WO2024130007A2 - Promoteurs de htert synthétiques et utilisations associées - Google Patents

Promoteurs de htert synthétiques et utilisations associées Download PDF

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Publication number
WO2024130007A2
WO2024130007A2 PCT/US2023/084083 US2023084083W WO2024130007A2 WO 2024130007 A2 WO2024130007 A2 WO 2024130007A2 US 2023084083 W US2023084083 W US 2023084083W WO 2024130007 A2 WO2024130007 A2 WO 2024130007A2
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cancer
sequence
aspects
synthetic
seq
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PCT/US2023/084083
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English (en)
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WO2024130007A3 (fr
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Spencer KNIGHT
Nachiketa Gupta
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Kriya Therapeutics, Inc.
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Publication of WO2024130007A2 publication Critical patent/WO2024130007A2/fr
Publication of WO2024130007A3 publication Critical patent/WO2024130007A3/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • 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

Definitions

  • the present disclosure pertains to synthetic promoters, expression vectors, and use of the same for gene transcription.
  • telomerase reverse transcriptase telomerase reverse transcriptase
  • hTERT Human telomerase reverse transcriptase
  • the catalytic subunit of telomerase functions to stabilize telomere length during chromosomal replication.
  • the naturally occurring hTERT promoter is active in most tumor and immortal cell lines but inactive in normal somatic cell types.
  • the wild-type hTERT promoter is also limited by its inability to direct high level and cancer cell-specific expression, e.g., for effective targeted gene therapy.
  • naturally occurring hTERT promoter can provide high expression in certain tumor microenvironments, but has significantly weaker expression levels than promoters such as CMV or EFl -alpha.
  • hTERT promoters have been described (See, e.g., Kim et al., Hum Gene Ther, 14(15): 1415-28 (2003); Li C, et al., Oncotarget, 6(23): 19542-51 (2015); Liu T, et al., Endocr Relat Cancer, 21(3):427-34 (2014)).
  • Kim et al. generated a modified hTERT promoter in which additional copies of c-Myc and Spl binding sites were incorporated adjacent to the natural promoter sequence.
  • Li et al. disclosed that the C228T mutation of TERT promoter, which frequently occurs in bladder cancer stem cells (BCSCs), triggers TERT expression leading to increased telomerase activity.
  • BCSCs bladder cancer stem cells
  • C250T TERT promoter mutations were found in many cancer types.
  • a fusion including hTERT promoter and CMV promoter elements was also disclosed in Sakaguchi et al., Oncol Rep. 38(2): 1108-1114 (2017).
  • these modified hTERT promoters show variable expression depending on the cancer cell type. Thus, there remains a need for improved promoters.
  • Certain aspects of the disclosure are directed to a synthetic promoter comprising a nucleotide sequence corresponding to any of SEQ ID NOs: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • Certain aspects of the disclosure are directed to a synthetic promoter sequence, wherein the synthetic promoter sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-185 or any sequence listed in Table 1.
  • Certain aspects of the disclosure are directed to a polynucleotide comprising a synthetic promoter sequence, wherein the synthetic promoter sequence comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-185 or any sequence listed in Table 1.
  • the nucleotide sequence has at least 80% sequence identity to any of SEQ ID NOs: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • the synthetic promoter sequence is derived from a naturally occurring human telomerase reverse transcriptase (hTERT) promoter sequence (e.g., SEQ ID NO: 188).
  • hTERT human telomerase reverse transcriptase
  • the synthetic promoter sequence comprises mutations C228T and C250T relative to a naturally occurring hTERT promoter sequence.
  • the synthetic promoter sequence comprises additional copies of transcription factor (e.g., c-Myc and Spl) binding sites relative to a naturally occurring hTERT promoter sequence.
  • transcription factor e.g., c-Myc and Spl
  • the synthetic promoter sequence or the polynucleotide promotes expression of an operably linked nucleotide sequence of interest (e.g., gene of interest).
  • the synthetic promoter sequence or the polynucleotide is positioned upstream of the operably linked nucleotide sequence of interest. [0014] In some aspects, the synthetic promoter sequence or the polynucleotide comprises an enhancer sequence.
  • the enhancer sequence is selected from the group consisting of a CMV enhancer, a SV40 enhancer or combinations thereof.
  • the enhancer sequence is a synthetic enhancer.
  • the nucleotide sequence of interest encodes a therapeutic protein.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3- 185 or any sequence listed in Table 1.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of SEQ ID NOs: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3-185 or any sequence listed in Table 1.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-185 or any sequence listed in Table 1.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 3-185 or any sequence listed in Table 1.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 3-185 or any sequence listed in Table 1.
  • Certain aspects of the disclosure are directed to an expression construct comprising any of the synthetic promoter sequences or the polynucleotide sequences disclosed herein.
  • the expression construct further comprises an untranslated region (UTR), a microRNA binding site, polyA sequence, an intron sequence, or any combination thereof. [0026] In some aspects, the expression construct further comprises a poly(A) (pA) sequence.
  • the pA sequence is a synthetic pA sequence.
  • the pA sequence is a bovine growth hormone (bGH) pA sequence
  • the pA sequence is a human growth hormone (hGH) pA sequence.
  • Certain aspects of the disclosure are directed to a delivery vector comprising any of the synthetic promoter sequences disclosed herein, any of the polynucleotides disclosed herein or any of the expression constructs disclosed herein.
  • the delivery vector is selected from the group consisting of a viral vector, a plasmid, a lipid, a protein particle, a bacterial vector, a lysosome, a virus-like particle, a polymeric particle, and an exosome.
  • the vector is a viral vector.
  • the vector is a lentiviral vector, an adenoviral vector, an adeno- associated viral (AAV) vector, or a retroviral vector.
  • the delivery vector comprises inverted terminal repeats (ITRs) flanking the expression construct.
  • ITRs inverted terminal repeats
  • the vector is an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • the delivery vector is a recombinant AAV (rAAV) vector comprising an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, and AAV12.
  • rAAV recombinant AAV
  • Certain aspects of the disclosure are directed to a cell comprising any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, any of the delivery vectors disclosed herein, or any of the viral vectors disclosed herein.
  • Certain aspects of the disclosure are directed to a viral particle comprising any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, or any of the delivery vectors disclosed herein.
  • the viral particle comprises an adeno-associated viral (AAV) capsid.
  • AAV adeno-associated viral
  • the AAV capsid is a recombinant AAV (rAAV) capsid has an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, and AAV12.
  • rAAV recombinant AAV
  • the AAV capsid is modified relative to the wild-type serotype.
  • Certain aspects of the disclosure are directed to a pharmaceutical composition comprising any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, any of the delivery vectors disclosed herein, any of the viral particle disclosed herein, or any of the cells disclosed herein.
  • compositions further comprise a pharmaceutically acceptable excipient.
  • Certain aspects of the disclosure are directed to a method of modulating transcription of a nucleic acid sequence in a cell by introducing any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, any of the delivery vectors disclosed herein, or any of the viral particle disclosed herein into the cell.
  • the cell is a tumor cell.
  • the tumor cell is derived from a cancer selected from the group consisting of hepatocellular carcinoma, colon carcinoma, melanoma, and any combination thereof.
  • the tumor cell is derived from a cancer selected from the group consisting of a skin cancer; a breast cancer; a brain cancer; a bone cancer; a head and neck cancer; a salivary gland cancer; a gynecologic cancer; a urologic cancer; a gastrointestinal cancer; an ocular cancer; a thoracic cancer; a blood cancer; a lymphoma, a cancer of the endocrine system; a sarcoma of soft tissue; a neoplasm of the central nervous system; and any combination thereof.
  • the tumor cell is derived from a cancer selected from the group consisting of metastatic melanoma, cutaneous malignant melanoma, cutaneous squamous cell carcinoma, basal cell carcinoma, invasive breast cancer, triple-negative breast cancer, inflammatory breast cancer, glioblastoma multiforme, medulloblastoma, pituitary carcinoma, brain stem gliomas, astrocytomas, oligodendrogliomas, hemangiopericytomas, germ cell tumors, pineal tumors, chordomas, chondrosarcomas, osteosarcomas, Ewing sarcomas, fibrosarcomas, adamantiomas, giant cell tumors, head and neck squamous cell carcinoma (HNSCC), salivary gland cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, nasopharyngeal cancer, thyroid cancer, cancer of the group consisting of metastatic
  • Certain aspects of the disclosure are directed to a use of any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, any of the delivery vectors disclosed herein, or any of the viral particles for the manufacture of a medicament for use in therapy or prophylaxis.
  • the therapy or prophylaxis comprises any of the methods disclosed herein.
  • FIG. 1 shows a schematic for engineering synthetic promoters to boost transcription.
  • FIG. 2 shows exemplary synthetic hTERT promoters.
  • FIGs. 3A-3C show boosted luciferase expression in A549 (lung adenocarcinoma) cells (FIGs. 3A and 3C), and in Huh7 (HCC) cells (FIGs. 3B and 3C) transfected with reporter constructs comprising engineered hTERT promoters compared to luciferase expression in A549 and Huh7 cells transfected with reporter constructs comprising the unmodified hTERT promoter.
  • FIG. 4 shows a schematic representing the experimental design of enhancer libraries couple with the synthetic hTERT promoters of the present disclosure.
  • FIG. 5 shows a schematic representing in vitro screening of the synthetic promoters.
  • FIG. 6 shows a schematic representing in vivo screening of the synthetic promoters.
  • Certain aspects of the disclosure are directed to a synthetic promoter sequence, wherein the synthetic promoter sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-185 or any sequence listed in Table 1.
  • Certain aspects of the disclosure are directed to a polynucleotide comprising a synthetic promoter sequence, wherein the synthetic promoter sequence comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 3-185 or any sequence listed in Table 1.
  • Certain aspects of the disclosure are directed to a synthetic promoter comprising a nucleotide sequence corresponding to any of SEQ ID NOs: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • Certain aspects of the disclosure are directed to an expression construct comprising any of the synthetic promoters or any of the polynucleotide disclosed herein.
  • Certain aspects of the disclosure are directed to a delivery vector comprising any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein or any of the expression constructs disclosed herein.
  • Certain aspects of the disclosure are directed to a cell comprising any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, any of the delivery vectors disclosed herein.
  • Certain aspects of the disclosure are directed to a virus particle comprising any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, or any of the delivery vectors disclosed herein.
  • Certain aspects of the disclosure are directed to a pharmaceutical composition comprising any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, any of the delivery vectors disclosed herein, any of the viral particles, or any of the cells disclosed herein.
  • Certain aspects of the disclosure are directed to a use of any of the synthetic promoters disclosed herein, any of the polynucleotides disclosed herein, any of the expression constructs disclosed herein, any of the delivery vectors disclosed herein, or any of the viral particles disclosed herein for the manufacture of a medicament for use in therapy or prophylaxis.
  • a or “an” entity refers to one or more of that entity; for example, “a nucleic acid sequence,” is understood to represent one or more nucleic acid sequences, unless stated otherwise.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • the term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context.
  • the number of nucleotides in a nucleic acid molecule must be an integer.
  • "at least 18 nucleotides of a 21- nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property.
  • At least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.
  • “At least” is also not limited to integers (e.g., "at least 5%” includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).
  • no more than or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range.
  • nucleic acid sequence e.g., a modified hTERT promoter
  • reference nucleic acid sequence e.g., a naturally occurring hTERT promoter
  • a tumor that is derived from a specified cancer includes a tumor that shares a lineage from the specified cancer.
  • composition represents a composition comprising a compound or molecule described herein, e.g., a vector disclosed herein, formulated with a pharmaceutically acceptable excipient, and can be manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal.
  • a "pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and noninflammatory in a patient.
  • operatively linked means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • operably linked means that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • nucleic acid refers only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • nucleic acid refers only to the primary structure of the molecule. Thus, these
  • Polynucleotides can be made recombinantly, enzymatically, or synthetically, e.g., by solid-phase chemical synthesis followed by purification.
  • sequence of the polynucleotide or nucleic acid reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides.
  • mRNA refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.
  • a "coding sequence” or a sequence "encoding" a particular molecule is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of mRNA) into polypeptide, in vitro or in vivo, when operably linked to an appropriate regulatory sequence, such as a promoter (e.g., a synthetic promoter).
  • a promoter e.g., a synthetic promoter.
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and comprises any chain or chains of two or more amino acids.
  • a “peptide,” a “peptide subunit,” a “protein,” an “amino acid chain,” an “amino acid sequence,” or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of a "polypeptide,” even though each of these terms can have a more specific meaning.
  • the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
  • polypeptides which have undergone post-translational or post-synthesis modifications, for example, conjugation of a palmitoyl group, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • the term "peptide,” as used herein encompasses full length peptides and fragments, variants or derivatives thereof.
  • a "peptide” as disclosed herein can be part of a fusion polypeptide comprising additional components such as, e.g., an Fc domain or an albumin domain, to increase half-life.
  • a peptide as described herein can also be derivatized in a number of different ways.
  • a peptide described herein can comprise modifications including e.g., conjugation of a palmitoyl group.
  • Percent (%) sequence identity or “Percent (%) identity” or “sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST.
  • modified refers to a changed state or structure of a molecule of the disclosure. Molecules can be modified in many ways including chemically, structurally, and functionally.
  • synthetic means produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present disclosure can be chemical or enzymatic. In some aspects, the synthetic promoter of the disclosure is not naturally occurring.
  • promoter refers to a DNA sequence recognized by the machinery of the cell, or introduced synthetic machinery, that can initiate the transcription of a nucleic acid (e.g., a gene of interest).
  • the term “promoter” can also encompass those nucleic acid elements sufficient for promoter-dependent gene expression controllable for cell-type specific, tissue-specific or inducible by external signals or agents; such elements can be located in the 5' or 3' regions of the native gene.
  • the promoter is a constitutive promoter, a cell-type specific promoter, or an inducible promoter.
  • Enhancers are a cis-acting element that stimulates or inhibits transcription of adjacent genes.
  • An enhancer that inhibits transcription is also referred to as a “silencer.”
  • Enhancers can function (e.g., can be associated with a coding sequence) in either orientation, over distances of up to several kilobase pairs (kb) from the coding sequence and from a position downstream of a transcribed region.
  • transcriptional regulatory protein refers to a nuclear protein that binds a DNA response element and thereby transcriptionally regulates the expression of an associated gene or genes.
  • Transcriptional regulatory proteins generally bind directly to a DNA response element, however in some cases binding to DNA can be indirect by way of binding to another protein that in turn binds to, or is bound to a DNA response element.
  • expression vector or "expression construct” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the term "delivery vector” or “vector” refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc.
  • a vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment.
  • a “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control.
  • delivery vector includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
  • a large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses.
  • insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.
  • Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector.
  • selectable markers or reporters allows identification and/or selection of host cells that incorporate and express other coding regions contained on the vector.
  • selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.
  • the delivery vector is selected from the group consisting of a viral vector (e.g., an AAV vector), a plasmid, a lipid, a protein particle, a bacterial vector, and a lysosome.
  • a viral vector e.g., an AAV vector
  • a plasmid e.g., a lipid, a protein particle, a bacterial vector, and a lysosome.
  • Some aspects of the disclosure are directed to biological vectors, which can include viruses, particularly attenuated and/or replication-deficient viruses.
  • a "viral vector” refers to a sequence that comprises one or more polynucleotide regions encoding or comprising a molecule of interest, e.g., a protein, a peptide, and an oligonucleotide or a plurality thereof.
  • Viral vectors can be used to deliver genetic materials into cells. Viral vectors can be modified for specific applications.
  • the delivery vector of the disclosure is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.
  • AAV adeno-associated viral
  • AAV vector or "adeno-associated viral vector” as used herein refers to any vector that comprises or derives from components of an adeno-associated vector and is suitable to infect mammalian cells, preferably human cells.
  • AAV vector typically designates an AAV-type viral particle or virion comprising a payload.
  • the AAV vector can be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary).
  • the AAV vector can be replication defective and/or targeted.
  • AAV adeno-associated virus
  • AAV includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh8, AAVrhlO, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al.
  • an "AAV vector” includes a derivative of a known AAV vector.
  • an "AAV vector” includes a modified or an artificial AAV vector.
  • an AAV vector includes a hybrid vector (e.g, AAV-DJ, AAV-PHP.B, AAV2-ESGHGYF, AAVM41, AAV- LK03, AAV2-BR1, AAV587MTP, AAV-Anc80L65, AAV2-7m8, AAV2HBKO, AAV2YF, AAV6-RGD or AAV6.2).
  • a hybrid vector e.g, AAV-DJ, AAV-PHP.B, AAV2-ESGHGYF, AAVM41, AAV- LK03, AAV2-BR1, AAV587MTP, AAV-Anc80L65, AAV2-7m8, AAV2HBKO, AAV2YF, AAV6-RGD or AAV6.2.
  • the terms "AAV genome” and "AAV vector” can be used interchangeably.
  • the AAV vector is modified relative to the wild-type AAV serotype sequence.
  • the modified AAV vector is a modified AAV6, e.g, an AAV6 vector comprising the RGD peptide (an AAV6-RGD vector) or an AAV6 vector comprising mutations of surface exposed tyrosine residues as described, for example, in Sayroo et al. Gene Ther. 2016 Jan; 23(1): 18-25.
  • the AAV6-RGD vector further comprises modified amino acids corresponding to Y705, Y731, T492, and K531, (e.g., Y705, Y731F, T492V, and K53 IE; also referred to as AAV-RGD-Y705-731F+T492V+K53 IE).
  • a “recombinant AAV particle”, “recombinant AAV vector”, “rAAV particle”, or “rAAV vector” refers to an AAV virus that comprises a capsid protein and a vector genome (or an AAV genome) comprising at least one heterologous polynucleotide encoding a protein of interest and at least one inverted terminal repeat (ITR) region.
  • ITR inverted terminal repeat
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
  • in vivo refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).
  • a wild type or “naturally occurring” sequence refers to a sequence (e.g., polynucleotide or polypeptide) which occurs in nature or is identical to the native sequence.
  • the naturally occurring sequence refers to a full length promoter sequence (e.g., hTERT promoter).
  • mutant is used to mean a sequence (e.g., polynucleotide or polypeptide) having a primary sequence which differs from the wild type sequence by one or more nucleic acid or amino acid additions, substitutions or deletions.
  • a mutant may arise naturally, or may be created artificially (for example by site-directed mutagenesis).
  • activator sequence refers to a nucleotide sequence that is bound by a transcription factor that increases transcription levels of the nucleotide sequence.
  • repressor sequence refers to a nucleotide sequence that is bound by a transcription factor that decreases the transcription levels of the nucleotide sequence.
  • the naturally occurring hTERT promoter can drive gene expression in a tumor microenvironment but is significantly weaker in absolute expression levels than common promoters such as CMV or EFl -alpha.
  • the present disclosure provides synthetic, engineered hTERT promoters with improved expression across multiple cancer cell lines.
  • the synthetic hTERT promoters disclosed herein can be engineered for context specific activation and/or reduced off-site expression (e.g., cancer cell specific expression) relative to the wild-type hTERT promoter.
  • the synthetic hTERT promoters disclosed herein include one or more enhancer sequences.
  • the synthetic hTERT promoters disclosed herein include one or more mutations in the hTERT promoter which are found in human cancers (e.g., C228T and/or C250T). In some aspects, the synthetic hTERT promoters disclosed herein include one or more enhancer sequences and one or more mutations in the hTERT promoter which are found in human cancers (e.g., C228T and/or C250T).
  • the synthetic hTERT promoters are shorter than the wild-type hTERT promoter, e.g., less than 500 base pairs, less than 450 base pairs, or less than 400 base pairs (e.g., 200-500 base pairs, 250-500 base pairs, 300-500 base pairs, 200-400 base pairs, 250-400 base pairs, or 300-400 base pairs).
  • the hTERT synthetic promoter can be operably linked to a synthetic enhancer constructed from pseudorepeats of dual transcription factor (TF) binding sites.
  • the synthetic enhancers disclosed herein are shorter than full length CMV enhancers (i.e. shorter than the CMV immediate early enhancer, 304 base pairs).
  • the synthetic enhancers disclosed herein are less than 300 base pairs, less than 280 base pairs, less than 260 base pairs, less than 240 base pairs, less than 220 base pairs, or less than 200 base pairs. In some aspects, the synthetic enhancers disclosed herein are 100-300 base pairs, 150-300 base pairs, 100-250 base pairs, 150- 250 base pairs, 200-300 base pairs, or 250-300 base pairs. In some aspects, the synthetic enhancers disclosed herein are more active in tumor cells as compared to normal cells (non-cancer cells).
  • the synthetic hTERT promoter disclosed here can drive expression at high levels in multiple cancer types (e.g., at least two different cancer cell lines (Huh7 and A549)) relative to unmodified hTERT promoter or unmodified hTERT promoter with a full length CMV enhancer sequence.
  • the synthetic hTERT promoters disclosed herein can comprise an enhancer (e.g., a synthetic enhancer comprising pseudorepeats of a dual transcription factor (TF) binding site).
  • the synthetic hTERT promoters disclosed herein can comprise a synthetic enhancer comprising pseudorepeats of a dual transcription factor (TF) binding site and a mutation in the hTERT promoter (e.g., C228T and/or C250T).
  • TF dual transcription factor
  • transcription factors regulating hTERT expression include activators such as c-Myc, Spl, HIF1A, F-l, AP2, ER, and Ets.
  • transcription factors regulating hTERT expression include repressors such as WT1, E2F1, VDR and MADE
  • transcription factors regulating hTERT expression include SP3, MZF1, KLF4, NFATC1, PAX5, ETS1, STAT3, and NFKB.
  • E2F1 can negatively regulate Myc-driven activation.
  • ETS can work cooperatively with Myc and NFKB signaling.
  • HIF1 A can be an activator in the hypoxia conditions.
  • HIF1A can be an activator in stress conditions.
  • c-Myc can be an oncogenic activator across multiple cancers.
  • MZF2 can drive competitive inhibition via occupation of activator binding sites.
  • NFATC1 can act as a direct and/or indirect activator via cooperative Myc signaling.
  • NFKB1 can act as an inflammatory factor, e.g., activated under cellular stress.
  • SP1 can act as a general transcription factor, e.g., with a dual role in hTERT.
  • STAT3 can act as an inflammatory factor, e.g., that works cooperatively with NFKB1.
  • VDR can repress hTERT via direct binding.
  • WT1 can repress hTERT via direct binding.
  • the synthetic promoter comprises a E2F1 consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 195-200.
  • the synthetic promoter comprises a E2F1 consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of TTTGGCGC (“Sequence 189"), TTTCGCGC ("Sequence 190"), TTTGCCGC (“Sequence 191”), GCGCCAAA (“Sequence 192”), GCGCGAAA (“Sequence 193"), and GCGGCAAA (“Sequence 194").
  • the synthetic promoter comprises a ETS1 consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 201-206.
  • the synthetic promoter comprises a HIF1A consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 207-212.
  • the synthetic promoter comprises a MYC consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 213-218.
  • the synthetic promoter comprises a MZF1 consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 219-224.
  • the synthetic promoter comprises a NFATCl consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 225-230.
  • the synthetic promoter comprises a NFKB1 consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 231-236.
  • the synthetic promoter comprises a SP1 consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 237-248.
  • the synthetic promoter comprises a STAT3 consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 249-254.
  • the synthetic promoter comprises a VDR consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of TGAGTTCA ("Sequence 255"), TGGGTTCA ("Sequence 256"), TAAGTTCA ("Sequence 257”), TGAACTCA (“Sequence 258”), TGAACCCA (“Sequence 259”), and TGAACTTA (“Sequence 260").
  • the synthetic promoter comprises a Wtl consensus sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 261-266.
  • the consensus sequence is 5-20 (e.g., 8-15) nucleic acids in length.
  • the synthetic promoter comprises an hTERT promoter comprising at least one mutation relative to the naturally occurring hTERT promoter sequence. In some aspects, the synthetic promoter comprises an hTERT promoter comprising at least two mutations relative to the naturally occurring hTERT promoter sequence. In some aspects, the synthetic promoter comprises an hTERT promoter comprising a mutation selected from the group consisting of C228T, C250T, or the combination thereof.
  • the synthetic hTERT promoter comprises a Kozak sequence (e.g., GCCAC (SEQ ID NO: 358) or GCCGCCACC (SEQ ID NO: 359) at the 3’-end of the promoter). In some aspects, synthetic hTERT promoter does not comprises a Kozak sequence (e.g., GCCAC (SEQ ID NO: 358) or GCCGCCACC (SEQ ID NO: 359) at the 3’-end of the promoter).
  • the synthetic promoter comprises a nucleotide sequence corresponding to any one of SEQ ID NOs: 3-185 or any sequence in Table 1.
  • the synthetic promoter comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NO: 3-185 or any sequence in Table 1.
  • the synthetic promoter comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 3-185 or any sequence in Table 1.
  • the synthetic promoter comprises a nucleotide sequence corresponding to any of SEQ ID NOs: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • the synthetic promoter comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NO: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • the synthetic promoter comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 18.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 27.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 33.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 35.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 60.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 79.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 82.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 95.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 95.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 126.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 126.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 143.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 143.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 145.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 145.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 148.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 152.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 154.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 154.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 156.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 158.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 158.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 160.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 161.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 162.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 175.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 175.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 176.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 176.
  • the synthetic promoter sequence or the polynucleotide comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 177.
  • the synthetic promoter consists of a nucleotide sequence corresponding to SEQ ID NO: 177.
  • the synthetic promoter comprises or is flanked by a CMV enhancer.
  • the CMV enhancer comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 356.
  • the synthetic promoter comprises a mutated VDR binding site.
  • the synthetic promoter comprises a CMV enhancer. In some aspects, the synthetic promoter comprises a nucleotide sequence corresponding to any one of SEQ ID NOs: 3-18, 63-84, 129-172, 174-185, and SEQ ID NO. 187.
  • the synthetic promoter comprises a 3x, 5x, lOx, or 15x (e.g., lOx) activator pair of pseudorepeats.
  • the synthetic promoter comprises a 3x, 5x, lOx, or 15x (e.g., lOx) activator pair of pseudorepeats and a CMV enhancer.
  • the synthetic promoter comprises a nucleotide sequence corresponding to any one of SEQ ID NOs: 19-62.
  • the synthetic promoter comprises a dual SV40 enhancer.
  • the synthetic promoter comprises an activator sequence selected from the group consisting of Myc, HIF1A, ETS1, NFATC1, NFKB1, SP1, STAT3, and combinations thereof.
  • the synthetic promoter comprises an activator sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 267-354.
  • the first activator sequence is a HIF1A binding sequence and the second binding sequence is a Myc binding sequence. In some aspects, the first activator sequence is a HIF1 A binding sequence and the second binding sequence is a SP1 binding sequence. In some aspects, the first activator sequence is a HIF1 A binding sequence and the second binding sequence is an ETS1 binding sequence. In some aspects, the first activator sequence is a HIF1A binding sequence and the second binding sequence is a NFATC1 binding sequence. In some aspects, the first activator sequence is a HIF 1 A binding sequence and the second binding sequence is a NFKB 1 binding sequence. In some aspects, the first activator sequence is a HIF1A binding sequence and the second binding sequence is a STAT3 binding sequence.
  • the first activator sequence is an ET SI binding sequence and the second binding sequence is a Myc binding sequence
  • the first activator sequence is an ETS1 binding sequence and the second binding sequence is a HIF1A binding sequence
  • the first activator sequence is an ETS1 binding sequence and the second binding sequence is a SP1 binding sequence.
  • the first activator sequence is an ETS1 binding sequence and the second binding sequence is a NFKB 1 binding sequence.
  • the first activator sequence is an ETS1 binding sequence and the second binding sequence is a STAT3 binding sequence.
  • the first activator sequence is aNFATCl binding sequence and the second binding sequence is a HIF1A binding sequence. In some aspects, the first activator sequence is a NFATC1 binding sequence and the second binding sequence is a Myc binding sequence.
  • the first activator sequence is a NFKB1 binding sequence and the second activator sequence is a STAT3 binding sequence.
  • the enhancer is a natural enhancer.
  • the enhancer is an engineered enhancer.
  • the enhancer is identified by a motif analysis and tiling (e.g., for transcription factors) for the genomic region proximal (e.g., upstream) to the hTERT promoter, e.g., by using cancer genomic databases.
  • the enhancers are cancerspecific enhancers.
  • the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • the polynucleotide comprises a synthetic promoter sequence derived from a naturally occurring human telomerase reverse transcriptase (hTERT) gene sequence (SEQ ID NO: 188).
  • hTERT human telomerase reverse transcriptase
  • the synthetic promoter sequence comprises additional copies of c- Myc and Spl binding sites relative to a naturally occurring hTERT sequence.
  • the polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3-185, or any one of the sequences in Table 1. [0174] In some aspects, the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 3-185, or any one of the sequences in Table 1. [0175] In some aspects, the polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 3-185, or any one of the sequences in Table 1. [0176] In some aspects, the polynucleotide comprises a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 3-185, or any one of the sequences in Table 1.
  • the polynucleotide comprises a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • the polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 18, 27, 33, 35, 60, 79, 82, 95, 126, 143, 145, 148, 152, 154, 156, 158, 160, 161, 162, 175, 176, and 177.
  • the nucleotide sequence of interest encodes a therapeutic protein.
  • the synthetic promoter sequence or the polynucleotide comprises an enhancer sequence.
  • the enhancer sequence is selected from the group consisting of a CMV enhancer, a SV40 enhancer or combinations thereof.
  • the enhancer sequence comprises a synthetic enhancer disclosed herein.
  • Certain aspects of the disclosure are directed to synthetic enhancer sequences.
  • the synthetic enhancer sequences have been derived from cancer-specific enhancer sequence.
  • the synthetic enhancer sequence have been bioinformatically designed to include naturally occurring mutations in aggressive cancers.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to any one of SEQ ID NOs: 195-254 or 261-354, any one of Sequences 189-194 or 255-260, any one of the sequences in Table 3, or any combination of sequences thereof.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 195-254 or 261-354, any one of Sequences 189-194 or 255-260, any one of the sequences in Table 3, or any combination of sequences thereof.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 311.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 311.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 312.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 312.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 315.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 315.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 315.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 316.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 317.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 317.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 317.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 318.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 318.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 318.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 320.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 320.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 322.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 323.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 323.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 323.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 324.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 324.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 324.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 325.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 325.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 326.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 326.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 326.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 327.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 327.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 328.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 328.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 328.
  • the synthetic enhancer sequence comprises a nucleotide sequence corresponding to SEQ ID NO: 332.
  • the synthetic enhancer sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 332.
  • the expression construct comprises a synthetic hTERT promoter disclosed herein operably linked to a payload (e.g., a nucleic acid of interest encoding a therapeutic protein).
  • a payload e.g., a nucleic acid of interest encoding a therapeutic protein
  • the pA sequence is a synthetic pA sequence.
  • the expression construct comprises any of the nucleic acid sequences shown in Table 4.
  • the polynucleotides can be complexed with polycationic substances such as poly-L-lysine or DEAC-dextran, targeting ligands, and/or DNA binding proteins (e.g., histones).
  • DNA- or RNA-liposome complex formulations comprise a mixture of lipids which bind to genetic material (DNA or RNA) and facilitate delivery of the nucleic acid into the cell.
  • Liposomes which can be used in accordance with the disclosure include DOPE (dioleyl phosphatidyl ethanol amine), CUDMEDA (N-(5-cholestrum-3-P-ol 3-urethanyl)- N',N'- dimethylethylene diamine).
  • AAV a parvovirus belonging to the genus Dependovirus
  • AAV has several attractive features not found in other viruses. For example, AAV can infect a wide range of host cells, including non-dividing cells. Furthermore, AAV can infect cells from different species. Importantly, AAV has not been associated with any human or animal disease, and does not appear to alter the physiological properties of the host cell upon integration. Finally, AAV is stable at a wide range of physical and chemical conditions, which lends itself to production, storage, and transportation requirements.
  • the AAV vector can comprise one or more filler sequences between one of more regions of the AAV vector.
  • the filler region can be located before a region such as, but not limited to, a payload region, an ITR, a promoter region, an intron region, an enhancer region, and/or a polyadenylation signal sequence region.
  • the filler region can be located after a region such as, but not limited to, a payload region, an ITR, a promoter region, an intron region, an enhancer region, and/or a polyadenylation signal sequence region.
  • the filler region can be located before and after a region such as, but not limited to, a payload region, an ITR, a promoter region, an intron region, an enhancer region, and/or a polyadenylation signal sequence region.
  • AAV particles are produced in mammalian cells wherein all three VP proteins are expressed at a stoichiometry approaching 1 : 1 : 10 (VP1 :VP2:VP3).
  • the regulatory mechanisms that allow this controlled level of expression include the production of two mRNAs, one for VP1, and the other for VP2 and VP3, produced by differential splicing.
  • compositions are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.

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Abstract

La présente invention concerne une séquence de promoteur synthétique dérivée d'une séquence de promoteur de transcriptase inverse de la télomérase humaine (hTERT).
PCT/US2023/084083 2022-12-16 2023-12-14 Promoteurs de htert synthétiques et utilisations associées WO2024130007A2 (fr)

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