WO2024124243A2 - Chimeric antigen receptor (car) t cells and uses thereof - Google Patents

Chimeric antigen receptor (car) t cells and uses thereof Download PDF

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WO2024124243A2
WO2024124243A2 PCT/US2023/083396 US2023083396W WO2024124243A2 WO 2024124243 A2 WO2024124243 A2 WO 2024124243A2 US 2023083396 W US2023083396 W US 2023083396W WO 2024124243 A2 WO2024124243 A2 WO 2024124243A2
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cell
cells
car
modified
aav
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WO2024124243A3 (en
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Dean Lee
Meisam NAEIMI KARAROUDI
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Research Institute At Nationwide Children's Hospital
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • T cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying T cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity.
  • CAR chimeric antigen receptor
  • T cells comprising plasmids, nucleic acids, and/or constructs for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR- associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (such as, for example, a CAR comprising a 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 CD8 transmembrane domain, a CD28 transmembrane domain, and/or a CD3 ⁇ transmembrane domain), a costimulatory domain (e.g., a 2B4 domain, a
  • CRISPR clustered regularly inters
  • the costimulatory domain comprises a CD28 co-stimulatory domain and/or a 4-1 BB co-stimulatory domain.
  • the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain.
  • T cells comprising plasmids, nucleic acids, and/or constructs for use with CRISPR/ Cas9 integration systems of any preceding aspect, wherein the left homology arm and right homology arm are the same length or different lengths.
  • the homology arms specifically hybridize to the Adeno-Associated Virus Integration Site 1 (AAVS1) of chromosome 19 of humans.
  • AAVS1 Adeno-Associated Virus Integration Site 1
  • T cells comprising plasmids, nucleic acids, and/or constructs for use with CRISPR/ Cas9 integration systems of any preceding aspect, wherein the plasmid, nucleic acid, or construct further comprises a murine leukemia virus-derived (MND) promoter.
  • MND murine leukemia virus-derived
  • the plasmid, nucleic acid, or construct of any preceding aspect is contained within and/or delivered into the T cell Adeno-associated viral (AAV) vectors (such as, for example, an AAV vector comprising the AAV6 serotype) comprising the plasmid, nucleic acid, or construct of any preceding aspect.
  • AAV Adeno-associated viral
  • 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 can be a single stranded AAV (ssAAV) or a self-complimentary AAV (scAAV).
  • Also disclosed herein are methods of treating, decreasing, reducing, inhibiting, ameliorating, and/or preventing a cancer and/or metastasis such as, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and/or myelodysplastic syndromes (MDS)) in a subject comprising administering to a subject with a cancer the modified cell of any preceding aspect.
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • HCL hairy cell leukemia
  • MDS myelodysplastic syndromes
  • 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 (such as, for example, a chimeric antigen receptor for a tumor antigen); wherein the transgene is flanked by homology arms; and wherein the homology arms are 1000bp in length or less; and b) introducing the transgene and the RNP complex into a T cell; wherein the transgene (such as, for example, a chimeric antigen receptor for a tumor antigen) is introduced into the T cell via infection with the Adeno-associated virus (AAV); wherein the RNP complex hybridizes to a target
  • RNP ribonucleoprotein
  • the RNP complex can be introduced into the cell via electroporation. In some aspects, the RNP complex can be introduced into the cell via viral delivery in the same or a different AAV (i.e., superinfection).
  • methods of genetically modifying a 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 chimeric antigen receptor (CAR) polynucleotide sequence; wherein the polynucleotide sequence is flanked by homology arms; and wherein the homology arms are 1000 bp in length or less; and b) introducing the polynucleotide sequence and the RNP complex
  • a T cell of any preceding aspect wherein the T cell is infected with about 5 to 500K multiplicity of infection (MOI) of the AAV disclosed herein.
  • MOI multiplicity of infection
  • methods of genetically modifying a T cell of any preceding aspect wherein the primary cells are incubated for about 4 to 10 days in the presence of IL-2, IL-15, and or IL-7 and/or irradiated feeder, plasma membrane particles, or exosomes cells prior to or after infection and/or electroporation.
  • a T cell of any preceding aspect further comprising expanding the primary T cells for about 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, or membrane-bound IL-15, or any combination thereof.
  • a cancer and/or metastasis such as, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and/or myelodysplastic syndromes (MDS)
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • HCL hairy cell leukemia
  • MDS myelodysplastic syndromes
  • T cell comprises a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (such as, for example a CD33 targeting CAR
  • a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is adjacent to one protospacer adjacent motif (PAM) and one sequence encoding crispr RNA (crRNA) or flanked by two PAMs and sequences encoding crRNAs.
  • CRISPR clustered regularly interspaced short palindromic repeat
  • Cas9 integration systems wherein the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is adjacent to one protospacer adjacent motif (PAM) and one sequence encoding crispr
  • the disclosed plasmid, nucleic acid, or construct can be used in any of the methods of treating, decreasing, reducing, inhibiting, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect; methods of creating a CAR T cell of any preceding aspect; and/or genetically modifying a T cell of any preceding aspect.
  • BRIEF DESCRIPTION OF FIGURES [0014] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [0015] Figure 1 shows CD33CAR-T cells generated by the disclosed technology.
  • Figure 2 shows the distribution of events by chromosome from the dGH assay in Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23.
  • Figure 3 shows the distribution of event rates by number of cells in Sample KromaTiD- CD33CARNK/8AM-dGH/8/1/23 from the dGH assay.
  • Figure 4 shows the inversion and sister chromatid exchange (SCE) event summary in Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23 from the dGH assay.
  • SCE sister chromatid exchange
  • Figure 5 shows the size difference summary counted by chromosome in Sample KromaTiD- CD33CARNK/8AM-dGH/8/1/23 from the dGH assay.
  • Figure 6 shows an example karyogram. There are SCE events present on Chromosome1p (Chr1p), Chromosome 5q (Chr5q), Chr7q, Chr8q, Chr9q, Chr10q x 2, Chr14q, ChrXp, and ChrXq. There is a size difference between homologs observed on Chr2. The small inversion on Chr8p is present Chr16q has a whole arm deletion.
  • Figure 7 shows an example karyogram.
  • FIG. 8 shows Next Generation Sequencing (NGS) coverage (grey) across the vector. Black arrows indicate the primer location. The vector map is shown on the bottom. Y-axes are limited to 100X.
  • Figure 9 shows the TLA sequence coverage across the human genome using primer set 2. The chromosomes are indicated on the y-axis, the chromosomal position on the x-axis. Identified integration site is circled.
  • Figure 10 shows the TLA sequence coverage (grey) across the vector integration locus, chr4:15,737,375-15,822,498.
  • the top arrow of Set 1 indicates the location of the breakpoint sequences.
  • the bottom arrow of Set 1 indicates the position of primer set 1.
  • the bar of Set 2 indicates the location of the homology arms. Y-axes are limited to 2,500X.
  • Figure 11 shows a schematic of NK cell genetic modification.
  • Figures 12A and 12B show successful generation of CD33 CAR expressing NK cells using combination of Cas9/RNP and AAV6.
  • Figure 13 shows successful generation of CD38 KO CD33 CAR expressing NK cells using combination of Cas9/RNP and AAV6.
  • Figures 14A, 14B, 14C, 14D, 14E, and 14F show the CD33 CAR expressing NK cells demonstrate efficient cytotoxicity against CD33 + tumors.
  • Figures 34B and 34C show that expressing CD33 CAR on NK cells also enhances antitumor activity of NK cells against Kasumi-1 as shown in representative cytotoxicity assay performed in 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 against HL-60 only in CD33 CAR-Gen2 NK cells.
  • Figure 15 shows the successful generation of CD38 KO NK cells from ex vivo expanded PB- NK cells using Cas9/RNP. CD38 expression in NK cells before and after Cas9/RNP-mediated CD38 deletion (n 5); Representative fluorescence-activated cell sorter (FACS) analyses of the purified CD38 KO NK cells.
  • Figures 16A, 16B, 16C, and 16D show the favorable metabolic reprogramming of CD38 KO NK cells.
  • Figure 36B shows the principal components analysis (PCA) of DEGs, showing consistent effect of CD38 deletion for each donor despite wide interdonor variability.
  • Figure 36D shows the graphical analysis of basal OCR, ECAR, OCR/ECAR, and spare respiratory capacity (SRC) derived from Figure 36C. All experiments were performed in quintuplicate.
  • FIG. 17 shows the relative expression in wild-type (WT) expanded human NK cells of mutated genes identified in CD38 KO NK cells.
  • Figure 18 shows the relative expression in WT expanded human NK cells of mutated genes identified in CD38 KO CD33 CAR NK cells.
  • Figure 19 shows the OCI-AML-3 xenograft model showing survival in mice receiving CAR- NK cells compared to non-modified NK cells.
  • Figure 20 shows the CD33 CAR construct. Sig-leucel utilizes a second generation anti-CD33 CAR.
  • the extracellular binding domain is an anti- CD33 scFv based on heavy and light chain sequences derived from the humanized monoclonal antibody HuM195 (lintuzumab), connected by a Whitlow linker.
  • HuM195 humanized monoclonal antibody
  • the hinge, stalk, and transmembrane domain are derived from human CD8 ⁇ .
  • the intracellular signaling domain is comprised of a CD3 ⁇ activation domain and a 4- 1BB costimulatory domain.
  • Figure 21 shows the AAV6 vector sequence map.
  • Figure 22 shows the schematic of clinical CD38 KO CD33 CAR NK treatment.
  • Figure 23 shows the flow diagram outlining the CD38 KO CD33 CAR NK manufacturing and testing.
  • Figures 24A and 24B show CD33 CAR expressing NK cells demonstrating improved effector function compared to wildtype (WT) expanded peripheral blood NK cells.
  • Figure 6A shows CD38 expressing on AML cells.
  • AML cell co-culture with WT-NK or CD33 CAR-NK cells induces AML cell death as shown by viability assessment or in SPADE plots (colored for pRb expression indicative of viable cycling cell), green arrows indicate live AML cells while red arrows indicate dead/dying AML cells.
  • CD33 CAR-NK cells demonstrate increased AML cells killing, surviving AML cells have reduced CD33 surface expression and increased CD38 expression.
  • Figure 6B shows bioplex results showing higher IFN- ⁇ and TNF- ⁇ secretion from CD33 CAR NK cells co-cultured with AML cells.
  • Figure 25 shows a schematic of targeted probes used in the directed Genomic Hybridization (dGH) assay.
  • Figure 26 shows a cell with no transgene signal. The bottom panel has the telomeric signal layer removed. Note that the overlay of telomeric and centromeric bracketing probes can appear as a transgene probe as seen in the top panel. Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23.
  • Figure 27 shows a cell with an inversion/SCE event in the target region of the centromeric probe (circled).
  • telomeric and centromeric bracketing probe signals can appear as a transgene probe.
  • Figure 28 shows a cell with an inversion/SCE event in the target region of the telomeric probe (circled). The right panel shows increased magnification and has the centromeric layer removed to better visualize the telomeric probe signal pattern. Note that the overlay of telomeric and centromeric bracketing probe signals can appear as a transgene probe as seen in the left panel.
  • Figure 29 shows a cell with transgene inserts on both copies of CD38 (circled). The right panel shows the transgene insert signal layer.
  • Figure 30 shows a cell with transgene inserts on both copies of CD38 (circled). The right panel has the telomeric probe signal layer removed. Note that the overlay of telomeric and centromeric bracketing probe signals can appear as a transgene probe signal in a composite image. Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23.
  • Figure 31 shows a cell with one transgene insert on-target at CD38 (circled) and one off-target (circled).
  • FIG. 32 shows the design of CD33CAR-Gen2 and CD33CAR-Gen4v2.
  • Figure 33 shows the anti-AML activity of CD33CAR-NK.
  • Figure 34 shows that CD33CAR NK cells have enhanced anti-AML activity.
  • Figure 35 shows a schematic indicating generation of fratricide resistant CD38KO/CD33- CAR NK cells.
  • Figure 36 shows the fratricide resistant CD38KO/CD33-CAR NK cells to target residual AML.
  • DETAILED DESCRIPTION [0051] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure.
  • the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
  • 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, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • administering to a subject includes any route of introducing or delivering to a subject an agent.
  • Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like.
  • parenteral e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques
  • Constant administration means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.
  • Systemic administration refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems.
  • local administration refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount.
  • locally administered agents are easily detectable in the local vicinity of the point of administration, but are undetectable or detectable at negligible amounts in distal parts of the subject’s body.
  • Administration includes self-administration and the administration by another.
  • Biocompatible generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
  • a “control” is an alternative subject or sample used in an experiment for comparison purposes.
  • a control can be "positive” or “negative.”
  • “Complementary” or “substantially complementary” refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid.
  • Complementary nucleotides are, generally, 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, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%.
  • substantial complementarity exists when an RNA or DNA strand hybridizes under selective hybridization conditions to its complement.
  • selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary. See Kanehisa (1984) Nucl. Acids Res.12:203.
  • composition refers to any agent that has a beneficial biological effect. Beneficial biological effects include both 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.
  • composition also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • composition includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • a DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA.
  • a DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, microRNA (miRNA), a "non-coding" RNA (ncRNA), a guide RNA, etc.).
  • Expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises 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.)
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • the “fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein.
  • the term "gene” or “gene sequence” refers to the coding sequence or control sequence, or fragments thereof.
  • a gene may include any combination of coding sequence and control sequence, or fragments thereof.
  • a “gene” as referred to herein may be all or part of a native gene.
  • a polynucleotide sequence as referred to herein may be used interchangeably with the term “gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof.
  • the term “gene” or “gene sequence” includes, for example, control sequences upstream of the coding sequence (for example, the ribosome binding site).
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (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 maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • 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.
  • percent (%) nucleotide sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the nucleotides in 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 achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • sequence comparisons typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J.
  • HSPs high scoring sequence pairs
  • 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, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • 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 by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
  • nucleic acid refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is wild type (and naturally occurring).
  • An "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, composition in a statistically significant amount. Thus, the 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.
  • a "decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease 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% decrease so long as the decrease is statistically significant.
  • nucleic acid means a polymer composed of nucleotides, e.g., deoxyribonucleotides (DNA) or ribonucleotides (RNA).
  • ribonucleic acid and RNA as used herein mean a polymer composed of ribonucleotides.
  • deoxyribonucleic acid and DNA as used herein mean a polymer composed of deoxyribonucleotides.
  • “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • operatively linked can indicate that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence.
  • This same definition is sometimes applied to the arrangement of coding sequences and/or transcription control elements (e.g. promoters, enhancers, and termination elements), and/or selectable markers in an expression vector.
  • the term "operatively linked” can also refer to the arrangement of polypeptide segments within a single polypeptide chain, where the individual polypeptide segments can be, without limitation, a protein, fragments thereof, linking peptides, and/or signal peptides.
  • operatively linked can refer to direct fusion of different individual polypeptides within the single polypeptides or fragments thereof where there are no intervening amino acids between the different segments as well as when the individual polypeptides are connected to one another via one or more intervening amino acids.
  • “Primers” are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically, a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.
  • a "protein coding sequence” or a sequence that encodes a particular protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • a coding sequence can include, but is 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 will usually be located 3' to the coding sequence.
  • polynucleotide refers to a single or double stranded polymer composed of nucleotide monomers.
  • polypeptide refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.
  • promoter as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • "Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the 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 components of the formulation in which it is contained.
  • “Pharmaceutically acceptable carrier” means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • 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 as described further herein.
  • “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
  • Effective amount refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like.
  • an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.
  • An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum 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. [0088] “Therapeutic agent” refers to any composition that has a beneficial biological effect.
  • Beneficial biological effects include both 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 (e.g., a cancer).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • “therapeutic agent” refers to an amount that is effective to achieve a desired therapeutic result.
  • a desired therapeutic result is the control of cancer.
  • a desired therapeutic result is the control of metastasis.
  • a desired therapeutic result is the reduction of tumor size.
  • a desired therapeutic result is the prevention and/or treatment of relapse.
  • Therapeutically effective amounts 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 and the age, gender, and weight of the subject.
  • the term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief.
  • the precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • 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 herein disclosed polypeptides that are the subject of the present disclosure.
  • transgene refers to a sequence (e.g., polynucleotide or polypeptide sequence) which does not occur in the cell to be artificially provided with the sequence, or is linked to another polynucleotide in an arrangement which does not occur in the cell to be artificially provided with the sequence.
  • artificial refers to non-natural occurrence in the host cell and includes manipulation by man, machine, exogenous factors (e.g., enzymes, viruses, etc.), other non-natural manipulations, or combinations thereof.
  • a transgene can comprise a gene operably linked to a promoter (e.g., an open reading frame), although is not limited thereto.
  • the transgene may integrate into the host cell chromosome, exist extrachromosomally, or exist in any combination thereof.
  • 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 individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
  • Plasmids, nucleic acids, and/or constructs for use and methods of genetically modifying cells comprising plasmids, nucleic acids, and/or constructs for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems, wherein the plasmid, nucleic acid, or construct comprises in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (such as, for example, a CAR comprising a 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 CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3 ⁇ transmembrane domain
  • CRISPR clustered regularly interspaced
  • the costimulatory domain comprises a CD28 co-stimulatory domain and/or a 4-1 BB co-stimulatory domain.
  • the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain.
  • CRISPR system or “CRISPR integration system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated “Cas” genes.
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. CRISPR systems are known in the art.
  • Endonuclease/RNPs are comprised of three components, recombinant endonuclease protein (for example, a Cas9 endonuclease) complexed with a CRISPR loci.
  • the endonuclease complexed to the CRISPR loci can be referred to as a CRISPR/Cas guide RNA.
  • the CRISPR loci comprises a synthetic single-guide RNA (gRNA) comprised of a RNA that can hybridize to a target sequence complexed complementary repeat RNA (crRNA) and trans complementary repeat RNA (tracrRNA). Accordingly, the CRISPR/Cas guide RNA hybridizes to a target sequence within the genomic DNA of the cell.
  • the class 2 CRISPR/Cas endonuclease is a type II CRISPR/Cas endonuclease.
  • the class 2 CRISPR/Cas endonuclease is a Cas9 polypeptide and the corresponding CRISPR/Cas guide RNA is a Cas9 guide RNA.
  • Cas9/RNPs are capable of cleaving genomic targets with higher efficiency as compared to foreign DNA-dependent approaches due to their delivery as functional complexes. Additionally, rapid clearance of Cas9/RNPs from the cells can reduce the off-target effects such as induction of apoptosis.
  • crRNA and tracrRNA can be mixed at a 1:1, 2:1, or 1:2 ratio of concentrations between about 50 ⁇ M and about 500 ⁇ M (for example, 50 ⁇ M, 60 ⁇ M, 70 ⁇ M, 80 ⁇ M, 90 ⁇ M, 100 ⁇ M, 125 ⁇ M, 150 ⁇ M, 175 ⁇ M, 200 ⁇ M, 225 ⁇ M, 250 ⁇ M, 275 ⁇ M, 300 ⁇ M, 325 ⁇ M, 350 ⁇ M, 375 ⁇ M, 400 ⁇ M, 425 ⁇ M, 450 ⁇ M, 475 ⁇ M, or 500 ⁇ M), preferably between 100 ⁇ M and about 300 ⁇ M, most preferably about 200 ⁇ M at 95 °C for about 5 min to form a crRNA:tracrRNA complex (i.e., the guide RNA).
  • a crRNA:tracrRNA complex i.e., the guide RNA
  • the crRNA:tracrRNA complex can then be mixed with between about 20 ⁇ M and about 50 ⁇ M (for example 21 ⁇ M, 22 ⁇ M, 23 ⁇ M, 24 ⁇ M, 25 ⁇ M, 26 ⁇ M, 27 ⁇ M, 28 ⁇ M, 29 ⁇ M, 30 ⁇ M, 31 ⁇ M, 32 ⁇ M, 33 ⁇ M, 34 ⁇ M, 35 ⁇ M, 36 ⁇ M, 37 ⁇ M, 38 ⁇ M, 39 ⁇ M, 40 ⁇ M, 41 ⁇ M, 42 ⁇ M, 43 ⁇ M, 44 ⁇ M, 45 ⁇ M, 46 ⁇ M, 47 ⁇ M, 48 ⁇ M, 49 ⁇ M, or 50 ⁇ M) final dilution of a Cas endonuclease (such as, for example, Cas9).
  • a Cas endonuclease such as, for example, Cas9
  • the CRISPR loci can modify the genome by introducing into the target DNA insertion or deletion of one or more base pairs, by insertion of a heterologous DNA fragment (e.g., the donor polynucleotide), by deletion of an endogenous DNA fragment, by inversion or translocation of an endogenous DNA fragment, or a combination thereof.
  • a heterologous DNA fragment e.g., the donor polynucleotide
  • the disclosed methods can be used to generate knock-outs, or knock-ins when combined with DNA for homologous recombination.
  • transduction via Adeno-associated viral (AAV) of Cas9/RNPs is a relatively efficient method that overcomes previous constraints of genetic modification in cells (such as, for example, T cells, B cells, macrophages, NK cells, NK T cells, fibroblasts, osteoblasts, hepatocytes, neuronal cells, epithelial cells, and/or muscle cells).
  • AAV Adeno-associated viral
  • any transcript above 100bp and any transgene is to have homology arms that are at least 800bp for each arm with many systems employing asymmetric arms of 800bp and 1000bp for a total of 1800bp.
  • the AAV vector cannot deliver a transgene larger than ⁇ 2.5 kb.
  • AAV CRISPR/CAS9 nucleotide delivery systems comprising a donor construct plasmid with homology arms between 30bp and 1000bp, including, but not limited to 30bp, 50bp, 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, 200bp, 210bp, 220bp, 230bp, 240bp, 250bp, 260bp, 270bp, 280bp, 290bp, 300bp, 310bp, 320bp, 330bp, 340bp, 350bp, 360bp, 370bp, 380bp, 390bp, 400bp, 410bp, 420bp, 430bp, 440bp, 450bp, 460bp, 470bp, 480bp, 490bp, 500bp, 510bp,
  • the homology arms can be symmetrical 30bp homology arms, symmetrical 300bp homology arms, symmetrical 500bp homology arms, symmetrical 600bp homology arms, symmetrical 800bp homology arms, symmetrical 1000bp homology arms, or asymmetrical 800bp homology arms comprising a 800bp left homology arm (LHA) and a 1000bp right homology arm (RHA) for homologous recombination (HR) or no homology arms at all for non-homologous end joining using homology-independent targeted integration (HITI) plasmids.
  • LHA left homology arm
  • RHA right homology arm
  • HR homologous recombination
  • HITI homology-independent targeted integration
  • the plasmids with or without homology arms are those disclosed in International Publication Number WO2020/198675, which is incorporated herein by reference in its entirety.
  • the plasmids have clinically approved splice acceptor (SA) (SEQ ID NO: 10) and clinically approved polyadenylation terminator (PA) (such as, for example BGH polyA terminator SEQ ID NO: 11).
  • SA splice acceptor
  • PA polyadenylation terminator
  • homology arms can be symmetrical (same length on each side) or asymmetrical (different lengths on each side) to accommodate differing transgene lengths.
  • homology arm lengths can have any combination of left homology arm (LHA) length and right homology arm (RHA) length including but not limited to LHA 30bp (SEQ ID NO: 2) and RHA 30bp (SEQ ID NO: 1), LHA 30bp and RHA 100bp, LHA 30bp and RHA 300bp (SEQ ID NO: 3), LHA 30bp and RHA 500bp (SEQ ID NO: 5), LHA 30bp and RHA 800bp (SEQ ID NO: 7), LHA 30bp and RHA 1000bp, LHA 100bp and RHA 30bp, LHA 100bp and RHA 100bp, LHA 100bp and RHA 300bp, LHA 100bp and RHA 500bp, LHA 100bp and RHA 800bp, LHA 100bp and RHA 1000bp, LHA 300bp (SEQ ID NO: 4) and RHA 30bp, LHA 300bp and RHA 100bp, LHA 300bp and RHA 100
  • AAV adeno-associated viruses
  • ssAAV linear single-stranded DNA with a length of approximately 4.7 kb
  • scAAV linear self-complementary DNA
  • the benefit of the scAAV vector is that it contains a mutated inverted terminal repeat (ITR), which is required for replication and helps to bypass rate-limiting steps of second strand generation in comparison to ssDNA vectors.
  • ITR inverted terminal repeat
  • CAR chimeric antigen receptor
  • both ssAAV and scAAV were designed and tested, which provides a wide range of options for gene insertion in primary T cells.
  • HA left and right homology arm
  • the CRISPaint approach has also been investigated, a homology-independent method for gene insertion or tagging.
  • the same Cas9 targeting site including the sequence encoding crRNA and PAM sequence (herein also termed as PAMg, e.g., SEQ ID NO: 9), is provided in the DNA template encoding the gene of interest.
  • PAMg e.g., SEQ ID NO: 9
  • both template and genomic DNA are cut simultaneously.
  • the CRISPaint template is presented as a linearized double-stranded DNA that can be integrated through non- homology repair machinery.
  • plasmids, nucleic acids, and/or constructs for delivering donor transgene to a cell and integrating said transgene (e.g., CAR) into the cell in combination with CRISPR/Cas9.
  • CAR transgene
  • plasmids, nucleic acids, and/or constructs for use with CRISPR/ Cas9 integration systems of any preceding aspect wherein the left homology arm and right homology arm are the same length or different lengths.
  • the homology arms specifically hybridize to the Adeno-Associated Virus Integration Site 1 (AAVS1) of chromosome 19 of humans.
  • the LHA is 600 bp in length.
  • the LHA comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 31 or a fragment thereof.
  • the RHA is 600 bp in length.
  • the RHA comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 32 or a fragment thereof.
  • the plasmid, nucleic acid, or construct disclosed herein comprises a polynucleotide sequence encoding a chimeric antigen receptor CAR polypeptide.
  • chimeric antigen receptor refers to a chimeric receptor that targets a cancer antigen and serves to bring the cell expressing the receptor to a cancer cell expressing the target antigen.
  • the CAR comprises a molecule that recognizes peptides derived from the tumor antigen presented by major histocompatibility (MHC) molecules, or an antibody or fragment thereof (such as for example, a Fab’, scFv, Fv) expressed on the surface of the CAR cell that targets a cancer antigen.
  • MHC major histocompatibility
  • the receptor is fused to a signaling domain (such as, for example, the CD3 ⁇ domain, NKG2C, or NKp44 domain) via a linker.
  • Tumor antigen targets are proteins that are produced by tumor cells that elicit an immune response. The selection of the antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-llRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, ⁇ -human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin Bl, 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, Globo
  • tumor antigens include the following: 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, pi 5; 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 translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • Differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, G
  • the CAR polypeptide can also comprise a transmembrane domain (such as, for example, an NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and/or a CD3 ⁇ transmembrane domain) and a co-stimulatory domain (such as, for example, a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination of a 2B4 domain, a CD28 co-stimulatory domain and/or a 4-1 BB co- stimulatory domain).
  • a transmembrane domain such as, for example, an NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and/or a CD3 ⁇ transmembrane domain
  • a co-stimulatory domain such as
  • the costimulatory domain comprises a CD28 co- stimulatory domain and/or a 4-1 BB co-stimulatory domain.
  • the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain.
  • the CAR polypeptide comprises a IgG4 hinge domain, a CD4 transmembrane domain, a CD28 co-stimulatory domain, a CD3zeta 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 (for example, CD33).
  • a target antigen for example, CD33
  • the CAR polypeptide comprises a IgG4 hinge domain, a NKG2D transmembrane domain, a 2B4 domain, a CD3zeta 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 (for example, CD33).
  • the CAR polypeptides are those shown in FIG. 6B.
  • the polynucleotide encoding the CAR polypeptide described herein comprises a sequence at least about 70% (for example, 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.
  • the polynucleotide encoding the scFV described herein comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 18 or a fragment thereof.
  • the polynucleotide encoding the scFv described herein comprises SEQ ID NO: 18, or a fragment thereof.
  • the polynucleotide encoding the IgG4-hinge described herein comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 19 or a fragment thereof.
  • the polynucleotide encoding the IgG4-hinge described herein comprises SEQ ID NO: 19, or a fragment thereof.
  • the polynucleotide encoding the CD28 co-stimulatory domain described herein comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 20 or a fragment thereof. In some embodiments, the polynucleotide encoding the CD28 co-stimulatory domain described herein comprises SEQ ID NO: 20, or a fragment thereof.
  • the polynucleotide encoding the CD3zeta (CD3z or CD3 ⁇ ) described herein comprises a sequence at least about 70% (for example, 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.
  • the polynucleotide encoding the CD3zeta described herein comprises SEQ ID NO: 21, SEQ ID NO: 28, or a fragment thereof.
  • the polynucleotide encoding the NKG2D transmembrane domain described herein comprises a sequence of at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 24 or a fragment thereof.
  • the polynucleotide encoding the NKG2D transmembrane domain comprises SEQ ID NO: 24, or a fragment thereof.
  • the polynucleotide encoding the 2B4 domain described herein comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 26 or a fragment thereof.
  • the polynucleotide encoding the 2B4 domain comprises SEQ ID NO: 26, or a fragment thereof.
  • the polynucleotide encoding the anti-CD33 scFV comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 29 or a fragment thereof.
  • the polynucleotide encoding the anti- CD33 scFv comprises SEQ ID NO: 29, or a fragment thereof.
  • the MND promoter described herein comprises a sequence of at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 30 or a fragment thereof.
  • the MND promoter comprises SEQ ID NO: 30.
  • the expression vector described herein comprises one or more linker sequences, wherein the linker sequence comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 25 or a fragment thereof.
  • the expression vector described herein comprises one or more linker sequences, wherein the linker sequence comprises SEQ ID NO: 25, or a fragment thereof.
  • the T cell disclosed herein comprises 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 CD3 ⁇ transmembrane domain), a costimulatory domain (e.g., a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination of a 2B4 domain, a CD28 co- stimulatory domain and/or a 4-1 BB co-stimulatory domain), CD3zeta, and a single-chain variable fragment (scFV) that specifically binds to a receptor on target cell (for example a cancer
  • a transmembrane domain
  • the CAR polypeptide specifically binds CD33.
  • plasmids, nucleic acids, and/or constructs that can be integrated into the genome of the transduced T cells via HITI, CRISPaint, or other nonhomologous end joining (NHEJ). As such, they have an advantage of integrating with higher efficiency.
  • the plasmids, nucleic acids, and/or constructs for NHEJ are those disclosed in International Publication Number WO2020/198675, which is incorporated herein by reference in its entirety.
  • the plasmids, nucleic acids, and/or constructs comprise one or more PAMg sequences (i.e., the protospacer adjacent motif (PAM) and the sequence encoding crRNA (i.e., the gRNA)) (SEQ ID NO: 9) to target the donor transgene integration.
  • PAMg sequences i.e., the protospacer adjacent motif (PAM) and the sequence encoding crRNA (i.e., the gRNA)
  • SEQ ID NO: 9 to target the donor transgene integration.
  • a single (PAMg) or a double (PAMgPAMg) Cas9-targeting sequences are incorporated around the transgene (e.g., a polynucleotide encoding the CAR, such as CD33 CAR, disclosed herein) but within the ITRs. Therefore, Cas9 can simultaneously cut gDNA and the CRISPaint DNA template, enabling integration at the genomic DSB.
  • the transgene e.g., a polynucleotide encoding the CAR, such as CD33 CAR, disclosed herein
  • T cell comprising a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems
  • CRISPR clustered regularly interspaced short palindromic repeat
  • Cas9 CRISPR-associated 9
  • the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is adjacent to one protospacer adjacent motif (PAM) and one polynucleotide sequence encoding crispr RNA (crRNA) or flanked by two PAMs and two polynucleotide sequences encoding crRNAs.
  • PAM protospacer adjacent motif
  • crRNA crispr RNA
  • T cell comprising a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises in order one protospacer adjacent motif (PAM) sequence and one polynucleotide sequence encoding crRNA, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and one PAM sequence and one polynucleotide sequence encoding crRNA.
  • CRISPR clustered regularly interspaced short palindromic repeat
  • Cas9 CRISPR-associated 9
  • SS plasmids, nucleic acids, and/or constructs may need more time to fold and serve as a double stranded DNA inside the cells prior to the integration which increases the DNA-sensing mechanism and cytotoxicity in some cells (such as, for example, T cells, B cells, macrophages, NK cells, fibroblasts, osteoblasts, hepatocytes, neuronal cells, epithelial cells, and/or muscle cells).
  • SC self-complementary
  • gRNA guide RNA
  • the disclosed plasmids, nucleic acids, and/or constructs use AAV integration, intron 1 of the protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, which is referred to the AAVS1, as the target site for the integration of the transgene.
  • This locus is a “safe harbor gene” and allows stable, long-term transgene expression in many cell types. As 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 being used as the target location, the CRSPR RNA (crRNA) must target said DNA.
  • the guide RNA disclosed herein comprises GGGGCCACTAGGGACAGGAT (SEQ ID NO: 17) or any 10 nucleotide sense or antisense contiguous fragment thereof.
  • the PAM+the sequence encoding crRNA comprises SEQ ID NO: 9.
  • AAVS1 is used for exemplary purposes here, it is understood and herein contemplated 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 being transfected or the transgene. Examples of other safe harbor genes, include but are not limited to C-C chemokine receptor type 5 (CCR5), the ROSA26 locus, and TRAC.
  • the plasmid, nucleic acid, or construct disclosed herein further comprise a murine leukemia virus-derived (MND) promoter.
  • MND murine leukemia virus-derived
  • the use of the AAV as a vector to deliver the disclosed CRISPR/Cas9 plasmid and any donor transgene is limited to a maximum of ⁇ 4.5kb. It is understood and herein contemplated that one method of increasing the allowable size of the transgene is to create additional room by exchanging the Cas (e.g., Cas9 of Streptococcus pyogenes (SpCas9) typically used for a synthetic Cas9, or Cas9) from a different bacterial source.
  • Cas e.g., Cas9 of Streptococcus pyogenes (SpCas9) typically used for a synthetic Cas9, or Cas9
  • the Cas can be derived from Staphylococcus aureus (SaCas9), Acidaminococcus sp.
  • Cas protein or “Cas” refers to a polypeptide encoded by a Cas (CRISPR- associated) gene.
  • a Cas protein includes proteins encoded by a gene in a cas locus and includes adaptation molecules as well as interference molecules.
  • An interference molecule of a bacterial adaptive immunity complex includes endonucleases.
  • a Cas endonuclease described herein comprises one or more nuclease domains.
  • RNA 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 (such as, for example, 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 the Adeno-associated virus (AAV); wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell.
  • AAV Adeno-associated virus
  • the method can further comprise introducing the RNP complex into the T cell via electroporation (such as when modifying a T cell).
  • the method can further comprise superinfecting the target cell (e.g., a T cell) with a second AAV virus comprising the RNP complex.
  • the target cell e.g., a T cell
  • the same AAV can comprise both the transgene and the RNP complex.
  • the transgene and RNP complex can be encoded on the same plasmid, nucleic acid, or construct.
  • RNA-binding protein (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 (such as, for example, a chimeric antigen receptor for a tumor antigen); wherein the transgene is adjacent to one PAM and crRNA or flanked by two PAMs and two sequences encoding crRNAs; 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 the AAV; wherein in the ribonucleoprotein (RNP) complex hybridizes to the target sequence within the genomic DNA of the T cell, and the T cell’s DNA repair enzymes insert the trans
  • the method can further comprise introducing the RNP complex into the T cell via electroporation (such as when modifying a T cell).
  • the method can further comprise superinfecting the target cell (e.g., T cell) with a second AAV virus comprising the RNP complex.
  • the target cell e.g., T cell
  • the same AAV can comprise both the transgene and the RNP complex.
  • the transgene and RNP complex can be encoded on the same plasmid, nucleic acid, or construct.
  • the AAV described herein can be used as a vector to deliver the disclosed a prime-editing plasmid and any donor transgene described herein (e.g., a polynucleotide encoding CAR).
  • Prime-editing is a “search-and-replace” genome editing technology that mediates targeted insertions, deletions base-to-base conversions, and combinations thereof in human cells without requiring DSBs or donor DNA templates.
  • Prime-editing can uses a fusion protein that comprises a catalytically impaired Cas9 endonuclease, an engineered reverse transcriptase enzyme, an RNA- programmable nickase, and/or a prime editing guide RNA (pegRNA), to copy genetic information directly from an extension on the pegRNA into the target genomic locus.
  • a prime editing guide RNA pegRNA
  • Methods for designing and using prime-editing are known in the art. See, e.g., Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149– 157 (2019), , incorporated by reference herein in its entity.
  • the T cells can be primary T cells from a donor source, such as, for example, an allogeneic donor source for an adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified cells), a T cell line, or from a source of expanded T cells derived a primary T cell source or T cell line.
  • a donor source such as, for example, an allogeneic donor source for an adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified cells), a T cell line, or from a source of expanded T cells derived a primary T cell source or T cell line.
  • the T cell Prior to the transduction of the T cells, the T cell can be incubated in a media suitable for the propagation of the T cells. It is understood and herein contemplated that the culturing conditions can comprise the addition of cytokines, antibodies, and/or feeder cells.
  • the media further comprises cytokines, antibodies, and/or feeder cells.
  • the media can comprise IL-2, IL-7, IL-12, IL-15, IL-18, and/or IL-21.
  • the media can also comprise anti-CD3 antibody.
  • the feeder cells can be purified from feeder cells that stimulate T cells.
  • T cell stimulating feeder cells for use in the claimed invention can be either irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or nonirradiated autologous or PBMCs; RPMI8866; HFWT, K562; K562 cells transfected with membrane bound IL-15, and 41BBL, or IL-21 or any combination thereof; or EBV-LCL.
  • the feeder cells are provided in combination with a solution of IL-21, IL-15, and/or 41BBL.
  • Feeder cells can be seeded in the culture of cells at a 1:2, 1:1, or 2:1 ratio.
  • the period of culturing can be between 1 and 14 days post AAV infection (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), preferably between 3 and 7 days, most preferably between 4 and 6 days.
  • the media can comprise IL-2, IL- 7, IL-12, IL-15, IL-18, and/or IL-21.
  • the incubation conditions for primary cells and expanded cells can be different.
  • the culturing of primary T cells prior to AAV infection comprises media and cytokines (such as, for example, IL-2, IL-7, IL-12, IL-15, IL-18, and/or IL-21) and/or anti-CD3 antibody for less than 5 days (for example 1, 2, 3, or 4 days).
  • cytokines such as, for example, IL-2, IL-7, IL-12, IL-15, IL-18, and/or IL-21
  • anti-CD3 antibody for less than 5 days (for example 1, 2, 3, or 4 days).
  • the culturing can occur in the presence of feeder cells (at for example, a 1:1 ratio) in addition to or in lieu of cytokines (such as, for example, IL-2, IL-7, IL-12, IL-15, IL-18, and/or IL-21) and/or anti- CD3 antibody.
  • Culturing of expanded T cells can occur for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to transduction.
  • a T cell comprising incubating primary T cells for 4 days in the presence of IL-2, IL-7, and/or IL-15 prior to infection with an AAV vector and/or electroporation (when the RNP complex is introduced via electroporation) or incubating expanded T cells in the presence of irradiated feeder cells for 4, 5, 6, or 7 days prior to infection with AAV and/or electroporation when the RNP complex is introduced via electroporation.
  • the now modified T cell can be propagated in a media comprising feeder cells that stimulate the modified T cells.
  • T cell stimulating feeder cells for use in the claimed invention, disclosed herein can be either irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or nonirradiated autologous or PBMCs; RPMI8866; HFWT, K562; K562 cells transfected with membrane bound IL-15, and 41BBL, or IL-21 or any combination thereof; or EBV-LCL.
  • PBMCs peripheral blood mononuclear cells
  • RPMI8866 HFWT, K562; K562 cells transfected with membrane bound IL-15, and 41BBL, or IL-21 or any combination thereof
  • EBV-LCL EBV-LCL
  • 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 in the culture of T cells at a 1:2, 1:1, or 2:1 ratio. It is understood and herein contemplated that the period of culturing can be between 1 and 14 days post infection and/or electroporation (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), preferably between 3 and 7 days, most preferably between 4 and 6 days.
  • the media 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.
  • modified T cells made by the disclosed methods.
  • modified T cells comprising any of the plasmids, nucleic acids, constructs, or vectors disclosed herein.
  • anti-CD33 CAR T cells wherein the anti-CD33 CAR-T comprises an scFv that targets CD33, a transmembrane domain (such as, for example, a NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and/or a CD3 ⁇ transmembrane domain) and a co-stimulatory domain (such as, for example, a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination of a 2B4 domain, a CD28 co-stimulatory domain and/or a 4-1 BB co-stimulatory domain).
  • a transmembrane domain such as, for example, a NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and
  • the costimulatory domain comprises a CD28 co-stimulatory domain and/or a 4-1 BB co-stimulatory domain.
  • the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain.
  • 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 (such as, for example, a chimeric antigen receptor for a tumor antigen); wherein the transgene is adjacent to one PAM and crRNA or flanked by two PAMs and crRNAs; 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 the Adeno- associated virus (AAV) into a target cell; wherein in the ribonucleoprotein (RNP) complex hybridizes to a target sequence within the genomic DNA of the T
  • RNP ribonucleoprotein
  • the method can further comprise introducing the RNP complex into the T cell via electroporation. In one aspect, the method can further comprise superinfecting the target cell (e.g., T cell) with a second AAV virus comprising the RNP complex. In one aspect, where the transgene is sufficiently small, the same AAV can comprise both the transgene and the RNP complex. In still further aspect, the transgene and RNP complex can be encoded on the same plasmid, nucleic acid, or construct.
  • a method of genetically modifying a 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 polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is flanked by homology arms; and wherein the homology arms are 800 bp in length or less; and b) introducing the polynucleotide sequence and the RNP complex into the T cell; wherein the polynucleotide sequence is introduced into the T cell via infection with the AAV into the T cell; wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell and the T
  • RNP ribonucleoprotein
  • the modified T cells used in the disclosed immunotherapy methods and created by the disclosed modification methods can be primary T cells from a donor source (such as, for example, an allogeneic donor source for an adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified T cells), a T cell line, or from a source of expanded cells derived a primary T cell source or T cell line. Because primary T cells can be used, it is understood and herein contemplated that the disclosed modifications of the T cell can occur ex vivo or in vitro.
  • the T cells used herein can be primary T cell or expanded T cells.
  • the primary T cells may be incubated for about 4 to 10 days in the presence of IL-2, IL-7, and/or IL-15 prior to infection of AAV vectors.
  • the 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.
  • the irradiated feeder cells, plasma membrane particles, or exosomes express membrane bound 4-1BBL, membrane-bound IL-21, or membrane-bound -15 or any combination thereof.
  • the modified T cells can be expanded and stimulated prior to administration of the modified T cells to the subject.
  • irradiated feeder cells plasma membrane (PM) particles, or exosomes (EX) expressing membrane bound IL-21 (mbIL-21)
  • PM21 particles and EX21 exosomes are referred to herein as PM21 particles and EX21 exosomes, respectively
  • expansion can further comprise irradiated feeder cells, plasma membrane (PM) particles, or exosomes expressing membrane bound IL-15 (mbIL-15) and/or membrane bound 4- 1BBL (mb4-1BBL).
  • the stimulation and expansion of the modified T cells can occur in vivo following or concurrent with the administration of the modified cells to the subject.
  • immunotherapy methods wherein the T cells are expanded in the subject following transfer of the T cells to the subject via the administration of IL-21 or PM particles with mbIL-21, exosomes with mbIL-21, and/or irradiated mbIL-21 expressing feeder cells.
  • the expansion further comprises the administration of IL-15 and/or 4-1BBL or PM particles, exosomes, and/or irradiated feeder cells that express membrane bound IL-15 and/or 4-1BBL.
  • the method disclosed herein comprises infecting the T cell with a range of MOI of AAV from about 1 to about 1000K MOI (e.g., about 5 to 500K MOI) of AAV.
  • the method disclosed herein comprises infecting the T cell with at least 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 MOI of AAV.
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • Sequence driven interaction means 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 are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art.
  • selective hybridization conditions can be defined as stringent hybridization conditions.
  • stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps.
  • the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12- 25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm.
  • Tm the melting temperature at which half of the molecules dissociate from their hybridization partners
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies.
  • Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations.
  • a preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C.
  • Stringency of hybridization and washing if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
  • selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid.
  • selective hybridization conditions would be 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 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid.
  • the non-limiting primer is in for example, 10 or 100 or 1000-fold excess.
  • This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10-fold or 100-fold or 1000-fold below their kd, or where only one of the nucleic acid molecules is 10-fold or 100-fold or 1000-fold or where one or both nucleic acid molecules are above their k d .
  • Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation.
  • selective hybridization conditions would be 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 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be 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 percent of the primer molecules are extended.
  • Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.
  • Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.
  • homology it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein.
  • nucleic acids There are a variety of molecules disclosed herein that are nucleic acid based. The disclosed nucleic acids are made up of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, the expressed mRNA will typically be made up of A, C, G, and U.
  • nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage.
  • the base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • a non-limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are many varieties of these types of molecules available in the art and available herein.
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties. There are many varieties of these types of molecules available in the art and available herein.
  • Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA).
  • Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. There are many varieties of these types of molecules available in the art and available herein. [0146] It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs.
  • Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are many varieties of these types of molecules available in the art and available herein. [0147] 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, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
  • a Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
  • the Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.
  • b) Sequences There are a variety of sequences related to the protein molecules involved in the signaling pathways disclosed herein, for example CD33, 4-1BB, NKG2D, or 2B4, all of which are encoded by nucleic acids or are nucleic acids.
  • the sequences for the human analogs of these genes, as well as other analogs, and alleles of these genes, and splice variants and other types of variants, are available in a variety of protein and gene databases, including Genbank.
  • Primers and/or probes can be designed for any given sequence given the information disclosed herein and known in the art.
  • Primers and probes can be designed for any given sequence given the information disclosed herein and known in the art.
  • Primers and probes [0150] Disclosed are compositions including primers and probes, which are capable of interacting with the disclosed nucleic acids, such as the CD33 as disclosed herein.
  • the primers are used to support DNA amplification reactions.
  • the primers will be capable of being extended in a sequence specific manner.
  • Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer.
  • Extension of the primer in a sequence specific manner therefore 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.
  • the primers are used for the DNA amplification reactions, such as PCR or direct sequencing.
  • the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner.
  • the disclosed primers hybridize with the disclosed nucleic acids or region of the nucleic acids or they hybridize with the complement of the nucleic acids or complement of a region of the nucleic acids.
  • the size of the primers or probes for interaction with the nucleic acids in certain embodiments can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification or the simple hybridization of the probe or primer.
  • a typical primer or probe would be 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, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700
  • a primer or probe can be less than or equal to 6, 7, 8, 9, 10, 11, 1213, 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, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,
  • the primers for the CD33 gene typically will be used to produce an amplified DNA product that contains a region of CD33 gene or the complete gene. In general, typically the size of the product will be such that the size can be accurately determined to within 3, or 2 or 1 nucleotides.
  • this product is 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
  • the product is less than or equal to 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
  • compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems.
  • the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
  • Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res.53:83-88, (1993)).
  • the plasmid descried herein can be a DNA template or a nucleotide construction that comprises the polynucleotide sequences provided herein.
  • plasmid, nucleic acid, or construct or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered.
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors.
  • Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector.
  • Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells.
  • Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells.
  • Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.
  • a preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens.
  • Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.
  • Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells.
  • viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome.
  • viruses When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material.
  • the necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.
  • Adeno-associated viral vectors Another type of viral vector is based on an 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 about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19 (such as, for example at AAV integration site 1 (AAVS1)). Vectors which contain this site-specific integration property are preferred.
  • AAVs 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) such as, for example AAV-Rh74, and/or synthetic AAV (such as, for example AAV-DJ, Anc80).
  • AAV serotypes can be selected based on cell or tissue tropism.
  • AAV vectors for use in the disclosed compositions and methods can be single stranded (SS) or self- complementary (SC).
  • the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene.
  • ITRs inverted terminal repeats
  • Heterologous refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.
  • the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector.
  • the AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression.
  • the disclosed vectors thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.
  • the inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard 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.
  • One method to overcome the loading capacity of an AAV vector is through the use of two vectors, wherein the transgene is split between the two plasmids and a 3’ splice donor and 5’ splice acceptor are used to join the two sections of transgene into a single full-length transgene.
  • the two transgenes can be made with substantial overlap and homologous recombination will join the two segments into a full-length transcript.
  • D. Expression systems [0166]
  • the nucleic acids that are delivered to cells typically contain expression controlling systems.
  • the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard 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.
  • a) Viral Promoters and Enhancers Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g., beta actin promoter.
  • viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g., beta actin promoter
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad.
  • Enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers f unction to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription.
  • Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus 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. [0169] The promoter and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone.
  • 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.
  • the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time.
  • a preferred promoter of this type is the CMV promoter (650 bases).
  • Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.
  • GFAP glial fibrillary acetic protein
  • Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites.
  • the transcription unit also contains a polyadenylation region.
  • a polyadenylation region One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA.
  • the identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs.
  • the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.
  • the viral vectors can include nucleic acid sequence encoding a marker product.
  • marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed.
  • Preferred marker genes are the E. Coli lacZ gene, which encodes ß-galactosidase, and green fluorescent protein.
  • the marker may be a selectable marker.
  • suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes.
  • the first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media.
  • Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the 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)).
  • Protein variants and derivatives are well understood to those of skill in the art and in can involve 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 as well as intrasequence insertions of single or multiple amino acid residues.
  • Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Immunogenic fusion protein derivatives such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro 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 from 2 to 6 residues are deleted at any one site within the protein molecule.
  • variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
  • Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
  • substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
  • substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 5 and 6 and are referred to as conservative substitutions.
  • substitutions that are less conservative than those in Table 6, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain.
  • the substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • an electropositive side chain e.g., lysyl, arginyl, or histidyl
  • an electronegative residue e.g., glutamyl or aspartyl
  • substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.
  • Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
  • Deletions of cysteine or other labile residues also may be desirable.
  • Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide.
  • 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 hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H.
  • variants and derivatives of the disclosed proteins herein are through defining the variants and derivatives in terms of homology/identity to specific known sequences. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • nucleic acid sequences related to a specific protein sequence i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences.
  • each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein is also known and herein disclosed and described.
  • a particularly preferred non-peptide linkage is --CH 2 NH--. 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.
  • Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. 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 in place of L-lysine) can be used to generate more stable peptides.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • parenterally e.g., intravenously
  • intramuscular injection by intraperitoneal injection
  • transdermally by intracorporeally, topically or the like
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions 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 and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. [0192] Parenteral administration of the composition, if used, is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine 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)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced.
  • receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration.
  • the plasmids, nucleic acids, constructs, vectors, and modified T cells disclosed herein can be used to treat, inhibit, reduce, decrease, ameliorate, and/or prevent any disease where uncontrolled cellular proliferation occurs such as cancers.
  • Cancer immunotherapy has been advanced in recent years; genetically-modified chimeric antigen receptor (CAR) T cells are an excellent example of engineered immune cells successfully deployed in cancer immunotherapy.
  • CAR genetically-modified chimeric antigen receptor
  • the disclosed plasmids, nucleic acids, constructs and methods can be used to generate CAR-T cells to target a cancer.
  • methods of treating, decreasing, reducing, inhibiting, ameliorating, and/or preventing a cancer and/or metastasis such as, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and/or myelodysplastic syndromes (MDS)
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • HCL hairy cell leukemia
  • MDS myelodysplastic syndromes
  • a cancer and/or metastasis such as, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and/or myelodysplastic syndromes (MDS)
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • HCL hairy cell leukemia
  • MDS myelodysplastic syndromes
  • the modified T cell comprises a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (such as, for example a CD33 targeting CAR),
  • “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 ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. [0197] By “reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth).
  • tumor growth means reducing the rate of growth of a tumor relative to a standard or a control.
  • “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • the plasmids, nucleic acids, constructs, vectors, and modified T cells disclosed herein can be used to treat, inhibit, reduce, decrease, ameliorate, and/or prevent cancer.
  • a representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: 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 head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer,
  • RNA-binding protein (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 (such as, for example, a chimeric antigen receptor for a tumor antigen); wherein the transgene is flanked by homology arms; and wherein the homology arms are less than 1000bp; and c) introducing the transgene and the RNP complex into the T cell; wherein the transgene is introduced into the T cell via infection with the Adeno-associated virus (AAV) into the T cell; wherein the RNP complex hybridizes to a target sequence within the
  • RNP ribonucleoprotein
  • the transgene can be comprised on the same plasmid, nucleic acid, or construct as the Cas9 endonuclease or encoded on a second plasmid, nucleic acid, or construct in the same or different AAV vector.
  • the target cell can be transduced with the RNP complex via electroporation before or concurrently with the infection of the cell with the transgene comprising AAV.
  • the modified T cells used in the disclosed immunotherapy methods can be primary cells from a donor source (such as, for example, an allogeneic donor source for an adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified cells), a T cell line, or from a source of expanded T cells derived a primary T cell source or cell line.
  • a donor source such as, for example, an allogeneic donor source for an adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified cells
  • a T cell line or from a source of expanded T cells derived a primary T cell source or cell line.
  • primary T cells can be used, it is understood and herein contemplated that the disclosed modifications of the T cell can occur ex vivo or in vitro.
  • a plasmid, nucleic acid, or construct comprising in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and a right homology arm; wherein the left and right homology arms are each 1000bp in length or less.
  • a use of a plasmid, nucleic acid, or construct, an AAV vector or a modified T cell as disclosed herein for the manufacture of a medicament are also disclosed herein.
  • a plasmid, nucleic acid, or construct, an AAV vector or a modified cell as disclosed herein for use in the treatment of cancer Also disclosed herein are a use of a plasmid, nucleic acid, or construct, an AAV vector, or a modified cell as disclosed herein for the manufacture of a medicament for the treatment of cancer.
  • a CAR T cell created by using a method of creating a chimeric antigen receptor (CAR) T cell as disclosed herein, for use in the treatment of cancer.
  • CAR T cell created by using a method of creating a chimeric antigen receptor (CAR) T cell as disclosed herein, for the manufacture of a medicament for the treatment of cancer.
  • CAR chimeric antigen receptor
  • Example 1 KromaTiD dGH SCREEN Whole Genome Analysis Report - Sample KromaTiD- CD33CARNK/8AM-dGH/8/1/23.
  • Assay Standard dGH SCREEN AS-0002.1 (50 cells)
  • Metaphase and Karyotype Qualification Sample were prepared and qualified for dGH analysis prior to running the assay.
  • dGH SCREEN is designed for samples with grossly normal karyotypes and has not been qualified for highly rearranged genome analysis.
  • the five-color whole genome assay (5CWG or dGH SCREEN) is a dGH paint combination assay for all 24 human chromosomes.
  • the assay is composed of unique sequence, high-density (HD) dGH chromosome paints in five color panels such that chromosomes painted in the same color can be differentiated by size, shape, and centromere position.
  • Results include per-chromosome attribution of inter- and intra- chromosomal structural events including inversion, translocations, aneuploidy (gain and loss), insertions, centromere abnormalities, and complex events across a sample.
  • Table 7 shows the summary of the integration sequencing.
  • This analysis showed: 1) Vector integrity by: a. Determining the presence of sequence variants and their allele frequency. b. Determining the presence of vector-vector breakpoints that represent concatemerization of multiple copies of the vector and/or structural rearrangements in a single vector sequence. 2) Identification of vector integration site(s) and breakpoint sequences between the vector and genome. 3) Assessment of the presence of the abundance of off-target integration sites. 4) Assessment of the presence of structural variants surrounding the vector integration site(s). [0223] The data generated shows that the vector integrated correctly at the targeted location in a subset of cells. Additionally, 3 sequence variants and 7 structural variants were detected in the vector.
  • TLA Targeted Locus Amplification
  • S sequencing
  • data mapping Viable frozen Human Primary NK cells were used and processed according to Cergentis’ TLA protocol. TLA was performed with two independent primer sets specific for the vector sequence and the genome (Table 8). The next generation sequencing (NGS) reads were aligned to the vector sequence and host genome. The human hg38 genome was used as a host reference genome sequence. Results CD38 KO /CD33CAR V4 NK [0225] Figure 8 depicts the NGS coverage across the vector sequence using primer sets 1 and 2. [0226] Coverage is observed across the complete vector sequence Vector: 1-6,346, indicating that the vector integrated into the backbone.
  • Detected sequence variants are presented in Table 9. Please note that all variants located at the homology arm are contemplated to be the genomic variants detected due to incomplete integration of this homology arm in a subset of the sample.
  • Vector concatemerization and structural variants The identified vector-vector breakpoint sites are shown in Table 10. In total, 7 structural variants were identified. Intact reads were also found at all positions of the breakpoints, indicating that (partial) vector sequences have concatemerized. Using TLA, it is not possible to determine the exact order of (partial) copies and to confirm the presence of at least one complete copy. Moreover, all breakpoint sequences are contemplated to represent concatemerization of the ectopic (non-integrated) copies.
  • Breakpoint sites 1 & 6, 2 & 7, and 4 & 10 represent the same breakpoint sequence but are not reported 2 times due to homology within the vector. The breakpoint sequence can therefore have originated from both indicated vector positions. Due to the heterogenous nature of the sample, it is expected that these fusions are present in a subset of the sample.
  • Example 3 Siglenkcabtagene unileucel (Sig-leucel) for the treatment of relapsed/refractory CD33 + acute myeloid leukemia Summary [0232] Sig-leucel is comprised of universal donor peripheral blood natural killer (NK) cells that have been genetically modified to express a chimeric antigen receptor (CAR) targeting CD33.
  • NK peripheral blood natural killer
  • CAR chimeric antigen receptor
  • the CD33 CAR NK cells are generated via a combination of methods that include feeder-cell expansion of NK cells, CRISPR/Cas9 gene editing, and adenovirus associated virus (AAV) gene delivery.
  • the electroporation of Cas9/RNP initiates disruption of the CD38 locus combined with CAR delivery by AAV with homology arms for site-directed gene insertion.
  • the sig-leucel second generation CAR protein consists of an extracellular portion that has a humanized anti-CD33 single chain antibody fragment (scFv) and an intracellular portion that contains cell signaling (CD3- ⁇ ) and co-stimulatory (4-1BB) domains. Based on current knowledge, this combination of gene editing has not been used in NK cells to generate a clinical product.
  • sig-leucel is for the treatment of patients with CD33 + relapsed/refractory acute myeloid leukemia (AML).
  • AML relapsed/refractory acute myeloid leukemia
  • Background Disease Background – Relapsed/Refractory AML [0233]
  • CR complete response rate
  • HSCT hematopoietic stem cell transplant
  • cytosine arabinoside cytarabine, Ara-C
  • fludarabine The importance of high-dose cytarabine as an integral agent in primary and salvage regimens for the treatment of AML has been well-established.
  • Fludarabine has been widely used to lymphodeplete patients prior to infusion of lymphocytes, and fludarabine-containing regimens usually combined with cytarabine with or without an anthracycline, have been used for reinduction of primary refractory or relapsed AML.
  • fludarabine potentiates in AML blasts an increase in intracellular retention of Ara-CTP, the active metabolite of cytarabine.
  • Best response in patients with primary refractory or first relapse included 54% CR and 14% CR with partial recovery of platelet count and 13.5% CR with incomplete blood count recovery.
  • the relapse studies described above report CR rates after first relapse. Patients in their second and subsequent relapse have increasingly poor response rates to traditional chemotherapy regimens with 25% CR rate after a 3rd treatment attempt and 17% after 4-6th treatment attempt.
  • the depth of response prior to HSCT is an important prognostic marker in AML.
  • the outcomes cited above refer to morphologic CR rates and strategies to improve MRD negative response rates prior to HSCT are critical to improving outcomes in this high-risk patient population.
  • NK cell therapy for AML Natural killer (NK) cells are cytotoxic lymphocytes that play a key role in recognizing malignant and virus infected cells and serve as a bridge between the innate and adaptive immune response. In hematologic malignancies, there is a qualitative and quantitative dysfunction of innate NK cells and defective NK cells at diagnosis portends a poor prognosis. NK cell phenotypes at diagnosis of AML can be stratified into highly functional and dysfunctional groups with distinct transcriptional modification in pathways involved in cytotoxicity, intracellular signaling and metabolism. Patients with a dysfunctional NK cell profile at diagnosis had a higher risk of relapse. In addition, patients with “hypomaturation” NK cell profiles have reduced overall and relapse free survival.
  • NK cell activation and cytotoxicity are tumor antigen independent and are instead regulated by a balance of activating and inhibitory NK receptor signaling.
  • Activating receptors recognize ligands on the surface of cancer or viral infected cells that signal danger, and inhibitory receptors are responsible for recognition of self.
  • NK receptor classes include natural cytotoxicity receptors (NCR), C-type lectin receptors, and killer cell immunoglobulin like receptors (KIRs).
  • NCR natural cytotoxicity receptors
  • C-type lectin receptors C-type lectin receptors
  • KIRs killer cell immunoglobulin like receptors
  • the inhibitory effect of stimulation of killer immunoglobulin receptors (KIR) by class I HLA may limit the clinical efficacy of autologous NK cells.
  • NK cell doses produced by this approach were typically limited to a single dose of ⁇ 10 7 /kg.
  • a genetically-modified K562 cell line was developed that, when used as an irradiated feeder cell, enables large numbers of clinical-grade NK cells to be generated from normal donors, patients, cord blood, and embryonic/ pluripotent stem cells. Peripheral blood NK cells were infused and expanded using this approach in Phase I/II trials for hematologic malignancies and pediatric solid tumors.
  • Updated results from the Phase II continued to yield 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.
  • the KIR genes can be categorized for the inhibitory (KIR A-haplotype) or the activating haplotypes (KIR B haplotype) based on their gene content. Individuals with KIR-B genotype have a higher number of activating NK cell receptors, which confers a higher alloreactivity and anti-tumor function. 2) NK cells are licensed (acquire enhanced killing ability) when they express inhibitory killer immunoglobulin receptors (KIR) for self-HLA class I molecules. This enables NK cells to recognize “self” and spare autologous cells from killing. Targets lacking self- HLA class I molecules are thus more likely to elicit recognition by licensed NK cells.
  • KIR inhibitory killer immunoglobulin receptors
  • the inhibitory KIR genes known to be relevant for NK alloreactivity are: (i) 2DL1 which binds to HLA-C group 2 alleles, (ii) 2DL2 and 2DL3 which bind to HLA-C group 1 alleles, (iii) and 3DL1 which binds to HLA-B Bw4 alleles.
  • 2DL1 which binds to HLA-C group 2 alleles
  • 2DL2 and 2DL3 which bind to HLA-C group 1 alleles
  • 3DL1 which binds to HLA-B Bw4 alleles.
  • activating KIRs recognize activating ligands that promote NK cell lysis. Inheritance of activating KIR is widely variable- 0 to 7 aKIR are possible in any one individual. Data from patients undergoing stem cell transplantation show that patients receiving allografts from donors with more activating KIRs have a better outcome than patients receiving allograft from donors with fewer activating KIR.
  • NKG2C expression is induced in patients with CMV infection and correlates with an adaptive NK cell phenotype and improved leukemia-free survival.
  • the optimal Universal Donor was defined as one who has an HLA genotype consisting of C1, C2, and Bw4 alleles, has a KIR genotype possessing the inhibitory KIR that bind to C1, C2, and [0253] Bw4 (leading to maximum licensing), has a high proportion of activating KIR, and has been exposed to CMV resulting in high NKG2C expression. Considering data available for Caucasian donors, C1/C2/Bw4 alleles occur in 32% of the population.
  • NK cell donor can be identified in approximately 1 out of 16 healthy individuals.
  • apheresis products were identified and collected as NK cell starting material from 10 different donors.
  • Six patients were enrolled in a phase 1 study investigating the safety of universal donor NK cells combined with chemotherapy for treatment of adult patients with primary refractory or relapsed AML or MDS (NCT04220684), in which universal donor NK cells were manufactured by the expansion technique utilizing irradiated CSTX002 feeder cells as described above.
  • NK cell dose 1 x 10 7 /kg/dose, with a total of 6 planned NK cell infusions over 2 weeks (day 0, 2, 4, 7, 9, and 11).
  • CD33 is a sialic acid-binding immunoglobulin-related lectin (siglec) that is present on the surface of hematopoietic cells.
  • siglec immunoglobulin-related lectin
  • CD33 is expressed on myeloid derived cells and leukemic blasts but importantly, is not present on pluripotent hematopoietic stem cells or non-hematopoietic cells.
  • CD33 is expressed in over 80% of patients with AML. Given the high expression of CD33 on AML blasts and leukemia progenitor cells, there has been a long precedence to try to exploit this target using immunotherapy approaches.
  • Gemtuzumab-ozogamicin is a humanized anti-CD33 monoclonal antibody conjugated to the DNA-binding cytotoxin calicheamicin that is FDA approved for use in pediatric and adult CD33 positive AML. After binding to CD33, GO is internalized and calicheamicin is released causing DNA double strand breaks and subsequent cell death. GO combined with chemotherapy is effective at reducing minimal residual disease in patients with relapsed and refractory AML. Commonly cited toxicities include myelosuppression related on-target off-tumor toxicity and sinusoidal obstruction syndrome (SOS) from calicheamicin hepatotoxicity.
  • SOS sinusoidal obstruction syndrome
  • Lintuzumab (HuM195) is a humanized anti-CD33 monoclonal antibody with a high binding avidity and cytotoxic activity against CD33-positive cells.
  • CAR NK Cells [0260] Historically, genetic modification of NK cells was unsuccessful due to NK cell resistance to viral transduction. In contrast to T cells, the innate function of NK cells as the first anti-viral defense renders them relatively resistant to traditional methods of gene modification through viral transduction. Alternative NK cell sources and newer methods of genetic engineering 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 were treated with a single dose of “off the shelf” cord blood derived CD19 CAR-NK cells. The CAR-NK cells were equipped with CD19 CAR, IL-15, and a caspase suicide gene.
  • CAR-NK cells were well tolerated with no dose limiting toxicity and no report of cytokine release syndrome or neurologic toxicity with a response rate of 73%.
  • CAR-NK cells expanded in vivo and were detectable for at least a year after infusion. Similar to data reported in CAR-T cell trials, patients who responded to therapy had a higher peak expansion of CAR-NK cells than those with no response.
  • CAR NK cell targets being developed for hematologic malignancies, including CD33, CD123, CD20, CD19/20, and BCMA.
  • Daratumumab is an FDA approved monoclonal antibody against CD38 that has changed the therapeutic landscape for multiple myeloma with overall response rates of greater than 80% when combined with chemotherapy. Pre-clinical and clinical reports have indicated that there may also be 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-to-NK ADCC in a process labeled fratricide.
  • CD38 negative or low NK cells are resistant to daratumumab- induced fratricide and have improved tumor cytotoxicity when combined with daratumumab compared to CD38+ NK cells.
  • CD38 knock out NK cells were generated using CRISPR/Cas9. These CD38 KO NK cells are resistant to daratumumab induced fratricide, have a superior 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. The present work has demonstrated sub- populations of AML blasts that are dim for CD33 but highly express CD38. [0262] The present disclosure provides site-directed insertion of a CD33 CAR into the CD38 locus. The purpose of this two-part gene editing is three-fold.
  • the present disclosure also provides the safety of CD38 KO CD33 CAR NK cells in patients with relapsed/refractory AML. As described above, CD33 is highly expressed on AML blasts and leukemia progenitor cells and there is a long history demonstrating the safety of this target for cancer immunotherapy.
  • NK cells have the added ability to recognize tumors through innate NK cell receptors, preventing relapse due to antigen escape. Additionally, allogenic HLA-mismatched NK cells have been given safely without causing GVHD, highlighting the ability to produce universal donor or “off-the-shelf” CAR-NK cells to circumvent cost and timing constraints seen with manufacturing CAR-T cell therapy.
  • the use of a standardized healthy-donor cell bank as the cell source also circumvents the issues seen with collecting and manufacturing autologous cell therapy products from heavily pre-treated patients with active leukemia.
  • NK cells are safe and cytokine release syndrome and neurologic toxicity have been minimal in NK cell trials to date.
  • NK cells are much more difficult to transduce efficiently with lentiviral vectors.
  • NK cells were engineered using CRISPR gene editing delivered as Cas9/Ribonucleoprotein (Cas9/RNP) via electroporation for the introduction of double strand break (DSB) in the CD38 locus, followed by AAV6 transduction for delivery of the CAR DNA.
  • the CAR is placed between ITRs of an AAV backbone, and is flanked by 600bp homology arms that target the CAR to the DSB in the CD38 locus.
  • CD38 KO CD33 CAR NK cells were successfully generated using the methodology described. In vitro data demonstrated successful depletion of CD38 and expression of CD33 CAR, which resulted in higher anti-AML activity than non-modified NK cells. Mechanism of Action and Efficacy Studies Generation of CD33 CAR NK cells [0266] CD33 CAR NK cells were generated by targeted insertion of the CD33 CAR construct into the AAVS1 locus on human chromosome 19.
  • AAVS1 is a well validated “safe harbor” for integrating DNA transgenes. This approach reliably produced CAR NK cells with a transduction efficiency of >60% in peripheral blood NK cells ( Figures 12A and 12B) [0267]
  • the same approach was used for delivery of the CAR DNA to the CD38 locus. Instead of targeting AAVS1, the homology arms were designed for CD38 locus on chromosome 4. This results in simultaneous knock-out of the CD38 and knock-in of the CD33 CAR into this locus (13).
  • Cas9/RNP targeting the CD38 locus was electroporated followed by AAV6 transduction of a CD33 CAR construct containing homology arms to the CD38 targeted region.
  • CD33 CAR-NK cells are highly effective against several AML cell lines and AML patient samples when assessed by NK cell degranulation ( Figures 14A and 14B) and three separate cytotoxicity assays: calcein 4h cytotoxicity ( Figures 14C, 14D, and 14E), real-time cell analysis (RTCA, xCELLigence) cytotoxicity assay ( Figure 14F), and CyTOF analysis ( Figure 5A). CyTOF analysis showed upregulation of CD38 in survivng AML post incubation with CD33 CAR NK cells ( Figure 24A).
  • CD38 knockout NK cells CD38 is a transmembrane glycoprotein that plays an important role in cellular metabolism. Targeting CD38 in human peripheral blood NK cells using Cas9/RNP not only improves the antibody dependent cytotoxicity of NK cells combined with daratumumab, but it also enhances the metabolic fitness of NK cells. Metabolic fitness plays a crucial role in NK cell function within the tumor microenvironment.
  • CD38 KO NK cells also had higher spare respiratory capacity and mitochondrial respiratory capacity compared with CD38 WT NK cells ( Figure 16D).
  • Anti-AML activity of CD38 KO CD33 CAR NK cells [0270] CD38 KO CD33 CAR NK cells generated from a healthy donor by the method described in Figure 33 were cocultured with AML cell line Kasumi-1 for 4 hours. To evaluate the anti-tumor efficacy, AML cells were labeled with calcein-AM and CD38 KO CD33 CAR NK (v4) cells were added at differing effector-to-target (E:T) ratios. Cytotoxicity of the labeled tumor cells was quantified by measuring calcein fluorescence released into the supernatant.
  • the data of the CD38 KO CD33 CAR NK (v4) cells shows enhanced anti-AML activity of these novel cells in Figure 8.
  • Genome Safety Studies Conducted [0271] As mentioned above, the present example demonstrates safe manufacturing of expanded NK cells derived from allogeneic, and specifically universal-donor sources. In particular, an approach for which there is less formal guidance and less prior experience on which to build- that of ex vivo targeted gene insertion using RNP electroporation and AAV vectors is utilized herein. This approach can increase the safety profile of a genetically- modified product.
  • Electroporation of pre-complexed Cas9/gRNA (RNP) enables tighter control of the genomic exposure to editing enzymes, and no possibility of sustained expression, compared to vector-based expression.
  • CD33 CAR NK cells Initially in CD33 CAR NK cells, the CD33 CAR was inserted into the AAVS1 locus, a human safe harbor locus. Using targeted locus amplification (TLA), low off-target CAR integration was demonstrated with this gene editing approach.
  • TLA targeted locus amplification
  • TLA targeted locus amplification
  • a similar TLA analysis was performed on CD38 KO CD33 CAR NK cells. Similar to the data for the safe-harbor AAVS1 site, only a single hotspot for integration was identified at the intended CD38 locus on Chromosome 4.
  • Knockout-exclusive single nucleotide polymorphisms and insertion-deletion mutations were detected when compared with wild-type (WT) NK cells. Because repair of the DNA breaks generated by Cas9/RNP will vary between cells and be close to the region of guide RNA homology, clustered events were not excluded as is typically done for somatic genomic analysis. Therefore, the Mutect2 filters were applied and those occurring at any frequency in the CD38 KO cells but not present in WT NK cells were included, and included only those that passed the applied filters or were clustered events, were nonsynonymous mutations, and were in coding regions.
  • RNA-seq only 4 of the off-target genes (CC2D1B, DENND4B, KMT2C, and WDR89) with possible high impact mutations are expressed at meaningful levels in NK cells ( Figure 17).
  • CD38 knockout CD33 CAR NK cells Given the benefit of targeting CD38 in NK cells and low off-target effect of using Cas9/RNP, NK cells with the CD33 CAR gene inserted into the CD38 locus were generated. The same approach was used for insertion of the CARs (Cas9/RNP + AAV6) by designing the homology arms for CD38 targeting site, resulting in simultaneous knock-out of the CD38 and knock-in of the CD33 CAR into this locus ( Figure 11). An AAV6 vector was utilized to generate CD38 KO CD33 CAR NK cells. Flow cytometry used to determine CAR transduction and CD38 knock out efficiency ( Figure 13). Pre-clinical studies [0285] In vitro studies.
  • CD38 KO CD33 CAR NK cells generated from at least three healthy donors are cocultured with AML cell lines and patient samples for 4 and 48 hours and then supernatants are collected to measure cytokine production by Luminex multiplex cytokine analysis.
  • AML cells are labeled with calcein- AM and CD38 KO CD33 CAR NK cells are added at differing effector-to-target (E:T) ratios. Cytotoxicity of the labeled tumor cells is quantified by measuring calcein fluorescence released into the supernatant.
  • the real time potency is measured using the xCELLigence RTCA MP instrument (ACEA Biosciences). Assays are performed to compare wild type NK cells with CD38 KO CD33 CAR NK cells, and also use CD33 CAR-NK cells inserted into the AAVS1 locus to assess the impact of CD38 deletion. [0286] In vivo studies. To further evaluate the efficacy of CD38 KO CD33 CAR NK, in vivo studies are performed using a xenograft mouse model. Briefly, NSG mice are inoculated with 1 x 10 6 luciferase- transduced MOLM-13, MV-4-11, or OCI-AML3 AML cells by tail vein injection.
  • Animals in the treatment group receive 10 7 CD38 KO CD33 CAR NK cells by tail vein injection on days 7 and 14 after tumor injection.
  • Low-dose IL-2 is given with NK cells while the mouse dose is analogous to 4 x 10 8 NK cells/kg in human studies, this is a setting lacking endogenous homeostatic cytokines.
  • Animals in the two control groups receive non-modified expanded NK cells, or do not receive any NK cells.
  • weekly bioluminesence imaging and peripheral blood samples for flow cytometry assessment of leukemia cells is performed. Survival curves are generated. All animal experiments are performed on a protocol approved by the Nationalwide Children’s Hospital Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • CD33 is a documented target with well-established safety in humans for antibody and CAR T cell therapeutics. Established models do not exist for testing the safety of targeting human CD33 in murine models, and there are no methods and materials for generating the equivalent murine anti- CD33 expanded CAR NK cells for immune-competent murine models. As described above, safety data in humans is established for expanded universal-donor NK cells. The pre-clinical and product release safety testing is focused on generating additional genomic safety data. [0288] Evaluation of on- and off-target integration frequencies.
  • dGH In-Site A targeted fluorescence-based genomic hybridization assay is being developed in collaboration with KromaTiD. This assay identifies site-specific insertions of the CAR into the CD38 locus, using a CAR-specific probe, and two CD38- locus-specific probes on the flanking ends of the target insertion site. G- banding results are reported from 200 metaphase spreads on each CD38 KO CD33 CAR NK cell product.
  • dGH Screen results do not support a release criteria, but supplement G-banding data and be for information only. (See Figures 2-10 and 26-32).
  • the AAV6 virus is intended for use only in providing the template for ex vivo insertion of the CAR gene. Given the low vector copy number used for transduction (10 4 – 10 5 MOI), followed by dilution in culture, two subsequent weeks of NK cell propagation (expected 2,000-fold expansion), and final wash steps, the final AAV infectious particles are not expected to be present in biologically-meaningful levels (typical gene therapy doses of 10 13 – 10 14 vector genomes/kg).
  • a digital droplet PCR assays was developed and validated for AAV2 ITR as a measure of viral genomes, which was used in release testing of the AAV6-pseudotyped vector.
  • viral genome copy number is reported to quantify residual virus in validations runs and clinical products.
  • the proposed drug product is comprised of NK cells derived from healthy donors selected for optimal universal-donor criteria, expanded in vitro with CSTX-002 feeder cells in media containing IL-2, and gene modified by electroporation of Cas9/RNP and transduction with AAV6, resulting in CD38 KO CD33 CAR NK cells.
  • CD33CAR V4 (LHCD8-41) CD38_ssAAV AAV Serotype: Serotype #AAV6
  • the CD33 CAR encoding DNA along with homology arms (HA) for the CD38 locus are cloned between ITRs of an AAV2 back-bone as: CD38 Right HA, BGHpA, MND Promotor, CD33 CAR, BGHpA and CD38 Left HA. See Figure 21.
  • Vector Production and Purification [0298] The AAV viral vector CD33CAR V4 (LHCD8-41) CD38_ssAAV is manufactured by Andelyn Biosciences at the Clinical Manufacturing Facility located at 575 Children’s Crossroad in Columbus, Ohio.
  • the viral vector wase produced at Andelyn Biosciences by co-transfection of human embryonic kidney (HEK) 293 cells with three DNA plasmids: Vector plasmid, AAV Helper plasmid, and Ad Helper plasmid.
  • the plasmids used to manufacture the viral vector were produced by Andelyn Biosciences and Aldevron. Plasmids are listed and production is described in Section [0300] The manufacturer aliquots the vector product into single-use vials which are preserved until use in cell manufacturing.
  • the vector is supplied in frozen form ( ⁇ -60oC) and transported to the Abigail Wexner Research Institute at National Children’s Hospital (AWRI- NCH) Cell-Based Therapy Core facility (CBT) where it will be stored at ⁇ -60oC until manufacturing use.
  • AWRI- NCH Abigail Wexner Research Institute at National Children’s Hospital
  • CBT Cell-Based Therapy Core facility
  • AAV2-ITR ddPCR assay was used to measure Physical Titer.
  • a research batch of the vector was produced by Andelyn Biosciences using the same final plasmids as the final clinical vector. This material was used for process development and for some initial pre-clinical studies.
  • a single batch of GMP-like vector product was manufactured under the toxicology process plan, but produced in the Andelyn GMP cleanrooms and with expanded clinical- grade testing.
  • Plasmid Production [0303] The AAV viral vector CD33CAR V4 (LHCD8-41) CD38_ssAAV was produced using the following plasmids: Vector plasmid CD33CAR V4 (LHCD8-41) CD38_ssAAV BackBone Kan AAV Helper Plasmid pNLRepcap6-Kan Ad Helper Plasmid pHelp Kan V4 [0304] Vector plasmid CD33CAR V4 (LHCD8-41) CD38_ssAAVBackBone Kan is a toxicology- grade plasmid produced by Andelyn Biosciences in their GMP facility.
  • the vector plasmid contains the Kanamycin resistance gene.
  • AAV Helper Plasmid pNLRepcap6-Kan is a Research HD grade plasmid produced by Aldevron. The helper plasmid contains the Kanamycin resistance gene.
  • Ad Helper Plasmid pHelp-KanV4 is a GMP-S grade plasmid produced by Aldevron and the Ad helper plasmid contains the Kanamycin resistance gene.
  • Sequencing in the form of NextGen PacBio sequencing was also performed for each plasmid to confirm identity and plasmid sequence purity.
  • the CoA of the Vector Plasmid includes PacBio sequencing results
  • the CoA of the AAV Helper Plasmid and the CoA of the Ad Helper Plasmid include only Sanger sequencing. Post-release PacBio sequencing results of these two plasmids are provided below.
  • the NextGen PacBio Sequencing covered the Ad Helper Plasmid (pHelp Kan V4) at an average read depth of 152,383x. Sequence analysis revealed four positions differing from the expected plasmid map, all of which were all insertions of a single nucleotide at frequencies ranging from10.38 – 16.27%.
  • the variants were present in the following regions, relative to sequencing from the PsiI restriction site used for linearization prior to sequencing: 47: Insertion of a T in a poly-T(9) region of the E4 gene 834: Insertion of an A in a poly-A(13) region of the E4 gene 2641: Insertion of a G in toward the end of the VA gene 5933: Insertion of an A in the ColE1 origin site [0309]
  • These variants may impact AAV replication efficiency during production of the vector, but should not impact production or structure of the AAV viral vector or its transgene.
  • Nine other variants in the sequence were present at 0.1 - 0.21%, and all other variants present at ⁇ 0.01%, which are within the statistical error distribution for this deep sequencing approach.
  • the NextGen PacBio Sequencing covered the AAV Helper Plasmid pNLRepcap6-Kan at an average read depth of 247,055x. Sequence analysis revealed one position differing from the expected plasmid map, which was insertion of a single nucleotide at a frequency of 11.79%. The variant was present in the following region, relative to sequencing from the AsiSI restriction site used for linearization prior to sequencing: 830: Insertion of an A within the pUC origin of replication site. [0311] This variant impacts plasmid replication but not production or structure of the AAV viral vector or its transgene.
  • CRISPR gene editing of NK cells are delivered as Cas9/Ribonucleoprotein (Cas9/RNP) via electroporation for the introduction of double strand breaks prior to AAV6 transduction.
  • the Cas9 protein and single guide RNA (sgRNA) are complexed together to form the ribonucleoprotein (RNP complex).
  • a sgRNA targets exon 1 of the CD38 gene.
  • 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 are used for preclinical testing.
  • GMP grade sgRNA and CTS TrueCut Cas9 Protein are used, which are complexed together prior to electroporation.
  • the GMP CD38-sgRNA is produced by Synthego.
  • the GMP CTS TrueCut Cas9 Protein is produced by Thermo Fisher Scientific.
  • the sgRNA is resuspended in PBS and then 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 may be used within 20 minutes of completion of incubation or may be stored on ice until use.
  • the Cas9/RNP complex is added to NK cells suspended in TheraPEAK P3 Primary Cell Nucleofector Solution set (Lonza), which has been produced according to applicable GMP standards and is intended to support GMP manufacturing. Electroporation is performed using the 4D- Nucleofector System (Lonza). [0316] All procedures for gene editing are performed in the Abigail Wexner Research Institute at Nationalwide Children’s Hospital (AWRI-NCH) Cell-Based Therapy Core facility (CBT), operating under applicable GMP guidelines.
  • AWRI-NCH Abigail Wexner Research Institute at National Children’s Hospital
  • CBT Cell-Based Therapy Core facility
  • Donors undergo infectious disease testing and screening as required for HCT/P donors at BTMB institutions compliant with 21 CFR Part 1271, the FDA Guidance document “Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps)” and any supplemental guidance documents issued. Donors are tested for KIR and HLA genotypes and infectious disease markers, NK cell proliferation and NKG2C content. [0318] Donors who meet all selection criteria have MNC(A) collected at a BTMB-qualified collection center. MNC(A) products are then shipped to the CBT Core facility, where they undergo additional processing.
  • CD38 KO CD33 CAR NK Cell Manufacturing All CD38 KO CD33 CAR NK cell products are manufactured in the Abigail Wexner Research Institute at Nationalwide 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 produce biologics under cGMP conditions.
  • the CBT Core facility comprises multiple dedicated laboratory suites used for Research and Development, Cell Manufacturing, Quality Control, and Temperature-Controlled Storage.
  • the CD38 KO CD33 CAR NK cell product are manufactured from CD-depleted MNC(A) and expanded by recursive stimulation with irradiated feeder cells expressing membrane-bound IL- 21 and 4-1BBL (clone CSTX002).
  • the NK cells are genetically modified following 1 week of expansion, rested for 2 days, and expanded in culture for another 14 days.
  • the product is cryopreserved at various cell concentrations, then released/dispensed for infusion at the prescribed dose into individual patients.
  • Cell Culture On Day 0 of NK expansion, the CD3-depleted MNC are thawed and washed and then propagated by recursive weekly stimulation with irradiated CSTX002 feeder cells (IFCs). Briefly, IFCs are added at an approximate 1:2 IFC-to-viable TNC ratio (also known as Stimulation 1 or Stim 1) in cell culture media containing 5% Immune Cell Serum Replacement and recombinant human IL- 2 at 100 IU/mL. Additional fresh IL-2 is added at 100 IU/mL every 1-3 days.
  • IFCs irradiated CSTX002 feeder cells
  • NK cells are aliquoted for storage in cryobags in liquid nitrogen vapor phase until they are thawed for the remaining manufacturing steps.
  • the NK cells are thawed and then genetically modified using CRISPR/Cas9 combined with AAV6.
  • the CD33 CAR is inserted into the CD38 locus via site-directed insertion utilizing CRISPR/Cas9.
  • CD38 is targeted using gRNA via electroporation of precomplexed Cas9/RNP into the expanded NK cells.
  • expanded NK cells are harvested, washed, and resuspended in 20ul of the TheraPEAK P3 Primary Cell 4D-Nucleofector Solution.
  • Cas9/RNP targeting CD38 5ul of pre- complexed Cas9/RNP targeting CD38 is added to the cell suspension.
  • the Cas9/RNP is then electroporated into NK cells utilizing a 4D Nucleofector System. After electroporation, the cells are rested for 30 minutes in media containing IL-2. The cells are then counted and AAV6 is added at a multiplicity of infection (MOI) of 75,000.
  • MOI multiplicity of infection
  • the cells rest for 2 days in culture and then are stimulated with irradiated feeder cells at a 1:1 IFC-to-viable TNC ratio (also known as Stimulation 2 or Stim 2) and cultured for another 7 days in the presence of 100 IU/mL of IL-2.
  • the cells are transferred to the Xuri Cell Expansion System for further expansion.
  • the cells are re-stimulated with irradiated feeder cells at a 1:1 IFC-to-viable TNC ratio (also known as Stimulation 3 or Stim 3) and cultured for another 7 days in the presence of IL-2.
  • Harvest and Cryoprotection [0326] On Day 23, cells are harvested and cryopreserved in aliquots.
  • CD38 KO CD33 CAR NK Cell products are cryopreserved in media containing DMSO, human serum albumin (HSA), and Plasma- Lyte A. The product is released/dispensed for infusion at the prescribed dose into individual patients.
  • HSA human serum albumin
  • CD38 KO CD33 CAR+ NK cell are administered in 4 dose levels as follows: a. Dose level 1: 1x10 7 CAR-NK cell/kg ( ⁇ 20%) Dose level 2: 3x10 7 CAR-NK cell/kg ( ⁇ 20%) Dose level 3: 1x10 8 CAR-NK cell/kg ( ⁇ 20%) b. Dose level 4: 2 doses of 1x10 8 CAR-NK cell/kg ( ⁇ 20%), one week apart Safety Testing using TLA, Churchill, dGH screens, custom dGH probes, and G-banding [0331] Targeted Locus Amplification.
  • Next-generation sequencing data is processed through Churchill, in which reads are aligned using BWA MEM (v0.7.15) to the GRCh37 reference genome. Variants are called using the Mutect2 tool of the Genome Analysis Toolkit (GATK v4.0.5.1, Broad Institute) and annotated using SnpEff (v4.3). Knockout-exclusive single nucleotide polymorphisms and insertion-deletion mutations (indels) are identified by comparing with expanded NK cells from the same donor that have not been electroporated or transduced. Because repair of the DNA breaks generated by Cas9/RNP vary between cells and be close to the region of guide RNA homology, clustered events are not excluded as is typically done for somatic genomic analysis.
  • the Mutect2 filters are applied and those occurring at any frequency in the CD38 KO CD33 CAR NK cells but not present in expanded NK cells from the same donor that have not been electroporated or transduced is included, and include only those that passed all the applied filters or clustered events, nonsynonymous mutations, and in coding regions.
  • dGH Screens a. Samples of final CD38 KO CD33 CAR NK cells are prepared with KromaTiD dGH cell prep kit as recommended to preserve metaphase spreads for hybridization. Samples are sent to KromaTiD for hybridization with fluorescent probes across the genome.
  • NK cell therapy in this context has yet to be realized.
  • One major obstacle for adoptive NK cell immunotherapy is obtaining sufficient cell numbers and having them readily available for infusions for patients with leukemia.
  • large numbers of NK cells are propagated ex vivo and have a bank of universal donor- derived NK cells was generated for “off-the-shelf” NK cell therapy.
  • Phase I trials in adult and pediatric patients with relapsed/refractory AML utilizing this universal donor NK cell bank are NCT04220684 and NCT05503134.
  • SEQ ID NO: 19 IgG4-Hinge 20.
  • SEQ ID NO: 24 NKG2D Transmembrane domain 25.
  • SEQ ID NO: 25 Linker 26.
  • SEQ ID NO: 26 – 2B4 tggcggagaaagcggaaggagaagcagagcgagacctcccctaaggagtttctgacaatctatgaggacgtgaaggatctgaagaccaggc 27.
  • SEQ ID NO: 27 – Linker 28 SEQ ID NO: 28 – CD3z 29.
  • SEQ ID NO: 48 PAMg RNA mCherry construct CCAATCCTGTCCCTAGTGGCCCCCACTAGGGACAGCGATCGGGTACATCGATCGCAGG 49.
  • SEQ ID NO: 49 CD33CAR V4 (LHCD8-41) CD38 ssAAV-Backbone 50.
  • SEQ ID NO: 50 CD33CAR V4 (LHCD8-41) CD38 ssAAV-Backbone

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Abstract

The present disclosure provides plasmids, nucleic acids, or constructs for use with a CRISPR/CAS9 system to genetically engineer T cells. In some aspects, disclosed herein are method of using such engineering T cells for treating cancers.

Description

CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS AND USES THEREOF RELATED APPLICATION This PCT application claims priority to, and the benefit of, U.S. Provisional Patent Application No.63/431,354, filed December 9th, 2022, entitled “CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS AND USES THEREOF,” which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on December 11th, 2023, as an .XML file entitled “10935- 020WO1_ST26.xml” created on December 7th, 2023, and having a file size of 115,746 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). FIELD The present disclosure relates to engineered T cells, compositions, and methods of use thereof. BACKGROUND [0001] T cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying T cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Generation of CAR-T cells using lentiviral transduction have some limitations, including random integration of the transgene which can have unintended consequences such as oncogene activation, gene silencing, or negative effects on the CAR-T antitumor efficacy. What are needed are new methods and vectors for engineering T cells. SUMMARY [0002] Disclosed are methods and compositions related to delivery of a CRISPR/CAS9 gene editing system to a T cell. [0003] In one aspect, disclosed herein are T cells comprising plasmids, nucleic acids, and/or constructs for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR- associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (such as, for example, a CAR comprising a 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 CD8 transmembrane domain, a CD28 transmembrane domain, and/or a CD3ξ transmembrane domain), a costimulatory domain (e.g., a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination of a 2B4 domain, a CD28 co- stimulatory domain, and/or a 4-1 BB co-stimulatory domain), and a CD3ξ signaling domain), and a right homology arm; wherein the left and right homology arms are each 1000bp in length or less (for example, 30 bp in length, 300 bp in length, 600 bp in length). In some embodiments, the costimulatory domain comprises a CD28 co-stimulatory domain and/or a 4-1 BB co-stimulatory domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain. [0004] Also disclosed herein are T cells comprising plasmids, nucleic acids, and/or constructs for use with CRISPR/ Cas9 integration systems of any preceding aspect, wherein the left homology arm and right homology arm are the same length or different lengths. In some aspects, the homology arms specifically hybridize to the Adeno-Associated Virus Integration Site 1 (AAVS1) of chromosome 19 of humans. [0005] In some embodiments, disclosed herein are T cells comprising plasmids, nucleic acids, and/or constructs for use with CRISPR/ Cas9 integration systems of any preceding aspect, wherein the plasmid, nucleic acid, or construct further comprises a murine leukemia virus-derived (MND) promoter. [0006] In some embodiments, the plasmid, nucleic acid, or construct of any preceding aspect is contained within and/or delivered into the T cell Adeno-associated viral (AAV) vectors (such as, for example, an AAV vector comprising the AAV6 serotype) comprising 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 can be a single stranded AAV (ssAAV) or a self-complimentary AAV (scAAV). [0007] Also disclosed herein are methods of treating, decreasing, reducing, inhibiting, ameliorating, and/or preventing a cancer and/or metastasis (such as, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and/or myelodysplastic syndromes (MDS)) in a subject comprising administering to a subject with a cancer the modified cell of any preceding aspect. [0008] In one aspect, disclosed herein are methods creating 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 (such as, for example, a chimeric antigen receptor for a tumor antigen); wherein the transgene is flanked by homology arms; and wherein the homology arms are 1000bp in length or less; and b) introducing the transgene and the RNP complex into a T cell; wherein the transgene (such as, for example, a chimeric antigen receptor for a tumor antigen) is introduced into the T cell via infection with the Adeno-associated virus (AAV); wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell and the DNA repair enzymes of the T cell insert the transgene into the host genome (for example, by homologous repair) at the target sequence, thereby creating a CAR T cell. In some aspects, the RNP complex can be introduced into the cell via electroporation. In some aspects, the RNP complex can be introduced into the cell via viral delivery in the same or a different AAV (i.e., superinfection). [0009] In one aspect, disclosed herein are methods of genetically modifying a T cell (including, but not limited to primary or expanded 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 chimeric antigen receptor (CAR) polynucleotide sequence; wherein the polynucleotide sequence is flanked by homology arms; and wherein the homology arms are 1000 bp in length or less; and b) introducing the polynucleotide sequence and the RNP complex into the T cell; wherein the polynucleotide sequence is introduced into the cell via infection with the AAV into the cell; wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell and the T cell’s DNA repair enzymes insert the transgene into the host genome at the target sequence within the genomic DNA of the T cell thereby creating a modified T cell. [0010] In some embodiments, disclosed herein are methods of genetically modifying a T cell of any preceding aspect, wherein the T cell is infected with about 5 to 500K multiplicity of infection (MOI) of the AAV disclosed herein. [0011] Also disclosed herein are methods of genetically modifying a T cell of any preceding aspect, wherein the primary cells are incubated for about 4 to 10 days in the presence of IL-2, IL-15, and or IL-7 and/or irradiated feeder, plasma membrane particles, or exosomes cells prior to or after infection and/or electroporation. In some embodiments, disclosed herein are methods of genetically modifying a T cell of any preceding aspect further comprising expanding the primary T cells for about 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, or membrane-bound IL-15, or any combination thereof. Also disclosed herein are methods of genetically modifying a T cell of any preceding aspect, further comprising expanding the modified T cell with irradiated feeder cells, plasma membrane particles, or exosomes following 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. [0012] In some aspects, disclosed herein is a method of treating, decreasing, reducing, inhibiting, ameliorating, and/or preventing a cancer and/or metastasis (such as, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and/or myelodysplastic syndromes (MDS)) in a subject comprising administering to the subject a therapeutically effective amount of a T cell, wherein the T cell comprises a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (such as, for example a CD33 targeting CAR), and a right homology arm; wherein the left and right homology arms are each 1000bp in length or less (for example, 600bp). [0013] In some aspects, disclosed herein is a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is adjacent to one protospacer adjacent motif (PAM) and one sequence encoding crispr RNA (crRNA) or flanked by two PAMs and sequences encoding crRNAs. It some aspects, the disclosed plasmid, nucleic acid, or construct can be used in any of the methods of treating, decreasing, reducing, inhibiting, ameliorating, and/or preventing a cancer and/or metastasis of any preceding aspect; methods of creating a CAR T cell of any preceding aspect; and/or genetically modifying a T cell of any preceding aspect. BRIEF DESCRIPTION OF FIGURES [0014] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [0015] Figure 1 shows CD33CAR-T cells generated by the disclosed technology. [0016] Figure 2 shows the distribution of events by chromosome from the dGH assay in Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23. [0017] Figure 3 shows the distribution of event rates by number of cells in Sample KromaTiD- CD33CARNK/8AM-dGH/8/1/23 from the dGH assay. [0018] Figure 4 shows the inversion and sister chromatid exchange (SCE) event summary in Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23 from the dGH assay. [0019] Figure 5 shows the size difference summary counted by chromosome in Sample KromaTiD- CD33CARNK/8AM-dGH/8/1/23 from the dGH assay. [0020] Figure 6 shows an example karyogram. There are SCE events present on Chromosome1p (Chr1p), Chromosome 5q (Chr5q), Chr7q, Chr8q, Chr9q, Chr10q x 2, Chr14q, ChrXp, and ChrXq. There is a size difference between homologs observed on Chr2. The small inversion on Chr8p is present Chr16q has a whole arm deletion. [0021] Figure 7 shows an example karyogram. There are SCE events present on Chr2p, Chr4q, Chr7q, and Chr12q. Size difference between homologs is observed for Chr2, Chr5, and Chr11. The recurrent small inversion on Chr8p is present. Chr2 has a broken chromatid. [0022] Figure 8 shows Next Generation Sequencing (NGS) coverage (grey) across the vector. Black arrows indicate the primer location. The vector map is shown on the bottom. Y-axes are limited to 100X. [0023] Figure 9 shows the TLA sequence coverage across the human genome using primer set 2. The chromosomes are indicated on the y-axis, the chromosomal position on the x-axis. Identified integration site is circled. [0024] Figure 10 shows the TLA sequence coverage (grey) across the vector integration locus, chr4:15,737,375-15,822,498. The top arrow of Set 1 indicates the location of the breakpoint sequences. The bottom arrow of Set 1 indicates the position of primer set 1. The bar of Set 2 indicates the location of the homology arms. Y-axes are limited to 2,500X. [0025] Figure 11 shows a schematic of NK cell genetic modification. [0026] Figures 12A and 12B show successful generation of CD33 CAR expressing NK cells using combination of Cas9/RNP and AAV6. Figure 12A shows representative flow cytometry showing the expression level of CD33 CAR on NK cells, 7 days after Cas9/RNP electroporation targeting AAVS1 and AAV6 transduction (MOI = 7.5 x 104). Figure 12B shows CD33 CAR expression level on NK cells remained stable at seven and fourteen days after transduction and electroporation (n=3). [0027] Figure 13 shows successful generation of CD38KO CD33 CAR expressing NK cells using combination of Cas9/RNP and AAV6. Representative flow cytometry showing the expression level of CD33 CAR on NK cells, 21 days after Cas9/RNP electroporation and AAV6 transduction (MOI = 7.5 x 104) (n=1). [0028] Figures 14A, 14B, 14C, 14D, 14E, and 14F show the CD33 CAR expressing NK cells demonstrate efficient cytotoxicity against CD33+ tumors. Figures 34A and 34D show that CD33 CAR NK cells degranulate significantly higher than wildtype NK cells when cocultured with Kasumi-1, ** adjusted P value= 0.004 and HL60, * adjusted P value= 0.01. Figures 34B and 34C show that expressing CD33 CAR on NK cells also enhances antitumor activity of NK cells against Kasumi-1 as shown in representative cytotoxicity assay performed in 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 against HL-60 only in CD33 CAR-Gen2 NK cells. [0029] Figure 15 shows the successful generation of CD38KO NK cells from ex vivo expanded PB- NK cells using Cas9/RNP. CD38 expression in NK cells before and after Cas9/RNP-mediated CD38 deletion (n 5); Representative fluorescence-activated cell sorter (FACS) analyses of the purified CD38KO NK cells. [0030] Figures 16A, 16B, 16C, and 16D show the favorable metabolic reprogramming of CD38KO NK cells. Figure 36A shows the heat map of DEGs of significantly altered pathways (cholesterol biosynthesis and OXPHOS) as determined by IPA, based on normalized RNA-seq data of paired CD38WT and CD38KO NK cells (n = 6). Figure 36B shows the principal components analysis (PCA) of DEGs, showing consistent effect of CD38 deletion for each donor despite wide interdonor variability. Figure 36C shows the summarized data of metabolic analysis of paired CD38WT and CD38KO NK cells (n = 3; mean ± SD). Figure 36D shows the graphical analysis of basal OCR, ECAR, OCR/ECAR, and spare respiratory capacity (SRC) derived from Figure 36C. All experiments were performed in quintuplicate. FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; ROT/AA, rotenone and antimycin A. [0031] Figure 17 shows the relative expression in wild-type (WT) expanded human NK cells of mutated genes identified in CD38KO NK cells. [0032] Figure 18 shows the relative expression in WT expanded human NK cells of mutated genes identified in CD38KO CD33 CAR NK cells. [0033] Figure 19 shows the OCI-AML-3 xenograft model showing survival in mice receiving CAR- NK cells compared to non-modified NK cells. [0034] Figure 20 shows the CD33 CAR construct. Sig-leucel utilizes a second generation anti-CD33 CAR. The extracellular binding domain is an anti- CD33 scFv based on heavy and light chain sequences derived from the humanized monoclonal antibody HuM195 (lintuzumab), connected by a Whitlow linker. The hinge, stalk, and transmembrane domain are derived from human CD8α. The intracellular signaling domain is comprised of a CD3ζ activation domain and a 4- 1BB costimulatory domain. [0035] Figure 21 shows the AAV6 vector sequence map. [0036] Figure 22 shows the schematic of clinical CD38KOCD33 CAR NK treatment. [0037] Figure 23 shows the flow diagram outlining the CD38KOCD33 CAR NK manufacturing and testing. [0038] Figures 24A and 24B show CD33 CAR expressing NK cells demonstrating improved effector function compared to wildtype (WT) expanded peripheral blood NK cells. Figure 6A shows CD38 expressing on AML cells. AML cell co-culture with WT-NK or CD33 CAR-NK cells induces AML cell death as shown by viability assessment or in SPADE plots (colored for pRb expression indicative of viable cycling cell), green arrows indicate live AML cells while red arrows indicate dead/dying AML cells. CD33 CAR-NK cells demonstrate increased AML cells killing, surviving AML cells have reduced CD33 surface expression and increased CD38 expression. Figure 6B shows bioplex results showing higher IFN-γ and TNF-α secretion from CD33 CAR NK cells co-cultured with AML cells. [0039] Figure 25 shows a schematic of targeted probes used in the directed Genomic Hybridization (dGH) assay. [0040] Figure 26 shows a cell with no transgene signal. The bottom panel has the telomeric signal layer removed. Note that the overlay of telomeric and centromeric bracketing probes can appear as a transgene probe as seen in the top panel. Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23. [0041] Figure 27 shows a cell with an inversion/SCE event in the target region of the centromeric probe (circled). Note that the telomeric and centromeric bracketing probe signals can appear as a transgene probe. Sample KromaTiD-WT-ctrl/8AM-dGH-8/1/23. [0042] Figure 28 shows a cell with an inversion/SCE event in the target region of the telomeric probe (circled). The right panel shows increased magnification and has the centromeric layer removed to better visualize the telomeric probe signal pattern. Note that the overlay of telomeric and centromeric bracketing probe signals can appear as a transgene probe as seen in the left panel. [0043] Figure 29 shows a cell with transgene inserts on both copies of CD38 (circled). The right panel shows the transgene insert signal layer. Note that the overlay of the telomeric and centromeric bracketing probe signals can appear as a transgene probe in a composite image. Sample KromaTiD- CD33CARNK/8AM-dGH/8/1/23. [0044] Figure 30 shows a cell with transgene inserts on both copies of CD38 (circled). The right panel has the telomeric probe signal layer removed. Note that the overlay of telomeric and centromeric bracketing probe signals can appear as a transgene probe signal in a composite image. Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23. [0045] Figure 31 shows a cell with one transgene insert on-target at CD38 (circled) and one off-target (circled). The bottom panel has the telomeric probe signal removed. Note that the overlay of telomeric and centromeric bracketing probe signals can appear as a transgene probe signal in a composite image. Sample KromaTiD-CD33CARNK/8AM-dGH/8/1/23. [0046] Figure 32 shows the design of CD33CAR-Gen2 and CD33CAR-Gen4v2. [0047] Figure 33 shows the anti-AML activity of CD33CAR-NK. [0048] Figure 34 shows that CD33CAR NK cells have enhanced anti-AML activity. [0049] Figure 35 shows a schematic indicating generation of fratricide resistant CD38KO/CD33- CAR NK cells. [0050] Figure 36 shows the fratricide resistant CD38KO/CD33-CAR NK cells to target residual AML. DETAILED DESCRIPTION [0051] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof. [0052] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Terminology [0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise. [0054] The following definitions are provided for the full understanding of terms used in this specification. [0055] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non- limiting embodiment, the terms are defined to be within 1%. [0056] 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, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. [0057] “Administration” to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. "Concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration, but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. Administration includes self-administration and the administration by another. [0058] "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject. [0059] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." [0060] “Complementary” or “substantially complementary” refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid. Complementary nucleotides are, generally, 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, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Alternatively, substantial complementarity exists when an RNA or DNA strand hybridizes under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary. See Kanehisa (1984) Nucl. Acids Res.12:203. [0061] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. [0062] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both 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. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. [0063] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, microRNA (miRNA), a "non-coding" RNA (ncRNA), a guide RNA, etc.). [0064] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises 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.) [0065] The “fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property, such as regulating the transcription of the target gene. [0066] The term "gene" or "gene sequence" refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a "gene" as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term "gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, control sequences upstream of the coding sequence (for example, the ribosome binding site). [0067] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (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 maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the 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 are identical to the nucleotides in 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 achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods. [0068] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. [0069] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul 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, which either match or satisfy some positive-valued threshold score T when aligned with a word 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, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
[0070] 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 by which 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 to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
[0071] The term "naturally-occurring" or "unmodified" or "wild type" as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is wild type (and naturally occurring).
[0072] An "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, composition in a statistically significant amount. Thus, the 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. [0073] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease 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% decrease so long as the decrease is statistically significant. [0074] The term "nucleic acid" as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides (DNA) or ribonucleotides (RNA). The terms "ribonucleic acid" and "RNA" as used herein mean a polymer composed of ribonucleotides. The terms "deoxyribonucleic acid" and "DNA" as used herein mean a polymer composed of deoxyribonucleotides. [0075] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. [0076] As used herein, "operatively linked" can indicate that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and/or transcription control elements (e.g. promoters, enhancers, and termination elements), and/or selectable markers in an expression vector. The term "operatively linked" can also refer to the arrangement of polypeptide segments within a single polypeptide chain, where the individual polypeptide segments can be, without limitation, a protein, fragments thereof, linking peptides, and/or signal peptides. The term operatively linked can refer to direct fusion of different individual polypeptides within the single polypeptides or fragments thereof where there are no intervening amino acids between the different segments as well as when the individual polypeptides are connected to one another via one or more intervening amino acids. [0077] “Primers” are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation. [0078] “Probes” are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically, a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. [0079] A "protein coding sequence" or a sequence that encodes a particular protein or polypeptide, is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. 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). A coding sequence can include, but is 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 will usually be located 3' to the coding sequence. [0080] The term "polynucleotide" refers to a single or double stranded polymer composed of nucleotide monomers. [0081] The term "polypeptide" refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds. [0082] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. [0083] As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. [0084] "Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the 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 components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration. [0085] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) 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 as described further herein. [0086] “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. [0087] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum 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. [0088] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both 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 (e.g., a cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. [0089] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g., a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of cancer. In some embodiments, a desired therapeutic result is the control of metastasis. In some embodiments, a desired therapeutic result is the reduction of tumor size. In some embodiments, a desired therapeutic result is the prevention and/or treatment of relapse. Therapeutically effective amounts 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 and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. [0090] As used herein, “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 herein disclosed polypeptides that are the subject of the present disclosure. The term “recombinant” refers to a sequence (e.g., polynucleotide or polypeptide sequence) which does not occur in the cell to be artificially provided with the sequence, or is linked to another polynucleotide in an arrangement which does not occur in the cell to be artificially provided with the sequence. It is understood that “artificial” refers to non-natural occurrence in the host cell and includes manipulation by man, machine, exogenous factors (e.g., enzymes, viruses, etc.), other non-natural manipulations, or combinations thereof. A transgene can comprise a gene operably linked to a promoter (e.g., an open reading frame), although is not limited thereto. Upon artificially providing a transgene to a cell, the transgene may integrate into the host cell chromosome, exist extrachromosomally, or exist in any combination thereof. [0091] 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 individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Plasmids, nucleic acids, and/or constructs for use and methods of genetically modifying cells [0092] Disclosed herein are modified T cells comprising plasmids, nucleic acids, and/or constructs for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems, wherein the plasmid, nucleic acid, or construct comprises in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (such as, for example, a CAR comprising a 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 CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3ξ transmembrane domain), a costimulatory domain (e.g., a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination of a 2B4 domain, a CD28 co-stimulatory domain, and/or a 4-1 BB co- stimulatory domain), and a CD3ξ signaling domain) and a right homology arm; wherein the left and right homology arms are each 1000bp in length or less (for example, about 30 bp in length, about 300 bp in length, or about 600 bp in length). In some embodiments, the costimulatory domain comprises a CD28 co-stimulatory domain and/or a 4-1 BB co-stimulatory domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain. [0093] In general, “CRISPR system” or “CRISPR integration system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated “Cas” genes. In some embodiments, one or more elements of a CRISPR system is 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 by reference herein in its entirety. [0094] Endonuclease/RNPs (for example, a Cas9/RNP) are comprised of three components, recombinant endonuclease protein (for example, a Cas9 endonuclease) complexed with a CRISPR loci. The endonuclease complexed to the CRISPR loci can be referred to as a CRISPR/Cas guide RNA. The CRISPR loci comprises a synthetic single-guide RNA (gRNA) comprised of a RNA that can hybridize to a target sequence complexed complementary repeat RNA (crRNA) and trans complementary repeat RNA (tracrRNA). Accordingly, the CRISPR/Cas guide RNA hybridizes to a target sequence within the genomic DNA of the 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 are capable of cleaving genomic targets with higher efficiency as compared to foreign DNA-dependent approaches due to their delivery as functional complexes. Additionally, rapid clearance of Cas9/RNPs from the cells can reduce the off-target effects such as induction of apoptosis. [0095] To make the RNP complex, crRNA and tracrRNA can be mixed at a 1:1, 2:1, or 1:2 ratio of concentrations between about 50µM and about 500 µM (for example, 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 min to form a crRNA:tracrRNA complex (i.e., the guide RNA). The crRNA:tracrRNA complex can then be mixed with between about 20 µM and about 50 µM (for example 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) final dilution of a Cas endonuclease (such as, for example, Cas9). [0096] Once bound to the target sequence in the target cell, the CRISPR loci can modify the genome by introducing into the target DNA insertion or deletion of one or more base pairs, by insertion of a heterologous DNA fragment (e.g., the donor polynucleotide), by deletion of an endogenous DNA fragment, by inversion or translocation of an endogenous DNA fragment, or a combination thereof. Thus, the disclosed methods can be used to generate knock-outs, or knock-ins when combined with DNA for homologous recombination. It is shown herein that transduction via Adeno-associated viral (AAV) of Cas9/RNPs is a relatively efficient method that overcomes previous constraints of genetic modification in cells (such as, for example, T cells, B cells, macrophages, NK cells, NK T cells, fibroblasts, osteoblasts, hepatocytes, neuronal cells, epithelial cells, and/or muscle cells). [0097] The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using Adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ~4.5 kilobases limits the donor size which includes homology arms. There are recommendations that any transcript above 100bp and any transgene is to have homology arms that are at least 800bp for each arm with many systems employing asymmetric arms of 800bp and 1000bp for a total of 1800bp. Thus, the AAV vector cannot deliver a transgene larger than ~2.5 kb. In one aspect, disclosed herein are AAV CRISPR/CAS9 nucleotide delivery systems comprising a donor construct plasmid with homology arms between 30bp and 1000bp, including, but not limited to 30bp, 50bp, 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, 200bp, 210bp, 220bp, 230bp, 240bp, 250bp, 260bp, 270bp, 280bp, 290bp, 300bp, 310bp, 320bp, 330bp, 340bp, 350bp, 360bp, 370bp, 380bp, 390bp, 400bp, 410bp, 420bp, 430bp, 440bp, 450bp, 460bp, 470bp, 480bp, 490bp, 500bp, 510bp, 520bp, 530bp, 540bp, 550bp, 560bp, 570bp, 580bp, 590bp, 600bp, 610bp, 620bp, 630bp, 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 symmetrical 30bp homology arms, symmetrical 300bp homology arms, symmetrical 500bp homology arms, symmetrical 600bp homology arms, symmetrical 800bp homology arms, symmetrical 1000bp homology arms, or asymmetrical 800bp homology arms comprising a 800bp left homology arm (LHA) and a 1000bp right homology arm (RHA) for homologous recombination (HR) or no homology arms at all for non-homologous end joining using homology-independent targeted integration (HITI) plasmids. In some examples, the plasmids with or without homology arms are those disclosed in International Publication Number WO2020/198675, which is incorporated herein by reference in its entirety. In some embodiments, the plasmids have clinically approved splice acceptor (SA) (SEQ ID NO: 10) and clinically approved polyadenylation terminator (PA) (such as, for example BGH polyA terminator SEQ ID NO: 11). It is understood and herein contemplated that homology arms can be symmetrical (same length on each side) or asymmetrical (different lengths on each side) to accommodate differing transgene lengths. That is, homology arm lengths can have any combination of left homology arm (LHA) length and right homology arm (RHA) length including but not limited to LHA 30bp (SEQ ID NO: 2) and RHA 30bp (SEQ ID NO: 1), LHA 30bp and RHA 100bp, LHA 30bp and RHA 300bp (SEQ ID NO: 3), LHA 30bp and RHA 500bp (SEQ ID NO: 5), LHA 30bp and RHA 800bp (SEQ ID NO: 7), LHA 30bp and RHA 1000bp, LHA 100bp and RHA 30bp, LHA 100bp and RHA 100bp, LHA 100bp and RHA 300bp, LHA 100bp and RHA 500bp, LHA 100bp and RHA 800bp, LHA 100bp and RHA 1000bp, LHA 300bp (SEQ ID NO: 4) and RHA 30bp, LHA 300bp and RHA 100bp, LHA 300bp and RHA 300bp, LHA 300bp and RHA 500bp, LHA 300bp and RHA 800bp, LHA 300bp and RHA 1000bp, LHA 500bp (SEQ ID NO: 6) and RHA 30bp, LHA 500bp and RHA 100bp, LHA 500bp and RHA 300bp, LHA 500bp and RHA 500bp, LHA 500bp and RHA 800bp, LHA 500bp and RHA 1000bp, LHA 800bp (SEQ ID NO: 8) and RHA 30bp, LHA 800bp and RHA 100bp, LHA 800bp and RHA 300bp, LHA 800bp and RHA 500bp, LHA 800bp and RHA 800bp, LHA 800bp and RHA 1000bp, LHA 1000bp and RHA 30bp, LHA 1000bp and RHA 100bp, LHA 1000bp and RHA 300bp, LHA 1000bp and RHA 500bp, LHA 1000bp and RHA 800bp, and LHA 1000bp and RHA 1000bp. [0098] There are several ways to provide the DNA template, including viral and non-viral methods. In non-viral approaches, the single-stranded or double-stranded DNA template is typically electroporated along with Cas9/RNP, however, it has a lower efficiency in comparison to viral transduction. For viral gene delivery, adeno-associated viruses (AAV), including AAV6, were used safely in clinical trials and are useful as vectors for sensitive primary immune cells, including T cells. [0099] Transcripts that are delivered via AAV vectors can be packaged as a linear single-stranded (ss) DNA with a length of approximately 4.7 kb (ssAAV) or as linear self-complementary (sc) DNA (scAAV). The benefit of the scAAV vector is that it contains a mutated inverted terminal repeat (ITR), which is required for replication and helps to bypass rate-limiting steps of second strand generation in comparison to ssDNA vectors. Due to the limitation in the packaging capacity of scAAV, 30bp, 300bp, 500bp, and 800-1000 bps of HAs for the right and left side of the Cas9-targeting site were designed to find the most optimal length of HAs and to provide possible lengths of HAs to be chosen based on the size of transgenes by researchers. Additionally, due to limitations in packaging capacity compared to ssAAV, scAAV may not be suitable for larger transgenes such as chimeric antigen receptor (CAR) targeting CD33. Therefore, based on the size of transgenes, both ssAAV and scAAV were designed and tested, which provides a wide range of options for gene insertion in primary T cells. [0100] It has been shown that the efficiency of recombination increases as the length of HAs increases. Therefore, for the ssAAV backbone, the longest possible length of the left and right homology arm (HA) was used for either mCherry (e.g., 800bp-1000bp of HAs) and CD33 CAR-T (e.g., 600bp of HAs). Since designing homology arms is a time-consuming procedure and requires multiple optimizations, the CRISPaint approach has also been investigated, a homology-independent method for gene insertion or tagging. In this method, the same Cas9 targeting site, including the sequence encoding crRNA and PAM sequence (herein also termed as PAMg, e.g., SEQ ID NO: 9), is provided in the DNA template encoding the gene of interest. Upon the introduction of the Cas9 complex, both template and genomic DNA are cut simultaneously. As a result, the CRISPaint template is presented as a linearized double-stranded DNA that can be integrated through non- homology repair machinery. Accordingly, in one aspect, disclosed herein are plasmids, nucleic acids, and/or constructs for delivering donor transgene to a cell and integrating said transgene (e.g., CAR) into the cell in combination with CRISPR/Cas9. Thus, disclosed herein are plasmids, nucleic acids, and/or constructs for use with CRISPR/ Cas9 integration systems of any preceding aspect, wherein the left homology arm and right homology arm are the same length or different lengths. [0101] In some aspects, the homology arms specifically hybridize to the Adeno-Associated Virus Integration Site 1 (AAVS1) of chromosome 19 of humans. In some embodiments, the LHA is 600 bp in length. In some embodiment, the LHA comprises a sequence at least about 70% (for example, 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 embodiment, the RHA comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 32 or a fragment thereof. [0102] The plasmid, nucleic acid, or construct disclosed herein comprises 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 serves to bring the cell expressing the receptor to a cancer cell expressing the target antigen. Typically, the CAR comprises a molecule that recognizes peptides derived from the tumor antigen presented by major histocompatibility (MHC) molecules, or an antibody or fragment thereof (such as for example, a Fab’, scFv, Fv) expressed on the surface of the CAR cell that targets a cancer antigen. The receptor is fused to a signaling domain (such as, for example, the CD3ζ domain, NKG2C, or NKp44 domain) via a linker. Tumor antigen targets are proteins that are produced by tumor cells that elicit an immune response. The selection of the antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-llRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin Bl, 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-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RUl, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70- 2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2/neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR- beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate-carcinoma tumor antigen- 1 (PCTA-1), ML-IAP, MAGE, MAGE-A1,MAD-CT-1, MAD-CT-2, MelanA/MART 1, XAGE1 , ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. Non-limiting examples of tumor antigens include the following: 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, pi 5; 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 translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the 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, CA 19-9, CA 72-4, CAM 17.1, NuMa, K- ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCASl, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET. [0103] The CAR polypeptide can also comprise a transmembrane domain (such as, for example, an NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and/or a CD3ξ transmembrane domain) and a co-stimulatory domain (such as, for example, a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination of a 2B4 domain, a CD28 co-stimulatory domain and/or a 4-1 BB co- stimulatory domain). In some embodiments, the costimulatory domain comprises a CD28 co- stimulatory domain and/or a 4-1 BB co-stimulatory 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 a IgG4 hinge domain, a CD4 transmembrane domain, a CD28 co-stimulatory domain, a CD3zeta 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 (for example, CD33). In some embodiments, the CAR polypeptide comprises a IgG4 hinge domain, a NKG2D transmembrane domain, a 2B4 domain, a CD3zeta 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 (for example, CD33). In some embodiments, the CAR polypeptides are those shown in FIG. 6B. In some embodiments, the polynucleotide encoding the CAR polypeptide described herein comprises a sequence at least about 70% (for example, 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. [0104] In some embodiments, the polynucleotide encoding the scFV described herein comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 18 or a fragment thereof. In some embodiments, the polynucleotide encoding the scFv described herein comprises SEQ ID NO: 18, or a fragment thereof. [0105] In some embodiments, the polynucleotide encoding the IgG4-hinge described herein comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 19 or a fragment thereof. In some embodiments, the polynucleotide encoding the IgG4-hinge described herein comprises SEQ ID NO: 19, or a fragment thereof. [0106] In some embodiments, the polynucleotide encoding the CD28 co-stimulatory domain described herein comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 20 or a fragment thereof. In some embodiments, the polynucleotide encoding the CD28 co-stimulatory domain described herein comprises SEQ ID NO: 20, or a fragment thereof. [0107] In some embodiments, the polynucleotide encoding the CD3zeta (CD3z or CD3ζ) described herein comprises a sequence at least about 70% (for example, 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, the polynucleotide encoding the CD3zeta described herein comprises SEQ ID NO: 21, SEQ ID NO: 28, or a fragment thereof. [0108] In some embodiments, the polynucleotide encoding the NKG2D transmembrane domain described herein comprises a sequence of at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 24 or a fragment thereof. In some embodiments, the polynucleotide encoding the NKG2D transmembrane domain comprises SEQ ID NO: 24, or a fragment thereof. [0109] In some embodiments, the polynucleotide encoding the 2B4 domain described herein comprises a sequence at least about 70% (for example, at least about 75%, 80%, 85%, 90%, 95%, 97%, or 99%) identical to SEQ ID NO: 26 or a fragment thereof. In some embodiments, the polynucleotide encoding the 2B4 domain comprises SEQ ID NO: 26, or a fragment thereof. [0110] In some embodiments, the polynucleotide encoding the anti-CD33 scFV comprises a sequence at least about 70% (for example, 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. [0111] In some embodiments, the MND promoter described herein comprises a sequence of at least about 70% (for example, 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. [0112] In some embodiments, the expression vector described herein comprises one or more linker sequences, wherein the linker sequence comprises a sequence at least about 70% (for example, 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 vector described herein comprises one or more linker sequences, wherein the linker sequence comprises SEQ ID NO: 25, or a fragment thereof. [0113] Accordingly, in some embodiments, the T cell disclosed herein comprises 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 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination of a 2B4 domain, a CD28 co- stimulatory domain and/or a 4-1 BB co-stimulatory domain), CD3zeta, and a single-chain variable fragment (scFV) that specifically binds to a receptor on target cell (for example a cancer cell expressing CD33). In some embodiments, the CAR polypeptide specifically binds CD33. [0114] Also disclosed herein are plasmids, nucleic acids, and/or constructs that can be integrated into the genome of the transduced T cells via HITI, CRISPaint, or other nonhomologous end joining (NHEJ). As such, they have an advantage of integrating with higher efficiency. In some examples, the plasmids, nucleic acids, and/or constructs for NHEJ are those disclosed in International Publication Number WO2020/198675, which is incorporated herein by reference in its entirety. To aid in the identification of cleavage site to remove the transgene for integration, the plasmids, nucleic acids, and/or constructs comprise one or more PAMg sequences (i.e., the protospacer adjacent motif (PAM) and the sequence encoding crRNA (i.e., the gRNA)) (SEQ ID NO: 9) to target the donor transgene integration. In some examples, for the NHEJ DNA templates (e.g., CRISPaint DNA templates), a single (PAMg) or a double (PAMgPAMg) Cas9-targeting sequences are incorporated around the transgene (e.g., a polynucleotide encoding the CAR, such as CD33 CAR, disclosed herein) but within the ITRs. Therefore, Cas9 can simultaneously cut gDNA and the CRISPaint DNA template, enabling integration at the genomic DSB. [0115] Accordingly, in some aspects, disclosed herein is T cell comprising a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is adjacent to one protospacer adjacent motif (PAM) and one polynucleotide sequence encoding crispr RNA (crRNA) or flanked by two PAMs and two polynucleotide sequences encoding crRNAs. In some aspects, disclosed herein is T cell comprising a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises in order one protospacer adjacent motif (PAM) sequence and one polynucleotide sequence encoding crRNA, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and one PAM sequence and one polynucleotide sequence encoding crRNA. [0116] Additionally, despite the benefit of using the single stranded (SS) plasmids, nucleic acids, and/or constructs to insert the larger transgenes, SS plasmids, nucleic acids, and/or constructs may need more time to fold and serve as a double stranded DNA inside the cells prior to the integration which increases the DNA-sensing mechanism and cytotoxicity in some cells (such as, for example, T cells, B cells, macrophages, NK cells, fibroblasts, osteoblasts, hepatocytes, neuronal cells, epithelial cells, and/or muscle cells). This problem is overcome herein by the use of self-complementary (SC)(double stranded) constructs in order to decrease the time of exposure to the exogenous DNA in cells. [0117] It is understood and herein contemplated that to target the Cas9 nuclease activity to the target site and also cleave the donor plasmid to allow for recombination of the donor transgene into the host DNA, a crispr RNA (crRNA) is used. In some cases, the crRNA is combined with a tracrRNA to form guide RNA (gRNA). The disclosed plasmids, nucleic acids, and/or constructs use AAV integration, intron 1 of the protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, which is referred to the AAVS1, as the target site for the integration of the transgene. This locus is a “safe harbor gene” and allows stable, long-term transgene expression in many cell types. As 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 being used as the target location, the CRSPR RNA (crRNA) must target said DNA. Herein, the guide RNA disclosed herein comprises GGGGCCACTAGGGACAGGAT (SEQ ID NO: 17) or any 10 nucleotide sense or antisense contiguous fragment thereof. Accordingly, in some examples, the PAM+the sequence encoding crRNA comprises SEQ ID NO: 9. While AAVS1 is used for exemplary purposes here, it is understood and herein contemplated 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 being transfected or the transgene. Examples of other safe harbor genes, include but are not limited to C-C chemokine receptor type 5 (CCR5), the ROSA26 locus, and TRAC. [0118] In one example, the plasmid, nucleic acid, or construct disclosed herein further comprise a murine leukemia virus-derived (MND) promoter. [0119] As noted above, the use of the AAV as a vector to deliver the disclosed CRISPR/Cas9 plasmid and any donor transgene is limited to a maximum of ~4.5kb. It is understood and herein contemplated that one method of increasing the allowable size of the transgene is to create additional room by exchanging the Cas (e.g., Cas9 of Streptococcus pyogenes (SpCas9) typically used for a synthetic Cas9, or Cas9) from a different bacterial source. Substitution of the Cas can also be used to increase the targeting specificity so less gRNA needs to be used. Thus, for example, the Cas can be derived from Staphylococcus aureus (SaCas9), Acidaminococcus sp. (AsCpf1), Lachnospiracase bacterium (LbCpf1), Neisseria meningitidis (NmCas9), Streptococcus thermophilus (StCas9), Campylobacter jejuni (CjCas9), enhanced SpCas9 (eSpCas9), SpCas9-HF1, Fokl-Fused dCas9, expanded Cas9 (xCas9), and/or catalytically dead Cas9 (dCas9). [0120] The term “Cas protein” or “Cas” refers to a polypeptide encoded by a Cas (CRISPR- associated) gene. A Cas protein includes proteins encoded by a gene in a cas locus and includes adaptation molecules as well as interference molecules. An interference molecule of a bacterial adaptive immunity complex includes endonucleases. A Cas endonuclease described herein comprises one or more nuclease domains. [0121] It is understood and herein contemplated that the use of a particular Cas9 can change the PAM sequence which the Cas9 endonuclease (or alternative) uses to screen for targets. As used herein, suitable PAM sequences comprises NGG (SpCas9 PAM) NNGRRT (SaCas9 PAM) NNNNGATT (NmCAs9 PAM), NNNNRYAC (CjCas9 PAM), NNAGAAW (St), TTTV (LbCpf1 PAM and AsCpf1 PAM); TYCV (LbCpf1 PAM variant and AsCpf1 PAM variant); where N can be any nucleotide; V = A, C, or G; Y = C or T; W = A or T; and R = A or G. [0122] In one aspect, disclosed here are methods of genetically modifying a T cell comprising 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 (such as, for example, 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 the Adeno-associated virus (AAV); wherein 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 (such as when modifying a T cell). In one aspect, the method can further comprise superinfecting the target cell (e.g., a T cell) with a second AAV virus comprising the RNP complex. In one aspect, where the transgene is sufficiently small, the same AAV can comprise both the transgene and the RNP complex. In still further aspects, the transgene and RNP complex can be encoded on the same plasmid, nucleic acid, or construct. [0123] In one aspect, disclosed herein are methods of genetically modifying a 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 (such as, for example, a chimeric antigen receptor for a tumor antigen); wherein the transgene is adjacent to one PAM and crRNA or flanked by two PAMs and two sequences encoding crRNAs; 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 the AAV; wherein in the ribonucleoprotein (RNP) complex hybridizes to the target sequence within the genomic DNA of the T cell, and the T cell’s DNA repair enzymes insert the transgene into the host genome at the target sequence (for example by non-homologous end joining), thereby creating a modified T cell. In one aspect, the method can further comprise introducing the RNP complex into the T cell via electroporation (such as when modifying a T cell). In one aspect, the method can further comprise superinfecting the target cell (e.g., T cell) with a second AAV virus comprising the RNP complex. In one aspect, where the transgene is sufficiently small, the same AAV can comprise both the transgene and the RNP complex. In still further aspect, the transgene and RNP complex can be encoded on the same plasmid, nucleic acid, or construct. [0124] In some examples, the AAV described herein can be used as a vector to deliver the disclosed a prime-editing plasmid and any donor transgene described herein (e.g., a polynucleotide encoding CAR). Prime-editing is a “search-and-replace” genome editing technology that mediates targeted insertions, deletions base-to-base conversions, and combinations thereof in human cells without requiring DSBs or donor DNA templates. Prime-editing can uses a fusion protein that comprises a catalytically impaired Cas9 endonuclease, an engineered reverse transcriptase enzyme, an RNA- programmable nickase, and/or a prime editing guide RNA (pegRNA), to copy genetic information directly from an extension on the pegRNA into the target genomic locus. Methods for designing and using prime-editing are known in the art. See, e.g., Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149– 157 (2019), , incorporated by reference herein in its entity. [0125] In one aspect, the T cells can be primary T cells from a donor source, such as, for example, an allogeneic donor source for an adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified cells), a T cell line, or from a source of expanded T cells derived a primary T cell source or T cell line. [0126] Prior to the transduction of the T cells, the T cell can be incubated in a media suitable for the propagation of the T cells. It is understood and herein contemplated that the culturing conditions can comprise the addition of cytokines, antibodies, and/or feeder cells. Thus, in one aspect, disclosed herein are methods of genetically modifying T cell, further comprising incubating the T cells for at least 1, 2, 3, 4, 5, 6, 7 ,89, 10, 11, 12, 13, or 14 days prior to transducing the T cells in media that supports the propagation of T cells; wherein the media further comprises cytokines, antibodies, and/or feeder cells. For example, the media can comprise IL-2, IL-7, IL-12, IL-15, IL-18, and/or IL-21. In one aspect, the media can also comprise anti-CD3 antibody. In one aspect, the feeder cells can be purified from feeder cells that stimulate T cells. For example, T cell stimulating feeder cells for use in the claimed invention, disclosed herein can be either irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or nonirradiated autologous or PBMCs; RPMI8866; HFWT, K562; K562 cells transfected with membrane bound IL-15, and 41BBL, or IL-21 or any combination thereof; or EBV-LCL. In some aspects, the feeder cells are provided in combination with a solution of IL-21, IL-15, and/or 41BBL. Feeder cells can be seeded in the culture of cells at a 1:2, 1:1, or 2:1 ratio. It is understood and herein contemplated that the period of culturing can be between 1 and 14 days post AAV infection (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), preferably between 3 and 7 days, most preferably between 4 and 6 days. For example, the media can comprise IL-2, IL- 7, IL-12, IL-15, IL-18, and/or IL-21. [0127] It is understood and herein contemplated that the incubation conditions for primary cells and expanded cells can be different. In one aspect, the culturing of primary T cells prior to AAV infection comprises media and cytokines (such as, for example, IL-2, IL-7, IL-12, IL-15, IL-18, and/or IL-21) and/or anti-CD3 antibody for less than 5 days (for example 1, 2, 3, or 4 days). For expanded T cells the culturing can occur in the presence of feeder cells (at for example, a 1:1 ratio) in addition to or in lieu of cytokines (such as, for example, IL-2, IL-7, IL-12, IL-15, IL-18, and/or IL-21) and/or anti- CD3 antibody. Culturing of expanded T cells can occur for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to transduction. Thus, in one aspect, disclosed herein are methods of genetically modifying a T cell comprising incubating primary T cells for 4 days in the presence of IL-2, IL-7, and/or IL-15 prior to infection with an AAV vector and/or electroporation (when the RNP complex is introduced via electroporation) or incubating expanded T cells in the presence of irradiated feeder cells for 4, 5, 6, or 7 days prior to infection with AAV and/or electroporation when the RNP complex is introduced via electroporation. [0128] Following transduction (e.g., via AAV infection or electroporation) of the T cell, the now modified T cell can be propagated in a media comprising feeder cells that stimulate the modified T cells. Thus, the modified T cells retain viability and proliferative potential, as they are able to be expanded post-AAV infection and/or electroporation (when the RNP complex is introduced via electroporation) using irradiated feeder cells. For example, T cell stimulating feeder cells for use in the claimed invention, disclosed herein can be either irradiated autologous or allogeneic peripheral blood mononuclear cells (PBMCs) or nonirradiated autologous or PBMCs; RPMI8866; HFWT, K562; K562 cells transfected with membrane bound IL-15, and 41BBL, or IL-21 or any combination thereof; or EBV-LCL. In some aspects, 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 in the culture of T cells at a 1:2, 1:1, or 2:1 ratio. It is understood and herein contemplated that the period of culturing can be between 1 and 14 days post infection and/or electroporation (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), preferably between 3 and 7 days, most preferably between 4 and 6 days. In some aspect, the media 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. [0129] In one aspect, it is understood and herein contemplated that one goal of the disclosed methods of genetically modifying a cell is to produce a modified cell. Accordingly, disclosed herein are modified T cells made by the disclosed methods. Thus, 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, wherein the anti-CD33 CAR-T comprises an scFv that targets CD33, a transmembrane domain (such as, for example, a NKG2D transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and/or a CD3ξ transmembrane domain) and a co-stimulatory domain (such as, for example, a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination of a 2B4 domain, a CD28 co-stimulatory domain and/or a 4-1 BB co-stimulatory domain). In some embodiments, the costimulatory domain comprises a CD28 co-stimulatory domain and/or a 4-1 BB co-stimulatory domain. In some embodiments, the transmembrane domain is a CD8 transmembrane domain, a CD28 transmembrane domain, or an NKG2D transmembrane domain. [0130] In one aspect, disclosed herein are methods of creating 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 (such as, for example, a chimeric antigen receptor for a tumor antigen); wherein the transgene is adjacent to one PAM and crRNA or flanked by two PAMs and crRNAs; 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 the Adeno- associated virus (AAV) into a target cell; wherein in the ribonucleoprotein (RNP) complex hybridizes to a target sequence within the genomic DNA of the T cell, and the T cell’s DNA repair enzymes insert the transgene into the host genome at the target sequence (for example by non-homologous end joining), thereby creating a modified T cell. In one aspect, the method can further comprise introducing the RNP complex into the T cell via electroporation. In one aspect, the method can further comprise superinfecting the target cell (e.g., T cell) with a second AAV virus comprising the RNP complex. In one aspect, where the transgene is sufficiently small, the same AAV can comprise both the transgene and the RNP complex. In still further aspect, the transgene and RNP complex can be encoded on the same plasmid, nucleic acid, or construct. [0131] In some aspect, disclosed herein is a method of genetically modifying a 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 polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is flanked by homology arms; and wherein the homology arms are 800 bp in length or less; and b) introducing the polynucleotide sequence and the RNP complex into the T cell; wherein the polynucleotide sequence is introduced into the T cell via infection with the AAV into the T cell; wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell and the T cell’s DNA repair enzymes insert the transgene into the host genome at the target sequence within the genomic DNA of the cell thereby creating a modified T cell. [0132] In one aspect, the modified T cells used in the disclosed immunotherapy methods and created by the disclosed modification methods can be primary T cells from a donor source (such as, for example, an allogeneic donor source for an adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified T cells), a T cell line, or from a source of expanded cells derived a primary T cell source or T cell line. Because primary T cells can be used, it is understood and herein contemplated that the disclosed modifications of the T cell can occur ex vivo or in vitro. [0133] The T cells used herein can be primary T cell or expanded T cells. The primary T cells may be incubated for about 4 to 10 days in the presence of IL-2, IL-7, and/or IL-15 prior to infection of AAV vectors. In one example, the 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 -15 or any combination thereof. [0134] Following transduction of the T cells, the modified T cells can be expanded and stimulated prior to administration of the modified T cells to the subject. For example, disclosed herein are methods of adoptively transferring T cells to a subject in need thereof wherein the T cell is expanded with irradiated feeder cells, plasma membrane (PM) particles, or exosomes (EX) expressing membrane bound IL-21 (mbIL-21) (PM particles and EX exosomes expressing mbIL-21 are referred to herein as PM21 particles and EX21 exosomes, respectively) prior to administration to the subject. In some aspects, expansion can further comprise irradiated feeder cells, plasma membrane (PM) particles, or exosomes expressing membrane bound IL-15 (mbIL-15) and/or membrane bound 4- 1BBL (mb4-1BBL). In some aspects, it is understood and herein contemplated that the stimulation and expansion of the modified T cells can occur in vivo following or concurrent with the administration of the modified cells to the subject. Accordingly disclosed herein are immunotherapy methods wherein the T cells are expanded in the subject following transfer of the T cells to the subject via the administration of IL-21 or PM particles with mbIL-21, exosomes with mbIL-21, and/or irradiated mbIL-21 expressing feeder cells. In some aspect, the expansion further comprises the administration of IL-15 and/or 4-1BBL or PM particles, exosomes, and/or irradiated feeder cells that express membrane bound IL-15 and/or 4-1BBL. [0135] In some embodiments, the method disclosed herein comprises infecting the T cell with a range of MOI of AAV from about 1 to about 1000K MOI (e.g., about 5 to 500K MOI) of AAV. For example, the method disclosed herein comprises infecting the T cell with at least 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 MOI of AAV. A. Hybridization/selective hybridization [0136] The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. Sequence driven interaction means 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 are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize. [0137] Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12- 25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art. [0138] Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be 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 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000-fold excess. This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10-fold or 100-fold or 1000-fold below their kd, or where only one of the nucleic acid molecules is 10-fold or 100-fold or 1000-fold or where one or both nucleic acid molecules are above their kd. [0139] Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be 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 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be 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 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation. [0140] Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein. [0141] It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein. B. Nucleic acids [0142] There are a variety of molecules disclosed herein that are nucleic acid based. The disclosed nucleic acids are made up of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantageous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment. a) Nucleotides and related molecules [0143] A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are many varieties of these types of molecules available in the art and available herein. [0144] A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties. There are many varieties of these types of molecules available in the art and available herein. [0145] Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. There are many varieties of these types of molecules available in the art and available herein. [0146] It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are many varieties of these types of molecules available in the art and available herein. [0147] 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, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute. [0148] A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides. b) Sequences [0149] There are a variety of sequences related to the protein molecules involved in the signaling pathways disclosed herein, for example CD33, 4-1BB, NKG2D, or 2B4, all of which are encoded by nucleic acids or are nucleic acids. The sequences for the human analogs of these genes, as well as other analogs, and alleles of these genes, and splice variants and other types of variants, are available in a variety of protein and gene databases, including Genbank. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating 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. c) Primers and probes [0150] Disclosed are compositions including primers and probes, which are capable of interacting with the disclosed nucleic acids, such as the CD33 as disclosed herein. In certain embodiments the primers are used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore 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 the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the disclosed nucleic acids or region of the nucleic acids or they hybridize with the complement of the nucleic acids or complement of a region of the nucleic acids. [0151] The size of the primers or probes for interaction with the nucleic acids in certain embodiments can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification or the simple hybridization of the probe or primer. A typical primer or probe would be 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, 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 long. [0152] In other embodiments a primer or probe can be less than or equal to 6, 7, 8, 9, 10, 11, 1213, 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, 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 long. [0153] The primers for the CD33 gene typically will be used to produce an amplified DNA product that contains a region of CD33 gene or the complete gene. In general, typically the size of the product will be such that the size can be accurately determined to within 3, or 2 or 1 nucleotides. [0154] In certain embodiments this product is 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 long. [0155] In other embodiments the product is less than or equal to 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 long. C. Delivery of the compositions to cells [0156] There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, 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 readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier. a) Nucleic acid based delivery systems [0157] Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res.53:83-88, (1993)). In some examples, the plasmid descried herein can be a DNA template or a nucleotide construction that comprises the polynucleotide sequences provided herein. [0158] As used herein, plasmid, nucleic acid, or construct or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10. [0159] Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans. (1) Adeno-associated viral vectors [0160] Another type of viral vector is based on an 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 about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19 (such as, for example at AAV integration site 1 (AAVS1)). Vectors which contain this site-specific integration property are preferred. AAVs 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) such as, for example AAV-Rh74, and/or synthetic AAV (such as, for example AAV-DJ, Anc80). AAV serotypes can be selected based on cell or tissue tropism. AAV vectors for use in the disclosed compositions and methods can be single stranded (SS) or self- complementary (SC). [0161] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus. [0162] Typically, the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. [0163] The disclosed vectors thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity. [0164] The inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard 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. [0165] It is understood and herein contemplated that the packaging capacity of an AAV is limited. One method to overcome the loading capacity of an AAV vector is through the use of two vectors, wherein the transgene is split between the two plasmids and a 3’ splice donor and 5’ splice acceptor are used to join the two sections of transgene into a single full-length transgene. Alternatively, the two transgenes can be made with substantial overlap and homologous recombination will join the two segments into a full-length transcript. D. Expression systems [0166] The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard 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. a) Viral Promoters and Enhancers [0167] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g., beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers 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. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein. [0168] Enhancer generally refers to a sequence of DNA that functions 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 Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers f unction to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus 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. [0169] The promoter and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs. [0170] 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 be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR. [0171] 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. [0172] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct. b) Markers [0173] The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. Coli lacZ gene, which encodes ß-galactosidase, and green fluorescent protein. [0174] In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media. [0175] The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the 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)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin. E. Peptides a) Protein variants [0176] Protein variants and derivatives are well understood to those of skill in the art and in can involve 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 as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro 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 from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 5 and 6 and are referred to as conservative substitutions. [0177] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 6, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation. [0178] 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 for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein. [0179] Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues. [0180] Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. 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 hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl. [0181] It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology/identity to specific known sequences. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level. [0182] Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol.48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection. [0183] The same types of homology can be obtained for nucleic acids by for example 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. [0184] It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations. [0185] As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein is also known and herein disclosed and described. [0186] It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 5 and Table 6. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way. [0187] Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH2NH-- , --CH2S--, --CH2-CH2 --, --CH=CH-- (cis and trans), --COCH2 --, --CH(OH) CH2--, and --CHH2SO— (These and others can be found 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 Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (-- CH2NH--, CH2CH2--); Spatola et al. Life Sci 38:1243-1249 (1986) (--CH H2--S); Hann J. Chem. Soc Perkin Trans. I 307-314 (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)CH2--); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH2--); and Hruby Life Sci 31:189-199 (1982) (--CH2--S--); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is --CH2NH--. 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. [0188] Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. [0189] D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. 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 in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. F. Pharmaceutical carriers/Delivery of pharmaceutical products [0190] As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. [0191] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions 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 and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. [0192] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. [0193] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type 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, K.D., 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., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine 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)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). G. Method of treating cancer [0194] The plasmids, nucleic acids, constructs, vectors, and modified T cells disclosed herein can be used to treat, inhibit, reduce, decrease, ameliorate, and/or prevent any disease where uncontrolled cellular proliferation occurs such as cancers. Cancer immunotherapy has been advanced in recent years; genetically-modified chimeric antigen receptor (CAR) T cells are an excellent example of engineered immune cells successfully deployed in cancer immunotherapy. It is understood and herein contemplated that the disclosed plasmids, nucleic acids, constructs and methods can be used to generate CAR-T cells to target a cancer. [0195] Thus, disclosed herein are methods of treating, decreasing, reducing, inhibiting, ameliorating, and/or preventing a cancer and/or metastasis (such as, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and/or myelodysplastic syndromes (MDS)) in a subject comprising administering to a subject with a cancer a modified T cell disclosed herein. For example, disclosed herein are methods of treating, decreasing, reducing, inhibiting, ameliorating, and/or preventing a cancer and/or metastasis (such as, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and/or myelodysplastic syndromes (MDS)) in a subject comprising administering to the subject a therapeutically effective amount of a modified T cell, wherein the modified T cell comprises a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems wherein the plasmid, nucleic acid, or construct comprises in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide (such as, for example a CD33 targeting CAR), and a right homology arm; wherein the left and right homology arms are each 1000bp in length or less (for example, 600bp). [0196] "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 ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. [0197] By “reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. [0198] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. [0199] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. [0200] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. [0201] As noted above, the plasmids, nucleic acids, constructs, vectors, and modified T cells disclosed herein can be used to treat, inhibit, reduce, decrease, ameliorate, and/or prevent cancer. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: 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 head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostatic cancer, or pancreatic cancer. [0202] In one aspect, disclosed herein are methods of adoptively transferring a modified T cells to a subject in need thereof said method comprising a) obtaining a T cell to be modified; 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 (such as, for example, a chimeric antigen receptor for a tumor antigen); wherein the transgene is flanked by homology arms; and wherein the homology arms are less than 1000bp; and c) introducing the transgene and the RNP complex into the T cell; wherein the transgene is introduced into the T cell via infection with the Adeno-associated virus (AAV) into the T cell; wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell and the T cell’s DNA repair enzymes insert the transgene into the host genome (for example, by homologous repair) at the target sequence within the genomic DNA of the target cell thereby creating a modified T cell; and d) transferring the modified T cell into the subject. In one aspect the transgene can be comprised on the same plasmid, nucleic acid, or construct as the Cas9 endonuclease or encoded on a second plasmid, nucleic acid, or construct in the same or different AAV vector. In one aspect, the target cell can be transduced with the RNP complex via electroporation before or concurrently with the infection of the cell with the transgene comprising AAV. [0203] In one aspect, the modified T cells used in the disclosed immunotherapy methods can be primary cells from a donor source (such as, for example, an allogeneic donor source for an adoptive transfer therapy or an autologous donor source (i.e., the ultimate recipient of the modified cells), a T cell line, or from a source of expanded T cells derived a primary T cell source or cell line. Because primary T cells can be used, it is understood and herein contemplated that the disclosed modifications of the T cell can occur ex vivo or in vitro. [0204] Also disclosed herein is a plasmid, nucleic acid, or construct comprising in order a left homology arm, a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide, and a right homology arm; wherein the left and right homology arms are each 1000bp in length or less. [0205] In another aspect, disclosed herein are a plasmid, nucleic acid, or construct, an AAV vector or a modified T cell as disclosed herein for use as a medicament. Also disclosed herein are a use of a plasmid, nucleic acid, or construct, an AAV vector or a modified T cell as disclosed herein for the manufacture of a medicament. [0206] Also disclosed herein are a plasmid, nucleic acid, or construct, an AAV vector or a modified cell as disclosed herein for use in the treatment of cancer. Also disclosed herein are a use of a plasmid, nucleic acid, or construct, an AAV vector, or a modified cell as disclosed herein for the manufacture of a medicament for the treatment of cancer. [0207] Also disclosed herein are a CAR T cell, created by using a method of creating a chimeric antigen receptor (CAR) T cell as disclosed herein, for use in the treatment of cancer. Also disclosed herein are a use of a CAR T cell, created by using a method of creating a chimeric antigen receptor (CAR) T cell as disclosed herein, for the manufacture of a medicament for the treatment of cancer. [0208] A number of embodiments of the disclosure have been described. Nevertheless, 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. [0209] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. EXAMPLES [0210] The following examples are set forth below to illustrate the compositions, devices, methods, and results according to 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 which are apparent to one skilled in the art. Example 1: KromaTiD dGH SCREEN Whole Genome Analysis Report - Sample KromaTiD- CD33CARNK/8AM-dGH/8/1/23. [0211] Assay: Standard dGH SCREEN AS-0002.1 (50 cells) [0212] Metaphase and Karyotype Qualification: Sample were prepared and qualified for dGH analysis prior to running the assay. [0213] dGH SCREEN is designed for samples with grossly normal karyotypes and has not been qualified for highly rearranged genome analysis. [0214] Spread resolution of 350+ (G-band equivalent) is selected for analysis. [0215] Assay Description: The five-color whole genome assay (5CWG or dGH SCREEN) is a dGH paint combination assay for all 24 human chromosomes. [0216] The assay is composed of unique sequence, high-density (HD) dGH chromosome paints in five color panels such that chromosomes painted in the same color can be differentiated by size, shape, and centromere position. [0217] Results include per-chromosome attribution of inter- and intra- chromosomal structural events including inversion, translocations, aneuploidy (gain and loss), insertions, centromere abnormalities, and complex events across a sample. [0218] Prior to analysis, images of dGH SCREEN painted metaphase spreads are qualified, processed, and sorted into karyograms for rapid, consistent reading of the assay. [0219] Per-cell event assessment is performed to leverage population-level analysis of events ranging from random to clonal. [0220] See Table 2 – Table 6 and Figure 2 – Figure 7. Example 2: Transgene analysis and integration site sequencing of 1 sample of human cells containing vector 33_CD33CAR V4 (LHCD8-41) CD38_ssAAV-BackBone Kan. [0221] One sample of transgenic human cells with 33_CD33CAR V4 (LHCD8-41) CD38_ssAAV- BackBone Kan vector sequence was analyzed. Table 7 shows the summary of the integration sequencing. [0222] This analysis showed: 1) Vector integrity by: a. Determining the presence of sequence variants and their allele frequency. b. Determining the presence of vector-vector breakpoints that represent concatemerization of multiple copies of the vector and/or structural rearrangements in a single vector sequence. 2) Identification of vector integration site(s) and breakpoint sequences between the vector and genome. 3) Assessment of the presence of the abundance of off-target integration sites. 4) Assessment of the presence of structural variants surrounding the vector integration site(s). [0223] The data generated shows that the vector integrated correctly at the targeted location in a subset of cells. Additionally, 3 sequence variants and 7 structural variants were detected in the vector. Finally, 7 random integration events were detected at the sgRNA cut site. Targeted Locus Amplification (TLA), sequencing, and data mapping [0224] Viable frozen Human Primary NK cells were used and processed according to Cergentis’ TLA protocol. TLA was performed with two independent primer sets specific for the vector sequence and the genome (Table 8). The next generation sequencing (NGS) reads were aligned to the vector sequence and host genome. The human hg38 genome was used as a host reference genome sequence. Results CD38KO/CD33CAR V4 NK [0225] Figure 8 depicts the NGS coverage across the vector sequence using primer sets 1 and 2. [0226] Coverage is observed across the complete vector sequence Vector: 1-6,346, indicating that the vector integrated into the backbone. Lower coverage on the backbone shows that it has integrated in a subset of the cells. Also, coverage is observed at the ITRs, indicating that next to the integration through the homology arms ITR based integrations also occurred in the sample. The coverage on the ITR regions and backbone also indicated that episomal (non-integrated) copies of the vector are present in this sample. [0227] The borders of the homology arms at positions 145 and 3,666 allow quantification of the alleles containing the correctly integrated TG in the data of primer set 2. At position 145, 34% of the reads show a successful targeting event, while at position 3,666 this is 38%. [0228] Sequence variants and structural variants were called in the covered regions. [0229] Sequence variants. Detected sequence variants are presented in Table 9. Please note that all variants located at the homology arm are contemplated to be the genomic variants detected due to incomplete integration of this homology arm in a subset of the sample. [0230] Vector concatemerization and structural variants. The identified vector-vector breakpoint sites are shown in Table 10. In total, 7 structural variants were identified. Intact reads were also found at all positions of the breakpoints, indicating that (partial) vector sequences have concatemerized. Using TLA, it is not possible to determine the exact order of (partial) copies and to confirm the presence of at least one complete copy. Moreover, all breakpoint sequences are contemplated to represent concatemerization of the ectopic (non-integrated) copies. [0231] Breakpoint sites 1 & 6, 2 & 7, and 4 & 10 represent the same breakpoint sequence but are not reported 2 times due to homology within the vector. The breakpoint sequence can therefore have originated from both indicated vector positions. Due to the heterogenous nature of the sample, it is expected that these fusions are present in a subset of the sample. Example 3: Siglenkcabtagene unileucel (Sig-leucel) for the treatment of relapsed/refractory CD33+ acute myeloid leukemia Summary [0232] Sig-leucel is comprised of universal donor peripheral blood natural killer (NK) cells that have been genetically modified to express a chimeric antigen receptor (CAR) targeting CD33. The CD33 CAR NK cells are generated via a combination of methods that include feeder-cell expansion of NK cells, CRISPR/Cas9 gene editing, and adenovirus associated virus (AAV) gene delivery. The electroporation of Cas9/RNP initiates disruption of the CD38 locus combined with CAR delivery by AAV with homology arms for site-directed gene insertion. The sig-leucel second generation CAR protein consists of an extracellular portion that has a humanized anti-CD33 single chain antibody fragment (scFv) and an intracellular portion that contains cell signaling (CD3-ζ) and co-stimulatory (4-1BB) domains. Based on current knowledge, this combination of gene editing has not been used in NK cells to generate a clinical product. The purpose of sig-leucel is for the treatment of patients with CD33+ relapsed/refractory acute myeloid leukemia (AML). Background Disease Background – Relapsed/Refractory AML [0233] Despite advances in understanding of the biology of AML, there has been a plateau in survival outcomes for patients with relapsed or refractory disease. With current standard relapse regimens, complete response (CR) rates remain between 50-70% even with the addition of anti- CD33 antibody therapy. In patients who have a morphologic response to re-induction and are consolidated with hematopoietic stem cell transplant (HSCT), 2-year OS is <50%. The current accepted standard of care for relapsed AML is chemotherapy with cytosine arabinoside (cytarabine, Ara-C) and fludarabine. The importance of high-dose cytarabine as an integral agent in primary and salvage regimens for the treatment of AML has been well-established. [0234] Fludarabine has been widely used to lymphodeplete patients prior to infusion of lymphocytes, and fludarabine-containing regimens usually combined with cytarabine with or without an anthracycline, have been used for reinduction of primary refractory or relapsed AML. Ghandi et al. demonstrated that fludarabine potentiates in AML blasts an increase in intracellular retention of Ara-CTP, the active metabolite of cytarabine. This led to 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). [0235] In a study by the International Berlin-Frankfurt-Munster Study group, pediatric patients with first relapse or primary refractory AML that received FLAG chemotherapy had a CR rate of 59% after 2 courses of therapy (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). [0236] The median absolute percentages of leukemic blasts on day 28 was 4%. A retrospective analysis by the Japanese Pediatric Leukemia/Lymphoma Study Group studying outcomes of relapsed pediatric AML demonstrated a 65% CR rate 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). [0237] Even with most patients proceeding to transplant, 5-year overall survival was 36.1%. In the most recent COG study for 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). [0238] Best response in patients with primary refractory or first relapse included 54% CR and 14% CR with partial recovery of platelet count and 13.5% CR with incomplete blood count recovery. The relapse studies described above report CR rates after first relapse. Patients in their second and subsequent relapse have increasingly poor response rates to traditional chemotherapy regimens with 25% CR rate after a 3rd treatment attempt and 17% after 4-6th treatment attempt. Similar to acute lymphoblastic leukemia, the depth of response prior to HSCT is an important prognostic marker in AML. The outcomes cited above refer to morphologic CR rates and strategies to improve MRD negative response rates prior to HSCT are critical to improving outcomes in this high-risk patient population. NK cell therapy for AML [0239] Natural killer (NK) cells are cytotoxic lymphocytes that play a key role in recognizing malignant and virus infected cells and serve as a bridge between the innate and adaptive immune response. In hematologic malignancies, there is a qualitative and quantitative dysfunction of innate NK cells and defective NK cells at diagnosis portends a poor prognosis. NK cell phenotypes at diagnosis of AML can be stratified into highly functional and dysfunctional groups with distinct transcriptional modification in pathways involved in cytotoxicity, intracellular signaling and metabolism. Patients with a dysfunctional NK cell profile at diagnosis had a higher risk of relapse. In addition, patients with “hypomaturation” NK cell profiles have reduced overall and relapse free survival. [0240] NK cell activation and cytotoxicity are tumor antigen independent and are instead regulated by a balance of activating and inhibitory NK receptor signaling. Activating receptors recognize ligands on the surface of cancer or viral infected cells that signal danger, and inhibitory receptors are responsible for recognition of self. NK receptor classes include natural cytotoxicity receptors (NCR), C-type lectin receptors, and killer cell immunoglobulin like receptors (KIRs). The inhibitory effect of stimulation of killer immunoglobulin receptors (KIR) by class I HLA may limit the clinical efficacy of autologous NK cells. Clinical evidence demonstrating the benefit of alloreactive NK cells in AML came from studying the graft versus leukemia (GVL) effect in allogeneic stem cell transplant. Early NK cell recovery post- stem cell transplant 22 and increased NK cells in the graft are associated with improved transplant outcomes in leukemia. [0241] Additional evidence of NK cell-mediated GVL was demonstrated in the setting of HLA- mismatched HSCT. Ruggeri et al. observed that patients with AML undergoing haploidentical HSCT had decreased relapse rates when HLA differences between the donor and recipient were present in the GVL direction in a missing-ligand model for NK cells (Ruggeri L, Capanni M, Urbani E, et al: Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295:2097-100, 2002). [0242] This concept was termed “ligand–ligand mismatch” and similar studies confirmed the importance of NK alloreactivity in AML patients undergoing HSCT. Similarly, decreased relapse and increased survival were seen in patients receiving HLA-mismatched transplants in which the donor-recipient pair was also mismatched for KIR genes. These clinical observations have also been supported by murine models demonstrating superior anti-tumor activity and survival with allogeneic NK cells compared to autologous NK cells. [0243] Supported by this early clinical evidence, adoptive NK cell therapy to augment the GVL effect was investigated. The earliest trials were performed with NK cells isolated from healthy donor leukapheresis products using immunomagnetic cell selection and overnight IL-2 activation. Using this approach, Miller et al. demonstrated that infusion of up to 2 x 107 haploidentical NK cells/kg after chemotherapy could 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). [0244] In a similar study, Rubnitz et al. reported the safety of KIR-mismatched NK cell infusion as post-remission consolidation therapy for children with AML, with no relapses reported in the 10 patients treated (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). [0245] Other studies using NK cells derived by this approach in the allogeneic HSCT setting in patients with lymphoid and myeloid malignancies have also demonstrated that NK cell infusions were safe and not associated with severe infusions reactions, GVHD, or graft rejection. However, the response rates in these studies were variable (OS from 29% to 73%) and the NK cell doses produced by this approach were typically limited to a single dose of ≤ 107 /kg. [0246] A genetically-modified K562 cell line was developed that, when used as an irradiated feeder cell, enables large numbers of clinical-grade NK cells to be generated from normal donors, patients, cord blood, and embryonic/ pluripotent stem cells. Peripheral blood NK cells were infused and expanded using this approach in Phase I/II trials for hematologic malignancies and pediatric solid tumors. These trials have collectively delivered over 300 infusions of expanded and activated NK cells to over 100 patients at doses up to 3x108 cells/kg and demonstrated early evidence of efficacy with no dose-limiting toxicities. The K562-derived feeder cell was genetically modified to express membrane-bound IL-21 and 4-1BBL. [0247] In a Phase I/II study (NCT01904136), 25 patients with myeloid malignancies (AML, MDS, CML) undergoing a haploidentical donor stem cell transplant (SCT) received three infusions of NK cells (on day −2, +7, and +28) expanded from peripheral blood of the donor in doses ranging from 104 to 108/kg. Thirteen patients treated during the Phase I dose escalation were reported, wherein only one patient had experienced relapse and relapse‐free survival and viral reactivations were unexpectedly low. No patients developed more than Grade II GvHD, which was less than the historic rate for this regimen without NK cells. One graft failure—associated with infection—was observed in the patient treated at the lowest dose level, 104/kg. No dose‐ limiting toxicities (DLTs) were observed in the Phase I, nor in the Phase II expansion of the study, and non‐DLT adverse events have been minimal, such as transient fever, rash, and tachycardia. Updated results from the Phase II continued to yield 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. [0248] Twenty‐two patients with myeloid leukemias undergoing matched allogeneic SCT received infusions of NK cells expanded from either cord blood (N = 10) or peripheral blood of related matched (N = 9) or haploidentical (N = 3) donors in doses ranging from 106 to 108/kg (NCT01823198). No dose‐limiting toxicities were observed, and non‐DLT adverse events were minimal, such as transient fever, rash, and tachycardia. [0249] In completed Phase I/II studies (NCT01787474 in USA and NCT02809092 in Brazil), 28 adult patients with relapsed/refractory AML were treated with reinduction chemotherapy (fludarabine/ cytarabine +/− GCSF) followed by six infusions of expanded haploidentical NK cells (thrice weekly for 2 weeks) in doses ranging from 106 to 107/kg. In a report of the first 13 patients, 78.6% achieved a response with 50% achieving a complete response. The first subject treated in this trial began therapy with 91% blasts in the bone marrow. Twenty days after completing the NK cell infusions, his bone marrow contained 5% blasts by flow cytometry. No further therapy was offered, and by 40 days after completion of therapy another marrow was obtained which showed only 0.6% blasts. One hundred days after treatment, the blasts had further declined to 0.07%, suggestive of an ongoing immunologic anti‐leukemic response induced by the NK cells. [0250] In the clinical trials mentioned above, subjects received patient-specific NK cell products (autologous or related allogeneic donors). As with autologous CAR T cell, these studies experienced high costs, delays, and subject withdrawal because of the manufacturing required after enrollment. [0251] To solve this problem, an approach was developed for defining optimal “universal” donors, and partnered with Be-the-Match Biotherapies (BTMB) to identify these donors and collect peripheral blood mononuclear cells by apheresis (MNC(A)). Using a step-wise algorithm, ideal alloreactive donors are selected from individuals with KIR-B genotype, licensed by group C1, C2, and Bw4 HLA, and cytomegalovirus (CMV)+ serostatus. The rationale for the universal donor selection criteria is: 1) Killer-cell immunoglobulin-like receptors are genetically highly polymorphic and inherited independently from HLA. The KIR genes can be categorized for the inhibitory (KIR A-haplotype) or the activating haplotypes (KIR B haplotype) based on their gene content. Individuals with KIR-B genotype have a higher number of activating NK cell receptors, which confers a higher alloreactivity and anti-tumor function. 2) NK cells are licensed (acquire enhanced killing ability) when they express inhibitory killer immunoglobulin receptors (KIR) for self-HLA class I molecules. This enables NK cells to recognize “self” and spare autologous cells from killing. Targets lacking self- HLA class I molecules are thus more likely to elicit recognition by licensed NK cells. The inhibitory KIR genes known to be relevant for NK alloreactivity are: (i) 2DL1 which binds to HLA-C group 2 alleles, (ii) 2DL2 and 2DL3 which bind to HLA-C group 1 alleles, (iii) and 3DL1 which binds to HLA-B Bw4 alleles. According to the missing- ligand model, for each NK cell expressing an inhibitory KIR gene there will be alloreactivity only if the corresponding ligand is absent in the recipient, and present in the donor- e.g., any donor possessing a Group C1 allele will be alloreactive to any individual lacking a Group C1 allele. Thus, donors who possess HLA in the C1, C2, and Bw4 families are predicted by this model to be alloreactive against any recipient lacking C1, or C2, or Bw4. 3) Whereas inhibitory KIRs prevent alloreactivity, activating KIRs (aKIR) recognize activating ligands that promote NK cell lysis. Inheritance of activating KIR is widely variable- 0 to 7 aKIR are possible in any one individual. Data from patients undergoing stem cell transplantation show that patients receiving allografts from donors with more activating KIRs have a better outcome than patients receiving allograft from donors with fewer activating KIR. 4) Lastly, NKG2C expression is induced in patients with CMV infection and correlates with an adaptive NK cell phenotype and improved leukemia-free survival. [0252] Thus, the optimal Universal Donor was defined as one who has an HLA genotype consisting of C1, C2, and Bw4 alleles, has a KIR genotype possessing the inhibitory KIR that bind to C1, C2, and [0253] Bw4 (leading to maximum licensing), has a high proportion of activating KIR, and has been exposed to CMV resulting in high NKG2C expression. Considering data available for Caucasian donors, C1/C2/Bw4 alleles occur in 32% of the population. Of the 23 KIR genotypes that account for 80% of the population, 25.3% meet all of these criteria. Approximately 90% of adults will have been exposed to CMV. Thus, the “ideal” NK cell donor can be identified in approximately 1 out of 16 healthy individuals. In collaboration with BTMB, apheresis products were identified and collected as NK cell starting material from 10 different donors. [0254] Six patients were enrolled in a phase 1 study investigating the safety of universal donor NK cells combined with chemotherapy for treatment of adult patients with primary refractory or relapsed AML or MDS (NCT04220684), in which universal donor NK cells were manufactured by the expansion technique utilizing irradiated CSTX002 feeder cells as described above. All 6 patients received an NK cell dose of 1 x 107/kg/dose, with a total of 6 planned NK cell infusions over 2 weeks (day 0, 2, 4, 7, 9, and 11). Five patients tolerated all 6 doses of NK cells administered within 2 weeks. No infusion-related reactions, neurotoxicity or graft versus host disease were observed. One patient developed symptoms concerning for cytokine release syndrome (CRS) coincident with streptococcus bacteremia after the first dose of NK cells, and therefore subsequent doses were held. Attribution could not be absolutely assigned to the bacteremia, so attribution to the NK cells was determined as Possible. Symptoms associated of CRS resolved with a course of steroids. [0255] These early trials demonstrate the safety and early clinical efficacy of delivering multiple doses of allogeneic expanded NK cells for AML. However, treatment of bulky relapsed disease may require a more potent NK cell product as leukemic blasts utilize mechanisms to promote NK cell dysfunction including altered activating receptor expression, and tumor downregulation of NK receptor ligands. Improving NK cell function and recognition of tumor antigens are critical to the success of cell therapy in AML. CD33 as a target for AML [0256] The safety of CAR directed cell therapy products is largely dependent on the CAR target molecule. An ideal CAR target is a tumor associated antigen that is expressed highly on tumor cells but is not expressed on other normal tissues in the body. CD33 is a sialic acid-binding immunoglobulin-related lectin (siglec) that is present on the surface of hematopoietic cells. CD33 is expressed on myeloid derived cells and leukemic blasts but importantly, is not present on pluripotent hematopoietic stem cells or non-hematopoietic cells. CD33 is expressed in over 80% of patients with AML. Given the high expression of CD33 on AML blasts and leukemia progenitor cells, there has been a long precedence to try to exploit this target using immunotherapy approaches. [0257] Gemtuzumab-ozogamicin (GO) is a humanized anti-CD33 monoclonal antibody conjugated to the DNA-binding cytotoxin calicheamicin that is FDA approved for use in pediatric and adult CD33 positive AML. After binding to CD33, GO is internalized and calicheamicin is released causing DNA double strand breaks and subsequent cell death. GO combined with chemotherapy is effective at reducing minimal residual disease in patients with relapsed and refractory AML. Commonly cited toxicities include myelosuppression related on-target off-tumor toxicity and sinusoidal obstruction syndrome (SOS) from calicheamicin hepatotoxicity. In the largest pediatric GO study to date, 1,022 pediatric patients with newly diagnosed AML were enrolled onto the Children’s Oncology Group trial AAML0531 and were randomly assigned to receive standard induction chemotherapy or standard induction chemotherapy with GO. While the addition of GO did not impact post induction remission rates, there was an improvement in disease free survival in patients receiving GO related to reduced relapse risk (HR, 0.73; 95% CI, 0.58 to 0.91; P=.006; 3- year RR: 32.8%±4.6% v 41.3%±4.9%), consistent with adult data. Grade 3-5 adverse events in this trial were similar between study arms, including incidence of hematologic toxicity and time to neutrophil recovery. Of note, however, posthoc analysis found a higher proportion of GO patients during INT2 with prolonged (> 59 days) neutrophil recovery times (12.0% v 6.3%; P=.01), which may have contributed to higher treatment related mortality in the low-risk group. Clinical efficacy of GO may be limited by pharmacokinetics and mechanisms of resistance including overexpression of drug efflux pumps in AML cells. However, the acceptable safety profile and lack of off target toxicity seen with GO supports the further exploration of CD33 as an immunotherapy target. [0258] Lintuzumab (HuM195) is a humanized anti-CD33 monoclonal antibody with a high binding avidity and cytotoxic activity against CD33-positive cells. While there were early reports of clinical efficacy in adults with AML, a large phase III randomized study using lintuzumab combined with chemotherapy in adults with relapsed AML failed to demonstrate a significant improvement in response rates or survival in patients who received the antibody. Importantly, these studies all provided further support of the safety of CD33 as a therapeutic target with only infusion related adverse events were noted with the addition of lintuzumab to chemotherapy. Due to concerns that antibody therapy alone was not sufficient for leukemia disease control, the development of Lintuzumab was shifted toward a radioisotope labeled HuM195 to increase potency. [0259] Following the success of CD19 CAR T cell therapy, there has been a recent push to develop CAR T cells for AML. Ten adults with relapsed/refractory AML were enrolled in a phase I trial using autologous CD33 CAR T cells. Only 3 patients were able to receive CAR T cell infusion – 4 patients had a CD33-CAR T cell product that failed to meet release criteria, 2 had rapidly progressive AML prior to apheresis, 1 patient died before receiving cells. Two patients had cytokine release syndrome and one patient had neurologic toxicity associated with the CAR T cell infusion. There were no dose limiting toxicities. None of the patients had a clinical response to therapy and all three died of progressive disease. This study highlights the feasibility issues using autologous T cells in patients with relapsed/refractory leukemia, as most of the patients (7/10) enrolled on this trial were not able to receive study therapy due to issues with collection and manufacturing of 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 now enrolling (NCT03971799). This study has also experienced numerous subject withdrawals, manufacturing difficulties, and delays in therapy. These studies demonstrate the need for “off-the-shelf” cell therapy in this patient population to improve the speed and efficacy of adoptive cell therapy. CAR NK Cells [0260] Historically, genetic modification of NK cells was unsuccessful due to NK cell resistance to viral transduction. In contrast to T cells, the innate function of NK cells as the first anti-viral defense renders them relatively resistant to traditional methods of gene modification through viral transduction. Alternative NK cell sources and newer methods of genetic engineering 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 were treated with a single dose of “off the shelf” cord blood derived CD19 CAR-NK cells. The CAR-NK cells were equipped with CD19 CAR, IL-15, and a caspase suicide gene. CAR-NK cells were well tolerated with no dose limiting toxicity and no report of cytokine release syndrome or neurologic toxicity with a response rate of 73%. CAR-NK cells expanded in vivo and were detectable for at least a year after infusion. Similar to data reported in CAR-T cell trials, patients who responded to therapy had a higher peak expansion of CAR-NK cells than those with no response. There are many other CAR NK cell targets being developed for hematologic malignancies, including CD33, CD123, CD20, CD19/20, and BCMA. CD38 Knockout [0261] Daratumumab is an FDA approved monoclonal antibody against CD38 that has changed the therapeutic landscape for multiple myeloma with overall response rates of greater than 80% when combined with chemotherapy. Pre-clinical and clinical reports have indicated that there may also be 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-to-NK ADCC in a process labeled fratricide. CD38 negative or low NK cells are resistant to daratumumab- induced fratricide and have improved tumor cytotoxicity when combined with daratumumab compared to CD38+ NK cells. To overcome NK fratricide induced by daratumumab, = CD38 knock out NK cells (CD38KO NK) were generated using CRISPR/Cas9. These CD38KO NK cells are resistant to daratumumab induced fratricide, have a superior 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. The present work has demonstrated sub- populations of AML blasts that are dim for CD33 but highly express CD38. [0262] The present disclosure provides site-directed insertion of a CD33 CAR into the CD38 locus. The purpose of this two-part gene editing is three-fold. First, to avoid the random transgene insertion and subsequent insertional mutagenesis that is seen with viral transduction techniques while maintaining CD33 antigen specific targeting by the CAR. Second, to enhance NK cell metabolic fitness. Third, to open the door for clinical trials combining these CD38KO CD33 CAR NK cells with anti-CD38 monoclonal antibodies to enhance therapeutic efficacy and prevent NK cell fratricide. [0263] The present disclosure also provides the safety of CD38KO CD33 CAR NK cells in patients with relapsed/refractory AML. As described above, CD33 is highly expressed on AML blasts and leukemia progenitor cells and there is a long history demonstrating the safety of this target for cancer immunotherapy. There are advantages to utilizing NK cells over autologous T cells for CAR- modified cellular therapy. While T and NK cell effector functions are similar, a CAR NK cell has the added ability to recognize tumors through innate NK cell receptors, preventing relapse due to antigen escape. Additionally, allogenic HLA-mismatched NK cells have been given safely without causing GVHD, highlighting the ability to produce universal donor or “off-the-shelf” CAR-NK cells to circumvent cost and timing constraints seen with manufacturing CAR-T cell therapy. The use of a standardized healthy-donor cell bank as the cell source also circumvents the issues seen with collecting and manufacturing autologous cell therapy products from heavily pre-treated patients with active leukemia. Finally, NK cells are safe and cytokine release syndrome and neurologic toxicity have been minimal in NK cell trials to date. [0264] Unlike T cells, NK cells are much more difficult to transduce efficiently with lentiviral vectors. To solve this problem, NK cells were engineered using CRISPR gene editing delivered as Cas9/Ribonucleoprotein (Cas9/RNP) via electroporation for the introduction of double strand break (DSB) in the CD38 locus, followed by AAV6 transduction for delivery of the CAR DNA. The CAR is placed between ITRs of an AAV backbone, and is flanked by 600bp homology arms that target the CAR to the DSB in the CD38 locus. This method is used to generate CD38KO CD33 CAR NK cells for clinical use. In addition to improved transduction efficiency, this approach also improves safety by limiting transgene copy number, reducing insertional mutagenesis, and improving uniformity of gene transcription. [0265] Herein, CD38KO CD33 CAR NK cells were successfully generated using the methodology described. In vitro data demonstrated successful depletion of CD38 and expression of CD33 CAR, which resulted in higher anti-AML activity than non-modified NK cells. Mechanism of Action and Efficacy Studies Generation of CD33 CAR NK cells [0266] CD33 CAR NK cells were generated by targeted insertion of the CD33 CAR construct into the AAVS1 locus on human chromosome 19. AAVS1 is a well validated “safe harbor” for integrating DNA transgenes. This approach reliably produced CAR NK cells with a transduction efficiency of >60% in peripheral blood NK cells (Figures 12A and 12B) [0267] The same approach was used for delivery of the CAR DNA to the CD38 locus. Instead of targeting AAVS1, the homology arms were designed for CD38 locus on chromosome 4. This results in simultaneous knock-out of the CD38 and knock-in of the CD33 CAR into this locus (13). To generate CD33 CAR NK cells, Cas9/RNP targeting the CD38 locus was electroporated followed by AAV6 transduction of a CD33 CAR construct containing homology arms to the CD38 targeted region. Anti-tumor efficacy of CD33 CAR NK cells [0268] The CD33 CAR-NK cells are highly effective against several AML cell lines and AML patient samples when assessed by NK cell degranulation (Figures 14A and 14B) and three separate cytotoxicity assays: calcein 4h cytotoxicity (Figures 14C, 14D, and 14E), real-time cell analysis (RTCA, xCELLigence) cytotoxicity assay (Figure 14F), and CyTOF analysis (Figure 5A). CyTOF analysis showed upregulation of CD38 in survivng AML post incubation with CD33 CAR NK cells (Figure 24A). Additionally, the CAR-expressing NK cells demonstrate significantly higher cytokine secretion when compared with wildtype (WT) NK cells (Figure 24B). CD38 knockout NK cells [0269] CD38 is a transmembrane glycoprotein that plays an important role in cellular metabolism. Targeting CD38 in human peripheral blood NK cells using Cas9/RNP not only improves the antibody dependent cytotoxicity of NK cells combined with daratumumab, but it also enhances the metabolic fitness of NK cells. Metabolic fitness plays a crucial role in NK cell function within the tumor microenvironment. RNA-seq was performed on WT and CD38KO NK cells and significant changes were observed in pathways involved in cholesterol biosynthesis (P = .00001) and oxidative phosphorylation (P = .00001) in CD38KO NK cells (Figure 16A). Analysis of genes in those pathways identified a modest but significant increase in expression of mitochondrial genes specifically associated with ATP synthesis, NAD recycling, and electron transport in CD38KO NK cells. Examination of the cellular metabolism of CD38KO NK cells demonstrated a significantly higher OCR:ECAR ratio in CD38KO NK cells compared with CD38WT NK cells (Figures 16C and 16D). These results show that deletion of CD38 induces NK cells to preferentially use OXPHOS to achieve their bioenergetic demands. Importantly, CD38KO NK cells also had higher spare respiratory capacity and mitochondrial respiratory capacity compared with CD38WT NK cells (Figure 16D). Anti-AML activity of CD38KO CD33 CAR NK cells [0270] CD38KO CD33 CAR NK cells generated from a healthy donor by the method described in Figure 33 were cocultured with AML cell line Kasumi-1 for 4 hours. To evaluate the anti-tumor efficacy, AML cells were labeled with calcein-AM and CD38KO CD33 CAR NK (v4) cells were added at differing effector-to-target (E:T) ratios. Cytotoxicity of the labeled tumor cells was quantified by measuring calcein fluorescence released into the supernatant. The data of the CD38KO CD33 CAR NK (v4) cells shows enhanced anti-AML activity of these novel cells in Figure 8. Genome Safety Studies Conducted [0271] As mentioned above, the present example demonstrates safe manufacturing of expanded NK cells derived from allogeneic, and specifically universal-donor sources. In particular, an approach for which there is less formal guidance and less prior experience on which to build- that of ex vivo targeted gene insertion using RNP electroporation and AAV vectors is utilized herein. This approach can increase the safety profile of a genetically- modified product. [0272] Electroporation of pre-complexed Cas9/gRNA (RNP) enables tighter control of the genomic exposure to editing enzymes, and no possibility of sustained expression, compared to vector-based expression. [0273] Site-directed insertion reduces the random and unknown nature of insertion sites common with retroviral and lentiviral gene insertion. [0274] The application of this method onto the universal-donor platform allows for full testing and release of an off-the-shelf product, compared to autologous products. [0275] With this in mind, the following genomic assays were developed: 1. Evaluation of CD33 CAR integration [0276] Random integration of genetic material into the host genome has been linked to poor outcomes in patients treated with genetically engineered T cells and gene therapy. Cas9/RNP site directed CAR insertion approach resulted in highly efficient CAR expression while overcoming the safety concerns associated with random integration of CARs in the human genome via lentivirus. [0277] Initially in CD33 CAR NK cells, the CD33 CAR was inserted into the AAVS1 locus, a human safe harbor locus. Using targeted locus amplification (TLA), low off-target CAR integration was demonstrated with this gene editing approach. The vector integrated as intended in human chromosome chr19: 55,115,754- 55,115,767, the expected integration at intron 1 of PPP1R12C. [0278] Here, a similar TLA analysis was performed on CD38KO CD33 CAR NK cells. Similar to the data for the safe-harbor AAVS1 site, only a single hotspot for integration was identified at the intended CD38 locus on Chromosome 4. [0279] Overall, targeted locus amplification analysis demonstrates that this approach results in high targeted insertion of the CD33 CAR at the intended site. 2. Identifying off-target effects CD38-directed CRISPR using Churchill [0280] Churchill is a bioinformatics approach developed and utilized by the clinical genomic laboratory at Nationwide Children’s Hospital to streamline whole genome sequencing analysis. Churchill fully automates the analytical process required to take raw sequencing data through the complex and computationally intensive processes of alignment, post-alignment processing, local realignment, recalibration and variant discovery, with the goal of identifying mutations of clinical relevance. This approach was used to analyze the whole genome sequencing data of the CD38KO NK cells and CD38KO CD33 CAR NK cells compared it with non-modified expanded NK cells from the same donor. [0281] Whole-genome sequencing (WGS) was performed and used to identify the off-target effects of Cas9/RNP targeted to CD38. Next-generation sequencing data were processed through Churchill, in which reads were aligned using BWA MEM (v0.7.15) to the GRCh37 reference genome. Variants were called using the Mutect2 tool of the Genome Analysis Toolkit (GATK v4.0.5.1, Broad Institute) and annotated using SnpEff (v4.3). Knockout-exclusive single nucleotide polymorphisms and insertion-deletion mutations (indels) were detected when compared with wild-type (WT) NK cells. Because repair of the DNA breaks generated by Cas9/RNP will vary between cells and be close to the region of guide RNA homology, clustered events were not excluded as is typically done for somatic genomic analysis. Therefore, the Mutect2 filters were applied and those occurring at any frequency in the CD38KO cells but not present in WT NK cells were included, and included only those that passed the applied filters or were clustered events, were nonsynonymous mutations, and were in coding regions. [0282] Twenty-six genes with single nucleotide polymorphisms and indels were found that were unique to the CD38KO NK cells and of moderate or high potential impact. In all, 18 genes had mutations categorized as moderate impact (missense and non-frameshift) and 7 genes (including CD38) had mutations categorized as high impact (startloss, stopgain, and frameshift) by SnpEff (Table 11). By RNA-seq, only 4 of the off-target genes (CC2D1B, DENND4B, KMT2C, and WDR89) with possible high impact mutations are expressed at meaningful levels in NK cells (Figure 17). [0283] Using the same approach on CD38KO CD33 CAR NK cells 13 genes (including CD38) with high-impact mutations were found compared to matched expanded but non-modified NK cells from the same donor (Table 12). Seven were expressed at detectable levels in expanded NK cells by RNAseq (Figure 18), but none overlapped with those identified in the CD38KO NK cells as described above. These WGS data show the ability to detect mutational events at high sensitivity, and have not identified any recurring off-target effects of high-impact induced by the CD38-targeted Cas9/RNP, with or without subsequent CD33 CAR insertion. Clinical Studies Generation of CD38 knockout CD33 CAR NK cells [0284] Given the benefit of targeting CD38 in NK cells and low off-target effect of using Cas9/RNP, NK cells with the CD33 CAR gene inserted into the CD38 locus were generated. The same approach was used for insertion of the CARs (Cas9/RNP + AAV6) by designing the homology arms for CD38 targeting site, resulting in simultaneous knock-out of the CD38 and knock-in of the CD33 CAR into this locus (Figure 11). An AAV6 vector was utilized to generate CD38KO CD33 CAR NK cells. Flow cytometry used to determine CAR transduction and CD38 knock out efficiency (Figure 13). Pre-clinical studies [0285] In vitro studies. To assess the CD38KO CD33 CAR NK cell function in vitro, effector function is measured against CD33+ tumor cell lines. CD38KO CD33 CAR NK cells generated from at least three healthy donors are cocultured with AML cell lines and patient samples for 4 and 48 hours and then supernatants are collected to measure cytokine production by Luminex multiplex cytokine analysis. To evaluate the anti-tumor efficacy, AML cells are labeled with calcein- AM and CD38KO CD33 CAR NK cells are added at differing effector-to-target (E:T) ratios. Cytotoxicity of the labeled tumor cells is quantified by measuring calcein fluorescence released into the supernatant. The real time potency is measured using the xCELLigence RTCA MP instrument (ACEA Biosciences). Assays are performed to compare wild type NK cells with CD38KO CD33 CAR NK cells, and also use CD33 CAR-NK cells inserted into the AAVS1 locus to assess the impact of CD38 deletion. [0286] In vivo studies. To further evaluate the efficacy of CD38KO CD33 CAR NK, in vivo studies are performed using a xenograft mouse model. Briefly, NSG mice are inoculated with 1 x 106 luciferase- transduced MOLM-13, MV-4-11, or OCI-AML3 AML cells by tail vein injection. Animals in the treatment group receive 107 CD38KO CD33 CAR NK cells by tail vein injection on days 7 and 14 after tumor injection. Low-dose IL-2 is given with NK cells while the mouse dose is analogous to 4 x 108 NK cells/kg in human studies, this is a setting lacking endogenous homeostatic cytokines. Animals in the two control groups receive non-modified expanded NK cells, or do not receive any NK cells. To follow the anti-tumor response, weekly bioluminesence imaging and peripheral blood samples for flow cytometry assessment of leukemia cells is performed. Survival curves are generated. All animal experiments are performed on a protocol approved by the Nationwide Children’s Hospital Institutional Animal Care and Use Committee (IACUC). Safety Evaluations of CD38KOCD33 CAR NK cells [0287] CD33 is a documented target with well-established safety in humans for antibody and CAR T cell therapeutics. Established models do not exist for testing the safety of targeting human CD33 in murine models, and there are no methods and materials for generating the equivalent murine anti- CD33 expanded CAR NK cells for immune-competent murine models. As described above, safety data in humans is established for expanded universal-donor NK cells. The pre-clinical and product release safety testing is focused on generating additional genomic safety data. [0288] Evaluation of on- and off-target integration frequencies. The Churchill assay above is performed on each CD38KO CD33 CAR NK cell product to verify on-target alteration of CD38 and identify off-target Cas9/RNP effects. Whole genome sequencing data of the CD38KO CD33 CAR NK cells is compared to non-modified expanded NK cells from the same donor. Release of the product for clinical use requires that at least 50% of the CD38 reads are identified as mutated. The remaining mutations are cataloged and accessed as baseline product information if ever needed for patient safety investigations. [0289] TLA is used to broadly identify integration sites of the CAR. As this data is orthogonal to the dGH In-Site assay but not a fully qualified assay with the CAR probe, it ia reported on at least three validation runs to collect baseline data on dominant integrations sites. [0290] dGH In-Site: A targeted fluorescence-based genomic hybridization assay is being developed in collaboration with KromaTiD. This assay identifies site-specific insertions of the CAR into the CD38 locus, using a CAR-specific probe, and two CD38- locus-specific probes on the flanking ends of the target insertion site. G- banding results are reported from 200 metaphase spreads on each CD38KO CD33 CAR NK cell product. Release criteria will be that ≥ 90% of CAR insertions are at the intended CD38 locus. [0291] Classic G-banding: To assess overall genomic integrity G-banding results are reported from 20 metaphase spreads on each CD38KO CD33 CAR NK cell product (performed by KromaTiD). G- banding results do not support a release criteria, but are for information only. (See Figures 2-10 and 26-32). [0292] dGH Screen: To further assess genomic integrity dGH Screen results are reported from 50 metaphase spreads on each CD38KO CD33 CAR NK cell product (performed by KromaTiD). The data report summarizes results, on vs off target integration, and structural variation associated with the CD38 site. dGH Screen results do not support a release criteria, but supplement G-banding data and be for information only. (See Figures 2-10 and 26-32). [0293] Evaluation of residual AAV6 vector. The AAV6 virus is intended for use only in providing the template for ex vivo insertion of the CAR gene. Given the low vector copy number used for transduction (104 – 105 MOI), followed by dilution in culture, two subsequent weeks of NK cell propagation (expected 2,000-fold expansion), and final wash steps, the final AAV infectious particles are not expected to be present in biologically-meaningful levels (typical gene therapy doses of 1013 – 1014 vector genomes/kg). Nonetheless, the presence of vector copy number is contemplated to be as very low and capsid as undetectable in order to demonstrate safety. [0294] A digital droplet PCR assays was developed and validated for AAV2 ITR as a measure of viral genomes, which was used in release testing of the AAV6-pseudotyped vector. Here, viral genome copy number is reported to quantify residual virus in validations runs and clinical products. Drug Product and Manufacturing Product Description [0295] The proposed drug product is comprised of NK cells derived from healthy donors selected for optimal universal-donor criteria, expanded in vitro with CSTX-002 feeder cells in media containing IL-2, and gene modified by electroporation of Cas9/RNP and transduction with AAV6, resulting in CD38KO CD33 CAR NK cells. Viral Vector [0296] Product Name: CD33CAR V4 (LHCD8-41) CD38_ssAAV AAV Serotype: Serotype #AAV6 [0297] The CD33 CAR encoding DNA along with homology arms (HA) for the CD38 locus are cloned between ITRs of an AAV2 back-bone as: CD38 Right HA, BGHpA, MND Promotor, CD33 CAR, BGHpA and CD38 Left HA. See Figure 21. Vector Production and Purification [0298] The AAV viral vector CD33CAR V4 (LHCD8-41) CD38_ssAAV is manufactured by Andelyn Biosciences at the Clinical Manufacturing Facility located at 575 Children’s Crossroad in Columbus, Ohio. [0299] The viral vector wase produced at Andelyn Biosciences by co-transfection of human embryonic kidney (HEK) 293 cells with three DNA plasmids: Vector plasmid, AAV Helper plasmid, and Ad Helper plasmid. The plasmids used to manufacture the viral vector were produced by Andelyn Biosciences and Aldevron. Plasmids are listed and production is described in Section [0300] The manufacturer aliquots the vector product into single-use vials which are preserved until use in cell manufacturing. The vector is supplied in frozen form (≤ -60ºC) and transported to the Abigail Wexner Research Institute at Nationwide Children’s Hospital (AWRI- NCH) Cell-Based Therapy Core facility (CBT) where it will be stored at ≤ -60ºC until manufacturing use. [0301] The sequence of AAV viral vector CD33CAR V4 (LHCD8-41) CD38_ssAAV is provided in SEQ ID NO: 50. AAV2-ITR ddPCR assay was used to measure Physical Titer. [0302] A research batch of the vector was produced by Andelyn Biosciences using the same final plasmids as the final clinical vector. This material was used for process development and for some initial pre-clinical studies. A single batch of GMP-like vector product was manufactured under the toxicology process plan, but produced in the Andelyn GMP cleanrooms and with expanded clinical- grade testing. Plasmid Production [0303] The AAV viral vector CD33CAR V4 (LHCD8-41) CD38_ssAAV was produced using the following plasmids: Vector plasmid CD33CAR V4 (LHCD8-41) CD38_ssAAV BackBone Kan AAV Helper Plasmid pNLRepcap6-Kan Ad Helper Plasmid pHelp Kan V4 [0304] Vector plasmid CD33CAR V4 (LHCD8-41) CD38_ssAAVBackBone Kan is a toxicology- grade plasmid produced by Andelyn Biosciences in their GMP facility. The vector plasmid contains the Kanamycin resistance gene. [0305] AAV Helper Plasmid pNLRepcap6-Kan is a Research HD grade plasmid produced by Aldevron. The helper plasmid contains the Kanamycin resistance gene. [0306] Ad Helper Plasmid pHelp-KanV4 is a GMP-S grade plasmid produced by Aldevron and the Ad helper plasmid contains the Kanamycin resistance gene. [0307] Sequencing in the form of NextGen PacBio sequencing was also performed for each plasmid to confirm identity and plasmid sequence purity. While the CoA of the Vector Plasmid includes PacBio sequencing results, the CoA of the AAV Helper Plasmid and the CoA of the Ad Helper Plasmid include only Sanger sequencing. Post-release PacBio sequencing results of these two plasmids are provided below. [0308] The NextGen PacBio Sequencing covered the Ad Helper Plasmid (pHelp Kan V4) at an average read depth of 152,383x. Sequence analysis revealed four positions differing from the expected plasmid map, all of which were all insertions of a single nucleotide at frequencies ranging from10.38 – 16.27%. The variants were present in the following regions, relative to sequencing from the PsiI restriction site used for linearization prior to sequencing: 47: Insertion of a T in a poly-T(9) region of the E4 gene 834: Insertion of an A in a poly-A(13) region of the E4 gene 2641: Insertion of a G in toward the end of the VA gene 5933: Insertion of an A in the ColE1 origin site [0309] These variants may impact AAV replication efficiency during production of the vector, but should not impact production or structure of the AAV viral vector or its transgene. Nine other variants in the sequence were present at 0.1 - 0.21%, and all other variants present at <0.01%, which are within the statistical error distribution for this deep sequencing approach. [0310] The NextGen PacBio Sequencing covered the AAV Helper Plasmid pNLRepcap6-Kan at an average read depth of 247,055x. Sequence analysis revealed one position differing from the expected plasmid map, which was insertion of a single nucleotide at a frequency of 11.79%. The variant was present in the following region, relative to sequencing from the AsiSI restriction site used for linearization prior to sequencing: 830: Insertion of an A within the pUC origin of replication site. [0311] This variant impacts plasmid replication but not production or structure of the AAV viral vector or its transgene. Genome Editing Component [0312] CRISPR gene editing of NK cells are delivered as Cas9/Ribonucleoprotein (Cas9/RNP) via electroporation for the introduction of double strand breaks prior to AAV6 transduction. The Cas9 protein and single guide RNA (sgRNA) are complexed together to form the ribonucleoprotein (RNP complex). A sgRNA targets exon 1 of the CD38 gene. [0313] 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 are used for preclinical testing. For manufacturing of the CAR NK cell investigational product needed for clinical use under this IND, GMP grade sgRNA and CTS TrueCut Cas9 Protein are used, which are complexed together prior to electroporation. The GMP CD38-sgRNA is produced by Synthego. The GMP CTS TrueCut Cas9 Protein is produced by Thermo Fisher Scientific. [0314] The sgRNA is resuspended in PBS and then 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 may be used within 20 minutes of completion of incubation or may be stored on ice until use. [0315] The Cas9/RNP complex is added to NK cells suspended in TheraPEAK P3 Primary Cell Nucleofector Solution set (Lonza), which has been produced according to applicable GMP standards and is intended to support GMP manufacturing. Electroporation is performed using the 4D- Nucleofector System (Lonza). [0316] All procedures for gene editing are performed in the Abigail Wexner Research Institute at Nationwide Children’s Hospital (AWRI-NCH) Cell-Based Therapy Core facility (CBT), operating under applicable GMP guidelines. Universal Donor NK cells [0317] Donors undergo infectious disease testing and screening as required for HCT/P donors at BTMB institutions compliant with 21 CFR Part 1271, the FDA Guidance document “Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps)” and any supplemental guidance documents issued. Donors are tested for KIR and HLA genotypes and infectious disease markers, NK cell proliferation and NKG2C content. [0318] Donors who meet all selection criteria have MNC(A) collected at a BTMB-qualified collection center. MNC(A) products are then shipped to the CBT Core facility, where they undergo additional processing. Apheresis collections are washed to remove platelets, and magnetic immunodepletion of CD3+ cells is performed on the apheresis product using the Miltenyi CliniMACS. The CD3-depleted MNCs are aliquoted for storage in cryobags in liquid nitrogen vapor phase until they are thawed and utilized for NK Cell manufacturing. [0319] This serves as the cellular source material for manufacturing the drug product. CD38KO CD33 CAR NK Cell Manufacturing [0320] All CD38KO CD33 CAR NK cell products are manufactured in 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 produce biologics under cGMP conditions. The CBT Core facility comprises multiple dedicated laboratory suites used for Research and Development, Cell Manufacturing, Quality Control, and Temperature-Controlled Storage. [0321] The CD38KO CD33 CAR NK cell product are manufactured from CD-depleted MNC(A) and expanded by recursive stimulation with irradiated feeder cells expressing membrane-bound IL- 21 and 4-1BBL (clone CSTX002). The NK cells are genetically modified following 1 week of expansion, rested for 2 days, and expanded in culture for another 14 days. The product is cryopreserved at various cell concentrations, then released/dispensed for infusion at the prescribed dose into individual patients. Cell Culture [0322] On Day 0 of NK expansion, the CD3-depleted MNC are thawed and washed and then propagated by recursive weekly stimulation with irradiated CSTX002 feeder cells (IFCs). Briefly, IFCs are added at an approximate 1:2 IFC-to-viable TNC ratio (also known as Stimulation 1 or Stim 1) in cell culture media containing 5% Immune Cell Serum Replacement and recombinant human IL- 2 at 100 IU/mL. Additional fresh IL-2 is added at 100 IU/mL every 1-3 days. Day 7 expanded NK cells are aliquoted for storage in cryobags in liquid nitrogen vapor phase until they are thawed for the remaining manufacturing steps. [0323] On Day 7, the NK cells are thawed and then genetically modified using CRISPR/Cas9 combined with AAV6. The CD33 CAR is inserted into the CD38 locus via site-directed insertion utilizing CRISPR/Cas9. First, CD38 is targeted using gRNA via electroporation of precomplexed Cas9/RNP into the expanded NK cells. Briefly, expanded NK cells are harvested, washed, and resuspended in 20ul of the TheraPEAK P3 Primary Cell 4D-Nucleofector Solution. 5ul of pre- complexed Cas9/RNP targeting CD38 is added to the cell suspension. The Cas9/RNP is then electroporated into NK cells utilizing a 4D Nucleofector System. After electroporation, the cells are rested for 30 minutes in media containing IL-2. The cells are then counted and AAV6 is added at a multiplicity of infection (MOI) of 75,000. [0324] After transfection, the cells rest for 2 days in culture and then are stimulated with irradiated feeder cells at a 1:1 IFC-to-viable TNC ratio (also known as Stimulation 2 or Stim 2) and cultured for another 7 days in the presence of 100 IU/mL of IL-2. [0325] The cells are transferred to the Xuri Cell Expansion System for further expansion. The cells are re-stimulated with irradiated feeder cells at a 1:1 IFC-to-viable TNC ratio (also known as Stimulation 3 or Stim 3) and cultured for another 7 days in the presence of IL-2. Harvest and Cryoprotection [0326] On Day 23, cells are harvested and cryopreserved in aliquots. CD38KO CD33 CAR NK Cell products are cryopreserved in media containing DMSO, human serum albumin (HSA), and Plasma- Lyte A. The product is released/dispensed for infusion at the prescribed dose into individual patients. [0327] Product distribution, thawing, and infusion are performed according to the protocol and/or institutional clinical standard operating procedures for effector cell therapy. The product is infused immediately after thaw with no additional manipulations such as washing or culturing. Flow Diagram Outlining Product Testing [0328] See the production flow chart (Figure 23) for a summary of the manufacturing process and testing. Critical Materials [0329] Critical materials that impact the quality, safety, potency or purity of the CD38KO CD33 CAR NK cell product are listed in Table 13. Shown in bold italics are excipients present in the final product. CD38KO CD33 CAR NK cell release Criteria and Additional Testing [0330] Testing is performed according to existing CBT Core facility SOPs. See flow diagram (Figure 23) for an overview of processing and testing. CD38KO CD33 CAR+ NK cell are administered in 4 dose levels as follows: a. Dose level 1: 1x107 CAR-NK cell/kg (±20%) Dose level 2: 3x107 CAR-NK cell/kg (±20%) Dose level 3: 1x108 CAR-NK cell/kg (±20%) b. Dose level 4: 2 doses of 1x108 CAR-NK cell/kg (±20%), one week apart Safety Testing using TLA, Churchill, dGH screens, custom dGH probes, and G-banding [0331] Targeted Locus Amplification. For the whole-genome mapping of CD38KO CD33 CAR integration, the TLA technology (Cergentis B.V.) is used. The genomic DNA from CD38KO CD33 CAR-expressing NK cells are isolated from each validation run and crosslinked, fragmented, and re- ligated using the kit provided by Cergentis, then submitted to Cergentis for sequencing. [0332] Next Generation Sequencing (NGS) Data Processed through Churchill. High-fidelity Cas9 has been shown to have low off-target editing because of its rapid degradation after electroporation. To study the off-target effects in CRISPR-modified NK cells, WGS is peformed on each single production of CD38KO CD33 CAR NK cells. Next-generation sequencing data is processed through Churchill, in which reads are aligned using BWA MEM (v0.7.15) to the GRCh37 reference genome. Variants are called using the Mutect2 tool of the Genome Analysis Toolkit (GATK v4.0.5.1, Broad Institute) and annotated using SnpEff (v4.3). Knockout-exclusive single nucleotide polymorphisms and insertion-deletion mutations (indels) are identified by comparing with expanded NK cells from the same donor that have not been electroporated or transduced. Because repair of the DNA breaks generated by Cas9/RNP vary between cells and be close to the region of guide RNA homology, clustered events are not excluded as is typically done for somatic genomic analysis. The Mutect2 filters are applied and those occurring at any frequency in the CD38KO CD33 CAR NK cells but not present in expanded NK cells from the same donor that have not been electroporated or transduced is included, and include only those that passed all the applied filters or clustered events, nonsynonymous mutations, and in coding regions. [0333] dGH Screens. a. Samples of final CD38KO CD33 CAR NK cells are prepared with KromaTiD dGH cell prep kit as recommended to preserve metaphase spreads for hybridization. Samples are sent to KromaTiD for hybridization with fluorescent probes across the genome. High resolution karyotyping is performed with image analysis, reporting (Figures 6 and 7): i.reciprocal, balanced and allelic translocations ii.orientation events like inversions and sister chromatid exchanges iii. chromosomal gain and loss events including sister chromatid fusions, dicentrics/acentrics, fragmentation/chromothrypsis, polypoidy and aneuploidy. [0334] Custm dGH In-site. Samples of final CD38KO CD33 CAR NK cells are prepared and submitted to KromaTiD as for dGH Screen, but are hybridized with 2-color probes for the CD38 knock-in site and the CD33 CAR. Analysis reports the total number and distribution of cells with CAR insertions, and the proportion inserted into the CD38 locus. [0335] G-banding. Samples of final CD38KO CD33 CAR NK cells are prepared as standard for G- banding analysis and submitted to KromaTiD. Standard metaphase karyotyping is performed and reported. Safety testing for residual AAV6 [0336] Based on the initial MOI of the transduction event, washing steps, and media dilution steps, a residual AAV6 MOI is contemplated to be in the drug product of less than 2.5 x 105 viral particles/mL in a product containing 108 NK cells/mL. At the highest dose level, this represents a dose that is almost 1 billion-fold lower than that of a typical dose of AAV6 systemic gene therapy (e.g., Zolgensma, dosed at 2 x 1014 viral particles/kg). Outline of Clinical Trials [0337] Current treatment options for relapsed and/or refractory AML are limited and overall survival in this patient population is poor. The best chance for durable remission in this high-risk group remains allogeneic HSCT, however patients need to obtain a complete response prior to HSCT for optimal success. Given the critical role of NK alloreactivity in mediating the anti-leukemia effect in AML, current translational efforts in AML are geared towards adoptive immunotherapy with functionally active NK cells. The safety of adoptive transfer of ex vivo expanded haploidentical NK cells has been demonstrated in patients with AML and other cancers, however, NK cell therapy in this context has yet to be realized. One major obstacle for adoptive NK cell immunotherapy is obtaining sufficient cell numbers and having them readily available for infusions for patients with leukemia. Herein, it is demonstrated that large numbers of NK cells are propagated ex vivo and have a bank of universal donor- derived NK cells was generated for “off-the-shelf” NK cell therapy. Phase I trials in adult and pediatric patients with relapsed/refractory AML utilizing this universal donor NK cell bank are NCT04220684 and NCT05503134. [0338] The purpose of this trial is to determine the safety and estimate the efficacy of universal donor derived CD38 knock out CD33 CAR NK cells in pediatric patients with relapsed/refractory AML. The trial is a nonrandomized, open label, Phase I dose escalation study of induction chemotherapy (fludarabine/cytarabine) followed by universal donor derived CD38KO CD33 CAR NK cells administered by intravenous infusion to relapsed/refractory AML patients. [0339] See Figure 22 for study design and Table 15 for Protocol Outline. [0340] 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 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 a true scope and spirit of the invention being indicated by the following claims.
TABLES Table 1. Breakout of Transgene Probe Events by sample, where each category is the total number of events.
Figure imgf000077_0001
Table 2.
Figure imgf000077_0002
Table 3. Structural Events T
Figure imgf000077_0003
Table 5. Aneuploidy detail, by chromosome identity and cell number
Figure imgf000077_0004
Figure imgf000078_0001
Table 6. Complex Events Summary. There were three cells that each contained a single complex event.
Figure imgf000078_0002
Table 7. Summary of Transgene analysis and integration site sequencing of vector 33_CD33CAR V4 (LHCD8-41) CD38_ssAAV-BackBone Kan.
Figure imgf000078_0003
Table 8. Primers used in TLA analysis
Figure imgf000078_0004
Table 9. Identified sequence variants in the vector
Figure imgf000078_0005
Table 10. Vector-vector breakpoints in the vector
Figure imgf000078_0006
Figure imgf000079_0001
*This structural variant can alternatively represent random integration of the vector. Table 11. List of high-impact off-target mutations in CRISPR edited CD38KO NK cells, identified by Churchill.
Figure imgf000079_0002
C 4 4 F F
Figure imgf000080_0001
Table 11 (continued) 4 4 F F
Figure imgf000080_0002
_ Table 12. List of high-impact off-target mutations in CD38KO CD33 CAR NK cells, as identified by Churchill. P P 9 3 F
Figure imgf000081_0001
Figure imgf000082_0001
Table 12 (Continued) P P 9 3 F P P
Figure imgf000082_0002
T
Figure imgf000082_0003
_
Figure imgf000083_0001
Figure imgf000084_0001
Table 14. CD38KO CD33 CAR NK lot release criteria
Figure imgf000084_0002
Table 15. Protocol Outline
Figure imgf000084_0003
Figure imgf000085_0001
Figure imgf000086_0001
Table 16. Amino Acid Abbreviations
Figure imgf000086_0002
Table 17. Amino Acid Substitutions
Figure imgf000086_0003
Figure imgf000087_0001
SEQUENCES 1. SEQ ID NO: 1 – 30bp right homology arm (RHA) gattggtgacagaaaagccccatccttagg
Figure imgf000088_0001
2. SEQ ID NO: 2 - 30bp left homology arm (LHA)
Figure imgf000088_0002
3. SEQ ID NO: 3 – 300bp RHA
Figure imgf000088_0003
4. SEQ ID NO: 4 – 300bp LHA
Figure imgf000088_0004
5. SEQ ID NO: 5 – 500bp RHA
Figure imgf000088_0005
6. SEQ ID NO: 6 – 500bp LHA
Figure imgf000088_0006
7. SEQ ID NO: 7 - 800bp RHA
Figure imgf000089_0001
8. SEQ ID NO: 8 – 800bp LHA
Figure imgf000089_0002
9. SEQ ID NO: 9 – PAMg (PAM + the sequence encoding crRNA)
Figure imgf000089_0003
10. SEQ ID NO: 10 – Splice acceptor
Figure imgf000089_0004
g 11. SEQ ID NO: 11 – BGH polyA terminator
Figure imgf000089_0005
12. SEQ ID NO: 12 – mCherry
Figure imgf000090_0001
13. SEQ ID NO: 13 – 30bp plasmid with mCherry transgene
Figure imgf000090_0002
14. SEQ ID NO: 14 – 300bp plasmid with mCherry transgene
Figure imgf000090_0003
Figure imgf000091_0001
15. SEQ ID NO: 15 – 500bp plasmid with mCherry transgene t tttt ttt ttt tt ttt ttttt t ttt ttt tt t t tttt t
Figure imgf000091_0002
16. SEQ ID NO: 16 – 800bp plasmid with mCherry transgene
Figure imgf000092_0001
17. SEQ ID NO: 17 – crRNA
Figure imgf000092_0002
18. SEQ ID NO: 18 – scFv
Figure imgf000093_0001
19. SEQ ID NO: 19 – IgG4-Hinge
Figure imgf000093_0002
20. SEQ ID NO: 20 – CD28
Figure imgf000093_0003
21. SEQ ID NO: 21 – CD3z
Figure imgf000093_0004
22. SEQ ID NO: 22 – CD33CAR-Gen2-Cloned in ssAAV Backbone
Figure imgf000093_0005
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
23. SEQ ID NO: 23 – CD33CAR-Gen4v2
Figure imgf000096_0002
Figure imgf000097_0001
Figure imgf000098_0001
24. SEQ ID NO: 24 – NKG2D Transmembrane domain
Figure imgf000098_0002
25. SEQ ID NO: 25 – Linker
Figure imgf000098_0003
26. SEQ ID NO: 26 – 2B4 tggcggagaaagcggaaggagaagcagagcgagacctcccctaaggagtttctgacaatctatgaggacgtgaaggatctgaagaccaggc
Figure imgf000098_0004
27. SEQ ID NO: 27 – Linker
Figure imgf000098_0005
28. SEQ ID NO: 28 – CD3z
Figure imgf000098_0006
29. SEQ ID NO: 29 – anti-CD33 scFv
Figure imgf000099_0001
30. SEQ ID NO: 30 – MND Promoter
Figure imgf000099_0002
31. SEQ ID NO: 31 – 600bp, LHA, AAVS1 (gen4v2 and gen2)
Figure imgf000099_0003
32. SEQ ID NO: 32 – 600bp, RHA, AAVS1 (gen4v2 and gen2)
Figure imgf000099_0004
Figure imgf000100_0001
33. SEQ ID NO: 33 - gRNA
Figure imgf000100_0002
34. SEQ ID NO: 34- gRNA
Figure imgf000100_0003
35. SEQ ID NO: 35- gRNA
Figure imgf000100_0004
36. SEQ ID NO: 36- gRNA GAGCTGCAGAAGGACAAGAT
Figure imgf000100_0005
37. SEQ ID NO: 37- gRNA
Figure imgf000100_0006
38. SEQ ID NO: 38- gRNA
Figure imgf000100_0007
39. SEQ ID NO: 39- gRNA G
Figure imgf000100_0008
40. SEQ ID NO: 40- gRNA
Figure imgf000100_0009
41. SEQ ID NO: 41- gRNA
Figure imgf000100_0010
42. SEQ ID NO: 42- gRNA
Figure imgf000100_0011
43. SEQ ID NO: 43 - promoter
Figure imgf000101_0001
44. SEQ ID NO: 44- promoter
Figure imgf000101_0002
45. SEQ ID NO: 45- promoter
Figure imgf000101_0003
46. SEQ ID NO: 46- promoter
Figure imgf000101_0004
47. SEQ ID NO: 47 – PAMgPAMg mCherry construct
Figure imgf000101_0005
Figure imgf000102_0001
48. SEQ ID NO: 48 – PAMg RNA mCherry construct CCAATCCTGTCCCTAGTGGCCCCCACTAGGGACAGCGATCGGGTACATCGATCGCAGG
Figure imgf000102_0002
49. SEQ ID NO: 49 – CD33CAR V4 (LHCD8-41) CD38 ssAAV-Backbone
Figure imgf000102_0003
Figure imgf000103_0001
50. SEQ ID NO: 50 – CD33CAR V4 (LHCD8-41) CD38 ssAAV-Backbone
Figure imgf000103_0002
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
51. SEQ ID NO: 51 – Off-target reference sequence
Figure imgf000106_0002
52. SEQ ID NO: 52 – Off-target altered sequence
Figure imgf000106_0003
53. SEQ ID NO: 53 – Off-target altered sequence TTTATTAGTAG
Figure imgf000106_0004
54. SEQ ID NO: 54 – Off-target altered sequence AAACTCTAGG
Figure imgf000106_0005
55. SEQ ID NO: 55 – Off-target altered sequence A
Figure imgf000106_0006
56. SEQ ID NO: 56 – Off-target altered sequence
Figure imgf000106_0007
57 SEQ ID NO: 57 – Off-target altered sequence
Figure imgf000106_0008
58 SEQ ID NO: 58 – Off-target reference sequence A
Figure imgf000106_0009
59. SEQ ID NO: 59 - IgG Hing CD4
Figure imgf000106_0010
60. SEQ ID NO: 60 – 41BB-L
Figure imgf000106_0011
61. SEQ ID NO: 61 – CD3z RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY N
Figure imgf000107_0001

Claims

CLAIMS What is claimed is: 1. A modified T cell comprising a plasmid, nucleic acid, and/or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems 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 and right homology arms are each 1000bp in length or less.
2. The modified T cell of claim 1, wherein the CAR polypeptide comprises a transmembrane domain, a co-stimulatory 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 specifically binding to CD33 comprises a sequence at least 90% identical to SEQ ID NO: 29 or a fragment thereof.
5. The modified T cell of claim 2, wherein the scFv specifically binding to CD33 comprises SEQ ID NO: 29, or a fragment thereof.
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. The modified T cell of any one of claims 2-5, wherein the co-stimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination thereof.
8. The modified T cell of any one of claims 1-7, further comprising a polyadenylation signal between the transgene and the right homology arm.
9. The modified T cell of any one of claims 1-8, wherein the left homology arm and right homology arm are the same length.
10. The modified T cell of claim 9, wherein the homology arms are each 30bp in length.
11. The modified T cell of claim 9, wherein the homology arms are each 300bp in length.
12. The modified T cell of claim 9, wherein the homology arms are each 600bp in length.
13. The modified T cell of claim 9, wherein the homology arms are each 1000bp in length.
14. The modified T cell of any one of claims 1-8, wherein the left homology arm and right homology arm are different lengths.
15. The modified T cell of any one of claims 1-14, wherein the homology arms specifically hybridize to the Adeno-Associated Virus Integration Site 1 (AAVS1) of chromosome 19 of humans.
16. The modified T cell of any one of claims 1-15, further comprising a murine leukemia virus- derived (MND) promoter.
17. The modified T cell of any one of claims 1-16, wherein the plasmid, nucleic acid, or construct is contained inside an Adeno-associated viral (AAV) vector.
18. The modified T cell of claim 17, wherein a serotype of the AAV comprises AAV6.
19. The modified T cell of claim 17 or 18, wherein the vector further comprises a plasmid, nucleic acid, or construct encoding a crRNA, tracer RNA (trcrRNA), and a CAS endonuclease.
20. The modified T cell of any of claims 17-19, wherein the vector is a single stranded AAV (ssAAV).
21. The modified T cell of any of claims 17-19, wherein the vector is a self-complimentary AAV (scAAV).
22. The modified T cell of any of claims 17-21, wherein the vector comprises a sequence at least 90% identical to SEQ ID NO: 22 or SEQ ID NO: 23 a fragment thereof.
23. The modified T cell of any of claims 1-22, wherein the T cell has been expanded in the presence of irradiated feeder cells, plasma membrane particles, or exosomes expressing membrane bound IL-21, membrane bound 4-1BBL, and/or membrane bound IL-15 or any combination thereof.
24. A method of treating a cancer in a subject comprising administering to the subject the modified T cell of any one of claims 1-23.
25. A method of treating a cancer in a subject comprising administering to the subject a modified T cell comprising a plasmid, nucleic acid, and/or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems 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 and right homology arms are each 1000bp in length or less.
26. The method of claim 24 or 25, wherein the cancer comprises leukemia.
27. A method of genetically modifying a 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 polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is flanked by homology arms; and wherein the homology arms are 800 bp in length or less; 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 with the AAV into the cell; wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the cell and the T cell’s DNA repair enzymes insert the polynucleotide sequence encoding the CAR polypeptide into the host genome at the target sequence within the genomic DNA of the cell thereby creating a modified T cell.
28. The method of claim 27, wherein the T cell is a primary T cell or an expanded T cell.
29. The method of claim 28, wherein the primary T cell is incubated for about 4 to 10 days in the presence of IL-2, IL-7, and/or IL-15 prior to infection.
30. The method of claim 28 or 29, wherein the primary T cell is expanded for about 4 to 10 days in the presence of irradiated feeder cells, plasma membrane particles, or exosomes prior to infection.
31. The method of claim 30, 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.
32. The method of any one of claims 27-30, further comprising expanding the modified T cell with irradiated feeder cells, plasma membrane particles, or exosomes following 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.
33. The method of any one of claims 27-31, further comprising expanding the modified T cell with IL-2, IL-7, and/or IL-15 following infection.
34. The method of any one of claims 27-32, wherein the T cell is infected with about 5 to 500,000 multiplicity of infection (MOI) of the AAV.
35. The method of any one of claims 27-33, wherein the RNP complex is introduced into the T cell via electroporation.
36. The method of any one of claims 27-34, wherein the RNP complex is introduced into the T cell via transfection; and wherein the RNP complex is encoded on the same or a different AAV.
37. The method of any one of claims 27-35, wherein the CAR polypeptide comprises a transmembrane domain, a co-stimulatory domain, a CD3ζ signaling domain, and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell.
38. The method of claim 37, wherein the receptor comprises CD33.
39. The method of claim 37, wherein the scFV specifically binding to CD33 comprises a sequence at least 90% identical to SEQ ID NO: 29 or a fragment thereof.
40. The method of claim 37, wherein the scFv specifically binding to CD33 comprises SEQ ID NO: 29, or a fragment thereof.
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. The method of any one of claims 27-41, wherein the co-stimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination thereof.
43. The method of any one of claims 27-42, wherein the left homology arm and right homology arm are the same length.
44. The method of claim 43, wherein the homology arms are each 600bp in length.
45. The method of any one of claims 27-42, wherein the left homology arm and right homology arm are different lengths.
46. The method of any one of claims 27-45, wherein the homology arms specifically hybridize to the Adeno-Associated Virus Integration Site 1 (AAVS1) of chromosome 19 of humans.
47. The method of any one of claims 27-46, wherein the plasmid, nucleic acid, or construct further comprises a murine leukemia virus-derived (MND) promoter.
48. The method of any one of claims 27-47, wherein a serotype of the AAV comprises AAV6.
49. The method of any one of claims 27-48, wherein the vector is a single stranded AAV (ssAAV) or a self-complimentary AAV (scAAV).
50. The method of any of claims 27-49, 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. A method of creating 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 polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is flanked by homology arms; and wherein the homology arms are 1000bp in length or less; 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 with the AAV into the T cell; wherein the RNP complex hybridizes to a target sequence within the genomic DNA of the T cell and the DNA repair enzymes of 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 T cell thereby creating a CAR T cell.
52. The method of claim 51, wherein the T cells are primary or expanded T cells.
53. The method of claim 52, wherein the primary T cells are incubated for about 4 to 10 days in the presence of IL-2, IL-7, and/or IL-15 prior to infection.
54. The method of claim 52 or 53, wherein the primary T cells are expanded for about 4 to 10 days in the presence of irradiated feeder cells, plasma membrane particles, or exosomes prior to infection.
55. The method of claim 54, 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.
56. The method of any one of claims 51-55, further comprising expanding the CAR T cell with irradiated feeder cells, plasma membrane particles, or exosomes following 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.
57. The method of any one of claims 51-56, further comprising expanding the CAR T cell with IL-2, IL-7, and/or IL-15 following infection.
58. The method of any one of claims 51-57, wherein the T cell is infected with about 5 to 500K MOI of the AAV.
59. The method of any one of claims 51-58, wherein the RNP complex is introduced into the T cell via electroporation.
60. The method of any one of claims 51-59, wherein the RNP complex is introduced into the T cell via transfection; and wherein the RNP complex is encoded on the same or a different AAV.
61. The method of any one of claims 51-60, wherein the CAR polypeptide comprises a transmembrane domain, a co-stimulatory domain, a CD3ζ signaling domain, and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell.
62. The method of claim 61, wherein the receptor comprises CD33.
63. The method of claim 61, wherein the scFV specifically binding to CD33 comprises a sequence at least 90% identical to SEQ ID NO: 29 or a fragment thereof.
64. The method cell of claim 61, wherein the scFv specifically binding to CD33 comprises SEQ ID NO: 29, or a fragment thereof.
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 a NKG2D transmembrane domain.
66. The method of any one of claims 51-65, wherein the co-stimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination thereof.
67. The method of any one of claims 51-66, wherein the left homology arm and right homology arm are the same length.
68. The method of claim 67, wherein the homology arms are each 600bp in length.
69. The method of any one of claims 51-66, wherein the left homology arm and right homology arm are different lengths.
70. The method of any one of claims 51-69, wherein the homology arms specifically hybridize to the Adeno-Associated Virus Integration Site 1 (AAVS1) of chromosome 19 of humans.
71. The method of any one of claims 51-70, wherein the plasmid, nucleic acid, or construct further comprises a murine leukemia virus-derived (MND) promoter.
72. The method of any one of claims 51-71, wherein a serotype of the AAV comprises AAV6.
73. The method of any one of claims 51-72, wherein the vector is a single stranded AAV (ssAAV) or a self-complimentary AAV (scAAV).
74. The method of any one of claims 51-73, 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. A method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of the CAR T cell created by using the method of any one of claims 51-74.
76. A method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of a T cell, wherein the T cell comprises a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR- associated 9 (Cas9) integration systems 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 and right homology arms are each 1000bp in length or less.
77. The method of claim 75 or 76, wherein the CAR polypeptide comprises a transmembrane domain, a co-stimulatory domain, a CD3ζ signaling domain, and a single-chain variable fragment (scFV) that specifically binds to a receptor on a target cell.
78. The method of claim 77, wherein the receptor comprises CD33.
79. The method of claim 77, wherein the scFV specifically binding to CD33 comprises a sequence at least 90% identical to SEQ ID NO: 29 or a fragment thereof.
80. The method of claim 77, wherein the scFv specifically binding to CD33 comprises SEQ ID NO: 29, or a fragment thereof.
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. The method of any one of claims 75-81, wherein the co-stimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination thereof.
83. The method of any one of claims 75-82, further comprising a polyadenylation signal between the transgene and the right homology arm.
84. The method of any one of claims 75-83, wherein the left homology arm and right homology arm are the same length.
85. The method of claim 84, wherein the homology arms are each 30bp in length.
86. The method of claim 84, wherein the homology arms are each 300bp in length.
87. The method of claim 84, wherein the homology arms are each 600bp in length.
88. The method of claim 84, wherein the homology arms are each 1000bp in length.
89. The method of any one of claims 75-83, wherein the left homology arm and right homology arm are different lengths.
90. The method of any one of claims 75-89, wherein the homology arms specifically hybridize to the Adeno-Associated Virus Integration Site 1 (AAVS1) of chromosome 19 of humans.
91. The method of any one of claims 75-90, further comprising a murine leukemia virus-derived (MND) promoter.
92. The method of any one of claims 75-91, wherein the plasmid, nucleic acid, or construct is transduced into the T cell by an Adeno-associated viral (AAV) vector.
93. The method of claim 92, wherein a serotype of the AAV comprises AAV6.
94. The method of claim 92 or 93, wherein the vector further comprises a plasmid, nucleic acid, or construct encoding a crRNA, tracer RNA (trcrRNA), and a CAS endonuclease.
95. The method of any one of claims 92-94, wherein the vector is a single stranded AAV (ssAAV).
96. The method of any one of claims 92-95, wherein the vector is a self-complimentary AAV (scAAV).
97. The method of any one of claims 92-96, 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. 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 syndromes (MDS).
99. A modified T cell comprising a plasmid, nucleic acid, or construct for use with clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 (Cas9) integration systems, wherein the plasmid, nucleic acid, or construct comprises a polynucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide; wherein the polynucleotide sequence is adjacent to one protospacer adjacent motif (PAM) and one polynucleotide sequence encoding crispr RNA (crRNA) or flanked by two PAMs and two polynucleotide sequences that encode crRNAs.
100. The modified T cell of claim 99, the plasmid, nucleic acid, or construct comprises in order one PAM sequence and one polynucleotide sequence that encodes crRNA, the polynucleotide sequence encoding the CAR polypeptide, and one PAM sequence and one polynucleotide sequence that encodes crRNA.
101. The modified T cell of claim 99 or 100, wherein the CAR polypeptide comprises a transmembrane domain, a co-stimulatory 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 scFV specifically binding to CD33 comprises a sequence at least 90% identical to SEQ ID NO: 29 or a fragment thereof.
104. The modified T cell of claim 101, wherein the scFv specifically binding to CD33 comprises SEQ ID NO: 29, or a fragment thereof.
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. The modified T cell of any one of claims 99-105, wherein the co-stimulatory domain of the CAR polypeptide comprises a 2B4 domain, a CD28 co-stimulatory domain, a 4-1 BB co-stimulatory domain, or any combination thereof.
107. The modified T cell of any one of claims 99-106, further comprising a murine leukemia virus- derived (MND) promoter.
108. The modified T cell of any one of claims 99-107, wherein the plasmid, nucleic acid, or construct is contained inside an Adeno-associated viral (AAV) vector.
109. The modified T cell of claim 108, wherein a serotype of the AAV comprises AAV6.
110. The modified T cell of claim 108 or 109, wherein the vector further comprises a plasmid, nucleic acid, or construct encoding a crRNA, tracer RNA (trcrRNA), and a CAS endonuclease.
111. The modified T cell of any of claims 108-110, wherein the vector is a single stranded AAV (ssAAV) or a self-complimentary AAV (scAAV).
112. The modified T cell of any of claims 99-111, wherein the T cell has been expanded in the presence of irradiated feeder cells, plasma membrane particles, or exosomes expressing membrane bound IL-21, membrane bound 4-1BBL, and/or membrane bound IL-15 or any combination thereof.
113. A method of treating a cancer in a subject comprising administering to a subject with a cancer the modified T cell of any one of claims 99-112.
114. The method of claim 113, wherein the cancer comprises leukemia.
115. A method of creating 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 polynucleotide sequence encoding a chimeric antigen receptor (CAR); wherein the polynucleotide sequence is adjacent to one protospacer adjacent motif (PAM) and one polynucleotide sequence encoding crispr RNA (crRNA) or flanked by two PAMs and two polynucleotide sequences that encode crRNAs; and b) introducing the polynucleotide sequence encoding the CAR polypeptide and the RNP complex into a T cell; wherein the plasmid, nucleic acid, or construct is introduced into the T cell via infection with the Adeno-associated virus (AAV) into a target cell; wherein in the ribonucleoprotein (RNP) complex hybridizes to a target sequence within the genomic DNA of the T cell, and the T cell’s DNA repair enzymes insert the polynucleotide encoding the CAR into the host genome at the target sequence, thereby creating a CAR T cell.
116. The method of claim 115, wherein the plasmid, nucleic acid, or construct comprises in order one PAM sequence and one polynucleotide sequence that encodes crRNA, the polynucleotide sequence encoding the CAR polypeptide, and one PAM sequence and one polynucleotide sequence that encodes crRNA.
117. A method of genetically modifying a 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 polynucleotide sequence encoding a chimeric antigen receptor (CAR); wherein the polynucleotide sequence is adjacent to one PAM and one polynucleotide sequence encoding crRNA or flanked by two PAMs and two polynucleotide sequences encoding crRNAs; 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 with the Adeno-associated virus (AAV) into a target cell; wherein in the ribonucleoprotein (RNP) complex hybridizes to a target sequence within the genomic DNA of the T cell, and the cell’s DNA repair enzymes insert the polynucleotide sequence encoding the chimeric antigen receptor (CAR) into the host genome at the target sequence, thereby creating a modified T cell.
118. The method of claim 117, wherein the plasmid, nucleic acid, or construct comprises in order one PAM sequence and one polynucleotide sequence encoding crRNAs, the polynucleotide sequence encoding the CAR polypeptide, and one polynucleotide sequence that encodes crRNA, and one PAM sequence.
119. The method of claim 117, wherein the plasmid, nucleic acid, or construct comprises in order the polynucleotide sequence encoding the CAR polypeptide, one polynucleotide sequence that encodes crRNA, and one PAM sequence.
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