Methods and compositions for using alternating electric fields in gene therapy

Abstract

Compositions are disclosed for methods of transporting vectors, therapeutics, detectables, or combinations thereof across the plasma membrane of a cell. Methods of detecting and / or treating tumor or cancer cells using the vectors, therapeutics, detectables, or combinations thereof, alone or in combination with the application of an alternating electric field, are also disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 130,449, filed December 24, 2020, which is incorporated by reference in its entirety. [Background technology]

[0002] It has previously been shown that when cells are exposed to alternating electric fields (AEFs) of a certain frequency range while they are undergoing mitosis, the AEFs can disrupt the mitotic process and cause apoptosis. This phenomenon has been successfully used to treat tumors (e.g., glioblastoma, mesothelioma, etc.), as described in U.S. Patent Nos. 7,016,725 and 7,565,205, each of which is incorporated herein by reference in its entirety. In the context of tumor treatment, these alternating electric fields are referred to as "TTFields" (or "tumor treating electric fields"). One reason that TTFields therapy is well suited to treat tumors is that it is believed that TTFields selectively destroys cells that are dividing during mitosis and has no effect on cells that are not dividing. Because tumor cells divide more frequently than other cells in a patient's body, applying TTFields to a subject selectively attacks tumor cells while leaving other cells unharmed. The same phenomenon has also been successfully shown to be useful for destroying bacteria, as described in U.S. Patent No. 9,750,934, which is incorporated herein by reference in its entirety. Here again, one of the reasons this approach is well suited for destroying bacteria is that bacterial cells divide more rapidly than other cells in the human body.

[0003] AC electric fields have also been shown to increase the permeability of cell membranes. Numerous studies have been conducted on viral gene therapy, and many ongoing clinical trials have been conducted with viral-based therapy for cancer. Currently, there is no efficient and specific viral therapy being used in cancer patients. In order to treat cancer in subjects, there is a need for the combination of using AC electric fields to help deliver therapy to cells.

Prior technical literature

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[0005] [Non-licensed document 1] Ramら、Cancer Res. 53:83~88, (1993) [Non-licensed document 2] Laimins, L., Proc. Natl. Acad. Sci. 78: 993 (1981) [Non-licensed document 3] Lusky, ML, Mol. Cell Bio. 3: 1108 (1983)

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[0006] Combining TTFields with viral-based therapies, or viruses overexpressing proteins for theragnostic delineation of cancer cells from normal tissues, can be specific and enhanced in cancer cells compared to healthy tissue or circulating cells.

[0007] The difference in the effect of TTFields application on viral load in cancer cells compared to healthy cells indicates that viral gene therapy and / or viruses carrying detectable markers to delineate cancer cells from normal tissues may be used in combination with TTFields to enable specific and enhanced effects in cancer cells but not in healthy cells.

[0008] Enhancing viral load in cancer cells while decreasing viral load in healthy cells could impact treatments using viral gene therapy and also provide better tools for visualizing cancer tissue or metastatic cells infected with vectors expressing detectable markers. [Means for solving the problem]

[0009] A method for transporting a composition across a cell membrane of a cell is disclosed, comprising the steps of: applying an alternating electric field to a cell for a period of time, where the application of the alternating electric field increases the permeability of the cell membrane; and introducing a composition into the cell, where the increase in the permeability of the cell membrane allows the composition to cross the cell membrane. In some aspects, the composition can be a vector, a therapeutic, a detectable label, or a combination thereof.

[0010] A method for transporting a vector across a cell membrane is disclosed, comprising the steps of: applying an alternating electric field to a cell for a period of time, where application of the alternating electric field increases the permeability of the cell membrane; and introducing the vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane.

[0011] Disclosed is a method of reducing cell viability, comprising: applying a first alternating electric field at a first frequency to a cell for a first period of time, where application of the first alternating electric field at the first frequency to the cell for the first period of time increases the permeability of the cell membrane of the cell; introducing a composition into the cell, where the increased permeability of the cell membrane allows the composition to cross the cell membrane; and applying a second alternating electric field at a second frequency to the cell for a second period of time, where the second frequency is different from the first frequency, and where the second alternating electric field at the second frequency reduces the viability of the cell. In some embodiments, the composition can be a vector, a treatment, a detectable label, or a combination thereof. In some embodiments, the cell is a tumor or cancer cell.

[0012] Disclosed are methods of reducing cell viability, comprising: applying a first alternating electric field at a first frequency to a cell for a period of time, where application of the first alternating electric field increases the permeability of the cell membrane; introducing a vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane, where the vector comprises a nucleic acid sequence capable of encoding a therapeutic agent, where once the vector crosses the cell membrane, the vector encodes the therapeutic agent, and where the presence of the therapeutic agent reduces the viability of the cell. In some embodiments, the cell is a tumor or cancer cell.

[0013] Disclosed is a method for detecting tumor or cancer cells, comprising the steps of: applying a first alternating electric field at a first frequency to the tumor or cancer cells for a period of time, where application of the first alternating electric field increases the permeability of the tumor or cancer cell membrane; introducing a vector into the tumor or cancer cell, where the increased permeability of the tumor or cancer cell membrane allows the vector to cross the tumor or cancer cell membrane, where the vector comprises a nucleic acid sequence capable of encoding a selectable marker, where once the vector has crossed the tumor or cancer cell membrane, the vector encodes the selectable marker; and detecting the selectable marker, where presence of the selectable marker indicates the presence of the tumor or cancer cell.

[0014] Disclosed are methods for detecting and treating tumor or cancer cells, comprising the steps of: applying a first alternating electric field at a first frequency to the tumor or cancer cell for a period of time, where the application of the alternating electric field increases the permeability of the tumor or cancer cell membrane; introducing a vector into the tumor or cancer cell, where the increased permeability of the tumor or cancer cell membrane allows the vector to cross the tumor or cancer cell membrane, the vector comprising a nucleic acid sequence capable of encoding a therapeutic agent and a selectable marker, where upon entry of the vector into the cell, the vector encodes the therapeutic agent and the selectable marker; and detecting the selectable marker, where the presence of the selectable marker indicates the presence of the tumor or cancer cell, and where the therapeutic agent encoded by the vector induces apoptosis of the tumor or cancer cell.

[0015] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows and in part will be understood from the description or may be obtained by the practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not limitations of the invention as claimed.

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the effect of TTFields application for 24 hours to A549 cells infected with PR8 virus on virus infection and proliferation. [Diagram 2] FIG. 1 shows the effect of TTFields application for 48 hours to A549 cells infected with PR8 virus on virus infection and proliferation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The disclosed methods and compositions will be more readily understood by reference to the following detailed description of specific embodiments and examples contained herein, as well as the drawings and their preceding and following description.

[0019] It is understood that the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or particular reagents, as these may vary, unless otherwise specified. It is also understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0020] Disclosed are materials, compositions and components that may be used for, used in conjunction with, used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, specific references to the various individual and collective combinations and permutations of each of these compounds may not be expressly disclosed, but each is specifically contemplated and described herein. Thus, when a class of molecules A, B and C is disclosed along with a class of molecules D, E and F, and an example of a combination of molecules A-D is disclosed, each is individually and collectively contemplated, even if each is not individually listed. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F should be specifically contemplated and considered disclosed from the disclosure of A, B and C; D, E, and F; and the example combinations A-D. Similarly, any subset or combination of these is specifically contemplated and disclosed. Thus, for example, subgroups A-E, B-F, and C-E should be considered specifically contemplated and disclosed from the disclosure of A, B, and C; D, E, and F; and combination examples A-D. 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, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed with any specific embodiment or combination of embodiments of the disclosed methods, and each such combination should be considered specifically contemplated and disclosed.

[0021] A.Definition It is understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0022] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise, so that, for example, a reference to "a vector" includes one or more such vectors, and a reference to "the vector" is a reference to one or more vectors and equivalents thereof known to those skilled in the art, and so forth.

[0023] As used herein, a "target site" is a specific site or location within or present in a subject or patient. For example, a "target site" may refer to, but is not limited to, a cell (e.g., cancer cell), a group of cells, an organ, a tissue, or a tumor. Thus, the phrase "target cell" may be used to refer to a target site, where the target site is a cell. In some aspects, organs that may be target sites include, but are not limited to, lung, brain, pancreas, abdominal organs (e.g., stomach, intestine), ovaries, breast, uterus, prostate, bladder, liver, colon, or kidney. In some aspects, cells or groups of cells that may be target sites or target cells include, but are not limited to, lung cells, brain cells, pancreatic cells, abdominal cells, ovarian cells, liver cells, colon cells, or kidney cells. In some aspects, a "target site" may be a tumor target site. In some aspects, a "target site" may be a virally infected cell. In some aspects, a "target cell" may be a cancer. In some aspects, a "target cell" may be a virally infected cell.

[0024] A "tumor target site" is a site or location within or present in a subject or patient that contains or is adjacent to one or more cancer cells, previously contained one or more tumor cells, or is suspected of containing one or more tumor cells. For example, a tumor target site may refer to a site or location within or present in a subject or patient that is prone to metastasis. Additionally, a target site or tumor target site may refer to a site or location of a resection of a primary tumor within or present in a subject or patient. Additionally, a target site or tumor target site may refer to a site or location adjacent to a resection of a primary tumor within or present in a subject or patient.

[0025] As used herein, one or more "alternating electric fields" refer to a very low intensity directional mid-frequency alternating electric field delivered to a subject, a sample obtained from a subject, or a specific location (e.g., a target site such as a cell) within a subject or patient. In some embodiments, the alternating electric field may be unidirectional or multidirectional. In some embodiments, the alternating electric field may be delivered through two pairs of transducer arrays that generate a perpendicular field within the target site. For example, for the Optune™ system (alternating electric field delivery system), one pair of electrodes is placed on the left and right (LR) of the target site, and the other pair of electrodes is placed on the anterior and posterior (AP) of the target site. Cycling the field in these two directions (i.e., LR and AP) ensures that the widest range of cell orientations are targeted.

[0026] As used herein, an "alternating electric field" applied to a tumor target site may be referred to as a "tumor treating electric field" or "TTFields." TTFields are established as an anti-mitotic cancer treatment modality because they disrupt correct microtubule assembly during metaphase, ultimately destroying cells in telophase, cytokinesis, or subsequent quiescence. TTFields target solid tumors and are described in U.S. Patent No. 7,565,205, the teachings of which are incorporated herein by reference in their entirety.

[0027] In-vivo and in-vitro studies have shown that the efficacy of TTFields treatment increases as the strength of the electric field increases. Therefore, optimizing the array placement on a subject to increase the intensity at the target site or target cells is a standard practice for the Optune system. Optimization of the array placement can be performed by "rules of thumb" (e.g., placing the array on the subject as close to the target site or target cells as possible), measurements representative of the patient's body shape, the size of the target site, and / or the target site or cell location. The measurements used as inputs can be from imaging data. The imaging data is intended to include any type of imaging data, such as, for example, single photon emission computed tomography (SPECT) imaging data, X-ray computed tomography (X-ray CT) data, magnetic resonance imaging (MRI) data, positron emission tomography (PET) data, data that can be captured by an optical instrument (e.g., a photographic camera, a charge-coupled device (CCD) camera, an infrared camera, etc.). In certain implementations, the imaging data can include 3D data obtained from or generated by a 3D scanner (e.g., point cloud data). Optimization may depend on understanding how the electric field is distributed within a target site or cell as a function of array position, and in some aspects takes into account the variability in the distribution of electrical properties within different patient heads.

[0028] The term "subject" refers to a target of administration, e.g., an animal. Thus, the subject of the disclosed method may be a vertebrate, such as a mammal. For example, the subject may be a human. The term does not denote a particular age or sex. Subject may be used interchangeably with "individual" or "patient." For example, the subject of administration may refer to the recipient of an alternating electric field.

[0029] By "treatment" is meant administering or applying a therapy, such as alternating electric fields and vectors, to a subject, such as a human or other mammal (e.g., an animal model) having an infection (e.g., viral) or disease (e.g., cancer) or having an increased susceptibility to developing an infection or disease, to prevent or slow the progression of the disease or infection, or to partially or completely reverse the effects of the infection or disease. For example, treating a subject with cancer can include delivering a therapy to the cells of the subject.

[0030] By "prevention" is meant minimizing or reducing the chance that a subject will develop an infection or disease.

[0031] As used herein, the terms "administer" and "administration" refer to any method for providing a treatment, such as an antiviral or anticancer therapeutic, to a target site or subject. Such methods are well known to those of skill in the art and include, but are not limited to: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections, such as intravenous, intraarterial, intramuscular, and subcutaneous administration. Administration may be continuous or intermittent. In various embodiments, the preparation may be administered therapeutically; i.e., administered to treat an existing disease or condition. In further various embodiments, the preparation may be administered prophylactically; i.e., administered for the prevention of a disease or condition. In one embodiment, one of skill in the art can determine an effective dose, an effective schedule, or an effective route of administration to treat a subject. In some embodiments, administering includes exposing or applying. Thus, in some aspects, exposing a target site or subject to an alternating electrical field or applying an alternating electrical field to a target site or subject refers to administering an alternating electrical field to the target site or subject.

[0032] "Optionally" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present as well as instances where it does not occur or is not present.

[0033] Ranges may be expressed herein as "about" one particular value and / or "about" another particular value. When such ranges are expressed, the range from one particular value and / or to the other particular value is also considered to be specifically contemplated and disclosed, unless the context clearly indicates otherwise. Similarly, when values ​​are expressed as approximately by use of the antecedent "about," it is understood that the particular value forms another specifically contemplated embodiment that is to be considered disclosed, unless the context clearly indicates otherwise. It is further understood that each endpoint of the range is significant both in relation to the other endpoint and independently of the other endpoint, unless the context clearly indicates otherwise. Finally, it is to be understood that all individual values ​​within an expressly disclosed range, and subranges of values ​​therein, are also to be considered to be specifically contemplated and disclosed, unless the context clearly indicates otherwise. The foregoing applies regardless of whether some or all of these embodiments are expressly disclosed in a particular instance.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present methods and compositions, particularly useful methods, devices and materials are described. Publications cited herein and the materials for which they are cited are specifically incorporated herein by reference. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. No reference is admitted to constitute prior art. The discussion of references states what their authors assert, and applicants reserve the right to verify the accuracy and pertinence of the cited documents. Although a number of publications have been referenced herein, it is expressly understood that such references do not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0035] Throughout the description and claims, the words "comprise" and variations of words such as "comprising" and "comprises" mean "including but not limited to" and are not intended to exclude, for example, other additives, ingredients, integers or steps. In particular, in a method described as including one or more steps or operations, each step is specifically contemplated to include what is recited (unless the step includes a limiting term such as "consisting of") and each step is not intended to exclude, for example, other additives, ingredients, integers or steps not recited in the step.

[0036] B. Vector Disclosed are vectors comprising a nucleic acid sequence capable of encoding a sequence of interest. The disclosed methods can include administration of one or more of the vectors described herein.

[0037] In some aspects, the vector for use in the methods disclosed herein can be an expression vector. The term "expression vector" includes any vector (e.g., plasmid, cosmid, phagemid, phage, virus) that contains a gene construct (e.g., linked to a transcriptional regulatory element) in a form suitable for expression by a cell.

[0038] The vector for use in the methods disclosed herein can be a non-viral vector or a viral vector.In some aspects, the vector can be a viral vector.For example, the viral vector can be an adeno-associated virus vector, an adeno-associated virus, a lentivirus or a herpes virus.In some aspects, the vector can be a non-viral vector, such as a DNA-based vector (e.g., a plasmid, a cosmid, a phagemid).

[0039] 1. Viral and non-viral vectors There are numerous vectors and methods that can be used to deliver the vectors of the present disclosure either in vitro or in vivo. These methods and compositions are broadly divided into two classes: viral-based delivery systems and non-viral-based delivery systems. For example, a nucleic acid capable of encoding a sequence of interest can be delivered across the cell membrane, the method comprising: applying an alternating electric field to the cell for a period of time, where the application of the alternating electric field increases the permeability of the cell membrane; and introducing the vector into the cell, where the increase in the permeability of the cell membrane allows the vector to cross the cell membrane. Such vectors can be in the form of plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or cationic liposomes. In certain cases, the methods can be modified to function specifically with large DNA molecules. Furthermore, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

[0040] An expression vector can be any nucleotide construct used to deliver a gene or gene fragment to a cell (e.g., a plasmid) or as part of an overall strategy for delivering a gene or gene fragment, e.g., as part of a recombinant retrovirus or adenovirus (Ram et al., Cancer Res. 53:83-88, (1993)). Disclosed herein are expression vectors that include a nucleic acid sequence that can encode a sequence of interest, such as a cancer therapeutic agent.

[0041] In some aspects, the vectors of the present disclosure contain regulatory elements. The "regulatory elements" present in an expression vector are the untranslated regions of the vector--enhancers, promoters, 5' and 3' untranslated regions--that interact with host cell proteins to effect transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) may be used. When it is necessary to generate a cell line containing multiple copies of the sequence encoding the sequence of interest, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.

[0042] "Enhancer" generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and may be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, ML et al., Mol. Cell Bio. 3: 1108 (1983)) of a transcription unit. Enhancers can also be located within introns (Banerji, JL et al., Cell 33: 729 (1983)) and within the coding sequence itself (Osborne, TF et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers often also contain response elements that mediate the regulation of transcription. Promoters may also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of genes. Although many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin), for general expression typically enhancers from eukaryotic cell viruses are used. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin and adenovirus enhancers.

[0043] Promoters or enhancers can be specifically activated, either by light or by specific chemical events that trigger their function. The system can be controlled by agents such as tetracycline and dexamethasone. There are also methods to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0044] In some aspects, the promoter or enhancer region may act as a constitutive promoter or enhancer to maximize expression of the polynucleotide of the invention. In certain constructs, the promoter or enhancer region is active in all eukaryotic cell types, even if it is only expressed in certain types of cells at certain times.

[0045] Expression vectors for use in the disclosed methods in eukaryotic host cells (yeast, fungus, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription that may affect mRNA expression. These regions are transcribed as polyadenylation segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated region also contains a transcription termination site. It is preferred that the transcription unit also contains a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcribed unit will be processed and transported like an mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that a homologous polyadenylation signal is used in the transgene construct. In one particular transcription unit, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases.

[0046] Expression vectors for use in the methods of the present disclosure may contain a nucleic acid sequence encoding a marker product that can be used to determine whether a gene has been delivered to a cell and is expressed upon delivery. Marker genes include, but are not limited to, the E. coli lacZ gene, which encodes β-galactosidase, and the gene encoding green fluorescent protein.

[0047] In some embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. For in vitro use, cells containing the vector after using the methods described herein can be isolated. If such a selectable marker is successfully transferred to a mammalian host cell, the transformed mammalian host cell can survive when placed under selection pressure. There are two widely used and distinct classifications of selection methods. The first classification is based on the metabolism of the cell and the use of mutant cell lines that lack the ability to grow independently from a supplemented medium. Two examples are CHO DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented medium. An alternative to supplementing the medium is to introduce intact DHFR or TK genes into cells lacking the respective genes, thereby altering their growth requirements: individual cells not transformed with the DHFR or TK genes will not be able to survive in unsupplemented medium.

[0048] Another type of selection that can be used with the compositions and methods disclosed herein is dominant selection, which refers to a selection scheme used in any cell type and does not require the use of mutant cell lines. These schemes typically use a drug that stops the growth of the host cells. Those cells with the novel gene express a protein that conveys drug resistance and survive the selection. Examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid (Mulligan, RC and Berg, P. Science 209: 1422 (1980)) or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). Three examples use bacterial genes under eukaryotic control that convey resistance to the appropriate drugs G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Others include the neomycin analogues G418 and puromycin.

[0049] As used herein, a plasmid or viral vector is an agent that transports a nucleic acid sequence, such as a nucleic acid sequence that can code for a sequence of interest (e.g., one or more proteins or peptides of interest), to a cell without degradation, and includes a promoter that results in expression of the gene in the cell to which it is delivered. In some embodiments, the vector is a viral vector. Viral vectors are, for example, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, polioviruses, lentiviruses (AIDS viruses), neuronal trophic viruses, Sindbis, and other RNA viruses, including viruses with an HIV backbone. Any viral family that shares the properties that make these viruses suitable for use as vectors is similarly preferred. Retroviruses include mouse Maloney leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors can carry a larger genetic payload, i.e., transgenes or marker genes, than other viral vectors, and for this reason are commonly used vectors. However, they are not as useful in non-proliferating cells. Adenoviral vectors are relatively stable, easy to work with, have high titers, may be delivered in aerosol formulations, and can infect non-dividing cells. Poxvirus vectors are large, have several sites for inserting genes, and they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector engineered to suppress the immune response of the host organism elicited by viral antigens. A preferred vector of this type carries the coding region for interleukin 8 or 10.

[0050] Viral vectors may have a higher processivity (i.e., ability to introduce genes) than chemical or physical methods of introducing genes into cells. Typically, viral vectors contain nonstructural early genes necessary for replication and encapsidation, structural late genes, RNA polymerase III transcripts, inverted terminal sequences, and promoters to regulate transcription and replication of the viral genome. When engineered as vectors, viruses are typically stripped of one or more early genes and a gene or gene / promoter cassette is inserted into the viral genome in place of the deleted viral DNA. This type of construct can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by a cell line engineered to express the gene products of the early genes in trans.

[0051] Retroviral vectors are generally described in Verma, IM, Retroviral vectors for gene transfer. In Microbiology, Amer. Soc. for Microbiology, pp. 229-232, Washington, (1985), which is incorporated by reference in its entirety. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO 90 / 02806 and WO 89 / 07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated by reference in their entirety for teaching methods for using retroviral vectors for gene therapy.

[0052] Retroviruses are essentially packages that are packaged with a nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal that ensures that the replicated daughter molecules are efficiently packaged within the package coat. In addition to the packaging signal, there are a number of molecules required in cis for replication and packaging of the replicated virus. Typically, a retroviral genome contains the gag, pol, and env genes, which are involved in making the protein coat. The gag, pol, and env genes are typically replaced by the foreign DNA that is transferred to the target cell. Retroviral vectors typically contain a packaging signal for incorporation into the packaging coat, a sequence that conveys the start of the gag transcription unit, elements required for reverse transcription including a primer binding site for binding to the tRNA primer for reverse transcription, a terminal repeat sequence that induces RNA strand switching during DNA synthesis, a purine-rich sequence 5' to 3' LTR that acts as a priming site for synthesis of the second strand of DNA synthesis, and a specific sequence near the end of the LTR that allows insertion of the DNA state of the retrovirus to insert into the host genome. This amount of nucleic acid is sufficient for delivery of 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.

[0053] Since most retroviral vectors have the replication machinery and packaging proteins removed (gag, pol and env), the vectors are typically generated by placing them into packaging cell lines. Packaging cell lines are cell lines that have been transfected or transformed with a retrovirus that contains the replication and packaging machinery but lacks any packaging signals. When vectors carrying sequences of interest are transfected into these cell lines, the vectors containing the sequences of interest are replicated and packaged into new retroviral particles by the machinery provided in cis by the helper cells. The genomes for the machinery are not packaged because they lack the necessary signals.

[0054] 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 advantage of using these viruses as vectors is that they can replicate in the initially infected cells but are unable to form new infectious viral particles, limiting their ability to spread to other cell types.Recombinant adenoviruses have been shown to achieve highly efficient gene transfer following direct in vivo delivery to airway epithelia, hepatocytes, vascular endothelium, CNS parenchyma, and numerous 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)), the teachings of which are incorporated herein by reference in their entirety for their teachings of methods of using retroviral vectors for gene therapy.Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors in the same manner as wild-type or replication-deficient adenoviruses, followed by internalization of the virus by receptor-mediated endocytosis (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)).

[0055] Viral vectors for use in the methods of the present disclosure may be based on adenoviruses in which the E1 gene has been removed and these virions are produced in cell lines such as the human 293 cell line. Optionally, both the E1 and E3 genes are removed from the adenoviral genome.

[0056] Another type of viral vector for use in the disclosed methods that can be used to introduce the polynucleotide of the invention into cells is based on the adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is non-pathogenic to humans. AAV-type vectors can transport approximately 4 to 5 kb, and wild-type AAV is known to stably insert into chromosome 19. Vectors with this site-specific integration property are preferred. A particularly preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which may contain a marker gene such as the herpes simplex virus thymidine kinase gene, HSV-tk, or a gene encoding green fluorescent protein, GFP.

[0057] In another type of AAV virus for use in the disclosed method, the AAV contains a pair of inverted terminal repeats (ITRs) flanking at least one cassette containing a promoter that is operably linked to a heterologous gene and directs cell-specific expression. Heterologous in this context refers to any nucleotide sequence or gene that is not native to AAV or B19 parvovirus. Typically, the AAV and B19 coding regions are removed, resulting in a safe, non-cytotoxic vector. The AAV ITRs or modifications thereof confer infectivity and site-specific integration, but do not confer cytotoxicity and promoter-directed cell-specific expression. U.S. Patent No. 6,261,834 is incorporated by reference in its entirety for material related to AAV vectors.

[0058] Other useful systems include, for example, replicative and host-limited non-replicative vaccinia virus vectors.In addition, the nucleic acid sequences of the present disclosure can be delivered to target cells by non-nucleic acid-based systems.For example, the polynucleotides of the present disclosure can be delivered via electroporation, or via lipofection, or via calcium phosphate precipitation.The delivery mechanism selected depends in part on the type of cells to be targeted and whether delivery occurs, for example, in vivo or in vitro.

[0059] In some aspects, the vector for use in the method of the present disclosure may further comprise a targeting moiety. In some aspects, the vector may comprise a nucleic acid sequence that can encode the targeting moiety. Any one or more known targeting moieties may be used. For example, the targeting moiety can direct the vector to a specific cell type, such as a cancer cell. In some aspects, the one or more targeting moieties may be, but are not limited to, a nucleic acid sequence, a peptide, an antibody, an antibody fragment, or a therapeutic agent.

[0060] 2. Nucleic acid sequences and sequences of interest The vectors of the present disclosure for use in the methods of the present disclosure comprise a nucleic acid sequence. In some aspects, the nucleic acid sequence can encode a sequence of interest.

[0061] In some aspects, the nucleic acid sequence is a gene of interest. The term "gene of interest" can refer to a nucleic acid sequence that is partially or completely heterologous, i.e., foreign, to the cell into which it is introduced (e.g., a therapeutic gene).

[0062] The term "gene of interest" can also refer to a nucleic acid sequence that is partially or completely complementary to an endogenous gene of the cell into which it is introduced. In some aspects, the term "gene of interest" does not include the complete gene sequence.

[0063] In some embodiments, the nucleic acid sequence may be a nucleic acid sequence that has a therapeutic property itself. For example, the nucleic acid sequence may be a microRNA, shRNA, or siRNA, where the microRNA, shRNA, or siRNA is a therapeutic agent.

[0064] In some aspects, the nucleic acid sequences in the disclosed vectors for use in the methods disclosed herein can also include one or more transcription control sequences and any other nucleic acid, such as introns, that may be required for optimal expression of a selected nucleic acid.

[0065] In some aspects, a nucleic acid sequence or gene of interest in a vector of the present disclosure for use in the methods disclosed herein can encode a sequence of interest, where "sequence of interest" refers to a peptide or polypeptide sequence (e.g., a therapeutic protein) expressed from a nucleic acid sequence.

[0066] In some aspects, the sequence of interest is a selectable marker, detectable marker, or therapeutic agent. In some aspects, the vectors of the present disclosure include a selectable or detectable marker separate from the nucleic acid sequence capable of encoding a sequence of interest or a nucleic acid sequence that is itself a therapeutic agent.

[0067] As discussed above, when considering vectors, examples of suitable selectable markers can be, but are not limited to, dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, puromycin, hygromycin resistance, neomycin resistance or β-galactosidase (β-gal). The selectable marker may be present in the vector backbone or may be a sequence of interest.

[0068] Detectable markers can be used for the purpose of tracking or visualizing the location of vectors expressing the detectable marker. Exemplary detectable markers include, but are not limited to: various fluorescent entities such as green fluorescent protein (GFP), blue fluorescent protein, cyan fluorescent protein, red fluorescent protein and various derivatives thereof; other fluorescent proteins such as (but are not limited to) dsRed, eqFP611, Dronpa, tag RFPs, KFP, EosFP, Dendra, IrisFP; other similar molecules known in the art, including but not limited to quantum dots and other fluorescent dyes; and any derivative or combination thereof. Detectable markers can be present in the vector backbone or can be sequences of interest. In some aspects, detectable markers can be selectable markers.

[0069] In some embodiments, the therapeutic agent may be, but is not limited to, cDNA, DNA, mRNA, microRNA, shRNA, siRNA, ribozyme, guide RNA molecule (gRNA), RNA-guided endonuclease protein or Cas9 protein or Cpf1 protein CRISPR-Cas. In some embodiments, the therapeutic agent may be a chemotherapeutic agent, an antiviral agent, an antibacterial agent, an immunosuppressant, or an immune activator.

[0070] In some aspects, the chemotherapeutic agent may be, but is not limited to, an alkylating agent, antimetabolite, antineoplastic antibiotic, and an antimitotic.

[0071] In a further aspect, the antineoplastic antibiotic agent is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or a pharmaceutically acceptable salt thereof.

[0072] In a further aspect, the antimetabolite is selected from gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or a pharmaceutically acceptable salt thereof.

[0073] In a further aspect, the alkylating agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or a pharma- ceutically acceptable salt thereof.

[0074] In a further aspect, the mitotic inhibitor is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etoposide, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or a pharma- ceutically acceptable salt thereof.

[0075] In some aspects, a vector of the present disclosure can include two nucleic acid sequences, where one nucleic acid sequence can encode a sequence of interest, where the sequence of interest is a selectable or detectable marker, and a second nucleic acid sequence can encode a sequence of interest, where the sequence of interest is a therapeutic agent, such that in some aspects, a vector of the present disclosure can include two sequences of interest, where one sequence of interest is a selectable or detectable marker, and a second sequence of interest is a therapeutic agent.

[0076] In some embodiments, the vector of the present disclosure can include two nucleic acid sequences, where one nucleic acid sequence can encode a sequence of interest, where the sequence of interest is a selectable or detectable marker, and the second nucleic acid sequence is a therapeutic agent. Thus, in some embodiments, one of the two nucleic acid sequences does not encode a therapeutic agent, but instead the nucleic acid sequence is a therapeutic agent.

[0077] 3. Pharmaceutical Compositions and Delivery Disclosed herein is a pharmaceutical composition comprising one or more vectors described herein.In some aspects, the vectors described herein can be provided in pharmaceutical compositions.For example, the vectors described herein can be formulated with pharmaceutically acceptable carriers.

[0078] In some embodiments, the pharmaceutical composition may include a therapeutic agent. In some embodiments, the pharmaceutical composition may include a therapeutic agent and one or more vectors described herein. For example, disclosed herein are pharmaceutical compositions including one or more vectors described herein and a therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent may be, but is not limited to, an anti-cancer drug, a cytotoxic drug, a pain management drug, Pseudomonas exotoxin A, a non-radioactive isotope (e.g., boron-10 for boron neutron capture therapy), and / or a photosensitizer (e.g., photofrin, foscan, 5-aminolevulinic acid, mono-L-aspartyl chlorin e6, phthalocyanine, metatetra(hydroxyphenyl)porphyrin, texaphyrin, ethyl etiopurinse). In some embodiments, the chemotherapeutic agent may be, but is not limited to, an alkylating agent, antimetabolite, antineoplastic antibiotic, a mitotic inhibitor, a checkpoint inhibitor, and a kinase inhibitor. Any of the therapeutic agents listed above as examples of sequences of interest may be provided separately from the vector as part of a pharmaceutical composition.

[0079] Disclosed herein are compositions comprising one or more vectors described herein, further comprising a carrier, such as a pharma- ceutically acceptable carrier. For example, disclosed are pharmaceutical compositions comprising a vector disclosed herein and a pharma- ceutically acceptable carrier.

[0080] For example, pharmaceutical compositions comprising the vectors described herein may include a pharma- ceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material or carrier well known to one skilled in the art that is selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. Examples of carriers include dimyristoyl phosphatidylcholine (DMPC), phosphate buffered saline, or multivesicular liposomes. For example, PG:PC:cholesterol:peptide or PC:peptide may be used as a carrier in the present invention. Other suitable pharma- ceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharma- ceutical acceptable salt is used in the formulation to make the formulation isotonic. Other examples of pharma- ceutical acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution may be about 5 to about 8 or about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, such as films, stents (implanted in blood vessels during revascularization procedures), liposomes or microparticles.It will be clear to those skilled in the art that certain carriers may be more preferable, for example, depending on the route of administration and the concentration of the vector to be administered.Most typically, these are standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0081] Pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives, and the like, so long as the intended activity of the polypeptides, peptides, nucleic acids, and vectors of the invention is not impaired. Pharmaceutical compositions may also include one or more active ingredients (in addition to the compositions of the invention), such as antibacterial agents, anti-inflammatory agents, anesthetic agents, and the like. Pharmaceutical compositions may be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated.

[0082] 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 antibacterial agents, antioxidants, chelating agents, and inert gases.

[0083] Formulations for optimal administration 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.

[0084] 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. Some compositions may also be administered as pharma-ceutically acceptable acid or salt addition salts 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 inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl, and aryl amines, and substituted ethanol amines.

[0085] In the methods described herein, delivery (or administration or introduction) of the vectors or pharmaceutical compositions disclosed herein to a subject may be via a variety of mechanisms.

[0086] The disclosed method includes a step of introducing a vector into a cell. In some aspects, the method includes introducing a vector into a target site. In some aspects, the method includes introducing a vector into a subject. Thus, the subject includes a cell that is a target site for the disclosed vector. In some aspects, all disclosed methods that include introducing a vector into a cell also include introducing a vector into a subject.

[0087] C. AC electric field The methods disclosed herein include an alternating electric field. In some embodiments, the alternating electric field used in the methods disclosed herein is a tumor treatment electric field. In some embodiments, the alternating electric field can vary depending on the type of cell or condition to which the alternating electric field is applied. In some embodiments, the alternating electric field can be applied through one or more electrodes placed on the subject's body. In some embodiments, there may be more than one pair of electrodes. For example, an array can be placed on the front / back and side of the patient and used with the systems and methods disclosed herein. In some embodiments, when two pairs of electrodes are used, the alternating electric field can be alternating between the pair of electrodes. For example, a first pair of electrodes can be placed on the front and back of the subject, and a second pair of electrodes can be placed on either side of the subject, and then the alternating electric field can be applied, alternating between the front and back electrodes, and then side to side electrodes.

[0088] In some embodiments, the frequency of the alternating electric field is between 50 kHz and 1 MHz. In some embodiments, the frequency of the alternating electric field is between 100 and 500 kHz. The frequency of the alternating electric field can be, but is not limited to, between 50 and 500 kHz, between 100 and 500 kHz, between 25 kHz and 1 MHz, between 50 and 190 kHz, between 25 and 190 kHz, between 180 and 220 kHz, or between 210 and 400 kHz. In some embodiments, the frequency of the alternating electric field can be 50 kHz, 100 kHz, 150 kHz, 200 kHz, 250 kHz, 300 kHz, 350 kHz, 400 kHz, 450 kHz, 500 kHz, 600 kHz, 700 kHz, 800 kHz, 900 kHz, 1 MHz, or any frequency therebetween. In some embodiments, the frequency of the alternating electric field is from about 200 kHz to about 400 kHz, from about 250 kHz to about 350 kHz, and can be about 300 kHz.

[0089] In some aspects, the field strength of the alternating electric field can be 1 to 4 V / cm RMS. In some aspects, a range of field strengths can be used (e.g., between 0.1 and 10 V / cm). In some aspects, the field strength can be 1.75 V / cm RMS. In some embodiments, the field strength is at least 1 V / cm RMS. In other embodiments, a combination of field strengths is applied, e.g., combining two or more frequencies simultaneously and / or applying two or more frequencies at different times.

[0090] In some embodiments, the AC electric field can be applied for various intervals ranging from 0.5 hours to 72 hours. In some embodiments, various durations can be used (e.g., between 0.5 hours and 14 days). In some embodiments, the application of the AC electric field can be repeated periodically. For example, the AC electric field can be applied daily for a duration of 2 hours.

[0091] In some embodiments, the exposure may last for at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours or more.

[0092] The method of the present disclosure comprises applying one or more alternating electric fields to cells or to a subject.In some embodiments, the alternating electric field is applied to a target site or a tumor target site.When applying an alternating electric field to a cell, this can often refer to applying the alternating electric field to the subject that contains the cell.Therefore, applying an alternating electric field to a target site of a subject results in applying an alternating electric field to a cell.

[0093] D. Transportation method Disclosed is a method for transporting a composition across a cell membrane comprising applying an alternating electric field to a cell for a period of time, where application of the alternating electric field increases the permeability of the cell membrane; and introducing a composition into the cell, where the increased permeability of the cell membrane allows the composition to cross the cell membrane. In some aspects, the composition is a vector, comprises a vector, or is a pharmaceutical composition comprising a vector.

[0094] In some aspects, any of the vectors or pharmaceutical compositions disclosed herein can be used in the disclosed methods for transporting a composition (e.g., a vector) across the plasma membrane of a cell.

[0095] A method for transporting a vector across a cell membrane is disclosed, comprising the steps of: applying an alternating electric field to a cell for a period of time, where application of the alternating electric field increases the permeability of the cell membrane; and introducing the vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane.

[0096] Also disclosed is a method for improving transport of a vector across a cell membrane, comprising the steps of: applying an alternating electric field to a cell for a period of time, where application of the alternating electric field increases the permeability of the cell membrane; and introducing the vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane.

[0097] In some aspects, the cell is a target cell. In some aspects, the cell is a cancer or tumor cell. In some aspects, the cell is not a cancer or tumor cell. In some aspects, the cell is a virus-infected cell. In some aspects, the cancer cell can be, but is not limited to, a glioblastoma cell, a uterine sarcoma cell, a breast adenocarcinoma cell, a pancreatic cancer cell, a non-small cell lung cancer, a hepatocyte, a gastric cancer cell, a brain cancer cell. In some aspects, the cell can be any cell in need of gene therapy. For example, a cell that is deficient in a particular protein can be targeted by an alternating electric field, whereby a vector can be used to deliver the deficient protein to the cell. In some aspects, the cell can be a virus-infected cell, where the cell is infected with a virus selected from, but not limited to, influenza virus, adenovirus, coronavirus, and lentivirus.

[0098] In some embodiments, the method for transporting a composition or vector across a cell membrane of a cell includes, in part, applying an alternating electric field to the cell for a period of time. In some embodiments, the alternating electric field can be used to introduce the vector only into cancer cells. In some embodiments, the alternating electric field can be applied at a frequency of about 200 kHz. In some embodiments, the alternating electric field can be applied at a frequency between 50 and 190 kHz. In some embodiments, the alternating electric field can be applied at a frequency between 210 and 400 kHz. In some embodiments, the alternating electric field can be applied at a frequency between 250 and 350 kHz. In some embodiments, the alternating electric field has a field strength of at least 1 V / cm RMS. In some embodiments, the alternating electric field has a frequency between 50 and 1 MHz. In some embodiments, the alternating electric field has a frequency between 50 and 190 kHz. In some embodiments, the alternating electric field has a frequency between 210 and 400 kHz. In some embodiments, the alternating electric field has a frequency between 250 and 350 kHz. In some embodiments, the alternating electric field has a field strength of at least 1 V / cm RMS, hi some embodiments, the alternating electric field has a field strength of between 1 and 4 V / cm RMS.

[0099] In some embodiments, the step of introducing the composition or vector begins at a given time, and the step of applying the AC electric field ends at least 12 hours after the given time. In some embodiments, the step of applying the AC electric field begins at least 1 hour before the given time. In some embodiments, the step of applying the AC electric field begins at least 1 to about 24 hours before the given time.

[0100] In some aspects, applying the AC electric field and introducing the composition or vector occur simultaneously. In some aspects, applying the AC electric field and introducing the composition or vector occur sequentially. In some aspects, applying the AC electric field occurs prior to introducing the composition or vector.

[0101] In some embodiments, the cells are cancer cells, where the cancer cells comprise glioblastoma cells and the alternating electric field has a frequency that is between 250 kHz and 350 kHz.

[0102] In some embodiments, the cells are cancer cells, where the cancer cells comprise uterine sarcoma cells and the alternating electric field has a frequency that is between 125 kHz and 175 kHz.

[0103] In some embodiments, the cells are cancer cells, where the cancer cells comprise breast adenocarcinoma cells, and the alternating electric field has a frequency that is between 75 kHz and 175 kHz.

[0104] In some aspects, the composition or vector is introduced intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intradurally, intravascularly, intravenously (targeted or non-targeted), intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, via intratumoral injection (e.g., under computed tomography guidance during surgery or biopsy), or via inhalation.

[0105] In some aspects, the composition or vector is introduced into the cell in a targeted or non-targeted manner.

[0106] In some aspects, the composition or vector is introduced once, twice, three times or more. The time between administrations can be hours, days, weeks or years.

[0107] In some embodiments, the composition or vector comprises a nucleic acid sequence that can code for a sequence of interest. In some embodiments, the sequence of interest is a selectable marker, a detectable marker, or a therapeutic agent. In some embodiments, the vector comprises a nucleic acid sequence, where the nucleic acid sequence is a therapeutic agent. For example, the nucleic acid sequence can be a microRNA, shRNA, or siRNA, where the microRNA, shRNA, or siRNA is a therapeutic agent.

[0108] In some aspects, the selectable or detectable marker can be, but is not limited to, dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, puromycin, hygromycin resistance, neomycin resistance, β-galactosidase (β-gal), fluorescent proteins (e.g., green, blue, red fluorescent proteins).

[0109] In some aspects, a composition or vector of the present disclosure can include two nucleic acid sequences, where one nucleic acid sequence can encode a sequence of interest, where the sequence of interest is a selectable or detectable marker, and a second nucleic acid sequence can encode a sequence of interest, where the sequence of interest is a therapeutic agent. Thus, in some aspects, a composition or vector of the present disclosure can include two sequences of interest, where one sequence of interest is a selectable or detectable marker, and a second sequence of interest is a therapeutic agent.

[0110] In some embodiments, a composition or vector of the present disclosure can include two nucleic acid sequences, where one nucleic acid sequence can encode a sequence of interest, where the sequence of interest is a selectable or detectable marker, and the second nucleic acid sequence is a therapeutic agent. Thus, in some embodiments, one of the two nucleic acid sequences does not encode a therapeutic agent, but instead the nucleic acid sequence is a therapeutic agent.

[0111] In some aspects, the vector is a plasmid, cosmid, phagemid, or viral vector. In some aspects, the vector is a viral vector. For example, the viral vector can be, but is not limited to, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus. In some aspects, the vector can be any of the vectors described herein.

[0112] In some embodiments, the composition or vector is provided in a pharmaceutical composition. In some embodiments, the pharmaceutical composition can be one or more of the pharmaceutical compositions disclosed herein. In some embodiments, the pharmaceutical composition can further comprise a therapeutic agent, such as a chemotherapeutic agent.

[0113] In some embodiments, the composition or vector may further comprise a targeting moiety. In some embodiments, the vector may comprise a nucleic acid sequence that can encode the targeting moiety. Any one or more of known targeting moieties may be used. For example, the targeting moiety may direct the composition or vector to a specific cell type, such as a cancer cell. In some embodiments, the one or more targeting moieties may be, but are not limited to, a nucleic acid sequence, a peptide, an antibody, an antibody fragment, or a therapeutic agent.

[0114] In some embodiments, the methods of PCT / US19 / 40479 filed July 3, 2019 may be used in the methods disclosed herein. For example, PCT / US19 / 40479 filed July 3, 2019, which is incorporated herein by reference for its teachings, describes a method and process for delivering a substance across a cell membrane of a cell. For example, PCT / US19 / 40479 filed July 3, 2019 describes a method and process that includes applying an alternating electric field to a cell for a period of time, where the application of the alternating electric field increases the permeability of the cell membrane; and introducing a substance into the vicinity of the cell, where the increased permeability of the cell membrane allows the substance to cross the cell membrane.

[0115] In some embodiments, the composition can be transported across cell membranes in vivo or in vitro. In some embodiments, the composition can be administered to a subject that includes cells, and once inside the subject, the composition can be transported across cell membranes in combination with the application of an AC electric field. In some embodiments, the composition can be administered to one or more cells in cell culture and with the application of an AC electric field. In some embodiments, once the composition is transported across cell membranes in combination with the application of an AC electric field, the cells can be introduced or reintroduced into a subject.

[0116] E. Treatment Method The above methods of delivering vectors to cells can be used to treat a subject in need thereof. In some aspects, the use of an alternating electric field enables or enhances the transport of the composition or vector across the cell membrane, and once inside the cell, the composition or vector can provide therapy, thereby treatment, to a subject in need thereof.

[0117] The treatment method of the present disclosure may include the application of an alternating electric field in vivo or in vitro. Thus, in some aspects, applying an alternating electric field to a cell includes applying an alternating electric field to a subject that includes the cell. In some aspects, applying an alternating electric field to a cell includes applying an alternating electric field to a cell in culture, and the cell can then be introduced into a subject to be treated. In some aspects, the cell can be introduced or reintroduced into a subject once the composition is transported across the cell membrane in combination with the application of an alternating electric field.

[0118] In some aspects, an additional treatment or therapeutic regimen (e.g., chemotherapy or radiation therapy) may be administered to the subject before, simultaneously with, or after the composition (e.g., vector) is delivered to the subject's cells.

[0119] 1. Treatment with vector Disclosed is a method for treating a subject in need thereof, comprising the steps of: applying an alternating electric field to a cell for a period of time, where the application of the alternating electric field increases the permeability of the cell membrane; and introducing a composition into the cell, where the increased permeability of the cell membrane allows the composition to cross the cell membrane and, upon entering inside the cell, the composition provides a therapeutic effect to the subject. In some embodiments, the composition is a vector or a pharmaceutical composition comprising the vector.

[0120] In some aspects, any of the vectors or pharmaceutical compositions disclosed herein may be used in the disclosed methods for transporting a vector across the plasma membrane of a cell.

[0121] A method for treating a subject in need thereof is disclosed, comprising the steps of: applying an alternating electric field to a cell for a period of time, where the application of the alternating electric field increases the permeability of the cell membrane; and introducing a vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane and, once inside the cell, the vector provides a therapeutic effect to the subject.

[0122] In some embodiments, the subject in need thereof may be a subject with any diagnosed medical condition that can be treated with a vector carrying a therapeutic agent. For example, a subject that benefits from gene therapy is a subject in need thereof. In some embodiments, a subject in need of one or more genes that need to be knocked out or down is a subject in need thereof. In some embodiments, the subject may be a subject diagnosed with cancer. In some embodiments, the subject may be a subject diagnosed with an autoimmune disorder. In some embodiments, the subject may be a subject diagnosed with a protein deficiency.

[0123] In some embodiments, the therapeutic agent may be, but is not limited to, cDNA, DNA, mRNA, microRNA, shRNA, siRNA, ribozyme, guide RNA molecule (gRNA), RNA-guided endonuclease protein, Cas9 protein, or Cpf1 protein, CRISPR-Cas. In some embodiments, the therapeutic agent may be a chemotherapeutic agent, an antiviral agent, an antibacterial agent, an immunosuppressant, or an immune activator.

[0124] In some embodiments, the therapeutic agent is a particular protein that the subject may be deficient in. In some embodiments, the therapeutic agent may be one or more elements of the CRISPR-Cas9 system. For example, in some embodiments, the therapeutic agent may be a gRNA that targets a particular gene to be cleaved, or an endonuclease that cleaves at the gRNA target site. In one example, the vector may contain a gRNA that targets PD-1. Using CRISPR to reduce or eliminate PD-1 expression in T cells can allow for better killing of cancer cells. In some embodiments, PD-1 on T cells binds to PD-L1 on the cancer cells, signaling to the T cells that the cells are healthy cells, so that the T cells do not kill the cancer cells. Using the methods of the present disclosure to introduce vectors that contain gRNA that targets PD-1 aids in the process of cleaving the PD-1 gene, reducing expression, and thereby resulting in greater killing of cancer cells that express PD-L1.

[0125] In some aspects, an additional treatment or therapeutic regimen (e.g., chemotherapy or radiation therapy) may be administered to the subject before, simultaneously with, or after the composition (e.g., vector) is delivered to the subject's cells.

[0126] 2. Treatment with a second AC electric field in addition to the vector A method for treating a subject in need thereof is disclosed, comprising the steps of: applying a first alternating electric field at a first frequency to a cell for a first period of time, where application of the first alternating electric field at the first frequency to the cell for the first period of time increases the permeability of a cell membrane of the cell; introducing a vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane, where the vector comprises a nucleic acid sequence capable of encoding a therapeutic agent; and applying a second alternating electric field at a second frequency to the cell for a second period of time, where the second frequency is different from the first frequency.

[0127] In some aspects, the cell may be a virally infected cell. In some aspects, the vector is a therapeutic agent or comprises a nucleic acid sequence capable of encoding a therapeutic agent. For example, the vector may comprise a nucleic acid sequence capable of encoding an antiviral agent or an immunomodulator.

[0128] In some embodiments, the second alternating electric field prevents viral replication and / or infection. Thus, the treatment is a dual treatment of the vector and a therapeutic agent provided by the alternating electric field that prevents viral replication and / or infection.

[0129] In some embodiments, the first AC field and the second AC field are different. The first and second AC fields can be one or more of those disclosed herein. In some embodiments, the first or second AC field or both can have a frequency between 50kHz and 1MHz. The AC fields can vary based on cell type. For example, the first AC field can have a first frequency between 250kHz and 350kHz, and the second AC field can have a frequency between 150kHz and 250kHz; the first AC field can have a first frequency between 125kHz and 175kHz, and the second AC field can have a frequency between 75kHz and 125kHz; or the first AC field can have a first frequency between 75kHz and 175kHz, and the second AC field can have a frequency between 100kHz and 300kHz. In some embodiments, the alternating electric field has a field strength of at least 1 V / cm RMS, hi some embodiments, the alternating electric field has a field strength of between 1 and 4 V / cm RMS.

[0130] In some embodiments, the second period of time includes a plurality of non-consecutive intermittent periods during which the second alternating electric field at a second frequency is applied to the cells, the plurality of non-consecutive intermittent periods being applied for a total period of at least one week.

[0131] In some embodiments, the first and second alternating electric fields can both be generated in vivo. In some embodiments, the first and second alternating electric fields can both be generated in vitro. In some embodiments, the first alternating electric field is generated in vitro and the second alternating electric field is generated in vivo. In some embodiments, when the composition is transported across the cell membrane in combination with the application of an alternating electric field, the cell can be introduced or reintroduced into the subject.

[0132] In some aspects, an additional treatment or therapeutic regimen (e.g., chemotherapy or radiation therapy) may be administered to the subject before, simultaneously with, or after the composition (e.g., vector) is delivered to the subject's cells.

[0133] F. Methods of killing cells The above methods of delivering vectors to cells can be used to kill cells. In some aspects, the use of an alternating electric field enables or enhances the transport of vectors across the cell membrane, and once inside the cell, the vector can provide a therapy, which results in cell death.

[0134] The above methods of delivering vectors to cells can be used to induce apoptosis in cells. In some aspects, the use of an alternating electric field enables or enhances the transport of vectors across the cell membrane, and once inside the cell, the vector can provide a therapy, which results in apoptosis of the cell.

[0135] In some aspects, an additional treatment or therapeutic regimen (e.g., chemotherapy or radiation therapy) may be administered to the subject before, simultaneously with, or after the composition (e.g., vector) is delivered to the subject's cells.

[0136] 1. Reducing cell viability using vector therapy A method of reducing cell viability is disclosed, comprising: applying a first alternating electric field at a first frequency to a cell for a period of time, where the application of the first alternating electric field increases the permeability of the cell membrane; introducing a vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane, where the vector comprises a nucleic acid sequence capable of encoding a therapeutic agent, where the vector crosses the cell membrane, where the vector encodes a therapeutic agent, and where the presence of the therapeutic agent reduces the viability of the cell. In some embodiments, the cell is a tumor or cancer cell. In some embodiments, the cancer cell is a glioblastoma cell, a uterine sarcoma cell, a breast adenocarcinoma cell, a pancreatic cancer cell, a non-small cell lung cancer, a hepatic cell, a gastric cancer cell, or a brain cancer cell.

[0137] In some embodiments, the therapeutic agent may be a pro-apoptotic factor, hi some embodiments, the therapeutic agent may be, but is not limited to, a cytokine, p53, suicide genes (e.g., cytosine deaminase / 5-fluorocytosine and herpes simplex virus / ganciclovir), or an activator of a known apoptotic gene.

[0138] In some aspects, the alternating electric field can be any of those described for transporting vectors into cells.

[0139] In some aspects, the cell is a tumor or cancer cell. In some aspects, the cancer cell is a glioblastoma cell, a uterine sarcoma cell, a breast adenocarcinoma cell, a pancreatic cancer cell, a non-small cell lung cancer cell, a hepatic cell, a gastric cancer cell, or a brain cancer cell.

[0140] In some embodiments, the step of introducing the vector begins at a given time, and the step of applying an AC electric field ends at least 12 hours after the given time. In some embodiments, the step of applying an AC electric field begins at least 1 hour before the given time. In some embodiments, the step of applying an AC electric field begins at least 1 to about 24 hours before the given time.

[0141] In some aspects, an additional treatment or therapeutic regimen (e.g., chemotherapy or radiation therapy) may be administered to the subject before, simultaneously with, or after the composition (e.g., vector) is delivered to the subject's cells.

[0142] 2. Reducing cell viability using a second AC electric field in addition to vector treatment In some aspects, the disclosed methods of reducing cell viability can further include administering a second alternating electric field at a second frequency.

[0143] Disclosed is a method of reducing cell viability, comprising the steps of: applying a first alternating electric field at a first frequency to a cell for a first period of time, where application of the first alternating electric field at the first frequency to the cell for the first period of time increases the permeability of a cell membrane of the cancer cell; introducing a vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane; and applying a second alternating electric field at a second frequency to the cell for a second period of time, where the second frequency is different from the first frequency, where the second alternating electric field at the second frequency reduces the viability of the cell.

[0144] Thus, in some embodiments, the second alternating electric field is a tumor treatment electric field. In some embodiments, the first and second alternating electric fields are different. The first and second alternating electric fields may be one or more of those disclosed herein. In some embodiments, the first or second alternating electric field or both may have a frequency between 50 kHz and 1 MHz. For example, in some embodiments, the cancer cells include glioblastoma cells, the first alternating electric field may have a first frequency between 250 kHz and 350 kHz, and the second alternating electric field may have a frequency between 150 kHz and 250 kHz. In some embodiments, the cancer cells include uterine sarcoma cells, the first alternating electric field may have a first frequency between 125 kHz and 175 kHz, and the second alternating electric field may have a frequency between 75 kHz and 125 kHz. In some embodiments, the cancer cells include breast adenocarcinoma cells, the first alternating electric field may have a first frequency between 75 kHz and 175 kHz, and the second alternating electric field may have a frequency between 100 kHz and 300 kHz. The alternating electric field may vary based on cell type.

[0145] In some embodiments, the first alternating electric field has a field strength of at least 1 V / cm RMS.

[0146] In some embodiments, the second alternating electric field is a treatment. In some embodiments, the second alternating electric field can kill cells, such as cancer cells.

[0147] The disclosed method of reducing cell viability by using an alternating electric field to transport vectors inside cells can be combined with the use of an alternating electric field for an even more efficient response (increased reduction in cell viability or increased apoptosis).

[0148] applying a first alternating electric field at a first frequency to a cell for a first period of time, where application of the first alternating electric field at the first frequency to the cell for the first period of time increases the permeability of a cell membrane of the cell; introducing a vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane; and A method of reducing cell viability is disclosed that includes applying a second alternating electric field at a second frequency to the cell for a second period of time, where the second frequency is different from the first frequency, and where the second alternating electric field at the second frequency reduces cell viability.

[0149] In some aspects, the cell is a tumor or cancer cell. In some aspects, the cancer cell is a glioblastoma cell, a uterine sarcoma cell, a breast adenocarcinoma cell, a pancreatic cancer cell, a non-small cell lung cancer cell, a hepatic cell, a gastric cancer cell, or a brain cancer cell.

[0150] In some embodiments, the second alternating electric field is a tumor treatment electric field. In some embodiments, the first and second alternating electric fields are different. The first and second alternating electric fields may be one or more of those disclosed herein. In some embodiments, the first or second alternating electric field or both may have a frequency between 50 kHz and 1 MHz. The alternating electric field may vary based on cell type. For example, regardless of cell type, the first alternating electric field may have a first frequency between 250 kHz and 350 kHz and the second alternating electric field may have a frequency between 150 kHz and 250 kHz; the first alternating electric field may have a first frequency between 125 kHz and 175 kHz and the second alternating electric field may have a frequency between 75 kHz and 125 kHz; or the first alternating electric field may have a first frequency between 75 kHz and 175 kHz and the second alternating electric field may have a frequency between 100 kHz and 300 kHz. In some embodiments, the alternating electric field has a field strength of at least 1 V / cm RMS, hi some embodiments, the alternating electric field has a field strength of between 1 and 4 V / cm RMS.

[0151] In some embodiments, the step of introducing the vector begins at a given time, and the step of applying the AC electric field ends at least 12 hours after the given time. In some embodiments, the step of applying the AC electric field begins at least 1 hour before the given time. In some embodiments, the step of applying the AC electric field begins at least 1 to about 24 hours before the given time.

[0152] In some embodiments, the second period of time includes multiple non-consecutive intermittent periods when the second alternating electric field is applied to the cells at the second frequency, the multiple non-consecutive intermittent periods totaling at least one week.

[0153] In some embodiments, the cells are present in the body of a living subject, wherein the first alternating electric field is applied to the cells by applying the first alternating electric field to the subject's body, and the second alternating electric field is applied to the cells by applying the second alternating electric field to the subject's body, and wherein the introducing step comprises administering the vector to the subject.

[0154] In some aspects, an additional treatment or therapeutic regimen (e.g., chemotherapy or radiation therapy) may be administered to the subject before, simultaneously with, or after the composition (e.g., vector) is delivered to the subject's cells.

[0155] G. Detection Methods 1. Detection The above methods of delivering vectors to cells can be used to detect cells. In some aspects, the use of an alternating electric field allows or enhances the transport of the vector across the cell membrane, and once inside the cell, the vector can encode a detectable marker, which allows for detection of the cell.

[0156] Disclosed is a method of detecting a specific cell type, comprising the steps of: applying an alternating electric field at a first frequency for a period of time to one or more cells having the specific cell type, where the application of the first alternating electric field increases the permeability of the cell membrane; introducing a vector into the one or more cells, where the increased permeability of the cell membrane allows the vector to cross the one or more cell membranes, and the vector comprises a nucleic acid sequence capable of encoding a detectable or selectable marker, whereby once the vector has crossed the one or more cell membranes, the vector encodes the detectable or selectable marker; and detecting the detectable or selectable marker, where the presence of the detectable or selectable marker indicates the presence of the one or more specific cells.

[0157] In some aspects, the cell is a tumor or cancer cell. In some aspects, the cancer cell is a glioblastoma cell, a uterine sarcoma cell, a breast adenocarcinoma cell, a pancreatic cancer cell, a non-small cell lung cancer cell, a hepatic cell, a gastric cancer cell, or a brain cancer cell.

[0158] In some embodiments, the particular cell type is a diseased cell. For example, in some embodiments, the particular cell type is a cancer cell. In some embodiments, the diseased cell may be identified as a cell expressing a particular cell surface marker that is not present in a healthy cell. In some embodiments, the vector is thereby targeted to a particular cell type. Adding targeting molecules to a vector is well known in the art, for example, a molecule that targets a particular ligand may be incorporated into the viral surface and / or may modify the viral envelope protein. In some embodiments, a cancer-specific antibody (e.g., a CD20 antibody) may be incorporated into a viral vector, whereby the CD20 antibody directs the vector to CD20-positive cells, allowing the virus to transduce the CD20-positive cells. When the virus enters a cell, the detectable marker may be detected, thereby identifying the cell as a CD20-positive cell.

[0159] In some aspects, the alternating electric field can be any of those described for the delivery of vectors into cells.

[0160] In some embodiments, the step of introducing the vector begins at a given time, and the step of applying the AC electric field ends at least 12 hours after the given time. In some embodiments, the step of applying the AC electric field begins at least 1 hour before the given time. In some embodiments, the step of applying the AC electric field begins at least 1 to about 24 hours before the given time.

[0161] 2. Detection and Treatment In some aspects, the method of detecting a particular cell type may further include treating the cells. In some aspects, the vector further includes a therapeutic agent. In some aspects, the therapeutic agent may be "on" in that a nucleic acid sequence capable of encoding the therapeutic agent may be activatable. In some aspects, once a particular cell type (e.g., a cancer cell) is detected, then a therapy may be administered. The therapy may thereby be administered as part of the vector or separately once detection is confirmed. In some aspects, administering the therapy may be performed using any technique in which a vector is administered.

[0162] Disclosed are methods of detecting and treating a cell in need thereof, comprising: applying a first alternating electric field at a first frequency to the cell for a period of time, where the application of the alternating electric field increases the permeability of the cell membrane; introducing a vector into the cell, where the increased permeability of the cell membrane allows the vector to cross the cell membrane, where the vector comprises a nucleic acid sequence capable of encoding a therapeutic agent and a detectable / selectable marker, where the vector encodes a therapeutic agent and a detectable / selectable marker upon entry of the vector into the cell, and detecting the detectable / selectable marker, where the presence of the detectable / selectable marker indicates the presence of a cell in need of treatment, and where the therapeutic agent encoded by the vector treats the cell. In some embodiments, "treating a cell" can refer to inducing apoptosis of the cell.

[0163] In some aspects, the cell is a tumor or cancer cell. In some aspects, the cancer cell is a glioblastoma cell, a uterine sarcoma cell, a breast adenocarcinoma cell, a pancreatic cancer cell, a non-small cell lung cancer cell, a hepatic cell, a gastric cancer cell, or a brain cancer cell.

[0164] In some embodiments, the disclosed method of detecting cells may further include administering a second alternating electric field at a second frequency. Thus, in some embodiments, the second alternating electric field is a tumor treatment electric field. In some embodiments, the first alternating electric field and the second alternating electric field are different. In some embodiments, the first or second alternating electric field or both may have a frequency between 50 kHz and 1 MHz. The first and second alternating electric fields may be one or more of those disclosed herein. For example, in some embodiments, the cancer cells include glioblastoma cells, the first alternating electric field may have a first frequency between 250 kHz and 350 kHz, and the second alternating electric field may have a frequency between 150 kHz and 250 kHz. In some embodiments, the cancer cells include uterine sarcoma cells, the first alternating electric field may have a first frequency between 125 kHz and 175 kHz, and the second alternating electric field may have a frequency between 75 kHz and 125 kHz. In some embodiments, the cancer cells include breast adenocarcinoma cells, and the first alternating electric field may have a first frequency between 75 kHz and 175 kHz, and the second alternating electric field may have a frequency between 100 kHz and 300 kHz. The alternating electric field may vary based on cell type. In some embodiments, the alternating electric field has a field strength of at least 1 V / cm RMS. In some embodiments, the alternating electric field has a field strength between 1 and 4 V / cm RMS.

[0165] In some embodiments, the second alternating electric field is a treatment. In some embodiments, the second alternating electric field can kill cells, such as cancer cells.

[0166] In some embodiments, the step of introducing the vector begins at a given time, and the step of applying the AC electric field ends at least 12 hours after the given time. In some embodiments, the step of applying the AC electric field begins at least 1 hour before the given time. In some embodiments, the step of applying the AC electric field begins at least 1 to about 24 hours before the given time.

[0167] In some embodiments, the cells are present in the body of a living subject, the first alternating electric field is applied to the cells by applying the first alternating electric field to the subject's body, the second alternating electric field is applied to the cells by applying the second alternating electric field to the subject's body, and the introducing step comprises administering the vector to the subject.

[0168] In some aspects, an additional treatment or therapeutic regimen (e.g., chemotherapy or radiation therapy) may be administered to the subject before, simultaneously with, or after the composition (e.g., vector) is delivered to the subject's cells.

[0169] H.Kit The above-mentioned materials and other materials can be packaged together in any suitable combination as a useful kit for carrying out or assisting in carrying out the method of the present disclosure. The kit components in a given kit are useful when designed and adapted for combined use in the method of the present disclosure. For example, kits for imaging and / or treatment are disclosed. In some embodiments, the kit can include one or more vectors of the present disclosure. The kit can also include an instrument for applying an alternating electric field.

[0170] Disclosed herein is a kit comprising one or more vectors described herein and a device capable of applying an alternating electric field. For example, disclosed herein is a kit comprising one or more vectors described herein and a TTFields device (e.g., Optune®, Novocure Ltd.). EXAMPLES

[0171] I. Working Example

[0172] The ability to transport viruses across cell membranes was studied using A549 (ATCC® CCL-185™) and influenza (PR8) was evaluated.

[0173] A549 (ATCC® CCL-185™) and influenza (PR8) were used. Cells were grown in DMEM (Biological Industries, 01-055-1A) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, 04-007-1A).

[0174] A549 cells were plated on a cover glass (diameter 22 mm) at 1.5 × 10 cells. 5 Cells were seeded at a density of 1 / coverslip. After 24 h of culture, cells were transferred to invitro dishes containing 2 ml DMEM supplemented with 2% FBS. Cells were infected with influenza (PR8) virus at 1% or 0.01% concentration for 6 h. After infection, cells were washed with PBS and maintained in DMEM supplemented with 2% FBS for an additional 18 or 42 h. TTFields were applied either during infection and during the growth phase. 24 or 48 h after infection, growth medium was collected and cells were trypsinized, resuspended and counted using a Scepter™ 2.0 Cell Counter (Merck, Millipore). After resuspension, cells were washed with PBS. Supernatants and pellets were frozen at -80°C and transferred to Sheba for viral genome extraction and qRT-PCR.

[0175] TTFields (150 kHz, 1.5 V / cm RMS) were applied using the inovitro™ system (Novocure Ltd) with the following modifications: the inovitro plate was covered with parafilm and the medium was not replaced during cell culture.

[0176] Data from one experiment. Data are expressed as mean ± SD. Statistical significance was analyzed by Student's t-test.

[0177] Compared to untreated cultures, there was a trend towards a decrease in cell number after 24 h of TTFields treatment with 0.01% virus and a significant decrease in cell number with 1% virus (Figure 1). After 48 h of TTFields treatment, there was a significant decrease in cell number with both 0.01% and 1% virus (Figure 2). After 24 h of TTFields treatment with both virus concentrations, there was a trend towards an increase in virus copies / cell, RQ and SN (Figure 1). After 48 h of TTFields treatment, there was a significant increase in virus copies in SN with both virus concentrations and with 1% virus. In addition, there was a trend towards an increase in virus copies / cell with 0.01% virus and in RQ with both concentrations (Figure 2).

[0178] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical composition comprising a vector for use in a method for transporting the vector across a plasma membrane of a cell for gene therapy in a subject, the method comprising: applying an alternating electric field to the cells for a period of time, the application of the alternating electric field increasing the permeability of the cell membrane; and Introducing a vector into a cell, where an increase in the permeability of the cell membrane allows the vector to cross the cell membrane.

13. A pharmaceutical composition comprising:

2. An in vitro method for transporting a vector across a plasma membrane of a cell, the method comprising: applying an alternating electric field to the cells for a period of time, the application of the alternating electric field increasing the permeability of the cell membrane; and Introducing a vector into a cell, where an increase in the permeability of the cell membrane allows the vector to cross the cell membrane. A method comprising:

3. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the vector comprises a nucleic acid sequence capable of encoding a sequence of interest.

4. 4. The pharmaceutical composition of claim 3 or the method of claim 3, wherein the sequence of interest is a selectable marker or a therapeutic agent.

5. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the vector comprises two nucleic acid sequences, one nucleic acid sequence capable of encoding a selectable marker and a second nucleic acid sequence capable of encoding or is a therapeutic agent.

6. the therapeutic agent is a cDNA, a DNA, an mRNA, a microRNA, an shRNA, an siRNA, a ribozyme, a guide RNA molecule (gRNA), an RNA-guided endonuclease protein or a Cas9 protein or a Cpf1 protein CRISPR-Cas, and / or 5. The pharmaceutical composition of claim 4 or the method of claim 4, wherein the selectable marker is dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, puromycin, green fluorescent protein (GFP), hygromycin resistance (Hyg), neomycin resistance (Neo) or β-galactosidase (β-gal).

7. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the vector is a plasmid, cosmid, phagemid or viral vector.

8. the vector is a viral vector, 8. The pharmaceutical composition of claim 7 or the method of claim 7, optionally wherein the viral vector is an adenovirus, an adeno-associated virus, a lentivirus or a herpesvirus.

9. the cell is a cancer or tumor cell; 3. The pharmaceutical composition of claim 1 or the method of claim 2, optionally wherein the cancer cells are glioblastoma cells, uterine sarcoma cells, breast adenocarcinoma cells, pancreatic cancer cells, non-small cell lung cancer cells, hepatic cells, gastric cancer cells, or brain cancer cells.

10. 3. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the alternating electric field is applied at a frequency between 250 kHz and 350 kHz, between 50 and 190 kHz, or between 210 and 400 kHz.

11. 3. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the alternating electric field has a field strength of at least 1 V / cm RMS, or the alternating electric field has a field strength of between 1 and 4 V / cm RMS.

12. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the step of introducing the vector begins at a given time, and the step of applying an alternating electric field ends at least 12 hours after the given time, and optionally, the step of applying an alternating electric field begins at least 1 hour before the given time.

13. the cancer cells include glioblastoma cells and the alternating electric field has a frequency that is between 250 kHz and 350 kHz; or the cancer cells include uterine sarcoma cells and the alternating electric field has a frequency that is between 125 kHz and 175 kHz; or 10. The pharmaceutical composition of claim 9 or the method of claim 9, wherein the cancer cells include breast adenocarcinoma cells and the alternating electric field has a frequency between 75 kHz and 175 kHz.

14. the vector is introduced intratumorally, intracranially, intraventricularly, intrathecally, epidurally, intradurally, intravascularly, intravenously (targeted or non-targeted), intraarterially, intramuscularly, subcutaneously, intraperitoneally, orally, intranasally, via intratumoral injection (e.g., under computed tomography guidance during surgery or biopsy) or via inhalation, 3. The pharmaceutical composition of claim 1 or the method of claim 2, optionally wherein the vector is introduced into the cell in a targeted or non-targeted manner.

15. 3. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the vector is introduced once, twice, three or more times.

16. The pharmaceutical composition of claim 1, further comprising a chemotherapeutic agent.

17. the vector further comprises one or more targeting moieties; Optionally, one or more targeting moieties are specific to a cell; 3. The pharmaceutical composition of claim 1 or the method of claim 2, further optionally wherein one or more targeting moieties is a peptide, an antibody, an antibody fragment or a therapeutic agent.

18. The cell is a tumor or cancer cell, The method is for reducing the viability of a tumor or cancer cell, the alternating electric field is a first alternating electric field at a first frequency; 3. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the vector comprises a nucleic acid sequence capable of encoding a therapeutic agent, and when the vector crosses the tumor or cancer cell membrane, the vector encodes the therapeutic agent, and the presence of the therapeutic agent reduces the viability of the tumor or cancer cell.

19. The method further comprising administering a second alternating electric field at a second frequency; 19. The pharmaceutical composition of claim 18 or the method of claim 18, optionally wherein the second alternating electric field is a tumor treating electric field.

20. The cell is a tumor or cancer cell, the method further comprising detecting a tumor or cancer cell; the alternating electric field is a first alternating electric field at a first frequency; the vector comprises a nucleic acid sequence capable of encoding a selectable marker, and when the vector crosses the tumor or cancer cell membrane, the vector encodes the selectable marker; 3. The pharmaceutical composition of claim 1 or the method of claim 2, wherein the method further comprises the step of detecting a selectable marker, the presence of which indicates the presence of tumor or cancer cells.

21. administering a second alternating electric field at a second frequency; 21. The pharmaceutical composition of claim 20 or the method of claim 20, optionally wherein the second alternating electric field is a tumor treating electric field.

22. the cancer cells are glioblastoma cells, uterine sarcoma cells, breast cancer cells, pancreatic cancer cells, non-small cell lung cancer cells, hepatic cells, gastric cancer cells, or brain cancer cells; optionally, the cancer cells include glioblastoma cells and the first frequency is between 250 kHz and 350 kHz; or the cancer cells include uterine sarcoma cells and the first frequency is between 125 kHz and 175 kHz; or 22. The pharmaceutical composition of claim 21 or the method of claim 21, wherein the cancer cells comprise breast adenocarcinoma cells and the first frequency is between 75 kHz and 175 kHz.

23. the step of introducing the vector begins at a given time and the step of applying the first alternating electric field ends at least 12 hours after the given time; and / or 21. The pharmaceutical composition of claim 20 or the method of claim 20, wherein the step of applying the first alternating electric field begins at least 1 hour before the given time.

24. 21. The pharmaceutical composition of claim 20 or the method of claim 20, further comprising administering a therapeutic agent.

25. The cell is a tumor or cancer cell, The method further comprises detecting and treating tumor or cancer cells; the alternating electric field is a first alternating electric field at a first frequency; The vector comprises a nucleic acid sequence capable of encoding a therapeutic agent and a selectable marker, and when the vector enters a cell, the vector encodes the therapeutic agent and the selectable marker, The pharmaceutical composition of claim 1 or the method of claim 2, wherein the presence of the selectable marker indicates the presence of tumor or cancer cells, and the therapeutic agent encoded by the vector induces apoptosis of the tumor or cancer cells.

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