WO2020091958A1

WO2020091958A1 - Multi-component vector systems, methods of making, and uses thereof

Info

Publication number: WO2020091958A1
Application number: PCT/US2019/055029
Authority: WO
Inventors: Lukas DC GRUENERT
Original assignee: Onconetics Pharmaceuticals, Inc.
Priority date: 2018-11-01
Filing date: 2019-10-07
Publication date: 2020-05-07
Also published as: EP3874041A1

Abstract

Provided herein are vectors, vector systems, compositions and methods for treating cancer. In an exemplary embodiment, the present disclosure provides a vector system comprising a transgene, the expression of which is regulated by an inducible promoter and a microRNA binding domain (MBD). In some embodiments, the inducible promoter is activated using a tetracycline-on/tetracycline-off expression system. The presence of the MBD in the vector system facilitates the expression in a specific cell type. Also provided is a vector for the expression of an antigen or an immunogenic epitope of an antigen, wherein the vector comprises a MBD that facilitates the expression of the antigen or the immunogenic epitope specifically in cancer cells. The present disclosure also provides compositions comprising the vector system, compositions comprising the vector, and methods of using the vector systems and the vectors for treating cancer.

Description

MULTI-COMPONENT VECTOR SYSTEMS, METHODS OF MAKING, AND USES

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present International Application claims the benefit of priority to U.S. Provisional Application No. 62/754,334, filed on November 1, 2018, the contents of which are hereby incorporated by reference in their entireties.

BACKGROUND

[0002] Current treatments for cancer have side effects that reduce the efficacy of treatments. One of the side effects is the toxicity of the treatment on healthy cells. Thus, there is an unmet need to provide therapeutic compositions and methods where the therapeutic agent is specifically expressed in target cancer cells but not in healthy cells.

BRIEF SUMMARY

[0003] Provided herein are vectors, vector systems, compositions and methods for treating and diagnosing cancer. For example, the present disclosure provides a vector system for the expression of a therapeutic protein, wherein the vector system comprises at least two DNA sequences: one sequence comprising a transgene encoding a therapeutic protein under the control of an inducible promoter and a microRNA binding domain (MBD) that facilitates the expression of the therapeutic protein in cancer cells and inhibits the expression of the therapeutic protein in non-cancer cells and a second sequence comprising a gene encoding an inducer or a component of an inducer of the inducible promoter of the transgene.

[0004] Accordingly, in one aspect, provided herein is a vector system comprising: (a) a first deoxyribonucleic acid (DNA) sequence comprising an inducible promoter, a gene encoding an inducer or a component of an inducer of the inducible promoter, and a first microRNA binding domain (MBD), wherein the first MBD comprises one or more microRNA binding sites (MBSs), wherein each MBS is specific for a microRNA (miR) that is present in a non-cancer cell and is not present or is downregulated in a cancer cell; and (b) a second DNA sequence comprising the same inducible promoter, a transgene, and a second MBD, wherein the second MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell.

[0005] In another aspect, the vector system comprises (a) a first DNA sequence comprising a tetracycline-inducible promoter, a gene encoding a tetracycline-controlled transactivator (tTA), and a first microRNA binding domain (MBD), wherein the first MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell; and (b) a second DNA sequence comprising the tetracycline-inducible promoter, a transgene, and a second MBD, wherein the second MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non cancer cell and is not present or is downregulated in a cancer cell.

[0006] In yet another aspect, provided herein is a vector comprising: (a) a first deoxyribonucleic acid (DNA) sequence comprising a transgene, wherein the transgene encodes for an antigen or an immunogenic epitope of an antigen; and (b) a second deoxyribonucleic (DNA) acid sequence comprising a microRNA binding domain (MBD); wherein the MBD comprises one or more microRNA binding sites (MBSs), wherein each MBS is specific for a microRNA (miR) that is present in a non-cancer cell and is not present or is downregulated in a cancer cell.

[0007] The present disclosure also provides compositions comprising the vectors and methods of using the vectors for treating and/or diagnosing cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 depicts an exemplary construct that may be present in a vector system according to the disclosure.

[0009] FIG. 2 depicts an exemplary structure of the first, second, third, or the fourth DNA sequence of the vector system according to the disclosure.

[0010] FIGs. 3-10 depict a series of events that is expected to occur in a cancer cell pre-treated with doxycycline and transfected with an exemplary vector system shown in FIG. 1. Specifically, FIGs. 3, 4, and 5 depict the expression status of the genes from the vector system expected to be transcribed during the first about 24 hours after transfection of doxycycline- treated cancer cells with the exemplary vector system of the disclosure. FIG. 6 depicts the expected status of the expressed proteins about 24 hours post-transfection of doxycycline-treated cancer cells with the exemplary vector system. FIG. 7 depicts the expected expression status of the tTA gene about 24-30 hours post-transfection of doxycycline-treated cancer cells with the exemplary vector system. FIGs. 8 and 9 depict the expected initiation of positive feedback loop about 30-72 hours post-transfection of doxycycline-treated cancer cells with the exemplary vector system. FIG. 10 depicts the expected continuation of the positive feedback loop after about 72 hours post-transfection of doxycycline-treated cancer cells with the exemplary vector system.

[0011] FIG. 11 depicts the expected expression status of the genes from the vector system about 30 hours after transfection of healthy cells with the exemplary vector system.

[0012] FIG. 12 depicts an exemplary transgene expression construct that may be present in a vector according to the disclosure.

[0013] FIG. 13 shows am image of PANC1 cells expressing RFP 48 hours after transfection of the cells with 1 pg of pCAELION vector as described in Example 2. The cells were treated with doxycycline and IPTG for a total of about 60 hours starting before transfection.

[0014] FIG. 14 shows am image of PANC1 cells expressing GFP 48 hours after transfection of the cells with 1 pg of pCAELION vector as described in Example 2. The cells were treated with doxycycline and IPTG for a total of about 60 hours starting before transfection.

[0015] FIG. 15 is a graph showing the number of cells expressing the RFP and the GFP from the images shown in FIGs. 13 and 14.

[0016] FIG. 16 shows am image of MCF10A cells expressing RFP 48 hours after transfection of the cells with 1 pg of pCAELION vector as described in Example 2. The cells were treated with doxycycline and IPTG for a total of about 60 hours starting before transfection.

[0017] FIG. 17 shows am image of MCF10A cells expressing GFP 48 hours after transfection of the cells with 1 pg of pCAELION vector as described in Example 2. The cells were treated with doxycycline and IPTG for a total of about 60 hours starting before transfection.

[0018] FIG. 18 is a graph showing the number of cells expressing the RFP and the GFP from the images shown in FIGs. 16 and 17.

DETAILED DESCRIPTION

[0019] Provided herein are vectors, vector systems, compositions and methods for treating and diagnosing cancer. By way of example, the present disclosure provides a vector system for the expression of a therapeutic protein, wherein the vector system is designed to initiate a positive feedback loop that drives a continuous expression of a protein (e.g. a therapeutic protein for treatment, or a detectable protein for diagnostics) once induced. For example, in some embodiments, the vector system comprises (1) a gene encoding an inducer or a component of an inducer of an inducible promoter, wherein the gene is under the control of the inducible promoter, and (2) a transgene encoding a therapeutic protein under the control of the inducible promoter and a microRNA binding domain (MBD) that facilitates the expression of the therapeutic protein in cancer cells and does not facilitate the expression of the therapeutic protein in non-cancer cells. The activation of the inducible promoter of the gene encoding the inducer, or the component of the inducer, of the inducible promoter, and the activation of the inducible promoter of the transgene initiates a positive feedback loop that drives a continuous expression of the therapeutic protein.

[0020] The present disclosure also provides a vector comprising a transgene that encodes for an antigen or an immunogenic epitope of an antigen, wherein the transgene is under the control of a MBD that facilitates the expression of the antigen or the immunogenic epitope of an antigen in cancer cells and inhibits the expression of the antigen or the immunogenic epitope of an antigen in non-cancer cells.

[0021] Also provided herein are compositions comprising the vectors or vector systems and methods of using the vectors or vector systems for treating and/or diagnosing cancer.

[0022] Before describing certain embodiments in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular illustrative embodiments only, and is not intended to be limiting. The terms used in this specification generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them. The scope and meaning of any use of a term will be apparent from the specific context in which the term is used. As such, the definitions set forth herein are intended to provide illustrative guidance in ascertaining particular embodiments of the disclosure, without limitation to particular compositions or biological systems. [0023] As used in the present disclosure and the appended claims, the singular forms“a,”“an” and“the” include plural references unless the content clearly dictates otherwise.

[0024] Throughout the present disclosure and the appended claims, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or group of elements but not the exclusion of any other element or group of elements.

[0025] As used herein, the terms“microRNA,”“miRNA,” and“miR” are used interchangeably and refers to a non-coding RNA that is about 20 to 35 nucleotides long and that post- transcriptionally regulates the cleavage of a target mRNA or represses the translation of the target mRNA. Throughout this disclosure, the effect of binding of miRNAs to the miRNA binding sites (MBSs) of the MBD is described. In so describing, the effects include preventing, and/or inhibiting/repressing the cleavage of the transgene mRNA and/or inhibition of the translation of the transgene mRNA.

[0026] The term“non-cancer cell” as used herein encompasses a heathy cell or a cell that is non- cancerous. The terms “healthy” and “normal” are used interchangeably throughout the disclosure.

[0027] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery. Vectors

Vector systems for a positive feedback looy-based expression of a transsene

[0028] The present application provides a vector system comprising at least two DNA sequences: (a) a first DNA sequence comprising an inducible promoter, a gene encoding an inducer or a component of an inducer of the inducible promoter (“a first inducer gene”), and a first microRNA binding domain (MBD); and (b) a second DNA sequence comprising the same inducible promoter, a transgene, and a second MBD. The first and the second MBDs comprise one or more microRNA binding sites (MBSs), wherein each MBS is specific for a microRNA that is endogenously expressed in a non-cancer cell and is not expressed or is downregulated in a cancer cell. In the presence of specific microRNAs for which the MBSs are present in the first and the second MBDs of the vector system, the transgene and the first inducer gene are not expressed. Without being bound by theory, the microRNAs can bind to the MBSs and inhibit or prevent the translation of the transgene mRNA (e.g. by inducing cleavage of the transgene mRNA or repressing the translation of the transgene mRNA) and the mRNA of the first inducer gene. On the other hand, the transgene and the first inducer gene can be expressed in cells where the specific microRNAs are not present or are downregulated. For example, the transgene and the first inducer gene can be expressed in cancer cells where the specific microRNAs are not present or are downregulated. This vector system may be referred to as a“miRNA-regulated vector system”. Additionally, the first inducer gene and the transgene of the vector system are under the control of an inducible promoter which is activated by the inducer, or the component of the inducer, encoded by the first inducer gene. The activation of the inducible promoter of the first inducer gene and the transgene starts a positive feedback loop, wherein the inducer binds and activates the first inducer gene to make more inducer and the inducer binds and activates the transgene to express more therapeutic protein.

[0029] In some embodiments, the inducible promoter of the first inducer gene may be activated initially by supplying the inducer, or the component of the inducer, externally. Once the first inducer gene is activated, it will activate the inducer, or the component of the inducer, in vivo , which in turn will activate the expression of the first inducer gene and the transgene. The expression of the inducer, or the component of the inducer, in vivo and the activation of the inducible promoters of the first inducer gene and the transgene by the inducer create a positive feedback loop that provides a continuous expression of the transgene.

[0030] In some embodiments, the inducible promoter of the first inducer gene may be activated by including in the vector system a third DNA sequence comprising a constitutively active promoter, a gene encoding an inducer or a component of an inducer of the inducible promoter (“a second inducer gene”), and a third MBD, wherein the third MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell. In this embodiment, the expression of the second inducer gene provides the inducer, or the component of the inducer, that activates the inducible promoter of the first inducer gene.

[0031] In an exemplary embodiment, the vector system provides an on/off type of an expression system for the expression of the transgene. For example, in some embodiments, the vector system provides a tetracycline-on/tetracycline-off (tet-on/tet-off) type of an expression system for the expression of a transgene. The on/off type expression of the transgene is further regulated by the MBDs that allow the expression of the transgene specifically in cancer cells while providing minimal or no expression of the transgene in non-cancer cells.

[0032] In an exemplary embodiment of a tet-on/tet-off type expression system, the vector system comprises (a) a first DNA sequence comprising a tetracycline-inducible promoter, a gene encoding a tetracycline-controlled transactivator (tTA) (“tTA gene”), and a first microRNA binding domain (MBD), wherein the first MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell; and (b) a second DNA sequence comprising a tetracycline- inducible promoter, a transgene, and a second MBD, wherein the second MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell. In this embodiment, since both the tTA gene and the transgene are under the control of the tetracycline-inducible promoter, the inducer of the tetracycline-inducible promoter need to be supplied initially for the vector system to activate the expression of the tTA gene and the transgene; once the expression of the tTA gene is induced, the tTA protein continues the function of induction. The inducer of the tetracycline-inducible promoter to be supplied initially can be administered in the form of a purified protein or in the form of a gene encoding the inducer that is under the control of a constitute promoter as described below. The presence of the first and the second MBD facilitates the expression of the tTA gene and the transgene specifically in cancer cells and provides minimal or no expression of these two genes in healthy cells.

[0033] In some embodiments, the expression of the tTA gene can be induced by contacting cancer cells with tetracycline or doxycycline and a reverse tetracycline-controlled transactivator (rtTA). It is expected that the rtTA would bind tetracycline or doxycycline and the rtTA- tetracycline or rtTA-doxycy cline complex would activate the tetracycline-inducible promoters of the tTA gene and the transgene thereby initiating the transcription and translation of the tTA gene and the transgene. It is expected that as the concentration of tetracycline or doxycycline in the cancer cell decreases, the concentration of rtTA-tetracycline or rtTA-doxycycline complex in the cancer cell would also decrease leading to less activation of tetracycline-inducible promoters of the tTA gene and the transgene by rtTA. However, because the tTA gene has already been activated and expressed in the cancer cell, it is expected that tTA would bind to its own tetracycline-inducible promoter in the first DNA sequence initiating a positive feedback loop by making more tTA. It is expected that as the concentration of tetracycline or doxycycline continues to decrease and the concentration of tTA increases, there could be a period of time where tTA binds to either tetracycline or doxycycline thereby creating a transient complex of tTA-tetracycline or tTA-doxycycline. This transient complex is expected to directly compete with the binding of tTA to tetracycline-inducible promoters. During this period of time, however, it is expected that the sum of the molar concentrations of activating complexes (i.e rtTA-tetracycline or rtTA-doxycycline complexes and free tTA) will match or exceed the molar concentrations of competing complexes (i.e. tTA-tetracycline or tTA-doxycycline complexes), and as more tTA is expressed, the molar concentration of tTA will increase over time thus accelerating the rate of transcription by the tetracycline-inducible promoters. It is expected that the positive feedback loop continues to accelerate generating a large amount of tTA that binds to its own tetracycline-inducible promoter in the first DNA sequence and the tetracycline-inducible promoter of the transgene leading to a continuous expression of the transgene.

[0034] In another embodiment, the expression of the tTA gene can be induced by including in the vector system a third DNA sequence comprising a constitutively active promoter, a gene encoding a reverse tetracycline-controlled transactivator (rtTA), and a third MBD, wherein the third MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell. In this embodiment, it is expected that rtTA will be constitutively expressed by the cancer cells because in the cancer cells, the microRNAs that bind the MBSs in the third MBD are absent or are present at very low levels thereby allowing translation of the rtTA mRNA. It is expected that once the cancer cell is contacted with tetracycline or doxycycline, the rtTA would bind tetracycline or doxycycline and the rtTA-tetracy cline or rtTA-doxycycline complex would activate the tetracycline-inducible promoters of the tTA gene and the transgene thereby initiating the transcription and translation of the tTA gene and the transgene. It is expected that as the concentration of tetracycline or doxycycline in the cancer cell decreases, the concentration of rtTA-tetracycline or rtTA-doxycycline complex in the cancer cell would also decrease leading to less activation of tetracycline-inducible promoters of the tTA gene and the transgene by rtTA. However, because the tTA gene has already been activated and expressed in the cancer cell, it is expected that tTA would bind to its own tetracycline-inducible promoter in the first DNA sequence initiating a positive feedback loop by making more tTA. It is expected that as the concentration of tetracycline or doxycycline continues to decrease and the concentration of tTA increases, there could be a period of time where tTA binds to either tetracycline or doxycycline thereby creating a transient complex of tTA-tetracy cline or tTA-doxycycline. This transient complex is expected to directly compete with the binding of tTA to tetracycline-inducible promoters. During this period of time, however, it is expected that the sum of the molar concentrations of activating complexes (i.e rtTA-tetracy cline or rtTA-doxycycline complexes and free tTA) will match or exceed the molar concentrations of competing complexes (i.e. tTA- tetracycline or tTA-doxycycline complexes), and as more tTA is expressed, the molar concentration of tTA will increase over time thus accelerating the rate of transcription by the tetracycline-inducible promoters. It is expected that the positive feedback loop continues to accelerate generating a large amount of tTA that binds to its own tetracycline-inducible promoter in the first DNA sequence and the tetracycline-inducible promoter of the transgene leading to a continuous expression of the transgene.

[0035] In some embodiments, the expression of the transgene can be further regulated by including in the second DNA sequence one or more binding sites for a transcription repressor 3’ or 5’ to the transgene and including in the vector system a fourth DNA sequence comprising a constitutively active promoter, a gene encoding the transcription repressor, and a fourth MBD, wherein the fourth MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a cancer cell. In the presence of specific microRNAs for which the MBSs are present in the fourth MBD of the vector system, the gene encoding the transcription repressor is not expressed. Since, the MBSs in the fourth MBD are specific for microRNAs present in the cancer cells, the gene encoding the transcription repressor is not expressed in cancer cells but is expressed in non-cancer cells. An exemplary miRNA that is present in a cancer cell includes, but is not limited to, miR-l55.

[0036] In an exemplary embodiment, the second DNA sequence of the vector system comprises one or more binding sites for a lac repressor 3’ or 5’ to the start codon of the transgene and the vector system comprises a fourth DNA sequence comprising a constitutively active promoter, a lacl gene encoding the lactose operon (lac) repressor, and a fourth MBD; wherein the fourth MBD comprises one or MBSs, wherein each MBS is specific for a miR that is present in a cancer cell. Since the lacl gene is under the control of an MBD (the fourth MBD) that is specific for microRNAs present in the cancer cells, it is expected that the lacl gene is not expressed or minimally expressed in the cancer cells.

[0037] In an exemplary embodiment, the vector system comprises (a) a first DNA sequence comprising a tetracycline-inducible promoter, a gene encoding a tetracycline-controlled transactivator (tTA), and a first microRNA binding domain (MBD), wherein the first MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non cancer cell and is not present or is downregulated in a cancer cell; (b) a second DNA sequence comprising a tetracycline-inducible promoter, a transgene , one or more binding sites for a lac repressor 3’ or 5’ to the start codon of the transgene, and a second MBD, wherein the second MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell; (c) a third DNA sequence comprising a constitutively active promoter, a gene encoding a reverse tetracycline-controlled transactivator (rtTA), and a third MBD, wherein the third MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell; and (d) a fourth DNA sequence comprising a constitutively active promoter, a lacl gene encoding a lactose operon repressor, and a fourth MBD; wherein the fourth MBD comprises one or MBSs, wherein each MBS is specific for a miR that is present in a cancer cell. This exemplary construct is shown in FIG. 1 [0038] In some embodiments, the first, second, third, and/or the fourth MBDs may be linked to the corresponding gene sequence (for e.g., the corresponding gene sequence for the first MBD is the gene sequence for the first inducer gene or the tTA gene, the corresponding gene sequence for the second MBD is the gene sequence for the transgene, and so on) in tandem, i.e., there is no overlap between the gene sequence and the MBD sequence. In some embodiments, the first, second, third, and/or the fourth MBDs could be located within the corresponding gene sequence. In some embodiments, some MBDs could be located in tandem with the corresponding gene sequence while the other MBDs could be located within the corresponding gene sequence.

[0039] The promoter, the gene sequence, and the MBD of each of the first, second, third, and the fourth DNA sequences can be considered to be operably linked. By“operably linked,” it is meant that the activation of the promoter is expected to activate the transcription of the corresponding gene sequence and the MBD.

[0040] The first, second, third, and/or the fourth DNA sequences can be present on one vector or they can be present on more than one vector. In some embodiments, the first and the second DNA sequences are on the same vector. In some embodiments, the first and the second DNA sequences are on different vectors. In some embodiments, the first and the second DNA sequences are on the same vector and the third DNA sequence is on a different vector. In some embodiments, the first and the third DNA sequences are on the same vector and the second DNA sequence is on a different vector. In some embodiments, the first DNA sequence is on one vector, and the second and third DNA sequences are on a second vector. In some embodiments, the first, second, and third DNA sequences are on the same vector. In some embodiments, the first and the second DNA sequences are on one vector and the third and the fourth DNA sequences are on a second vector. In some embodiments, the first, second, and third DNA sequences are on one vector, and the fourth DNA sequence is on a second vector. In some embodiments, the first, third, and fourth DNA sequences are on one vector, and the second DNA sequence is on a second vector. In some embodiments, the first, second, and fourth DNA sequences are on one vector, and the third DNA sequence is on a second vector. In some embodiments, the first and third DNA sequences are on one vector, and the second and fourth DNA sequences are on a second vector. In some embodiments, the first and fourth DNA sequences are on one vector, and the second and third DNA sequences are on a second vector. In some embodiments, the first DNA sequence is on one vector, and the second, third, and fourth DNA sequences are on a second vector.

[0041] When more than one DNA sequence is present on the same vector, any of the DNA sequences can be 3’ or 5’ to the other DNA sequences. For example, if the first and the second DNA sequences are present on the same vector, the first DNA sequence can be 3’ or 5’ to the second DNA sequence and so on.

[0042] In some embodiments, the transgene present in the vector systems of the present disclosure encodes a therapeutic protein. In some other embodiments, the transgene encodes an siRNA or a shRNA sequence, the expression of which silences a gene in target cells, e.g. cancer cells.

[0043] As provided herein, a therapeutic protein is any protein that inhibits the proliferation and/or metastasis of cancer cells. Examples of therapeutic proteins include, but are not limited to, an antigen or an immunogenic epitope of an antigen, apoptosis inducers, growth regulators, tumor suppressors, ion channels, cell-surface or internal antigens, or any protein mutated in cancer cells (e.g. BRCA1, BRCA2, etc.).

[0044] In certain embodiments, the therapeutic protein is an apoptosis-inducing protein, such as a caspase, thymidine kinase, e.g., Herpes Simplex Virus thymidine kinase (HSV-tk), a granzyme, an exotoxin, or a proapoptotic member of the Bcl-2 family. Expression of the HSV- tk mediates phosphorylation of the prodrug gancyclovir (GCV), thus inhibiting DNA replication in rapidly dividing cancer cells. As phosphorylated GCV is also toxic in normal cells, tumor cell-specific expression of HSV-tk is required and can be accomplished using the vectors of the present disclosure.

[0045] In certain embodiments, the therapeutic protein is an antigen or an immunogenic epitope of an antigen. In some embodiments, the therapeutic protein is an antigen or an immunogenic epitope of an antigen that a subject has been exposed to via a previous vaccination or infection. Exemplary therapeutic proteins in this embodiment include antigens or antigenic epitopes of Varicella-Zoster Virus (chickenpox or shingles virus), Herpes Simplex Virus, Hepatitis B virus, Measles virus (morbillivirus), Mumps virus (paramyxovirus), Rubella virus, Human Immunodeficiency Virus (HIV), Influenza virus, Rhinovirus, Corynebacterium diphtheriae , Clostridium tetani , and/or Bordetella pertussis. By using the vector system described herein, it is expected that the antigen or the immunogenic epitope of an antigen would be selectively expressed on the surface of the cancer cell transduced with the vector system of the present disclosure.

[0046] The antigen or the immunogenic epitope of an antigen expressed on the cancer cell surface can then be recognized by immune cells such as T cells or B cells of the subject, activating an immune response against the cancer cell. If the antigen or the immunogenic epitope of an antigen is the one that the subject has been previously exposed to via vaccination or infection, the expression of such antigen or the immunogenic epitope on cancer cells can trigger a memory T cell and/or a memory B cell response and promote faster elimination of the cancer cell expressing the antigen or the immunogenic epitope. Additionally, the vector may be introduced in together with other immuno-stimulatory agents, including checkpoint inhibitors (e.g. PD-l or PD-L1 inhibitors) in order to enhance the therapeutic effect of the antigen.

[0047] In some embodiments, the transgene present in the vector systems of the present disclosure can be a reporter gene encoding for a reporter protein, e.g. useful for in vitro, in vivo, or ex vivo diagnostics or medical imaging. In some embodiments, the reporter transgene encodes a fluorescent protein or a bioluminescent protein. For example, the transgene may encode a fluorescent protein selected from the group consisting of: green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, far-red fluorescent protein, orange fluorescent protein, and ultraviolet-excitable green fluorescent protein. These reporter proteins are known and are summarized by Shaner et ah, Nat Methods., 2005 Dec; 2(l2):905-9. In some embodiments, the reporter transgene may encode for a bioluminescent protein including a firefly luciferase such as Renilla luciferase.

[0048] In some embodiments, the first, second, and the third MBDs of the vector system comprise MBSs that are specific for microRNAs present in a cancer cell selected from the group consisting of: breast cancer cell, colon cancer cell, brain cancer cell, pancreatic cancer cell, lung cancer cell, cervical cancer cell, uterine cancer cell, prostate cancer cell, ovarian cancer cell, melanoma cancer cell, lymphoma cancer cell, myeloma cancer cell, and/or leukemic cancer cell. In some embodiments, the cancer cell is an early stage breast cancer cell. In some embodiments, the cancer cell is a late stage breast cancer cell.

[0049] FIG. 2 shows an exemplary structure of the first, second, third, or the fourth DNA sequence of the vector system. [0050] As provided herein and illustrated in FIG. 2, each of the first, second, third, and the fourth MBD comprises MBSs. In some embodiments, the MBD can comprise 1 to 12 MBSs, e.g. the MBD comprises MBSs that are specific for 1 to 12 microRNAs. In various embodiments, the MBD may comprise about 1-12, about 1-10, about 1-8, about 2-12, about 2- 10, about 2-8, about 2-6, about 2-5, about 3-12, about 3-12, about 3-10, about 3-8, about 3-6, about 4-12, about 4-10, about 4-8, about 4-6, about 5-10, or about 5-8 MBSs. For example, FIG. 2 exemplifies a vector system where the MBD comprises 2 MBSs; each MBS being specific for a different microRNA.

[0051] In some embodiments, the MBSs could be specific for the ~6 to 8-nucleotide“seed” sequence at the 5’ end of a miRNA. In some embodiments, the MBSs could be specific for other regions of miRNAs such as the sequence at the 3’ end of a miRNA. In yet some other embodiments, the MBSs could be specific for a sequence of a miRNA that forms the central loop in the miRNA:mRNA duplexes. In some embodiments, the MBSs could be specific for combinations of these features.

[0052] Multiple copies of each MBS may be present in the MBD. In various embodiments, 1- 12, 2-10, 4-8, or 3-6 copies of each MBS may be present in the MBD. In some embodiments, the MBD comprises at least 2 copies of each MBS. In some other embodiments, the MBD comprises 3, 4, 5, or 6 copies of each MBS. When the MBD comprises more than one MBS and multiple copies of each MBS are present, the multiple copies of each MBS may be present as a single cluster or the multiple copies may be scattered throughout the MBD, i.e. one or more copies of each MBS may alternate with one or more copies of other MBSs. For example, the exemplary expression construct shown in FIG. 2 comprises 2 MBSs in the MBD and 2 copies of each MBS are present in the MBD.

[0053] The length of each copy of an MBS can be selected based on desired binding aspects. In some embodiments, the length of each copy of an MBS may range from about 6 to 33 nucleotides. For example, the length of each copy of a MBS could be about 6 to about 33 nucleotides, about 6 to 30 nucleotides, about 6 to 27 nucleotides, about 6 to 25 nucleotides, about 6 to 23 nucleotides, about 6 to 20 nucleotides, about 6 to 18 nucleotides, about 6 to 15 nucleotides, about 6 to 13 nucleotides, about 6 to 11 nucleotides, or about 6 to 8 nucleotides. In some embodiments, the length of each copy of a MBS is about 6 to 13 nucleotides. [0054] As provided herein, an MBD can comprise multiple MBSs that are specific for different microRNAs. Also contemplated herein, an MBD can comprise multiple MBSs that are specific for the same microRNA, but bind to different regions of the microRNA.

[0055] Multiple copies of each MBS may be separated from each other by spacer sequences that are about 5 to 50 nucleotides long. For example the spacer sequences could be about 5 to 45, about 5 to 40, about 5 to 35, about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 10 to 50, about 10 to 45, about 10 to 40, about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10 to 15, about 15 to 50, about 15 to 45, about 15 to 40, about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20 nucleotides long. The individual MBSs may be separated from each other by about the same number of nucleotides.

[0056] The MBD can be located on either the 3’ or the 5’ side of the corresponding gene sequence (for e.g., the corresponding gene sequence for the first MBD is the gene sequence for the first inducer gene or the tTA gene, the corresponding gene sequence for the second MBD is the gene sequence for the transgene, and so on). In the exemplary expression construct shown in FIG. 2, the MBD is on the 3’ side of the corresponding gene sequence.

[0057] As provided herein, the transgene of the described vector system can encode a therapeutic protein or a reporter protein. In the protein encoding DNA sequences provided in the vector systems described herein, the DNA sequences need not contain any introns, unless the introns are determined to be required for the proper transcription of the transgene, proper functioning of the therapeutic protein, regulation of the transgene expression by miRNAs or any other aspect of the expression of the transgene

[0058] In some embodiments, vector systems may comprise components that may increase the translational efficiency of the transgene mRNA. For example, in some embodiments, 3’ UTRs that provide a high translational efficiency to an mRNA may be included in the vector systems. In some embodiments, a translational efficiency can be quantified as the ratio of ribosome protected fragments (RPF) to the abundance of ribonucleic acids (RNA) (Cottrell et al., Sci Rep. 2017 Nov 2;7(l): 14884). In some embodiments, the present disclosure provides vector systems that comprise 3’ UTRs that provide a translational efficiency of about -0.25 to about -0.8, about - 0.28 to about -0.8, about -0.3 to about -0.8, about -0.25 to about -0.75, -0.25 to about -0.7, about -0.25 to about -0.6, about -0.3 to about -0.75, including values and ranges therebetween. In these embodiments, the first, second, third, and/or the fourth DNA sequences of the vector system may comprise a DNA sequence comprising one or more 3’ UTRs of one or more genes (3’ UTR DNA sequence), wherein the 3’ UTR DNA sequence is 3’ of the coding sequence of the gene.

[0059] In some embodiments, the 3’ UTR DNA sequence comprises 3’ UTRs of one or more housekeeping genes, e.g., GAPDH, or cytoskeleton genes, e.g., a-tubulin, and b-tubulin. In some other embodiments, the 3’ UTR DNA sequence comprises 3’ UTRs of one or more genes selected from the group consisting of: Rpl32, HSP70a, and CrebA.

[0060] In some embodiments, the constitutive promoter is specifically expressed in cancer cells. In some embodiments, the constitutive promoter is selected from the group consisting of: SV40, CMV, EFla, PGK1, Ubc, human b actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal 1/10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, and U6.

[0061] The MBD may start from about 1 to 50 nucleotides after the last nucleotide of the stop codon of the corresponding gene. In various embodiments, the MBD may start from about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30, about 1 to 25, about 1 to 20, about 1 to 15, or about 1 to 10 nucleotides after the last nucleotide of the stop codon of the corresponding gene.

[0062] In some embodiments, the vector system may comprise a DNA sequence encoding a repressor element that is 5’ of the gene sequence. This repressor element may facilitate further repression of the expression of the transgene. In some embodiments, the DNA sequence encodes a hemagglutinin-A epitope as the repressor element.

[0063] The vectors of the vector system can be viral DNA vectors or non-viral DNA vectors. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex virus vectors. Non-viral vectors include plasmids and cosmids.

[0064] Vector systems of the present disclosure are used to express a transgene specifically in cancer cells. A transgene encodes a protein of interest, e.g., a therapeutic protein or a detectable marker protein. The expression of the protein of interest is regulated by endogenously expressed miRNAs. The MBSs present in the second MBD are specific for one or more miRNAs that are present in non-cancer cells and are absent or are down-regulated in cancer cells. Upon transcription of the second DNA sequence containing the transgene and the MBD, target miRNAs present in non-cancer cells are intended to bind to their corresponding MBSs present on the MBD of the transgene mRNA and inhibit the translation of the transgene mRNA thereby inhibiting the expression of the protein of interest in non-cancer cells. In cancer cells, the transgene mRNA is intended to be translated and the protein of interest would be expressed since target miRNAs that bind the MBSs of the second MBD are absent or are down-regulated in cancer cells. Additionally, the expression of the transgene is further regulated by including in the vector system an inducible promoter and a system to activate or turn-off the induction of the inducible promoter as described above.

Vectors for expression of a transsene encoding an antisen or antigenic epitopes

[0065] The present application provides a vector comprising a transgene, wherein the transgene encodes for an antigen or an immunogenic epitope of an antigen and a microRNA binding domain (MBD). The MBD comprises one or more microRNA binding sites (MBSs), wherein each MBS is specific for a microRNA that is endogenously expressed in a non-cancer cell and is not expressed or is downregulated in a cancer cell. In the presence of specific microRNAs for which the MBSs are present in the vector, the transgene is not expressed. Without being bound by theory, the microRNAs can bind to the MBSs and inhibit or prevent the translation of the transgene mRNA (e.g. by inducing cleavage of the transgene mRNA or repressing the translation of the transgene mRNA). On the other hand, the transgene can be expressed in cells where the specific microRNAs are not present or are downregulated. For example, the transgene can be expressed in cancer cells where the specific microRNAs are not present or are downregulated. In some embodiments, the therapeutic protein is an antigen or an immunogenic epitope of an antigen that a subject has been exposed to via a previous vaccination or infection.

[0066] In some embodiments, the vector comprises a first deoxyribonucleic acid (DNA) sequence comprising a transgene encoding an antigen or an immunogenic epitope of an antigen and a second DNA sequence comprising a MBD, wherein the MBD comprises one or more MBSs, wherein each MBS is specific for a microRNA that is present in a non-cancer cell and is not present or is downregulated in a cancer cell. This vector may be referred to as a“miRNA- regulated expression vector” or“miRNA-regulated vector”.

[0067] In some embodiments, the second DNA sequence comprising a MBD is linked to the first DNA sequence comprising a transgene in tandem, i.e., there is no overlap between the first and the second DNA sequences. In another embodiment, the second DNA sequence comprising a MBD is located within the first DNA sequence comprising a transgene. [0068] In some embodiments, the MBD comprises one or more MBSs that are specific for microRNAs present in a cancer cell selected from the group consisting of: breast cancer cell, colon cancer cell, brain cancer cell, pancreatic cancer cell, lung cancer cell, cervical cancer cell, uterine cancer cell, prostate cancer cell, ovarian cancer cell, melanoma cancer cell, lymphoma cancer cell, myeloma cancer cell, and/or leukemic cancer cell.

[0069] In some embodiments, the cancer cell is an early stage breast cancer cell. The term “early stage breast cancer” as used herein refers to cancer that has not spread beyond the breast or axillary lymph nodes. This includes ductal carcinoma in situ and stage IA, stage IB, stage IIA, stage IIB and stage IIIA breast cancers as defined by the American Joint Committee on Cancer (AJCC) in the AJCC Cancer Staging Manual, 7^th Edition. In such an embodiment, the vector comprises a first DNA sequence comprising a transgene encoding an antigen or an immunogenic epitope of an antigen; and a second DNA sequence comprising a MBD; wherein the MBD comprises one or more MBSs, wherein each MBS is specific for a microRNA that is present in a non early-stage breast cancer cell and is not present or is downregulated in an early stage breast cancer cell.

[0070] In another embodiment, the cancer cell is a late stage breast cancer cell. The term“late stage breast cancer” as used herein refers to cancer originating in the breast that is far along in its growth and has spread beyond the axillary lymph nodes and other areas in the body. This includes stage MB, stage IIIC and stage IV breast cancer as defined by the American Joint Committee on Cancer (AJCC) in the AJCC Cancer Staging Manual, 7^th Edition. In such an embodiment, the vector comprises a first DNA sequence comprising a transgene encoding an antigen or an immunogenic epitope of an antigen; and a second DNA sequence comprising a MBD; wherein the MBD comprises one or more MBSs, wherein each MBS is specific for a microRNA that is present in a non late-stage breast cancer cell and is not present or is downregulated in a late stage breast cancer cell.

[0071] FIG. 12 shows an exemplary transgene expression construct that may be present in a vector of the present disclosure.

[0072] As provided herein and illustrated in FIG. 12, the MBD comprises MBSs. In some embodiments, the MBD can comprise 1 to 12 MBSs, e.g. the MBD comprises MBSs that are specific for 1 to 12 microRNAs. In various embodiments, the MBD may comprise about 1-12, about 1-10, about 1-8, about 2-12, about 2-10, about 2-8, about 2-6, about 2-5, about 3-12, about 3-12, about 3-10, about 3-8, about 3-6, about 4-12, about 4-10, about 4-8, about 4-6, about 5-10, or about 5-8 MBSs. For example, FIG. 12 exemplifies a vector where the MBD comprises 2 MBSs; each MBS being specific for a different microRNA.

[0073] In some embodiments, the MBSs could be specific for the ~6 to 8-nucleotide“seed” sequence at the 5’ end of a miRNA. In some embodiments, the MBSs could be specific for other regions of miRNAs such as the sequence at the 3’ end of a miRNA. In yet some other embodiments, the MBSs could be specific for a sequence of a miRNA that forms the central loop in the miRNA:mRNA duplexes. In some embodiments, the MBSs could be specific for combinations of these features.

[0074] Multiple copies of each MBS may be present in the MBD. In various embodiments, 1- 12, 2-10, 4-8, or 3-6 copies of each MBS may be present in the MBD. In some embodiments, the MBD comprises at least 2 copies of each MBS. In some other embodiments, the MBD comprises 3, 4, 5, or 6 copies of each MBS. When the MBD comprises more than one MBS and multiple copies of each MBS are present, the multiple copies of each MBS may be present as a single cluster or the multiple copies may be scattered throughout the MBD, i.e. one or more copies of each MBS may alternate with one or more copies of other MBSs. For example, the exemplary expression construct shown in FIG. 12 comprises 2 MBSs in the MBD and 2 copies of each MBS are present in the MBD.

[0075] The length of each copy of an MBS can be selected based on desired binding aspects. In some embodiments, the length of each copy of an MBS may range from about 6 to 33 nucleotides. For example, the length of each copy of a MBS could be about 6 to about 33 nucleotides, about 6 to 30 nucleotides, about 6 to 27 nucleotides, about 6 to 25 nucleotides, about 6 to 23 nucleotides, about 6 to 20 nucleotides, about 6 to 18 nucleotides, about 6 to 15 nucleotides, about 6 to 13 nucleotides, about 6 to 11 nucleotides, or about 6 to 8 nucleotides. In some embodiments, the length of each copy of a MBS is about 6 to 13 nucleotides.

[0076] As provided herein, an MBD can comprise multiple MBSs that are specific for different microRNAs. Also contemplated herein, an MBD can comprise multiple MBSs that are specific for the same microRNA, but bind to different regions of the microRNA.

[0077] Multiple copies of each MBS may be separated from each other by spacer sequences that are about 5 to 50 nucleotides long. For example the spacer sequences could be about 5 to 45, about 5 to 40, about 5 to 35, about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 10 to 50, about 10 to 45, about 10 to 40, about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10 to 15, about 15 to 50, about 15 to 45, about 15 to 40, about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20 nucleotides long. The individual MBSs may be separated from each other by about the same number of nucleotides.

[0078] In various embodiments, the second DNA sequence comprising the MBD can be located on either the 3’ or the 5’ side of the first DNA sequence. For example, in the exemplary expression construct shown in FIG. 12, the second DNA sequence is on the 3’ side of the first DNA sequence.

[0079] The promoter, the gene sequence, and the MBD of the vector can be considered to be operably linked. By“operably linked,” it is meant that the activation of the promoter is expected to activate the transcription of the gene sequence and the MBD.

[0080] As provided herein, the transgene of the described vector encodes an antigen or an immunogenic epitope of an antigen. The first DNA sequence comprising a transgene comprises the coding sequence of the antigen or the immunogenic epitope of an antigen encoded by the transgene, i.e., the DNA sequence does not contain any introns, unless the introns are determined to be required for the proper transcription of the transgene, proper functioning of the antigen or the immunogenic epitope of an antigen, regulation of the transgene expression by miRNAs or any other aspect of the expression of the transgene. The first DNA sequence comprises a terminator sequence that marks the end of the coding sequence and mediates transcription termination.

[0081] In some embodiments, vectors may comprise components that enhance the translational efficiency of the transgene mRNA. For example, in some embodiments, 3’ UTRs that provide a high translational efficiency to an mRNA may be included in the vectors. In some embodiments, a translational efficiency can be quantified as the ratio of ribosome protected fragments (RPF) to the abundance of ribonucleic acids (RNA) (Cottrell et ak, Sci Rep. 2017 Nov 2;7(l): 14884). In some embodiments, the present disclosure provides vectors that comprise 3’ UTRs that provide a translational efficiency of about -0.25 to about -0.8, about -0.28 to about - 0.8, about -0.3 to about -0.8, about -0.25 to about -0.75, -0.25 to about -0.7, about -0.25 to about -0.6, about -0.3 to about -0.75, including values and ranges therebetween. In these embodiments, the vector comprises a third DNA sequence that comprises one or more 3’ UTRs of one or more genes. [0082] The first, second, and third DNA sequences can be linked in any order. For example, in some embodiments, the first, second, and third DNA sequences are linked such that the second DNA sequence is 3’ of the first DNA sequence, and the third DNA sequence is 3’ of the second DNA sequence. In other embodiments, the second DNA sequence is 5’ of the first DNA sequence, and the third DNA sequence is 3’ of the first DNA sequence. In some other embodiments, the second DNA sequence is within the first DNA sequence and the third DNA sequence is 3’ of the first DNA sequence.

[0083] In some embodiments, the third DNA sequence comprises 3’ UTRs of one or more housekeeping genes, e.g., GAPDH, or cytoskeleton genes, e.g., a-tubulin, and b-tubulin. In some other embodiments, the third DNA sequence comprises 3’ UTRs of one or more genes selected from the group consisting of: Rpl32, HSP70a, and CrebA.

[0084] In some embodiments, the vector comprises a fourth DNA sequence 5’ of the first DNA sequence, wherein the fourth DNA sequence comprises a promoter, optionally with an enhancer. The first DNA sequence comprising a transgene, the second DNA sequence comprising a MBD, and the third DNA sequence comprising 3’ UTRs of one or more genes, if present, are under the control of an identical promoter (and enhancer, if present).

[0085] In some embodiments, the promoter is specifically expressed in cancer cells. In some embodiments, the promoter is specifically expressed in a breast cell, colon cell, brain cell, pancreatic cell, lung cell, cervical cell, uterine cell, and/or ovarian cell. In some embodiments, the promoter is selected from the group consisting of: SV40, CMV, EFla, PGK1, Ubc, human b actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal 1/10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, and U6.

[0086] The second DNA sequence comprising the MBD may start from about 1 to 50 nucleotides after the last nucleotide of the stop codon of the transgene. In various embodiments, the second DNA sequence may start from about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30, about 1 to 25, about 1 to 20, about 1 to 15, or about 1 to 10 nucleotides after the last nucleotide of the stop codon of the transgene.

[0087] In some embodiments, the vector may comprise a fifth DNA sequence containing a repressor element that is 5’ of the first DNA sequence. This repressor element facilitates further repression of the expression of the transgene. In some embodiments, the fifth DNA sequence encodes a hemagglutinin-A epitope. [0088] The first DNA sequence, the second DNA sequence, the third DNA sequence, the fourth DNA sequence, and/or the fifth DNA sequence together may be referred to as an expression cassette or an expression construct. The sequences can be linked in any order.

[0089] As provided herein, the transgene present in the vectors of the present disclosure encodes for an antigen or an immunogenic epitope of an antigen. In some embodiments, the transgene encodes for an antigen or an immunogenic epitope of an antigen that a subject has been exposed to via a previous vaccination or infection. In exemplary embodiments, the transgene encodes for an antigen or an antigenic epitope of Varicella-Zoster Virus (chickenpox or shingles virus), Herpes Simplex Virus, Hepatitis B virus, Measles virus (morbillivirus), Mumps virus (paramyxovirus), Rubella virus, Human Immunodeficiency Virus (HIV), Influenza virus, Rhinovirus, Corynebacterium diphtheriae , Clostridium tetani , and/or Bordetella pertussis.

[0090] As noted above, the antigen or the immunogenic epitope of an antigen is expressed on the surface of the cancer cell transduced with the vector of the present disclosure. The antigen or the immunogenic epitope of an antigen expressed on the cancer cell surface can be recognized by immune cells such as T cells or B cells of the subject, activating an immune response against the cancer cell. If the antigen or the immunogenic epitope of an antigen is the one that the subject has been previously exposed to via vaccination or infection, the expression of such antigen or the immunogenic epitope on cancer cells can trigger a memory T cell and/or a memory B cell response and promote faster elimination of the cancer cell expressing the antigen or the immunogenic epitope. Additionally, the vector may be introduced in together with other immuno-stimulatory agents, including checkpoint inhibitors (e.g. PD-l or PD-L1 inhibitors) in order to enhance the therapeutic effect of the antigen.

[0091] In some embodiments, the vectors may comprise an additional second set of DNA sequences comprising a second transgene that is under the control of a second MBD containing one or more MBSs as described above.

[0092] The vectors can be viral DNA vectors or non-viral DNA vectors. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex virus vectors. Non-viral vectors include plasmids and cosmids.

[0093] Vectors of the present disclosure are used to express a transgene encoding an antigen or an immunogenic epitope of an antigen specifically in cancer cells. The expression of the antigen or the immunogenic epitope of an antigen is regulated by endogenously expressed miRNAs. The MBSs present in the MBD are specific for one or more miRNAs that are present in non-cancer cells and are absent or are down-regulated in cancer cells. Upon transcription of the first and second DNA sequences containing the transgene and the MBD respectively, target miRNAs present in non-cancer cells are intended to bind to their corresponding MBSs present on the MBD of the transgene mRNA and inhibit the translation of the transgene mRNA thereby inhibiting the expression of the antigen or the immunogenic epitope of an antigen in non-cancer cells. In cancer cells, the transgene mRNA is intended to be translated and the antigen or the immunogenic epitope of an antigen would be expressed since target miRNAs are absent or are down-regulated in cancer cells.

Exemplary Embodiments

[0094] In an exemplary embodiment, the MBSs specific for miRs not present or down-regulated in a cancer cell are specific for miRs not present or down-regulated in a breast cancer cell. The breast cancer cell can be an early stage breast cancer cell or a late stage breast cancer cell. In some embodiments, the MBSs specific for miRs not present or down-regulated in a breast cancer cell are selected from one of the combinations listed in Table 1.

Table 1

Exemplary MBDs in the vector systems that provide a positive feedback loop-based expression of a transsene

[0095] In some embodiments, the vector system comprises (1) a first DNA sequence under the control of an inducible promoter comprising a first inducer gene and a first MBD and (2) a second DNA sequence under the control of the same inducible promoter comprising a transgene and a second MBD, wherein the first and the second MBDs comprise one or more MBSs, wherein each MBS is specific for a microRNA that is present in a non-breast cancer cell and is not present or is downregulated in a breast cancer cell, and wherein the one or more MBSs are specific for one or more microRNAs selected from miR-629, miR-200C, miR-203A, miR-4760, miR-429, miR-95, and miR-489 (Combination 1). If the vector system comprises a third DNA sequence comprising a second inducer gene and a third MBD, the third MBD may also comprise MBSs specific for microRNAs in Combination 1. Using this vector system, the transgene can be expressed in a late stage breast cancer cell.

[0096] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-452, miR-224, miR-lOO, miR-3 l, and miR-lOA (Combination 2). Using this vector system, the transgene can be expressed in an early stage breast cancer cell.

[0097] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-224, miR-577, miR-452, miR-22l, miR-lOO, miR-205, and miR-3 l (Combination 3). Using this vector system, the transgene can be expressed in an early stage breast cancer cell.

[0098] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-205, miR-200C, and miR-5l0 (Combination 4). Using this vector system, the transgene can be expressed in a late stage breast cancer cell.

[0099] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-200C and miR-203C-3p (Combination 5). Using this vector system, the transgene can be expressed in a late stage breast cancer cell. [0100] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-452, miR-224, miR-lOO, and miR-3 l (Combination 6). Using this vector system, the transgene can be expressed in an early stage breast cancer cell.

[0101] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-lOO, miR-l38, miR-22l, miR-222, and miR- 205 (Combination 7). Using this vector system, the transgene can be expressed in a breast cancer cell.

[0102] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-205-5p and miR-34c-5p (Combination 8). Using this vector system, the transgene can be expressed in a breast cancer cell.

[0103] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-205-5p, miR-34c-5p, and miR-203c-3p, and miR-200C (Combination 9). Using this vector system, the transgene can be expressed in a late stage breast cancer cell.

[0104] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-629, miR-200C, miR-203A, miR-4760, miR-429, miR-95, miR-489, miR- 205, miR-5l0, miR-34c-5p, and miR-203c-3p (Combination 10). Using this vector system, the transgene can be expressed in a late stage breast cancer cell.

[0105] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-452, miR-224, miR-lOO, miR-3 l, miR-lOA, miR-577, miR-22l, miR-205, and miR-34c-5p (Combination 11). Using this vector system, the transgene can be expressed in an early stage breast cancer cell.

[0106] In some embodiments, the first and the second MBDs, and if present, the third MBD may comprise one or more MBSs that are specific for one or more microRNAs selected from a group consisting of: miR-629, miR-200C, miR-203A, miR-4760, miR-429, miR-95, miR-489, miR- 452, miR-224, miR-lOO, miR-3 l, miR-lOA, miR-577, miR-22l, miR-205, miR-5l0, miR-l38, miR-222, miR-205-5p, miR-34c-5p, and miR-203c-3p (Combination 12). Exemplary MBDs in the vectors expressing an antisen or an immunogenic eyitoye of an antisen

[0107] In an exemplary embodiment, the vector comprises a first DNA sequence comprising a transgene encoding an antigen or an immunogenic epitope of an antigen and a second DNA sequence comprising a MBD, wherein the MBD comprises one or more MBSs, wherein each MBS is specific for a microRNA that is present in a non-breast cancer cell and is not present or is downregulated in a breast cancer cell, and wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-629, miR-200C, miR-203A, miR-4760, miR-429, miR-95, and miR-489 (Combination 1). Using this vector, the transgene can be expressed in a late stage breast cancer cell.

[0108] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-452, miR-224, miR-lOO, miR-3 l, and miR-lOA (Combination 2). Using this vector, the transgene can be expressed in an early stage breast cancer cell.

[0109] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-224, miR-577, miR-452, miR-22l, miR-lOO, miR-205, and miR-3 l (Combination 3). Using this vector, the transgene can be expressed in an early stage breast cancer cell.

[0110] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-205, miR-200C, and miR-5l0 (Combination 4). Using this vector, the transgene can be expressed in a late stage breast cancer cell.

[0111] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-200C and miR-203C-3p (Combination 5). Using this vector, the transgene can be expressed in a late stage breast cancer cell.

[0112] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-452, miR-224, miR-lOO, and miR-3 l (Combination 6). Using this vector, the transgene can be expressed in an early stage breast cancer cell.

[0113] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-lOO, miR-l38, miR-22l, miR-222, and miR- 205 (Combination 7). Using this vector, the transgene can be expressed in a breast cancer cell.

[0114] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-205-5p and miR-34c-5p (Combination 8). Using this vector, the transgene can be expressed in a breast cancer cell.

[0115] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-205-5p, miR-34c-5p, and miR-203c-3p, and miR-200C (Combination 9). Using this vector, the transgene can be expressed in a late stage breast cancer cell.

[0116] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-629, miR-200C, miR-203A, miR-4760, miR-429, miR-95, miR-489, miR-205, miR-5l0, miR-34c- 5p, and miR-203c-3p (Combination 10). Using this vector, the transgene can be expressed in a late stage breast cancer cell.

[0117] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-452, miR-224, miR-lOO, miR-3 l, miR-lOA, miR-577, miR-22l, miR-205, and miR-34c-5p (Combination 11). Using this vector, the transgene can be expressed in an early stage breast cancer cell.

[0118] In some embodiments, the MBD present in the vector may comprise one or more MBSs that are specific for one or more microRNAs selected from the group consisting of: miR-629, miR-200C, miR-203A, miR-4760, miR-429, miR-95, miR-489, miR-452, miR-224, miR-lOO, miR-3 l, miR-lOA, miR-577, miR-22l, miR-205, miR-5l0, miR-l38, miR-222, miR-205-5p, miR-34c-5p, and miR-203c-3p (Combination 12).

Compositions

[0119] The present disclosure also provides pharmaceutical compositions.

[0120] An exemplary pharmaceutical composition comprises any vector system or the vectors according to the invention and one or more pharmaceutically acceptable excipients. Kits

[0121] The present disclosure also provides treatment kits.

[0122] An exemplary treatment kit of the disclosure can comprise any one of the vector systems or the vectors of the disclosure or pharmaceutical compositions comprising any one of the vector systems or the vectors of the disclosure.

[0123] Kits generally further comprise instructions for use.

Treatment methods

[0124] The present application also provides methods for treating cancer. A method for treating cancer comprises administering a therapeutically effective amount of any one of the vector systems or the vectors of the present disclosure to a subject in need thereof. In some embodiments, the method comprises administering the vector system or the vector in combination with a second therapeutic agent such as gancyclovir. As referred to herein, a subject can be any animal, including a mammal, e.g. a human, a monkey (e.g. a cynomolgus monkey), companion animals (e.g. cats, dogs) etc.

[0125] In some embodiments, cancers that can be treated using the vector systems or the vectors of the present disclosure include, but are not limited to, breast cancer, colon cancer, brain cancer, pancreatic cancer, lung cancer, cervical cancer, uterine cancer, prostate cancer, ovarian cancer, melanoma, lymphoma, myeloma, and/or leukemia.

[0126] The breast cancer treated according to the methods of the disclosure include an early stage breast cancer or a late stage breast cancer. The guidelines for various stages of breast cancer can be found at the AJCC Cancer Staging Manual, 7^th Edition.

[0127] In some embodiments, the treatment methods may comprise administering an inducer or a component of an inducer of the inducible promoter to the subject. For example, in the embodiment where the vector system comprises tet-on/tet-off system, the method may comprise administering tetracycline and/or doxycycline and/or a rtTA protein to the subject.

[0128] In some embodiments where the vector system or the vector expresses an antigen or an immunogenic epitope of an antigen, the treatment methods may comprise introducing the vector system or the vector with other immuno-stimulatory agents, including checkpoint inhibitors (e.g. PD-l or PD-L1 inhibitors) in order to enhance the therapeutic effect of the antigen or the immunogenic epitope of an antigen.

[0129] Various routes of administration can be used, e.g. parenteral (e.g. intravenous, intramuscular, subcutaneous, etc.), oral, nasal (e.g. nebulizer, inhaler, etc.), transmucosal (buccal, nasal mucosal, etc.), and transdermal.

[0130] In some embodiments, the present disclosure allows for the development of patient- specific therapeutic compositions and methods. For example, a vector system or a vector can be designed based on a patient’s specific genetic profile, e.g., by screening a sample obtained from the patient, to determine the microRNAs down-regulated in specific target cells (e.g., breast cancer cells) compared to non-target cells. In such embodiments, the vector system or the vector can then include a MBD that comprises one or more MBSs specific for the microRNAs down- regulated or absent in that particular patient’s target cells (e.g., breast cancer cells) but not down- regulated in the patient’s non-target cells (e.g. non-breast cancer cells). Similarly, the vector system or the vector can include a transgene that encodes for an antigen or an immunogenic epitope of an antigen that the subject has previously been exposed to via vaccination or infection. For example, if the subject has received vaccination for chicken pox, the transgene can encode for the antigen or the antigenic epitope of the chicken pox virus that is present in the chicken pox vaccine. The expression of such antigen or the antigenic epitope can activate the memory immune response and since the antigen or the antigenic epitope is specifically expressed in cancer cells, the activated immune response will only eliminate the cancer cell but not the non-cancer cell. Such a vector can be considered to be a personalized therapeutic agent that can then be delivered to the patient to facilitate the expression of a transgene specifically in target cells (e.g., cancer cells) while providing minimal or no expression of the transgene in non target cells.

[0131] In some embodiments, to obtain a patient’s genetic profile (e.g. a miRNA profile), biopsies from previously diagnosed or undiagnosed tissue samples can be obtained. If the tissue is undiagnosed, diagnosis can be achieved using standard pathological methods in-house. Once diagnosed, the biopsied tissue can be processed in many ways. For example, in some embodiments the biopsied tissue can be sectioned. In this embodiment, one portion of the biopsy can be stored in long term storage (i.e. frozen at -80°C or stored in liquid nitrogen), one portion can be put into cell culture, and one portion can be analyzed for its genetic profile. This genetic profile can be confirmed and compared against biopsies of surrounding, healthy tissue, and tissue from other major and accessible tissue in the body, which may also include the vital organs of the patient.

EXAMPLES

Example 1 : Positive feedback loop-based expression of a transgene

[0132] In this exemplary embodiment, a vector system comprising an inducible transgene, tetracycline-regulatable elements, lac-repressor elements, and microRNA binding domains (MBDs) is described. Specifically, the vector system comprises the following elements:

[0133] First DNA sequence: tTA (tet-OFF)

[0134] The tTA protein is expressed using a tTA gene that is under the control of a tetracycline- inducible (“tet-inducible”) promoter, and a first MBD comprising MBSs specific for miRs present in non-cancer cells but absent or down-regulated in cancer cells. This provides a decreased expression, if any, of the tTA gene in non-cancer cells.

[0135] Second DNA sequence: Therapeutic Transgene

[0136] The transgene protein is expressed using a transgene that is under the control of the tet- inducible promoter with Lac repressor binding sites at the 3’ end of the promoter but 5’ to the start codon of the transgene. The transgene is also under the control of a second MBD comprising MBSs specific for miRs present in non-cancer cells but absent or down-regulated in cancer cells. The expression of the transgene is“double gated” by the inducible promoter and the lac repressor binding sites.

[0137] Third DNA sequence: rtTA (tet-ON)

[0138] The rtTA protein is expressed using a rtTA gene under the control of a constitutively active mammalian promoter (e.g. CMV, SV40, CAG, EF1 alpha, etc). The rtTA gene is also under the control of a third MBD comprising MBSs specific for miRs present in non-cancer cells but absent or down-regulated in cancer cells. This provides a decreased expression, if any, of the tTA gene in non-cancer cells.

[0139] Fourth DNA sequence: Lac I

[0140] The lac I protein is expressed using a lad gene that is under the control of a constitutively active mammalian promoter (e.g. CMV, SV40, CAG, EF1 alpha, etc). The lad gene can be under the control of a fourth MBD comprising MBSs specific for miRs present in cancer cells but absent or down-regulated in non-cancer cells, leading to its decreased expression, if any, in cancer cells.

[0141] When non-cancer cells and cancer cells are transfected with the above vector system, the vector system is expected to operate as follows in non-cancer cells and cancer cells:

[0142] In non-cancer cells pretreated with doxycycline (doxy): (1) The lac I protein (lac repressor) is expected to express following transfection at normal levels because there are no cancer cell-specific microRNAs to downregulate it. Also, the rtTA protein is expected to be expressed but at low levels due to non-cancer cell-specific miRNAs targeting the third MBD. (2) It is expected that the lac repressor would bind the lac repressor binding sites in the second DNA sequence, blocking expression of the therapeutic transgene. (3) The rtTA that may be expressed after non-cancer cell-specific miRNA downregulation binds to doxy. (4) It is expected that the rtTA/doxy conjugate would bind to the tet-inducible promoters in the first and second DNA sequences, catalyzing transcription of both tTA and the transgene; however, the transgene transcription is expected to be blocked by lac repressor sites. (5) Transcription and translation is expected to begin for tTA, but tTA expression is expected to be downregulated by non-cancer cell-specific miRNAs binding to the first MBD. (6) Doxy will be removed from the medium. (7) rtTA/doxy conjugates are expected to decrease in number as doxy concentration decreases, leading to less activation of the tet-inducible promoter. This is expected to enhance the effect of the non-cancer cell-specific MBD on the expression of tTA, leading to complete silencing of tTA. (8) Treatment with IPTG can be started. (9) It is expected that as IPTG binds the lac repressor, it will compete with the lac repressor for its binding to the lac repressor binding sites. (10) As there is no induction of tet-inducible promoter; therefore, it is expected that the transgene will not be expressed. Any leaky transgene can be further downregulated by non-cancer cells specific MBSs in the second MBD.

[0143] In cancers cells pretreated with doxycycline (doxy): (1) It is expected that the rtTA protein will express following transfection at normal levels because there are non-cancer cell specific microRNAs are nor present to downregulate the expression of rtTA. Also, the lac repressor may be expressed at low levels due to cancer cell-specific miRNAs targeting the fourth MBD. (2) Whatever lac repressor is expressed, after the cancer cell-specific miRNA downregulation, it is expected to bind to lac repressor binding sites in the second DNA sequence, blocking expression of the therapeutic transgene. (3) it is expected that rtTA would bind doxy. (4) It is expected that the rtTA/doxy conjugate would bind to tet-inducible promoters in the first and the second DNA sequences, catalyzing transcription of both tTA and the transgene (the transgene transcription may be blocked by the lac repressor). (5) Transcription and translation is expected to begin for tTA. (6) Doxy is removed from medium. (7) rtTA/doxy conjugates are expected to decrease in number as doxy concentration in cytosol decreases, leading to less activation of tet-inducible promoter by rtTA. However, because tTA is already expressed in the cell, it is expected to bind to its own tet-inducible promoter in the first DNA sequence, starting a positive feedback loop. (8) Treatment with IPTG can be started. (9) It is expected that as IPTG binds the lac repressor, it will compete with the lac repressor for its binding to the lac repressor binding sites in the second DNA sequence. (10) Abundant tTA in the cancer cell is expected to bind to the tet-inducible promoter in the second DNA sequence, initiating transcription and translation of transgene.

Example 2: Positive feedback loop-based expression of a transgene encoding GFP

[0144] In this experiment, a vector system as described herein was designed to test whether the vector system can elicit a differential expression of a reporter transgene between a normal breast cell line (MCF10A) and cancerous pancreatic cell line (PANC1) in-vitro. The vector system, pCAELION, was custom synthesized by Genscript, Piscataway, New Jersey, per the inventor’s instructions. Additionally, a variant of pCAELION containing Herpes Thymidine Kinase as a transgene was designed.

[0145] Identification of relevant miRNAs: A miRNA sequencing data of MCFlOa (normal breast cell line), BT549 (triple negative breast cancer) and PANC1 (pancreatic cancer cell line) was analyzed and the profiles were compared. It was found that miR-205-5p and miR-200C were expressed at extremely low levels or were completely silenced in both BT549 and PANC1 but expressed with abundance in MCF10A, whereas miR-l55 was highly expressed in BT549 and PANC1 but silenced in MCF10A.

[0146] Plasmid design and generation: A plasmid vector as described in paragraph [0030] of the present application was designed. A reporter transgene encoding a GFP (green fluorescent protein) was used in place of a therapeutic transgene as a readout to measure the expression of the vector system. Additionally, the mCherry gene encoding a constitutively active red fluorescent protein (RFP) was included in the vector and used to normalize for the transfection efficiency. Four copies of a fully complementary sequence of the entirety of miR-205-5p and four copies of the seed region of miR-200C-3p were inserted as the MBSs into the first, second, and the third MBDs. Four copies of a fully complementary region of the entirety of miRNA-l55 were inserted as the MBSs into the fourth MBD. Additionally, a sequence encoding a GAPDH 3’ UTR was included 3’ of the first and the second MBD.

[0147] Cell Culture: PANC1 cells were cultured in DMEM high glucose with supplemented glutamine, 10% FBS, andlx pen-strep. MCFlOa cells were cultured in DMEM:F/l2 medium containing 5% Horse Serum, 20ng/ml EGF, 0.5 mg/ml Hydrocortisone, lOOng/ml Cholera Toxin, lOug/ml Insulin, with lx pen strep. All cells were fed every other day when not treated with IPTG and/or doxycycline, and every day when treated with IPTG and/or doxycycline.

[0148] Transfection and Optimization: Cells were transfected with Lipofectamine® 3000 from Thermo Fisher into 12 well dishes at -70% confluence. Conditions for transfection were optimized by altering the concentration of Lipofectamine® against the ratio of DNA concentration to cell number transfected (Data not shown). Optimal conditions were determined based on the transfection efficiency using a GFP construct that was not regulated by the presence of MBDs. Following optimization, MCF10A and PANC1 were transfected with the miRNA- regulated vector system described above and a control vector system where the MBSs for the target miRNAs are flipped. BT549 were also transfected, but the RFP/GFP readout was not strong enough to quantify. When the cells were transfected with the pCAELION vector comprising Herpes Thymidine Kinase as the transgene and treated with gancyclovir, the inventor observed differential killing of PANC1 cells compared to MCF10A cells. However, this data was not quantified.

[0149] The amount of the vector DNA was determined via titration (lpg/transfection; data not shown).

[0150] Inducing the expression of the reporter transgene: Transfected cells, when treated with Isopropyl b-D-l-thiogalactopyranoside (IPTG), Doxycycline, or both, a medium containing 250mM IPTG and/or a medium containing 2pg/pl Doxycycline was used. The expression of the transgene was induced as follows:

• One day prior to transfection to day 3 of Transfection: treat all cells with both IPTG and Doxycycline containing medium • Day 3 onward: treat only with IPTG-containing medium

[0151] The dosing of IPTG and doxycycline and the treatment schedule were empirically optimized to enhance differential expression of GFP relative to RFP (data not shown).

[0152] Analysis: Transfected cells were analyzed via image analysis and quantification using Keyence BX 710 series fluorescence microscope and software. The edges of the wells were defined and pictures were taken of the entire well to capture both GFP and RFP readouts. Exposures and aperture settings remained consistent between samples. Pictures were stitched together using Keyence software, and were altered to cut noise using both the“Haze Reduction” and“Black Balance” commands in the software. Cells were counted using the software’s“Hybrid cell count” feature. To determine the efficiency of inducing the expression of the vector system, total RFP positive cells were compared to total GFP positive cells. Three conditions were analyzed per experiment: non-transfected, transfected but non-induced, and transfected and induced. The data is shown in FIGs. 13-18.

Claims

1. A vector system comprising:

(a) a first deoxyribonucleic acid (DNA) sequence comprising an inducible promoter, a gene encoding an inducer or a component of an inducer of the inducible promoter, and a first microRNA binding domain (MBD), wherein the first MBD comprises one or more microRNA binding sites (MBSs), wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell; and

(b) a second DNA sequence comprising the same inducible promoter, a transgene, and a second MBD, wherein the second MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell.

2. The vector system of claim 1, comprising:

(c) a third DNA sequence comprising a constitutively active promoter, a gene encoding an inducer or a component of an inducer of the inducible promoter, and a third MBD, wherein the third MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell.

3. The vector system of claim 1 or 2, wherein the second DNA sequence comprises one or more binding sites for a transcription repressor 3’ or 5’ to the transgene.

4. The vector system of claim 3, comprising:

(d) a fourth DNA sequence comprising a constitutively active promoter, a gene encoding the transcription repressor, and a fourth MBD, wherein the fourth MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a cancer cell.

5. The vector system of any one of claims 1-4, wherein the inducible promoter is a tetracycline-inducible promoter.

6. The vector system of any one of claims 1-5, wherein the component of an inducer of the inducible promoter is a tetracycline-controlled transactivator (tTA) or a reverse tetracycline- controlled transactivator (rtTA).

7. The vector system of any one of claims 1-6, wherein the gene encoding an inducer or a component of an inducer of the inducible promoter in the first DNA sequence encodes for a tTA.

8. The vector system of any one of claims 2-7, wherein the gene encoding an inducer or a component of an inducer of the inducible promoter in the third DNA sequence encodes for a rtTA.

9. The vector system of any one of claims 3-8, wherein the one or more binding sites for a transcription repressor in the second DNA sequence are the binding sites for a lac repressor.

10. The vector system of any one of claims 4-9, wherein the gene encoding the transcription repressor is lacl.

11. The vector system of any one of claims 1-10, wherein the first and the second DNA sequences are on the same vector.

12. The vector system of any one of claims 1-10, wherein the first and the second DNA sequences are on different vectors.

13. The vector system of any one of claims 2-11, wherein the first and the second DNA sequences are on the same vector and the third DNA sequence is on a different vector.

14. The vector system of any one of claims 2-10, wherein the first and the third DNA sequences are on the same vector and the second DNA sequence is on a different vector.

15. The vector system of any one of claims 2-10, wherein the first DNA sequence is on one vector, and the second and third DNA sequences are on a second vector.

16. The vector system of any one of claims 2-11, wherein the first, second, and third DNA sequences are on the same vector.

17. The vector system of any one of claims 4-11, wherein the first and the second DNA sequences are on one vector and the third and the fourth DNA sequence is on a second vector.

18. The vector system of any one of claims 4-11, wherein the first, second, and third DNA sequences are on one vector, and the fourth DNA sequence is on a second vector.

19. The vector system of any one of claims 4-10, wherein the first, third, and fourth DNA sequences are on one vector, and the second DNA sequence is on a second vector.

20. The vector system of any one of claims 4-11, wherein the first, second, and fourth DNA sequences are on one vector, and the third DNA sequence is on a second vector.

21. The vector system of any one of claims 4-10, wherein the first and third DNA sequences are on one vector, and the second and fourth DNA sequences are on a second vector.

22. The vector system of any one of claims 4-10, wherein the first and fourth DNA sequences are on one vector, and the second and third DNA sequences are on a second vector.

23. The vector system of any one of claims 4-10, wherein the first DNA sequence is on one vector, and the second, third, and fourth DNA sequences are on a second vector.

24. The vector system of any one of claims 4-11, wherein the first and the second DNA sequences are on one vector, the third DNA sequence is on a second vector, and the fourth DNA sequence is on a third vector.

25. The vector system of any one of claims 4-10, wherein the first DNA sequence is on one vector, the second and third DNA sequences are on a second vector, and the fourth DNA sequence is on a third vector.

26. The vector system of any one of claims 4-10, wherein the first and fourth DNA sequences are on one vector, the second DNA sequence is on a second vector, and the third DNA sequence is on a third vector.

27. The vector system of any one of claims 4-10, wherein the first and third DNA sequences are on one vector, the second DNA sequence is on a second vector, and the fourth DNA sequence is on a third vector.

28. The vector system of any one of claims 4-10, wherein the second and fourth DNA sequences are on one vector, the first DNA sequence is on a second vector, and the third DNA sequence is on a third vector.

29. The vector system of any one of claims 4-10, wherein first DNA sequence is on one vector, the second DNA sequence is on a second vector, and the third and fourth DNA sequences are on a third vector.

30. The vector system of any one of claims 4-11, wherein the first, second, third, and the fourth DNA sequences are on the same vector.

31. A vector system comprising:

(a) a first DNA sequence comprising a tetracycline-inducible promoter, a gene encoding a tetracycline-controlled transactivator (tTA), and a first microRNA binding domain (MBD), wherein the first MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell; and

(b) a second DNA sequence comprising the tetracycline-inducible promoter, a transgene, and a second MBD, wherein the second MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non-cancer cell and is not present or is downregulated in a cancer cell.

32. The vector system of claim 31, comprising:

(c) a third DNA sequence comprising a constitutively active promoter, a gene encoding a reverse tetracycline-controlled transactivator (rtTA), and a third MBD, wherein the third MBD comprises one or more MBSs, wherein each MBS is specific for a miR that is present in a non cancer cell and is not present or is downregulated in a cancer cell.

33. The vector system of claim 31 or 32, wherein the second DNA sequence comprises one or more binding sites for a lac repressor 3’ or 5’ to the start codon of the transgene.

34. The vector system of claim 33, comprising:

(d) a fourth DNA sequence comprising a constitutively active promoter, a lacl gene encoding a lactose operon repressor, and a fourth MBD; wherein the fourth MBD comprises one or MBSs, wherein each MBS is specific for a miR that is present in a cancer cell.

35. The vector system of any one of claims 31-34, wherein the first and the second DNA sequences are on the same vector.

36. The vector system of any one of claims 31-34, wherein the first and the second DNA sequences are on different vectors.

37. The vector system of any one of claims 32-34, wherein the first and the second DNA sequences are on the same vector and the third DNA sequence is on a different vector.

38. The vector system of any one of claims 32-34, wherein the first and the third DNA sequences are on the same vector and the second DNA sequence is on a different vector.

39. The vector system of any one of claims 32-34, wherein the first DNA sequence is on one vector, and the second and third DNA sequences are on a second vector.

40. The vector system of any one of claims 32-34, wherein the first, second, and third DNA sequences are on the same vector.

41. The vector system of claim 34, wherein the first and the second DNA sequences are on one vector and the third and the fourth DNA sequence is on a second vector.

42. The vector system of claim 34, wherein the first, second, and third DNA sequences are on one vector, and the fourth DNA sequence is on a second vector.

43. The vector system of claim 34, wherein the first, third, and fourth DNA sequences are on one vector, and the second DNA sequence is on a second vector.

44. The vector system of claim 34, wherein the first, second, and fourth DNA sequences are on one vector, and the third DNA sequence is on a second vector.

45. The vector system of claim 34, wherein the first and third DNA sequences are on one vector, and the second and fourth DNA sequences are on a second vector.

46. The vector system of claim 34, wherein the first and fourth DNA sequences are on one vector, and the second and third DNA sequences are on a second vector.

47. The vector system of claim 34, wherein the first DNA sequence is on one vector, and the second, third, and fourth DNA sequences are on a second vector.

48. The vector system of claim 34, wherein the first and the second DNA sequences are on one vector, the third DNA sequence is on a second vector, and the fourth DNA sequence is on a third vector.

49. The vector system of claim 34, wherein the first DNA sequence is on one vector, the second and third DNA sequences are on a second vector, and the fourth DNA sequence is on a third vector.

50. The vector system of claim 34, wherein the first and fourth DNA sequences are on one vector, the second DNA sequence is on a second vector, and the third DNA sequence is on a third vector.

51. The vector system of claim 34, wherein the first and third DNA sequences are on one vector, the second DNA sequence is on a second vector, and the fourth DNA sequence is on a third vector.

52. The vector system of claim 34, wherein the second and fourth DNA sequences are on one vector, the first DNA sequence is on a second vector, and the third DNA sequence is on a third vector.

53. The vector system of claim 34, wherein first DNA sequence is on one vector, the second DNA sequence is on a second vector, and the third and fourth DNA sequences are on a third vector.

54. The vector system of claim 34, wherein the first, second, third, and the fourth DNA sequences are on the same vector.

55. The vector system of any one of claims 1-54, wherein the first, second, third, and/or fourth MBD comprises 1-12 MBSs.

56. The vector system of any one of claims 1-55, wherein the first, second, third, and/or fourth MBD comprises at least 2 copies of each MBS.

57. The vector system of any one of claims 1-56, wherein the first, second, third, and/or fourth MBD comprises 3 to 5 copies of each MBS.

58. The vector system of any one of claims 1-57, wherein each MBS is 6-33 nucleotides long.

59. The vector system of any one of claims 1-58, wherein the length of each MBS is about 6- 30 nucleotides, about 6-27 nucleotides, about 6-25 nucleotides, about 6 to 23 nucleotides, about 6 to 20 nucleotides, about 6 to 18 nucleotides, about 6 to 15 nucleotides, about 6 to 13 nucleotides, about 6 to 11 nucleotides, or about 6 to 8 nucleotides.

60. The vector system of any one of claims 1-59, wherein the cancer cell is selected from the group consisting of: breast cancer cell, colon cancer cell, brain cancer cell, pancreatic cancer cell, lung cancer cell, cervical cancer cell, uterine cancer cell, and/or ovarian cancer cell.

61. The vector system of any one of claims 1-60, wherein the one or more MBSs in the first, second, and/or third MBD are specific for one or more microRNAs selected from the group consisting of: miR-629, miR-200C, miR-203A, miR-4760, miR-429, miR-95, miR-489, miR- 452, miR-224, miR-lOO, miR-3 l, miR-lOA, miR-577, miR-22l, miR-205, miR-5l0, miR-l38, miR-222, miR-205-5p, miR-34c-5p, miR-203c-3p, and miR-l55.

62. The vector system of any one of claims 1-61, wherein the cancer cell is a late stage breast cancer cell.

63. The vector system of claim 62, wherein the one or more MBSs in the first, second, third, and/or fourth MBD are specific for one or more microRNAs selected from the group consisting of: miR-629, miR-200C, miR-203A, miR-4760, miR-429, miR-95, and miR-489.

64. The vector system of any one of claims 1-61, wherein the cancer cell is an early stage breast cancer cell.

65. The vector system of claim 64, wherein the one or more MBSs in the first, second, third, and/or fourth MBD are specific for one or more microRNAs selected from the group consisting of: miR-452, miR-224, miR-lOO, miR-3 l, and miR-lOA.

66. The vector system of claim 64, wherein the one or more MBSs in the first, second, third, and/or fourth MBD are specific for one or more microRNAs selected from the group consisting of: miR-224, miR-577, miR-452, miR-22l, miR-lOO, miR-205, and miR-3 l.

67. The vector system of claim 62, wherein the one or more MBSs in the first, second, third, and/or fourth MBD are specific for one or more microRNAs selected from the group consisting of: miR-205, miR-200C, and miR-5l0.

68. The vector system of claim 62, wherein the one or more MBSs in the first, second, third, and/or fourth MBD are specific for miR-200C.

69. The vector system of claim 64, wherein the one or more MBSs in the first, second, third, and/or fourth MBD are specific for one or more microRNAs selected from the group consisting of: miR-452, miR-224, miR-lOO, and miR-3 l.

70. The vector system of any one of claims 1-61, wherein the one or more MBSs in the first, second, and/or third MBD are specific for one or more microRNAs selected from the group consisting of: miR-205-5p, miR-34c-5p, and miR-203c-3p.

71. The vector system of any one of claims 1-61, wherein the one or more MBSs in the first, second, and/or third MBD are specific for one or more microRNAs selected from one of the combinations presented in Table 1.

72. The vector system of any one of claims 4-30 and 34-54, wherein the one or more MBSs in the fourth MBD are specific for miR-l55.

73. The vector system of any one of claims 1-72, wherein the first DNA sequence is 3’ to the second DNA sequence.

74. The vector system of any one of claims 1-72, wherein the second DNA sequence is 3’ to the first DNA sequence.

75. The vector system of any one of claims 4-30 and 34-54, wherein the first, second, third, and the fourth DNA sequences are on the same vector, and the first DNA sequence is 3’ of the second, third, and/or fourth DNA sequence.

76. The vector system of any one of claims 4-30 and 34-54, wherein the first, second, third, and the fourth DNA sequences are on the same vector, and the second DNA sequence is 3’ of the first, third, and/or fourth DNA sequence.

77. The vector system of any one of claims 4-30 and 34-54, wherein the first, second, third, and the fourth DNA sequences are on the same vector, and the third DNA sequence is 3’ of the first, second, and/or fourth DNA sequence.

78. The vector system of any one of claims 4-30 and 34-54, wherein the first, second, third, and the fourth DNA sequences are on the same vector, and the fourth DNA sequence is 3’ of the first, second, and/or third DNA sequence.

79. The vector system of any one of claims 1-78, wherein the first, second, third, and/or fourth DNA sequences comprise a DNA sequence 3’ of the first, second, third, and/or fourth MBD, wherein the DNA sequence comprises one or more 3’ untranslated regions (3’-UTRs) of one or more genes, wherein the 3’-UTR provides a translation efficiency of -0.3 to -.0.8 where the translation efficiency is defined as the ratio of ribosome protected fragments (RPF) to the abundance of ribonucleic acids (RNA).

80. The vector system of claim 79, wherein the one or more genes are one or more housekeeping or cytoskeleton genes.

81. The vector system of claim 80, wherein the one or more housekeeping or cytoskeleton genes are selected from the group consisting of: GAPDH, a-tubulin, and b-tubulin.

82. The vector system of claim 79, wherein the one or more genes are selected from the group consisting of: Rpl32, HSP70a, and CrebA.

83. The vector system of any one of claims 1-82, wherein the first, second, third, and/or fourth MBD starts from about 1 to 50 nucleotides after the last nucleotide of the stop codon of the gene in the first, second, third, and/or fourth DNA sequences.

84. The vector system of any one of claims 1-83, wherein the transgene encodes a therapeutic protein that inhibits proliferation and/or metastasis of cancer cells.

85. The vector system of claim 84, wherein the therapeutic protein is an apoptosis inducing protein.

86. The vector system of claim 85, wherein the apoptosis inducing protein is thymidine kinase, a caspase, a granzyme, an exotoxin, or a proapoptotic member of the Bcl-2 family.

87. The vector system of any one of claims 1-83, wherein the therapeutic protein is an antigen or an immunogenic epitope of an antigen.

88. The vector system of claim 87, wherein the antigen or the immunogenic epitope of an antigen is an antigen or an immunogenic epitope of an antigen that a subject has been exposed to via a previous vaccination or infection.

89. The vector system of claim 87 or 88, wherein the antigen or the immunogenic epitope of an antigen is from an infectious agent selected from the group consisting of: Varicella-Zoster Virus (chickenpox or shingles virus), Herpes Simplex Virus, Hepatitis B virus, Measles virus (morbillivirus), Mumps virus (paramyxovirus), Rubella virus, Human Immunodeficiency Virus (HIV), Influenza virus, Rhinovirus, Corynebacterium diphtheriae , Clostridium tetani , and Bordetella pertussis.

90. The vector system of any one of claims 87-89, wherein the antigen or the immunogenic epitope is expressed on the surface of the infectious agent or cells infected with the infectious agent.

91. The vector system of any one of claims 1-83, wherein the transgene encodes an siRNA or a shRNA sequence, the expression of which silences a gene in target cells.

92. The vector system of any one of claims 1-91, wherein the vector is a viral vector or a plasmid.

93. The vector system of claim 92, wherein the viral vector is selected from the group consisting of: a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a herpes simplex virus vector.

94. The vector system of any one of claims 1-91, wherein the vector is a non-viral vector.

95. A pharmaceutical composition comprising the vector system of any one of claims 1-94, and one or more pharmaceutically acceptable excipients.

96. A method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the vector system of any one of claims 1-94 or the pharmaceutical composition of claim 95 to the subject.

97. The method of claim 68, comprising administering an inducer or a component of an inducer of the inducible promoter to the subject.

98. The method of claim 96, comprising administering tetracycline, doxycycline or a derivative thereof to the subject prior to administration of the vector or the pharmaceutical composition.

99. The method of claim 96 or 98, comprising administering tetracycline, doxycycline or a derivative thereof to the subject after administration of the vector or the pharmaceutical composition.

100. A vector compri sing :

(a) a first deoxyribonucleic acid (DNA) sequence comprising a transgene, wherein the transgene encodes for an antigen or an immunogenic epitope of an antigen; and

(b) a second deoxyribonucleic (DNA) acid sequence comprising a microRNA binding domain (MBD); wherein the MBD comprises one or more microRNA binding sites (MBSs), wherein each MBS is specific for a microRNA (miR) that is present in a non-cancer cell and is not present or is downregulated in a cancer cell.

101. The vector of claim 100, wherein the MBD comprises 1-12 MBSs.

102. The vector of claim 100 or 101, wherein the MBD comprises at least 2 copies of each MBS.

103. The vector of any one of claims 100-102, wherein the MBD comprises 3 to 5 copies of each MBS.

104. The vector of any one of claims 100-103, wherein each MBS is 6-33 nucleotides long.

105. The vector of any one of claims 100-104, wherein the length of each MBS is about 6-30 nucleotides, about 6-27 nucleotides, about 6-25 nucleotides, about 6 to 23 nucleotides, about 6 to 20 nucleotides, about 6 to 18 nucleotides, about 6 to 15 nucleotides, about 6 to 13 nucleotides, about 6 to 11 nucleotides, or about 6 to 8 nucleotides.

106. The vector of any one of claims 100-105, wherein the cancer cell is selected from the group consisting of: breast cancer cell, colon cancer cell, brain cancer cell, pancreatic cancer cell, lung cancer cell, cervical cancer cell, uterine cancer cell, and/or ovarian cancer cell.

107. The vector of any one of claims 100-106, wherein the cancer cell is a breast cancer cell.

108. The vector of any one of claims 100-107 wherein the cancer is a late stage breast cancer cell.

109. The vector of claim 107 or 108, wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-629, miR-200C, miR-203 A, miR- 4760, miR-429, miR-95, and miR-489.

110. The vector of any one of claims 100-107, wherein the cancer cell is an early stage breast cancer cell.

111. The vector of claim 107 or 110, wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-452, miR-224, miR-lOO, miR-3 l, and miR-lOA.

112. The vector of any one of claims 107, and 110-111, wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-224, miR-577, miR-452, miR-22l, miR-lOO, miR-205, and miR-3 l.

113. The vector of claim 107 or 108, wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-205, miR-200C, and miR-5l0.

114. The vector of claim 107 or 108, wherein the second nucleic acid sequence comprises a MBS specific for miR-200C.

115. The vector of claim 107 or 108, wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-452, miR-224, miR-lOO, and miR- 31.

116. The vector of claim 107, wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-lOO, miR-l38, miR-22l, miR-222, and miR- 205.

117. The vector of any one of claims 107, 108, and 110, wherein the one or more MBSs are specific for miR-205-5p and miR-34c-5p.

118. The vector of claim 107 or 108, wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-205-5p, miR-34c-5p, and miR- 203c-3p.

119. The vector of claim 107 wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-629, miR-200C, miR-203 A, miR-4760, miR-429, miR-95, miR-489, miR-205, miR-5l0, miR-34c-5p, and miR-203 c-3p.

120. The vector of claim 107, wherein the one or more MBSs are specific for one or more microRNAs selected from the group consisting of: miR-452, miR-224, miR-lOO, miR-3 l, miR- 10A, miR-577, miR-22l, miR-205, and miR-34c-5p.

121. The vector of any one of claims 100-120, wherein the second deoxyribonucleic acid sequence is 3’ to the first deoxyribonucleic acid sequence.

122. The vector of any one of claims 100-120, wherein the second deoxyribonucleic acid sequence is 5’ to the first deoxyribonucleic acid sequence.

123. The vector of claim 121, comprising a third deoxyribonucleic acid sequence 3’ of the second deoxyribonucleic acid sequence, wherein the third deoxyribonucleic acid sequence comprises one or more 3’ untranslated regions (3’-UTRs) of one or more genes, wherein the 3’- UTR provides a translation efficiency of -0.3 to -.0.8 where the translation efficiency is defined as the ratio of ribosome protected fragments (RPF) to the abundance of ribonucleic acids (RNA).

124. The vector of claim 123, wherein the one or more genes are one or more housekeeping or cytoskeleton genes.

125. The vector of claim 124, wherein the one or more housekeeping or cytoskeleton genes are selected from the group consisting of: GAPDH, a-tubulin, and b-tubulin.

126. The vector of claim 124, wherein the one or more genes are selected from the group consisting of: Rpl32, HSP70a, and CrebA.

127. The vector of any one of claims 100-126, comprising a fourth deoxyribonucleic acid sequence 5’ of the first deoxyribonucleic acid sequence, wherein the fourth deoxyribonucleic acid sequence comprises a promoter.

128. The vector of any one of claims 100-121 and 123-126, comprising a fifth

deoxyribonucleic acid sequence 5’ of the first deoxyribonucleic acid sequence, wherein the fifth deoxyribonucleic acid sequence comprises a repressor element that facilitates further inhibition of the expression of the transgene in non-cancer cells.

129. The vector of claim 128, wherein the fifth deoxyribonucleic acid sequence encodes a hemagglutinin-A epitope.

130. The vector of any one of claims 100-121 and 123-129, wherein the second

deoxyribonucleic acid sequence starts from about 1 to 50 nucleotides after the last nucleotide of the stop codon of the transgene.

131. The vector of claim 127, wherein the first deoxyribonucleic acid sequence and the second deoxyribonucleic acid sequence are under the control of the same promoter.

132. The vector of claim 131, wherein the first deoxyribonucleic acid sequence, the second deoxyribonucleic acid sequence, and the third deoxyribonucleic acid sequence are under the control of an identical promoter.

133. The vector of claim 127, 131 or 132, wherein the promoter is specifically expressed in a cancer cell.

134. The vector of any one of claims 100-133, wherein the antigen or the immunogenic epitope of an antigen is from an infectious agent selected from the group consisting of:

Varicella-Zoster Virus (chickenpox or shingles virus), Herpes Simplex Virus, Hepatitis B virus, Measles virus (morbillivirus), Mumps virus (paramyxovirus), Rubella virus, Human

Immunodeficiency Virus (HIV), Influenza virus, Rhinovirus, Corynebacterium diphtheriae , Clostridium tetani , and Bordetella pertussis.

135. The vector of any one of claims 100-134, comprising a second transgene.

136. The vector of claim 135, comprising a second microRNA binding domain (MBD) wherein the second transgene is under the control of the second MBD; wherein the second MBD comprises one or more microRNA binding sites (MBSs), wherein each MBS is specific for a microRNA that is present in a non-cancer cell and is not present or is downregulated in a cancer cell.

137. The vector any one of claims 100-136, wherein the vector is a viral vector or a plasmid.

138. The vector of claim 137, wherein the viral vector is selected from the group consisting of: a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a herpes simplex virus vector.

139. The vector any one of claims 100-136, wherein the vector is a non-viral vector.

140. The vector of any one of claims 100-139, wherein the antigen or the immunogenic epitope of an antigen is an antigen or an immunogenic epitope of an antigen that a subject has been exposed to via a previous vaccination or infection.

141. A pharmaceutical composition comprising the vector of any one of claims 100-140, and one or more pharmaceutically acceptable excipients.

142. A method for treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the vector of any one of claims 100-140 or the

pharmaceutical composition of claim 141 to the subject.

143. The method of claim 142, wherein the cancer is selected from the group consisting of: breast cancer, colon cancer, brain cancer, pancreatic cancer, lung cancer, cervical cancer, uterine cancer, prostate cancer, ovarian cancer, melanoma, lymphoma, myeloma, and leukemia.

144. The method of claim 142, wherein the cancer is a breast cancer.

145. The method of claim 142 or 144, wherein the cancer is an early stage breast cancer.

146. The method of claim 142 or 144, wherein the cancer is a late stage breast cancer.