherein the homology arms are each 1000 bp in length.

89. The method of any one of claims 75 to 83, wherein the left homologous arm and the right homologous arm are of different lengths.

90. 90. The method of any one of claims 75 to 89, wherein the homologous arms specifically hybridize to the adeno-associated virus integration site 1 (AAVS1) of human chromosome 19.

91. 91. The method of any one of claims 75 to 90, further comprising a murine leukemia virus-derived (MND) promoter.

92. 92. The method of any one of claims 75 to 91, wherein the plasmid, the nucleic acid, or the construct is transduced into the T cells by an adeno-associated virus (AAV) vector.

93. 93. The method of claim 92, wherein the AAV serotype comprises AAV6.

94. 94. The method of claim 92 or 93, wherein the vector further comprises a plasmid, nucleic acid, or construct encoding a crRNA, a tracer RNA (trcrRNA), and a CAS endonuclease.

95. 95. The method of any one of claims 92 to 94, wherein the vector is a single-stranded AAV (ssAAV).

96. 96. The method of any one of claims 92 to 95, wherein the vector is a self-complementary AAV (scAAV).

97. 97. The method of any one of claims 92 to 96, wherein the vector comprises a sequence at least 90% identical to SEQ ID NO: 22 or SEQ ID NO: 23 or a fragment thereof.

98. 98. The method of any one of claims 75-97, wherein the cancer comprises acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or myelodysplastic syndrome (MDS).

99. 1. An engineered T cell comprising a plasmid, nucleic acid, or construct for use in a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated 9 (Cas9) integrated system, wherein the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, wherein the polynucleotide sequence is flanked by a protospacer adjacent motif (PAM) and a polynucleotide sequence encoding a crisprRNA (crRNA), or is flanked by two polynucleotide sequences encoding two PAMs and the crRNA.

100. 100. The modified T cell of claim 99, wherein the plasmid, nucleic acid, or construct comprises, in order, a polynucleotide sequence encoding a PAM sequence and a crRNA, the polynucleotide sequence encoding the CAR polypeptide, and a polynucleotide sequence encoding a PAM sequence and a crRNA.

101. 101. The modified T cell of claim 99 or 100, wherein the CAR polypeptide comprises a transmembrane domain, a costimulatory domain, a CD3ζ signaling domain, and a single-chain variable fragment (scFv) that specifically binds to a receptor on a target cell.

102. The modified T cell of claim 101, wherein the receptor comprises CD33.

103. The modified T cell of claim 101, wherein the scFV that specifically binds to CD33 comprises a sequence that is at least 90% identical to SEQ ID NO: 29 or a fragment thereof.

104. The modified T cell of claim 101, wherein the scFv that specifically binds to CD33 comprises SEQ ID NO: 29 or a fragment thereof.

105. 105. The modified T cell of any one of claims 99-104, wherein the transmembrane domain of the CAR polypeptide comprises a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3ζ transmembrane domain, or an NKG2D transmembrane domain.

106. 106. The modified T cell of any one of claims 99-105, wherein the costimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, or any combination thereof.

107. 107. The modified T cell of any one of claims 99-106, further comprising a murine leukemia virus-derived (MND) promoter.

108. 108. The modified T cell of any one of claims 99-107, wherein the plasmid, the nucleic acid, or the construct is comprised within an adeno-associated virus (AAV) vector.

109. The modified T cell of claim 108, wherein the AAV serotype comprises AAV6.

110. 110. The modified T cell of claim 108 or 109, wherein the vector further comprises a plasmid, nucleic acid, or construct encoding a crRNA, a tracer RNA (trcrRNA), and a CAS endonuclease.

111. The modified T cell of any one of claims 108 to 110, wherein the vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

112. 112. The modified T cell of any one of claims 99-111, wherein the T cell is expanded in the presence of irradiated feeder cells, plasma membrane particles, or exosomes expressing membrane-bound IL-21, membrane-bound 4-1BBL, membrane-bound IL-15, or any combination thereof.

113. 113. A method of treating cancer in a subject, comprising administering to a subject having cancer the modified T cell of any one of claims 99-112.

114. 114. The method of claim 113, wherein the cancer comprises leukemia.

115. 1. A method for generating chimeric antigen receptor (CAR) T cells, comprising: a) obtaining a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA and an AAV vector comprising a plasmid, nucleic acid, or construct comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the polynucleotide sequence is flanked by a protospacer adjacent motif (PAM) and a polynucleotide sequence encoding a crisprRNA (crRNA) or is flanked by two polynucleotide sequences encoding two PAMs and the crRNA; b) introducing the polynucleotide sequence encoding the CAR polypeptide and the RNP complex into a T cell, wherein the plasmid, the nucleic acid, or the construct is introduced into the T cell via infection of a target cell with the adeno-associated virus (AAV), the ribonucleoprotein (RNP) complex hybridizes to a target sequence within the genomic DNA of the T cell, and DNA repair enzymes in the T cell insert the polynucleotide encoding the CAR into the host genome at the target sequence, thereby generating a CAR T cell.

116. 116. The method of Claim 115, wherein the plasmid, nucleic acid, or construct comprises, in order, a PAM sequence and a polynucleotide sequence encoding a crRNA, the polynucleotide sequence encoding the CAR polypeptide, and a PAM sequence and a polynucleotide sequence encoding a crRNA.

117. 1. A method for genetically modifying T cells, comprising: a) obtaining a ribonucleoprotein (RNP) complex comprising a Class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA and an AAV vector comprising a plasmid, nucleic acid, or construct comprising a polynucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the polynucleotide sequence is adjacent to one polynucleotide sequence encoding a PAM and a crRNA or flanked by two polynucleotide sequences encoding two PAMs and a crRNA; and b) introducing the polynucleotide sequence encoding the CAR polypeptide and the RNP complex into the T cell, wherein the polynucleotide sequence encoding the CAR polypeptide is introduced into the cell via infection of the target cell with the adeno-associated virus (AAV), the ribonucleoprotein (RNP) complex hybridizes to a target sequence within the genomic DNA of the T cell, and DNA repair enzymes of the cell insert the polynucleotide sequence encoding the chimeric antigen receptor (CAR) into the host genome at the target sequence, thereby generating a modified T cell.

118. 118. The method of Claim 117, wherein the plasmid, nucleic acid, or construct comprises, in order, one PAM sequence and one polynucleotide sequence encoding a crRNA, the polynucleotide sequence encoding the CAR polypeptide, one polynucleotide sequence encoding a crRNA, and one PAM sequence.

119. 118. The method of Claim 117, wherein the plasmid, the nucleic acid, or the construct comprises, in order, the polynucleotide sequence encoding the CAR polypeptide, one polynucleotide sequence encoding a crRNA, and one PAM sequence.

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