Methods and compositions for activation of gamma-delta t-cells

Abstract

To provide methods and compositions for the activation of gamma delta T-cells.SOLUTION: The present invention relates generally to methods and compositions for gene therapy and immunotherapy that activate gamma delta T-cells, and in particular, can be used in the treatment of various cancers and infectious diseases. In one aspect, a method of activating a GD T-cell is provided. The method includes infecting, in the presence of the GD T-cell, a target cell with a viral delivery system that encodes at least one genetic element. In embodiments, the at least one genetic element includes a small RNA capable of inhibiting production of an enzyme involved in the mevalonate pathway.SELECTED DRAWING: None

Description

Technical Field

[0001] This application was filed on January 15, 2016, claims priority to U.S. Provisional Patent Application No. 62 / 279,474, entitled "Methods and Compositions for Activation of Gamma Delta T Cells", which is hereby incorporated by reference herein.

[0002] The present disclosure generally relates to the fields of gene therapy and immunotherapy, particularly as related to increased activation of gamma delta ("GD") T cells.

Background Art

[0003] Human T cells are distinguished based on the T cell receptor structure. The major populations, including the CD4+ and CD8+ subsets, express receptors composed of an alpha chain and a beta chain. A smaller subset expresses T cell receptors composed of a gamma chain and a delta chain. Gamma delta ("GD") T cells constitute 3 - 10% of circulating lymphocytes, and the Vδ2+ subset constitutes 75% of the GD T cells in the blood. Vδ2+ cells recognize non - peptide epitopes and do not require antigen presentation by the major histocompatibility complex ("MHC") or human leukocyte antigen ("HLA"). Most Vδ2+ T cells also express the Vγ9 chain and are stimulated by exposure to 5 - carbon pyrophosphate compounds, which are intermediates in the mevalonate and non - mevalonate sterol / isoprenoid synthesis pathways. The response to isopentenyl pyrophosphate (5 - carbon) is universal in healthy humans.

[0004] Another subset of GD T cells, Vδ1+, constitutes a fairly small percentage of the T cells circulating in the blood, but Vδ1+ cells are commonly found in epithelial mucosa and skin.

[0005] Generally, γδ T cells have several functions including the killing of tumor cells and pathogen-infected cells. Stimulation via their unique T cell receptor (the "TCR") composed of two glycoprotein chains γ and δ improves the ability of cytotoxicity, cytokine secretion and other effector functions. The TCR of γδ T cells has unique specificities, and the cells themselves exist at high clonal frequencies, thus enabling a rapid innate immune-like response against tumors and pathogens.

[0006] Aminobisphosphonate drugs (the "ABP"), and other inhibitors of farnesyl diphosphate synthase (the "FDPS") downstream of isopentenyl pyrophosphate (the "IPP") in the mevalonate pathway (see FIG. 1 for example) have been used to treat various diseases including cancer, particularly those involving bone metastasis. ABP includes trade names such as Zometa® (Novartis) and Fosamax® (Merck).

[0007] ABP has also been used to stimulate γδ T cells. This may be because FDPS is inhibited in bone marrow cells, IPP begins to accumulate, and geranylgeranyl pyrophosphate (the "GGPP"), a downstream product of FDPS that suppresses the activation of the inflammasome pathway, is reduced. The reduction in GGPP removes the inhibitor of the caspase-dependent inflammasome pathway, enabling the secretion of mature cytokines including interleukin-beta and interleukin-18, the latter of which is particularly important for gamma delta T cell activation.

[0008] Therefore, when FDPS is blocked, the increased IPP and decreased GGPP combine to activate Vδ2+ T cells. Vδ2+ cells activated by IPP or ABP can rapidly proliferate, express several cytokines and chemokines, and function to cytolytically destroy tumor cells or cells infected with pathogenic microorganisms.

[0009] However, ABP is not only associated with inflammation and osteonecrosis, but also has poor bioavailability due to their chemistry. Similarly, IPP has a very short half-life and is difficult to synthesize. Both types of compounds require systemic administration in an individual. Thus, both ABP in general and specifically IPP are often inadequate for therapeutic purposes. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0010] In one aspect, a method of activating γδ T cells is provided. The method includes infecting a target cell with a viral delivery system encoding at least one genetic element in the presence of γδ T cells. In embodiments, the at least one genetic element includes a small molecule RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. In embodiments, the enzyme is FDPS. In embodiments, when the enzyme is inhibited in the target cell, the target cell subsequently activates γδ T cells. In embodiments, the target cell is a cancer cell or a cell infected with an infectious agent. In preferred embodiments, activation of the γδ T cells results in γδ T cells that kill cancer cells or cells infected with an infectious agent. In embodiments, the at least one encoded genetic element includes a microRNA or shRNA. In further embodiments, the target cell is also contacted with an aminobisphosphonate drug. In embodiments, the aminobisphosphonate drug is zoledronic acid.

[0011] In another aspect, a method of treating cancer in a subject is provided. The method includes administering to the subject a therapeutically effective amount of a viral delivery system encoding at least one genetic element. In embodiments, the at least one genetic element includes a small molecule RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. In further embodiments, when the enzyme is inhibited in cancer cells in the presence of γδ T cells, the cancer cells activate the γδ T cells, thereby treating the cancer. In embodiments, the enzyme is FDPS. In embodiments, the at least one encoded genetic element includes a microRNA or shRNA. In further embodiments, the target cells are also contacted with an aminobisphosphonate drug. In embodiments, the aminobisphosphonate drug is zoledronic acid.

[0012] In another aspect, a method of treating an infectious disease in a subject is provided. The method includes administering to the subject a therapeutically effective amount of a viral delivery system encoding at least one genetic element. In embodiments, the at least one genetic element includes a small molecule RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. In further embodiments, when the enzyme is inhibited in cells infected with an infectious agent in the presence of γδ T cells, the infected cells activate the γδ T cells, thereby treating the infected cells and the infectious disease. In embodiments, the enzyme is FDPS. In embodiments, the at least one encoded genetic element includes a microRNA or shRNA. In further embodiments, the target cells are also contacted with an aminobisphosphonate drug. In embodiments, the aminobisphosphonate drug is zoledronic acid.

[0013] In another aspect, the at least one encoded genetic element is

Chemical Formula

Chem.

[0014] In another aspect, at least one encoded genetic element is

Chem.

Chem.

Chem.

[0015] In another aspect, a viral vector comprising at least one encoded genetic element is provided. The at least one encoded genetic element comprises a small molecule RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. In an embodiment, the enzyme involved in the mevalonate pathway is farnesyl diphosphate synthase (FDPS). In an embodiment, the at least one encoded genetic element comprises microRNA or shRNA.

[0016] In another aspect, at least one encoded genetic element comprises shRNA having at least 80%, or at least 85%, or at least 90%, or at least 95% percent identity to v. In a preferred embodiment, the shRNA comprises SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4.

[0017] In another aspect, at least one of the encoded genetic elements comprises a microRNA having at least 80%, or at least 85%, or at least 90%, or at least 95% percent identity with SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10. In a preferred embodiment, the microRNA comprises SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

[0018] In embodiments, the viral vector is composed of any vector capable of efficiently transducing small interfering RNAs into target cells. In embodiments, the viral vector is a lentiviral vector. In other embodiments, the viral vector is an adeno-associated viral vector.

[0019] In another aspect, the viral vector comprises a second encoded genetic element. In embodiments, the second genetic element comprises at least one cytokine or chemokine. In embodiments, the at least one cytokine is selected from the group consisting of IL-18, TNF-α, interferon-γ, IL-1, IL-2, IL-15, IL-17, and IL-12. In embodiments, the at least one chemokine is a CC chemokine or a CXC chemokine. In further embodiments, the at least one chemokine is RANTES.

[0020] In another aspect, a lentiviral vector system for expressing lentiviral particles is provided. This system includes a lentiviral vector, at least one envelope plasmid for expressing an envelope protein optimized to infect cells; and at least one helper plasmid for expressing the gag, pol, and rev genes. When the lentiviral vector, at least one envelope plasmid, and at least one helper plasmid are transfected into packaging cells, lentiviral particles are produced by the packaging cells. In embodiments, the lentiviral particles can infect target cells and inhibit enzymes involved in the mevalonate pathway within the target cells. In embodiments, the enzyme involved in the mevalonate pathway is FDPS. In embodiments, the lentiviral vector system includes a first helper plasmid for expressing the gag and pol genes and a second helper plasmid for expressing the rev gene. In embodiments, the envelope protein is preferably optimized to infect target cells. In embodiments, the target cells are cancer cells. In other embodiments, the target cells are cells infected with an infectious agent.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0036] Summary of the Disclosure The present disclosure relates to gene therapy constructs and their delivery to cells that result in the inhibition of farnesyl diphosphate synthase (“FDPS”) required to convert isopentenyl phosphate (IPP) to farnesyl diphosphate (FDP), such as shown in FIG. 1. In embodiments, one or more viral vectors are provided with microRNAs or short hairpin RNAs (shRNAs) that target FDPS, thereby reducing the expression level of this enzyme. Viral vectors include lentiviral vectors and AAV vectors. The result of modulating the expression of FDPS is to increase the accumulation of IPP, a stimulator of the proliferation and differentiation of γδ T cells. Accordingly, the constructs provided herein are used to activate γδ T cells and are used to treat cancer and infectious diseases. Definitions and Interpretations

[0037] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure have the meanings that are commonly understood by one of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. In general, the nomenclature used in conjunction with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and chemistry of proteins and nucleic acids, as well as hybridization described herein, and their techniques are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods described in a variety of general and more specific references cited and discussed throughout this specification, unless otherwise noted, which are well known in the art. For example, Sambrook J. and Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Any enzymatic reaction or purification technique is performed as generally accomplished in the art or according to the manufacturer's specifications described herein. The nomenclature used in conjunction with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their laboratory procedures and techniques, are those well known and commonly used in the art.

[0038] As used in the specification and the appended claims, the singular forms "a," "an," and "the" are used interchangeably and are intended to include the plural forms as well, unless the context clearly dictates otherwise. Also, as used herein, "and / or" refers to any and all possible combinations of one or more of the listed items, as well as the absence of a combination when alternatively construed ("or"), and includes these.

[0039] All numerical designations, including ranges, pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) in increments of 0.1. Although not necessarily explicitly stated, it should be understood that the term "about" precedes all numerical designations. The term "about" also includes the exact value "X" in addition to minor increments of "X" such as "X + 0.1" or "X - 0.1". Although not necessarily explicitly stated, it should also be understood that the reagents described herein are merely exemplary and that their equivalents are known in the art.

[0040] As used herein, the term "about" is understood by those of ordinary skill in the art and varies to some extent depending on the context in which it is used. Where the use of a term is not obvious to those of ordinary skill in the art, considering the context in which it is used, "about" means plus or minus 10% of the particular term.

[0041] The term "administration" of an active agent or "administering" an active agent is to be understood to mean providing an active agent to a subject in need of treatment in a therapeutically useful form and in a form that can be introduced into the body of the individual in a therapeutically effective amount.

[0042] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude others. "Consisting essentially of" means, when used to define compositions and methods, that other elements of any essential significance to the composition or method are excluded. "Consisting of" means excluding other elements that are not trace amounts of other components for the claimed compositions and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of this disclosure. Thus, the methods and compositions are intended to include (comprising) additional steps and components, or to include (including) steps and compositions of no significance (consisting essentially of), or to be intended to be only the recited method steps or compositions (consisting of).

[0043] As used herein, "expression," "expressed," or "encoding" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Expression can include splicing of mRNA or other forms of post-transcriptional or post-translational modification in eukaryotic cells.

[0044] The term "farnesyl diphosphate synthase" may also be referred to herein as FDPS and may also be referred to herein as farnesyl pyrophosphate synthase or FPPS.

[0045] The term "gamma delta T cells" can be referred to as γδ T cells in this specification and can also be referred to as GD T cells. The term "gamma delta T cell activation" refers to any measurable biological phenomenon associated with gamma delta T cells that represent such activated T cells. Non-limiting examples of such biological phenomena include an increase in cytokine production, a change in the qualitative or quantitative composition of cell surface proteins, an increase in T cell proliferation, and / or an increase in T cell effector function, such as killing target cells or assisting other effector cells to kill target cells.

[0046] The terms "individual", "subject" and "patient" are used interchangeably in this specification and refer to any individual mammalian subject, such as a cow, dog, cat, horse or human.

[0047] The term "miRNA" refers to microRNA and can also be referred to as "miR" in this specification.

[0048] The term "packaging cell line" refers to any cell line that can be used to express lentiviral particles.

[0049] As used herein, the term "percent identity" refers to the percentage of nucleotides or amino acid residues that are identical when two or more nucleic acid or polypeptide sequences are aligned for maximum correspondence using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, "percent identity" may exist over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared. For sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, sub-sequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0050] Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Ausubel et al., infra generally).

[0051] An example of an appropriate algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website.

[0052] Percent identity between two nucleotide sequences can be determined using the NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70 or 80, and length weights of 1, 2, 3, 4, 5 or 6, using the GAP program in the GCG software package (available at http: / / www.gcg.com). Percent identity between two nucleotide sequences or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Further, percent identity between two amino acid sequences can be determined using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6 or 4, and length weights of 1, 2, 3, 4, 5 or 6, using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com).

[0053] The nucleic acid and protein sequences of the present disclosure can further be used, for example, as "query sequences" to perform searches against public databases to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. A BLAST nucleotide search can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules provided in the present disclosure. A BLAST protein search can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, to obtain gapped alignments for comparison purposes. When using the BLAST and Gapped BLAST programs, the default parameters of the representative programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0054] As used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment, with no excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio, and are suitable for use in contact with human and animal tissues, organs and / or body fluids.

[0055] As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible and include these. Compositions can include pharmaceutically acceptable salts, such as acid addition salts or base addition salts (see, for example, Berge et al. (1977) J Pharm Sci 66:1-19). As used herein, the term "SEQ ID NO." is synonymous with the term "Array ID No."

[0056] As used herein, "small RNA" refers to a non-coding RNA that has a silencing or interfering function and is generally shorter than about 200 nucleotides in length. In other embodiments, the small RNA is about 175 nucleotides in length or less, about 150 nucleotides in length or less, about 125 nucleotides in length or less, about 100 nucleotides in length or less, or about 75 nucleotides in length or less. Such RNAs include microRNA (miRNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), and small hairpin RNA (shRNA). The "small RNA" of the present disclosure must be capable of inhibiting or knocking down the gene expression of a target gene, generally via a pathway that results in the destruction of the target gene mRNA.

[0057] The term "therapeutically effective amount" refers to the amount of an active agent of the present disclosure in a suitable composition and in a suitable dosage form that is sufficient to treat the symptoms of a complication found in a patient suffering from a given discomfort, injury, disease, or condition, or to prevent its progression or onset. The therapeutically effective amount varies depending on the condition or severity of the patient, and conditions such as the age and weight of the subject being treated. The therapeutically effective amount can vary depending on any of several factors, including, for example, the route of administration, the condition of the subject, and other factors understood by those of ordinary skill in the art.

[0058] As used herein, the term "therapeutic vector" includes, without limitation, reference to a lentiviral vector or an AAV vector.

[0059] "Treatment" is intended to target and combat a disease state, i.e., to relieve or prevent a disease state. Thus, a particular treatment depends on the disease state being targeted, as well as the current and future state of drug therapies and treatment approaches. Treatment can be associated with toxicity.

[0060] The term "treatment" or "treating" generally refers to an intervention that attempts to alter the natural course of the subject being treated and can be performed for prophylaxis or during the course of clinical pathology. Desired effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, suppressing, attenuating or inhibiting any direct or indirect pathological consequence of a disease, remitting or alleviating a disease state, and causing a favorable or improved prognosis. Description of Aspects of the Present Disclosure

[0061] In one aspect, a method of activating γδ T cells is provided. The method includes infecting a target cell with a viral delivery system encoding at least one genetic element in the presence of γδ T cells. In embodiments, the at least one encoded genetic element includes a small interfering RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. In embodiments, the enzyme is FDPS. In embodiments, when the enzyme is inhibited in the target cell, the target cell activates γδ T cells. In embodiments, the target cell is a cancer cell or a cell infected with an infectious agent. In embodiments, the at least one encoded genetic element includes a microRNA or shRNA.

[0062] In embodiments, the at least one encoded genetic element

Chemical formula

Chemical formula

[0063] In another aspect, at least one encoded genetic element

Chem.

Chem.

[0064] In another aspect, the target cells are also contacted with an aminobisphosphonate drug. In a preferred embodiment, the aminobisphosphonate drug is zoledronic acid.

[0065] In another aspect, a method of treating cancer in a subject is provided. The method includes administering to the subject a therapeutically effective amount of a viral delivery system encoding at least one genetic element. In an embodiment, the at least one encoded genetic element comprises a small molecule RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. In a further embodiment, when the enzyme is inhibited in cancer cells in the presence of gd T cells, the cancer cells activate the gd T cells, thereby treating the cancer. In an embodiment, the enzyme is FDPS. In an embodiment, the at least one encoded genetic element comprises a microRNA or shRNA.

[0066] In another aspect, a method of treating an infectious disease in a subject is provided. The method includes administering to the subject a therapeutically effective amount of a viral delivery system encoding at least one genetic element. In embodiments, the at least one encoded genetic element includes a small molecule RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. In further embodiments, when the enzyme is inhibited in cells infected with an infectious agent in the presence of γδ T cells, the infected cells activate the γδ T cells, thereby treating the infected cells and the infectious disease. In embodiments, the enzyme is FDPS. In embodiments, the at least one encoded genetic element includes a microRNA or shRNA.

[0067] In embodiments, the at least one encoded genetic element includes an shRNA having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or higher percent identity to SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4. In preferred embodiments, the shRNA includes SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4.

[0068] In other embodiments, the at least one encoded genetic element includes a microRNA having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or higher percent identity to SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10. In preferred embodiments, the microRNA includes SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

[0069] In another aspect, a viral vector is provided that includes at least one encoded genetic element. The at least one encoded genetic element includes a small molecule RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. In embodiments, the enzyme involved in the mevalonate pathway is farnesyl diphosphate synthase (FDPS). In embodiments, the at least one encoded genetic element includes a microRNA or shRNA.

[0070] In another aspect, the at least one encoded genetic element includes an shRNA having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or higher percent identity to SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4. In preferred embodiments, the shRNA includes SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; or SEQ ID NO: 4.

[0071] In another aspect, the at least one encoded genetic element includes a microRNA having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or higher percent identity to SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10. In preferred embodiments, the microRNA includes SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; or SEQ ID NO: 10.

[0072] In an embodiment, the viral vector includes any vector capable of efficiently transducing small interfering RNAs. In an embodiment, the viral vector is a lentiviral vector. In other embodiments, the viral vector is an adeno-associated virus (AAV) vector.

[0073] In another aspect, the viral vector includes a second encoded genetic element. In an embodiment, the second genetic element includes at least one cytokine or chemokine. In an embodiment, the at least one cytokine is selected from the group consisting of IL-18, TNF-α, interferon-γ, IL-1, IL-2, IL-15, IL-17, and IL-12. In an embodiment, the at least one chemokine is a CC chemokine, a CXC chemokine, a cCX3 chemokine, or an XC chemokine. In a further embodiment, the at least one chemokine is the CC chemokine RANTES.

[0074] In another aspect, a lentiviral vector system for expressing lentiviral particles is provided. This system includes a lentiviral vector, at least one envelope plasmid for expressing an envelope protein optimized to infect cells; and at least one helper plasmid for expressing the gag, pol, and rev genes. When the lentiviral vector, at least one envelope plasmid, and at least one helper plasmid are transfected into packaging cells, lentiviral particles are produced by the packaging cells. In embodiments, the lentiviral particles are capable of infecting target cells and inhibiting an enzyme involved in the mevalonate pathway within the target cells. In embodiments, the enzyme involved in the mevalonate pathway is FDPS. In embodiments, the lentiviral vector system includes a first helper plasmid for expressing the gag and pol genes and a second helper plasmid for expressing the rev gene. In embodiments, the envelope protein is preferably optimized to infect target cells. In embodiments, the target cells are cancer cells. In other embodiments, the target cells are cells infected with an infectious disease. cancer

[0075] The compositions and methods provided herein are used for treating cancer. The cell, tissue or target can be a cancer cell, a cancerous tissue, a tissue that can harbor a cancerous tissue, or a subject or patient diagnosed with or at risk of developing a disease or condition. In certain embodiments, the cell can be an epithelial, endothelial, mesothelial, glial, stromal or mucosal cell. Cancer cell populations can include, but are not limited to, brain, neuron, blood, endometrial, meningeal, esophageal, lung, cardiovascular, liver, lymphatic, breast, bone, connective tissue, adipose, retinal, thyroid, glandular, adrenal, pancreatic, gastric, intestinal, renal, bladder, colorectal, prostate, uterine, ovarian, cervical, testicular, splenic, skin, smooth muscle, myocardial or skeletal muscle cells. In still further embodiments, cancers include, but are not limited to, astrocytoma, acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphoblastic leukemia, angiosarcoma, B-cell lymphoma, Burkitt lymphoma, breast cancer, bladder cancer, head and neck carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal squamous cell carcinoma, Ewing sarcoma, fibrosarcoma, glioma, glioblastoma, gastrinoma, gastric cancer, hepatoblastoma, hepatocellular carcinoma, Kaposi sarcoma, Hodgkin lymphoma, laryngeal squamous cell carcinoma, larynx cancer, leukemia, leiomyosarcoma, lipoma, liposarcoma, melanoma, mantle cell lymphoma, medulloblastoma, mesothelioma, myxofibrosarcoma, myeloid leukemia, mucosa-associated lymphoid tissue B-cell lymphoma, multiple myeloma, high-risk myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, neurofibroma, high-grade non-Hodgkin lymphoma, non-Hodgkin lymphoma, lung cancer, non-small cell lung cancer, ovarian cancer, esophageal cancer, osteosarcoma, pancreatic cancer, pheochromocytoma, prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland tumor, Schwannomma, small cell lung cancer, head and neck squamous cell carcinoma, testicular tumor, thyroid cancer, urothelial cancer and Wilms tumor.

[0076] The compositions and methods provided herein are also used to treat NSCLC (non-small cell lung cancer), pediatric malignancies, cervical and other tumors caused or promoted by human papillomavirus (HPV), melanoma, Barrett's esophagus (pre-malignant syndrome), adrenal and skin cancers, as well as autoimmune and neoplastic skin diseases. Infectious diseases

[0077] The compositions and methods disclosed herein can be used to treat infectious diseases. The term "infectious disease" includes any disease caused by an infectious agent. "Infectious agent" includes any exogenous pathogen, including, without limitation, bacteria, fungi, viruses, mycoplasma, and parasites, including any recognized infectious organism in the art that causes pathogenesis in animals. Infectious agents that can be treated with the compositions provided in the present disclosure include organisms such as bacteria that are gram-negative or gram-positive cocci or bacilli, DNA viruses such as papillomavirus, parvovirus, adenovirus, herpesvirus, and vaccinia virus, and RNA viruses such as arenavirus, coronavirus, rhinovirus, respiratory syncytial virus, influenza virus, picomaviruses, paramyxovirus, reovirus, retrovirus, and rhabdovirus, including DNA and RNA viruses not limited to these. Examples of fungi that can be treated with the compositions and methods of the present disclosure include fungi that cause diseases such as, for example, ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidioidomycosis, and candidiasis, including fungi that grow as molds or are yeast-like. The compositions and methods provided herein can be utilized to treat parasitic infections including, but not limited to, infections caused by Trichinella spiralis, Schistosoma mansoni, tissue roundworms, amoebas, as well as Plasmodium, Trypanosoma, Leishmania, and Toxoplasma species. Methods of γδ T cell activation

[0078] Compositions and methods for activating γδ T cells in an individual, and methods for treating tumors and infectious diseases are provided herein. For example, in embodiments, since activated γδ T cells include natural mechanisms for tumor immune surveillance, the compositions and methods provided herein can be used in methods for treating all known cancers (see, for example, Pauza et al., Frontiers in Immunol. 5:687, 2014). Similarly, in embodiments, the compositions and methods provided herein can be used to treat infectious diseases including, but not limited to, flavivirus, influenza virus, human retrovirus, mycobacteria, Plasmodium malariae, and various other viral, fungal, and bacterial infections (see, for example, Pauza and Cairo, Cell Immunol. 296(1), 2015).

[0079] Generally, the vector system is administered to an individual for transfecting or transducing a target cell population using the disclosed constructs to decrease the expression of FDPS and, in other embodiments, to increase the expression of chemokines or cytokines. Administration and transfection / transduction can occur in vivo or ex vivo, and the transfected cells are subsequently readministered to the subject in a later scenario.

[0080] Administration of the disclosed vectors and transfection or transduction of the disclosed constructs into the cells of a subject result in decreased expression of FDPS, increased expression of cytokines or chemokines, accumulation of IPP, and, in many cases, a reduced growth rate for the genetically modified tumor cells. All of these features function together to activate γδ T cells and co-localize γδ T cells to the site of the tumor or infection.

[0081] The disclosed method can also increase the ability of NK cells to recognize and destroy tumor cells and / or infected cells. The crosstalk between γδ T cells and NK cells is an important aspect in regulating the immune response and inflammatory response. Furthermore, γδ T cells are known to induce dendritic cell maturation, recruit B cells and macrophages, and be involved in the secretion of various cytolytic activities, such as interferon-γ and TNF-α.

[0082] In embodiments, the disclosed compositions and methods provided herein constitute a form of gene therapy for activating γδ T cells at the site of tumor or infectious disease pathology. In one aspect, the compositions and methods provided herein support their proliferation, differentiation, and functional capabilities by activating γδ T cells and promoting the production of specific cytokines necessary for cytolytic activity capable of killing cancer cells or treating infectious diseases.

[0083] In embodiments, the gene therapy sequence (e.g., FDPS shRNA) is carried by a therapeutic vector including but not limited to viral vectors such as lentivirus or adeno-associated virus, although other viral vectors may also be suitable. The gene therapy construct can also be delivered in the form of DNA or RNA including but not limited to plasmid form. In embodiments, the disclosed gene therapy construct can also be delivered in the form of protein-nucleic acid complexes or lipid-nucleic acid complexes and mixtures of these formulations. For example, the protein-nucleic acid complex can contain the nucleic acid of interest in a complex with cationic peptides such as lysine and arginine. The lipid-nucleic acid complex can include lipid emulsions, micelles, liposomes, and / or mixtures of neutral and cationic lipids such as DOTMA, DOSPA, DOTAP, and DMRIE.

[0084] In embodiments, the therapeutic vector can include a single construct, or at least two, at least three, at least four or at least five different constructs. If more than one construct is present in the vector, these constructs can be identical or different. For example, the constructs can vary with respect to their promoters, the presence or absence of integration elements, and / or their sequences. In some embodiments, the therapeutic vector includes at least one construct encoding a small molecule RNA capable of knocking down the expression of FDPS. In embodiments, the therapeutic vector also encodes certain cytokine(s) and / or chemokine(s) including but not limited to TNF-α, interferon-γ, IL-1, IL-2, IL-15, IL-17, IL-18 or IL-12. In some embodiments, a single construct can encode both a small molecule RNA capable of knocking down the expression of FDPS and certain cytokines or chemokines including but not limited to TNF-α, interferon-γ, IL-1, IL-2, IL-15, IL-17, IL-18 or IL-12.

[0085] In embodiments, the viral vector can introduce a nucleic acid construct that integrates into the host chromosome. Alternatively, transient delivery vectors can be used to prevent chromosomal integration and limit the lifespan of the gene therapy construct.

[0086] In embodiments, the disclosed constructs and vectors contain short hairpin RNAs ("shRNAs"), microRNAs ("miRNAs") or siRNAs capable of reducing or knocking down the expression of the FDPS and / or geranyl pyrophosphate synthase ("GPPS") and / or farnesyl transferase ("FT") genes. By downregulating these genes that control steroid and isoprenoid synthesis, the isopentenyl pyrophosphate ("IPP") level is increased. The increase and accumulation of IPP is a known mechanism for increasing γδ T cell activation. Furthermore, the downregulation of these pyrophosphate synthase genes removes an important negative regulator of inflammasome function, which in turn results in increased expression of cytokines important for γδ T cell activation and effector cell function.

[0087] In embodiments, the disclosed constructs are regulated by specific promoters capable of producing interleukin-2 and / or interleukin-15 to sustain γδ T cell proliferation. Furthermore, the disclosed constructs can be regulated by specific promoters capable of producing interleukin-1 beta and / or interleukin-18 and / or interferon-gamma necessary for γδ T cell differentiation and acquisition of all effector cell functions. Desired effector cell functions include the ability to directly cause cytotoxic cell death of tumor and / or infected cells, secrete beneficial cytokines and / or chemokines, increased expression of NK receptors necessary to recognize cancerous or infected cells, and increased expression of Fc receptors necessary to bind targeting antibodies to co-localize γδ T cells with cancerous cell targets or infected cell targets.

[0088] In embodiments, the disclosed method has an indirect effect of activating γδ T cells and increasing the ability of natural killer (NK) cells to attack and destroy cancerous, tumor, or infected cells. Activation of NK cells requires γδ T cells that are stimulated to proliferate and differentiate and to express the 4-1BB ligand co-stimulatory ligand necessary to engage the 4-1BB co-stimulatory receptor on NK cells. This form of crosstalk is known as an important mechanism for activating NK cells and is achieved herein through the action of the disclosed methods and compositions.

[0089] In another aspect, crosstalk between γδ T cells and NK cells is an important mechanism for eliminating inflammatory dendritic cells that accumulate in diseased tissues. Alone, neither γδ T cells nor NK cells are capable of destroying dendritic cells, but when the above-described crosstalk interaction occurs, NK cells are altered to become cytotoxic to inflammatory dendritic cells. This immunomodulatory mechanism depends on the strong activation and proliferation of γδ T cells.

[0090] In embodiments, the disclosed method for activating γδ T cells further includes suppressing a pathological inflammatory response that can include atherosclerotic disease, chronic immune activation that stimulates tumor growth, autoimmune diseases including psoriasis and other conditions in the epidermis, inflammatory diseases of the central nervous system, and arthritis as well as other diseases of unregulated immune responses that can include cell proliferation.

[0091] In embodiments, the therapeutic vector is administered concurrently with an aminobisphosphonate (ABP) drug to achieve synergistic activation of gamma delta T cells. The synergy can allow for alternating, modified, or reduced dosages of the ABP and can reduce adverse reactions to the ABP, including acute inflammatory responses and chronic diseases. Constructs for γδ T Cell Activation

[0092] Inhibition of FDPS results in IPP accumulation, activation of Vδ2+ γδ T cells, and expression of IL-18, which is also important in the activation of γδ T cells. Inhibition of farnesyl transferase results in decreased prenylation of proteins. The disclosed constructs can be transfected or transduced into specific target cells such as tumor cells or infected cells, where they inhibit the translation of FDPS and can express RNA sequences (i.e., siRNA, shRNA, or microRNA) encoding and expressing cytotoxic cytokines or chemokines.

[0093] Constructs are disclosed herein for decreasing the expression of FDPS and / or FT, increasing the expression of cytokines, and increasing the expression of chemokines including RANTES. For example, in some embodiments, the construct can encode interferon-gamma, IL-1, IL-2, IL-15, IL-17, IL-18, or IL-12.

[0094] Expression of cytokines and chemokines such as those listed above results in localized cytotoxic destruction of tumor cells or cells infected with pathogenic organisms. Thus, expression of such constructs by tumor cells or infected cells results in unwanted cells that aid in their own destruction.

[0095] Similarly, when the disclosed constructs are expressed in tumor cells or infected cells, decreasing the expression of FDPS and FT results in activation of γδ T cells and recruitment of γδ T cells to the tumor site or the site of cell infection. Increasing the expression of RANTES further attracts γδ T cells to the intended tissue location. Recruitment of γδ T cells to the tumor site can be a particularly effective means of inducing anti-tumor immunity since γδ T cells can kill a wide range of tumors of epithelial origin as well as many leukemias and lymphomas and are further capable of producing high levels of the anti-tumor cytokine IFNγ.

[0096] Reduced expression of FDPS can be achieved via shRNA, microRNA, siRNA, or other means known in the art. For example, shRNAs according to SEQ ID NO: 1, 2, 3 or 4, or variants thereof, can be used in the disclosed constructs and methods, but this example is not limiting. The coding regions of RNA for reducing the expression of FDPS and FT, as well as the coding regions of cytokines and chemokines, can be in the same construct or on different constructs.

[0097] Classical approaches for the production of recombinant polypeptides, or gene regulatory molecules including small RNAs, are the use of stable expression constructs. These constructs are based on chromosomal integration of a transfected expression plasmid (or at least a portion thereof) into the genome of the host cell, short-term plasmid transfection, or non-integrating viral vectors that also have a limited half-life. The site of gene integration is generally random, and the number and ratio of gene integrations at any particular site are often unpredictable; similarly, non-integrating plasmids or viral vectors also produce nuclear DNA, but these species usually lack the sequences necessary for DNA replication and persistent maintenance. Thus, constructs that rely on chromosomal integration result in the permanent maintenance of the recombinant gene that can exceed the treatment interval.

[0098] An alternative to stable expression constructs for gene expression is transient expression constructs. The expression of the latter gene expression constructs is based on non-integrating plasmids, and thus, expression is typically lost when the cell undergoes division or when the plasmid vector is destroyed by endogenous nucleases.

[0099] The disclosed constructs are preferably episomal constructs that are transiently expressed. Episomal constructs are destroyed or diluted over time, and as a result, they do not cause permanent changes to the genome of the subject and are not incorporated into the chromosomes of the target cells. The process of episomal replication typically incorporates both the host cell replication machinery and viral trans-acting factors.

[0100] Avoiding chromosomal integration reduces certain barriers to in vivo gene delivery. However, even integration-deficient constructs can have background frequencies of integration, and any DNA molecule may be found to have low homology to recombine with host sequences; however, these rates of integration are exceptionally low and generally not clinically significant.

[0101] Thus, in some embodiments, the disclosed vectors support the delivery of active genes and / or small interfering RNAs over a period of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 weeks. In some embodiments, the disclosed vectors support the delivery of active genes and / or small interfering RNAs over a period of about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. Any combination of these periods, such as 1 month and 1 week, or 3 months and 2 weeks, may also be used in the methods of the invention.

[0102] However, in some embodiments, the construct contains an integration element that depends on the retroviral integrase gene, and as a result, this construct is integrated into the target chromosome. Retrotransposition and translocation are further examples of mechanisms by which mobile genetic elements are incorporated or inserted into chromosomes. Plasmids can be integrated into chromosomes by recombination, and gene editing technologies, including CRISPR and TALEN, utilize guide RNA sequences to alter chromosomal loci by gene conversion mechanisms.

[0103] The construct may include specific promoters for expressing cytokines involved in the maintenance of GD T cells (i.e., IL-2, IL-7, IL-17, and IL-15). For example, promoters that can be incorporated into the disclosed constructs include, but are not limited to, TATA-box promoter, CpG-box promoter, CCAAT-box promoter, TTGACA-box promoter, BRE-box promoter, INR-box promoter, AT-based promoter, CG-based promoter, ATCG-compact promoter, ATCG-balanced promoter, ATCG-middle promoter, ATCG-less promoter, AT-less promoter, CG-less promoter, AT-spike promoter, and CG-spike promoter. See Gagniuc and Ionescu-Tirgoviste, Eukaryotic genomes may exhibit up to 10 generic classes of gene promoters, BMC GENOMICS 13:512 (2012). Therapeutic vector

[0104] The construct can be delivered via known transfection and / or transduction vectors including, but not limited to, lentiviral vectors, adeno-associated viruses, poxviruses, herpesvirus vectors, protein and / or lipid complexes, liposomes, micelles, etc.

[0105] Viral vectors can be preferentially targeted to cell types useful for the disclosed methods (i.e., tumor cells or bone marrow cells). Viral vectors can be used to transduce genes into target cells due to specific viral envelope-host cell receptor interactions and viral machinery for gene expression. As a result, viral vectors have been used as vehicles for the transfer of genes into many different cell types, including whole embryos, fertilized eggs, isolated tissue samples, in situ tissue targets, and cultured cell lines. The ability to introduce and express foreign genes in cells is useful for the study of gene expression and the elucidation of cell lineages, as well as for providing the potential for therapeutic interventions such as gene therapy, somatic reprogramming of induced pluripotent stem cells, and various types of immunotherapy. Viral components derived from viruses such as the Papovaviridae (e.g., bovine papillomavirus, i.e., BPV) or the Herpesviridae (e.g., Epstein-Barr virus, i.e., EBV) or the Hepadnaviridae (e.g., hepatitis B virus, i.e., HBV) or poxvirus vectors including vaccinia can be used in the disclosed vectors.

[0106] Lentiviral vectors are a preferred type of vector for the disclosed compositions and methods, but the present disclosure is not specifically limited to lentiviral vectors. Lentiviruses are a genus of viruses that can deliver significant amounts of viral nucleic acid into host cells. Lentiviruses are characterized by their unique ability to infect / transduce non-dividing cells, and after transduction, lentiviruses integrate their nucleic acids into the chromosomes of host cells.

[0107] Infectious lentiviruses have three major genes gag, pol, and env that encode pathogenic proteins, as well as two regulatory genes tat and rev. Depending on the specific serotype and virus, additional accessory genes may be present that encode proteins involved in the regulation, synthesis, and / or processing of viral nucleic acids, as well as other replication functions.

[0108] Furthermore, lentiviruses contain long terminal repeat (LTR) regions that can be approximately 600 nt in length. The LTR can be segmented into U3, R, and U5 regions. The LTR can mediate the integration of retroviral DNA into the host chromosome via the action of integrase. Alternatively, when integrase is not functional, the LTR can be used to circularize the viral nucleic acid.

[0109] Viral proteins involved in the early stages of lentiviral replication include reverse transcriptase and integrase. Reverse transcriptase is an RNA-dependent DNA polymerase encoded by the virus. This enzyme uses the viral RNA genome as a template for the synthesis of a complementary DNA copy. Reverse transcriptase also has RNaseH activity for the destruction of the RNA-template. Integrase binds both the viral cDNA generated by reverse transcriptase and the host DNA. Integrase processes the LTR before inserting the viral genome into the host DNA. Tat acts as a trans-activator during transcription to enhance initiation and elongation. The rev-responsive element acts post-transcriptionally to regulate mRNA splicing and transport to the cytoplasm.

[0110] Viral vectors generally contain glycoproteins, and various glycoproteins can provide specific affinities. For example, the VSVG peptide can increase transfection into bone marrow cells. Alternatively, viral vectors can also have targeting moieties such as antibodies conjugated to their shell peptides. Targeting antibodies can be specific for antigens overexpressed on tumors such as HER-2, PSA, CEA, M2-PK, and CA19-9, for example.

[0111] Other viral vector specificities are also known in the art and can be used to target specific populations of cells. For example, poxvirus vectors target macrophages and dendritic cells. Lentiviral vector system

[0112] Lentiviral virions (particles) are expressed by a vector system that encodes viral proteins necessary to produce the virions (viral particles). There is at least one vector that contains a nucleic acid sequence encoding a lentiviral pol protein necessary for reverse transcription and integration, operably linked to a promoter. In another embodiment, the pol protein is expressed by multiple vectors. There is also a vector that contains a nucleic acid sequence encoding a lentiviral gag protein necessary to form the viral capsid, operably linked to a promoter. In certain embodiments, this gag nucleic acid sequence is on a vector separate from at least a portion of the pol nucleic acid sequence. In another embodiment, the gag nucleic acid is on a vector separate from all of the pol nucleic acid sequences encoding the pol protein.

[0113] Numerous modifications can be made to the vectors used to create the particles to further minimize the possibility of generating wild-type revertants. These include, but are not limited to, deletions in the U3 region of the LTR, tat deletions, and matrix (MA) deletions.

[0114] The gag, pol, and env vector(s) do not contain nucleotides from the lentiviral genome that package lentiviral RNA, called the lentiviral packaging sequence.

[0115] The vector(s) that form the particles preferably do not contain a nucleic acid sequence from the lentiviral genome that expresses an envelope protein. Preferably, a separate vector that contains a nucleic acid sequence encoding an envelope protein, operably linked to a promoter, is used. This env vector also does not contain the lentiviral packaging sequence. In one embodiment, the env nucleic acid sequence encodes a lentiviral envelope protein.

[0116] In another embodiment, the envelope protein is not derived from a lentivirus but from a different virus. The resulting particles are called pseudotyped particles. By appropriate selection of the envelope, virtually any cell can be “infected.” For example, an env gene encoding an envelope protein that targets the endocytic compartment can be used, such as those of influenza virus, VSV-G, alphavirus (Semliki Forest virus, Sindbis virus), arenavirus (lymphocytic choriomeningitis virus), flavivirus (tick-borne encephalitis virus, dengue virus, hepatitis C virus, GB virus), rhabdovirus (vesicular stomatitis virus, rabies virus), paramyxovirus (mumps or measles), and orthomyxovirus (influenza virus). Other envelopes that can preferably be used include those derived from Moloney leukemia virus, such as MLV-E, MLV-A, and GALV. These latter envelopes are particularly preferred when the host cell is a primary cell. Other envelope proteins can be selected depending on the desired host cell. For example, targeting a specific receptor such as a dopamine receptor can be used for brain delivery. Another target can be the vascular endothelium. These cells can be targeted using a filovirus envelope. For example, the GP of Ebola is converted to the GP and GP2 glycoproteins by post-transcriptional modification. In another embodiment, different lentiviral capsids with pseudotyped envelopes can be used (e.g., FIV or SHIV [U.S. Patent No. 5,654,195]). SHIV pseudotyped vectors can be readily used in animal models such as monkeys.

[0117] As detailed herein, a lentiviral vector system typically includes at least one helper plasmid that contains at least one of the gag, pol, or rev genes. Each of the gag, pol, and rev genes can be provided on an individual plasmid, or one or more genes can be provided together on the same plasmid. In one embodiment, the gag, pol, and rev genes are provided on the same plasmid (e.g., FIG. 2). In another embodiment, the gag and pol genes are provided on a first plasmid and the rev gene is provided on a second plasmid (e.g., FIG. 3). Thus, both 3-vector and 4-vector systems can be used to produce lentiviruses, as described in the Examples section and elsewhere in this specification. The therapeutic vector, envelope plasmid, and at least one helper plasmid are transfected into a packaging cell line. A non-limiting example of a packaging cell line is the 293T / 17 HEK cell line. When the therapeutic vector, envelope plasmid, and at least one helper plasmid are transfected into the packaging cell line, lentiviral particles are ultimately produced.

[0118] In another aspect, a lentiviral vector system for expressing lentiviral particles is disclosed. This system includes a lentiviral vector as described herein; an envelope plasmid for expressing an envelope protein optimized to infect cells; and at least one helper plasmid for expressing the gag, pol, and rev genes, wherein when the lentiviral vector, envelope plasmid, and at least one helper plasmid are transfected into a packaging cell line, lentiviral particles are produced by this packaging cell line, and these lentiviral particles are capable of inhibiting the production of the chemokine receptor CCR5 or targeting the HIV RNA sequence.

[0119] In another aspect, as detailed in Figure 2, the lentiviral vector, also referred to herein as a therapeutic vector, may include the following elements: a hybrid 5' long terminal repeat (RSV / 5'LTR) (SEQ ID NOs: 11-12), the psi sequence (RNA packaging site) (SEQ ID NO: 13), the RRE (Rev response element) (SEQ ID NO: 14), the cPPT (polypurine tract) (SEQ ID NO: 15), the H1 promoter (SEQ ID NO: 16), the FDPS shRNA (SEQ ID NOs: 1, 2, 3, 4), the woodchuck post-transcriptional regulatory element (WPRE) (SEQ ID NO: 17), and the 3' delta LTR (SEQ ID NO: 18). In another aspect, sequence variations by substitution, deletion, addition, or mutation may be used to modify the sequence references herein.

[0120] In another aspect, as detailed herein, the helper plasmid is designed to include the following elements: the CAG promoter (SEQ ID NO: 19); the HIV component gag (SEQ ID NO: 20); the HIV component pol (SEQ ID NO: 21); the HIV Int (SEQ ID NO: 22); the HIV RRE (SEQ ID NO: 23); and the HIV Rev (SEQ ID NO: 24). In another aspect, the helper plasmid may be modified to include a first helper plasmid for expressing the gag and pol genes and a second separate plasmid for expressing the rev gene. In another aspect, sequence variations by substitution, deletion, addition, or mutation may be used to modify the sequence references herein.

[0121] In another aspect, as detailed herein, the envelope plasmid is designed to include the following elements from left to right: the RNA polymerase II promoter (CMV) (SEQ ID NO: 25) and the vesicular stomatitis virus G glycoprotein (VSV-G) (SEQ ID NO: 26). In another aspect, sequence variations by substitution, deletion, addition, or mutation may be used to modify the sequence references herein.

[0122] In another aspect, the plasmids used for lentiviral packaging can be modified with similar elements, and the intron sequences can potentially be removed without loss of vector function. For example, the following elements can replace similar elements in the plasmids that make up the packaging system: elongation factor-1 (EF-1), phosphoglycerate kinase (PGK), and ubiquitin C (UbC) promoters can replace the CMV or CAG promoter. SV40 polyA and bGH polyA can replace the rabbit beta-globin polyA. The HIV sequences in the helper plasmid can be constructed from different HIV strains or clades. The VSV-G glycoprotein can be replaced with a membrane glycoprotein derived from feline endogenous virus (RD114), gibbon ape leukemia virus (GALV), rabies (FUG), lymphocytic choriomeningitis virus (LCMV), influenza A fowl pest virus (FPV), Ross River alphavirus (RRV), murine leukemia virus 10A1 (MLV), or Ebola virus (EboV).

[0123] Notably, lentiviral packaging systems can be commercially obtained (e.g., the Lenti-vpak packaging kit from OriGene Technologies, Inc., Rockville, MD) and can also be designed as described herein. Furthermore, it is within the skill of the art to substitute or modify the properties of the lentiviral packaging system to improve any number of related factors, including the production efficiency of lentiviral particles. Dosage and Dosage Form

[0124] The disclosed vectors enable short-term, medium-term, or long-term expression of the gene or sequence of interest, as well as episomal maintenance of the disclosed vectors. Accordingly, the dosing regimen can vary based on the condition being treated and the method of administration.

[0125] In one embodiment, the transduction vector can be administered to the subject in need at varying dosages. Specifically, the subject can receive about ≥10 6can be administered at an infectious dose (on average, one dose is required to transduce one target cell). More specifically, the subject is about ≧10 7 about ≧10 8 about ≧10 9 or about ≧10 10 of the infectious dose, or can be administered at any number of doses intermediate these values. The upper limit of transduction vector dosing is determined for each disease indicator and depends on the toxicity / safety profile for each individual product or product lot.

[0126] Furthermore, the vectors of the present disclosure can be administered periodically, e.g., once or twice a day, or any other appropriate period. For example, the vector can be administered to a subject in need thereof once a week, once every two weeks, once every three weeks, once a month, every two months, every three months, every six months, every nine months, once a year, every 18 months, every two years, every 30 months or every three years.

[0127] In one embodiment, the disclosed vectors are administered as a pharmaceutical composition. In some embodiments, pharmaceutical compositions containing the disclosed vectors can be formulated in a wide variety of dosage forms for clinical applications, including, but not limited to, nasal, pulmonary, oral, topical or parenteral dosage forms. Each of the dosage forms can include various solubilizing agents, disintegrants, surfactants, fillers, thickeners, binders, diluents, e.g., wetting agents or other pharmaceutically acceptable excipients. Pharmaceutical compositions containing the vectors can also be formulated for injection, insufflation, infusion or intradermal exposure. For example, injectable formulations can contain the disclosed vectors in aqueous or non-aqueous solutions at an appropriate pH and osmotic pressure.

[0128] The disclosed vectors can be administered to a subject via direct injection into the tumor site or at the site of infection. In some embodiments, the vectors can be administered systemically. In some embodiments, the vectors can be administered to the tissue directly surrounding the tumor or site of infection via guided cannula insertion.

[0129] The disclosed vector compositions can be administered using any pharmaceutically acceptable method, for example, intranasally, buccally, sublingually, orally, rectally, ophthalmically, parenterally (intravenously, intradermally, intramuscularly, subcutaneously, intraperitoneally), pulmonary, intravaginally, topically, can be topically administered, can be topically administered after laceration, via an aerosol, in a semi-solid medium such as agarose or gelatin, or can be mucosally administered via a buccal or nasal spray formulation.

[0130] Furthermore, the disclosed vector compositions can be formulated into any pharmaceutically acceptable dosage form, for example, solid dosage forms, tablets, pills, lozenges, capsules, liquid dispersions, gels, aerosols, pulmonary aerosols, nasal aerosols, ointments, creams, semi-solid dosage forms, solutions, emulsions and suspensions. Furthermore, the composition can be a controlled release formulation, a sustained release formulation, an immediate release formulation, or any combination thereof. Furthermore, the composition can be a transdermal delivery system.

[0131] In some embodiments, the pharmaceutical composition containing the vector can be formulated in a solid dosage form for oral administration, and the solid dosage form can be a powder, granule, capsule, tablet or pill. In some embodiments, the solid dosage form can contain one or more excipients, for example, calcium carbonate, starch, sucrose, lactose, microcrystalline cellulose or gelatin. Furthermore, the solid dosage form can contain a lubricant such as talc or magnesium stearate in addition to the excipient. In some embodiments, the oral dosage form can be an immediate release form or a modified release form. Modified release dosage forms include controlled release or sustained release, enteric release, etc. Excipients used in modified release dosage forms are generally known to those skilled in the art.

[0132] In a further embodiment, the pharmaceutical composition containing the vector can be formulated as a sublingual or buccal dosage form. Such dosage forms include sublingual tablets or solution compositions administered under the tongue, and buccal tablets placed between the cheek and the gum.

[0133] In some embodiments, a pharmaceutical composition comprising a vector can be formulated as a nasal dosage form. Such dosage forms of the present invention include solutions, suspensions, and gel compositions for nasal delivery.

[0134] In some embodiments, a pharmaceutical composition comprising a vector can be formulated as a liquid dosage form for oral administration, such as a suspension, emulsion, or syrup. In some embodiments, the liquid dosage form can include various excipients, such as humectants, sweeteners, flavorants, or preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. In certain embodiments, the composition comprising the vector can be formulated to be suitable for administration to pediatric patients.

[0135] In some embodiments, the pharmaceutical composition can be formulated as a dosage form for parenteral administration, such as a sterile aqueous solution, suspension, emulsion, non-aqueous solution, or suppository. In some embodiments, the solution or suspension can include vegetable oils such as propylene glycol, polyethylene glycol, olive oil, or injectable esters such as ethyl oleate.

[0136] The dosage of the pharmaceutical composition can vary depending on the patient's weight, age, gender, administration time and mode, excretion rate, and disease severity.

[0137] In some embodiments, the treatment of cancer is achieved by guided direct injection of the disclosed vector construct into the tumor using a needle or intravascular cannula insertion. In some embodiments, the disclosed vector is administered into the cerebrospinal fluid, blood, or lymph circulation by intravenous or arterial cannula insertion or injection, intradermal delivery, intramuscular delivery, or injection into an excretory organ near the site of the disease.

[0138] The following examples are provided to illustrate the present invention. However, it should be understood that the present invention is not limited to the specific conditions or details described in these examples. All publications mentioned herein are specifically incorporated by reference.

Examples

[0139] (Example 1) Development of a lentiviral vector system A lentiviral vector system as summarized in FIG. 4 was developed (circular form). Lentiviral particles were produced in 293T / 17 HEK cells (purchased from American Type Culture Collection, Manassas, VA) after transfection with a therapeutic vector, an envelope plasmid, and a helper plasmid. Transfection of 293T / 17 HEK cells that produced functional viral particles used the reagent poly(ethyleneimine) (PEI) to increase the efficiency of plasmid DNA uptake. The plasmid and DNA were first added separately in serum-free culture medium at a ratio of 3:1 (mass ratio of PEI to DNA). After 2 - 3 days, the cell culture medium was collected, and the lentiviral particles were purified by high-speed centrifugation and / or filtration followed by anion-exchange chromatography. The concentration of lentiviral particles can be expressed in terms of transduction units / ml (TU / ml). The determination of TU was achieved by measuring the HIV p24 level in the culture fluid (the p24 protein is incorporated into the lentiviral particles), measuring the number of viral DNA copies per cell by quantitative PCR, or by infecting cells and using light (when the vector encodes a luciferase or fluorescent protein marker).

[0140] As described above, a 3-vector system (i.e., a 2-vector lentiviral packaging system) was designed for the production of lentiviral particles. A schematic diagram of the 3-vector system is shown in FIG. 2. Briefly, referring to FIG. 2, the top vector is a helper plasmid containing Rev in this case. The vector appearing in the center of FIG. 2 is the envelope plasmid. The bottom vector is the therapeutic vector described herein.

[0141] Referring more specifically to FIG. 2, the helper + Rev plasmid contains the CAG enhancer (SEQ ID NO: 27); the CAG promoter (SEQ ID NO: 19); the chicken beta-actin intron (SEQ ID NO: 28); HIV gag (SEQ ID NO: 20); HIV Pol (SEQ ID NO: 21); HIV Int (SEQ ID NO: 22); HIV RRE (SEQ ID NO: 23); HIV Rev (SEQ ID NO: 24); and rabbit beta-globin polyA (SEQ ID NO: 29).

[0142] The envelope plasmid contains the CMV promoter (SEQ ID NO: 25); the beta-globin intron (SEQ ID NO: 30); VSV-G (SEQ ID NO: 28); and rabbit beta-globin polyA (SEQ ID NO: 31).

[0143] Synthesis of a 2-vector lentiviral packaging system containing the helper (+Rev) plasmid and the envelope plasmid. Materials and Methods:

[0144] Construction of the helper plasmid: The helper plasmid was constructed by first PCR amplifying DNA fragments from the pNL4-3 HIV plasmid (NIH Aids Reagent Program) containing the Gag, Pol and integrase genes. Primers were designed to amplify fragments with EcoRI and NotI restriction sites that could be used for insertion at the same site in the pCDNA3 plasmid (Invitrogen). The forward primer was

Chemical formula

[0145]

Chemical formula

Chemical formula

[0146] Next, a DNA fragment containing Rev, RRE, and the rabbit beta-globin polyA sequence, which has XbaI and XmaI flanking restriction sites, was synthesized by MWG Operon. The DNA fragment was then inserted into the plasmid at the XbaI and XmaI restriction sites. The DNA sequence was as follows:

[0147] [Chem.] [Chem.]

[0148] Finally, the CMV promoter of pCDNA3.1 was replaced with the CAG enhancer / promoter + chicken beta-actin intron sequence. A DNA fragment containing the CAG enhancer / promoter / intron sequence, which has MluI and EcoRI flanking restriction sites, was synthesized by MWG Operon. The DNA fragment was then inserted into the plasmid at the MluI and EcoRI restriction sites. The DNA sequence was as follows:

[0149] [Chem.] [Chem.] Construction of the VSV-G envelope plasmid:

[0150] The vesicular stomatitis Indiana virus glycoprotein (VSV-G) sequence having adjacent EcoRI restriction sites was synthesized by MWG Operon. The DNA fragment was then inserted into the pCDNA3.1 plasmid (Invitrogen) at the EcoRI restriction site, and the correct orientation was determined by sequencing using CMV-specific primers. The DNA sequence was as follows:

[0151]

Chem.

Chem.

[0152] A 4-vector system (i.e., a 3-vector lentiviral packaging system) was also designed and produced using the methods and materials described herein. A schematic diagram of the 4-vector system is shown in Figure 3. Briefly, referring to Figure 3, the top vector is a helper plasmid that does not contain Rev in this case. The second vector from the top is another Rev plasmid. The second vector from the bottom is an envelope plasmid. The bottom vector is the previously described therapeutic vector.

[0153] Referring in part to Figure 2, the helper plasmid contains the CAG enhancer (SEQ ID NO: 27); the CAG promoter (SEQ ID NO: 19); the chicken beta-actin intron (SEQ ID NO: 28); HIV gag (SEQ ID NO: 20); HIV Pol (SEQ ID NO: 21); HIV Int (SEQ ID NO: 22); HIV RRE (SEQ ID NO: 23); and the rabbit beta-globin polyA (SEQ ID NO: 29).

[0154] The Rev plasmid contains the RSV promoter (SEQ ID NO: 38); HIV Rev (SEQ ID NO: 39); and the rabbit beta-globin polyA (SEQ ID NO: 29).

[0155] The envelope plasmid contains the CMV promoter (SEQ ID NO: 25); the beta globin intron (SEQ ID NO: 30); VSV-G (SEQ ID NO: 28); and the rabbit beta globin polyA (SEQ ID NO: 29).

[0156] Synthesis of a 3-vector lentiviral packaging system comprising a helper plasmid, a Rev plasmid and an envelope plasmid Materials and methods: Construction of a helper plasmid without Rev:

[0157] A helper plasmid without Rev was constructed by inserting a DNA fragment containing the RRE and the rabbit beta globin polyA sequence. This sequence with adjacent XbaI and XmaI restriction sites was synthesized by MWG Operon. Subsequently, the RRE / rabbit polyA beta globin sequence was inserted into the helper plasmid at the XbaI and XmaI restriction sites. The DNA sequence is as follows:

[0158]

Chem.

Chem.

[0159] The RSV promoter and the HIV Rev sequence with adjacent MfeI and XbaI restriction sites were synthesized as a single DNA fragment by MWG Operon. Subsequently, the DNA fragment was inserted into the pCDNA3.1 plasmid (Invitrogen) at the MfeI and XbaI restriction sites, with the CMV promoter replaced by the RSV promoter. The DNA sequence was as follows:

[0160]

Chem.

[0161] Plasmids for 2-vector and 3-vector packaging systems can be modified with similar elements, and intron sequences could potentially be removed without loss of vector function. For example, the following elements could replace similar elements in 2-vector and 3-vector packaging systems:

[0162] Promoters: The elongation factor-1 (EF-1) (SEQ ID NO: 41), phosphoglycerate kinase (PGK) (SEQ ID NO: 42), and ubiquitin C (UbC) (SEQ ID NO: 43) can replace the CMV (SEQ ID NO: 25) or CAG promoter (SEQ ID NO: 19). These sequences can also be further varied by addition, substitution, deletion, or mutation.

[0163] PolyA sequences: The SV40 polyA (SEQ ID NO: 44) and bGH polyA (SEQ ID NO: 45) can replace the rabbit beta-globin polyA (SEQ ID NO: 29). These sequences can also be further varied by addition, substitution, deletion, or mutation.

[0164] HIV Gag, Pol, and integrase sequences: The HIV sequences in the helper plasmid can be constructed from different HIV strains or clades. For example, HIV from the Bal strain Gag (SEQ ID NO: 20); HIV Pol (SEQ ID NO: 21); and HIV Int (SEQ ID NO: 22) can be exchanged with the gag, pol, and int sequences contained in the helper / helper+Rev plasmids outlined herein. These sequences can also be further varied by addition, substitution, deletion, or mutation.

[0165] Envelope: The VSV-G glycoprotein can be replaced with membrane glycoproteins derived from feline endogenous virus (RD114) (SEQ ID NO: 46), simian leukemia virus (GALV) (SEQ ID NO: 47), rabies (FUG) (SEQ ID NO: 48), lymphocytic choriomeningitis virus (LCMV) (SEQ ID NO: 49), influenza A fowl pest virus (FPV) (SEQ ID NO: 50), Ross River alphavirus (RRV) (SEQ ID NO: 51), murine leukemia virus 10A1 (MLV) (SEQ ID NO: 52) or Ebola virus (EboV) (SEQ ID NO: 53). The sequences of these envelopes are identified in the sequence section of this specification. Furthermore, these sequences can also be further varied by addition, substitution, deletion or mutation.

[0166] In summary, the 3-vector system versus the 4-vector system can be compared and contrasted in part as follows. The 3-vector lentiviral vector system includes: 1. Helper plasmid: HIV Gag, Pol, Integrase and Rev / Tat; 2. Envelope plasmid: VSV-G / FUG envelope; and 3. Therapeutic vector: RSV 5’LTR, Psi packaging signal, Gag fragment, RRE, Env fragment, cPPT, WPRE and 3’δLTR. The 4-vector lentiviral vector system includes: 1. Helper plasmid: HIV Gag, Pol and Integrase; 2. Rev plasmid: Rev; 3. Envelope plasmid: VSV-G / FUG envelope; and 4. Therapeutic vector: RSV 5’LTR, Psi packaging signal, Gag fragment, RRE, Env fragment, cPPT, WPRE and 3’ delta LTR. The sequences corresponding to the above elements are identified in the sequence listing section of this specification. (Example 2) Development of a Lentiviral Vector Expressing FDPS

[0167] The purpose of this example was to develop an FDPS lentiviral vector.

[0168] Design of inhibitory RNA: Using the sequence of Homo sapiens farnesyl diphosphate synthase (FDPS) (NM_002004.3) mRNA, we searched for potential siRNA or shRNA candidates that knockdown FDPS levels in human cells. Potential RNA interference sequences were selected from siRNA or shRNA design programs such as the GPP Web Portal (http: / / portals.broadinstitute.org / gpp / public / ) or Thermo Scientific's BLOCK-iT RNAi Designer (https: / / rnaidesigner.thermofisher.com / rnaiexpress / ) from the selected candidates. Each selected shRNA sequence was inserted into a lentiviral vector immediately 3' to an RNA polymerase III promoter, such as H1 (SEQ ID NO: 16), U6 (SEQ ID NO: 54) or 7SK (SEQ ID NO: 55), to regulate shRNA expression. Using these lentiviral shRNA constructs, cells were transduced and changes in specific mRNA levels were measured. The most potent shRNAs for reducing mRNA levels were individually embedded within a microRNA backbone to enable expression by either the EF-1 alpha or CMV RNA polymerase II promoter. The microRNA backbone was selected from mirbase.org. The RNA sequences were also synthesized as synthetic siRNA oligonucleotides and introduced directly into cells without using a lentiviral vector.

[0169] Vector construction: For the FDPS shRNA, oligonucleotide sequences containing BamHI and EcoRI restriction sites were synthesized by Eurofins MWG Operon. Overlapping sense and antisense oligonucleotide sequences were mixed and annealed during cooling from 70 °C to room temperature. The lentiviral vector was digested with restriction enzymes BamHI and EcoRI at 37 °C for 1 hour. The digested lentiviral vector was purified by agarose gel electrophoresis and extracted from the gel using a DNA gel extraction kit from Thermo Scientific. The DNA concentration was determined, and the vector to oligo (3:1 ratio) was mixed, annealed, and ligated. The ligation reaction was carried out at room temperature for 30 minutes using T4 DNA ligase. 2.5 microliters of the ligation mix was added to 25 microliters of STBL3 competent bacterial cells. Transformation was achieved after heat shock at 42 °C. The bacterial cells were spread on an agar plate containing ampicillin, and drug-resistant colonies (indicating the presence of the ampicillin-resistant plasmid) were recovered and expanded in LB broth. To check for the insertion of the oligo sequence, plasmid DNA was extracted from the recovered bacterial culture using a Thermo Scientific DNA mini prep kit. The insertion of the shRNA sequence in the lentiviral vector was verified by DNA sequencing using primers specific to the promoter used to regulate shRNA expression. The following target sequences were used to determine exemplary shRNA sequences that knockdown FDPS: [Chemical formula]

[0170] Next, the shRNA sequences were assembled into synthetic microRNAs (miRs) under the control of the EF-1 alpha promoter. Briefly, miR hairpin sequences, such as miR30, miR21, or miR185 detailed below, were obtained from mirbase.org. Synthetic miR sequences were constructed using 19 - 22 mer shRNA target sequences. The miR sequences were aligned as antisense - target sequence - hairpin loop sequence (specific to each microRNA) - sense target sequence. The following miR sequences were developed:

Chemical formula

Chemical formula

[0171] This example shows that knockdown of FDPS in THP1 monocytic leukemia cells by the FDPS shRNA #4 expressing lentivirus (LV) stimulates TNF - α expression in gamma delta T cells, as shown in Figure 5.

[0172] THP1 cells (1×10 5 cells) were transduced with LV - control or LV - FDPS shRNA #4 for 3 days. Two days after transduction, the cells were either treated or not treated with 1 μM zoledronic acid. After 24 hours, the transduced THP - 1 cells were at 5×10 5PBMC cells were co-cultured with IL-2 in round-bottom 96-well plates for 4 hours. PBMC cells were pre-stimulated with zoledronic acid and IL-2 for 11 days to expand and proliferate Vγ9Vδ2 T cells. After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti-TCR-Vδ2 and anti-TNF-α antibodies, the cells were analyzed via flow cytometry. Live cells were gated, and Vδ2+ and TNF-α+ cells were selected on dot blots. Activated cytotoxic Vγ9Vδ2 T cells appeared in the upper right quadrant of the flow cytogram. In the absence of zoledronic acid, LV-control stimulated 3.1% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #4 stimulated 5%. In the presence of zoledronic acid treatment, LV-control stimulated 7.2% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #4 stimulated 56.2%. (Example 4) Knockdown of FDPS in THP1 leukemia cells over 14 days by shRNA #4

[0173] This example shows that knockdown of FDPS over 14 days in THP1 leukemia cells by FDPS shRNA #4-expressing lentivirus (LV) stimulates TNF-α expression in GD T cells, as shown in Figure 6.

[0174] THP1 cells (1×10 5 cells) were transduced with LV-control or LV-FDPS shRNA #4 for 14 days. Two days after transduction, the cells were either treated or not treated with 1 uM zoledronic acid. After 24 hours, the transduced THP-1 cells were 5×10 5PBMC cells were co-cultured with IL-2 in round-bottom 96-well plates for 4 hours. PBMC cells were pre-stimulated with zoledronic acid and IL-2 for 11 days to expand and proliferate Vγ9Vδ2 T cells. After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti-TCR-Vδ2 and anti-TNF-α antibodies, the cells were analyzed via flow cytometry. Live cells were gated, and Vδ2+ and TNF-α+ cells were selected on dot blots. Activated cytotoxic Vγ9Vδ2 T cells appeared in the upper right quadrant of the flow cytogram. In the absence of zoledronic acid, LV-control stimulated 0.9% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #4 (SEQ ID NO: 4) stimulated 15.9%. In the presence of zoledronic acid treatment, LV-control stimulated 4.7% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #4 (SEQ ID NO: 4) stimulated 76.2%. (Example 5) Knockdown of FDPS in PC3 prostate cancer cells over 3 days by shRNA #1

[0175] This example shows that knockdown of FDPS over 3 days in PC3 prostate cancer cells by FDPS shRNA #1-expressing lentivirus (LV) stimulates TNF-α expression in γδ T cells, as shown in Figure 7.

[0176] PC3 cells were transduced with LV-control or LV-FDPS shRNA #1 (SEQ ID NO: 1) for 3 days. Two days after transduction, the cells were either treated or not treated with 1 μM zoledronic acid. After 24 hours, the transduced PC3 cells were at 5×10 5PBMC cells were co-cultured with IL-2 in round-bottom 96-well plates for 4 hours. PBMC cells were pre-stimulated with zoledronic acid and IL-2 for 11 days to expand and proliferate Vγ9Vδ2 T cells. After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti-TCR-Vδ2 and anti-TNF-α antibodies, the cells were analyzed via flow cytometry. Gating on live cells, Vδ2+ and TNF-α+ cells were selected on dot blots. Activated cytotoxic Vγ9Vδ2 T cells appeared in the upper right quadrant of the flow cytogram. In the absence of zoledronic acid, LV-control stimulated 0.2% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #1 stimulated 0.5%. In the presence of zoledronic acid treatment, LV-control stimulated 1.7% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #1 (SEQ ID NO: 1) stimulated 32.2%. (Example 6) Knockdown of FDPS over 3 days in PC3 prostate cancer cells by shRNA #4

[0177] This example shows that knockdown of FDPS over 3 days in PC3 prostate cancer cells by FDPS shRNA #4-expressing lentivirus (LV) stimulates TNF-α expression in γδ T cells, as shown in Figure 8.

[0178] PC3 cells were transduced with LV-control or LV-FDPS shRNA #4 (SEQ ID NO: 4) for 3 days. Two days after transduction, the cells were either treated or not treated with 1 μM zoledronic acid. After 24 hours, the transduced PC3 cells were at 5×10 5PBMC cells were co-cultured with IL-2 in round-bottom 96-well plates for 4 hours. PBMC cells were pre-stimulated with zoledronic acid and IL-2 for 11 days to expand and proliferate Vγ9Vδ2 T cells. After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti-TCR-Vδ2 and anti-TNF-α antibodies, the cells were analyzed via flow cytometry. Live cells were gated, and Vδ2+ and TNF-α+ cells were selected on a dot blot. Activated cytotoxic Vγ9Vδ2 T cells appeared in the upper right quadrant of the flow cytogram. In the absence of zoledronic acid, LV-control stimulated 0.5% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #4 (SEQ ID NO: 4) stimulated 1.9%. In the presence of zoledronic acid treatment, LV-control stimulated 2.1% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #4 stimulated 28.7%. (Example 7) Knockdown of FDPS over 3 days in HepG2 liver cancer cells by shRNA #1 and #4

[0179] This example shows that knockdown of FDPS over 3 days in HepG2 liver cancer cells by FDPS shRNA #1 (SEQ ID NO: 1)-expressing lentivirus (LV) and shRNA #4 (SEQ ID NO: 4)-expressing lentivirus (LV) stimulates TNF-α expression in γδ T cells, as shown in Figure 9.

[0180] HepG2 cells were transduced with LV-control, LV-FDPS shRNA #1 (SEQ ID NO: 1) or LV-FDPS shRNA #4 (SEQ ID NO: 4) for 3 days. Two days after transduction, the cells were treated or not treated with 1 μM zoledronic acid. After 24 hours, the transduced HepG2 cells were at 5×10 5PBMC cells were co-cultured for 4 hours in round-bottom 96-well plates together with IL-2. PBMC cells were pre-stimulated with zoledronic acid and IL-2 for 11 days to expand and proliferate Vγ9Vδ2 T cells. After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti-TCR-Vδ2 and anti-TNF-α antibodies, the cells were analyzed via flow cytometry. Live cells were gated, and Vδ2+ and TNF-α+ cells were selected on dot blots. Activated cytotoxic Vγ9Vδ2 T cells appeared in the upper right quadrant of the flow cytogram. In the absence of zoledronic acid, LV-control stimulated 0.4% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #1 (SEQ ID NO: 1) and #4 (SEQ ID NO: 4) stimulated 0.7% and 0.9%, respectively. In the presence of zoledronic acid treatment, LV-control stimulated 6.9% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-FDPS shRNA #1 and #4 stimulated 7.6% and 21.1%, respectively. (Example 8) Knockdown of FDPS over 3 days in THP1 leukemia by microRNA-30

[0181] This example shows that knockdown of FDPS over 3 days in THP1 leukemia cells by FDPS-targeted synthetic microRNA-30-expressing lentivirus (LV) stimulates TNF-α expression in gamma delta T cells, as shown in Figure 10.

[0182] THP1 cells (1×10 5 cells) were transduced with LV-control or LV-miR30 FDPS #1 (SEQ ID NO: 5) for 3 days. Two days after transduction, the cells were treated or not treated with 1 uM zoledronic acid. After 24 hours, the transduced THP-1 cells were 5×10 5PBMC cells were co-cultured with IL-2 in round-bottom 96-well plates for 4 hours. PBMC cells were pre-stimulated with zoledronic acid and IL-2 for 11 days to expand and proliferate Vγ9Vδ2 T cells. After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti-TCR-Vδ2 and anti-TNF-α antibodies, the cells were analyzed via flow cytometry. Live cells were gated, and Vδ2+ and TNF-α+ cells were selected on dot blots. Activated cytotoxic Vγ9Vδ2 T cells appeared in the upper right quadrant of the flow cytogram. In the absence of zoledronic acid, LV-control stimulated 0.2% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-miR30 FDPS stimulated 8.1%. In the presence of zoledronic acid treatment, LV-control stimulated 5.3% of TNF-α-expressing Vγ9Vδ2 T cells, and LV-miR30 FDPS #1 (SEQ ID NO: 5) stimulated 67.3%. (Example 9) E:T ratio obtained from a mixture of THP-1 cells, cultured human gd T cells, and / or Zometa (Zol)

[0183] This example demonstrates, as shown in Figure 11, the results resulting from mixing treated THP-1 monocyte-like tumor cells with cultured human gd T cells.

[0184] The monocyte-like cell line THP-1 was treated with a control lentiviral vector (LV), an LV that suppresses farnesyl diphosphate synthase gene expression (LV-FDPS), zoledronic acid (Zol), or combinations. The legends shown in Figure 11 were as follows: lentiviral control vector (LV-control), a lentiviral vector expressing a microRNA that downregulates FDPS (LV-FPPS), Zometa (Zol), Zometa + lentiviral control (Zol + LV-control), or a lentiviral vector expressing a microRNA that downregulates Zometa + FPPS (Zol + LV-FPPS).

[0185] Human GD T cells derived from anonymous donors were cultured and added to treated THP-1 cells at a ratio of 4:1, 2:1, or 1:1 (GD T:THP-1) for 4 hours. Cell death was measured by a fluorescence assay. When THP-1 cells were treated with a combination of LV-FDPS and Zol, cytotoxic T cell death by GD T cells increased significantly compared to either treatment alone. When LV-FDPS treatment alone was compared to Zol treatment alone, LV-FDPS resulted in greater death but was >3-fold lower than tumor cell death after combination treatment. The combined LV-FDPS+Zol treatment caused nearly 70% tumor cell death at a ratio of 4:1; this was more than 3-fold higher than the second-best treatment (LV-FDPS alone). (Example 10) shRNA-based RNA interference targeting the human farnesyl diphosphate synthase (FDPS) gene delivered by lentivirus

[0186] As shown in Figure 12, HepG2 human hepatocellular carcinoma cells were infected with a lentiviral vector containing the H1 promoter and either a non-targeting or one of four different FDPS shRNA sequences. After 48 hours, RNA was extracted from the cells and converted to cDNA. FDPS The expression of cDNA was determined by quantitative PCR using SYBR Green and FDPS primers. FDPS expression was normalized to actin levels for each sample. H1 promoter and non-targeting sequence [Chemical formula] An FDPS-targeting lentiviral vector containing any one of them was produced in 293 T cells.

[0187] Next, HepG2 human hepatocellular carcinoma cells were infected with a lentiviral vector to determine the efficacy of FDPS knockdown. After 48 hours, RNA was extracted from the cells using the RNeasy RNA isolation kit (Qiagen) and converted to cDNA using the SuperScript VILO cDNA synthesis kit (Thermo Scientific). The expression of FDPS cDNA was determined by quantitative PCR on an Applied Biosystems StepOne qPCR instrument using SYBR Green PCR mix (Thermo Scientific) and FDPS primers (forward primer: 5’-AGGAATTGATGGCGAGAAGG-3’ (SEQ ID NO: 61) and reverse primer: 5’-CCCAAAGAGGTCAAGGTAATCA-3’ (SEQ ID NO: 62)). FDPS expression was normalized to actin levels for each sample using actin primers (forward primer: 5’-AGCGCGGCTACAGCTTCA-3’ (SEQ ID NO: 63) and reverse primer: 5’-GGCGACGTAGCACAGCTTCT-3’ (SEQ ID NO: 64)). The relative FDPS RNA expression in the shCon sample was set to 100%. There were decreases in FDPS expression of 85% (FDPS sequence #1), 89% (FDPS sequence #2), 46% (FDPS sequence #3), and 98% (FDPS sequence #4). (Example 11) miR-based RNA interference targeting the human farnesyl diphosphate synthase (FDPS) gene delivered by lentivirus

[0188] As shown in FIG. 13, HepG2 human hepatocellular carcinoma cells were infected with a lentiviral vector containing either the H1 promoter (SEQ ID NO: 16), the FDPS shRNA #4 (SEQ ID NO: 4) sequence, or the EF-1α promoter (SEQ ID NO: 41) and the miR30-based FDPS sequence. After 48 hours, the cells were lysed and immunoblots were performed using anti-FDPS (Thermo Scientific) and anti-actin (Sigma) antibodies as protein loading controls.

[0189] More specifically, HepG2 human hepatocellular carcinoma cells were infected with a lentiviral vector containing either the H1 promoter (SEQ ID NO: 16) and the FDPS shRNA sequence

Chem.

Chem.

[0190] (Example 12) 3-day knockdown of FDPS in HepG2 liver cancer cells by adeno-associated virus (AAV) expressing FDPS shRNA #4

[0191] ​This example shows that knockdown of FDPS over 3 days in HepG2 liver cancer cells by adeno-associated virus (AAV) expressing FDPS shRNA #4 (SEQ ID NO: 4) stimulates TNF-α expression in γδ T cells (Figure 14, panel B).

[0192] HepG2 cells were transduced with control or AAV-FDPS shRNA #4 (SEQ ID NO: 8) for 3 days. Two days after transduction, the cells were either treated or not treated with 1 μM zoledronic acid. After 24 hours, the transduced HepG2 cells were co-cultured for 4 hours in round-bottom 96-well plates with 5 × 10 5 PBMC cells and IL-2. PBMC cells were pre-stimulated with zoledronic acid and IL-2 for 11 days to expand and proliferate Vγ9Vδ2 T cells. After staining for Vγ9Vδ2 and TNF-α using fluorophore-conjugated anti-TCR-Vδ2 and anti-TNF-α antibodies, the cells were analyzed via flow cytometry. Live cells were gated, and Vδ2+ and TNF-α+ cells were selected on dot blots. Activated cytotoxic Vγ9Vδ2 T cells appeared in the upper right quadrant of the flow cytogram (Figure 14, panel B).

[0193] AAV vector construction. The FDPS shRNA sequence #4 (SEQ ID NO: 4) was inserted into the pAAV plasmid (Cell Biolabs). The FDPS oligonucleotide sequence containing BamHI and EcoRI restriction sites was synthesized by Eurofins MWG Operon. The overlapping sense and antisense oligonucleotide sequences were mixed and annealed during cooling from 70 °C to room temperature. pAAV was digested at 37 °C for 1 hour using the restriction enzymes BamHI and EcoRI. The digested pAAV plasmid was purified by agarose gel electrophoresis and extracted from the gel using the DNA gel extraction kit from Thermo Scientific. The DNA concentration was determined, and the vector to oligo (3:1 ratio) was mixed, annealed, and ligated. The ligation reaction was carried out at room temperature for 30 minutes using T4 DNA ligase. 2.5 microliters of the ligation mix was added to 25 microliters of STBL3 competent bacterial cells. Transformation was achieved after heat shock at 42 °C. The bacterial cells were spread on an agar plate containing ampicillin, and drug-resistant colonies (indicating the presence of the ampicillin-resistant plasmid) were recovered and grown in LB broth. To check for the insertion of the oligo sequence, plasmid DNA was extracted from the recovered bacterial culture using the Thermo Scientific DNA mini prep kit. The insertion of the shRNA sequence in the pAAV plasmid was verified by DNA sequencing using primers specific to the promoter used to regulate shRNA expression. An exemplary AAV vector having the H1 promoter (SEQ ID NO: 16), shFDPS sequence (e.g., SEQ ID NO: 4), left terminal inverted repeat (left ITR; SEQ ID NO: 65), and right terminal inverted repeat (right ITR; SEQ ID NO: 66) can be found in FIG. 14, panel A.

[0194] Production of AAV particles. The AAV-FDPS shRNA plasmid was combined with the plasmids pAAV-RC2 (Cell Biolabs) and pHelper (Cell Biolabs). The pAAV-RC2 plasmid contains the Rep and AAV2 capsid genes, and pHelper contains the adenoviral E2A, E4, and VA genes. To produce AAV particles, these plasmids were transfected into 293T cells at a ratio of 1:1:1 (pAAV-shFDPS:pAAV-RC2:pHelper). For transfection of cells in a 150 mm dish (BD Falcon), 10 micrograms of each plasmid were added together in 1 ml of DMEM. In a separate tube, 60 microliters of the transfection reagent PEI (1 microgram / ml) (Polysciences) was added to 1 ml of DMEM. The two tubes were mixed together and incubated for 15 minutes. The transfection mixture was then added to the cells, and the cells were collected 3 days later. The cells were lysed by freeze / thaw lysis in dry ice / isopropanol. Benzonase nuclease (Sigma) was added to the cell lysate at 37 °C for 30 minutes. The cell debris was then pelleted by centrifugation at 12,000 rpm for 15 minutes at 4 °C. The supernatant was collected and then added to the target cells. (Example 13) Reduced RAP1 prenylation in cells transduced with LV-shFDPS and treated with zoledronic acid

[0195] This example shows that a lentivirus-delivered shRNA targeting the human farnesyl diphosphate synthase (FDPS) gene and zoledronic acid act synergistically to inhibit farnesyl diphosphate production.

[0196] FDPS is an enzyme in the isoprenoid synthesis pathway that catalyzes the production of farnesyl diphosphate. Inhibition of FDPS enzyme activity by zoledronic acid or reduced protein expression by shRNA-mediated knockdown results in reduced farnesyl diphosphate levels. Farnesylation of cellular proteins requires farnesyl diphosphate. RAP1A is a protein that is modified by farnesylation and can be used as a biomarker for cellular farnesyl diphosphate levels. Antibodies that specifically recognize reduced RAP1A farnesylation were used to measure FDPS activity after transduction with LV-shFDPS alone or in combination with zoledronic acid. HepG2 human hepatocellular carcinoma cells were infected with a lentiviral vector containing the FDPS shRNA sequence #4. For zoledronic acid-treated cells, zoledronic acid (Sigma) was added over the last 24 hours. After 48 hours, cells were lysed in NP-40 lysis buffer and proteins were quantified using Bio-Rad protein assay reagent. 50 microgram protein samples were electrophoresed on a 4-12% Bis-Tris gel (Thermo Scientific) and transferred to a PVDF membrane (EMD Millipore). Immunoblots were performed using anti-FDPS (Thermo Scientific), anti-RAP1A (Santa Cruz) and anti-actin (Sigma) antibodies as protein loading controls. Antibodies were conjugated to HRP-conjugated secondary antibodies and detected using an Immobilon Western ECL reagent (EMD Millipore) with a Licor c-DiGit Blot scanner. An increase in RAP1A band intensity correlates with decreased farnesylation. RAP1A defarnesylation occurred only in cells transduced with LV-shFDPS and treated with zoledronic acid. (Example 14) Treatment of Subjects with Cancer LV-FDPS is a gene drug delivered by a lentiviral vector via local administration to sites of advanced unresectable hepatocellular carcinoma.

[0197] The Phase I clinical trial tests the safety and feasibility of delivering LV-FDPS to the site of hepatocellular carcinoma (HCC) using ultrasound-guided cannula insertion into the patient's liver, without concomitant radiotherapy or chemotherapy. This study is rationally expected to result in successful treatment of HCC. This study is a non-blind, 4 × 3 dose escalation (4 dose ranges, up to 3 subjects per dose) to identify the maximum tolerated dose of LV-FDPS in patients 18 years of age or older with stage III / IV unresectable HCC.

[0198] LV-FDPS is a gene therapy designed to reduce the expression of the enzyme farnesyl diphosphate synthase in tumor cells. Experimental studies have shown that tumor cells modified by LV-FDPS induce the anti-tumor activity of human gamma-delta T cells, including the ability to cause tumor death by cytotoxicity.

[0199] Subjects with target lesions having a longest diameter ≥ 1 cm (measured by helical CT) and a maximum diameter ≤ 4.9 cm and meeting the inclusion and exclusion criteria detailed below are enrolled in the following available dosing categories. Up to 3 subjects are enrolled in each dose group. The doses are several transduction units of LV-FDPS described in the product release criteria, delivered via intrahepatic cannula insertion as a single bolus having a volume not exceeding 25 mL. The minimum dose is 1×10 9 transduction units, and the escalation is 10-fold to the next dose of 1×10 10 transduction units, and the next dose is 1×10 11 transduction units, the maximum dose based on reported experience using recombinant adenovirus therapy for HCC of 1×10 12 transduction units (Sangro et al., A phase I clinic trial of thymidine kinase-based gene therapy In advanced hepatocellular carcinoma (Cancer Gene Ther. 17:837-43, 2010), subjects are enrolled, treated, and evaluated over 3 months. All safety evaluations are completed for each group before enrolling and treating subjects at the next higher dose level. Enrollment and dose escalation continue until the maximum tolerated dose is reached or the study is completed.

[0200] Catheter insertion is via the left subclavian artery until the tip of the catheter reaches the appropriate hepatic artery junction. Catheter insertion is guided by ultrasound as described (Lin et al., Clinical effects of intra-arterial infusion chemotherapy with cisplatin, mitomycin C, leucovor and 5-Fluorouracil for unresectable advanced hepatocellular carcinoma, J. Chin. Med. Assoc. 67:602-10, 2004). Primary evaluation items

[0201] Safety: Systemic and local adverse events are graded according to CTCAS and coded according to MedRA. Adverse event data for all subjects in a single dose range are evaluated before dose escalation. The final safety evaluation incorporates data from all dose ranges. Secondary evaluation items

[0202] · Lesion distribution and retention of LV-FDPS after local administration, and subsequent biopsies or autopsies to obtain tissue. · Response rate (ORR) at the target and measurable non-local lesions (if any) by physical analysis, medical imaging, or biopsy during the 3 months after treatment. · Levels of LV-FDPS in the bloodstream at 10 minutes, 30 minutes, 1 hour, and 1 day after local injection. · Changes in liver function markers including ALP, ALT, ASAT, total bilirubin and GGT during the 3 months after treatment. · Disease-free survival period exceeding that of historical control (without LV-FDPS) patients in the ad hoc analysis. Inclusion criteria

[0203] · Aged over 18 years, including both men and women. · Diagnosis confirmed based on currently accepted clinical standards of hepatocellular carcinoma of parenchymal cell origin that is not suitable for resection, transplantation or other potentially curative therapies, either by histology or cytology or at the time of screening. · The attending physician determines that the lesion is suitable for local regional targeted delivery. · The target lesion must show a measurable disease with a one-dimensional longest diameter of ≥1.0 cm by computed tomography; the maximum longest diameter is ≤5.0 cm. · Karnofsky Performance Score, 60 - 80% of ECOG value. · Life expectancy ≥12 weeks. · Hematopoietic function: WBC ≥2,500 / mm 3 ; ANC ≥1000 / mm 3 ; Hemoglobin ≥8 g / dL; Platelet count ≥50,000 / mm 3 ; Coagulation INR ≤1.3. · AST and ALT < 5 times the ULN; ALPS < 5 times the ULN. Bilirubin ≤1.5 times the ULV; Creatinine ≤1.5 times the ULN and eGFR ≥50. · Thyroid function: Total T3 or free T3, total T4 or free T4 and THC ≤ CTCAE grade 2 abnormalities. · Renal, cardiovascular and respiratory functions appropriate according to the opinion of the attending physician. · Immunological function: Circulating V gamma 9V delta 2+ T cells ≥30 / mm 3 ; No immunodeficiency diseases. · HIV negative by serological and viral RNA tests. · Written informed consent. Exclusion criteria

[0204] ·A target lesion adjacent to, surrounding, or infiltrating a blood vessel. ·Primary HCC suitable for resection, transplantation, or other potentially curative therapies. ·Liver surgery or chemoembolization within the past 4 months. ·Liver radiation or total body radiation therapy within the past 4 months. ·Chemotherapy within 4 weeks, or any use of nitrosourea, mitomycin C, or cisplatin. ·Current or within the past 4 weeks receipt of aminobisphosphonate therapy. ·Investigational agent within 4 weeks or <5 drug half-lives. ·Impaired wound healing due to diabetes. ·Significant psychiatric illness, alcohol dependence, or illegal drug use. ·Failure to comply with the study protocol and reporting requirements. ·Aminobisphosphonate treatment within the past 4 months. ·The presence of clinically significant cardiovascular, cerebrovascular (stroke), immunological (excluding hepatitis B or C virus infection, viral hepatitis, or cirrhosis), endocrine, or central nervous system disorders; current encephalopathy; aneurysm bleeding requiring hospitalization or transfusion within the past 4 months. ·History of HIV or acquired immunodeficiency syndrome. ·Current or previous treatment with antiretroviral drug therapy. ·Refusal to incorporate pregnancy, lactation, or use of barrier or chemical contraceptives throughout the study period and follow-up period.

[0205] LV-FDPS is a gene drug delivered by a lentiviral vector via local administration to sites of advanced unresectable hepatocellular carcinoma - adjuvant administration of aminobisphosphonate

[0206] The Phase I clinical trial tests the safety and feasibility of delivering LV-FDPS to the sites of hepatocellular carcinoma (HCC) using ultrasound-guided cannula insertion into the patients' livers, with concomitant aminobisphosphonate chemotherapy. This study is rationally predicted to result in a successful treatment of HCC. This study is a non-blinded 4 × 3 dose escalation (4 dose ranges, up to 3 subjects per dose) to identify the maximum tolerated dose of LV-FDPS in patients 18 years of age or older with stage III / IV unresectable HCC.

[0207] LV-FDPS is a gene therapy designed to reduce the expression of the enzyme farnesyl diphosphate synthase in tumor cells. Experimental studies have shown that tumor cells modified by LV-FDPS induce the anti-tumor activity of human gamma-delta T cells, including the ability to cause tumor death by cytotoxicity. Previous experimental studies have also shown the potential for a positive interaction between LV-FDPS and specific aminobisphosphonate drugs that can be prescribed in primary or metastatic disease. For this study, subjects receive escalating doses of LV-FDPS along with a continuous standard therapeutic dosing with Aredia® (pamidronate), Zometa® (zoledronic acid), or Actonel® (risedronate), according to the advice of the physician and the preference of the subject.

[0208] Have a target lesion with a longest diameter ≥ 1 cm (measured by helical CT) and a maximum diameter ≤ 4.9 cm, register subjects who meet the inclusion criteria and exclusion criteria detailed below, and initiate aminobisphosphonate treatment. Re-evaluate the size of the target lesion 30 days later to ensure that the subject still meets the start criteria for LV-FDPS. Register subjects who do not have an objective clinical response to aminobisphosphonate into the next available LV-FDPS dosing category. Adopt up to three subjects in each dosing group, and all continue aminobisphosphonate over the study duration unless otherwise advised by the attending physician. The LV-FDPS dose is several transduction units of LV-FDPS described in the product release criteria, delivered via intrahepatic cannula insertion in a single bolus with a volume not exceeding 25 mL. The minimum dose is 1×10 9 transduction units, and the increment is 10-fold up to the next dose of 1×10 10 transduction units, and the next dose is 1×10 11 transduction units, and based on the reported experience using recombinant adenovirus therapy for HCC, the maximum dose is 1×10 12 transduction units (Sangro et al., A phase I clinic trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma, 2010, Cancer Gene Ther. 17:837-43). Register and treat the subjects and evaluate them over 3 months. Complete all safety evaluations for each group before registering and treating subjects at the next higher dose level. Registration and dose escalation continue until the maximum tolerated dose is achieved or the study is completed.

[0209] The cannula insertion is via the left subclavian artery until the tip of the catheter reaches the appropriate hepatic artery junction. The cannula insertion is guided by ultrasonography as described (Lin et al., Clinical effects of intra-arterial infusion chemotherapy with cisplatin, mitomycin C, leucovor and 5-Fluorouracil for unresectable advanced hepatocellular carcinoma, 2004, J. Chin. Med. Assoc. 67:602-610). Primary evaluation items

[0210] Safety: Systemic and local adverse events are graded according to CTCAS and coded according to MedRA. Adverse event data for all subjects in a single dose range are evaluated prior to dose escalation. The final safety evaluation incorporates data from all dose ranges. Secondary evaluation items

[0211] · Lesion distribution and retention of LV-FDPS after regional administration, and subsequent biopsies or autopsies to obtain tissue. · Response rate (ORR) at the target and measurable non-local lesions (if present) by physical analysis, medical imaging or biopsy during the 3 months after treatment. · Levels of LV-FDPS in the blood stream at 10 minutes, 30 minutes, 1 hour and 1 day after local injection. · Changes in liver function markers including ALP, ALT, ASAT, total bilirubin and GGT during the 3 months after treatment. · Disease-free survival period exceeding that of historical control (without LV-FDPS) patients in the ad hoc analysis. Inclusion criteria

[0212] · Aged 18 years or older, including both males and females. ·Diagnosis confirmed by histology or cytology, or at the time of screening, based on the currently accepted clinical standard for hepatocellular carcinoma of parenchymal cell origin that is not suitable for resection, transplantation, or other potentially curative therapies. ·The treating physician determines that the lesion is suitable for local regional targeted delivery. ·The target lesion must demonstrate a measurable disease with a one-dimensional longest diameter of ≥ 1.0 cm by computed tomography; the maximum longest diameter is ≤ 5.0 cm. ·Karnofsky Performance Score, 60 - 80% of ECOG value. ·Life expectancy ≥ 12 weeks. ·Hematopoietic function: WBC ≥ 2,500 / mm 3 ; ANC ≥ 1000 / mm 3 ; Hemoglobin ≥ 8 g / dL; Platelet count ≥ 50,000 / mm 3 ; Coagulation INR ≤ 1.3. ·AST and ALT < 5 times ULN; ALPS < 5 times ULN. Bilirubin ≤ 1.5 times ULV; Creatinine ≤ 1.5 times ULN and eGFR ≥ 50. ·Thyroid function: Total T3 or free T3, total T4 or free T4 and THC ≤ CTCAE grade 2 abnormalities. ·Renal, cardiovascular and respiratory functions appropriate as per the opinion of the treating physician. ·Immunological function: Circulating V gamma 9V delta 2+ T cells ≥ 30 / mm 3 ; No immunodeficiency diseases. ·HIV negative by serology and viral RNA testing. ·Written informed consent. Exclusion criteria

[0213] ·Intolerance to aminobisphosphonate adjuvant therapy, or unwillingness to continue aminobisphosphonate adjuvant therapy. ·Objective clinical response after aminobisphosphonate treatment. ·Target lesions adjacent to, surrounding, or infiltrating blood vessels. ·Primary HCC suitable for resection, transplantation, or other potentially curative therapies. ·Liver surgery or chemoembolization within the past 4 months. ·Liver radiation or total body irradiation within the past 4 months. ·Chemotherapy other than aminobisphosphonates within 4 weeks, or any use of nitrosourea, mitomycin C, or cisplatin. ·Investigational agent within 4 weeks or <5 drug half-lives. ·Impaired wound healing due to diabetes. ·Significant mental illness, alcohol dependence, or illegal drug use. ·Failure to comply with the study protocol and reporting requirements. ·The presence of clinically significant cardiovascular, cerebrovascular (stroke), immunological (excluding hepatitis B or C virus infection, viral hepatitis, or cirrhosis), endocrine, or central nervous system disorders; current encephalopathy; aneurysm bleeding requiring hospitalization or transfusion within the past 4 months. ·History of HIV or acquired immunodeficiency syndrome. ·Current or previous treatment with antiretroviral drug therapy. ·Refusal to incorporate pregnancy, lactation, or use of barrier or chemical contraceptives throughout the study period and follow-up period. (Example 15) Treatment of subjects with chronic viral disease(s) of the liver LV-FDPS is a gene drug delivered by a lentiviral vector via local administration to the liver for the treatment of hepatitis B virus, hepatitis C virus, HIV, or other viral infections of the liver.

[0214] The Phase I clinical trial tests the safety and feasibility of delivering LV-FDPS to the virus-infected liver using ultrasound-guided cannula insertion. This study is rationally predicted to result in successful treatment of liver infection. This study is an open-label 4×3 dose escalation (4 dose ranges, up to 3 subjects per dose) to identify the maximum tolerated dose of LV-FDPS in patients 18 years of age or older with chronic viral disease of the liver resistant to chemotherapy.

[0215] LV-FDPS is a gene therapy designed to reduce the expression of the enzyme farnesyl diphosphate synthase in tumor cells. Experimental studies have shown that tumor cells modified by LV-FDPS induce human gamma delta T cells, including the ability to be cytotoxic to virus-infected cells.

[0216] Subjects with confirmed viral infection of the liver, including hepatitis B virus, hepatitis C virus, HIV or other viruses, are enrolled in the following available LV-FDPS dosing categories. Up to three subjects are employed in each dosing group. The LV-FDPS dose is some transducing units of LV-FDPS described in the product release criteria that are delivered via intrahepatic cannula insertion in a single bolus with a volume not exceeding 25 mL. The minimum dose is 1×10 9 transducing units, and the increment is 10-fold up to the next dose of 1×10 10 transducing units, and the next dose is 1×10 11 transducing units, which is the maximum dose of 1×10 12 transducing units based on the reported experience using recombinant adenovirus therapy for HCC (Sangro et al., A phase I clinic trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma, 2010, Cancer Gene Ther. 17:837-43). Subjects are enrolled, treated, and evaluated over 3 months. All safety evaluations are completed for each group before enrolling and treating subjects at the next higher dose level. Enrollment and dose escalation continue until the maximum tolerated dose is achieved or the study is completed.

[0217] The cannula insertion is via the left subclavian artery until the tip of the catheter reaches the appropriate hepatic artery junction. The cannula insertion is guided by ultrasound examination as described (Lin et al., Clinical effects of intra-arterial infusion chemotherapy with cisplatin, mitomycin C, leucovor and 5-Fluorouracil for unresectable advanced hepatocellular carcinoma, 2004, J. Chin. Med. Assoc. 67:602-10). Primary evaluation items

[0218] Safety: Systemic and local adverse events are graded according to CTCAS and coded according to MedRA. Adverse event data for all subjects in a single dose range are evaluated prior to dose escalation. The final safety evaluation incorporates data from all dose ranges. Secondary evaluation items

[0219] · Lesion distribution and retention of LV-FDPS after regional administration, and subsequent biopsies or autopsies to obtain tissue. · Efficacy rate (ORR) measured as sustained virological response (SVR) within the organ or systemically during the 3 months after treatment. · Levels of LV-FDPS in the bloodstream at 10 minutes, 30 minutes, 1 hour, and 1 day after local injection. · Changes in liver function markers including ALP, ALT, ASAT, total bilirubin, and GGT during the 3 months after treatment. · Disease-free survival period exceeding that of historical control (without LV-FDPS) patients in the ad hoc analysis. Inclusion criteria

[0220] · Aged 18 years or older, including both males and females. A diagnosis confirmed by histology or cytology or based on currently accepted clinical standards of chronic viral infection of the liver that is not suitable for resection, transplantation, or other potentially curative therapy at the time of screening. The treating physician determines that the lesion is suitable for locoregional targeted delivery. Karnofsky Performance Score, 60-80% of ECOG value. · Life expectancy ≥ 12 weeks. ·Hematopoietic function: WBC≧2,500 / mm 3 ;ANC≧1000 / mm 3 Hemoglobin ≥ 8 g / dL; platelet count ≥ 50,000 / mm 3 ;coagulation INR≦1.3. AST and ALT <ULNの5倍;ALPS<ULNの5倍。ビリルビン≦ULVの1.5倍;クレアチン≦ULNの1.5倍およびeGFR≧50。 Thyroid function: abnormalities of total or free T3, total or free T4 and THC ≤ CTCAE grade 2. Renal, cardiovascular and respiratory function as appropriate in the opinion of the treating physician. ·Immunological function: Circulating V gamma 9V delta 2+ T cells ≧30 / mm 3 ;No immunodeficiency disease. ·HIV negative by serology and viral RNA testing. · Written informed consent. Exclusion criteria

[0221] · Chronic viral diseases amenable to resection, transplantation or other potentially curative therapy. Liver surgery or chemoembolization within the past 4 months. Liver irradiation or total body radiation therapy within the past 4 months. Investigational drugs with a drug half-life of ≤4 weeks or <5 weeks. Current (within the past 4 weeks) or ongoing receipt of aminobisphosphonate treatment. Impaired wound healing due to diabetes. · Significant mental illness, alcoholism or illicit drug use. · Unwillingness to follow study protocols and reporting requirements. · The presence of clinically significant cardiovascular, cerebrovascular (stroke), immunological (excluding viral infections, viral hepatitis or cirrhosis), endocrine or central nervous system disorders; current encephalopathy; aneurysm bleeding requiring hospitalization or blood transfusion within the past 4 months. · Refusal to incorporate pregnancy, lactation, or the use of barrier or chemical contraceptives throughout the study period and follow-up period.

[0222] LV-FDPS is a gene drug delivered by a lentiviral vector via local administration to the liver for the treatment of hepatitis B virus, hepatitis C virus, HIV or other viral infections of the liver - combined adjunct aminobisphosphonate therapy

[0223] The Phase I clinical trial tests the safety and feasibility of delivering LV-FDPS to the virus-infected liver using ultrasound-guided cannulation. This study is rationally predicted to result in a successful treatment of liver infection. This study is an open-label 4 × 3 dose escalation (4 dose ranges, up to 3 subjects per dose) to identify the maximum tolerated dose of LV-FDPS in patients 18 years of age or older with chronic viral diseases of the liver resistant to chemotherapy.

[0224] LV-FDPS is a gene therapy designed to reduce the expression of the enzyme farnesyl diphosphate synthase in tumor cells. Experimental studies have shown that tumor cells modified by LV-FDPS induce human gamma delta T cells, including the ability to be cytotoxic to virus-infected cells. Previous experimental studies have also shown the possibility of a positive interaction between LV-FDPS and certain aminobisphosphonate drugs that can be prescribed between infectious diseases. For this study, subjects receive escalating doses of LV-FDPS along with continuous standard therapeutic dosing with Aredia® (pamidronate), Zometa® (zoledronic acid) or Actonel® (risedronate) according to the advice of the physician and the preference of the subject.

[0225] Subjects with confirmed viral infection of the liver, including hepatitis B virus, hepatitis C virus, HIV or other viruses, initiate aminobisphosphonate treatment for 45 days prior to re-screening to meet the enrollment criteria for LV-FDPS treatment of infectious diseases. Enroll eligible subjects into the following available LV-FDPS dosing categories. Adopt up to three subjects per dosing group. The LV-FDPS dose is several transduction units of LV-FDPS described in the product release criteria, delivered via intrahepatic cannula insertion in a single bolus with a volume not exceeding 25 mL. The minimum dose is 1×10 9 transduction units, and the increment is 10-fold up to the next dose of 1×10 10 transduction units, and the next dose is 1×10 11 transduction units, the maximum dose based on reported experience using recombinant adenovirus therapy for HCC is 1×10 12 transduction units (Sangro et al., A phase I clinic trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma, 2010, Cancer Gene Ther. Vol. 17: pp. 837-43). Enroll, treat, and evaluate subjects over 3 months. Complete all safety evaluations for each group before enrolling and treating subjects at the next higher dose level. Enrollment and dose escalation continue until the maximum tolerated dose is reached or the study is completed.

[0226] The cannula insertion is via the left subclavian artery until the tip of the catheter reaches the appropriate hepatic artery junction. The cannula insertion is guided by ultrasound as described (Lin et al., Clinical effects of intra-arterial infusion chemotherapy with cisplatin, mitomycin C, leucovor and 5-Fluorouracil for unresectable advanced hepatocellular carcinoma, 2004, J. Chin. Med. Assoc. 67:602-10). Primary evaluation items

[0227] Safety: Systemic and local adverse events are graded according to CTCAS and coded according to MedRA. Adverse event data for all subjects in a single dose range are evaluated prior to dose escalation. The final safety evaluation incorporates data from all dose ranges. Secondary evaluation items

[0228] · Lesion distribution and retention of LV-FDPS after regional administration, and subsequent biopsies or autopsies to obtain tissue. · Efficacy rate (ORR) measured as sustained virological response (SVR) within the organ or systemically during the 3 months after treatment. · Levels of LV-FDPS in the bloodstream at 10 minutes, 30 minutes, 1 hour and 1 day after local injection. · Changes in liver function markers including ALP, ALT, ASAT, total bilirubin and GGT during the 3 months after treatment. · Disease-free survival period exceeding that of historical control (without LV-FDPS) patients in the ad hoc analysis. Inclusion criteria

[0229] · Aged 18 years or older, including both males and females. ·A diagnosis confirmed by histology or cytology, or based on currently accepted clinical standards for chronic viral infection of the liver that is not suitable for resection, transplantation or other potentially curative therapies at the time of screening. ·The attending physician determines that the lesion is suitable for local regional targeted delivery. · Karnofsky Performance Score, 60 - 80% of ECOG value. · Median life expectancy ≥ 12 weeks. · Hematopoietic function: WBC ≥ 2,500 / mm 3 ; ANC ≥ 1000 / mm 3 ; Hemoglobin ≥ 8 g / dL; Platelet count ≥ 50,000 / mm 3 ; Coagulation INR ≤ 1.3. · AST and ALT < 5 times ULN; ALPS < 5 times ULN. Bilirubin ≤ 1.5 times ULV; Creatinine ≤ 1.5 times ULN and eGFR ≥ 50. · Thyroid function: Total T3 or free T3, total T4 or free T4 and THC ≤ CTCAE grade 2 abnormalities. · Renal, cardiovascular and respiratory functions appropriate according to the opinion of the attending physician. · Immunological function: Circulating V gamma 9 V delta 2 + T cells ≥ 30 / mm 3 ; No immunodeficiency diseases. · HIV negative by serology and viral RNA testing. · Written informed consent. Exclusion Criteria

[0230] · Chronic viral diseases suitable for resection, transplantation or other potentially curative therapies. · Liver surgery or chemoembolization within the past 4 months. · Liver radiation or total body radiation therapy within the past 4 months. · Investigational drug within 4 weeks or < 5 drug half-lives. · Impaired wound healing due to diabetes. · Severe mental illness, alcohol dependence or illegal drug use. · Not willing to comply with the study protocol and reporting requirements. · The presence of clinically significant cardiovascular, cerebrovascular (stroke), immunological (excluding viral infections, viral hepatitis or cirrhosis), endocrine or central nervous system disorders; current encephalopathy; venous aneurysm bleeding requiring hospitalization or blood transfusion within the past 4 months. · Refusal to incorporate pregnancy, lactation, or the use of barrier or chemical contraceptives throughout the test and follow-up periods. Sequence The following sequences are referred to herein:

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[0231] Specific ones of the preferred embodiments of the present invention have been described above and specifically listed, but the present invention is not intended to be limited to such embodiments. Various modifications can be made to the present invention without departing from the scope and spirit of the present invention. The present invention provides, for example, the following items. (Item 1) A viral vector comprising at least one encoded genetic element containing a small molecule RNA capable of inhibiting the production of an enzyme involved in the mevalonate pathway. (Item 2) The viral vector according to Item 1, wherein the enzyme is farnesyl diphosphate synthase (FDPS). (Item 3) The viral vector according to Item 1, wherein the at least one encoded genetic element contains a microRNA or shRNA. (Item 4) The shRNA is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Claims

【Claim 1】 The invention described in the drawings.

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