Methods of increasing NK cell efficacy through inhibition of v-domain IG suppressor of t cell activation (VISTA)

Abstract

Disclosed are V-domain Ig suppressor of T cell activation (VISTA) knock-out natural killer (NK) cells and methods of making the same. Also disclosed are methods of enhancing the efficacy of NK cell killing of cancer cells by blocking VISTA expression and / or signaling.

Description

[0001] METHODS OF INCREASING NK CELL EFFICACY THROUGH INHIBITION OF V- DOMAIN IG SUPPRESSOR OF T CELL ACTIVATION (VISTA)

[0002] I. BACKGROUND

[0003] Gliomas are the most violent primary brain tumor. Glioblastomas (GBM) which are the most common among glioma types, are the most invasive type with a significant cause of death. High-grade glioma (HGG) patients present a median overall survival (OS) of only 1. 164 years (y - hereafter). Contrasting to the 7.023y OS of the Low-grade glioma (LGG) group. Surprisingly, the expression of VSIR (V-set immunoregulatory receptor - also known as VISTA, B7-H5, PD- 1H, Gi24, Diesl, SISP1, and DDla) in LGG patients was associated with a poor prognosis (OS = 1.99y) (TCGA database). Given the association of VISTA expression with poor prognosis, what is needed are new cancer interventions and treatments that reduce the checkpoint inhibition of VISTA.

[0004] II. SUMMARY

[0005] Disclosed are methods and compositions related to the inhibition of VISTA on NK cells.

[0006] In one aspect, disclosed herein are genetically modified NK cell comprising a knockout of the gene encoding the V-domain immunoglobulin suppressor of T cell activation (VISTA) protein.

[0007] Also disclosed herein are methods of adoptively transferring an engineered NK cell to a subject in need thereof said method comprising: a) obtaining a target NK cell to be modified (including, but not limited to an autologous NK cell or NK cell for an allogenic donor source); b) obtaining gRNA that targets the VISTA gene V-set immunoregulatory receptor (VSIR); c) introducing into the target NK cell (such as introduction via electroporation or a viral vector), a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the genomic DNA of the target NK cell creating an engineered NK cell; and d) transferring the engineered NK cell into the subject. In some aspects, the subject has a caner (such as, for example, a glioblastoma (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, and melanoma).

[0008] In some aspects, disclosed herein are methods of adoptively transferring an engineered NK cell to a subject of any preceding aspect, wherein the NK cell is a primary NK cell that is modified ex vivo and after modification transferred to the subject. Also disclosed herein are methods of adoptively transferring an engineered NK cell to a subject of any preceding aspect, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject or wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mb IL-21 expressing feeder cells.

[0009] In one aspect, disclosed herein are methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis (such as for example, a glioblastoma (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, and melanoma) in a subject comprising administering to the subject the NK cell of claim of any preceding aspect or preforming the adoptive transfer method of any preceding aspect. For example, disclosed herein are methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis (such as for example, a glioblastoma (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, and melanoma) in a subject comprising: a) obtaining a target NK cell to be modified (including, but not limited to an autologous NK cell or NK cell for an allogenic donor source); b) obtaining gRNA (such as SEQ ID NOs:3 and 4) that targets the VISTA gene V-set immunoregulatory receptor (VSIR)(SEQ ID NO: 1); c) introducing into the target NK cell (such as introduction via electroporation or a viral vector), a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the VISTA gene of the target NK cell creating an engineered NK cell; and d) transferring the engineered NK cell into the subject.

[0010] In some aspects, disclosed herein are methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis of any preceding aspect, wherein the NK cell is a primary NK cell that is modified ex vivo and after modification transferred to the subject.

[0011] Also disclosed herein are methods of methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis of any preceding aspect, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject or wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mbIL-21 expressing feeder cells.

[0012] In one aspect, disclosed herein are methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis (such as for example, a glioblastoma (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, and melanoma) in a subject comprising administering to the subject a vector (such as, for example a viral vector, including, but not limited to an AAV vector) comprising a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the genomic DNA of the target NK cell.

[0013] Also disclosed herein are methods of enhancing the anti-tumor efficacy of a natural killer (NK) cell comprising a) obtaining a target NK cell to be modified (including, but not limited to an autologous NK cell or NK cell for an allogenic donor source); b) obtaining gRNA (such as SEQ ID NOs:3 and 4) that targets the VISTA gene V-set immunoregulatory receptor (VSIR)(SEQ ID NO: 1); and c) introducing into the target NK cell (such as introduction via electroporation or a viral vector), a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the VISTA gene in the NK cell thereby creating a NK cell with enhanced anti-tumor efficacy.

[0014] In some aspects, disclosed herein are methods of enhancing the anti-tumor efficacy of a natural killer (NK) cell of any preceding aspect, wherein the NK cell is a primary NK cell that is modified ex vivo and after modification transferred to the subject.

[0015] Also disclosed herein are methods of enhancing the anti-tumor efficacy of a natural killer (NK) cell of any preceding aspect, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject or wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mb IL-21 expressing feeder cells.

[0016] In one aspect, disclosed herein are methods of enhancing the anti-tumor efficacy of a natural killer (NK) cell comprising contacting an NK cell with an agent (such as, for example, an siRNA, RNAi, anti-sense oligonucleotide, and / or long non-coding RNA (IncRNA) that binds to the VISTA gene and / or an anti-VSITA antibody (such as, for example, a monoclonal antibody, polyclonal antibody, or any fragment or variant thereof including, but not limited to an anti- VISTA scFv, nanobody, or diabody) that reduces VISTA gene expression or VISTA protein signaling; whereby a reduction in VISTA gene expression or VISTA protein signaling reduces the inhibitory effect on the NK cell thereby enhancing the anti-tumor efficacy of the NK cell. III. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0018] Figure 1 shows a flowchart of NK cell isolation, expansion, and gene editing.

[0019] Figures 2A, 2B, 2C, 2D, 2E, and 2F show elevated expression of VSIR (VISTA) in both LGG and HGG is associated with poor prognosis. HGG vs LGG Overall survival (2 A). High expression level of VSIR (VISTA) is associated with poor OS (2B and 2C). C10orf54 (VISTA) expression and their correlation with SELPLG and IGSF11 (VISTA-Ligands 2D and 2E). PDL / PD-L1 correlation in LGG and HGG (2F).

[0020] Figure 3 shows that VISTA activation mediated by different agonists does not impair NK cell proliferation.

[0021] Figure 4 shows that VISTA activation mediated by VSIG3 does decrease target cell lysis by NK cells. Shown is the precent of cell lysis of wild-type NK cells in the presence of VISTA agonists VSIG8, PSGL1, Syndecan, LRG1, and VSIG3.

[0022] Figures 5A, 5B, and 5C show that C10orf54 (VISTA) expression is induced by expansion with feeder cells, CRISPR / Cas9 can reduce its expression. RNAseq analysis showed that naive NK cells express C10orf54(VISTA) and expansion mediated by mbIL21K562 was able to induce VISTA expression (5A). Quantification of VISTA expression by flow cytometry after CRISPR / Cas9 (5B and 5C)

[0023] Figures 6A, 6B, 6C, 6D, 6E, and 6F show that VISTANO NK cells showed improved killing ability against NK-resistant tumor cell lines. Both WT and VISTAKO NK cells were cocultured with U251 (6A and 6B) or U87 (6C and 6D) Glioma tumor cell lines; RAJI (6E) Burkitt’s lymphoma cell line; or SKMel30 (6F) Melanoma cell line for long-term killing assay by using xCELLigence (a Real-time cell analysis). Bar graph of T4hs (6B and 6D).

[0024] Figure 7 shows that VSIG3 still plays an inhibitor role in VISTAKO NK cells. Shown is the precent of cell lysis of wild-type NK cells and VISTAKO NK cells with and without VSIG3 agonist.

[0025] Figure 8 shows that VISTA deficient (ViDe) NK cells are able to kill multiple tumor cells better than wild-type NK cells.

[0026] IV. DETAILED DESCRIPTION

[0027] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0028] A. Definitions

[0029] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0030] 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.

[0031] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0032] “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. 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.

[0033] 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.

[0034] "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.

[0035] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant 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.

[0036] 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. 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.

[0037] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0038] 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.

[0039] "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.

[0040] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0041] “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.

[0042] “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.

[0043] 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.).

[0044] 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.

[0045] 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).

[0046] 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.

[0047] “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.

[0048] A "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 provided by the disclosure 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.

[0049] "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.

[0050] “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.

[0051] “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 non-immunogenic 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.

[0052] “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 type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. 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.

[0053] 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.

[0054] B. Compositions

[0055] Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular VISTA deficient or knockout natural killer (NK) cell is disclosed and discussed and a number of modifications that can be made to a number of molecules including the VISTA deficient or knockout natural killer (NK) cell are discussed, specifically contemplated is each and every combination and permutation of VISTA deficient or knockout natural killer (NK) cell and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B- D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0056] VSIR (V-set immunoregulatory receptor - also known as V-domain immunoglobulin suppressor of T cell activation (VISTA) , B7-H5, PD-1H, Gi24, Dies 1 , SISP1 , and DDl a) (SEQ ID NO: 2) is encoded by the VSIR gene (SEQ ID NO: 1) located on 10q22.1 within an intron of the CDH23 gene. The VISTA protein is expressed in a wide variety of tissues. VISTA has been shown to be upregulated in T cells upon activation of the immune cell. VISTA’s role within the immune system is as a negative checkpoint receptor associated with immune tolerance.

[0057] Gliomas are the most violent primary brain tumor. Glioblastomas (GBM) which are the most common among glioma types, are the most invasive type with a significant cause of death. High-grade glioma (HGG) patients present a median overall survival (OS) of only 1.164 years (y - hereafter). Contrasting to the 7.023y OS of the Low-grade glioma (LGG) group. Surprisingly, the expression of VSIR (V-set immunoregulatory receptor - also known as VISTA, B7-H5, PD- 1H, Gi24, Diesl, SISP1, and DDla) in LGG patients was associated with a poor prognosis (OS = 1.99y) (TCGA database).

[0058] Data from the literature showed that VISTA expression can inhibit T-cell responses in vitro and preclinical models of autoimmunity and cancer. VISTA was shown to interact with PSGL-l / SELPLG and VSIG3 / IGFS 11 ligands eliciting T cell suppression in vitro. Although many studies showed the importance of VISTA on T cells, prior to the disclosure herein, the role of VISTA on NK cells was still unexplored. The data herein showed that expansion mediated by K562-IL21 feeder cells induced -50% increase of VISTA expression on NK cell surface. Thus, escaping from a potential inhibition mediated by VISTA and its ligands can an improve NK cell recognition and killing ability against GBM. To accomplish this escaped inhibition, VISTAKO NK cells were created to target GBM cells.

[0059] Accordingly, disclosed herein are genetically modified NK cell comprising a knockout of the gene encoding the V-domain immunoglobulin suppressor of T cell activation (VISTA) protein.

[0060] Genetically reprogramming NK cells with plasmids has always been challenging due to difficulties in transgene delivery in a DNA dependent manner such as lentiviral and retroviral transduction causing substantial procedure-associated NK cell apoptosis and the limited production of genetically engineered NK cells. Described herein are methods for using a DNA- free genome editing of primary and expanded human NK cells utilizing endonuclease ribonucleoprotein complexes (such as, for example, Cas9 / RNPs) to reprogram (i.e., engineer or modify) NK cells.

[0061] Endonuclease / RNPs (for example, a Cas9 / RNP) are comprised of three components, recombinant endonuclease protein (for example, a Cas9 endonuclease, a Cast 3 endonucloease, and the like) complexed with a CRISPR loci. The endonuclease complexed to the CRISPR loci can be referred to as a CRTSPR / Cas guide RNA. The CRISPR loci comprises a synthetic singleguide 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. Accordingly, in one aspect, disclosed here are methods of genetically modifying an NK cell comprising obtaining guide RNA (gRNA) specific for a target DNA sequence in the NK cell; and b) transducing (for example, introducing via electroporation) into a target NK cell, a ribonucleoprotein (RNP) complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to the target sequence within the genomic DNA of the NK cell.

[0062] 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) (such as SEQ ID NOs:3 and 4). The plasmids / vectors delivering the RNP compliex can target the VISTA gene located on 10q22.1 within an intron of the CDH23 gene.

[0063] It is understood and herein contemplated that there can be size limits on the donor transgene construct size delivered to the target genome. One method of increasing the allowable size of the transgene is to create additional room by exchanging the Cas9 of Streptococcus pyogenes (SpCas9) typically used for a synthetic Cas9, or Cas9 from a different bacterial source. Substitution of the Cas9 can also be used to increase the targeting specificity so less gRNA needs to be used. Thus, for example, the Cas9 can be derived from Staphylococcus aureus (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiracase bacterium (LbCpfl), Neisseria meningitidis (NmCas9), Streptococcus thermophilus (StCas9), Campylobacter jejuni (CjCas9), enhanced SpCas9 (eSpCas9), SpCas9-HFl, Fokl-Fused dCas9, expanded Cas9 (xCas9), and / or catalytically dead Cas9 (dCas9).

[0064] 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 (LbCpfl PAM and AsCpfl PAM); TYCV (LbCpfl PAM variant and AsCpfl 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.

[0065] 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 50 pM and about 500 DM (for example, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 35, 375, 400, 425, 450, 475, or 500pM), preferably between 100 p and about 300 pM, most preferably about 200 pM at 95C 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 20pM and about 50pM (for example 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 48, 49, or 50DM) final dilution of a Cas endonuclease (such as, for example, Cas9).

[0066] 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 electroporation of Cas9 / RNPs is an easy and relatively efficient method that overcomes the previous constraints of genetic modification in NK cells.

[0067] Human NK cells are a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of T cell receptor (CD3). NK cells sense and kill target cells that lack major histocompatibility complex (MHC)-class I molecules. NK cell activating receptors include, among others, the natural cytotoxicity receptors (NKp30, NKp44 and NKp46), and lectin-like receptors NKG2D and DNAM-1. Their ligands are expressed on stressed, transformed, or infected cells but not on normal cells, making normal cells resistant to NK cell killing. NK cell activation is negatively regulated via inhibitory receptors, such as killer immunoglobin (Ig)— like receptors (KIRs), NKG2A / CD94, TGFD, and leukocyte Ig-like receptor- 1 (LIR-1). In one aspect, the target cells can be primary NK 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 NK cells), NK cell line (including, but not limited to NK RPMI8866; HFWT, K562, and EBV-LCL ), or from a source of expanded NK cells derived a primary NK cell source or NK cell line.

[0068] Prior to the transduction of the NK cells, the NK cell can be incubated in a media suitable for the propagation of NK 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 an NK cell, further comprising incubating the NK cells for 1, 2, 3, 4, 5, 6,7 ,8 9, 10, 11, 12, 13, or 14 days prior to transducing the cells in media that supports the propagation of NK cells; wherein the media further comprises cytokines, antibodies, and / or feeder cells. For example, the media can comprise IL-2, 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 NK cells. NK 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 NK cell feeder cells provided in combination with a solution of IL-21, IL- 15, and / or 41BBL. Feeder cells can be seeded in the culture of NK cells at a 1 :2, 1: 1, or 2:1 ratio. The It is understood and herein contemplated that the period of culturing can be between 1 and 14 days post 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.

[0069] It is understood and herein contemplated that the incubation conditions for primary NK cells and expanded NK cells can be different. In one aspect, the culturing of primary NK cells prior to electroporation comprises media and cytokines (such as, for example, IL-2, 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 NK cells the culturing can occur in the presence of NK feeder cells (at for example, a 1:1 ratio) in addition to or in lieu of cytokines (such as, for example, IL-2, IL-12, IL- 15, IL-18, and / or IL-21) and / or anti-CD3 antibody. Culturing of expanded NK 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 an NK cell comprising incubating primary NK cells for 4 days in the presence of IL-2 prior to electroporation or incubating expanded NK cells in the presence of irradiated feeder cells for 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42, 48, 54, 60 hours, 3, 4, 5, 6, or 7 days prior to electroporation. It is understood and herein contemplated that methods of transduction to modify NK cells in the disclosed methods are limited. Due to their immune function, NK cells are resistant to viral and bacterial vectors and the induction of NK cell apoptosis by said vectors. Thus, prior to the present methods CRISPR / Cas modification of NK cells has been unsuccessful. To circumvent problems with viral vectors, the disclosed methods transform the target NK cells using electroporation. Electroporation is a technique in which an electric field is applied to cells to increase the permeability of the cell membrane. The application of the electric filed cause a charge gradient across the membrane which draws the charged molecules such as, nucleic acid, across the cell membrane. Thus, in one aspect, disclosed herein are methods of genetically modifying an NK cell comprising obtaining guide RNA (gRNA) specific for a target DNA sequence in the NK cell; and b) introducing via electroporation into a target NK cell, a ribonucleoprotein (RNP) complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to the target sequence within the genomic DNA of the NK cell.

[0070] Following transduction (e.g., electroporation) of the NK cell, the now modified NK cell can be propagated in a media comprising feeder cells that stimulate the modified NK cells. Thus, the modified cells retain viability and proliferative potential, as they are able to be expanded post-electroporation using irradiated feeder cells. NK 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 NK cell feeder cells provided in combination with a solution of IL-21, IL-15, and / or 41BBL. Feeder cells can be seeded in the culture of NK cells at a 1:2, 1: 1, or 2:1 ratio. The It is understood and herein contemplated that the period of culturing can be between 1 and 14 days post 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 NK cells can further comprise cytokines such as, for example, IL-2, IL-12, IL-15, IL-18, and / or IL-21.

[0071] As noted throughout the present disclosure, the disclosed modified NK cells are ideally suited for use in immunotherapy such as the adoptive transfer of modified (i.e, engineered NK cells to a subject in need thereof. Thus, in one aspect, disclosed herein are methods of adoptively transferring an engineered NK cells to a subject in need thereof said method comprising a) obtaining a target NK cell to be modified; b) obtaining gRNA specific for a target DNA sequence; c) introducing via electroporation into the target NK cell, a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas gRNA that hybridizes to the target sequence within the genomic DNA of the target NK cell creating an engineered NK cell; and d) transferring the engineered NK cell into the subject.

[0072] Accordingly, disclosed herein are methods of adoptively transferring an engineered NK cell to a subject in need thereof said method comprising: a) obtaining a target NK cell to be modified (including, but not limited to an autologous NK cell or NK cell for an allogenic donor source); b) obtaining gRNA (such as SEQ ID NOs:3 and 4) that targets the VISTA gene V-set immunoregulatory receptor (VSIR)(SEQ ID NO: 1); c) introducing into the target NK cell (such as introduction via electroporation or a viral vector), a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the genomic DNA of the target NK cell creating an engineered NK cell; and d) transferring the engineered NK cell into the subject. In some aspects, the subject has a cancer (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, renal cell carcinoma, pancreatic cancer (such as, for example, pancreatic adenocarcinoma), lung cancer (including, but not limited to non-small cell lung cancer), prostate cancer, colorectal cancer, acute myeloid leukemia, ovarian cancer, endometrial cancer, Esophageal adenocarcinoma, gastric cancer, hepatic caner, ovarian cancer, and melanoma).

[0073] Alternatively, VISTA expression in the NK cell can be reduced through use of an antisense oligonucleotide, siRNA, RNAi, or IncRNA that hybridizes to the VISTA gene. Thus, in one aspect, disclosed herein are methods of adoptively transferring an engineered NK cell to a subject in need thereof said method comprising: a) obtaining a target NK cell to be modified (including, but not limited to an autologous NK cell or NK cell for an allogenic donor source); b) contacting the NK cell with an antisense oligonucleotide, siRNA, RNAi, or IncRNA that hybridizes to the VISTA gene; and c) transferring the engineered NK cell into the subject. In some aspects, the subject has a caner (such as, for example, a glioblastoma (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, renal cell carcinoma, pancreatic cancer (such as, for example, pancreatic adenocarcinoma), lung cancer (including, but not limited to non-small cell lung cancer), prostate cancer, colorectal cancer, acute myeloid leukemia, ovarian cancer, endometrial cancer, Esophageal adenocarcinoma, gastric cancer, hepatic caner, ovarian cancer, and melanoma).

[0074] In one aspect, the modified NK cells used in the disclosed immunotherapy methods can be primary NK 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 NK cells), NK cell line (including, but not limited to NK RPMI8866; HFWT, K562, and EBV-LCL ), or from a source of expanded NK cells derived a primary NK cell source or NK cell line. Because primary NK cells can be used, it is understood and herein contemplated that the disclosed modifications of the NK cell can occur ex vivo or in vitro. In some aspects, disclosed herein are methods of adoptively transferring an engineered NK cell to a subject, wherein the NK cell is a primary NK cell that is modified ex vivo and after modification transferred to the subject.

[0075] Following transduction of the NK cells, the modified NK cells can be expanded and stimulated prior to administration of the modified (i.e., engineered) NK cells to the subject. For example, disclosed herein are methods of adoptively transferring NK cells to a subject in need thereof wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject. In some aspect, it is understood and herein contemplated that eh stimulation and expansion of the modified (i.e, engineered) NK cells can occur in vivo following or concurrent with the administration of the modified NK cells to the subject. Accordingly disclosed herein are immunotherapy methods wherein the NK cells are expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mbIL-21 expressing feeder cells. Thus, also disclosed herein are methods of adoptively transferring an engineered NK cell to a subject, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject or wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mbIL-21 expressing feeder cells.

[0076] 1. Homology / identity

[0077] It is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequences. Specifically disclosed are variants of these and other genes and proteins herein disclosed which have at least, 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 percent homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0078] 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.

[0079] 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 which are herein incorporated by reference for at least material related to nucleic acid alignment.

[0080] 2. Hybridization / selective hybridization

[0081] 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.

[0082] 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.

[0083] 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 nonlimiting 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 3. Nucleic acids

[0088] There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example the VISTA gene or gRNA (such as SEQ ID NOs:3 and 4) that target the VISTA gene, or any of the nucleic acids disclosed herein for making VISTA knockouts, or fragments thereof, as well as various functional nucleic acids. 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, that 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 advantagous 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

[0089] 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 intemucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-l-yl (C), guanin-9-yl (G), uracil-l-yl (U), and thymin-l-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. An 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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, Nl, 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.

[0094] 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

[0095] There are a variety of sequences related to VISTA that are disclosed herein, 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 anlogs, 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

[0096] Disclosed are compositions including primers and probes, which are capable of interacting with the disclosed nucleic acids, such as the VISTA gene 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.

[0097] 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,

[0098] 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,

[0099] 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,

[0100] 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750,

[0101] 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.

[0102] In other embodiments a primer or probe can be less than or equal to 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,

[0103] 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,

[0104] 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,

[0105] 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,

[0106] 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.

[0107] The primers for the VISTA gene typically will be used to produce an amplified DNA product that contains a region of the VISTA 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. 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,

[0108] 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,

[0109] 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225,

[0110] 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.

[0111] 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,

[0112] 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,

[0113] 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175,

[0114] 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.

[0115] 4. Delivery of the compositions to cells

[0116] 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

[0117] 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)).

[0118] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as the RNP complex targeting VISTA into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. In some embodiments the vectors facilitating delivery are derived from either a virus or a retrovirus. 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 pay load, 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.

[0119] 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.

[0120] (1) Retroviral Vectors

[0121] A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer.

[0122] A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3’ LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. The removal of the gag, pol, and env genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

[0123] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

[0124] (2) Adenoviral Vectors

[0125] The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61: 1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj- Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61: 1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92: 1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4: 154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73: 1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5: 1287- 1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor- mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650- 655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061- 6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0126] A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.

[0127] (3) Adeno-asscodated viral vectors

[0128] 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. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0129] 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.

[0130] 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 sitespecific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No. 6,261,834 is herein incorproated by reference for material related to the AAV vector. The disclosed vectors thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.

[0131] 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.

[0132] (4) Large payload viral vectors

[0133] Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter and Robertson, .Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

[0134] Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. b) Non-nucleic acid based systems

[0135] The disclosed compositions can be delivered to the target cells in a variety of ways. For example, the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.

[0136] Thus, the compositions can comprise, in addition to the disclosed vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0137] In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECT AMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECT AM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

[0138] 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)). These techniques can be used for a variety of other speciifc cell types. 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)).

[0139] Nucleic acids that are delivered to cells which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral intergration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can be come integrated into the host genome.

[0140] Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art. c) In vivo / ex vivo

[0141] As described above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject’s cells in vivo and / or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

[0142] If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

[0143] 5. Pharmaceutical carriers / Delivery of pharmaceutical products

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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, 1 104:179-187, (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)). a) Pharmaceutically Acceptable Carriers

[0148] The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0149] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0150] Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0151] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

[0152] The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0153] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0154] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0155] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

[0156] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. b) Therapeutic Uses

[0157] Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 pg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above.

[0158] 6. Antibodies

[0159] (1) Antibodies Generally

[0160] The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with VISTA such that VISTA is inhibited from interacting with Sdc-2, VSIG-3, VSIG-8, PSGL-1, LRIG-1, MMP-13, Syndecan, and / or Galectin-9. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and / or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0161] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0162] The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[0163] The monoclonal antibodies may also be made by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

[0164] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen. As used herein, the term “antibody or fragments thereof” encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab, Fv, sFv, scFv, and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain VISTA binding activity are included within the meaning of the term “antibody or fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0165] Also included within the meaning of “antibody or fragments thereof” are conjugates of antibody fragments and antigen binding proteins (single chain antibodies).

[0166] The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove / add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0167] As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and / or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

[0168] (2) Human antibodies

[0169] The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.

[0170] (3) Humanized antibodies

[0171] Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an sFv, Fv, Fab, Fab’, F(ab’)2, or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.

[0172] To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

[0173] Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No. 5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. Patent No. 5, 939,598 (Kucherlapati et al.), U.S. Patent No. 6,130,364 (Jakobovits et al.), and U.S. Patent No. 6,180,377 (Morgan et al.).

[0174] (4) Administration of antibodies

[0175] Administration of the antibodies can be done as disclosed herein. Nucleic acid approaches for antibody delivery also exist. The broadly neutralizing anti- VISTA antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient’s or subject’s own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment. The delivery of the nucleic acid can be by any means, as disclosed herein, for example.

[0176] C. Method of treating cancer

[0177] The disclosed NK cell knockout can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin’s Disease;, Burkitt’s lymphoma, myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and / or chronic myeloid leukemia (CML)); bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer; lung cancers such as small cell lung cancer, non-small cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LU AD); neuroblastoma / glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer including, but not limited to triple negative breast cancer; genitourinary cancer; pulmonary cancer; esophageal carcinoma; head and neck carcinoma; large bowel cancer; hematopoietic cancers; testicular cancer; and colon and rectal cancers.

[0178] In one aspect, the treatment of the cancer can include the administration NK cells that have been modified to have reduced expression of VISTA, the administration of a vector that comprises an RNP complex that targets VISTA, the administration of an antibody that blocks the interaction of VISTA with its ligand, as well as, siRNA, RNAi, and / or IncRNA that target VISTA. In some aspects, the contact of the NK cell with the vector, siRNA, RNA, and / or IncRNA can occur ex vivo.

[0179] In one aspect, disclosed herein are methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis (such as for example, a glioblastoma (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, renal cell carcinoma, pancreatic cancer (such as, for example, pancreatic adenocarcinoma), lung cancer (including, but not limited to non-small cell lung cancer), prostate cancer, colorectal cancer, acute myeloid leukemia, ovarian cancer, endometrial cancer, Esophageal adenocarcinoma, gastric cancer, hepatic caner, ovarian cancer, and melanoma) in a subject comprising administering to the subject any engineered / modified NK cell disclosed herein (such as, for example, an NK cell comprising a VISTA knockout) or preforming any adoptive cell transfer method (i.e., adoptive cell therapy) disclosed herein. For example, disclosed herein are methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis (such as for example, a glioblastoma (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, renal cell carcinoma, pancreatic cancer (such as, for example, pancreatic adenocarcinoma), lung cancer (including, but not limited to non-small cell lung cancer), prostate cancer, colorectal cancer, acute myeloid leukemia, ovarian cancer, endometrial cancer, Esophageal adenocarcinoma, gastric cancer, hepatic caner, ovarian cancer, and melanoma) in a subject comprising: a) obtaining a target NK cell to be modified (including, but not limited to an autologous NK cell or NK cell for an allogenic donor source); b) obtaining gRNA (such as SEQ ID NOs:3 and 4) that targets the VISTA gene V-set immunoregulatory receptor (VSIR)(SEQ ID NO: 1); c) introducing into the target NK cell (such as introduction via electroporation or a viral vector), a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the VISTA gene of the target NK cell creating an engineered NK cell; and d) transferring the engineered NK cell into the subject.

[0180] In some aspects, disclosed herein are methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis, wherein the NK cell is a primary NK cell that is modified ex vivo and after modification transferred to the subject.

[0181] Also disclosed herein are methods of methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject or wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mbIL-21 expressing feeder cells.

[0182] In one aspect, disclosed herein are methods of treating inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer and / or metastasis (including, but not limited to low grade glioblastoma (LGG) and high grade glioblastoma (HGG), Burkitt’s lymphoma, renal cell carcinoma, pancreatic cancer (such as, for example, pancreatic adenocarcinoma), lung cancer (including, but not limited to non-small cell lung cancer), prostate cancer, colorectal cancer, acute myeloid leukemia, ovarian cancer, endometrial cancer, Esophageal adenocarcinoma, gastric cancer, hepatic caner, ovarian cancer, and melanoma) in a subject comprising administering to the subject a vector (such as, for example a viral vector, including, but not limited to an AAV vector) comprising a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the genomic DNA of the target NK cell.

[0183] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC- T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S -Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil— Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL- PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S- Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-AB V, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR- U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil— Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EV ACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil— Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (FluorouraciL -Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRLBEVACIZUMAB, FOLFIRI- CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYC AMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa- 2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado-Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trilluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METH AZOL AS TONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin- stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nel arabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa- 2b, PEG-INTRON® (Peginterferon Alfa- 2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (SipuleuceLT), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa- 2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R-EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R-ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine 1 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISEN OX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XT ANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv- Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / or ZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT-011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHlgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS-986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS 118, MGD013, and Immutep).

[0184] It is understood and herein contemplated that inhibiting VISTA expression and / or signaling can block the inhibitor effects of VISTA on NK cell activation and as a consequence anti-tumor efficacy of NK cells can be increased and / or enhanced. Thus, in one aspect, disclosed herein are methods of increasing or enhancing the anti-tumor efficacy of a natural killer (NK) cell comprising a) obtaining a target NK cell to be modified (including, but not limited to an autologous NK cell or NK cell for an allogenic donor source); b) obtaining gRNA (such as SEQ ID NOs:3 and 4) that targets the VISTA gene V-set immunoregulatory receptor (VSIR)(SEQ ID NO: 1); and c) introducing into the target NK cell (such as introduction via electroporation or a viral vector), a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the VISTA gene in the NK cell thereby creating a NK cell with enhanced anti-tumor efficacy.

[0185] In some aspects, disclosed herein are methods of enhancing the anti-tumor efficacy of a natural killer (NK) cell, wherein the NK cell is a primary NK cell that is modified ex vivo and after modification transferred to the subject.

[0186] Also disclosed herein are methods of enhancing the anti-tumor efficacy of a natural killer (NK) cell, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject or wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mbIL-21 expressing feeder cells.

[0187] In one aspect, disclosed herein are methods of enhancing the anti-tumor efficacy of a natural killer (NK) cell comprising contacting an NK cell with an agent (such as, for example, an siRNA, RNAi, anti-sense oligonucleotide, and / or long non-coding RNA (IncRNA) that binds to the VISTA gene (SE ID NO: 1) and / or an anti-VSITA antibody (such as, for example, a monoclonal antibody, polyclonal antibody, or any fragment or variant thereof including, but not limited to an anti- VISTA scFv, nanobody, or diabody that can bind VISTA (SEQ ID NO: 2)) that reduces VISTA gene expression or VISTA protein signaling; whereby a reduction in VISTA gene expression or VISTA protein signaling reduces the inhibitory effect on the NK cell thereby enhancing the anti-tumor efficacy of the NK cell.

[0188] D. Examples

[0189] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0190] 1. Example 1

[0191] Gliomas are the most violent primary brain tumor. Glioblastomas (GBM) which are the most common among glioma types, are the most invasive type with a significant cause of death. High-grade glioma (HGG) patients present a median overall survival (OS) of only 1.164 years (y - hereafter); contrasting to the 7.023y OS of the Low-grade glioma (LGG) group.

[0192] VISTA a B7 family ligand is expressed on B, T, and NK cells with low to moderate expression. Data from the literature showed that VISTA expression can inhibit T-cell responses in vitro and preclinical models of autoimmunity and cancer. VISTA was shown to interact with PSGL-l / SELPLG and VSIG3 / IGFS11 ligands eliciting T cell suppression in vitro (Figure 2). As shown in Figure 2A, 2B, and 2C VISTA expression was elevated in both LGG and HGG. Surprisingly, the expression of VSIR (V-set immunoregulatory receptor - also known as VISTA, B7-H5, PD-1H, Gi24, Diesl, SISP1, and DDla) is not just associated with poor prognosis in HGG, but in LGG elevated VISTA was also associated with a poor prognosis in patience (OS = 1.99y) (TCGA database). VISTA expression was also shown to strongly correlate with the VISTA ligands SELPLG and IGSF11 (Figures 2D and 2E) and also correlate with PD1 / PDL1 expression (Figure 2F). Although many studies showed the importance of VISTA on T cells, prior to the disclosure herein, the role of VISTA on NK cells was still unexplored.

[0193] Investigating the impact of VISTA activation mediated by VISTA ligands LRIG1, PSGL1, Syndecan, VSIG3 and VSIG8, we found no impact on NK cell proliferation (Figure 3). Further, with the exception of VSIG3, VISTA activation did not decrease target cell lysis (Figure 4). The data herein shows that expansion mediated by K562-IL21 feeder cells induced -50% increase of VISTA expression on NK cell surface (Figure 5A). Thus, escaping from a potential inhibition mediated by VISTA and its ligands can an improve NK cell recognition and killing ability against GBM. To accomplish this escaped inhibition, VISTAKO NK cells were created to target GBM cells. A Cas9 / RNP methodology was used to generate stable VISTAKO NK cells. Specifically, NK cells were isolated from buffy coat and expanded in the presence of K562 feeder cells that expressed membrane bound IL-21 on their cell surface (mbIL21-K562). Following expansion, NK cells were electroporated to introduce a a ribonucleoprotein (RNP) complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to the gene encoding VISTA in the NK cell (Figure 1). The data showed that the successful generation of VISTAKO NK cells, by decreasing 54+14% of VISTA expression on NK cells (Figures 5B and 5C). Next, WT and VISTAKO NK cells were submitted to a long-term killing assay by using xCELLigence (a Realtime cell analysis). To test the efficacy of both WT and VISTA KO NK cells, we used U251MG (Figure 6A and 6B) and U87MG (Figure 6C and 6D) glioma tumor cell lines, RAJI (Figure 6E) a Burkitt’s lymphoma cell line, and SKMel30 (Figure 6F) a melanoma cell line. The data showed that VISTAKO NK cells showed better killing against all cell lines when compared to WT NK cells during all 46 hours of experiments. Unexpectedly, our data showed that in a lower E:T ratio (0.5: 1) WT N K cells failed to kill U87MG cells, presenting 0% of cell lysis. However, knocking out VISTA from NK cells improved killing by -30%. Thus, deleting VISTA with CRISPR / Cas9 can generate genetically modified NK cells with enhanced ability to target cancers and overcome the hostile tumor microenvironment improving the clinical outcomes of cancer immunotherapy in gliomas and other NK cell resistant cancers like Burkitt’s lymphoma and melanoma. Surprisingly, NK cell mediated lysis of target cells was not effected when VISTA was activated via VSIG3. a) Methods

[0194] (1) Human NK cell purification and expansion

[0195] Healthy donor buffy coats were obtained as source material from the Central Ohio Region American Red Cross. This research was determined to be exempt research by the Institutional Review Board of Nationwide Children’s Hospital.

[0196] Isolate PBMCs from Buffy Coat. Briefly, Layer 35 mL of buffy coat sample on 15 ml Ficol-Paque. Centrifuge at 400 x g for 20 minutes without brake. Wash the recovered PBMCs three times with PBS. NK cell can be isolated at this stage by RosettesSep. Expand NK cells by stimulating with irradiated 10 x 106 mblL21 -expressing feeder cells at Days 0, 7, and 14. Replace media with fresh AIMV or RPMI containing 10% FBS, 1% Glutamine, 1% Penicillin Streptomycin and 100 lU / mL of IL-2 for the entire media volume every other day.

[0197] (2) gRNA design and selection

[0198] CRISPR design web tools was used to design guide RNA. The VISTA gene (also known as the VSIR gene) located on 10q22.1 within an intron of the CDH23 gene was chosen as a target genome. CRISPR guides (20 nucleotides followed by a PAM sequence: NGG) were scanned from the sequence entered earlier. gRNAs which have the highest score were selected based on their on- target and off-target rates.

[0199] The CRISPR guide RNAs (gRNAs) disigned to target the VISTA gene indicated by CRISPR design web tools are gRNA 1 AAGUUCCUCUGCGCGUCCGA (SEQ ID NO: 3) and gRNA 2 UGCGCGUCCGACGGCGACAU (SEQ ID NO: 4). The CRISPR RNAs were ordered as synthetic sequence-specific crRNAs and a conserved, transactivating RNA (tracrRNA) was ordered to interact through partial homology with the crRNA.

[0200] (3) Transduction of Human Primary and Expanded NK cells

[0201] Transduction of Cas9 / RNPs elements into NK is done by electroporation using 4D- Nucleofector System as follows:

[0202] (4) Cell Preparation

[0203] For primary NK cells, incubate freshly isolated NK cells in RPMI or AIMV medium in the presence of 100 lU / mL of IL-2 for 4 days and perform the electroporation at Day 5 (Replace the media every other day as described earlier and the day before transduction). This can be modified for expanded NK cells. For expanded NK cells, Stimulate the cells at day 0 with irradiated feeder cells at a ratio of 1 : 1 and perform the electroporation at Day 5 or 6 or 7. (Replace the media every other day as described earlier and the day before transduction). At the day of electroporation prepare a T25 flask filled with 8 ml fresh RPMI containing 100 lU / mL of IL-2 for cells undergoing electroporation and pre-incubate flasks in a humidified 37 °C / 5 % CO2 incubator. Thawed cells or cells that have undergone 2nd or 3rd stimulation can be electroporated at any time after their recovery as described. Take 3-4 x 106 cells per condition for 26, u L transduction mix as a very high concentration of NK cells in Nucleofector Solution enhances the transduction rate. Cells 3 times can be washed with PBS to remove all FBS, which commonly contains RNase activity. Spin them down each time at 300g for 8 minutes. (5) Form the crRNA:tracerRNA / complex crRNAs and TracerRNA were resuspended in IX TE solution to final concentrations of 200pM. Mix 2.2pl of each 200pM gRNA with 200 pM TracerRNA as shown in table 1. Heat the samples at 95 °C for 5 min and allow to cool on the bench top to room temperature (15-25 °C). Resuspended RNAs and crRNA: tracerRNA / complex were stored at -20 °C for later use.

[0204] Table 1. Form the crRNA:tracerRNA / complex using 200 pM RNAs

[0205] (6) Form the RNP complex

[0206] To save time, the RNP complex can be formed during the washing step. For single crRNA:tracrRNA duplex reaction, dilute Cas9 Endonuclease to 36 pM as mentioned in Table 2.

[0207] Table 2. For single crRNA:tracrRNA duplex reaction, Dilute Cas9 endonuclease to 36 pM.

[0208] For combination transduction of crRNA:tracrRNA duplexes dilute Cas9 Endonuclease to 36 pM as shown in Table 3.

[0209] Table 3. For combination transduction of crRNA:tracrRNA duplexes were diluted Cas9 endonuclease to 36 pM.

[0210] Cas9 Endonuclease was to crRNA: tracrRNA duplexes slowly while swirling pipette tip, over 30 s to 1 minute. Incubate the mixture at room temperature for 15-20 min

[0211] (7) Electroporation

[0212] The entire supplement to the Nucleofector Solution P3 was added and the mixture kept at room temperature. Cell pellets (3-4 x 106 cells) were resuspended in 20pl of P3 Primary 4D Nucleofector Solution. 5pL of RNP complex was added to the cell suspension. Ipl of lOOpM Cas9 electroporation Enhancer was added to the Cas9 / RNPs / cell mix. Cas9 / RNPs / cell mix was transferred into 20pl Nucleocuvette Strips. (8) Post Transduction

[0213] Cells were rested for 3 minutes in strips. After resting, 80 pL of the pre-equilibrated culture media was added to the cuvette and the sample was gently transferred into flasks. 48 hours after transduction, genomic DNA was extracted from 5 x 105 cells for the gene deletions screening.

[0214] SEQ ID NO: 1 nucleic acid sequence for V-set immunoregulatory receptor (VSIR)- also known as V-domain immunoglobulin suppressor of T cell activation (VISTA) ccggccgcgt cccgcct ccc cggcaccaga agatcctctg cgcgtccgac ggcgacatgg 60 gcgtccccac ggccctggag gccggcagct ggcgctgggg atccctgct c ttcgctctct 120 t cctggctgc gtccctaggt ccggtggcag ccctcaaggt cgccacgccg t attccctgt 180 atgtctgt cc cgaggggcag aacgt caccc t cacctgcag gctcttgggc cctgtggaca 240 aagggcacga tgtgaccttc tacaagacgt ggc accgcag ctcgaggggc gaggtgcaga 300 cctgctcaga gcgccggccc atccgcaacc t cacgttcca ggat ctt cac ctgcaccatg 360 gaggccacca ggctgccaac accagccacg acctggctca gcgccacggg ctggagt cgg 420 ccL ccgacca cca Lggcaac L Lc Loca l. ca cca lgcgcaa cc LgacccLg c Lgga Lagcg 480 gcctctactg ctgcctggtg gtggagat ca ggcaccacca ctcggagcac agggtccatg 540 gtgccatgga gctgcaggtg cagacaggca aagatgcacc atccaactgt gtggtgt acc 600 cat cctcctc ccaggagagt gaaaacat ca cggctgcagc cctggct acg ggtgcctgca 660 t cgtaggaat cct ctgcctc cccct cat cc tgctcctggt ctacaagcaa aggcaggcag 720 cct ccaaccg ccgtgcccag gagctggtgc ggatggacag caacatt caa gggattgaaa 780 accccggctt tgaagcct ca ccacctgccc aggggatacc cgaggccaaa gtcaggcacc 840 ccctgtccta tgtggcccag cggcagcctt ctgagtctgg gcggcat ctg ctttcggagc 900 ccagcacccc cctgt ct cct ccaggccccg gagacgtctt cttcccatcc ctggaccctg 960 t ccctgactc tccaaacttt gaggt cat ct agcccagctg ggggacagtg ggctgttgtg 1020 gctgggtctg gggcaggtgc atttgagcca gggctggct c tgtgagtggc ctccttggcc 1080 t cggccctgg ttccctccct cctgctctgg gco cagatac tgtgacatcc caaaagccca 1140 gcccctcaac ccctctggat gct acatggg gacgctggac ggct cagccc ctgttccaag 1200 gat tttgggg tgctgagatt ctcccctaga gacctgaaat tcaccagct a cagatgccaa 1260 atgacttaca tct taagaag tct caaaacg t ccagccctt cagcagctct cgttctgaga 1320 catgagcctt gggatgtggc agcat cagtg ggacaagatg gacactgggc caccctccca 1380 ggcaccagac acagggcacg gtggagagac ttctcccccg tggccgcctt ggctcccccg 1440 ttttgcccga ggctgct ctt ctgtcagact t cctctttgt accacagtgg ctctggggcc 1500 aggcctgcct gcccactggc cat cgccacc t tccccagct gcct cct acc agcagtt tct 1560 ctgaagat ct gtcaacaggt taagt caatc tggggcttcc actgcctgca ttccagt ccc 1620 cagagcttgg tggtcccgaa acgggaagta cac attgggg catggtggcc t ccgtgagca 1680 aatggtgt ct tgggcaat ct gaggccagga cagatgttgc cccacccact ggagatggtg 1740 ctgagggagg tgggtggggc ctt ctgggaa ggcgagtgga gaggggcacc tgccccccgc 1800 cct ccccatc ccctact ccc actgctcagc gcgggccat t gcaagggtgc cacacaatgt 1860 cttgt ccacc ctgggacact tctgagtatg aagcgggatg ctattaaaaa ctacatgggg 1920 aaacaggtgc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1977

[0215] SEQ ID NO: 2 Amino acid sequence for VISTA

[0216] MGVPTALEAGSWRWGSLLFALFLAASLGPVAAFKVATPYSLYVCPFTEGQNVTLTCRLLGPVD

[0217] KGHDVTFYKTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHFTQAANTSHDLAQRHGLESA

[0218] SDHHGNFSITMRNLTLLDSGLYCCLVVEIRHHHSEHRVHGAFTMELQVQTGKDAPSNCVVYPSS

[0219] SQESENITAAALATGACIVGILCLPLILLLVYKQRQAAFTSNRRAQELVRMDSNIQGIENPGFEAS

[0220] PPAQGIPEAKVRHPLSYVAQRQPSESGRHLLSEFTPSTPLSPPGPGDVFFPSLDPVPDSPNFEVI

Claims

V. CLAIMSWhat is claimed is:

1. A genetically modified NK cell comprising a knockout of the gene encoding the V- domain immunoglobulin suppressor of T cell activation (VISTA) protein.

2. A method of adoptively transferring an engineered NK cell to a subject in need thereof said method comprising a) obtaining a target NK cell to be modified; b) obtaining gRNA that targets the VISTA gene V-set immunoregulatory receptor (VSIR); c) introducing into the target NK cell, a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the VISTA gene of the target NK cell creating an engineered NK cell; and d) transferring the engineered NK cell into the subject.

3. The method of claim 2, wherein the subject has a cancer.

4. The method of claim 3, wherein the cancer is selected from the group consisting of glioblastoma, Burkitt’s lymphoma, and melanoma.

5. The method of claim 2 or 3, wherein the NK cell is a primary NK cell that is modified ex vivo and after modification transferred to the subject.

6. The method of any one of claims 2-5, wherein the NK cell is an autologous NK cell.

7. The method of any one of claims 2-5, wherein the NK cell is from an allogeneic donor source.

8. The method of any one of claims 2-7, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject.

9. The method of any one of claims 2-8, wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mbIL-21 expressing feeder cells.

10. The method of any one of claims 2-9, wherein the RNP complex is introduced into the target NK cell vial electroporation.

11. A method of treating a cancer in a subject comprising administering to the subject the NK cell of claim 1 or preforming the adoptive transfer of an engineered NK cell method of any one of claims 2-10.

12. A method of treating a cancer in a subject comprising: a) obtaining a target NK cell to be modified; b) obtaining gRNA that targets the VISTA gene V-set immunoregulatory receptor (VSIR); c) introducing into the target NK cell, a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the VISTA gene of the target NK cell creating an engineered NK cell; and d) transferring the engineered NK cell into the subject.

13. The method of claim 12, wherein the NK cell is a primary that is modified ex vivo and after modification transferred to the subject.

14. The method of claim 12 or 13, wherein the NK cell is an autologous NK cell.

15. The method of any one of claims 12-14, wherein the NK cell is from an allogeneic donor source.

16. The method of any one of claims 12-15, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject.

17. The method of any one of claims 12-16, wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mbIL-21 expressing feeder cells.

18. The method of any one of claims 12-17, wherein the RNP complex is introduced into the target NK cell vial electroporation.

19. A method of treating a cancer in a subject comprising administering to the subject a vector comprising a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9)complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the genomic DNA of the target NK cell.

20. The method of claim 19, wherein the vector comprises an Adeno-associated virus (AAV) vector.

21. The method of any one of claims 11-20, wherein the cancer is selected from the group consisting of glioblastoma, Burkitt’s lymphoma, and melanoma.

22. A method of enhancing the anti-tumor efficacy of a natural killer (NK) cell comprising a) obtaining a target NK cell to be modified; b) obtaining gRNA that targets the VISTA gene V-set immunoregulatory receptor (VSIR); and c) introducing into the target NK cell, a RNP complex comprising a class 2 CRISPR / Cas endonuclease (Cas9) complexed with a corresponding CRISPR / Cas guide RNA that hybridizes to a target sequence within the VISTA gene in the NK cell thereby creating a NK cell with enhanced anti-tumor efficacy.

23. The method of claim 22, wherein the NK cell is a primary NK cell that is modified ex vivo and after modification transferred to the subject.

24. The method of claim 22 or 23, wherein the NK cell is an autologous NK cell.

25. The method of any one of claims 22-24 wherein the NK cell is from an allogeneic donor source.

26. The method of any one of claims 22-25, wherein the NK cell is expanded with irradiated mbIL-21 expressing feeder cells prior to administration to the subject.

27. The method of any one of claims 22-26, wherein the NK cell is expanded in the subject following transfer of the NK cells to the subject via the administration of IL-21 or irradiated mbIL-21 expressing feeder cells.

28. The method of any one of claims 22-27, wherein the RNP complex is introduced into the target NK cell vial electroporation.

29. A method of enhancing the anti-tumor efficacy of a natural killer (NK) cell comprising contacting an NK cell with an agent that reduces VISTA gene expression or VISTA proteinsignaling; whereby a reduction in VISTA gene expression or VISTA protein signaling reduces the inhibitory effect on the NK cell thereby enhancing the anti-tumor efficacy of the NK cell.

30. The method of claim 29, wherein the agent comprises an siRNA, RNAi, anti-sense oligonucleotide, and / or long non-coding RNA (IncRNA) that binds to the VISTA gene.

31. The method of claim 29, wherein the agent comprises an anti- VISTA antibody.

Images