WO2024255851A1 - Cassette d'expression spécifique d'une tumeur et ses utilisations - Google Patents

Cassette d'expression spécifique d'une tumeur et ses utilisations Download PDF

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WO2024255851A1
WO2024255851A1 PCT/CN2024/099233 CN2024099233W WO2024255851A1 WO 2024255851 A1 WO2024255851 A1 WO 2024255851A1 CN 2024099233 W CN2024099233 W CN 2024099233W WO 2024255851 A1 WO2024255851 A1 WO 2024255851A1
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sequence
promoter
cancer
seq
expression cassette
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Qi XIAO
Cuiying SHAO
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Nanjing Curegene Technology Co Ltd
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Nanjing Curegene Technology Co Ltd
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Priority to EP24822799.3A priority Critical patent/EP4728068A1/fr
Priority to CN202480040327.3A priority patent/CN121358860A/zh
Publication of WO2024255851A1 publication Critical patent/WO2024255851A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination

Definitions

  • the present invention relates to a tumor-specific expression vector, particularly to hybrid promoters to drive gene expression efficiently and specifically in cancer cells.
  • the invention further relates to expression cassettes and vectors containing the hybrid promoter and other transcriptional cis-elements.
  • the present invention also relates to the use of the vector in detection, diagnosis or treatment of various cancers.
  • One strategy of gene therapy against cancer involves new treatment modality that introduces genes into cancerous cell or surrounding tissue to cause cell death or slow the growth of the cancer.
  • transcriptomes of various cancer cells have been studied in these decades. Alteration in transcriptional gene pattern is well recognized essential for uncontrolled proliferation and escaping programmed cell death in cancer cells.
  • Dysregulated transcriptional machine usually consists of overexpression, defection of degeneration and hyperactivity of pro-oncogenic transcription factors and mutation of tumor suppressive ones.
  • cancer-specific killing could be achieved by delivering therapeutic genes under the control of cis elements that can be interacted and activated by overexpressed or constitutively activated tumoral transcription factors.
  • topical medication or systemic delivery of anti-cancer gene molecules it is a major problem whether the expression of therapeutic genes is controlled by tumor-specific promoters, which determines the success of cancer gene therapy.
  • Tumor specific promoters are considered promising genetic tools for cancer treatment.
  • Well used tumor specific promoters including alpha-fetoprotein promoter (AFP) , thyroid transcription factor 1 (TTF-1) , glypican-3 protein (GPC3) , human secretory leukocyte protease inhibitor (hSLPI) , ERBB2, Mucin 1 (MUC1) , L-plastin, ⁇ lactalbumin (LALBA) , cyclooxygenase 2 (COX2) , epithelial glycoprotein (EPG2) , A33, uPAR, carcinoembryonic antigen (CEA) , breast cancer 1 (BRCA1) , BRCA2 and survivin.
  • AFP alpha-fetoprotein promoter
  • TTF-1 thyroid transcription factor 1
  • GPC3 glypican-3 protein
  • hSLPI human secretory leukocyte protease inhibitor
  • MUC1 Mucin 1
  • LALBA ⁇ lactalbumin
  • these naive tumor-specific promoters are active in a limited class of cancer cells or the transcriptional activity varies widely in different tumors.
  • the activity of the human survivin promoter ranges from 0.3%to 16%of that of the CMV promoter. How to efficiently express therapeutic gene in cancer cells is the bottleneck for tumor targeting gene therapy.
  • Hybrid promoters may include combinations of known promoters with each other or with certain heterologous regulatory elements to increase the strength and specificity of expression in cancer cells.
  • researchers creating hybrid promoters prefer to maximize the efficiency and specificity of expression in a particular type of cancer cell.
  • the advantage of using promoters and other regulatory elements that are strictly specific for a certain type of tumor is the minimization of side effects that occur due to reduced expression of the therapeutic gene in normal tissues.
  • the disadvantage of such approaches is their non-universal nature.
  • telomere maintenance for which the activity of telomerase consisted of non-coding RNA component hTR and the catalytic subunit hTERT is essential. Consistently, hTERT expression is identified in almost 90%of human primary cancers. However, in normal tissues, hTERT was only detected in spermatogonia, colon cryptic stem or progenitor cells, and some transitional epithelial basal cells, but not in major functional organs, such as liver kidney muscle, or placenta. Similar with most tumor specific promoters, hTERT shows varied expression activity in different types of tumors. In general, advanced cancer cells frequently present higher telomerase activity and expression level. Some cancers in early stages only show weak transcriptional activity of hTERT, which may be resulted from lower level of oncogenic transcriptional factors, such as c-Myc and ETS in these cells.
  • ROS Reactive oxygen species
  • NRF2 expression is often enhanced in various tumor types, including lung, breast, colon, and ovarian cancer, high NRF2 level plays a critical role in tumor cell growth and chemoresistance by elevating reactive oxygen species (ROS) inhibiting enzymes, detoxifying factors, anti-apoptotic proteins and drug transporters.
  • ROS reactive oxygen species
  • an expression cassette comprising:
  • a chimeric promoter comprising an hTERT promoter and a minimal promoter
  • the hTERT promoter is an hTERT core promoter, preferably comprising at least one of the sequences of SEQ ID NOs: 1-7 or a sequence having at least 90%sequence identity thereto.
  • the minimal promoter is a minimal viral promoter or a minimal TATA box promoter.
  • the minimal promoter is a minimal CMV promoter, preferably comprising the sequence of SEQ ID NO: 16 or a sequence having at least 90%sequence identity thereto.
  • the minimal TATA box promoter is an E1F TATA box promoter, preferably comprising the sequence of SEQ ID NO: 17 or a sequence having at least 90%sequence identity thereto.
  • the expression cassette further comprises one or more anti-oxidative response elements.
  • the anti-oxidative response element is from NQO1 or GCLM.
  • the anti-oxidative response element comprises the sequence of SEQ ID NO: 8 or a sequence having at least 90%sequence identity thereto, or the anti-oxidative response element comprises the sequence of SEQ ID NO: 10 or a sequence having at least 90%sequence identity thereto.
  • the expression cassette further comprises one or more enhancers.
  • the enhancer is a SV40 enhancer, preferably comprising the sequence of SEQ ID NO: 12 or a sequence having at least 90%sequence identity thereto.
  • the coding sequence encodes a protein that is capable of inducing death of a cell once the protein is expressed by the cell.
  • the protein is selected from the group consisting of a cytotoxin, a tumor-associated antigen, a tumor specific antigen, a cytokine, amembrane penetrator, a checkpoint protein and an antibody.
  • the protein is neutrophile elastase, diphtheria toxin, IL-2 or granzyme B.
  • the expression cassette when introduced into a non-tumor cell, is not expressed in the non-tumor cell, or is expressed at a level lower than that in a tumor cell.
  • the present disclosure provides a nucleic acid molecule comprising the expression cassette.
  • the present disclosure provides a vector comprising the expression cassette or the nucleic acid molecule.
  • the vector is a viral vector, such as a lentivirus vector, aretrovirus vector, an adeno-associated virus (AAV) vector, or an adenovirus vector.
  • a viral vector such as a lentivirus vector, aretrovirus vector, an adeno-associated virus (AAV) vector, or an adenovirus vector.
  • the present disclosure provides a composition comprising
  • the deliver agent is a biodegradable polymer, preferably, a in vivo jetPEI (Polyethylenimine) , hyperbranched Poly ( ⁇ -amino ester) s (HPAEs) , lipid nanoparticle (LNP) , liposomes or exosome.
  • a biodegradable polymer preferably, a in vivo jetPEI (Polyethylenimine) , hyperbranched Poly ( ⁇ -amino ester) s (HPAEs) , lipid nanoparticle (LNP) , liposomes or exosome.
  • the composition further comprises one or more anti-cancer agents.
  • the present disclosure provides uses of the expression cassette, the nucleic acid molecule or the vector in the manufacture of a medicament for the treatment of a cancer.
  • the present disclosure provides a method of treating cancer, comprising administrating to a subject in need thereof a therapeutically effective amount of the nucleic acid molecule, the vector, or the composition.
  • the cancer is a solid tumor, preferably, a breast cancer, endometrial cancer, ovarian cancer, cervical cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, anal cancer, urothelial cancer, pancreatic cancer, salivary gland cancer, melanoma, skin cancer, head and neck cancer, sarcoma or brain cancer.
  • the cancer is a hematological malignancy, preferably, amultiple myeloma (MM) , lymphoma, chronic lymphocytic leukemia (CLL) , B-cell acute lymphoblastic leukemia (B-ALL) , or acute myelogenous leukemia (AML) , diffuse large B cell lymphoma (DLBCL) , non-Hodgkin lymphoma (NHL) , extranodal NK/T cell lymphoma (ENKL) , Hodgkin Lymphoma (HL) , plasmablastic lymphoma, Burkitt’s lymphoma, marginal zone lymphoma (MZL) , or mantle cell lymphoma (MCL) .
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • B-ALL B-cell acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • FIG. 1 schematically illustrates structures of the tumor specific regulatory elements.
  • upper panel depicts vector name/ID and its composition; lower panel shows brief map of individual regulatory elements.
  • FIG. 2 shows the relative transcription activity of vector comprised with hTERT and relative hybrid promoters in HEK293 cells.
  • Vectors containing hTERT elements showed stronger transcriptional activity in HEK293 cells than the ones with tissue specific elements.
  • FIGs. 3A-3B show the relative transcription activity of vector comprised with hTERT and relative hybrid promoters in MC38 cells (A) or HCT116 cells (B) .
  • Vectors containing ARE-hTERT hybrid promoters displayed comparable transcriptional activity with strong CMV promoters.
  • FIGs. 4A-4C show the relative transcription activity of vector comprised tumor specific promoters in A375 cells (A) , G361 cells (B) or G361 cells (C) .
  • Vectors containing ARE-hTERT hybrid promoters displayed comparable transcriptional activity with strong CMV promoters. In contrast, vectors containing tumor specific promoters used in clinical trials showed weak transcriptional activity, which is much lower than ARE-hTERT ones.
  • FIG. 5 shows the relative transcription activity of vector comprised tumor specific promoters in Pan02 cells.
  • Vectors containing ARE-hTERT hybrid promoters displayed comparable transcriptional activity with strong CMV promoters.
  • vectors containing tumor specific promoters used in clinical trials showed weak transcriptional activity, which is much lower than ARE-hTERT ones.
  • FIGs. 6A-6D show the relative transcription activity of vector comprised tumor specific promoters in Bel 7402 cells (A, C) or HepG2 cells (B, D) .
  • Vectors containing ARE-hTERT hybrid promoters displayed comparable transcriptional activity with strong CMV promoters.
  • vectors containing tumor specific promoters used in clinical trials showed weak transcriptional activity, which is much lower than ARE-hTERT ones.
  • FIGs. 7A-7B show the relative transcription activity of vector comprised tumor specific promoters in HUVECs cells (A) or smooth muscle cells (B) .
  • Vectors containing ARE-hTERT hybrid promoters displayed little or poor transcriptional activity in human normal cells.
  • a first option refers to the applicability of the first element without the second.
  • a second option refers to the applicability of the second element without the first.
  • a third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or. ”
  • any numerical value such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about. ”
  • a numerical value typically includes ⁇ 10%of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1 mg/mL to 10 mg/mL includes 0.9 mg/mL to 11 mg/mL.
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • expression cassette refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to a termination signal. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter sequence or of an inducible promoter sequence that initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue or organ or stage of development.
  • a gene expression cassette for expressing a protein of interest comprises at least a promoter sequence, a coding sequence (CDS) of the protein of interest, and a poly A addition sequence.
  • promoter or “promoter sequence” is a DNA sequence that RNA polymerase recognizes, binds and starts transcription. It generally contains conserved sequence required for RNA polymerase binding and transcription initiation, most of which are located upstream of the transcription start point, and the promoter itself is not transcribed. Examples of promoters include but are not limited to CMV, EF1A, CAG, CBh, and SFFV promoters.
  • chimeric promoter also known as “composite promoter” or “hybrid promoter” , refers to a promoter that comprises at least two parts from different natural promoters or artificially synthetic promoters. The two parts are not naturally adjacent to each other in one nucleic acid molecule.
  • a chimeric promoter comprises at least a first sequence from a tissue selective promoter sequence a tumor-selective promoter sequence and a second sequence from a minimal promoter.
  • a tumor-selective promoter sequence is defined herein as a promoter sequence that is capable of driving transcription of a gene in tumor cell while remaining largely “silent" or expressed at low or relatively low levels in other tissue types.
  • the tumor-selective promoter sequence may be an hTR promoter sequence, hTERT promoter sequence, CEA promoter sequence, a PSA promoter sequence, a probasin promoter sequence, a ARR2PB promoter sequence, or an AFP promoter sequence.
  • Telomerase is a ribonucleoprotein complex that is responsible for the complete replication of chromosomal ends. Many studies have demonstrated that a majority of malignant tumors express telomerase activity, whereas most normal cells do not.
  • telomere activity in humans Three major components associated with telomerase activity in humans have been identified: (a) the RNA component (hTER) ; (b)the telomerase-associated protein (hTEP1) ; and (c) the telomerase catalytic unit or human telomerase reverse transcriptase (hTERT) . Only hTER and hTERT, however, are required for the reconstitution of telomerase activity in vitro and therefore represent the minimal catalytic core of telomerase in humans. The promoter region of hTERT has been cloned and characterized previously.
  • telomerase-specific expression of cytotoxic or proapoptotic genes such as the diphtheria toxin A-chain, FADD, caspases, and Bax by the hTERT promoter has been achieved and reported in various gene transfer systems, such as plasmid and adenovirus.
  • the transcription promoting strength of the hTERT promoter like most other intrinsic mammalian promoters, is usually much weaker than commonly used viral promoters such as the CMV promoter and the SV40 early promoter. Consequently, it uses for gene therapy is hampered by the problem of low transgene expression.
  • the present inventors have developed a novel chimeric promoter composed of an hTERT promoter sequence fused to a minimal promoter sequence.
  • the hTERT promoter sequence comprises any one of the sequences of SEQ ID NOs: 1-7.
  • the hTERT promoter sequence comprises the sequence of SEQ ID NO: 1.
  • Promoter elements, particularly a TATA element or the like, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as minimal promoters.
  • the minimal promoter functions to permit transcription.
  • a minimal promoter thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator. Any minimal promoter sequence known to those of ordinary skill in the art is contemplated for inclusion in the promoter sequences of the present invention.
  • the minimal promoter sequence can be a minimal viral promoter, such as an adenoviral promoter sequence, a baculoviral promoter sequence, a CMV promoter sequence, a parvovirus promoter sequence, a herpesvirus promoter sequence, a poxvirus promoter sequence, an adeno-associated virus promoter sequence, a semiliki forest virus promoter sequence, an SV40 promoter sequence, a vaccinia virus promoter sequence, or a retrovirus promoter sequence.
  • the minimal promoter sequence is from a CMV promoter sequence, and thus referred to herein as “CMVmini promoter” .
  • the CMVmini promoter comprises the sequence of SEQ ID NO: 16.
  • the chimeric promoter comprises an hTERT promoter sequence of any one of the sequences of SEQ ID NOs: 1-7 operatively coupled to a CMVmini promoter of the sequence of SEQ ID NO: 16.
  • the minimal promoter sequence is a minimal TATA box promoter.
  • “minimal TATA box promoter” used herein refers to the TATA region of a promoter, exclusive of other cis-acting elements.
  • the minimal TATA box promoter is a E1F TATA box promoter.
  • the E1F TATA box promoter comprises the sequence of SEQ ID NO: 17. Therefore, in some embodiments, the chimeric promoter comprises an hTERT promoter sequence of any one of the sequences of SEQ ID NOs: 1-7 operatively coupled to an E1F TATA box promoter of the sequence of SEQ ID NO: 17.
  • operably linked refers to a linkage of two or more polynucleotides or two or more nucleic acid sequences in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
  • a “nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising nucleobases. Any of the tumor-selective promoter sequences and minimal promoter sequences set forth above can be incorporated into the nucleic acid sequences of the present invention.
  • the tissue-selective promoter sequence is an hTERT promoter sequence such as, for example, the sequence set forth in SEQ ID NO: 1.
  • the nucleic acids include one or more additional promoter sequences that may or may not be operatively coupled to the tumor-selective promoter sequence.
  • CDS coding sequence
  • the term “coding sequence (CDS) ” is a region or nucleic acid sequence that codes a protein or RNA (e.g., siRNA) of interest.
  • the region or sequence usually begins at the 5’ end by a start codon and ends at the 3’ end with a stop codon.
  • the protein of interest is a cytotoxic (or cytostatic) agent which directly or indirectly stimulates cell death.
  • the cytotoxic agent can be a cytotoxic protein or peptide or an apoptotic triggering protein or peptide.
  • the cytotoxic protein or peptide can be a bacterial cytotoxin such as an alpha-pore forming toxin (e.g., cytolysin A from E.
  • beta-pore-forming toxin e.g., ⁇ -Hemolysin, PVL-panton Valentine leukocidin, aerolysin, clostridial Epsilon-toxin, Clostridium perfringens enterotoxin
  • binary toxins anthrax toxin, edema toxin, C. botulinum C2 toxin, C spirofome toxin, C. perfringens iota toxin, C.
  • cyto-lethal toxins A and B) ) , prion, parasporin, a cholesterol-dependent cytolysins (e.g., pneumolysin) , a small pore-forming toxin (e.g., Gramicidin A) , a cyanotoxin (e.g., microcystins, nodularins) , ahemotoxin, a neurotoxin (e.g., botulinum neurotoxin) , a cytotoxin, cholera toxin, diphtheria toxin, pseudomonas exotoxin A, tetanus toxin, or an immunotoxin (idarubicin, ricin A, CRM9, Pokeweed antiviral protein, DT) .
  • a cholesterol-dependent cytolysins e.g., pneumolysin
  • small pore-forming toxin e.g., Gramicidin A
  • Exemplary apoptotic triggering proteins or peptides include apoptotic protease activating factor-1 (Apaf-1) , cytochrome-c, caspase initiator proteins (CASP2, CASP8, CASP9, CASP10) , apoptosis inducing factor (AIF) , p53, p73, p63, Bcl-2, Bax, granzyme B, poly-ADP ribose polymerase (PARP) , and P 21-activated kinase 2 (PAK2) .
  • the protein of interest can be any tumoristatic, i.e. of greater toxicity to tumor cells relative to non-tumor cells.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • A 3′poly (A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • the poly (A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal sequence or “poly (A) sequence” , for triggering the endonuclease cleavage of an mRNA and the additional of a series of adenosines to the 3′end of the cleaved mRNA.
  • the poly (A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • poly A addition sequence refers to a sequence downstream of a coding sequence for the addition of poly (A) sequence to a RNA transcript.
  • the poly A addition sequence may be a poly (A) sequence itself or a polyadenylation signal sequence described above.
  • the polyadenylation signal sequence is from a growth hormone gene-derived polyadenylation signal sequence, such as a bovine growth hormone gene-derived polyadenylation signal sequence and a human growth hormone gene-derived polyadenylation signal sequence, an SV40 virus-derived polyadenylation signal sequence, and a human or rabbit ⁇ -globin gene-derived polyadenylation signal sequence.
  • the polyadenylation signal sequence comprises the sequence of SEQ ID NO: . The incorporation of the poly A addition sequence into the gene expression cassette may increases transcription efficiency.
  • the term “enhancer” refers to a sequence having a promoting effect on the action of a promoter.
  • An enhancer can promote transcription irrespective of the direction of a sequence.
  • the enhancer to be used in the present invention may include one kind of enhancer, but a plurality of enhancers including two or more enhancers identical to each other may be used or a plurality of enhancers different from each other may be used in combination. Their order is not specifically limited.
  • a CMV enhancer, an SV40 enhancer, an hTERT (telomerase reverse transcriptase) enhancer, or the like may be used.
  • the hTERT enhancer, the SV40 enhancer, and the CMV enhancer may be linked in the stated order.
  • the enhancer can be arranged downstream of the poly A addition sequence.
  • anti-oxidative response element is a cis-acting enhancer sequence located in the regulatory regions of some antioxidant and detoxifying genes.
  • the ARE can be activated by redox-cycling phenols and electrophiles.
  • Nrf2 nuclear factor erythroid 2-related factor 2
  • Some studies have shown that a protein called nuclear factor erythroid 2-related factor 2 (Nrf2) may be the principal transcription factor necessary for ARE activation, even though many other transcription factors also bind to the ARE sequence.
  • Nrf2 nuclear factor erythroid 2-related factor 2
  • the transcription factor Nrf2 Upon toxic insult, glutathione depletion or chemical activation, the transcription factor Nrf2 translocates to the nucleus and dimerizes with small Maf proteins to form a trans-activation complex that binds to the ARE.
  • Nrf2-induced ARE activation coordinates the expression of many genes involved in combating oxidative stress and toxicity in a wide variety of tissues and cell types.
  • the ARE is from NQO1 gene.
  • the ARE comprises the sequence of SEQ ID NO: 8.
  • the ARE comprises the sequence of SEQ ID NO: 10.
  • vector is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) .
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors” ) .
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.
  • the term "subject” refers to a human or any non-human animal (e g, mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) .
  • a human includes pre and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term "subject” is used herein interchangeably with “individual” or "patient. " A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • pharmaceutically acceptable carrier refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid) , solvent or encapsulating material, involved in carrying or transporting a therapeutic compound for administration to the subject.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid) , solvent or encapsulating material, involved in carrying or transporting a therapeutic compound for administration to the subject.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent or encapsulating material involved in carrying or transporting a therapeutic compound for administration to the subject.
  • delivery agent refers to a transporter that carries a therapeutic agent (e.g., a nucleic acid molecule or a vector of the present invention) , whether attached to some element on the surface, attached directed to the surface, embedded onto the surface, or contained partially or completely within the surface, or within the interior of the agent.
  • a therapeutic agent e.g., a nucleic acid molecule or a vector of the present invention
  • Non-limiting examples of delivery agents include a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a microsphere, ananosphere, a microcapsules, a nanocapsule, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a micelle, or a polymeric compound, such as, poly-glycolic acid, poly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, pseudo-poly (amino acids) , polyhydroxybutyrate-related copolymers, polyanhydrides, polymethylmethacrylate, poly (ethylene oxide) .
  • a polymeric compound such as, poly-glycolic acid, poly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethanes, polyacryl
  • the deliver agent is a biodegradable polymer, such as, a in vivo jetPEI (Polyethylenimine) , hyperbranched Poly ( ⁇ -amino ester) s (HPAEs) , lipid nanoparticle (LNP) , liposomes or exosome.
  • a biodegradable polymer such as, a in vivo jetPEI (Polyethylenimine) , hyperbranched Poly ( ⁇ -amino ester) s (HPAEs) , lipid nanoparticle (LNP) , liposomes or exosome.
  • an effective amount refers to an amount of an agent or a combination of agents, sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms.
  • an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation.
  • an effective amount is an amount sufficient to delay development.
  • an effective amount is an amount sufficient to prevent or delay recurrence.
  • An effective amount can be administered in one or more administrations.
  • the effective amount of the drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • treatment is an approach for obtaining beneficial or desired results including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.
  • treatment is a reduction of pathological consequence of the disease.
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • Percent (%) amino acid sequence identity “homology” or “homologous to” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the present invention provides genetic engineering strategies implementing novel hybrid promoters with cancer cell selectivity. These hybrid promoters may be used in gene therapy to treat various types of cancers. These novel hybrid promoters are based on the combination of one or more anti-oxidative response enhancers/elements (ARE) operably linked to engineered human telomerase reverse transcriptase and other tumor specific core promoters. It is shown that the expression of a transgene is significantly increased in various cancer cells when hybrid promoter including ARE elements and enhancer element following poly (A) sequence. Cytotoxin driven by ARE-hTERT promoters efficiently kills cancer cells and presents good safety in primary normal cells.
  • ARE anti-oxidative response enhancers/elements
  • An aspect of invention relates to a nucleic acid molecule comprising one or a plurality of anti-oxidative response enhancers linker to an hTERT or other tumor specific promoter.
  • an hTERT promoter For simultaneously superior transcriptional efficiency and specificity in cancer cells, we comprised an hTERT promoter, anti-oxidative response elements from NQO1 or GCLM and SV40 enhancer to generate an expression cassette or expression construct.
  • An preferred hTERT promoter is a 243bp nucleotide fragment cloned from 5’flank region and partial transcript sequence of hTERT gene (-190...53bp referred to transcript initiating site as 0) .
  • anti-oxidative response cis elements amplify transcriptional activity of hTERT promoter producing sufficient mRNA for anti-cancer efficacy.
  • the nucleotide sequence of the hTERT promoter is show in SEQ ID NO: 1:
  • hTERT core promoter SEQ ID NO: 1
  • hTERT sequence is a homologue of SEQ ID NO: 1.
  • the hTERT sequence is a variant of SEQ ID NO: 1.
  • the hTERT sequence is a fragment of SEQ ID NO: 1. In another embodiment, the hTERT sequence is a homologue of a fragment of SEQ ID NO: 1. In another embodiment, the hTERT sequence is a variant of a fragment of SEQ ID NO: 1.
  • the hTERT sequence is at least 60%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 65%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 70%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 72%homologous to SEQ ID NO: 1.
  • the hTERT sequence is at least 74%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 76%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 78%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 80%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 82%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 84%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 88%homologous to SEQ ID NO: 1.
  • the hTERT sequence is at least 94%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 96%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 98%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 99%homologous to SEQ ID NO: 1. In another embodiment, the hTERT sequence is at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 88%, 94%, 96%, 98%, or 99%homologous to any one of SEQ ID NO: 2-7.
  • ARE1 The nucleotide sequence of an ARE cis element from NQO1 (hereafter referred to as “ARE1” ) is show in SEQ ID NO: 8:
  • the ARE1 sequence is a homologue of SEQ ID NO: 8. In another embodiment, the ARE1 sequence is a variant of SEQ ID NO: 8. In another embodiment, the ARE1 sequence is a fragment of SEQ ID NO: 8. In another embodiment, the ARE1 sequence is a homologue of a fragment of SEQ ID NO: 8. In another embodiment, the ARE1 sequence is a variant of a fragment of SEQ ID NO: 8. Each possibility represents a separate embodiment of the present invention. In another embodiment, the ARE1 sequence is at least 60%homologous to SEQ ID NO: 8.
  • the ARE1 sequence is at least 65%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 70%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 72%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 74%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 76%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 78%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 80%homologous to SEQ ID NO: 8.
  • the ARE1 sequence is at least 82%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 84%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 88%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 94%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 96%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 98%homologous to SEQ ID NO: 8. In another embodiment, the ARE1 sequence is at least 99%homologous to SEQ ID NO: 8.
  • the ARE1 sequence is at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 88%, 94%, 96%, 98%, or 99%homologous to SEQ ID NO: 9.
  • the ARE1 sequence comprises the sequence TCCAGTCA (ARE1-a) .
  • ARE2 The nucleotide sequence of an ARE cis element from GCLM (hereafter referred to as ARE2) is show in SEQ ID NO: 10.
  • the ARE2 sequence is a homologue of SEQ ID NO: 10. In another embodiment, the ARE2 sequence is a variant of SEQ ID NO: 10. In another embodiment, the ARE2 sequence is a fragment of SEQ ID NO: 10. In another embodiment, the ARE2 sequence is a homologue of a fragment of SEQ ID NO: 10. In another embodiment, the ARE2 sequence is a variant of a fragment of SEQ ID NO: 10. Each possibility represents a separate embodiment of the present invention. In another embodiment, the ARE2 sequence is at least 60%homologous to SEQ ID NO: 10.
  • the ARE2 sequence is at least 65%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 70%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 72%homologous to SEQ ID NO: 01. In another embodiment, the ARE2 sequence is at least 74%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 76%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 78%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 80%homologous to SEQ ID NO: 10.
  • the ARE2 sequence is at least 82%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 84%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 88%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 94%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 96%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 98%homologous to SEQ ID NO: 10. In another embodiment, the ARE2 sequence is at least 99%homologous to SEQ ID NO: 10.
  • the ARE2 sequence is at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 88%, 94%, 96%, 98%, or 99%homologous to SEQ ID NO: 11 (ARE2-b) .
  • the ARE1 sequence comprises the sequence TTAGTCA (ARE2-a) or TGAGTAA (ARE2-c) .
  • the nucleotide sequence of a SV40 enhancer element following poly (A) sequence or a polyadenylation signal sequence is show in SEQ ID NO: 12.
  • the SV40 enhancer sequence is a homologue of SEQ ID NO: 12.
  • the SV40 enhancer sequence is a variant of SEQ ID NO: 12.
  • the SV40 enhancer sequence is a fragment of SEQ ID NO: 12.
  • the SV40 enhancer sequence is a homologue of a fragment of SEQ ID NO: 12.
  • the SV40 enhancer sequence is a variant of a fragment of SEQ ID NO: 12.
  • the SV40 enhancer sequence is at least 60%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 65%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 70%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 72%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 74%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 76%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 78%homologous to SEQ ID NO: 12.
  • the SV40 enhancer sequence is at least 80%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 82%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 84%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 88%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 94%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 96%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 98%homologous to SEQ ID NO: 12. In another embodiment, the SV40 enhancer sequence is at least 99%homologous to SEQ ID NO: 12.
  • the nucleotide sequence of a SCGB2A2 enhancer element cloned from 5’ flank regulatory region of SCGB2A2 gene is show in SEQ ID NO: 13:
  • the SCGB2A2 enhancer sequence is a homologue of SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is a variant of SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is a fragment of SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is a homologue of a fragment of SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is a variant of a fragment of SEQ ID NO: 13. Each possibility represents a separate embodiment of the present invention. In another embodiment, the SCGB2A2 enhancer sequence is at least 60%homologous to SEQ ID NO: 13.
  • the SCGB2A2 enhancer sequence is at least 65%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 70%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 72%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 74%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 76%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 78%homologous to SEQ ID NO: 13.
  • the SCGB2A2 enhancer sequence is at least 80%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 82%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 84%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 88%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 94%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 96%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 98%homologous to SEQ ID NO: 13. In another embodiment, the SCGB2A2 enhancer sequence is at least 99%homologous to SEQ ID NO: 13.
  • the nucleotide sequence of a SCGB2A2 core promoter cloned from SCGB2A2 promoter is show in SEQ ID NO: 14:
  • SCGB2A2 promoter sequence is a homologue of SEQ ID NO: 14.
  • the SCGB2A2 promoter sequence is a variant of SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is a fragment of SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is a homologue of a fragment of SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is a variant of a fragment of SEQ ID NO: 14. Each possibility represents a separate embodiment of the present invention. In another embodiment, the SCGB2A2 promoter sequence is at least 60%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 65%homologous to SEQ ID NO: 14.
  • the SCGB2A2 promoter sequence is at least 70%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 72%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 74%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 76%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 78%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 80%homologous to SEQ ID NO: 14.
  • the SCGB2A2 promoter sequence is at least 82%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 84%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 88%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 94%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 96%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 98%homologous to SEQ ID NO: 14. In another embodiment, the SCGB2A2 promoter sequence is at least 99%homologous to SEQ ID NO: 14.
  • nucleotide sequence of a hCCRK promoter cloned from hCCRK promoter is show in SEQ ID NO: 15: ACCCAGGTACCTATGTTCAAAAGTGCCTCAATCCTAGTTAACAAGGGCAGAGACCACGAGAAACAACACTGTGTTTAGTAGCAACTTAACAACCAGCCAG CAGTTCTGTCCACACACACACCACCGGGCATGGTTCCAAAGCTAAAAAGGCACTAATTGCTTTTCTATAAGGAGGTAGAACACAGTCCCTCCGTGTTCTTTAGGCCTGATGGTCTGCATTATCGGATCTGTTACCGTGTTAATTGTTCCTGTCTCACACAGCCGGTTTGGGCTTCTCTGCATATGTCTGGGATGGTGACGGGTTCCTATATAGAGGAGTACTGGGGAAGCCTCTGTGTGTGTGTGTGTGTCCGTGCATATGTACACATGTGTGTAAAAAGCAGCCACACGCTGAAAATGGTTAACGGGTAGCCAGGCTGTCTGTACTGGGGCTGTACTGGGGGGTT
  • the hCCRK promoter sequence is a homologue of SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is a variant of SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is a fragment of SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is a homologue of a fragment of SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is a variant of a fragment of SEQ ID NO: 15. Each possibility represents a separate embodiment of the present invention. In another embodiment, the hCCRK promoter sequence is at least 60%homologous to SEQ ID NO: 15.
  • the hCCRK promoter sequence is at least 65%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 70%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 72%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 74%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 76%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 78%homologous to SEQ ID NO: 15.
  • the hCCRK promoter sequence is at least 80%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 82%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 84%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 88%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 94% homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 96%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 98%homologous to SEQ ID NO: 15. In another embodiment, the hCCRK promoter sequence is at least 99%homologous to SEQ ID NO: 15.
  • the nucleotide sequence of a CMV mini promoter is show in SEQ ID NO: 16: GGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCCCGCATTCGAGCTCGGTACCCGG(SEQ ID NO: 16) .
  • the CMVmini promoter sequence is a homologue of SEQ ID NO: 16.
  • the CMVmini promoter sequence is a variant of SEQ ID NO: 16.
  • the CMVmini promoter sequence is a fragment of SEQ ID NO: 16.
  • the CMVmini promoter sequence is a homologue of a fragment of SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is a variant of a fragment of SEQ ID NO: 16. Each possibility represents a separate embodiment of the present invention. In another embodiment, the CMVmini promoter sequence is at least 60%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 65%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 70%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 72%homologous to SEQ ID NO: 16.
  • the CMVmini promoter sequence is at least 74%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 76%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 78%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 80%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 82%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 84%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 88%homologous to SEQ ID NO: 16.
  • the CMVmini promoter sequence is at least 94%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 96%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 98%homologous to SEQ ID NO: 16. In another embodiment, the CMVmini promoter sequence is at least 99%homologous to SEQ ID NO: 16.
  • nucleotide sequence of one H19 814 promoter refers to patent US_9173964_B2; the nucleotide sequences of other tumor specific elements, including TYR, TRP1 and AFP refers to In vivo gene product documents.
  • the expression cassette comprises, from 5’ -3’ , a SV40 enhancer, an hTERT promoter, a CMVmini promoter, a CDS sequence and a SV40 poly A addition sequence (Fig. 1, CG010201) .
  • SV40-hTERT-CMVmini region of the expression cassette comprises the sequence of SEQ ID NO: 18 or a sequence having at least 90%sequence identity thereto.
  • the expression cassette comprises, from 5’ -3’ , an hTERT promoter, a CMVmini promoter, a CDS sequence and a SV40 poly A addition sequence (Fig. 1, CG010301) .
  • hTERT-CMVmini region of the expression cassette comprises the sequence of SEQ ID NO: 19 or a sequence having at least 90%sequence identity thereto.
  • the expression cassette comprises, from 5’ -3’ , an hTERT promoter, a E1F TATA promoter, a CDS sequence, a SV40 poly A addition sequence and a SV40 enhancer (Fig. 1, CG010401) .
  • hTERT-E1F TATA region of the expression cassette comprises the sequence of SEQ ID NO: 20 or a sequence having at least 90%sequence identity thereto.
  • the expression cassette comprises, from 5’ -3’ , an ARE1 sequence, an hTERT promoter, a CMVmini promoter, a CDS sequence, a SV40 poly A addition sequence and a SV40 enhancer (Fig. 1, CG010501) .
  • ARE1-hTERT-CMVmini region of the expression cassette comprises the sequence of SEQ ID NO: 21 or a sequence having at least 90%sequence identity thereto.
  • the expression cassette comprises, from 5’ -3’ , an ARE2 sequence, an hTERT promoter, a CMVmini promoter, a CDS sequence, a SV40 poly A addition sequence and a SV40 enhancer (Fig. 1, CG010601) .
  • ARE2-hTERT-CMVmini region of the expression cassette comprises the sequence of SEQ ID NO: 22 or a sequence having at least 90%sequence identity thereto.
  • the expression cassette of the present invention may comprise one or more ARE cis elements (ARE1 or ARE2) , such as 1, 2, 3 or more ARE1 sequences, which can be linked directly or spaced by short linker (s) .
  • ARE1 or ARE2 ARE cis elements
  • 1, 2, 3 or more ARE1 sequences which can be linked directly or spaced by short linker (s) .
  • the CDS sequence of any one of the expression cassette of the present invention may encode any gene product which can be used as a cytotoxic (or cytostatic) agent for tumor cells.
  • the gene product includes but not limited to: a cytotoxin, such as, DTA, tk-HSV, GZMA, GZMB, pro-and cleaved-caspase1, 3, 8 and 11, Bcl-2, FADD and its variants; a Membrane penetrator, such as, perforin, GSDMA, GSDMB, GSDMD, GSDME and relative proteins in various species and N terminal or active mutant variants, MLKL and its variants; a siRNA targeting, such as, EGFR, HER2, Kras, c-Myc, hTERT, IGFR, AKT1, AKT2, AKT3, mTOR, cAMPK, YAP, NRF2, ESCRT subunits, or NQO1; a Intracellular&membrane recombinant protein,
  • a site at which the CDS sequence or gene of interest is to be inserted may be present as a multiple cloning site.
  • the gene of interest may be inserted at the multiple cloning site (insertion site) by utilizing a sequence to be recognized by a restriction enzyme.
  • a gene expression cassette that does not contain DNA of the gene of interest itself but contains a portion at which the DNA is to be inserted as a multiple cloning site as described above is also encompassed in the present invention.
  • the expression cassette of the present invention may be utilized by being incorporated into a gene expression vector.
  • the present invention also encompasses a vector containing the gene expression cassette of the present invention.
  • the vector into which the gene expression cassette of the present invention is inserted examples include: a plasmid; viral vectors, such as an adenovirus (Ad) vector, an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a herpes virus vector, and a Sendai virus vector; and non-viral vectors, such as a biodegradable polymer.
  • the vector into which the gene expression cassette has been introduced may be introduced into cells by a known method, such as infection or electroporation. Further, a commercially available vector may be modified so as to contain the expression cassette of the present invention. Further, the present invention also encompasses a viral vector containing the expression cassette for the gene of interest described above.
  • an Ad vector and an AAV vector each enable specific diagnosis or treatment of a disease such as cancer
  • the gene expression cassette of the present invention can allow the gene to be expressed stably and sustainably, and hence the viral vectors are desirably used as appropriate for applications of gene expression.
  • the viral vector may be generated by inserting the expression cassette for the gene of interest described above onto a viral genome usable as a vector.
  • the present invention concerns formulation of one or more of the gene expression cassettes disclosed herein in pharmaceutically acceptable carrier or associated with a delivery agent for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
  • a composition comprising: 1) one or more of the gene expression cassettes and 2) a pharmaceutically acceptable carrier and/or a deliver agent.
  • the present invention contemplates the formulation of one or more viral vectors, virions, or virus particles (or pluralities thereof) that comprise one or more of the disclosed gene expression cassettes.
  • compositions it will also be understood that, if desired, the gene expression cassette or vectors containing the same as disclosed herein may be administered in combination with other agents as well, such as, e.g., peptides, proteins or polypeptides or various pharmaceutically-active agents.
  • agents such as, e.g., peptides, proteins or polypeptides or various pharmaceutically-active agents.
  • the viral vector compositions may thus be delivered along with various other agents as required in the particular instance.
  • Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein.
  • such compositions may further comprise substituted or derivatized RNA, DNA, or PNA compositions.
  • Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, topical, sublingual, subcutaneous, transdermal, parenteral, intravenous, intranasal, and intramuscular administration, intratumoral, lymph node administration and formulation.
  • Further embodiments of the present invention generally pertain to methods of treating a subject with a hyperproliferative disease, that include: (1) obtaining a pharmaceutical composition of a polynucleotide that includes any of the gene expression cassettes described above; and (2) administering a pharmaceutically effective amount of the composition to the subject.
  • the subject can be any subject, such as a mammal.
  • the mammal may be a human, such as a patient with a disease.
  • the disease can be any disease that can afflict a subject.
  • the subject is a human, and the disease is a hyperproliferative disease such as tumor or cancer.
  • the cancer may be any cancer, such breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colon cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia.
  • the subject is undergoing secondary anticancer therapy. Any secondary anti-cancer therapy known to those of ordinary skill in the art is contemplated, such as chemotherapy, surgical therapy, radiation therapy, immunotherapy, or additional gene therapy.
  • the present invention provides a method for treating a tumor in a subject in need thereof, comprising administering to the subject a gene expression cassette, a vector or a pharmaceutical composition of the present invention, thereby treating a tumor in a subject in need thereof.
  • the present invention provides a method for inhibiting tumor progression in a subject in need thereof, comprising administering to the subject a gene expression cassette, a vector or a pharmaceutical composition of the present invention, thereby inhibiting tumor progression in a subject in need thereof.
  • the present invention provides a method for inhibiting tumor metastasis in a subject in need thereof, comprising administering to the subject a gene expression cassette, a vector or a pharmaceutical composition of the present invention, thereby inhibiting tumor metastasis in a subject in need thereof.
  • the tumor is solid tumors include, but are not limited to, breast cancer, endometrial cancer, ovarian cancer, cervical cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, anal cancer, urothelial cancer, pancreatic cancer, salivary gland cancer, melanoma, skin cancer, head and neck cancer, sarcoma and brain cancer.
  • the tumor is a hematological malignancy include but not limited to, multiple myeloma (MM) , lymphoma, chronic lymphocytic leukemia (CLL) , B-cell acute lymphoblastic leukemia (B-ALL) , or acute myelogenous leukemia (AML) , diffuse large B cell lymphoma (DLBCL) , non-Hodgkin lymphoma (NHL) , extranodal NK/T cell lymphoma (ENKL) , Hodgkin Lymphoma (HL) , plasmablastic lymphoma, Burkitt’s lymphoma, marginal zone lymphoma (MZL) , or mantle cell lymphoma (MCL) .
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • B-ALL B-cell acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • DLBCL
  • the cis element or the expression cassette is constructed or packaged in a vector.
  • the vector is a viral vector, such as an adenovirus (Ad) vector, an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a herpes virus vector, or a Sendai virus vector.
  • the vector is a non-viral vector, such as in vivo jetPEI (Polyethylenimine) , hyperbranched Poly ( ⁇ -amino ester) s (HPAEs) , lipid nanoparticle (LNP) , liposomes or exosome.
  • HEK293FT immortalized cell line HEK293FT.
  • Transcript activity was determined by measurement of luminance value following after plasmid transfection with tumor specific promoters 24 hours referring manufacturer's (Vazyme, Bio-Lite TM Luciferase assay system) recommendations.
  • HEK293FT cells were seeded in each well of 96-well plate and transfection was performed when confluent is 60 to 80%. 0.1 ⁇ g of pDNA loaded with jetPrime in 100ul jetPRIME buffer were used for individual well.
  • Tumor specific plasmids are constructed via engineering tumor specific promoters (CG010201, CG010301, CG010401, CG010501, CG010601, CG010801, CG010A01, CG010C01, CG011001, CG011101, CG011201, CG011301, CG010D01, CG010E01 and CG010F01, synthesized and purified by GenScript. ) and Luciferase into pcDNA3.4 backbone (GenScript) via GIBSON recombination.
  • control group is described in FIG. 1 CMV-CDS-Poly (A) .
  • CG010501 ARE1-hTERT
  • CG010601 ARE2-hTERT
  • FIG. 2 The structure of control group is described in FIG. 1 CMV-CDS-Poly (A) .
  • CG010501 ARE1-hTERT
  • CG010601 ARE2-hTERT
  • FIG. 2 The structure of control group is described in FIG. 1 CMV-CDS-Poly (A) .
  • CG010501 ARE1-hTERT
  • CG010601 ARE2-hTERT
  • an hTERT hybrid promoter comprised vector efficiently drives gene expression in immortalized human cell line, showing potential for further analysis in cancer cells.
  • hTERT and its derived hybrid promoter candidates described in Example 1 were tested in vitro on mouse and human colorectal carcinoma cell lines, MC38 and HCT116. Experiment procedure is performed as described in EXAMPLE 1. As seen with the immortalized HEK293T kidney epithelial cells. CG010501 (ARE1-hTERT) and CG010601 (ARE2-hTERT) exhibited superior expressing efficiency in both colorectal carcinoma cell lines, relative to hTERT single promoter constructs (FIGS 3A-B, CG010301 and CG101401) .
  • an ARE-hTERT hybrid promoter comprised vector efficiently drives gene expression in colorectal carcinoma cell lines and shows potential for anti-tumor gene therapy.
  • hTERT and its derived hybrid promoter candidates described above were tested in vitro on human melanoma cell lines, A375 and G361. Experiment procedure is performed as described in above. As seen with the above cancer cell lines. CG010501 (ARE1-hTERT) and CG010601 (ARE2-hTERT) exhibited superior expressing efficiency in both melanoma cell lines, relative to hTERT single promoter constructs (FIGS 4A-B, CG010301 and CG101401) and melanoma specific promoters (FIG 4C, CG011001, CG011101, CG011201 and CG011301) .
  • hTERT and its derived hybrid promoter candidates described above were tested in vitro on mouse and human pancreatic adenocarcinoma cell lines, Pan02. Experiment procedure is performed as described in above. As seen with the above cancer cell lines, CG010601 (ARE2-hTERT) exhibited higher expressing efficiency in both pancreatic adenocarcinoma cell lines, relative to hTERT single promoter constructs and pancreatic cancer specific promoters (FIG 5, CG010A01 and CG010C01) .
  • an ARE-hTERT hybrid promoter comprised vector efficiently drives gene expression in pancreatic adenocarcinoma cell lines and shows potential for anti-tumor gene therapy.
  • hTERT and its derived hybrid candidates described in above were tested in vitro on mouse and human hepatocellular carcinoma cell lines, Bel7402 and HepG2. Experiment procedure is performed as described in above. As seen with the above cancer cell lines, CG010601 (ARE2-hTERT) exhibited higher expressing efficiency in both hepatocellular carcinoma cell lines, relative to hTERT single promoter constructs (FIGS 6A-B, CG010301 and CG101401) and liver cancer specific promoters (FIGS 6C-D, CG010D01, CG010E01 and CG010F01) .
  • an ARE-hTERT hybrid promoter comprised vector efficiently drives gene expression in hepatocellular carcinoma cell lines and shows potential for anti-tumor gene therapy.
  • hTERT and its derived hybrid candidates described in above were tested in primary cells.
  • Human umbilical vein endothelial cell and smooth muscle cell are used.
  • Experiment procedure is performed as described in above.
  • CG010601 exhibited little transcription activity in both primary cells (FIGS 7A-B) .
  • CG010501 ARE1-hTERT
  • CG010501 showed higher luciferase expression in human smooth muscle cell, but the expression level is still poor (FIG 7B) .
  • an ARE-hTERT hybrid promoter comprised vector shows wonderful tumor selectivity to prevent unwanted side-effect of interested genes in normal tissues, implying good safety of ARE-hTERT hybrid promoters in clinical application.
  • the anti-cancer therapeutic potential of hybrid promoter construct ARE2-hTERT-ELANE was further test in vitro by determining their ability to induce apoptosis in both mouse and human colorectal adenocarcinoma lines, relative to CMV promoter. Anti-tumor activity was determined by cell viability with CTG assay, measuring ATP level in liver cells, MC38 and HCT116 colorectal adenocarcinoma cancer cell lines were transfected with ARE2-hTERT-ELANE, ARE2-hTERT-luci and CMV-ELANE at the concentration of 1, 2ug and 5ug/ml for 24 hours.
  • ARE2-hTERT-ELANE exhibited comparable cell killing with CMV-ELANE in MC38 mouse colorectal cancer cell line, while ARE2-hTERT-luci showed no anti-tumor effect. Similar results were obtained when the experiment was repeated with human HCT116 colorectal cancer cell line. The result shows an ARE-hTERT hybrid promoter comprised vector, expressing neutrophile elastase, exhibited significantly superior ability to induce apoptosis in colorectal adenocarcinoma cell lines.
  • ARE-hTERT-DTA The anti-cancer therapeutic potential of hybrid promoter constructs ARE-hTERT-DTA were further test in vitro by determining their ability to lyse both mouse and human colorectal adenocarcinoma lines, MC38 and HCT116 colorectal adenocarcinoma cancer cell lines, relative to CMV promoter. Anti-tumor activity was determined by cell lysis with LDH assay, measuring released extracellular lactate dehydrogenase activity. The transfection procedure majorly refers to above EXAMPLE 7. The concentration of transfect plasmid was altered to 0.5ug/ml. ARE2-hTERT-ELANE exhibited comparable ability of cell lysis with CMV-ELANE in both colorectal carcinoma cell lines.
  • an ARE-hTERT hybrid promoter comprised vector, expressing diphtheria toxin, exhibited significantly superior ability to induce cell lysis in colorectal adenocarcinoma cell lines.
  • the anti-cancer therapeutic potential of hybrid promoter constructs ARE-hTERT-DTA were further test in vitro by determining their ability to lyse 2 human colorectal adenocarcinoma lines, G361 and A375 melanoma cell lines, relative to CMV promoter. Anti-tumor activity was determined by cell lysis with LDH assay, measuring released extracellular lactate dehydrogenase activity. The transfection procedure majorly refers to EXAMPLE 8. The ARE2-hTERT-DTA exhibited comparable ability of cell lysis with CMV-DTA in both colorectal carcinoma cell lines.
  • an ARE-hTERT hybrid promoter comprised vector, expressing diphtheria toxin, exhibited significantly superior ability to induce cell lysis in melanoma cell lines.
  • the anti-cancer therapeutic potential of hybrid promoter constructs ARE-hTERT-DTA were further test in vitro by determining their ability to lyse 2 human colorectal adenocarcinoma lines, Bel7042 and HepG2 hepatocellular carcinoma cell lines, relative to CMV promoter. Anti-tumor activity was determined by cell lysis with LDH assay, measuring released extracellular lactate dehydrogenase activity. The transfection procedure majorly refers to EXAMPLE 8. The ARE2-hTERT-DTA exhibited comparable ability of cell lysis with CMV-DTA in both hepatocellular carcinoma cell lines.
  • an ARE-hTERT hybrid promoter comprised vector, expressing diphtheria toxin, exhibited significantly superior ability to induce cell lysis in hepatocellular adenocarcinoma cell lines.
  • the anti-cancer therapeutic potential of hybrid promoter constructs ARE2-hTERT-hIL-2 were further test in vitro by determining their ability to secret functional recombinant hIL-2 in different cancer cell lines, relative to CMV promoter.
  • the expression level of IL-2 was determined by ELISA assay, measuring tagged extracellular recombinant human IL-2.
  • the transfection procedure majorly refers to above, while the concentration of IL-2 expressing vectors is altered to 5ug/ml.
  • the ARE2-hTERT-IL-2 exhibited comparable IL-2 level in culture medium with CMV-IL-2 in multiple cancer cell lines. For functional analysis, culture medium from transfected cancers were used for CSFE labeled isolated CD3 T cells proliferation.
  • T cell isolation and CSFE assay were performed according to manufacturers’ protocols. After co-culture 48 hours, the cells were collected for flow cytometry analysis. Compared to the CMV-IL-2 group, ARE2-hTERT-hIL-2 expressed similar capacity of facilitating T cell proliferation.
  • an ARE-hTERT hybrid promoter comprised vector, expressing recombinant human IL-2, exhibited significantly superior ability to active T cell immune response.
  • the anti-cancer therapeutic potential of hybrid promoter constructs ARE2-hTERT- ⁇ PD1 were further test in vitro by determining their ability to secret functional recombinant hIL-2 in different cancer cell lines, relative to CMV promoter.
  • the expression level of ⁇ PD1 antibody was determined by ELISA assay, measuring tagged extracellular recombinant ⁇ PD1 antibody.
  • the transfection procedure majorly refers to EXAMPLE 11.
  • the ARE2-hTERT- ⁇ PD1 exhibited comparable antibody level in culture medium with CMV- ⁇ PD1 in multiple cancer cell lines.
  • culture medium from transfected cancers were used for CSFE labeled isolated CD3 T cells proliferation.
  • the procedure of T cell isolation and CSFE assay were performed according to manufacturers’ protocols. After co-culture 48 hours, the cells were collected for flow cytometry analysis.
  • ARE2-hTERT- ⁇ PD1 expressed similar capacity of facilitating T cell proliferation.
  • an ARE-hTERT hybrid promoter comprised vector, expressing recombinant human ⁇ PD1, exhibited significantly superior ability to active T cell immune response.
  • ARE2-hTERT-GZMB The anti-cancer therapeutic potential of hybrid promoter constructs ARE2-hTERT-GZMB were further test in vitro by determining their ability to kill different cancer cell lines, relative to CMV promoter. Anti-tumor activity was determined by cell lysis with LDH assay, measuring released extracellular lactate dehydrogenase activity. The transfection procedure majorly refers to above EXAMPLE 7. ARE2-hTERT-GZMB exhibited comparable ability of cell lysis with CMV-GZMB in both colorectal carcinoma cell lines.
  • an ARE-hTERT hybrid promoter comprised vector, expressing recombinant human GZMB, exhibited significantly superior ability to kill cancer cells. Sequences mentioned or used in the present invention:
  • CG010201 SV40-hTERT-CMVmini:
  • CG010501 ARE1-hTERT-CMVmini:
  • CG010601 ARE2-hTERT-CMVmini:
  • CG011201 SV40-TYR:
  • CG010E01 AFP-AFP:
  • CG010F01 SV40-AFP:

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Abstract

L'invention concerne une cassette d'expression comprenant : 1) un promoteur chimérique, comprenant un promoteur hTERT et un promoteur minimal ; 2) une séquence codante (CDS) ; et 3) une séquence poly (A) ou une séquence signal de polyadénylation. La séquence CDS dans la cassette d'expression est exprimée uniquement dans des cellules tumorales ou cancéreuses ou est exprimée à un niveau beaucoup plus élevé dans des cellules tumorales ou cancéreuses que dans des cellules normales, et ainsi la cassette d'expression peut être utilisée dans le traitement du cancer.
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Citations (3)

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CN101993892A (zh) * 2009-08-31 2011-03-30 长沙赢润生物技术有限公司 肿瘤细胞中靶向性表达shRNA的真核表达载体
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