EP4698662A2 - Expression régulable et contrôlable d'un transgène chez des primates - Google Patents

Expression régulable et contrôlable d'un transgène chez des primates

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Publication number
EP4698662A2
EP4698662A2 EP24793343.5A EP24793343A EP4698662A2 EP 4698662 A2 EP4698662 A2 EP 4698662A2 EP 24793343 A EP24793343 A EP 24793343A EP 4698662 A2 EP4698662 A2 EP 4698662A2
Authority
EP
European Patent Office
Prior art keywords
antibody
inducer
administered
artemisinin
transgene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24793343.5A
Other languages
German (de)
English (en)
Inventor
Graham WHYTESIDE
Victoria Fiona TORRANCE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AskBio Inc
Original Assignee
AskBio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AskBio Inc filed Critical AskBio Inc
Publication of EP4698662A2 publication Critical patent/EP4698662A2/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

  • Indications that have approved treatments include spinal muscular atrophy (Zolgensma, Norvartis), lymphoma (Kymriah, Novartis), Haemophilia A and B (Roctavin and Hemgenix, CSL Behring LLC) and RPE65 mutation-associated retinal dystrophy (Luxterna).
  • the key component of GT is the delivery of genetic material to a cell; however, in early GT experiments it was discovered that this had the potential to cause toxicity at a cellular and systemic level. Therefore, innovations to improve the expression cassette such as reducing CpG content, codon optimisation, reduction of viral components and tissue specific promoters have been vital in enabling the development of safe and efficacious therapies.
  • the flavonoid compound is administered to the subject, e.g., primate subject following the prior administration of an inducer to the subject, e.g., primate subject, and optionally, the inducer is administered in an amount that is sufficient to express a measurable level of the transgene in the subject.
  • the inducer of the prior administration is different from the flavonoid compound administered to the subject.
  • the inducer of the prior administration is same as the flavonoid compound administered to the subject but at a different dose.
  • the first and second inducer are independently artemisinin, a derivative of artemisinin, or a flavonoid compound. In some embodiments, the first and second inducer are independently artemisinin or a derivative of artemisinin, [0011] It is noted that the first and second inducers can be same or different.
  • the first inducer is artemisinin or a derivative thereof.
  • the first inducer is artemisinin, artesunate, dihydroartemisinin, artemether or artemether.
  • the first inducer is artemisinin or artesunate.
  • the first inducer is a flavonoid compound.
  • the first inducer is flavone.
  • the first inducer is flavone.
  • the second inducer is artemisinin; derivative of artemisinin; a flavonoid compound, e.g. flavone, chrysin, baicalein, or galangin; l,4-Bis[2-(3,5- dichloropyridyloxy)]benzene (TCPOBOP); phenobarbital (PB); 6-(4Chlorophenyl)imidazo[2,l- b][l,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO); Acetaminophen; Buprenorphine; Phenytoin; Carbamazepine; Valproic Acid; Chlorpromazine; Efavirenz; Nevirapine; Rilpivirine; Etravirine; Diazepam; Cyclophosphamid; Ifosfamide; Cerivastatin; Simvastatin; lovastatin; substituted s
  • the second inducer is artemisinin or a derivative thereof.
  • the second inducer is artemisinin, artesunate, dihydroartemisinin, artemether or artemether.
  • the second inducer is artemisinin or artesunate.
  • the second inducer is a flavonoid compound.
  • the second inducer is flavone.
  • the second inducer is flavone.
  • the third inducer or a subsequent inducer is artemisinin; derivative of artemisinin; a flavonoid compound, e.g. flavone, chrysin, baicalein, or galangin; 1,4- Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP); phenobarbital (PB); 6-
  • the third or a subsequent inducer is artemisinin or a derivative thereof.
  • the or a subsequent third inducer is artemisinin, artesunate, dihydroartemisinin, artemether or artemether.
  • the third or a subsequent inducer is artemisinin or artesunate.
  • the third or a subsequent inducer is a flavonoid compound.
  • the third or a subsequent inducer is flavone.
  • the third inducer is flavone.
  • the time between administrations is at least 24 hours, 48 hours, 72 hours, 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks. In some embodiments of any of the aspects, the time between administrations is at least 24 hours, 48 hours, or 72 hours. In some embodiments of any of the aspects, the time between administrations is no more than 2 months, 3 months, 4 months, 5 months, or 6 months. In some embodiments of any of the aspects, the time between administrations is no more than 2 months.
  • the time between administrations is 5-7 days, 5-10 days, 7-10 days, 7-14 days, 10-14 days, 10-20 days, 14-21 days, 14-28 days, 21-28 days, or 21-34 days.
  • the time between administration is about 24 hours.
  • an inducer is administered the subject every day (e.g., about every 24 hours).
  • an inducer is administered the subject every day (e.g., about every 24 hours) for a period of about one week to about 6 months.
  • an inducer is administered the subject every day (e.g., about every 24 hours) for a period of about two weeks to about 3 months.
  • an inducer is administered to the subject every day (e.g., about every 24 hours) for a period of about two weeks, about three weeks, about 4 weeks, about 5 weeks or about 6 weeks.
  • an inducer is administered the subject every day (e.g., about every 24 hours) for a period of about 15 days, about 30 days, about 40 days, about 45 days, about 50 days, about 65 days, about 75 days, about 80 days, or about 90 days.
  • an inducer is administered to the subject every day (e.g., about every 24 hours) for a period of about 15 days, about 30 days, about 40 days, about 45 days, about 50 days, about 65 days, about 75 days, about 80 days, or about 90 days.
  • continuous dosing shows no toxicity in the subject.
  • an inducer is administered to the subject every day (i.e., continuous dosing) for a period of about 15 days, about 30 days, about 40 days, about 45 days, about 50 days, about 65 days, about 75 days, about 80 days, or about 90 days, and the subject does not show signs of toxicity. It is noted that the same inducer can be administered every day or different inducers can be administered on different days. Further, amount of the inducer can be same or different on different days.
  • the inducer at the second time point e.g., second inducer
  • a subsequent time point e.g. third inducer
  • the inducer at the second time point is administered when it is determined that the level of expression of the transgene is decreased to 75% or less of the level of expression of the transgene within the 24 hours after administering at the first time point (e.g., the first inducer), or the previous time point (e.g., second time inducer).
  • the inducer at the second time point e.g., second inducer
  • a subsequent time point e.g.
  • the inducer at the second time point e.g., second inducer
  • a subsequent time point e.g. third inducer
  • the inducer at the second time point is administered when it is determined that the level of expression of the transgene is decreased to a background level of expression of the transgene following the 24 hours after administering at the first time point (e.g., the first inducer), or the previous time point (e.g., second time inducer).
  • the inducer at the second time point e.g., second inducer
  • a subsequent time point e.g. third inducer
  • the inducer at the second time point is administered when it is determined that the level of expression of the transgene is decreased to 25% or less of the level of expression of the transgene within the 24 hours after administering at the first time point (e.g., the first inducer), or the previous time point (e.g., second time inducer).
  • the artemisinin derivative is of Formula (I) described herein, or a pharmaceutically acceptable salt thereof.
  • the artemisinin derivative is a compound of Formula (I), (I- A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • the artemisinin derivative is artesunate, dihydroartemisinin, artesunate, or artemether.
  • the artemisinin derivative is artesunate, or dihydroartemisinin.
  • the flavonoid compound is flavone.
  • the first and second inducer or a subsequent inducer are administered at about the same amount. For example, dose of the inducer in successive administrations remains same.
  • the first inducer and the second inducer are administered at different amounts.
  • the second inducer (or a subsequent inducer) is administered at a lower amount relative to the amount of the first inducer (or the previous inducer), e.g., dose of the inducer in successive administrations is reduced from its prior administration.
  • the second inducer (or a subsequent inducer) is administered in amount about 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, 1/15, 1/20, 1/25, 1/30, 1/35, 1/40, 1/45, 1/50, 1/55, 1/60, 1/65, 1/70, 1/75, 1/80, 1/85, 1/90, 1/95, 1/100, 1/125, 1/150, 1/175, 1/200, 1/500, 1/1000 or even less of the amount of first the inducer (or the previous inducer).
  • the second inducer (or a subsequent inducer) is administered in amount about 1/2, 1/4, 1/5, 1/8, 1/10, 1/20, 1/40, 1/80 or less of the amount of the first inducer (or the prior inducer).
  • the second inducer (or a subsequent inducer) is administered at a higher amount relative to the amount of the first inducer (or the previous inducer).
  • the amount of inducer administered at the second time point (or a subsequent time point) is at least about 25%, 50%, about 75%, about 1-fold, about 1.2-folds, about 1.25-folds, about 1.3-folds, about 1.5-folds, about 1.75-folds, about 2-folds, about 2.25-folds, about 2.5-folds, about 2.5-folds, about 2.75-folds, about 3-folds, 4-fold, 5-fold, 6-folds, 7-fold, 8-fold, 9-folds, 10-folds or higher than the amount of the first inducer (or previous time point).
  • the first inducer is administered at a dose from about 1 mg/kg to about 100 mg/kg.
  • the first inducer is administered at a dose of about 80mg/kg, about 70mg/kg, about 60mg/kg, about 50mg/kg, about 40mg/kg, about 30mg/kg, about 20mg/kg, about lOmg/kg, about 6mg/kg, about 5mg/kg, or about 1 mg/kg.
  • the third inducer is administered at a dose from about 1 mg/kg to about 100 mg/kg.
  • the third inducer is administered at a dose of 80mg/kg, about 70mg/kg, about 60mg/kg, about 50mg/kg, about 40mg/kg, about 30mg/kg, about 20mg/kg, about lOmg/kg, about 6mg/kg, about 5mg/kg, or about Img/kg.
  • the first inducer is administered at a dose of about 10 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point. In some embodiments, the first inducer is administered at a dose of about 6 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point. In some embodiments, the first inducer is administered at a dose of about 1 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point. In some embodiments, the second inducer is administered at a dose of about 80 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less. In some embodiments, the second inducer is administered at a dose of about 40 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • the second inducer is administered at a dose of about 20 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less. In some embodiments, the second inducer is administered at a dose of about 10 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less. In some embodiments, the second inducer is administered at a dose of about 6 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less. In some embodiments, the second inducer is administered at a dose of about 1 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • each dose is no more than 10 mg/kg.
  • the second dose, and/or a subsequent dose e.g., third dose
  • the first dose, the second dose, and/or a subsequent dose is 5 mg/kg or greater.
  • the first dose is 1-80 mg/kg
  • the second dose is 1-40 mg/kg
  • a subsequent dose e.g., third dose 1-40 mg/kg.
  • the second dose and subsequent dose is 1-10 mg/kg.
  • the first dose and each subsequent dose is independently 1 mg/kg to 80 mg/kg. In some embodiments of any of the aspects, the first dose and each subsequent dose is independently 5 mg/kg to 80 mg/kg. In some embodiments of any of the aspects, the second dose and subsequent dose (e.g., third dose) is independently 5 mg/kg to 20 mg/kg. In some embodiments of any of the aspects, the second dose and subsequent dose (e.g., third dose) is independently 5 mg/kg to 10 mg/kg. [0036] In some embodiments, the second time point is at least two days after the first time point. For example, the second time point is at least one month after the first time point, such as the second time point is at least two months of the first time point.
  • the expression of the transgene increases for a predetermined time after each administration. In some embodiments of any of the aspects, the expression of the transgene increases for a predetermined time after each administration and the magnitude of the increase is positively correlated with the size of the dose.
  • the method further comprises a step of determining the expression level of the transgene after administering the first, second, or subsequent (e.g., third) inducer.
  • the method further comprises a step of determining the expression level prior of the transgene to administering the first, second, or subsequent (e.g., third) inducer.
  • the method further comprises administering the exogenous nucleotide sequence to the subject prior to administering the first, second, or subsequent (e.g., third) inducer.
  • the method further comprises administering the exogenous nucleotide sequence to the subject prior to administering the first inducer.
  • the method is a method of therapy, optionally, the method of therapy is a method of gene therapy.
  • the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is a viral vector.
  • the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is adeno-associated viral (AAV), lentiviral, adenoviral, herpes viral, or hepatitis viral vector.
  • the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV vector, e.g., an AAV gene therapy vector.
  • the first and second inducers independently are administered orally or are intraperitoneally. In some embodiments, at least one of the first and second inducer is administered orally. For example, if the inducer is artemisinin or a derivative thereof, the artemisinin or a derivative thereof is administered orally. In some embodiments, the second inducer is administered orally. In some embodiments, the first inducer is orally. In some embodiments, both the first inducer and second inducer are administered orally.
  • the first and second inducer is administered intraperitoneally.
  • the inducer is artemisinin or a derivative thereof
  • the artemisinin or a derivative thereof is administered intraperitoneally.
  • the second inducer is administered intraperitoneally.
  • the first inducer is intraperitoneally.
  • both the first and second inducer are administered intraperitoneally.
  • artemisinin or a derivative of artemisinin for use as an inducer in a method of regulating expression of a transgene in a subject, e.g., a primate subject, wherein the subject, e.g., the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the synthetic inducible promoter comprises a CRE that is capable of being bound and activated by a heterodimer of CAR and RXR, wherein the inducer is administered to the subject at a dose which comprises an amount of the inducer that is sufficient to express a therapeutically effective level of the transgene in the subject.
  • a flavonoid compound for use as an inducer in a method of regulating expression of a transgene in a subject, e.g., a primate subject, wherein the subject, e.g., the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the synthetic inducible promoter comprises a CRE that is capable of being bound and activated by a heterodimer of CAR and RXR, wherein the inducer is administered to the subject at a dose which comprises an amount of the inducer that is sufficient to express a therapeutically effective level of the transgene in the subject.
  • a flavonoid compound e.g., flavone
  • the flavonoid compound e.g., flavone
  • the subject e.g., the primate subject following the prior administration of an inducer to the subject, e.g., the primate subject.
  • the inducer of the prior administration is a different inducer, or the inducer of the prior administration is the same inducer but at a different dose.
  • a first induction with artemisinin or a derivative thereof increases the transgene expression level to a maximum at about 24 hrs and declines thereafter, wherein, second induction and/or, successive inductions with artemisinin or a derivative thereof has similar induction profile as seen with the first induction, for at least about 1 month, or at least about 2 months, or, at least about 3 months, or, at least about 5 months or, at least about six months or, even more, and optionally, the method further comprises single administration of exogenous nucleotide sequence encoding the transgene operatively linked to the synthetic inducible promoter prior to first administering the inducer to induce said first induction.
  • flavonoid compound is administered at a dose of from about O.lmg/kg to 80mg/kg, from about O.lmg/kg to about 70mg/kg, from about O.lmg/kg to about 60mg/kg, from about O.lmg/kg to about 50mg/kg, from about O.lmg/kg to about 40mg/kg, from about O.lmg/kg to about 30mg/kg, from about O.lmg/kg to about 20mg/kg, from about 0. Img/kg to about lOmg/kg, from about 0. Img/kg to about 6mg/kg, from about O.lmg/kg to about 5mg/kg, or from about O.lmg/kg to about Img/kg.
  • a first induction with the flavonoid compound increases the transgene expression level to a maximum at about 24 hrs and declines thereafter, wherein, second induction and/or, successive inductions with the flavonoid compound has similar induction profile as seen with the first induction, for at least about 1 month, or at least about 2 months, or, at least about 3 months, or, at least about 5 months or, at least about six months or, even more, and optionally, the method further comprises single administration of exogenous nucleotide sequence encoding the transgene operatively linked to the synthetic inducible promoter prior to first administering the inducer to induce said first induction.
  • the transgene expression level at about 24 hrs in the first induction, or, second induction, or successive inductions is at least about 200-fold higher than that of prior to administering the inducer for the first time, or, the second time, or, any number of successive administrations.
  • the first induction, the second induction, and/or, the successive inductions are not complete until about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, about 144 hours, about 150 hours, 160 hours, about 168 hours, about 192 hours, about 216 hours, about 240 hours, about 264 hours, or about 290 hours after administering the inducer.
  • the artemisinin or a derivative there of is administered at a dose from about 0.5 mg/kg to about 150mg/kg.
  • artemisinin or a derivative there of is administered at a dose from about 1 mg/kg to about 100 mg/kg, from about 1 mg/kg to about 80 mg/kg, from about 1 mg/kg to about 40 mg/kg, from about 1 mg/kg to about 20 mg/kg, or from about 1 mg/kg to about 10 mg/kg.
  • the artemisinin or a derivative there of is administered at a dose of about 80mg/kg, about 70mg/kg, about 60mg/kg, about 50mg/kg, about 40mg/kg, about 30mg/kg, about 20mg/kg, about lOmg/kg, about 6mg/kg, about 5mg/kg, or about Img/kg.
  • the transgene encodes an antibody or antigen binding portion an antibody, a coagulation factor, a cytokine, a hormone, a growth factor, a virus protein, a cell receptor ligand, a chimeric antigen receptor (CAR) or a CRISPR-Cas nuclease.
  • the transgene encodes an antibody, optionally selected from the group consisting of an anti-TNFa, anti-PDl antibody, anti-HER2 antibody, anti-PCSK9 antibody, anti-VEGFR2 antibody, anti-IL17 antibody, anti-A03-42 antibody, or anti-Sema3A antibody or an antigen binding fragment thereof.
  • FIG. 1 is schematic representation of PK/PD of artemisinin and some exemplary derivatives thereof.
  • FIG. 2 is a bar-graph showing the induction of an exemplary inducible promoter by artemisinin, various exemplary derivatives of artemisinin and other compounds. Structures of artemisinin, exemplary derivatives of artemisinin, and other compounds are also shown.
  • FIG. 3A is a schematic representation of protocols for in vivo short-term induction in male mice via IP injection.
  • FIGS. 5B and 5C show live imaging (FIG. 5B) and ex vivo imaging (FIG. 5C) of differences in induction between administration via oral gavage and IP injection.
  • FIG. 6 shows dose response in female mice via oral gavage administration.
  • a method of regulating expression of a transgene in a primate subject comprises an exogenous nucleotide sequence comprising an inducible promoter.
  • the inducible promoter is operably linked to a transgene and comprises a cis-regulatory element (CRE) that is capable of being bound and activated by a heterodimer of CAR and RXR.
  • CRE cis-regulatory element
  • the data presented herein show that once the administration of the inducer is stopped, the expression of the transgene goes down to a background expression level rapidly. Further, the background expression level remains relatively steady even after repeated administration of the inducer. This is contrast to the current methods in the art. With the current methods in the art, the background expression levels are seen to be significantly higher even after induction has been stopped for a period of more than one, two, three, four or more weeks. In contrast, in the methods described herein, the expression of the transgene goes down to a background expression level in less than a week once the administration of the inducer is stopped.
  • the background expression level can be an expression level of the transgene prior to first induction or first administration of the inducer.
  • the transgene expression level after the first or a subsequent administration is at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400- fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 1100-fold, at least 1200-fold, at least 1300-fold, at least 1400-fold, at least 1500- fold, at least 1600-fold, at least 1700-fold, at least 1800-fold, at least about 1900-fold, at least 1000- fold, or higher than the level prior to the first or subsequent administration.
  • the transgene expression level at about 24 hours after the first or a subsequent administration is at least about 25-fold, at least about 50 fold, at least about 75- fold, at least about 100-fold, at least about 125-fold, at least about 150-fold, at least about 175-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 1100-fold, at least about 1200-fold, at least about 1300-fold, at least about 1400-fold, at least about 1500-fold, at least about 1600-fold, at least about 1700-fold, at least about 1800-fold, at least about 1900-fold, at least about 1000-fold, or higher than the level prior to the first or subsequent administration.
  • the transgene expression level at about 24 hours after the first or a subsequent administration is at least 25-fold, at least 50 fold, at least 75-fold, at least 100-fold, at least 125-fold, at least 150-fold, at least 175-fold, at least 200- fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 1100-fold, at least 1200-fold, at least 1300- fold, at least 1400-fold, at least 1500-fold, at least 1600-fold, at least 1700-fold, at least 1800-fold, at least about 1900-fold, at least 1000-fold, or higher than the level prior to the first or subsequent administration.
  • the second time point, or any subsequent time point is at least 12 hours after the inducer administration at the first time point, or the previous time point.
  • the second time point, or any subsequent time point is at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 66 hours, or at least 72 hours after the first time point, or the previous time point.
  • the second time point, or any subsequent time point is at least at least 1 week after the first time point, or the previous time point. In some embodiments, where more than one administration of the inducer(s) is administered, the second time point, or any subsequent time point is at least at least 2 weeks after the first time point, or the previous time point.
  • the inducer (or inducers) is administered two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) times.
  • the second time point, or any subsequent time point is at least after one week, at least two weeks, at least three weeks, at least four weeks, five weeks, six weeks, seven weeks, eight weeks or more after the first time point, or the previous time point.
  • the subsequent administration is based upon need of the subject such as a flare-up of a condition the transgene expression product treats.
  • a particular antibody such as a TNF-a antibody.
  • Some exemplary non-limiting examples where the subsequent administration is based upon need of the subject include, but are not limited to inducing anti-inflammatory antibodies when there is a flare up of the inflammatory condition, inducing a transgene product that has on-target toxicity; inducing transient expression of anti-cancer biologic, e.g., antibody, bispecific, or cytokine; inducing GLP1 for diabetes and/or, weight loss; inducing transient expression of growth factors to initiate nerve or other tissue regeneration in acute spinal cord injury or other injuries.
  • anti-cancer biologic e.g., antibody, bispecific, or cytokine
  • GLP1 for diabetes and/or, weight loss
  • transient expression of growth factors to initiate nerve or other tissue regeneration in acute spinal cord injury or other injuries.
  • the second time point, or any subsequent time point is at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, eleven months, a year or longer after the first time point, or the previous time point.
  • the second time point, or a subsequent time point is within two months of the first time point, or the previous time point.
  • the second time point, or any subsequent time point is within one month of the first time point, or the previous time point.
  • the second time point, or any subsequent time point is within seven weeks, within six weeks, within five weeks, within four weeks, within three weeks or within two weeks of the first time point, or the previous time point. Subsequent administrations can be longer.
  • the second time point, or a subsequent time point is within 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days of the first time point, or the previous time point. In some embodiments of any one of the aspects described herein, where more than one administration of the inducer(s) is administered, the second time point, or any subsequent time point is 96 hours, 90 hours, 84 hours, or 78 hours of the first time point, or the previous time point.
  • the second time point, or a subsequent time point is within 72 hours, within 66 hours, within 60 hours, within 54 hours, within 48 hours, within 42 hours, or within 36 hours of the first time point, or the previous time point.
  • the second time point, or a subsequent time point is within 12-36 hours, e.g., within 20-28 hours of the first time point, or the previous time point.
  • the second time point, or a subsequent time point is within about 24 hours of the first time point, or the previous time point.
  • the second time point, or a subsequent time point is least 24 hours after and within two weeks of the first time point in a given regimen, or the previous time point.
  • the second time point, or any subsequent time point is at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 66 hours, or at least 72 hours after and within 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days of the first time point, or the previous time point.
  • the second time point, or any subsequent time point is at least after 36 hours, e.g., at least 42 hours or at least 48, at least 54 hours, at least 66 hours, at least 72 hours, at least 80 hours, at least 88 hours or at least 96 hours of the first time point, or the previous time point.
  • the second time point, or any subsequent time point is at least 36 hours after the first time point, or the previous time point.
  • the second time point, or any subsequent time point is least 48 hours after or at least 72 hours after the first time point, or the previous time point.
  • the time between administrations is 5-7 days. In some embodiments, where more than one administration of the inducer(s)is administered, the time between administrations is 5-10 days.
  • the time between administrations is 7-10 days. In some embodiments, where more than one administration of the inducer(s)is administered, the time between administrations is 7-14 days. In some embodiments, where more than one administration of the inducer(s)is administered, the time between administrations is 10-14 days. In some embodiments, where more than one administration of the inducer(s)is administered, the time between administrations is 10-20 days. In some embodiments, where more than one administration of the inducer(s)is administered, the time between administrations is 14-21 days. In some embodiments, where more than one administration of the inducer(s)is administered, the time between administrations is 14-28 days. In some embodiments, where more than one administration of the inducer(s)is administered, the time between administrations is 21-28 days. In some embodiments, where more than one administration of the inducer(s)is administered, the time between administrations is 21-34 days.
  • Times of administration can be periodic, as needed, or pulsed. This permits tailoring of both administration and dosing amounts. Administration is designed to prevent accumulation of proteins in a cell that can be detrimental to the cell.
  • the at least second dose or at least third dose are administered when it is determined that the level of expression of the transgene is decreased to 75% or less, or 50% or less, or 25% or less of the level of expression of the transgene within the 24 hours after the first dose. In some embodiments, the at least second dose or at least third dose are administered when it is determined that the level of expression of the transgene is decreased to a background level of expression of the transgene. In some embodiments, the at least second dose or at least third dose are administered when it is determined that the level of expression of the transgene is decreased to a level of expression of the transgene prior to the first dose.
  • the inducer is administered in an amount sufficient to induce expression of the transgene from the expression cassette.
  • the inducer is administered at a dose from about 50 ⁇ g/kg to about 1000 mg/kg.
  • the inducer (or inducers) is used or given at a dose from about 75 ⁇ g/kg to about 900 mg/kg, from about 100 ⁇ g/kg to about 800 mg/kg, from about 150 ⁇ g/kg to about 700 mg/kg, from about 200 ⁇ g/kg to about 600 mg/kg, from about 250 ⁇ g/kg to about 250 mg/kg, from about 250 ⁇ g/kg to about 50 mg/kg, from about 100 ⁇ g/kg to about 1 mg/kg, or from about 1 mg/kg to about 80 mg/kg.
  • the inducer (or inducers) is administered at a dose from about 1 mg/kg to about 40 mg/kg.
  • the inducer is administered at a dose from about 1 mg/kg to about 20 mg/kg, from about 1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 6mg/kg. In some embodiments, the inducer (or inducers) is administered at a dose from about 1 mg/kg to about 80 mg/kg.
  • an amount of inducer(s) administered at the second time point, or any subsequent time point can be about the same or different from the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the second or subsequent dose of the inducer(s) can be determined using a dose-dependent relationship between the first or preceding dose of inducer(s) and the level of transgene expression from the expression cassette to achieve the desired level of expression of the transgene. For example, if the second administration, or any subsequent administration, is within a week or less of the prior administration, in one embodiment, the second or subsequent dose can be at a significant lower dose than the first dose, or the previous dose.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is about the same as the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is within about 10% (wt/wt or mol/mol) of the amount of the inducer administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is within about 7.5%, about 5%, about 2.5%, about 2%, about 1.5%, about 1%, or about 0.5%, (wt/wt or mol/mol) of the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is lower than the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is at least about 10% (wt/wt or mol/mol) lower than the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is at least about 50% (wt/wt or mol/mol) lower than the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is about 55%, at about 60%, about 65%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% (wt/wt or mol/mol) lower than the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is about half or less than half of the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is about 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, 1/15, 1/20, 1/25, 1/30, 1/35, 1/40, 1/45, 1/50, 1/55, 1/60, 1/65, 1/70, 1/75, 1/80, 1/85, 1/90, 1/95, 1/100, 1/125, 1/150, 1/175, 1/200, 1/500, 1/1000 or less of the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is about 1/2, 1/4, 1/5, 1/8, 1/10, 1/20, 1/40, 1/80 or less than the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s)administered at the second time point, or any subsequent time point is decreased by about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg or more compared to the amount of the inducer(s)administered at the first time point, or the previous time point. In some embodiments, the amount of inducer(s)administered at the second time point, or any subsequent time point is decreased by 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg or more compared to the amount of the inducer(s)administered at the first time point, or the previous time point.
  • the decreased subsequent dose is sufficient to attain the predetermined level of transgene expression.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is higher than the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is at least about 10% (wt/wt or mol/mol) higher than the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at the second time point, or any subsequent time point is at least 25%, 50%, about 75%, about 1-fold, about 1.2-folds, about 1.25- folds, about 1.3-folds, about 1.5-folds, about 1.75-folds, about 2-folds, about 2.25-folds, about 2.5- folds, about 2.5-folds, about 2.75-folds, about 3-folds, 4-fold, 5-fold, 6-folds, 7-fold, 8-fold, 9- folds, 10-folds or higher than the amount of the inducer(s) administered at the first time point, or the previous time point.
  • the amount of inducer(s) administered at each subsequent time point is lower than the amount of the inducer(s) administered at an immediate previous time point and the amount of inducer(s) administered at the last time point is higher than the amount of the inducer(s) administered at the immediate previous time point.
  • the inducer (or inducers) is administered in an amount sufficient to induce expression of the transgene from the expression cassette.
  • the inducer (or inducers) is administered at a dose from about 50 ⁇ g/kg to about 1000 mg/kg.
  • the inducer (or inducers) is used or given at a dose from about 75 ⁇ g/kg to about 900 mg/kg, from about 100 ⁇ g/kg to about 800 mg/kg, from about 150 ⁇ g/kg to about 700 mg/kg, from about 200 ⁇ g/kg to about 600 mg/kg, from about 250 ⁇ g/kg to about 250 mg/kg, from about 250 ⁇ g/kg to about 50 mg/kg, from about 100 ⁇ g/kg to about 1 mg/kg, or from about 1 mg/kg to about 80 mg/kg.
  • the inducer (or inducers) is administered at a dose from about 1 mg/kg to about 40 mg/kg.
  • the inducer is administered at a dose from about 1 mg/kg to about 20 mg/kg, from about 1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 6mg/kg. In some embodiments, the inducer (or inducers) is administered at a dose from about 1 mg/kg to about 80 mg/kg
  • the inducer(s) is administered at the second time point, or any subsequent time point at a dose from about 1 mg/kg to 80 mg/kg.
  • the inducer(s) can be administered at a dose of about 1 mg/kg, about 5 mg/kg, about 6 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, or about 80 mg/kg.
  • the inducer(s) is administered at the first time point, or the previous time point at a dose from about 0.5 mg/kg to 15 mg/kg.
  • the inducer(s) is administered at the first time point, or the previous time point at a dose from about 0.75 mg/kg to about 15 mg/kg or from about 1 mg/kg to 10 mg/kg.
  • the inducer(s) is administered at the first time point, or the previous time point at a dose of about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, about 10 mg/kg, about 10.5 mg/kg, about 11 mg/kg, about 11.5 mg/kg, about 12 mg/kg, 12.5 mg/kg, about 13 mg/kg, about 13.5 mg/kg, about 14 mg/kg, about 14.5 mg/kg, or about 15 mg/kg.
  • the inducer(s) is administered at each administration at a dose of no more than about 10 mg/kg.
  • the inducer(s) is administered at each administration at a dose of no more than about 10 mg/kg, no more than lOmg/kg, no more than about 5 mg/kg, or no more than 5 mg/kg.
  • the first dose, second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of at least about 5 mg/kg.
  • the first dose, second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of at least about 5 mg/kg, of at least 5 mg/kg, of at least about 6 mg/kg, or at least 6 mg/kg.
  • the second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of no more than about 10 mg/kg.
  • the second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of no more than about 10 mg/kg, no more than lOmg/kg, no more than about 5 mg/kg, or no more than 5 mg/kg.
  • the second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of about 10 mg/kg to about 80 mg/kg. In some embodiments, the second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of 10 mg/kg to 80 mg/kg. In some embodiments, the second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of about 5 mg/kg to about 20 mg/kg. In some embodiments, the second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of 5 mg/kg to 20 mg/kg.
  • the second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of about 5 mg/kg to about 10 mg/kg. In some embodiments, the second dose, third dose, and/or subsequent doses of the inducer(s) are administered at a dose of 5 mg/kg to 10 mg/kg.
  • the first dose is 1-80 mg/kg
  • the second dose is 1-40 mg/kg
  • the subsequent dose e.g., third dose
  • the first dose is 1-80 mg/kg
  • the second dose is 1-40 mg/kg
  • the subsequent dose is 1-40 mg/kg.
  • the administering comprises: administering a plurality of doses of a first inducer during a period of 2 to 4 weeks; administering 1 or 2 doses of a second inducer; and administering at least 1 dose of a third inducer.
  • the administering comprises: administering a plurality of doses of a first inducer during a period of 2 to 4 weeks, wherein each dose of the first inducer is 1-200 mg/kg; administering 1 or 2 doses of a second inducer, wherein each dose of the second inducer is 1-100 mg/kg; administering at least 1 dose of a third inducer, wherein each dose of the third inducer is 1-80 mg/kg.
  • the administering comprises: administering a plurality of doses of a first inducer during a period of 2 to 4 weeks, wherein each dose of the first inducer is 160 mg/kg; administering 1 or 2 doses of a second inducer, wherein each dose of the second inducer is 80 mg/kg; administering at least 1 dose of a third inducer, wherein each dose of the third inducer is 40 mg/kg.
  • a dose of the third inducer is administered until the subject no longer requires the expression of the transgene.
  • the method further comprises repeating the first and/or second administering step if the subject becomes non- responsive to the inducer(s).
  • the method further comprises repeating the administration of the first inducer and/or second inducer if the subject becomes non-responsive to the third inducer.
  • the amount of inducer(s) administered to the subject can be varied over time by adjusting the dose, adjusting the time period between doses, or both.
  • the dose can be increased while the time period between doses is kept constant, the dose can be kept constant while the time period between doses is reduced, or the dose can be increased and the time period between doses is reduced.
  • the dose can be decreased while the time period between doses is kept constant, the dose can be kept constant while the time period between doses is reduced, or the dose can be decreased and the time period between doses is increased.
  • the inducer(s) is administered at the second time point, or any subsequent time point can be same as the inducer(s) administered at the first time point, or the previous time point.
  • the inducer(s) administered at the first time point, or the previous time point can be artemisinin and the inducer(s) is administered at the second time point, or any subsequent time point can be an artemisinin derivative.
  • the inducer(s) administered at the first time point, or the previous time point can be an artemisinin derivative and the inducer(s) is administered at the second time point, or any subsequent time point can artemisinin.
  • the inducer(s) administered at the first time point, or the previous time point can be a flavonoid compound
  • the inducer(s) administered at the second time point, or any subsequent time point can be same or different.
  • the inducer(s) administered at the first time point, or the previous time point can be flavone, baicalein, chrysin, galangin, (OH)4 flavone, 6-hydroxyluteolin, acacetin, acerosin, alnetin, apigenin, apiin, artocarpetin, baicalein, beta-sitosterol, biochamin A, cannaflavin A, cannflavin A, cannflavin B, cannflavin C, catechin, catechin-3 -gallate, cerrosillin, chrysoeriol, chrysoeriol, cirsilineol, cirsiliol, cirsimaritin, corymbosin, cupress
  • the inducer(s) administered at the first time point, or the previous time point can be a flavonoid compound and the inducer(s) administered at the second time point, or any subsequent time point can be artemisinin.
  • the inducer(s) administered at the first time point, or the previous time point can be can be flavone, baicalein, chrysin, galangin, (OH)4 flavone, 6-hydroxyluteolin, acacetin, acerosin, alnetin, apigenin, apiin, artocarpetin, baicalein, beta-sitosterol, biochamin A, cannaflavin A, cannflavin A, cannflavin B, cannflavin C, catechin, catechin-3 -gallate, cerrosillin, chrysoeriol, chrysoeriol, cirsilineol, cirsiliol, cirsimaritin, corymbosin,
  • the inducer(s) administered at the first time point, or the previous time point can be artemisinin and the inducer(s) is administered at the second time point, or any subsequent time point can be a flavonoid compound, e.g., flavone.
  • the inducer(s) administered at the first time point, or the previous time point can be artemisinin and the inducer(s) is administered at the second time point, or any subsequent time point can be flavone, baicalein, chrysin, galangin, (OH)4 flavone, 6-hydroxyluteolin, acacetin, acerosin, alnetin, apigenin, apiin, artocarpetin, baicalein, beta-sitosterol, biochamin A, cannaflavin A, cannflavin A, cannflavin B, cannflavin C, catechin, catechin-3 -gallate, cerrosillin, chrysoeriol, chrysoeriol, cirsilineol, cirsiliol, cirsimaritin, corymbosin, cupressuflavone, cyanidine, daidzein, datiscetin, delphinidine, digidrokempferol
  • the inducer(s) administered at the first time point, or the previous time point can be can be flavone, baicalein, chrysin, galangin, (OH)4 flavone, 6-hydroxyluteolin, acacetin, acerosin, alnetin, apigenin, apiin, artocarpetin, baicalein, beta-sitosterol, biochamin A, cannaflavin A, cannflavin A, cannflavin B, cannflavin C, catechin, catechin-3 -gallate, cerrosillin, chrysoeriol, chrysoeriol, cirsilineol, cirsiliol, cirsimaritin, corymbosin, cupressuflavone, cyanidine, daidzein, datiscetin, delphinidine, digidrokempferol , dihydroquercetin, diosmetin, diosmin,
  • the inducer(s) administered at the first time point, or the previous time point can be an artemisinin derivative and the inducer(s) is administered at the second time point, or any subsequent time point can be flavone, baicalein, chrysin, galangin, (OH)4 flavone, 6-hydroxyluteolin, acacetin, acerosin, alnetin, apigenin, apiin, artocarpetin, baicalein, beta-sitosterol, biochamin A, cannaflavin A, cannflavin A, cannflavin B, cannflavin C, catechin, catechin-3 -gallate, cerrosillin, chrysoeriol, chrysoeriol, cirsilineol, cirsiliol, cirsimaritin, corymbosin, cupressuflavone, cyanidine, daidzein, datiscetin, delphinidine, digidrokempfe
  • the inducer(s) administered at the first time point, or the previous time point can be a flavonoid compound, e.g., flavone
  • the inducer(s) administered at the second time point, or any subsequent time point can be l,4-Bis[2-(3,5- dichloropyridyloxy)]benzene (TCPOBOP); phenobarbital (PB); 6-(4Chlorophenyl)imidazo[2,l- b][l,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO); Acetaminophen; Buprenorphine; Phenytoin; Carbamazepine; Valproic Acid; Chlorpromazine; Efavirenz; Nevirapine; Rilpivirine; Etravirine; Diazepam; Cyclophosphamid; Ifosfamide; Cerivastatin; Simvastat
  • administer refers to the placement of an inducer or expression cassette into a subject by a method or route which results in at least partial localization of the inducer or the expression cassette at a desired site such that desired effect, e.g., expression of the transgene is produced.
  • An inducer or exogenous nucleotide sequence (e.g., a vector comprising the exogenous nucleotide sequence) described herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.
  • routes known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.
  • Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.
  • “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
  • administration will generally be systemic. In some other embodiments, administration will be local.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection, infusion and other injection or infusion techniques, without limitation.
  • systemic administration means the administration of a pharmaceutical composition comprising an inducer such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • the inducer administration is intraperitoneal administration.
  • the administration of the inducer(s) administered at the first time point is intraperitoneal administration.
  • the administration of the inducer(s) administered at the second time point is intraperitoneal administration.
  • the administration of the inducer(s) administered at the first time point and the administration of the inducer(s) administered at the second time point are intraperitoneal administration. In some embodiments, the administration of the subsequent inducer(s) at subsequent time point(s) is intraperitoneal administration.
  • the inducer administration is oral administration.
  • the administration of the inducer(s) administered at the first time point is oral administration.
  • the administration of the inducer(s) administered at the second time point is oral administration.
  • the administration of the inducer(s) administered at the first time point and the administration of the inducer(s) administered at the second time point are oral administration.
  • the administration of the subsequent inducer(s) at subsequent time point(s) is oral administration.
  • oral administration can be in the form of solutions, suspensions, tablets, pills, capsules, sustained- release formulations, oral rinses, powders and the like.
  • Embodiments of the various aspects described herein include an inducible promoter.
  • the inducible promoter comprises a cis-regulatory motif (CRE) that is capable of being bound and activated by a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR).
  • CRE cis-regulatory motif
  • CAR constitutive androstane receptor
  • RXR retinoid X receptor
  • CAR Constitutive androstane receptor
  • CAR-RXR heterodimer When CAR is activated (by either direct or indirect activation), it translocates to the nucleus where CAR and RXR form a heterodimer (referred to herein as “CAR-RXR heterodimer” or “CAR-RXR”) that can bind to an activate genes comprising an appropriate target sequence.
  • the CAR-RXR heterodimer binds to and induces target genes via the PB-responsive enhancer module (PBREM).
  • PBREM PB-responsive enhancer module
  • the CRE that is capable of being bound and activated by a CAR-RXR heterodimer comprises a PBREM element/enhancer, or a functional variant thereof.
  • the inducible promoter comprises at least one PBREM enhancer, e.g., a wild-type PBREM enhancer.
  • the inducible promoter comprises at least two or more (e.g., 2, 3, 4, 5 or more) PBREM enhancers.
  • the inducible promoter comprises two PBREM enhancers.
  • Exemplary PBREM sequences include, but are not limited to, mouse PBREM sequence:
  • PBREM is a 51 -bp DNA inducible enhancer consisting of two nuclear receptor DR4 motifs (NR1 and NR2) flanking a nuclear factor -1 (NF1) binding site. It has been proposed in the literature that the NR1 sequence is the binding site for the CAR-RXR heterodimer, and the remaining sequences are partially or completely redundant. A triple repeat of NR1 (was shown to remain inducible in Sueyoshi, et al. (J. BIOL. CHEM. Vol. 274,10, pp. 6043-6046, 1999).
  • parts other than NR1 may play a role in making the PBREM motif more specific and inducible (e.g., reducing background, constitutive expression and allowing from a greater magnitude of induction).
  • the NR1 motif is underlined, the NF1 motif is in italics, and the NR2 motif is in bold.
  • the CRE that is capable of being bound and activated by a CAR-RXR heterodimer comprises each of an NR1 motif, an NF1 motif, and an NR2 motif. It is noted that the NR1 motif, the NF1 motif and the NR2 motif can be present in any order. For example, the CRE comprises the motifs in the order NR1-NF1- NR2.
  • a CRE that is capable of being bound and activated by a CAR-RXR heterodimer suitably comprises at least one NR1 motif.
  • the NR1 motif comprises the nucleotide sequence TGTACT-X-TGACC[C/T] (SEQ ID NO: 3), wherein X represents any sequence which is from 3 to 6 nucleotides in length.
  • X can be 3 to 5 nucleotides in length.
  • X is 4 nucleotides in length.
  • X is 5 nucleotides in length.
  • Some exemplary nucleotide sequences for X include, but are not limited to, TTCC and TTTCC.
  • X comprises the sequence TTCC.
  • X consists of the nucleotide sequence TTCC or TTTCC.
  • nucleotides are shown within square brackets with a slash, it indicates that one of the indicated nucleotides within the square brackets is present at that location.
  • the NR1 motif comprises a nucleotide sequence that is at least 90% identical to nucleotides 3 to 18 of SEQ ID NO: 1 or SEQ ID NO: 2
  • the NR1 motif comprises a nucleotide sequence that is at least 90% identical the sequence TGTACTTTCCTGACCT (SEQ ID NO: 4, mouse NR1) or TGTACTTTCCTGACCC (SEQ ID NO: 5, human NR1).
  • the NR1 motif comprises a sequence that is at least 95% identical to SEQ ID NO: 4 or SEQ ID NO: 5.
  • the NR1 motif comprises a sequence that is at least 99% identical to SEQ ID NO: 4 or SEQ ID NO: 5 In some embodiments of any one of the aspects described herein, the NR1 motif comprises a nucleotide sequence that is at least 90% identical, preferably at least 95% identical, more preferably at least 99%, and most preferably perfectly identical to SEQ ID NO: 4 or SEQ ID NO: 5 In some embodiments of any one of the aspects described herein, the NR1 motif comprises the nucleotide sequence TGTACTTTCCTGACCN (SEQ ID NO: 6) or CTGTACTTTCCTGACCN (SEQ ID NO: 7).
  • the functional variant of a motif can comprise two or more operably linked NR1 motifcontaining sequences.
  • the functional variant of a PBREM motif can comprise three, four, five or more operably linked NR1 motif-containing sequences.
  • two or more NR1 motifs are present, they can be immediately adjacent to one another or separated by a spacer.
  • a CRE that is capable of being bound and activated by a CAR-RXR heterodimer suitably comprises the general structure NR1-S-NR1, wherein NR1 represents any NR1 motif as discussed herein, and S represents an optional spacer.
  • each spacer can be independently from 2 to 50 nucleotides in length. In some embodiments, each spacer is from 3 to 40, from 4 to 30, from 5 to 20, from 6 to 10, or from 7 to 9 nucleotides in length. In some embodiments, each spacer is independently, 5, 6, 7, 8, 9, 10 or 11 nucleotides in length.
  • the NR1 motif comprises the nucleotide sequence [TGTACTTTCCTGACCN-S-] n (SEQ ID NO: 8), or [CTGTACTTTCCTGACCN-S-] n (SEQ ID NO: 9), wherein N is any nucleotide, n is an integer from 1 to 10, and S is absent or a spacer.
  • n is an integer from 1 to 6.
  • n is an integer from 2 to 4.
  • n is selected from 2, 3, or 4.
  • n is 1, 2 or 3.
  • n is 1.
  • n is 2.
  • n is 3.
  • each spacer, S can be independently from 2 to 50 nucleotides in length.
  • each spacer, S is from 3 to 40, from 4 to 30, from 5 to 20, from 6 to 10, or from 7 to 9 nucleotides in length.
  • each spacer, S is independently, 5, 6, 7, 8, 9, 10 or 11 nucleotides in length.
  • n 2 or 3.
  • the functional variant of a PBREM motif comprises the nucleotide sequence:
  • NCTGTACTTTCCTGACCNTG (SEQ ID NO: 15), wherein N is any nucleotide, and S is absent or a spacer.
  • n 3
  • the functional variant of a PBREM motif comprises the nucleotide sequence:
  • TCTGTACTTTCCTGACCTTG (SEQ ID NO: 16); or ACTGTACTTTCCTGACCCTG-S-ACTGTACTTTCCTGACCCTG-S- ACTGTACTTTCCTGACCCTG (SEQ ID NO: 17), wherein N is any nucleotide, and S is absent or a spacer.
  • the functional variant of a PBREM motif comprises the nucleotide sequence: gatcTCTGTACTTTCCTGACCTTGgatcgatcTCTGTACTTTCCTGACCTTGgatcgatcTC TGTACTTTCCTGACCTTGgatc (SEQ ID NO: 18); or gatcACTGTACTTTCCTGACCCTGgatcgatcACTGTACTTTCCTGACCCTGgatcgatcA CTGTACTTTCCTGACCCTGgatc (SEQ ID NO: 19).
  • the NF1 motif comprises a nucleotide sequence that varies by 7 or fewer nucleotides (e.g., 6, 5, 4, 3, 2, or 1 nucleotides) from nucleotides 19 to 31 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the NF1 motif comprises the nucleotide sequence TGGCACAGTGCCA (SEQ ID NO: 20) or TGAAGAGGTGGCA (SEQ ID NO: 21), or a nucleotide sequence that varies no more than 7 nucleotides from SEQ ID NO: 20 or SEQ ID NO: 21
  • the NF1 motif comprises a nucleotide sequence that varies by no more than 1 to 6 nucleotides from SEQ ID NO: 20 or SEQ ID NO: 21.
  • the NF1 motif comprises the nucleotide sequence TGNNNNNGTGNCA (SEQ ID NO: 22), where N is any nucleotide.
  • the NF1 motif consists of SEQ ID NO: 20 or SEQ ID NO: 21
  • the NR2 motif comprises a nucleotide sequence that varies by 5 or fewer nucleotides (e.g., 4, 3, 2, or 1 nucleotides) from nucleotides 35 to 50 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the NR2 motif suitably comprises the nucleotide sequence TCAACTTGCCTGACAC (SEQ ID NO: 23) or TGGACTTTCCTGAACC (SEQ ID NO: 24), or a nucleotide sequence that varies by no more than by 5 nucleotides from SEQ ID NO: 23 or SEQ ID NO: 24.
  • the NR2 motif consists of SEQ ID NO: 23 or SEQ ID NO: 24
  • the CRE that is capable of being bound and activated by a CAR- RXR heterodimer comprises a nucleotide sequence that is at least 60% identical to SEQ ID NO: 1 or SEQ ID NO: 2
  • the CRE that is capable of being bound and activated by a CAR- RXR heterodimer comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99 % identical to SEQ ID NO: 1 or SEQ ID NO: 2.
  • mouse PBREM SEQ ID NO: 1
  • human PBREM SEQ ID NO: 2
  • mouse PBREM mouse PBREM
  • human PBREM SEQ ID NO: 2
  • mouse PBREM remains functional in human cells
  • human function in PBREM remains functional in mouse cells.
  • at least this level of overall sequence difference across the PBREM motif can be tolerated.
  • the CRE comprises a nucleotide sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the CRE comprises a comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 1 or SEQ ID NO: 2
  • the CRE comprises a nucleotide sequence having at least 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2
  • the CRE comprises a nucleotide sequence having at least 95% identity to SEQ ID NO: 26. In some embodiments, the CRE comprises a nucleotide sequence having at least 99% identity to SEQ ID NO: 26. For example the CRE comprises a nucleotide sequence having 100% identity to SEQ ID NO: 26.
  • % identity is calculated with respect to the specifically defined nucleotides rather than the undefined “Ns”.
  • Functional variants of a PBREM sequence where one or more nucleotides identified as N are deleted are specifically contemplated as part of such embodiments of the invention, for example where up to 7, 6, 5, 4, 3, 2, or 1 nucleotides marked N are deleted.
  • functional variants of a PBREM sequence where one or more nucleotides are inserted are specifically contemplated as part of such embodiments of the invention, for example where up to 7, 6, 5, 4, 3, 2, or 1 nucleotides are inserted.
  • the rat PBREM element from the CYP2B2 gene comprise a T inserted between the T and C at position 10-11, i.e., within NR1 of the PBREM element. Replacement, deletion or insertion of nucleotides in regions outside of the NR1 motif are likely to be well-tolerated.
  • the PBREM element or functional variant thereof can be present in either orientation.
  • the reverse complement of the PBREM elements described herein thus form part of disclosure.
  • the human PBREM element is naturally present in the reverse orientation compared to the mouse PBREM element, and that the human PBREM element was shown to remain functional in the “reverse” orientation (i.e., the same orientation as mouse PBREM) in Sueyoshi, et al. (J. BIOL. CHEM. Vol. 274,10, pp. 6043-6046, 1999).
  • the inducible promoter comprises a plurality of CREs that are each capable of being bound and activated by a heterodimer of CAR and RXR. Accordingly, in some embodiments of any one of the aspects described herein, the inducible promoter comprises a plurality of CREs that are each capable of being bound and activated by a heterodimer of CAR and RXR. Stated in another way, the inducible promoter comprises a cis- regulatory module (CRM) that comprises a plurality of CREs that are each capable of being bound and activated by a heterodimer of CAR and RXR.
  • CCM cis- regulatory module
  • the CREs that are capable of being bound and activated by a heterodimer of CAR and RXR can be identical, or they can be different to one another.
  • the inducible promoter comprises from 2 to 10 CREs that are each capable of being bound and activated by a heterodimer of CAR and RXR.
  • the inducible promoter comprises from 2 to 7 CREs.
  • the inducible promoter comprises from 2 to 5 CREs.
  • inducible promoter comprises from 2 to 4 CREs.
  • inducible promoter comprises 2 or 3 CREs.
  • the inducible promoter comprises 2 or 3 CREs.
  • the inducible promoter comprises the general structure CRE-L-CRE, wherein CRE represents any CRE as discussed herein, and L represents an optional spacer.
  • each spacer, L can have any suitable length.
  • each spacer, L can be independently from 2 to 100 nucleotides in length.
  • each spacer, L can be independently from 3 to 50 nucleotides, or from 5 to 30 nucleotides, or from 10 to 25 nucleotides in length.
  • each spacer, L is independently from 15 to 25 nucleotides in length.
  • each spacer, L is independently 18, 19, 20, 21 or 22 nucleotides in length. A spacer of approximately 20 nucleotides in length has been found to be particularly suitable.
  • each spacer, L is a multiple of 5 nucleotides in length.
  • the CRM comprises the amino acid sequence:
  • L-] m (SEQ ID NO: 27), where m is an integer from 1 to 10, N is any nucleotide, and L is absent or a spacer.
  • m is an integer from 1 to 6.
  • m is an integer from 2 to 4.
  • m is selected from 2, 3, or 4.
  • m is 1, 2 or 3.
  • m is 1.
  • m is 2.
  • m is 3.
  • each spacer, L can be independently from 2 to 100 nucleotides in length. In some embodiments, each spacer, L, can be independently from 3 to 50 nucleotides, or from 5 to 30 nucleotides, or from 10 to 25 nucleotides in length. For example, each spacer, L, is independently from 15 to 25 nucleotides in length. In some embodiments, each spacer, L, is independently 18, 19, 20, 21 or 22 nucleotides in length. A spacer of approximately 20 nucleotides in length has been found to be particularly suitable. In some non-limiting examples, each spacer, L, is a multiple of 5 nucleotides in length.
  • the CRM comprises the nucleotide sequence:
  • the CRM comprises a nucleotide sequence having at least 60% identity to the nucleotide sequence:
  • the CRM comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 85%, or 90% identical to one of SEQ ID NO: 30-33.
  • the inducible promoter comprises a nucleotide sequence having at least 95% identity to one of SEQ ID NO: 30-33.
  • the inducible promoter comprises a nucleotide sequence having at least 99% identity to one of SEQ ID NO: 30-33.
  • the inducible promoter comprises a nucleotide sequence having 100% identity to one of SEQ ID NO: 30-33
  • the CRM comprises a nucleotide sequence having at least 60% identity to the nucleotide sequence:
  • the CRM comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 85%, or 90% identical to any one of SEQ ID NO: 34-38.
  • the inducible promoter comprises a nucleotide sequence having at least 95% identity to any one of SEQ ID NO: 34-38.
  • the inducible promoter comprises a nucleotide sequence having at least 99% identity to any one of SEQ ID NO: 34-38.
  • the inducible promoter comprises a nucleotide sequence having 100% identity to any one of SEQ ID NO: 34-38.
  • a CRE that is capable of being bound and activated by a CAR-RXR heterodimer comprises a portion of the PBREM elements (e.g., NR1, NF1 and NR2) from two or more different species.
  • a CRE that is capable of being bound and activated by a CAR-RXR heterodimer comprises PBREM elements (e.g., NR1, NF1 and NR2) from species in two or more different genera (e.g., two different mammals).
  • PBREM elements e.g., NR1, NF1 and NR2
  • Such motifs can be referred to as “hybrid PBREM motifs”.
  • a hybrid PBREM motif typically comprises all of the motifs of a wild-type PBREM motif (i.e., the NR1, NF1, and NR2 motifs), but these motifs are derived from two or more different species.
  • a hybrid PBREM motif comprises a portion (e.g., the NR1, NF1, and NR2 motifs) from a first species, and a corresponding portion from a second species (e.g., the corresponding NR1, NF1, and NR2 motifs).
  • a hybrid PBREM motif can comprise portions (e.g., the NR1, NF1, and NR2 motifs) of primate (e.g., human) and rodent (e.g., mouse) PBREM motifs.
  • a hybrid PBREM motif may comprise an NR1 motif from primate (e.g., human) and an NR2 motif from rodent (e.g., mouse), or an NR1 motif from rodent (e.g., mouse) and an NR2 motif from primate (e.g., human).
  • Primate or rodent NR1 and NR2 motifs can also be combined with an NF1 motif from the corresponding species.
  • the hybrid PBREM motif comprises one of the following combinations of PBREM motifs: hNRl-mNFl-hNR2; hNRl-mNFl-mNR2; mNRl-hNFl-mNR2; or mNRl-hNFl-hNR2 (wherein “h” indicates the human motif and m indicates the mouse motif).
  • the inducible primer comprises a nucleotide sequence having at least 60% identity to the sequence.
  • the inducible primer comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 85%, or 90% identical to one of SEQ ID NO: 39-42.
  • the inducible primer comprises a nucleotide sequence having at least 95% identity to one of SEQ ID NO: 39-42.
  • the inducible promoter comprises a nucleotide sequence having at least 99% identity to one of SEQ ID NO: 39-42.
  • the inducible promoter comprises a nucleotide sequence having 100% identity to one of SEQ ID NO: 39-42
  • the inducible promoter comprises no or only minimal nucleic acid sequences that would lead to constitutive expression or expression in cells which do not express CAR and/or RXR, e.g., non-liver cells.
  • constitutive expression or expression in cells which do not express CAR and/or RXR e.g., non-liver cells.
  • background expression and expression in cells which do not express CAR and RXR, e.g., non-liver cells is minimized or avoided entirely.
  • the inducible promoter comprises a CRE that is capable of being bound and activated by a heterodimer of CAR and RXR operably linked to a minimal promoter or proximal promoter.
  • the inducible promoter comprises a CRE that is capable of being bound and activated by a heterodimer of CAR and RXR operably linked to a minimal promoter.
  • a proximal promoter is used, it is a cell or organ specific promoter.
  • a minimal promoter is preferred, as a proximal promoter will tend to drive at least some degree of background expression. However, some amount of background expression may be desirable in some instances.
  • the minimal or proximal promoter is a liver-specific promoter.
  • the inducible promoter is a liver-specific inducible promoter.
  • the minimal promoter can be any suitable minimal promoter.
  • a wide range of minimal promoters are known in the art.
  • Some exemplary minimal promoters include, but are not limited to, HSV thymidine kinase minimal promoter (MinTK), CMV minimal promoter (CMVmp) and SV40 minimal promoter (SV40mp).
  • the minimal promoter can be a synthetic minimal promoter.
  • the inducible promoter comprises one or more (e.g., two or three) CREs that are capable of being bound and activated by a heterodimer of CAR and RXR operably linked to the MinTK minimal promoter. Without wishing to be bound by a theory, this combination provides a desirable combination of low background expression and inducibility.
  • the inducible promoter comprises one or more (e.g., two or three) CREs that are capable of being bound and activated by a heterodimer of CAR and RXR operably linked to the SV40 minimal promoter. This combination has been shown to provide high levels of inducibility, albeit with some increase in background expression levels.
  • the inducible promoter comprises one or more (e.g., two or three) CREs that are capable of being bound and activated by a heterodimer of CAR and RXR operably linked to the CMVmp minimal promoter.
  • the CRE that is capable of being bound and activated by a heterodimer of CAR and RXR or, where a plurality of CREs are present, the most proximal CRE
  • the minimal or proximal promoter can be immediately adj acent to one another or separated by a spacer.
  • the CRE that is capable of being bound and activated by a heterodimer of CAR and RXR or, where a plurality of CREs are present, the most proximal CRE
  • the minimal or proximal promoter are separated by a spacer.
  • spacer separating the CRE and the minimal or proximal promoter can have any suitable length.
  • spacer can be independently from 10 to 200 nucleotides in length.
  • spacer can be independently from 15 to 150 nucleotides, or from 20 to 125 nucleotides, or from 25 to 100 nucleotides in length.
  • spacer can be 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95 or 100 nucleotides in length.
  • the spacer is 20, 46, 80, or 100 nucleotides in length.
  • the inducible promoter comprises a nucleotide sequence having at least 60% identity to the sequence:
  • TTCCGGTACTGTTGGTAAAGCCACCC (SEQ ID NO: 45, PB1-SV40-MP);
  • GCAAAAAGCTTGGCATTCCGGTACTGTTGGTAAAGCCACCC (SEQ ID NO: 50, PB-1-SV40);
  • TTCCGGTACTGTTGGTAAAGCCACCC (SEQ ID NO: 52, PB1-3-SV40);
  • AAACATGGAAGATGCCAAAAACATTAAG SEQ ID NO: 56, human PBREM- MinTK
  • AAACATGGAAGATGCCAAAAACATTAAG SEQ ID NO: 59, mNRl-hNFl-mNR2- MinTK (MHM MinTK));
  • TTCCGGTACTGTTGGTAAAGCCACCC (SEQ ID NO: 61, hPB-SV40);
  • the inducible promoter comprises a nucleotide sequence that is at least 65%, 70%, 75%, 80%, 85%, or 90% identical to one of SEQ ID NO: 43-67.
  • the inducible promoter comprises a nucleotide sequence having at least 95% identity to one of SEQ ID NO: 43-67.
  • the inducible promoter comprises a nucleotide sequence having at least 99% identity to one of SEQ ID NO: 43-67.
  • the inducible promoter comprises a nucleotide sequence having 100% identity to one of SEQ ID NO: 43-67.
  • PBREM elements are underlines and minimal promoter is in bold.
  • transgene means a nucleic acid sequence (encoding, for example, a desired gene expression product such as a protein or RNA) that is partly or entirely heterologous, i.e., foreign, to the subject into which it is introduced, or is homologous to an endogenous gene of the subject into which it is introduced.
  • a transgene for use in methods described herein typically encodes a desired gene expression product such as a polypeptide (protein) or RNA.
  • the transgene can be a full-length cDNA or genomic DNA sequence, or any fragment, subunit or mutant thereof that has at least some desired biological activity.
  • the transgene encodes a protein
  • the protein can be essentially any type of protein.
  • the protein can be an enzyme, an antibody or antibody fragment (e.g., a monoclonal antibody), a hormone, a blood clotting factor, a growth factor, a cytokine, a viral protein (e.g., REP, CAP, REV, VSV-G, or RD114), a therapeutic protein, or a toxic protein (e.g., Caspase 3, 8 or 9).
  • the gene encodes a therapeutic expression product.
  • the gene encodes a therapeutic protein suitable for use in treating a disease or condition associated with the liver.
  • Such genes may be referred to as “therapeutic genes”.
  • the therapeutic expression product can be a protein, e.g., a secretable protein such as, e.g., a clotting factor (e.g., factor IX or factor VIII), GAA, a cytokine, a growth factor, an antibody or nanobody, a chemokine, a plasma factor, insulin, erythropoietin, lipoprotein lipase, or a toxic protein.
  • a secretable protein such as, e.g., a clotting factor (e.g., factor IX or factor VIII), GAA, a cytokine, a growth factor, an antibody or nanobody, a chemokine, a plasma factor, insulin, erythropoietin, lipoprotein lipase, or a toxic protein.
  • the therapeutic expression product can be RNA, such as an siRNA or miRNA.
  • Some exemplary therapeutic expression products include, but are not limited to, factor VIII, factor IX, factor VII, factor X, von Willebrand factor, erythropoietin (EPO), interferon-a, interferon-B, interferon-y, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4 ), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL- 10), interleukin 11 (IL-11 ), interleukin 12 (IL-12), interleukin-37 (IL-37), interleukin-28 (11-38), chemokine (C-X-C motif) ligand 5 (CXCL5), granulocyte-colon, interleukin 1 (IL
  • Proteins or polypeptides of interest can be, for example, antibodies, enzymes or fragments thereof, viral proteins, cytokines, lymphokines, adhesion molecules, receptors and derivatives or fragments thereof, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine like proteins or particles, process enzymes, growth factors, hormones, and any other polypeptides that can serve as agonists or antagonists and/or have therapeutic or diagnostic use.
  • the protein is an immunoglobulin, preferably an antibody or antibody fragment, most preferably a Fab or scFv antibody.
  • the protein is a viral protein.
  • the transgene encodes an anti-PDl antibody, anti-HER2 antibody, anti-PCSK9 antibody, anti-VEGFR2 antibody, anti-IL17 antibody, anti-A03-42 antibody, anti-Sema3A antibody, anti-4- IBB (CD137) antibody, anti-5*-nucleotidase antibody, anti-5T4 antibody, anti-activin receptor-like kinase 1 antibody, anti-ACVR2B antibody, antiadenocarcinoma antigen antibody, anti-amyloid antibody, anti-Ang-2 antibody, anti-angiopoietin 2 antibody, anti-angiopoietin 2 antibody, anti-angiopoietin 3 antibody, anti-anthrax toxin antibody, anti-AOC3 (VAP-1) antibody, anti-AXL antibody, anti-BAFF-R antibody, anti-B-cell activating factor (BAFF) antibody, anti-B-cell maturation antigen (BCMA) antibody, anti-B-
  • the transgene encodes an anti-PDl antibody. In some embodiments, the transgene encodes an anti-HER2 antibody. In some embodiments, the transgene encodes an anti-PCSK9 antibody. In some embodiments, the transgene encodes an anti-VEGFR2 antibody. In some embodiments, the transgene encodes an anti-IL17 antibody. In some embodiments, the transgene encodes an anti-Ap3-42 antibody. In some embodiments, the transgene encodes an anti-Sema3A antibody.
  • the transgene encodes an antibody selected from the group consisting of 3F8, Abagovomab, Abciximab, Abituzumab, Abrezekimab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afasevikumab, Afelimomab, Alacizumab pegol, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Amivantamab, Anatuniomab mafenatox, Andecaliximab, Anetumab ravtansine, Anifrolumab, Ansuvimab, Anrukinzumab, (IMA-638), Apolizumab, Aprutumab ixadotin, Arcitumomab, Ascrinvacumab, Ase
  • 3F8 Abagov
  • the transgene encodes a coagulation factor.
  • the transgene encodes coagulation factor VIII (FVIII).
  • the transgene encodes coagulation factor IX (FIX).
  • the transgene encodes coagulation factor VII (FVII).
  • the transgene encodes coagulation factor XI (FXI ).
  • the transgene encodes a cytokine, e.g., a protein or peptide cytokine.
  • the transgene encodes interleukin-la (IL-la), interleukin-1/3 (IL-1/3), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8/CXCL8), interleukin- 10 (IL- 10), interleukin- 12 (IL- 12), interleukin- 13 (IL-13), interleukin- 15 (IL-15), interleukin- 17 (IL-17), interleukin- 18 (IL-18), interleukin-37 (IL- 37), interleukin-38 (IL-38), tumor necrosis factor-a (TNF-a), interferon-a (INF-a), interferon-b (INF-b), interferon-y
  • TNF-a tumor necrosis
  • the transgene encodes IL-2. In some embodiments, the transgene encodes IL-37. In some embodiments, the transgene encodes IL-38. In some embodiments of any of the aspects, the subject comprises at least one cell, e.g., at least one liver cell, comprising a plurality of exogenous nucleotide sequences, each exogenous nucleotide sequence comprising a synthetic inducible promoter operably linked to a transgene, wherein the transgenes collectively encode IL-37 and IL- 38.
  • the exogenous nucleotide sequence further comprises a nucleic acid sequence encoding a posttranscriptional regulatory element. In some embodiments, the exogenous nucleotide sequence comprises a nucleic acid encoding a polyA element.
  • the compound of Formula I-C is 10 ⁇ -(Sulfamino)dihydroartemisinin or Bis[(10 ⁇ -dihydroartemisinyl)]sulfamide.
  • Compounds of Formula I-C and their synthesis are described, for example, in US Pat. Pub. No.2005/0119232, content of which is incorporated herein by reference in its entirety.
  • the artemisinin derivative is of Formula (I-D): (Formula I-D), wherein: R 4 is halogen (e.g., F, Cl or Br); and R 1 is H, –R 12 , –OR 12 , –NHR 12 , –NR 12 R 12 , or –SR 12 , where each R 12 is independently H, optionally substituted cycloalkyl, optionally substituted aryl, or C-linked heteroaryl or heterocyclylalkyl group.
  • R 4 is halogen (e.g., F, Cl or Br)
  • R 1 is H, –R 12 , –OR 12 , –NHR 12 , –NR 12 R 12 , or –SR 12 , where each R 12 is independently H, optionally substituted cycloalkyl, optionally substituted aryl, or C-linked heteroaryl or heterocyclylalkyl group.
  • R 1 is –OCH3, –OCH2CH3, –OCH2CH2OH, – NHPh, –CH2N(CH3)2, –SPh or –CH2Ph.
  • R 1 is –OCH3, –OCH2CH3, –OCH2CH2OH and R 4 is Br.
  • R 1 is –OCH3.
  • R 1 is –OCH 3 , –OCH 2 CH 3 , –OCH 2 CH 2 OH and R 4 is Cl.
  • R 1 is –OCH2CH3.
  • R 1 is –OCH 3 , –OCH 2 CH 3 , –OCH 2 CH 2 OH and R 4 is I.
  • R 1 is –OCH 3 .
  • R 1 is –OCH 3 , –OCH 2 CH 3 , –OCH 2 CH 2 OH and R 4 is F.
  • R 1 is –OCH 3 .
  • Compounds of Formula I-D and their synthesis are described, for example, in Chinese Pat. App. No.10135977 (Pub. No. CN 201310069722), content of which is incorporated herein by reference in its entirety.
  • the artemisinin derivative is of Formula (I-E):
  • a is 0 to 10
  • R 13 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, hydroxyalkyl, polyhydroxyalkyl, hydroxyaryl, polyhydroxyaryl, heterocyclic-alkyl, mercapto-alkyl, dithio-alkyl, carboxyalkyl, amidoalkyl, or guanidinoalkyl
  • R 14 is OR A3 , NR A3 R A4 , or peptide
  • R A3 and R A4 are independently H, optionally substituted alkyl, or optionally substituted aryl
  • the compound of Formula I-E is selected from the group consisting of:
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently H or OH.
  • the artemisinin derivative is of Formula (I-F): (Formula I-F), wherein: b is 0 or 1;
  • X 6 is H or OH
  • Y 6 is H, CH3 or phenyl
  • Z 6 is NH 2 , NHR 6N , N(R 6N ) 2 , NHCH2CH2OH, NHCH2CH2OH, NH(CH 2 )2-3NMe 2 , each R 6N is independently Ci-Cealkyl.
  • b is 0; X 6 and Y 6 are H; and Z 6 is NH2.
  • b is 1; X 6 is OH; Y 6 is H; and Z 6 is NH2, NHMe, [00284] In some compounds of Formula I-F, b is 1; X 6 is OH; Y 6 is CH3 or phenyl; and Z 6 is , where each R 6N is independently Ci-Cealkyl.
  • the compound of Formula I-F is selected from the group consisting of 2'-amino arteether, 3'-amino-2'-hydroxy- artemisinin propyl ether, 3'-methylamino-2'-hydroxy-artemisinin propyl ether, 3 '-methylamino- 2'-hydroxy-artemisinin butyl ether, 3'-amino-2'-hydroxy-artemisinin butyl ether, 3'- hydroxyethylamino-2'-hydroxy-artemisinin propyl ether, 3'-phenyl-3'-methylamino-2'-hydroxy- artemisinin propyl ether, 3'-phenyl-3'-amino-2'-hydroxy-artemisin propyl ether, 3'- hydroxyethylamino-2'-hydroxy-artemisinin butyl ether, 3'-dimethylamino-2'-hydroxy-artemis
  • the artemisinin derivative is of Formula (I-G): (Formula I-G), wherein:
  • X is - O- or -O-O-;
  • R 1 is H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, — (CH2)jR 2G , — (CH2)jO(CH2)kR 2G , — (CH 2 )jC(O)(CH 2 )kR 2G , — (CH 2 )jC(O)(CH 2 )kR 2G , — (CH 2 )jC(O)O(CH 2 )kR 2G — (CH 2 )jNR 3G R 4G , — (CH 2 )jC(O)(CH 2 )kNR 3G R 4G , — (CH 2 )jNR 5G C(O)(CH 2 )kR 2G — (CH 2 )jC(O)(CH 2 )
  • R 2G is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, perfluoroalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted (aryl or heteroaryl)-X G - (aryl or heteroaryl) wherein X G is O, S, NH or N(Ci-Ce)alkyl, cholesterol, or a substituted or unsubstituted monosaccharide, and wherein each R 2G is optionally independently substituted with 1 to 5 R 10G groups;
  • R 3G , R 4G , and R 5G are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, perfluoroalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl, wherein R 3G , R 4G , and R 5G , are each optionally independently substituted with 1 to 5 R 1OG groups; R 3G and R 4G , together with the N atom to which they are attached, form substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl, wherein substituted or unsubstituted heterocycloalkyl, and substituted or unsub
  • Formula I-G is of Formula I-GA:
  • the compound of Formula I-G is of Formula I-GB: (Formula I-GB).
  • R 1 is H, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl, substituted or unsubstituted benzyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted furyl, substituted or unsubstituted thienyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted isoxazolyl, substituted or unsubstituted 1,2,4-oxadiazolyl, substituted or unsubstituted 1,3,4-oxadiazolyl, substituted or unsubstituted tetrazolyl, substituted or unsubstituted thi
  • R 2G is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl, substituted or unsubstituted benzyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted furyl, substituted or unsubstituted thienyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted isoxazolyl, substituted or unsubstituted 1,2,4- oxadiazolyl, substituted or unsubstituted 1,3,4-oxadiazolyl, substituted or unsubstituted te
  • R 3G and R 4G are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl.
  • R 1 is — C(O)R 2G , — OC(O)R 2G , — C(O)OR 2G , or — C(O)NR 3G R 4G ;
  • R 2G is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl; and
  • R 3G and R 4G independently are hydrogen, or substituted or unsubstituted alkyl.
  • R 1 is — (CH2)jC(O)O(CH2)kR 2G ; and R 2G is substituted or unsubstituted glucose, substituted or unsubstituted galactose, substituted or unsubstituted mannose, substituted or unsubstituted fructose.
  • R 1 is — C(O)OR 2 ; and R 2G is tetraacetyl a-D glucopyranose, tetraacetyl 0-D glucopyranose, tetraacetyl a-D galactose, tetraacetyl 0-D galactose, tetraacetyl a-D mannose, tetraacetyl 0-D mannose, tetraacetyl a-D fructose, or tetraacetyl 0-D fructose.
  • the compound of Formula I-G is selected from the group consisting of:
  • the artemisinin derivative is of Formula (I-H): n is an integer from 1 to 10.
  • the compound of Formula I-H is selected from the group consisting of:
  • the artemisinin derivative is of Formula (I-I):
  • R 91 -R" are independently hydrogen, halogen, nitro, cyano group, trifluoromethyl, trifluoromethoxy, Ci-C4alkyl, Ci-C4alkoxymethyl, di(Ci-C4alkyl)amino, or C3- Crcycloalkyl.
  • the compound of Formula I-I is selected from the group consisting of: [00304] Compounds of Formula I-I and their synthesis are described, for example, in Chinese Pat. App. No.101580510 (Pub. No. CN 200910011753), content of which is incorporated herein by reference in its entirety. [00305] In some embodiments of any one of the aspects described herein, the artemisinin derivative is of Formula (I-J): (Formula I-J).
  • Y J is O or NR N ;
  • X J is H, —NR 1J R 2J , —CHR 8J R 9J or Ar;
  • R 1 is H, halogen, OH, an optionally substituted cycloalkyl, aryl, C-linked heteroaryl or heterocyclylalkyl, —NR 3J R 4J , —O—CO—R 5J or —OR 6J ;
  • R 1J and R 2J are independently an optionally substituted alkyl, cycloalkyl, aryl, or aralkyl; or R 1J and R 2J together with the interjacent nitrogen atom represent an optionally substituted heterocyclic group or an amino group derived from an optionally substituted amino acid ester;
  • R 3J is H or an optionally substituted alkyl, alkenyl or alkynyl group;
  • R 4J is
  • R 1 is a C3-8 cycloalkyl group, a C6-18 aryl group, a 5- to 10-membered C-linked heteroaryl group or a 5- to 10-membered heterocyclyl-C 1-6 alkyl group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C 1-4 alkyl, C 2-4 alkenyl, C 1-4 haloalkyl, C 1-4 alkoxy, amino, C1-4 alkylamino, di(C1-4 alkyl)amino, carboxyl, C6-10 aryl, 5 to 10-membered heterocyclic and C1-4 alkyl- or phenyl-substituted 5- to 10-membered heterocyclic groups.
  • R 1 is a C6-18 aryl group optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C 1-4 alkyl, C 2-4 alkenyl, C 1-4 haloalkyl, C 1-4 alkoxy, C 1- 4 haloalkoxy, amino, C 1-4 alkylamino, di (C 1-4 alkyl)amino and carboxyl groups.
  • the inducer is prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • the compounds can be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • these compounds may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
  • Such compositions and preparations should contain at least 0.1% of compound.
  • the percentage of the agent in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit.
  • the amount of compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients.
  • suitable nontoxic pharmaceutically acceptable ingredients are known to those skilled in the preparation of nasal dosage forms and some of these can be found in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005.
  • suitable carriers is dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels.
  • Nasal dosage forms generally contain large amounts of water in addition to the active ingredient.
  • liquid carriers In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • dosage unit refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • oral or enteral formulations tablets can be formulated in accordance with conventional procedures employing solid carriers well-known in the art.
  • Capsules employed for oral formulations to be used with the methods described herein can be made from any pharmaceutically acceptable material, such as gelatin or cellulose derivatives.
  • Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated, such as those described in U.S. Pat. No. 4,704,295, “Enteric Film- Coating Compositions,” issued Nov. 3, 1987; U.S. Pat. No. 4, 556,552, “Enteric Film- Coating Compositions,” issued Dec. 3, 1985; U.S. Pat. No. 4,309,404, “Sustained Release Pharmaceutical Compositions,” issued Jan. 5, 1982; and U.S. Pat. No. 4,309,406, “Sustained Release Pharmaceutical Compositions,” issued Jan.
  • the inducer can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.
  • Parenteral injections may involve bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspension, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Embodiment 1 A method of regulating expression of a transgene in a primate subject, the method comprising: (a) administering to the subject at a first time point a first inducer, wherein the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the inducible promoter comprises a cis-regulatory element (CRE) that is capable of being bound and activated by a heterodimer of CAR and RXR, wherein the first inducer is artemisinin or a derivative thereof, and wherein the first dose comprises an amount of the inducer that is sufficient to express a measurable level of the transgene in the subject; (b) administering to the subject at least at a second time point a second inducer to continue expression of the transgene at a predetermined level for
  • CRE cis-regulatory element
  • Embodiment 2 The method of Embodiment 1, wherein the artemisinin derivative is of Formula (I) or a pharmaceutically acceptable salt thereof.
  • Embodiment 3 The method of any one of Embodiments 1-2, wherein the first and second inducer are the same.
  • Embodiment 4 The method of Embodiment 3, wherein the first and second inducer are artemisinin or a derivative thereof.
  • Embodiment 5 The method of Embodiment 3, wherein the first inducer is artemisinin and second inducer is artemisinin or a derivative thereof.
  • Embodiment 6 The method of any one of Embodiments 1-2, wherein the first and second inducer are different.
  • Embodiment 7 The method of Embodiments 6, wherein the first inducer is a first artemisinin derivative, and the second inducer is artemisinin or a second artemisinin derivative.
  • Embodiment 8 The method of Embodiment 6, wherein the first inducer is an artemisinin derivative and the second inducer is artemisinin or a derivative thereof.
  • Embodiment 9 The method of Embodiment 6, wherein the first inducer is an artemisinin derivative, and the second inducer is artemisinin.
  • Embodiment 10 The method of any one of Embodiments 1-9, wherein the artemisinin derivative is a compound of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • the artemisinin derivative is a compound of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • Embodiment 11 The method of any one of Embodiments 1-10, wherein the artemisinin derivative is artesunate, dihydroartemisinin, artesunate, or artemether.
  • Embodiment 12 The method of any one of Embodiments 1-11, wherein the artemisinin derivative is artesunate, or dihydroartemisinin.
  • Embodiment 13 The method of any one Embodiments 1-12, wherein the first and second inducer are administered at about the same amount.
  • Embodiment 14 The method of Embodiment 13, wherein the first inducer and the second inducer are administered at different amounts.
  • Embodiment 15 The method of Embodiment 14, wherein the second inducer is administered at a lower amount relative to the amount of the first inducer.
  • Embodiment 16 The method of Embodiment 15, wherein the second inducer is administered in amount about 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, 1/15, 1/20, 1/25, 1/30, 1/35, 1/40, 1/45, 1/50, 1/55, 1/60, 1/65, 1/70, 1/75, 1/80, 1/85, 1/90, 1/95, 1/100, 1/125, 1/150, 1/175, 1/200, 1/500, 1/1000 or even less of the amount of first the inducer.
  • Embodiment 17 The method of Embodiment 16, wherein the second inducer is administered in amount about 1/2, 1/4, 1/5, 1/8, 1/10, 1/20, 1/40, 1/80 or less of the amount of the first inducer.
  • Embodiment 18 The method of Embodiment 14, wherein the second inducer is administered at a higher amount relative to the amount of the first inducer.
  • Embodiment 19 The method of Embodiment 18, wherein the amount of inducer administered at the second time point is at least about 25%, 50%, about 75%, about 1-fold, about 1.2-folds, about 1.25-folds, about 1.3-folds, about 1.5-folds, about 1.75-folds, about 2-folds, about 2.25-folds, about 2.5-folds, about 2.5-folds, about 2.75-folds, about 3-folds, 4-fold, 5-fold, 6-folds, 7-fold, 8-fold, 9-folds, 10-folds or higher than the amount of the first inducer.
  • Embodiment 20 The method of any one of Embodiments 1-19, wherein the first and second inducer are administered independently at a dose from about 0.5 mg/kg to about 150mg/kg.
  • Embodiment 21 The method of any one of Embodiments 1-20, wherein the first inducer is administered at a dose from about 1 mg/kg to about 100 mg/kg.
  • Embodiment 22 The method of any one of Embodiments 1-21, wherein the first inducer is administered at a dose of about 80mg/kg, about 70mg/kg, about 60mg/kg, about 50mg/kg, about 40mg/kg, about 30mg/kg, about 20mg/kg, about 10mg/kg, about 6mg/kg, about 5mg/kg, or about 1mg/kg.
  • Embodiment 23 The method of any one of Embodiments 1-22, wherein the second inducer is administered at a dose from about 1 mg/kg to about 100 mg/kg.
  • Embodiment 24 The method of any one of Embodiments 1-23, wherein the second inducer is administered at a dose of 80mg/kg, about 70mg/kg, about 60mg/kg, about 50mg/kg, about 40mg/kg, about 30mg/kg, about 20mg/kg, about 10mg/kg, about 6mg/kg, about 5mg/kg, or about 1mg/kg.
  • Embodiment 25 The method of any one of Embodiments 1-14, wherein the first inducer is administered at a dose of about 80 mg/kg or less and the second inducer is administered at a dose of about 20mg/kg or less at the second or subsequent time point.
  • Embodiment 26 The method of any one of Embodiments 1-14, wherein the first inducer is administered at a dose of about 40 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point.
  • Embodiment 27 The method of any one of Embodiments 1-14, wherein the first inducer is administered at a dose of about 20mg/kg and the second inducer is administered at a dose of about 80 mg/kg, 40mg/kg, 20mg/kg, 10 mg/kg, 6 mg/kg or 1 mg/kg at the second or subsequent time point.
  • Embodiment 28 The method of any one of Embodiments 1-14, wherein the first inducer is administered at a dose of about 10 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point.
  • Embodiment 29 The method of any one of Embodiments 1-14, wherein the first inducer is administered at a dose of about 6 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point.
  • Embodiment 30 The method of any one of Embodiments 1-14, wherein the first inducer is administered at a dose of about 1 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point.
  • Embodiment 31 The method of any one of Embodiments 1-14, wherein the second inducer is administered at a dose of about 80 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 32 The method of any one of Embodiments 1-14, wherein the second inducer is administered at a dose of about 40 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 33 The method of any one of Embodiments 1-14, wherein the second inducer is administered at a dose of about 20 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 34 The method of any one of Embodiments 1-14, wherein the second inducer is administered at a dose of about 10 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 35 The method of any one of Embodiments 1-14, wherein the second inducer is administered at a dose of about 6 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 36 The method of any one of Embodiments 1-14, wherein the second inducer is administered at a dose of about 1 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 37 The method of any one of Embodiments 1-36, wherein the second time point is at least two days after the first time point.
  • Embodiment 38 The method of any one of Embodiments 1-37, wherein the second time point is at least one month after the first time point.
  • Embodiment 39 The method of any one of Embodiments 1-38, wherein the second time point is at least two months of the first time point.
  • Embodiment 40 The method of any one of Embodiments 1-39, wherein the expression of the transgene goes down to a background expression level within one week of administering the inducer.
  • Embodiment 41 The method of any one of Embodiments 1-40, wherein the method further comprises a step of determining the expression level of the transgene after administering the first or second inducer.
  • Embodiment 42 The method of any one of Embodiments 1-41, wherein the method further comprises a step of determining the expression level prior of the transgene to administering the first or second inducer.
  • Embodiment 43 The method of any one of Embodiments 1-42, wherein the method further comprising administering the exogenous nucleotide sequence to the subject prior to administering the first or second inducer.
  • Embodiment 44 The method of any one of Embodiments 1-43, wherein the method is a method of therapy, optionally, the method of therapy is a method of gene therapy.
  • Embodiment 45 The method of any one of Embodiments 1-44, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is a viral vector.
  • Embodiment 46 The method of any one of Embodiments 1-45, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is adeno- associated viral (AAV), lentiviral, adenoviral, herpes viral, or hepatitis viral vector.
  • AAV adeno- associated viral
  • Embodiment 47 The method of any one of Embodiments 1-46, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV vector, e.g., an AAV gene therapy vector.
  • Embodiment 48 The method of any one of Embodiments 1-47, wherein the first and second inducers independently are administered orally or intraperitoneally.
  • Embodiment 49 The method of any one of Embodiments 1-48, wherein at least one of the first and second inducer is administered orally.
  • Embodiment 50 The method of Embodiment 49, wherein the second inducer is administered orally.
  • Embodiment 51 The method of Embodiment 49, wherein the first inducer is administered orally.
  • Embodiment 52 The method of any one of Embodiments 1-48, wherein at least one of the first and second inducer is administered intraperitoneally.
  • Embodiment 53 The method of Embodiment 52, wherein the second inducer is administered intraperitoneally.
  • Embodiment 54 The method of Embodiment 52, wherein the first inducer is administered intraperitoneally.
  • Embodiment 56 The first and at least a second inducer for use according to Embodiment 55, wherein the method is according to any one of Embodiments 3 to 47.
  • Embodiment 57 The first and at least a second inducer for use according to Embodiment 55 or 56, which is a method of therapy, suitably a method of gene therapy.
  • Embodiment 58 The first and at least a second inducer for use according to any one of Embodiments 55-57, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV gene therapy vector.
  • Embodiment 59 Artemisinin or a derivative of artemisinin, for use as an inducer in a method of regulating expression of a transgene in a primate subject, wherein the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the synthetic inducible promoter comprises a cis- regulatory element (CRE) that is capable of being bound and activated by a heterodimer of CAR and RXR, wherein the inducer is administered to the subject at a dose which comprises an amount of the inducer that is sufficient to express a therapeutically effective level of the transgene in the subject.
  • CRE cis- regulatory element
  • Embodiment 60 Artemisinin or a derivative of artemisinin for use according to Embodiment 59, wherein the method is a method of therapy, suitably a method of gene therapy.
  • Embodiment 61 Artemisinin or a derivative of artemisinin for use according to Embodiment 59 or 60, wherein the artemisinin derivative is a compound as defined in Embodiment 2, optionally wherein the artemisinin derivative is of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • Embodiment 62 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 59-61, wherein the derivative of artemisinin is artesunate, dihydroartemisinin, artesunate, or artemether.
  • Embodiment 63 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 59-62, wherein artemisinin or a derivative of artemisinin is administered to the primate subject following the prior administration of an inducer to the primate subject.
  • Embodiment 64 Artemisinin or a derivative of artemisinin for use according to Embodiment 63, wherein the inducer of the prior administration was a different inducer, or wherein the inducer of the prior administration was the same inducer but at a different dose.
  • Embodiment 65 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 59-64, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV gene therapy vector.
  • Embodiment 66 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 59-65, wherein the method comprises prior administration of the exogenous nucleotide sequence to the subject prior to administering the inducer.
  • Embodiment 67 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 59-66, wherein the artemisinin or a derivative of artemisinin is administered at a dose of from about 0.1mg/kg to 80mg/kg, from about 0.1mg/kg to about 70mg/kg, from about 0.1mg/kg to about 60mg/kg, from about 0.1mg/kg to about 50mg/kg, from about 0.1mg/kg to about 40mg/kg, from about 0.1mg/kg to about 30mg/kg, from about 0.1mg/kg to about 20mg/kg, from about 0.1mg/kg to about 10mg/kg, from about 0.1mg/kg to about 6mg/kg, from about 0.1mg/kg to about 5mg/kg, or from about 0.1mg/kg to about 1mg/kg.
  • Embodiment 68 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 55-67, wherein the inducer comprising artemisinin or a derivative of artemisinin is administered orally, optionally wherein the composition comprises artesunate.
  • Embodiment 69 The method of any one of Embodiments 1-54, wherein a first induction with artemisinin or a derivative thereof increases the transgene expression level to a maximum at about 24 hrs and declines thereafter, wherein, [00548] a second induction and/or, successive inductions with artemisinin or a derivative thereof has similar induction profile as seen with the first induction, for at least about 1 month, or at least about 2 months, or, at least about 3 months, or, at least about 5 months or, at least about six months or, even more, and wherein, [00549] the method further comprises single administration of exogenous nucleotide sequence encoding the transgene operatively linked to the synthetic inducible promoter prior to first administering the inducer to induce said first induction.
  • Embodiment 70 The method of Embodiment 69, the transgene expression level at about 24 hrs in the first induction, or, second induction, or successive inductions is at least about 200 fold higher than that of prior to administering the inducer for the first time, or, the second time, or, any number of successive administrations.
  • Embodiment 71 The method of Embodiment 69, the first induction, the second induction, and/or, the successive inductions are not complete until about 160 hours after administering the inducer.
  • Embodiment 72 The method of Embodiment 69, wherein dose of the inducer in successive administrations is reduced from its prior administration.
  • Embodiment 73 The method of Embodiment 69, wherein dose of the inducer in successive administrations remains same.
  • Embodiment 74 The method of Embodiment 69, wherein the dose of the inducer ranges from about 1mg/kg to about 80mg/kg.
  • Embodiment 75 The method of Embodiment 69, wherein the inducers are administered orally.
  • Embodiment 76 The method of Embodiment 69, wherein the inducers are administered intraperitoneally.
  • Embodiment 77 A method of regulating expression of a transgene in a primate subject, the method comprising: administering artemisinin or a derivative thereof to the primate subject, wherein the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the inducible promoter comprises a cis-regulatory element (CRE) that is capable of being bound and activated by a heterodimer of CAR and RXR, wherein the artemisinin or a derivative thereof is administered in an amount that is sufficient to express a measurable level of the transgene in the subject.
  • CRE cis-regulatory element
  • Embodiment 78 The method of Embodiment 77, wherein the artemisinin derivative is of Formula (I).
  • Embodiment 79 The method of Embodiment 77 or 78, wherein the artemisinin derivative is a compound of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • Embodiment 80 The method of any one of Embodiments 77-79, wherein the derivative of artemisinin is artesunate, dihydroartemisinin, artesunate, or artemether.
  • Embodiment 81 The method of any one of Embodiments 77-80, wherein the derivative of artemisinin is artesunate, or dihydroartemisinin.
  • Embodiment 82 The method of any one of Embodiments 77-81, wherein the artemisinin or a derivative there of is administered at a dose from about 0.5 mg/kg to about 150mg/kg.
  • Embodiment 83 The method of any one of Embodiments 77-82, wherein the artemisinin or a derivative there of is administered at a dose from about 1 mg/kg to about 100 mg/kg.
  • Embodiment 84 The method of any one of Embodiments 77-83, wherein the artemisinin or a derivative there of is administered at a dose from about 1 mg/kg to about 80 mg/kg.
  • Embodiment 85 The method of any one of Embodiments 77-84, wherein the artemisinin or a derivative there of is administered at a dose from about 1 mg/kg to about 40 mg/kg.
  • Embodiment 86 The method of any one of Embodiments 77-85, wherein the artemisinin or a derivative there of is administered at a dose from about 1 mg/kg to about 20 mg/kg.
  • Embodiment 87 The method of any one of Embodiments 77-86, wherein the artemisinin or a derivative there of is administered at a dose from about 1 mg/kg to about 10 mg/kg.
  • Embodiment 88 The method of any one of Embodiments 77-84, wherein the artemisinin or a derivative there of is administered at a dose of about 80mg/kg, about 70mg/kg, about 60mg/kg, about 50mg/kg, about 40mg/kg, about 30mg/kg, about 20mg/kg, about 10mg/kg, about 6mg/kg, about 5mg/kg, or about 1mg/kg.
  • Embodiment 89 The method of any one of Embodiments 77-88, wherein the method further comprises administering the exogenous nucleotide sequence to the subject prior to administering the artemisinin or a derivative thereof.
  • Embodiment 90 The method of any one of Embodiments 77-89, wherein the artemisinin or a derivative thereof is administered to the primate subject following the prior administration of an inducer to the primate subject, and optionally, the inducer is administered in an amount that is sufficient to express a measurable level of the transgene in the subject.
  • Embodiment 91 The method of Embodiment 90, wherein the inducer of the prior administration is different from the artemisinin or a derivative thereof administered to the subject.
  • Embodiment 92 The method of Embodiment 90, wherein the inducer of the prior administration is same the artemisinin or a derivative thereof administered to the subject but at a different dose.
  • Embodiment 93 The method of any one of Embodiments 77-92, wherein the method is a method of therapy, optionally, the method of therapy is a method of gene therapy.
  • Embodiment 94 The method of any one of Embodiments 77-93, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is a viral vector.
  • Embodiment 95 The method of any one of Embodiments 77-94, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is adeno-associated viral (AAV), lentiviral, adenoviral, herpes viral, or hepatitis viral vector.
  • AAV adeno-associated viral
  • Embodiment 96 The method of any one of Embodiments 77-95, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV vector, e.g., an AAV gene therapy vector.
  • Embodiment 97 The method of any one of Embodiments 77-96, wherein the artemisinin or a derivative thereof is administered orally.
  • Embodiment 98 The method of any one of Embodiments 77-96, wherein the artemisinin or a derivative thereof is administered intraperitoneally.
  • Embodiment 99 A method of regulating expression of a transgene in a primate subject, the method comprising: administering a flavonoid compound to the primate subject, wherein the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the inducible promoter comprises a cis-regulatory element (CRE) that is capable of being bound and activated by a heterodimer of CAR and RXR, wherein the flavonoid compound is administered in an amount that is sufficient to express a measurable level of the transgene in the subject.
  • CRE cis-regulatory element
  • Embodiment 100 The method of Embodiment 99, wherein the flavonoid compound is of Formula (II-A).
  • Embodiment 101 The method of Embodiment 99, wherein the flavonoid compound is of Formula (II-B).
  • Embodiment 102 The method of Embodiment 99, wherein the flavonoid compound is of Formula (II-C).
  • Embodiment 103 The method of Embodiment 99, wherein the flavonoid compound is flavone, baicalein, chrysin, galangin, (OH) 4 flavone, 6-hydroxyluteolin, acacetin, acerosin, alnetin, apigenin, apiin, artocarpetin, balcalein, beta-sitosterol, biochamin A, cannaflavin A, cannflavin A, cannflavin B, cannflavin C, catechin, catechin-3-gallate, cerrosillin, chrysoeriol, chrysoeriol, cirsilineol, cirsiliol, cirsimaritin, corymbosin, cupressuflavone, cyanidine, daidzein, datiscetin, delphinidine, digidrokempferol , dihydroquercetin, diosmetin, diosmin,
  • Embodiment 104 The method of any one of Embodiments 99-103, wherein the flavonoid compound is flavone.
  • Embodiment 105 The method of any one of Embodiments 99-104, wherein the flavonoid compound is administered at a dose from about 0.5 mg/kg to about 150mg/kg.
  • Embodiment 106 The method of any one of Embodiments 99-105, wherein the flavonoid compound is administered at a dose from about 1 mg/kg to about 100 mg/kg.
  • Embodiment 107 The method of any one of Embodiments 99-106, wherein the flavonoid compound is administered at a dose from about 1 mg/kg to about 80 mg/kg.
  • Embodiment 108 The method of any one of Embodiments 99-107, wherein the flavonoid compound is administered at a dose from about 1 mg/kg to about 40 mg/kg.
  • Embodiment 109 The method of any one of Embodiments 99-108, wherein the flavonoid compound is administered at a dose from about 1 mg/kg to about 20 mg/kg.
  • Embodiment 112 The method of any one of Embodiments 99-111, wherein the method further comprises administering the exogenous nucleotide sequence to the subject prior to administering the flavonoid compound.
  • Embodiment 113 The method of any one of Embodiments 99-112, wherein the flavonoid compound is administered to the primate subject following the prior administration of an inducer to the primate subject, and optionally, the inducer is administered in an amount that is sufficient to express a measurable level of the transgene in the subject.
  • Embodiment 114 The method of Embodiment 115, wherein the inducer of the prior administration is different from the flavonoid compound administered to the subject.
  • Embodiment 115 The method of Embodiment 115, wherein the inducer of the prior administration is same as the flavonoid compound administered to the subject but at a different dose.
  • Embodiment 116 The method of any one of Embodiments 99-115, wherein the method is a method of therapy, optionally, the method of therapy is a method of gene therapy.
  • Embodiment 117 The method of any one of Embodiments 99-116, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is a viral vector.
  • Embodiment 118 The method of any one of Embodiments 99-117, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is adeno-associated viral (AAV), lentiviral, adenoviral, herpes viral, or hepatitis viral vector.
  • AAV adeno-associated viral
  • Embodiment 119 The method of any one of Embodiments 99-118, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV vector, e.g., an AAV gene therapy vector.
  • Embodiment 120 The method of any one of Embodiments 99-119, wherein the flavonoid compound is administered orally.
  • Embodiment 121 The method of any one of Embodiments 99-120, wherein the flavonoid compound is administered intraperitoneally.
  • Embodiment 99 The method of any one of Embodiments 1-54 or 69-121, wherein the at least one cell comprising the exogenous nucleotide sequence is a liver cell.
  • Embodiment 1 A method of regulating expression of a transgene in the liver of a subject, the method comprising: administering to the subject at at least a first time point a dose of a first inducer; administering to the subject at at least a second time point a second dose of a second inducer, wherein the second inducer can be the same as or different than the first inducer; and administering to the subject at at least a third time point a third dose of a third inducer, wherein the third inducer can be the same as or different than the first inducer and the second inducer; wherein the subject comprises at least one liver cell comprising an exogenous nucleotide sequence comprising a synthetic inducible promoter operably linked to a transgene; wherein the synthetic inducible promoter comprises at least one PBREM enhancer; wherein the transgene is detrimental to the liver cell or to the
  • Embodiment 2 The method of Embodiment 1, wherein the expression of the transgene increases for a predetermined time after each administration.
  • Embodiment 3 The method of any one of Embodiments 1-2, wherein the expression of the transgene increases for a predetermined time after each administration and the magnitude of the increase is positively correlated with the size of the dose.
  • Embodiment 4 The method of any one of Embodiments 1-3, wherein the time between administrations is at least 24 hours, 48 hours, 72 hours, 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks.
  • Embodiment 5 The method of any one of Embodiments 1-4, wherein the time between administrations is at least 24 hours, 48 hours, or 72 hours.
  • Embodiment 6 The method of any one of Embodiments 1-5, wherein the time between administrations is no more than 2 months, 3 months, 4 months, 5 months, or 6 months.
  • Embodiment 7 The method of any one of Embodiments 1-6, wherein the time between administrations is no more than 2 months.
  • Embodiment 8 The method of any one of Embodiments 1-7, wherein the time between administrations is 5-7 days, 5-10 days, 7-10 days, 7-14 days, 10-14 days, 10-20 days, 14-21 days, 14-28 days, 21-28 days, or 21-34 days.
  • Embodiment 9 The method of any one of Embodiments 1-8, wherein the at least second dose or at least third dose are administered when it is determined that the level of expression of the transgene is decreased to 75% or less of the level of expression of the transgene within the 24 hours after the first dose.
  • Embodiment 10 The method of any one of Embodiments 1-9, wherein the at least second dose or at least third dose are administered when it is determined that the level of expression of the transgene is decreased to 50% or less of the level of expression of the transgene within the 24 hours after the first dose.
  • Embodiment 11 The method of any one of Embodiments 1-10, wherein the at least second dose or at least third dose are administered when it is determined that the level of expression of the transgene is decreased to 25% or less of the level of expression of the transgene within the 24 hours after the first dose.
  • Embodiment 12 The method of any one of Embodiments 1-11, wherein the subsequent dose is administered when it is determined that the level of expression of the transgene is decreased to a background level of expression of the transgene following the 24 hours after the first dose.
  • Embodiment 13 The method of any one of Embodiments 1-12, wherein each dose is no more than 10 mg/kg.
  • Embodiment 14 The method of any one of 13, wherein the second dose and/or third dose is no more than 10 mg/kg.
  • Embodiment 15 The method of any one of Embodiments 1-14, wherein the first dose, the second dose, and/or the third dose is 5 mg/kg or greater.
  • Embodiment 16 The method of any one of Embodiments 1-15, wherein the first dose is 1-80 mg/kg, the second dose is 1-40 mg/kg, and/or the third dose is 1-40 mg/kg.
  • Embodiment 17 The method of any one of Embodiments 1-16, wherein the second dose and subsequent dose is 1-10 mg/kg.
  • Embodiment 18 The method of any one of Embodiments 1-17, wherein the first dose and subsequent dose is 1 mg/kg to 80 mg/kg.
  • Embodiment 19 The method of any one of Embodiments 1-18, wherein the first dose and subsequent dose is 5 mg/kg to 80 mg/kg.
  • Embodiment 20 The method of any one of Embodiments 1-19, wherein the second dose and subsequent dose is 5 mg/kg to 20 mg/kg.
  • Embodiment 21 The method of any one of Embodiments 1-20, wherein the second dose and subsequent dose is 5 mg/kg to 10 mg/kg.
  • Embodiment 22 The method of any one of Embodiments 1-21, wherein the administering comprises: administering a plurality of doses of a first inducer during a period of 2 to 4 weeks, wherein each dose of the first inducer is 160 mg/kg;administering 1 or 2 doses of a second inducer, wherein each dose of the second inducer is 80 mg/kg; administering at least 1 dose of a third inducer, wherein each dose of the third inducer is 40 mg/kg.
  • Embodiment 23 The method of any one of Embodiments 1-22, wherein the administering comprises: administering a plurality of doses of a first inducer during a period of 2 to 4 weeks, wherein each dose of the first inducer is 160 mg/kg or less; administering 1 or 2 doses of a second inducer, wherein each dose of the second inducer is 80 mg/kg or less; administering at least 1 dose of a third inducer, wherein each dose of the third inducer is 40 mg/kg or less.
  • Embodiment 24 The method of claim 22 or 23, wherein a dose of the third inducer is administered until the subject no longer requires the expression of the transgene.
  • Embodiment 25 The method of any one of claims 22-24, wherein the method further comprises repeating the first or second administering step if the subject becomes non-responsive to the inducer.
  • Embodiment 26 The method of any one of Embodiments 1-25, wherein each of the first dose, second dose, and third dose comprises an amount of the inducer that is sufficient to express a measurable level of the transgene in the subject.
  • Embodiment 27 The method of any one of Embodiments 1-26, wherein the first inducer is artemisinin, a derivative of artemisinin, or flavone.
  • Embodiment 28 The method of any one of Embodiments 1-27, wherein the second inducer is artemisinin, a derivative of artemisinin, or flavone.
  • Embodiment 29 The method of any one of Embodiments 1-28, wherein the third inducer is artemisinin, a derivative of artemisinin, or flavone.
  • Embodiment 30 The method of any one of Embodiments 1-29, wherein the first and second inducer are the same.
  • Embodiment 31 The method of claim 30, wherein the first and second inducer are an artemisinin derivative.
  • Embodiment 32 The method of claim 30, wherein the first and second inducer are artemisinin.
  • Embodiment 33 The method of any one of Embodiments 1-32, wherein the first and second inducer are different.
  • Embodiment 34 The method of claims 33, wherein the first inducer is a first artemisinin derivative, and the second inducer is a second artemisinin derivative.
  • Embodiment 35 The method of claim 33, wherein the first inducer is artemisinin, and the second inducer is an artemisinin derivative.
  • Embodiment 36 The method of claim 33, wherein the first inducer is an artemisinin derivative, and the second inducer is artemisinin.
  • Embodiment 37 The method of any one of Embodiments 1-36, wherein the first inducer, second inducer, and third inducer are the same.
  • Embodiment 38 The method of claim 37, wherein the first inducer, second inducer, and third inducer are an artemisinin derivative.
  • Embodiment 39 The method of claim 37, wherein the first inducer, second inducer, and third inducer are artemisinin.
  • Embodiment 40 The method of any one of Embodiments 1-40, wherein the a) first inducer and b) second inducer and third inducer are different, and the second inducer and third inducer are the same.
  • Embodiment 41 The method of claims 40, wherein the first inducer is a first artemisinin derivative, and the second inducer and third inducer are a second artemisinin derivative.
  • Embodiment 42 The method of claim 40, wherein the first inducer is artemisinin, and the second inducer and third inducer are an artemisinin derivative.
  • Embodiment 43 The method of claim 40, wherein the first inducer is an artemisinin derivative, and the second inducer and third inducer are artemisinin.
  • Embodiment 44 The method of any one of Embodiments 1-43, wherein the artemisinin derivative is a compound of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • the artemisinin derivative is a compound of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (
  • Embodiment 45 The method of any one of Embodiments 1-44, wherein the derivative of artemisinin is artesunate, arteether, artemether, or dihydroartemisinin (DHA).
  • Embodiment 46 The method of any one of Embodiments 1-45, wherein the first or second inducer is, independently, artemisinin or artesunate.
  • Embodiment 47 The method of any one of Embodiments 1-46, wherein the first or second inducer is, independently, arteether, artemether, DHA, or flavone.
  • Embodiment 48 The method of any one of Embodiments 1-46, wherein the synthetic inducible promoter comprises at least two PBREM enhancers.
  • Embodiment 49 The method of any one of Embodiments 1-47, wherein the synthetic inducible promoter comprises at least three PBREM enhancers.
  • Embodiment 50 The method of any one of Embodiments 1-48, wherein the synthetic inducible promoter comprises three PBREM enhancers.
  • Embodiment 51 The method of any one of Embodiments 1-49, wherein the synthetic inducible promoter comprises a MinTK promoter.
  • Embodiment 52 The method of any one of Embodiments 1-50, wherein the synthetic inducible promoter comprises a MinTK minimal promoter.
  • Embodiment 53 The method of any one of Embodiments 1-51, wherein the transgene encodes a chimeric antigen receptor (CAR), hormone, antibody reagent, or nuclease.
  • Embodiment 54 The method of claim 53, wherein the hormone is selected from the group consisting of Erythropoietin (EPO) and parathyroid hormone (PTH).
  • EPO Erythropoietin
  • PTH parathyroid hormone
  • Embodiment 55 The method of claim 53, wherein the antibody reagent is selected from the group consisting of ⁇ - PD1, ⁇ -HER2, ⁇ -PCSK9, ⁇ -VEGFR2, ⁇ -IL17, and ⁇ -A ⁇ 3-42.
  • Embodiment 56 The method of claim 53, wherein the nuclease is selected from the group consisting of Cas9, Cas12, Cas Phi, CasX, and CasRx.
  • Embodiment 57 The method of any one of Embodiments 1-56, wherein the subject is a primate.
  • Embodiment 58 The method of any one of Embodiments 1-57, wherein the subject is a human.
  • Embodiment 59 The method of any one of Embodiments 1-58, wherein the administration of the first inducer, the second inducer, and/or the third inducer is oral administration.
  • Embodiment 60 The method of any one of Embodiments 1-59, wherein the administration of the first inducer, the second inducer, and/or the third inducer is intraperitoneal administration.
  • Embodiment 61 The method of any one of Embodiments 1-60, the method further comprising administering the exogenous nucleotide sequence to the subject prior to administering the first inducer.
  • Embodiment 62 A first, a second, and a third inducer for use in a method of regulating expression of a transgene in the liver of a subject, the method comprising: administering to the subject at at least a first time point a dose of a first inducer; administering to the subject at at least a second time point a second dose of a second inducer, wherein the second inducer can be the same as or different than the first inducer; and administering to the subject at at least a third time point a third dose of a third inducer, wherein the third inducer can be the same as or different than the first inducer and the second inducer; wherein the subject comprises at least one liver cell comprising an exogenous nucleotide sequence comprising a synthetic inducible promoter operably linked to a transgene; wherein the synthetic inducible promoter comprises at least one PBREM enhancer; wherein the transgene is detrimental to the liver cell or to the subject when constitutively expressed.
  • Embodiment 63 A first, a second and a third inducer for use according to claim 62, wherein the method is as defined in any one of claims 2 to 61.
  • Embodiment 64 A first, a second and a third inducer for use according to claim 62 or 63, wherein the method is a method of therapy, suitably a method of gene therapy.
  • Embodiment 65 Artemisinin or a derivative thereof for use as a first, a second and/or a third inducer in a method of regulating expression of a transgene in the liver of a subject, the method comprising: administering to the subject at at least a first time point a dose of a first inducer; administering to the subject at at least a second time point a second dose of a second inducer, wherein the second inducer can be the same as or different than the first inducer; and administering to the subject at at least a third time point a third dose of a third inducer, wherein the third inducer can be the same as or different than the first inducer and the second inducer; wherein the subject comprises at least one liver cell comprising an exogenous nucleotide sequence comprising a synthetic inducible promoter operably linked to a transgene; wherein the synthetic inducible promoter comprises at least one PBREM enhancer; wherein the transgene is detrimental to the liver cell or to
  • Embodiment 66 Artemisinin or a derivative thereof for use according to claim 65, wherein at least two of the first, second and third inducers are each, independently, artemisinin or a derivative thereof.
  • Embodiment 67 Artemisinin or a derivative thereof for use according to claim 65 or 66, wherein the first, second and third inducers are each, independently, artemisinin or a derivative thereof.
  • Embodiment 68 Artemisinin or a derivative thereof for use according to any one of claims 65 to 67, wherein the method is as defined in any one of claims 2 to 61.
  • Embodiment 69 Artemisinin or a derivative thereof for use according to any one of claims 65 to 68, wherein the method is a method of therapy, suitably a method of gene therapy.
  • Embodiment 1 A method of regulating expression of a transgene in a primate subject, the method comprising: (a) administering to the subject at a first time point a first inducer, wherein the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the inducible promoter comprises a PBREM enhancer, wherein the first inducer is artemisinin or a derivative thereof or a flavonoid compound, and wherein the inducer is administered in an amount that is sufficient to express a measurable level of the transgene in the subject; and (b) administer
  • Embodiment 2 The method of Embodiment 1, wherein the expression of the transgene increases for a predetermined time after each administration.
  • Embodiment 3 The method of Embodiment 1 or 2, wherein the first and second inducer are the same.
  • Embodiment 4 The method of Embodiment 3, wherein the first and second inducer are artemisinin, a derivative of artemisinin, or a flavonoid compound.
  • Embodiment 5 The method of Embodiment 3, wherein the first inducer and second inducer are artemisinin.
  • Embodiment 6 The method of Embodiment 3, wherein the first inducer and second inducer are a derivative of artemisinin.
  • Embodiment 7 The method of Embodiment 3, wherein the first inducer and second inducer are a flavonoid compound, optionally, the flavonoid compound is flavone.
  • Embodiment 8 The method of any one of Embodiments 1-2, wherein the first and second inducer are different.
  • Embodiment 9 The method of Embodiment 8, wherein the first inducer is artemisinin, and the second inducer is an artemisinin derivative or a flavonoid compound.
  • Embodiment 10 The method of Embodiments 8, wherein the first inducer is a first artemisinin derivative, and the second inducer is artemisinin, a second artemisinin derivative, or a flavonoid compound.
  • Embodiment 11 The method of Embodiment 8, wherein the first inducer is a flavonoid compound and the second inducer is artemisinin or a derivative of artemisinin.
  • Embodiment 12 The method of any one of Embodiments 1-11, wherein the artemisinin derivative is a compound of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • the artemisinin derivative is a compound of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • Embodiment 13 The method of any one of Embodiments 1-12, wherein the artemisinin derivative is artesunate, dihydroartemisinin (DHA), artesunate, or artemether, optionally, the artemisinin derivative is artesunate, or dihydroartemisinin, more optionally, the artemisinin derivative is artesunate.
  • DHA dihydroartemisinin
  • Embodiment 14 The method of any one of Embodiments 1-13, wherein the flavonoid compound is flavone, baicalein, chrysin, galangin, (OH) 4 flavone, 6-hydroxyluteolin, acacetin, acerosin, alnetin, apigenin, apiin, artocarpetin, balcalein, beta-sitosterol, biochamin A, cannaflavin A, cannflavin A, cannflavin B, cannflavin C, catechin, catechin-3-gallate, cerrosillin, chrysoeriol, chrysoeriol, cirsilineol, cirsiliol, cirsimaritin, corymbosin, cupressuflavone, cyanidine, daidzein, datiscetin, delphinidine, digidrokempferol , dihydroquercetin, diosmetin, dio
  • Embodiment 15 The method of any one of Embodiments 1-14, wherein the first and second inducer are administered at about the same amount.
  • Embodiment 16 The method of Embodiment 15, wherein the first inducer and the second inducer are administered at different amounts.
  • Embodiment 17 The method of Embodiment 16, wherein the second inducer is administered at a lower amount relative to the amount of the first inducer.
  • Embodiment 18 The method of Embodiment 17, wherein the second inducer is administered in amount about 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, 1/15, 1/20, 1/25, 1/30, 1/35, 1/40, 1/45, 1/50, 1/55, 1/60, 1/65, 1/70, 1/75, 1/80, 1/85, 1/90, 1/95, 1/100, 1/125, 1/150, 1/175, 1/200, 1/500, 1/1000 or even less of the amount of first the inducer.
  • Embodiment 18 The method of Embodiment 18, wherein the second inducer is administered in amount about 1/2, 1/4, 1/5, 1/8, 1/10, 1/20, 1/40, 1/80 or less of the amount of the first inducer.
  • Embodiment 20 The method of Embodiment 16, wherein the second inducer is administered at a higher amount relative to the amount of the first inducer.
  • Embodiment 21 The method of Embodiment 20, wherein the amount of inducer administered at the second time point is at least about 25%, 50%, about 75%, about 1-fold, about 1.2-folds, about 1.25-folds, about 1.3-folds, about 1.5-folds, about 1.75-folds, about 2-folds, about 2.25-folds, about 2.5-folds, about 2.5-folds, about 2.75-folds, about 3-folds, 4-fold, 5-fold, 6-folds, 7-fold, 8-fold, 9-folds, 10-folds or higher than the amount of the first inducer.
  • Embodiment 22 The method of any one of Embodiments 1-21, wherein the first and second inducer are administered independently at a dose from about 0.5 mg/kg to about 150mg/kg.
  • Embodiment 23 The method of any one of Embodiments 1-22, wherein the first inducer is administered at a dose from about 1 mg/kg to about 100 mg/kg.
  • Embodiment 24 The method of any one of Embodiments 1-23, wherein the first inducer is administered at a dose of about 80mg/kg, about 70mg/kg, about 60mg/kg, about 50mg/kg, about 40mg/kg, about 30mg/kg, about 20mg/kg, about 10mg/kg, about 6mg/kg, about 5mg/kg, or about 1mg/kg.
  • Embodiment 25 The method of any one of Embodiments 1-24, wherein the second inducer is administered at a dose from about 1 mg/kg to about 100 mg/kg.
  • Embodiment 26 The method of any one of Embodiments 1-25, wherein the second inducer is administered at a dose of 80mg/kg, about 70mg/kg, about 60mg/kg, about 50mg/kg, about 40mg/kg, about 30mg/kg, about 20mg/kg, about 10mg/kg, about 6mg/kg, about 5mg/kg, or about 1mg/kg.
  • Embodiment 27 The method of any one of Embodiments 1-16, wherein the first inducer is administered at a dose of about 80 mg/kg or less and the second inducer is administered at a dose of about 20mg/kg or less at the second or subsequent time point.
  • Embodiment 30 The method of any one of Embodiments 1-16, wherein the first inducer is administered at a dose of about 10 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point.
  • Embodiment 31 The method of any one of Embodiments 1-16, wherein the first inducer is administered at a dose of about 6 mg/kg and the second inducer is administered at a dose of about 80mg/kg or less at the second or subsequent time point.
  • Embodiment 34 The method of any one of Embodiments 1-16, wherein the second inducer is administered at a dose of about 40 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 35 The method of any one of Embodiments 1-16, wherein the second inducer is administered at a dose of about 20 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 36 The method of any one of Embodiments 1-16, wherein the second inducer is administered at a dose of about 10 mg/kg and the first inducer is administered at a dose of about 80 mg/kg or less.
  • Embodiment 42 The method of any one of Embodiments 1-41, wherein the second inducer is administered when it is determined that the level of expression of the transgene is decreased to 25% or less of the level of expression of the transgene attained after induction with the first inducer.
  • Embodiment 43 The method of any one of Embodiments 1-42, wherein the second inducer is administered when it is determined that the level of expression of the transgene is decreased to a background level of expression of the transgene.
  • Embodiment 48 The method of any one of Embodiments 1-47, wherein the time between administrations is no more than 2 months.
  • Embodiment 49 The method of any one of Embodiments 1-48, wherein the time between administrations is 1-2 days, 3-4 days, 5-7 days, 5-10 days, 7-10 days, 7-14 days, 10-14 days, 10-20 days, 14-21 days, 14-28 days, 21-28 days, or 21-34 days.
  • Embodiment 50 The method of any one of Embodiments 1-159, wherein the time between administrations is about a day, e.g., about 24 hours.
  • Embodiment 56 The method of any one of Embodiments 1-55, wherein the exogenous nucleotide sequence comprising the inducible promoter operably linked to a transgene is adeno- associated viral (AAV), lentiviral, adenoviral, herpes viral, or hepatitis viral vector.
  • AAV adeno- associated viral
  • Embodiment 57 The method of any one of Embodiments 1-56, wherein the exogenous nucleotide sequence comprising the inducible promoter operably linked to a transgene is an AAV vector, e.g., an AAV gene therapy vector.
  • Embodiment 62 The method of any one of Embodiments 1-58, wherein at least one of the inducers is administered intraperitoneally.
  • Embodiment 63 The method of Embodiment 62, wherein the second inducer is administered intraperitoneally.
  • Embodiment 64 The method of Embodiment 62, wherein the first inducer is administered intraperitoneally.
  • Embodiment 66 The method of Embodiment 65, wherein the transgene expression level at about 24 hrs after the first induction, or second induction, or successive inductions is at least about 200-fold higher than that of prior to administering the inducer for the first time, or, the second time, or any number of successive administrations.
  • Embodiment 67 The method of Embodiment 65, wherein the first induction, the second induction, and/or, the successive inductions are not complete until about 160 hours after administering the inducer.
  • Embodiment 68 The method of Embodiment 65, wherein dose of the inducer in successive administrations is reduced from its prior administration.
  • Embodiment 74 The method of Embodiment 73, wherein the method comprises administering a plurality of doses of the inducer.
  • Embodiment 75 The method of Embodiment 74, wherein the time between administrations is at least 12 hours, at least 24 hours, 48 hours, 72 hours, 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks.
  • Embodiment 76 The method of any one of Embodiments 74-75, wherein the time between administrations is at least 24 hours, 48 hours, or 72 hours.
  • Embodiment 80 The method of any one of Embodiments 74-79, wherein the time between administrations is about 1 day.
  • Embodiment 81 The method of any one of Embodiments 74-80, wherein the transgene expression level at about 24 hrs after an induction is at least about 200-fold higher than that of prior to administering the inducer for the first time or any number of prior administrations.
  • Embodiment 82 The method of any one of Embodiments 74-81, wherein an induction is not complete until about 160 hours after administering the inducer.
  • Embodiment 85 The method of any one of Embodiments 74-84, wherein dose in successive administrations remains same.
  • Embodiment 86 The method of any one of Embodiments 73-85, wherein the inducer is artemisinin or an artemisinin derivative.
  • Embodiment 100 The method of any one of Embodiments 73-99, wherein a second inducer or, subsequent inducer is administered to the primate subject following the prior administration of an inducer to the primate subject, and optionally, the inducer of the prior administration is administered in an amount that is sufficient to express a measurable level of the transgene in the subject.
  • Embodiment 101 The method of Embodiment 100, wherein the inducer of the prior administration is different from the second or subsequent inducer administered to the subject.
  • Embodiment 102 The method of Embodiment 101, wherein the inducer of the prior administration is same as the second or, subsequent inducer administered to the subject but at a different dose.
  • Embodiment 107 The method of any one of Embodiments 73-106, wherein each administration independently of other administrations is oral administration or intraperitoneal administration.
  • Embodiment 108 The method of any one of Embodiments73-107, wherein each administration is oral administration.
  • Embodiment 109 The method of any one of Embodiments 73-107, wherein each administration is intraperitoneal administration.
  • Embodiment 111 The method of Embodiment 110, wherein the transgene encodes an antibody or an antigen binding portion thereof, optionally the antibody is an anti-PD1 antibody, anti-HER2 antibody, anti-PCSK9 antibody, anti-VEGFR2 antibody, anti-IL17 antibody, anti-A ⁇ 3- 42 antibody, anti-Sema3A antibody, anti-4-1BB (CD137) antibody, anti-5'-nucleotidase antibody, anti-5T4 antibody, anti-activin receptor-like kinase 1 antibody, anti-ACVR2B antibody, anti- adenocarcinoma antigen antibody, anti-amyloid antibody, anti-Ang-2 antibody, anti-angiopoietin 2 antibody, anti-angiopoietin 2 antibody, anti-angiopoietin 3 antibody, anti-anthrax toxin antibody, anti-AOC3 (VAP-1) antibody, anti-AXL antibody, anti-BAFF-R antibody, anti-
  • Embodiment 113 The method of Embodiment 110, wherein the transgene encodes a coagulation factor, optionally the coagulation factor is Factor VIII, Factor IX, Factor VII, Factor X, or Factor XI, preferably the coagulation factor is Factor VIII or Factor IX.
  • the transgene encodes a coagulation factor, optionally the coagulation factor is Factor VIII, Factor IX, Factor VII, Factor X, or Factor XI, preferably the coagulation factor is Factor VIII or Factor IX.
  • Embodiment 114 The method of Embodiment 110, wherein the transgene encodes a hormone, optionally the hormone is selected from the group consisting of selected from erythropoietin (EPO), parathyroid hormone (PTH), adiponectin , adrenaline (or epinephrine), adrenocorticotropic hormone (ACTH or corticotropin), adropin, aldosterone, amylin (or islet amyloid polypeptide), androstenedione, angiotensin, angiotensinogen, antidiuretic hormone (or vasopressin), anti-müllerian hormone (or müllerian-inhibiting factor/hormone), arginine vasopressin), atrial natriuretic peptide (ANP or atriopeptin), brain natriuretic peptide, calcitonin, calcitriol, cholecystokinin (EPO), parat
  • Embodiment 115 The method of Embodiment 110, wherein the transgene encodes a growth factor, optionally, the growth factor is tumor necrosis factor-alpha (TNF- ⁇ ), adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), colony-stimulating factor (e.g., macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), or granulocyte macrophage colony-stimulating factor (GM-CSF)), epidermal growth factor (EGF), ephrin (e.g., ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ep
  • Embodiment 116 The method of Embodiment 110, the transgene encodes a CRISPR- Cas nuclease, optionally selected from the group consisting of Cas9, Cas12, Cas Phi, CasX, and CasRx.
  • Embodiment 117 The method of Embodiment 110, wherein the transgene encodes a cytokine, optionally, the cytokine is interleukin-l ⁇ (IL-l ⁇ ), interleukin-1/3 (IL-1/3), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8/CXCL8), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin- 13 (IL- 13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-37 (IL-37), interleukin-38 (IL-38), tumor necrosis factor- ⁇ (TNF- ⁇ ), interferon- ⁇ (INF- ⁇ ), interferon-b (INF- b), interferon- ⁇ (INF- ⁇ ), gran
  • the transgene encodes IL-2. In some embodiments, the transgene encodes IL-37. In some embodiments, the transgene encodes IL-38.
  • Embodiment 118 The method of Embodiment 110, wherein the transgene encodes a virus protein, optionally selected from the group consisting of viral envelop protein, viral nucleocapsid protein, viral membrane protein, REP, CAP, REV, VSV-G and RD114
  • Embodiment 119 The method of any one of clams 1-110, wherein the transgene is detrimental to the cell or to the subject when constitutively expressed.
  • Embodiment 120 The method of any one of Embodiments 1-119, wherein the subject is a non-human primate.
  • Embodiment 121 The method of any one of Embodiments 1-120, wherein the subject is a human.
  • Embodiment 122 The method of any one of Embodiments 1-121, wherein the cell comprising the cell comprising the exogenous nucleotide sequence is a liver cell.
  • Embodiment 123 A first and at least a second inducer for use in a method of regulating expression of a transgene in a primate subject, the method comprising: (a) administering to the subject at a first time point a first inducer, wherein the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the inducible promoter comprises a PBREM enhancer, wherein the first inducer is artemisinin, a derivative of artemisinin or a flavonoid compound, and wherein the first dose comprises an amount of the inducer that is sufficient to express a measurable level of the transgene in the subject; and (b) administering to the subject at least at a second time point a second inducer to continue expression of the transgene at a predetermined level for a predetermined time or to modulate expression of the transgene to a predetermined level, and wherein the second inducer
  • Embodiment 124 The first and at least a second inducer for use according to Embodiment 123, wherein the method is according to any one of Embodiments 1 to 122.
  • Embodiment 125 The first and at least a second inducer for use according to Embodiment 123 or 124, which is a method of therapy, suitably a method of gene therapy.
  • Embodiment 126 The first and at least a second inducer for use according to any one of Embodiments 123-125, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV gene therapy vector.
  • Embodiment 128 Artemisinin or a derivative of artemisinin for use according to Embodiment 127, wherein the method is a method of therapy, suitably a method of gene therapy.
  • Embodiment 129 Artemisinin or a derivative of artemisinin for use according to Embodiment 127 or 128, wherein the artemisinin derivative is a compound as defined in Embodiment 2, optionally wherein the artemisinin derivative is of Formula (I), (I-A), (I-B), (I-C), (I-D), (I-E), (I-F), (I-G), (I-H), (I-I), (I-J), (I-K), (I-L), (I-M), (I-M’), (I-N), (I-N’), (I-O) or (I-O’).
  • Embodiment 130 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 127-129, wherein the derivative of artemisinin is artesunate, dihydroartemisinin, artesunate, or artemether.
  • Embodiment 131 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 127-130, wherein artemisinin or a derivative of artemisinin is administered to the primate subject following the prior administration of an inducer to the primate subject.
  • Embodiment 132 Artemisinin or a derivative of artemisinin for use according to Embodiment 131, wherein the inducer of the prior administration was a different inducer, or wherein the inducer of the prior administration was the same inducer but at a different dose.
  • Embodiment 133 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 127-132, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV gene therapy vector.
  • Embodiment 134 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 127 to 133, wherein the method comprises prior administration of the exogenous nucleotide sequence to the subject prior to administering the inducer.
  • Embodiment 135 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 127-134, wherein the artemisinin or a derivative of artemisinin is administered at a dose of from about 0.1mg/kg to 80mg/kg, from about 0.1mg/kg to about 70mg/kg, from about 0.1mg/kg to about 60mg/kg, from about 0.1mg/kg to about 50mg/kg, from about 0.1mg/kg to about 40mg/kg, from about 0.1mg/kg to about 30mg/kg, from about 0.1mg/kg to about 20mg/kg, from about 0.1mg/kg to about 10mg/kg, from about 0.1mg/kg to about 6mg/kg, from about 0.1mg/kg to about 5mg/kg, or from about 0.1mg/kg to about 1mg/kg.
  • Embodiment 136 Artemisinin or a derivative of artemisinin for use according to any one of Embodiments 127-135, wherein the inducer comprising artemisinin or a derivative of artemisinin is administered orally, optionally wherein the composition comprises artesunate.
  • Embodiment 137 A flavonoid compound, for use as an inducer in a method of regulating expression of a transgene in a primate subject, wherein the primate subject comprises at least one cell comprising an exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene, wherein the inducible promoter comprises a PBREM enhnacer, wherein the inducer is administered to the subject at a dose which comprises an amount of the inducer that is sufficient to express a therapeutically effective level of the transgene in the subject.
  • Embodiment 138 Flavonoid compound for use according to Embodiment 137, wherein the method is a method of therapy, suitably a method of gene therapy.
  • Embodiment 139 Flavonoid compound for use according to Embodiment 137 or 138, wherein the flavonoid compound is flavone, baicalein, chrysin, galangin, (OH) 4 flavone, 6- hydroxyluteolin, acacetin, acerosin, alnetin, apigenin, apiin, artocarpetin, balcalein, beta-sitosterol, biochamin A, cannaflavin A, cannflavin A, cannflavin B, cannflavin C, catechin, catechin-3- gallate, cerrosillin, chrysoeriol, chrysoeriol, cirsilineol, cirsiliol, cirsimaritin, corymbosin
  • Embodiment 140 Flavonoid compound for use according to any one of Embodiments 137-139, wherein the flavonoid compound is flavone
  • Embodiment 141 Flavonoid compound for use according to any one of Embodiments 137-140, wherein the flaovonoid compound is administered to the primate subject following the prior administration of an inducer to the primate subject.
  • Embodiment 142 Flavonoid compound for use according to Embodiment 141, wherein the inducer of the prior administration was a different inducer, or wherein the inducer of the prior administration was the same inducer but at a different dose.
  • Embodiment 143 Flavonoid compound for use according to any one of Embodiments 137-142, wherein the exogenous nucleotide sequence comprising an inducible promoter operably linked to a transgene is an AAV gene therapy vector.
  • Embodiment 144 Flavonoid compound for use according to any one of Embodiments 137-143, wherein the method comprises prior administration of the exogenous nucleotide sequence to the subject prior to administering the inducer.
  • Embodiment 145 Flavonoid compound for use according to any one of Embodiments 137-144, wherein the flavonoid compound is administered at a dose of from about 0.1mg/kg to 80mg/kg, from about 0.1mg/kg to about 70mg/kg, from about 0.1mg/kg to about 60mg/kg, from about 0.1mg/kg to about 50mg/kg, from about 0.1mg/kg to about 40mg/kg, from about 0.1mg/kg to about 30mg/kg, from about 0.1mg/kg to about 20mg/kg, from about 0.1mg/kg to about 10mg/kg, from about 0.1mg/kg to about 6mg/kg, from about 0.1mg/kg to about 5mg/kg, or from about 0.1mg/kg to about 1mg/kg.
  • Embodiment 146 Flavonoid compound for use according to any one of Embodiments 137-145, wherein the flavonoid compound is administered orally.
  • Embodiment 147 Flavonoid compound for use according to any one of Embodiments 137-145, wherein the flavonoid compound is administered intraperitoneally.
  • Embodiment 148 A method of regulating exprcssion of a transgene in the liver of a subject, the method comprising: administering to the subject at least a first time point a dose of a first inducer; administering to the subject at least a second time point a second dose of a second inducer, wherein the second inducer can be the same as or different than the first inducer; and administering to the subject at least a third time point a third dose of a third inducer, wherein the third inducer can be the same as or different than the first inducer and the second inducer; wherein the subject comprises at least one liver cell comprising an exogenous nucleotide sequence comprising a synthetic inducible promoter operably linked to a transgene; wherein the synthetic inducible promoter comprises at least one PBREM enhancer; and wherein the transgene is detrimental to the liver cell or to the subject when constitutively expressed.
  • Embodiment 150 The method of Embodiment 148 or 149, wherein the second inducer is selected from the group consisting of artemisinin; derivative of artemisinin; a flavonoid compound, e.g. flavone, chrysin, baicalein, or galangin; 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP); phenobarbital (PB); 6-(4Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde- O-(3,4-dichlorobenzyl)oxime (CITCO); Acetaminophen; Buprenorphine; Phenytoin; Carbamazepine; Valproic Acid; Chlorpromazine; Efavirenz; Nevirapine; Rilpivirine; Etravirine; Diazepam; Cyclophosphamid; Ifosfamide
  • CRE cis-regulatory element
  • CREs typically regulate gene transcription by binding to TFs, i.e., they include TFBS.
  • a single TF may bind to many CREs, and hence control the expression of many genes (pleiotropy).
  • CREs are usually, but not always, located upstream of the transcription start site (TSS) of the gene that they regulate.
  • TSS transcription start site
  • Enhancers are CREs that enhance (i.e., upregulate) the transcription of genes that they are operably associated with, and can be found upstream, downstream, and even within the introns of the gene that they regulate. Multiple enhancers can act in a coordinated fashion to regulate transcription of one gene.
  • “Silencers” in this context relates to CREs that bind TFs called repressors, which act to prevent or downregulate transcription of a gene.
  • the term “silencer” can also refer to a region in the 3' untranslated region of messenger RNA, that bind proteins which suppress translation of that mRNA molecule, but this usage is distinct from its use in describing a CRE.
  • the CREs described herein are liver- specific inducible enhancers.
  • the CRE is located 1500 nucleotides or less from the transcription start site (TSS), more preferably 1000 nucleotides or less from the TSS, more preferably 500 nucleotides or less from the TSS, and suitably 250, 200, 150, or 100 nucleotides or less from the TSS.
  • CREs described herein are preferably comparatively short in length, preferably 100 nucleotides or less in length, for example they may be 90, 80, 70, 60 nucleotides or less in length.
  • the CRE is a PBREM enhancer.
  • the term “cis-regulatory module” or “CRM” means a functional module made up of two or more CREs.
  • a CRM typically comprises a plurality of liver-specific inducible CREs.
  • the multiple CREs within the CRM act together (e.g., additively or synergistically) to enhance the transcription of a gene that the CRM is operably associated with.
  • shuffle i.e., reorder
  • invert i.e., reverse orientation
  • spacing between CREs has been altered.
  • promoter refers to a region of DNA that generally is located upstream of a nucleic acid sequence to be transcribed that is needed for transcription to occur, i.e., which initiates transcription. Promoters permit the proper activation or repression of transcription of a coding sequence under their control.
  • a promoter typically contains specific sequences that are recognized and bound by plurality of TFs. TFs bind to the promoter sequences and result in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene. A great many promoters are known in the art.
  • the inducible described herein typically drive a low level of expression prior to being induced, and upon induction they drive a significantly higher level of expression (e.g., a 2, 3, 4, 5, 6, 7, 8, 9, or even 10-fold increase in expression after induction.
  • the inducible promoters described herein is a synthetic promoter.
  • the term “synthetic promoter” as used herein relates to a promoter that does not occur in nature. In the present context it typically comprises a synthetic CRE and/or CRM described herein operably linked to a minimal (or core) promoter or proximal promoter, e.g., liver-specific proximal promoter.
  • the CREs and/or CRMs described herein serve to provide inducible liver- specific transcription of a gene operably linked to the promoter.
  • Parts of the synthetic promoter can be naturally occurring (e.g., the minimal promoter or one or more CREs in the promoter), but the synthetic promoter as a complete entity is not naturally occurring.
  • “minimal promoter” also known as the “core promoter” refers to a short DNA segment which is inactive or largely inactive by itself, but can mediate transcription when combined with other transcription regulatory elements.
  • Minimum promoter sequence can be derived from various different sources, including prokaryotic and eukaryotic genes.
  • minimal promoters examples include the dopamine beta-hydroxylase gene minimum promoter, cytomegalovirus (CMV) immediate early gene minimum promoter (CMV- MP), SV40 minimal promoter (SV40-MP), and the herpes thymidine kinase minimal promoter (MinTK).
  • CMV cytomegalovirus
  • CMV- MP cytomegalovirus immediate early gene minimum promoter
  • SV40-MP SV40 minimal promoter
  • MinTK herpes thymidine kinase minimal promoter
  • a minimal promoter typically comprises the transcription start site (TSS) and elements directly upstream, a binding site for RNA polymerase II, and general transcription factor binding sites (often a TATA box).
  • TATA box transcription start site
  • proximal promoter relates to the minimal promoter plus the proximal sequence upstream of the gene that tends to contain primary regulatory elements.
  • the proximal promoter is suitably a naturally occurring liver-specific proximal promoter that can be combined with one or more CREs or CRMs described herein.
  • the proximal promoter can be synthetic.
  • a “functional variant” of a cis-regulatory element, cis-regulatory module, promoter or other nucleic acid sequence in the context of the present disclosure is a variant of a reference sequence that retains the ability to function in the same way as the reference sequence, e.g., as an inducible liver-specific cis-regulatory enhancer element, inducible liver-specific cis-regulatory module or inducible liver-specific promoter.
  • Alternative terms for such functional variants include “biological equivalents” or “equivalents”.
  • a functional variant of a cis-regulatory element will contain a suitable binding site for the CAR-RXR heterodimer.
  • the CAR-RXR heterodimer is believed to bind to the NR1 motif in the wild type PBREM element, and thus a sequence which can function as an NR1 motif is desired.
  • liver-specific expression is such that there is negligible expression in other (i.e., non-liver) tissues or cells, i.e., expression is highly liver-specific.
  • expression is highly liver-specific.
  • the ability of a promoter to function as a liver-specific inducible promoter can be readily assessed by the skilled person. The skilled person can thus easily determine whether any variant of the specific promoters exemplified herein remains functional (i.e., it is a functional variant as defined above).
  • a functional variant of a CRE should provide a level of inducibility that is at least 50%, preferably 75%, more preferably 80%, 85%, 90%, or 95% as inducible when compared to the equivalent construct comprising the wild type PBREM element, e.g. SEQ ID NO: 1 (measured in terms of fold increase in expression as a result of induction, i.e. a 2 fold increase in expression of a reported gene upon induction is considered to be 50% as inducible as a 4-fold increase).
  • SEQ ID NO: 1 measured in terms of fold increase in expression as a result of induction, i.e. a 2 fold increase in expression of a reported gene upon induction is considered to be 50% as inducible as a 4-fold increase.
  • a functional variant preferably results in a background expression level that is no more than three times as high, preferably no more than twice as high, and preferably no more than 1.5 times as high when compared to the equivalent construct comprising the wild-type mouse PBREM element (e.g. again in the PB1-MinTK construct and tested in ARE hepatocytes).
  • Liver-specificity can be identified wherein the expression of a gene (e.g., a therapeutic or reporter gene) when induced occurs preferentially or predominantly in liver-derived cells.
  • Preferential or predominant expression can be defined, for example, where the level of expression when induced is significantly greater in liver-derived cells than in other types of cells (i.e., non- liver-derived cells).
  • Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups.
  • Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof.
  • a modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property.
  • nucleic acid can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear. [00856]
  • identity and “identical” and the like refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules.
  • Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al.1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250).
  • BLAST Basic Local Alignment Search Tool
  • Methods for aligning sequences for comparison are well-known in the art.
  • Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A.
  • a global optimal alignment is suitably found by the Needleman-Wunsch algorithm with the following scoring parameters: Match score: +2, Mismatch score: -3; Gap penalties: gap open 5, gap extension 2.
  • the percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, where the alignment length includes both matches and mismatches, multiplied by 100.
  • synthetic means a nucleic acid molecule that does not occur in nature. Synthetic nucleic acid expression constructs described herein are produced artificially, typically by recombinant technologies.
  • “Therapeutically effective amount” and like phrases mean a dose or plasma concentration in a subject that provides the desired specific pharmacological effect, e.g., to express a therapeutic gene. It is emphasized that a therapeutically effective amount may not always be effective in treating the conditions described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the disease or condition being treated. [00864] The terms “treatment” or “treating” refer to reducing, ameliorating or eliminating one or more signs, symptoms, or effects of a disease or condition.
  • a subject can be a subject with a disease or condition in need of treatment. Typically, the subject in need thereof will display symptoms characteristic of a disease. The method typically comprises ameliorating the symptoms displayed by the subject in need thereof, by expressing a therapeutic amount of the expression product.
  • the term “primate” refers to a mammal that falls within the human, ape or monkey family. In some embodiments, the primate is human. In some embodiments, the subject is a non-human primate.
  • non-human primate refers to a mammal that falls within the ape or monkey family and includes, but is not limited to, a baboon, a chimpanzee, a capuchin, a pigtail macaque, a sooty mangabey, a squirrel monkey, a gibbon, a Rhesus monkey, a Cynomolgus monkey, a gorilla, an orangutan, and any other ape or monkey species. [00867] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed.
  • Example 1 Testing of artemisinin and some exemplary derivatives
  • the construct contained 3x human PBREM enhancers together with the MinTK promoter (3xhPB-minTK, SEQ ID NO: 66) to drive the expression of luciferase. This construct was tested in the presence and absence of the indicated drugs after transfection into AXOL ARE- hepatocytes.
  • Axol Assay-Ready Expanded Hepatocytes are primary human hepatocytes that have been expanded in vitro. Large batch sizes (up to 2000 vials) are available, ensuring a reliable, ready-to-use and consistent source of primary hepatocytes.
  • ARE Hepatocytes express CYP enzymes, are metabolically functional, are polarized and can be infected by the Hepatitis C virus.
  • AXOL ARE-hepatocytes express CAR negating the need to transfect with a CAR-expressing plasmid.
  • the inducers used are known to act by activating the CAR transcription factor and promoting the translocation to the nucleus.
  • ARE assay-ready expanded hepatocytes
  • ARE hepatocytes
  • ARE hepatocyte thawing medium
  • ARE maintenance medium Axol, ax3710
  • Virimer red transfection reagent Lipocalyx, VR04-02-15
  • Artemisinin Sigma-Aldrich, 1042747
  • Artesunate Sigma-Aldrich, PH2573
  • Arteether Sigma-Aldrich, SML2591
  • DHA Sigma-Aldrich, 1200520
  • Artemether Sigma-Aldrich, 1042780
  • Nicardipine Sigma-Aldrich, Y001924
  • Flavone Sigma-Aldrich, F2003
  • Rifampicin Tcoris 4121
  • oestradiol Tcoris 2824
  • Valproic acid Tcoris 2815
  • ARE hepatocytes were cultured and transfected as described in the manufacturer’s manual. 200,000 cells were seeded in 2 ml culture medium in collagen-coated 6 well plate. The cells were incubated for 4 h at 37°C and 5% CO2 for sufficient adhesion.
  • transfection mixture containing the DNA to be transfected (3xhPB-MinTK operably linked with luciferase and a ⁇ -galactosidase containing plasmid) was added and the cells were incubated on an orbital shaker at 100 rpm for three hours at 37°C and 5% CO 2 .3hrs after transfection the putative inducers were added to the appropriate wells. Cells were incubated under static conditions at 37°C and 5% CO2 overnight and in the morning the medium was replaced with fresh ARE Hepatocyte Maintenance Medium. Readout was 24hrs after induction. Luciferase expression was measured as per manufacturer’s instructions. Luciferase was normalised by total protein and transfection efficiency.
  • Artemisinin is metabolised by cyb2b6 which is the major enzyme involved in CAR activated detoxification, whereas the other drugs are metabolised by different cytochrome P450 enzymes.
  • the 2 most potent artemisinin compounds to use for induction of the 3xhPB promoter are artemisinin and artesunate, however each of the other derivatives could be used if lower expression levels were required (up to CMV-IE).
  • flavone also showed induction of the promoter. This induction was equivalent to the response observed for DHA and Arteether which was equivalent to CMV-IE. However, this drug was used at 6x the concentration of the anti-malarial compounds.
  • Example 2- Mice In vivo experiments using AAV comprising 3xhPB-Min-TK [00876] The 3xhPB-MinTK was taken in vivo to assess the following attributes: • Induction with artemisinin via IP injection • Induction with Artemisinin via oral gavage • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of artemisinin • Kinetics of induction, to obtain response times of the promoter to artemisinin • Different doses of AAV to assess maximum inducibility [00877] All of these experiments were performed using luciferase as a marker gene Methods: [00878] AAV production was performed as described in Greiger et al, 2015.
  • mice experiments [00879] The AAV serotype chosen was AAV9 as this has a tropism for most tissues and organs and would therefore give an idea of the specificity of our promoters (avoiding issues of AAV tropism). Output from the experiment was luciferase activity measured visually. Promoter activity from the mice were monitored weekly and after 14 – 21 days, once control vector AAV9 with CMV-IE had shown consistent steady results a baseline for induction profiling was established. After this point the inducer was added with measurements taken before and after induction. See results section for more details. Mice: [00880] Adults (8 weeks of age) CD1 male or female mice were injected with an AAV9 vector via tail vein with a total of 1x10 11 vg/mouse.
  • mice were imaged as follows. They were first anesthetized and received an intraperitoneal injection of luciferin (300 ⁇ l of luciferin stock 15mg/ml). After 5 minutes the mice were placed in the IVIS machine and images were acquired. The exposure time used for images were 1 and 10 seconds. Images were taken once every 7 days until a steady state baseline was achieved with an AAV9 containing CMV-IE. Induction was measured after artemisinin, dissolved in DMSO, was administered at an appropriate concentration depending on the experiment. Delivery was via IP or oral gavage. Ex vivo luciferase assays: [00881] The tissue samples were weighed to assure the same amount of tissue was being used for each sample.
  • 3xhPB-MinTK promoter 1
  • CMV-IE Con
  • After 14 days with weekly measurements of luciferase activity via the IVIS it was determined that the AAV expression from the CMV-IE group had stabilised.
  • the mice were then injected on day 15 with either 80mg/kg of artemisinin or vehicle and luciferase activity was measured at 9, 24 and 48hrs after induction.
  • 3xhPB-MinTK promoter 1
  • CMV-IE Con
  • After 28 days with weekly measurements of luciferase activity via the IVIS it was determined that the AAV expression from the CMV-IE group had stabilised.
  • the mice were then injected on day 32, 48 and day 134 with either 80mg/kg of artemisinin or vehicle and luciferase activity was measured at 9, 24 and 48hrs after induction.
  • 3xhPB-MinTK promoter 1
  • CMV-IE Con
  • the drug was delivered via oral gavage, with one group administered the vehicle.
  • mice were then treated on day 14 with either 80, 50, 20, 10, or 5mg/kg of artemisinin or vehicle and luciferase activity was measured at 9, 24 and 48hrs after induction. The results of this can be seen in figure 6.
  • Addition of artemisinin at all concentrations leads to a significant increase activity observed after 9-hrs, with a >30 – 5,000-fold induction observed. Again, this time point was peak induction with the level of luciferase activity decreasing at 24, and 48hrs with activity almost at non-induced levels by 48hrs.
  • the strength of activity observed at peak expression with 80mg/kg of artemisinin was 10x that of CMV-IE. Once again, the CMV-IE group activity remained constant and did not respond to artemisinin.
  • the drug was delivered via oral gavage, with one group administered the vehicle. After 14 days with weekly measurements of luciferase activity via the IVIS, it was determined that the AAV expression from the CMV-IE group had stabilised. The mice were then treated on day 14 with either 80mg/kg of artemisinin or vehicle and luciferase activity was measured at 3, 6, 9, 24 and 48hrs after induction. The results of this can be seen in Figure 7. Treatment with artemisinin leads to a significant increase in activity from 3hrs, with activity increasing until the peak was observed after 9-hrs. The level of luciferase activity then decreased at 24, and 48hrs with activity almost at non-induced levels by 48hrs.
  • 3xhPB-MinTK promoter 1
  • CMV-IE Con
  • All artemisinin treatment was at 80mg/kg.
  • the drug was delivered via oral gavage, with one group administered the vehicle. After 14 days with weekly measurements of luciferase activity via the IVIS, it was determined that the AAV expression from the CMV-IE group had stabilised.
  • mice were then treated on day 14 with either 80mg/kg of artemisinin or vehicle and luciferase activity was measured 9, 24 and 48hrs after induction.
  • luciferase activity was measured 9, 24 and 48hrs after induction.
  • Treatment with artemisinin leads to a significant increase in activity from with activity peaking after 9-hrs.
  • the level of luciferase activity then decreased at 24, and 48hrs with activity almost at non-induced levels by 48hrs.
  • the different AAV doses showed what was expected, that the lower the dose the lower the overall expression is, however, induction kinetics remained similar regardless of the dose.
  • Example 3- NHP In vivo experiments using AAV comprising 3xhPB-Min-TK [00889] The 3xhPB-MinTK packaged into AAV9 was taken in vivo in NHPs to assess the following attributes: • Induction with artemisinin via IP injection • Induction with Artemisinin via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of artemisinin • Kinetics of induction, to obtain response times of the promoter to artemisinin [00890] In this experiment chorionic gonadotropin was used as a marker gene.
  • NHPs male and female NHPs were injected with 1e13vg/kg of AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of the chorionic gonadotropin (cGT) gene.
  • LSP was chosen as the comparator promoter here as it has been used in gene therapy clinical trials and is therefore therapeutically relevant.
  • serum cGT concentrations After 28 days with weekly measurements of serum cGT concentrations it was determined that the AAV expression from the LSP group had stabilised.
  • the NHPs were then treated on day 30 with 80mg/kg of artemisinin via IP injection and serum levels of cGT were measured at 24, 48 and 72hrs after induction.
  • artemisinin was injected IP at 40, 20, 10 and 1mg/kg.
  • artemisinin is able to induce a response from the 3xhPB-MinTK promoter at every dose and in a dose dependent manner. Indeed 1mg/kg shows comparable activity to the LSP promoter.
  • the drug administered was the artemisinin derivative artesunate. This is because artemisinin has poor solubility which made it difficult to get into a form to be ingested by the NHPs. However, artesunate is more soluble and is amenable to oral administration. Also, as seen in Figure 2, artesunate is able to activate the promoters.
  • Figure 12 shows the collated results of induction in non-human primates using the protocol shown in Figure 11, i.e. two cycles of induction with 80 mg/kg IP artemisinin. Results for the LSP control promoter and saline negative control are also shown for comparison. Pre- induction gonadotropin levels are indicated by lower arrows and gonadotropin levels after induction are illustrated by upper arrows.
  • Figures 13A and 13B are bar-graph (Figure 13A) and line-graph (Figure 13B) showing individual results for each of the non-human primate in Figure 12.
  • M LSP and F LSP are the male and female LSP controls, respectively; M1 Ind and M2 Ind are males with the inducible promoter; F1 Ind and F2 Ind are females with the inducible promoter; M S and F S are male and female saline controls, respectively.
  • chorionic gonadotropin was used as a marker gene. This is a self-Ag which is not normally expressed in NHPs and which can be measured via ELISA from the serum.
  • Methods [00900] AAV production was performed as described in Greiger et al., 2015. NHP experiments [00901] Figure 14 depicts the study outline for this experiment.
  • NHPs [00902] NHP species used was Macaca fascicularis with each monkey pre-screened for neutralising Abs against AAV9 and gonadotropin expression. Each NHP weighed between 2 and 3kg at time of AAV injection.
  • Figure 15 shows an overview of all inductions that were performed in this study.
  • both IP induction via artemisinin and oral induction via artesunate produced a strong response from the promoters. Since it has poor solubility, it was difficult to get artemisinin into a form to be ingested by the NHPs.
  • artesunate was used for the oral inductions since artesunate is more soluble and is amenable to oral administration.
  • Figure 15 also shows that the promoters are dose responsive to IP and oral administration with activity observed with dose as low as 0.1mg/kg, which is far below the recommended dose of artemisinin and artesunate for treatment of malaria.
  • Figure 16 shows the kinetics of expression 24, 48, 72 and 168hrs hrs after each induction. The graph presented is an aggregate of all NHPs and there was no difference in response between male and female NHPs ( Figure 15). Pre-induction levels of cGT were equivalent to the no AAV NHP suggesting that the promoters had no background leak.
  • NHPs were dosed for 30 days continuously with 1mg/kg of artesunate orally. Measurements of cCG were taken every 7 days until final dose and then 24, 48, 72 and 168hrs later. The results of this experiment can be seen in Figure 17. As seen from Figure 17, the 1mg/kg dose elicited a strong expression from the promoters that was visible after 7 days and this expression remained constant for the time course of administration. However, once the dosing was stopped, the levels of serum cCG returned to baseline after 168hrs. There was no observed toxicity in the NHPs during this study. This demonstrates that long-term administration of the inducer is feasible and safe.
  • Example P0 In vivo induction of a marker gene in non-human primates with flavone
  • flavone is used as the inducer to induce the in vivo expression in NHPs of chorionic gonadotropin from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the chorionic gonadotropin to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0002] A vector as described herein, e.g., 3xhPB-MinTK and encoding chorionic gonadotropin is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the chorionic gonadotropin is produced as described in Greiger et al. 2015.
  • AAV9 containing LSP driving the expression of the chorionic gonadotropin is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of the chorionic gonadotropin are measured weekly, e.g., by ELISA or similar assays.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0004] Levels of the chorionic gonadotropin at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • chorionic gonadotropin levels are used as background or pre- induction level of the chorionic gonadotropin.
  • Pre-induction levels of the chorionic gonadotropin can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the chorionic gonadotropin, e.g., in the serum, with a subsequent return to background levels.
  • Example P1A In vivo induction of an anti-TNF ⁇ antibody in non-human primates
  • artemisinin is used as the inducer to induce the in vivo expression in NHPs of an anti-TNF ⁇ antibody (e.g., adalimumab) from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the antibody to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0006] A vector as described herein, e.g., 3xhPB-MinTK and encoding an anti-TNF ⁇ antibody is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the antibody is produced as described in Greiger et al. 2015.
  • AAV9 containing LSP driving the expression of the antibody is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of the antibody are pre-screened for neutralizing Abs against AAV9 and injected with 1e13vg/kg of AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of the antibody.
  • the levels of the antibody are measured weekly, e.g., by ELISA or similar assays.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0008] Levels of the antibody at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of the antibody from NHPs that had no AAV injected into them are used as background or pre-induction level of the antibody.
  • Pre- induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the antibody, e.g., in the serum, with a subsequent return to background levels.
  • Example P1B In vivo induction of an antibody in non-human primates [0009]
  • Example P1A is repeated with each of the following antibodies: anti-PD1 antibody, anti-HER2 antibody, anti-PCSK9 antibody, anti-VEGFR2 antibody, anti-IL17 antibody, anti-A ⁇ 3- 42 antibody, anti-Sema3A antibody, anti-4-1BB (CD137) antibody, anti-5'-nucleotidase antibody, anti-5T4 antibody, anti-activin receptor-like kinase 1 antibody, anti-ACVR2B antibody, anti- adenocarcinoma antigen antibody, anti-amyloid antibody, anti-Ang-2 antibody, anti-angiopoietin 2 antibody, anti-angiopoietin 2 antibody, anti-angiopoietin 3 antibody, anti-anthrax toxin antibody, anti-AOC3 (VAP-1) antibody, anti-AXL antibody, anti-BAFF-R antibody, anti-B-cell activating factor
  • Example P1A Materials and methods are as described in Example P1A, where the anti-TNF ⁇ is replaced by one of the above antibodies.
  • Levels of the antibody at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the antibody from NHPs that had no AAV injected into them are used as background or pre-induction level of the antibody.
  • Pre-induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the antibody, e.g., in the serum, with a subsequent return to background levels.
  • Example 1C In vivo induction of antibodies in non-human primates
  • Examples P1A and P1B are repeated with flavone as the inducer.
  • Materials and methods are as described in Examples P1A and P1B, where the artemisinin is replaced with flavone.
  • Levels of the antibody at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the antibody from NHPs that had no AAV injected into them are used as background or pre-induction level of the antibody. Pre-induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • Example 1D In vivo induction of antibodies in non-human primates
  • Examples P1A and P1B are repeated with each of the following inducers: artesunate, arteether, dihydroartemisinin, and artemether.
  • Materials and methods are as described in Examples P1A and P1B, where the artemisinin is replaced with one of the above inducers. Levels of the antibody at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the antibody from NHPs that had no AAV injected into them are used as background or pre-induction level of the antibody. Pre-induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak. However, treatment with the inducer leads to a significant increase in the antibody, e.g., in the serum, with a subsequent return to background levels.
  • Example P2A In vivo induction of coagulation factor VIII (FVIII) in non-human primates
  • artemisinin is used as the inducer to induce the in vivo expression in NHPs of coagulation factor VIII from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the FVIII to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0016] A vector as described herein, e.g., 3xhPB-MinTK and encoding the coagulation factor VIII is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the FVIII is produced as described in Greiger et al. 2015.
  • AAV9 containing LSP driving the expression of the cytokine is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of FVIII.
  • the levels of FVIII are measured weekly, e.g., by ELISA or similar assay.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0018] Levels of the FVIII at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of FVIII from NHPs that had no AAV injected into them are used as background or pre-induction level of FVIII.
  • Example P2B In vivo induction of coagulation factor VIII in non-human primates
  • Example P2A is repeated with flavone as the inducer.
  • Materials and methods are as described in Example P2A, where the artemisinin is replaced with flavone.
  • Levels of FVIII at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Example P2C In vivo induction of coagulation factor VIII in non-human primates [0021]
  • Example P2A is repeated with each of the following inducers: artesunate, arteether, dihydroartemisinin, and artemether.
  • Example P2A Materials and methods are as described in Example P2A, where the artemisinin is replaced with one of the above inducers.
  • Levels of FVIII at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of FVIII from NHPs that had no AAV injected into them are used as background or pre-induction level of FVIII.
  • Pre- induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in FVIII, e.g., in the serum, with a subsequent return to background levels.
  • Example P2D In vivo induction of coagulation factor IX (FIX) in non-human primates
  • artemisinin is used as an inducer to induce the in vivo expression in NHPs of coagulation factor IX from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the FIX to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0024] A vector as described herein, e.g., 3xhPB-MinTK and encoding the coagulation factor IX is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the FIX is produced as described in Greiger et al.2015.
  • AAV9 containing LSP driving the expression of the cytokine is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • Male and/or female NHPs are pre-screened for neutralizing Abs against AAV9 and injected with 1e13vg/kg of AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of FIX.
  • the levels of FIX are measured weekly, e.g., by ELISA or similar assay.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0026] Levels of the FIX at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of FIX from NHPs that had no AAV injected into them are used as background or pre-induction level of FIX.
  • FIX Pre-induction levels of FIX can be equivalent to the no AAV NHP suggesting that the promoters have no background leak. However, treatment with the inducer leads to a significant increase in the FIX, e.g., in serum with a subsequent return to background levels.
  • Example P2E In vivo induction of coagulation factor IX in non-human primates [0027]
  • Example P3A is repeated with each flavone as the inducer.
  • Materials and methods are as described in Example P2C, where the artemisinin is replaced with flavone. Levels of FIX at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • FIX levels from NHPs that had no AAV injected into them are used as background or pre-induction level of FIX.
  • Pre-induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in FIX, e.g., in the serum, with a subsequent return to background levels.
  • Example P2F In vivo induction of coagulation factor IX in non-human primates [0029]
  • Example P3A is repeated with each of the following inducers: artesunate, arteether, dihydroartemisinin, and artemether.
  • Example P2C Materials and methods are as described in Example P2C, where the artemisinin is replaced with one of the above inducers.
  • Levels of FIX at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of FIX from NHPs that had no AAV injected into them are used as background or pre-induction level of FIX.
  • Pre- induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in FIX, e.g., in the serum, with a subsequent return to background levels.
  • Example P2G In vivo induction of coagulation factor VII (FVII) in non-human primates
  • artemisinin is used as the inducer to induce the in vivo expression in NHPs of coagulation factor VII from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the FVII to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0032] A vector as described herein, e.g., 3xhPB-MinTK and encoding the coagulation factor VII is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the FVII is produced as described in Greiger et al.2015.
  • AAV9 containing LSP driving the expression of the cytokine is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • Male and/or female NHPs are pre-screened for neutralizing Abs against AAV9 and injected with 1e13vg/kg of AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of FVII.
  • the levels of FVII are measured weekly, e.g., by ELISA or similar assay.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0034] Levels of the FVII at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of FVII from NHPs that had no AAV injected into them are used as background or pre-induction level of FVII.
  • Pre-induction levels of FVII can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the FVII, e.g., in serum with a subsequent return to background levels.
  • Example P2H In vivo induction of coagulation factor VII in non-human primates
  • Example P4A is repeated with flavone as the inducer.
  • Materials and methods are as described in Example P2E, where the artemisinin is replaced with flavone. Levels of FVII at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Example P2I In vivo induction of coagulation factor VII in non-human primates [0037]
  • Example P4A is repeated with each of the following inducers: artesunate, arteether, dihydroartemisinin, and artemether.
  • Example P2E Materials and methods are as described in Example P2E, where the artemisinin is replaced with one of the above inducers.
  • Levels of FVII at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of FVII from NHPs that had no AAV injected into them are used as background or pre-induction level of FVII.
  • Pre- induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in FVII, e.g., in the serum, with a subsequent return to background levels.
  • Example P2J In vivo induction of coagulation factor XI (FXI) in non-human primates
  • artemisinin is used as the inducer to induce the in vivo expression in NHPs of coagulation factor XI from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the FXI to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0040] A vector as described herein, e.g., 3xhPB-MinTK and encoding the coagulation factor XI is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the FXI is produced as described in Greiger et al.2015.
  • AAV9 containing LSP driving the expression of the cytokine is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • Male and/or female NHPs are pre-screened for neutralizing Abs against AAV9 and injected with 1e13vg/kg of AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of FXI.
  • the levels of FXI are measured weekly, e.g., by ELISA or similar assay.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0042] Levels of the FXI at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of FXI from NHPs that had no AAV injected into them are used as background or pre-induction level of FXI.
  • Example P2K In vivo induction of coagulation factor XI in non-human primates
  • Example P4A is repeated with flavone as the inducer.
  • Materials and methods are as described in Example P2G, where the artemisinin is replaced with flavone.
  • Levels of FXI at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Example P2L In vivo induction of coagulation factor XI in non-human primates [0045]
  • Example P4A is repeated with each of the following inducers: artesunate, arteether, dihydroartemisinin, and artemether.
  • Example P2G Materials and methods are as described in Example P2G, where the artemisinin is replaced with one of the above inducers.
  • Levels of FXI at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of FXI from NHPs that had no AAV injected into them are used as background or pre-induction level of FXI.
  • Pre- induction levels of the antibody can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in FXI, e.g., in the serum, with a subsequent return to background levels.
  • Example P3A In vivo induction of interleukin-2 in non-human primates
  • artemisinin is used as the inducer to induce the in vivo expression in NHPs of interleukin-2 (IL-2) from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the IL-2 to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0048] A vector as described herein, e.g., 3xhPB-MinTK and encoding IL-2 is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the IL-2 is produced as described in Greiger et al.2015.
  • AAV9 containing LSP driving the expression of the cytokine is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • Male and/or female NHPs are pre-screened for neutralizing Abs against AAV9 and injected with 1e13vg/kg of AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of the IL-2.
  • the levels of the IL-2 are measured weekly, e.g., by ELISA or similar assay.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0050] Levels of the IL-2 at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of the IL-2 from NHPs that had no AAV injected into them are used as background or pre-induction level of the IL-2.
  • Pre-induction levels of the IL-2 can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the cytokine, e.g., in serum with a subsequent return to background levels.
  • Example P3B In vivo induction of a cytokine in non-human primates [0051] Example P3A is repeated with each of the following cytokines: interleukin-l ⁇ (IL-l ⁇ ), interleukin-1/3 (IL-1/3), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin- 6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8/CXCL8), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin- 13 (IL-13), interleukin- 15 (IL-15), interleukin- 17 (IL-17), interleukin- 18 (IL-18), tumor necrosis factor- ⁇ (TNF- ⁇ ), interferon- ⁇ (INF- ⁇ ), interferon-b (INF-b), interferon- ⁇ (INF- ⁇ ), granulocyte- monocyte colony stimulating factor (GM-CSF), granulocyte colony
  • Example P3A Materials and methods are as described in Example P3A, where the IL-2 replaced by one of the above cytokines Levels of the cytokine at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of the cytokine from NHPs that had no AAV injected into them are used as background or pre-induction level of the cytokine. Pre-induction levels of the cytokine can be equivalent to the no AAV NHP suggesting that the promoters have no background leak. However, treatment with the inducer leads to a significant increase in the cytokine, e.g., in the serum, with a subsequent return to background levels.
  • Example P3D In vivo induction of a cytokine in non-human primates
  • Examples P3A and P3B are repeated with flavone as the inducer.
  • Materials and methods are as described in Examples P3A and P3B, where the artemisinin is replaced with flavone.
  • Levels of the cytokine at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the cytokine from NHPs that had no AAV injected into them are used as background or pre-induction level of the cytokine. Pre-induction levels of the cytokinecan be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • Example P3D In vivo induction of a cytokine in non-human primates
  • Examples P3A and P3B are repeated with each of the following inducers: artesunate, arteether, dihydroartemisinin, and artemether.
  • Materials and methods are as described in Examples P3A and P3B, where the artemisinin is replaced with one of the above inducers. Levels of the cytokine at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • cytokine levels from NHPs that had no AAV injected into them are used as background or pre- induction level of the cytokine.
  • treatment with the inducer leads to a significant increase in the cytokine, e.g., in the serum, with a subsequent return to background levels.
  • Example P4A In vivo induction of erythropoietin in non-human primates
  • artemisinin is used as the inducer to induce the in vivo expression in NHPs of erythropoietin (EPO) from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the EPO to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0058] A vector as described herein, e.g., 3xhPB-MinTK and encoding EPO is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the EPO is produced as described in Greiger et al.2015.
  • AAV9 containing LSP driving the expression of the EPO is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • Male and/or female NHPs are pre-screened for neutralizing Abs against AAV9 and injected with 1e13vg/kg of AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of the EPO.
  • the levels of the EPO are measured weekly, e.g., by ELISA or similar assays.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0060] Levels of the EPO at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of the EPO from NHPs that had no AAV injected into them are used as background or pre-induction level of the EPO.
  • Pre-induction levels of the EPO can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the EPO, e.g., in the serum, with a subsequent return to background levels.
  • Example P4B In vivo induction of a hormone in non-human primates [0061]
  • Example P4A is repeated with each of the following hormones: parathyroid hormone (PTH), insulin-like growth factor (or somatomedin), adrenaline (or epinephrine), melatonin, noradrenaline (or norepinephrine), triiodothyronine, thyroxine, dopamine, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin (or islet amyloid polypeptide), anti-müllerian hormone (or müllerian-inhibiting factor/hormone), adiponectin , adrenocorticotropic hormone (or corticotropin), angiotensinogen, angiotensin, antidiuretic hormone (or vasopressin, arginine vasopressin), atrial natriuretic peptide (or
  • Example P4A Materials and methods are as described in Example P4A, where the EPO replaced by one of the above hormones.
  • Levels of the hormone at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the hormone from NHPs that had no AAV injected into them are used as background or pre-induction level of the hormone.
  • Pre-induction levels of the hormone can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the hormone, e.g., in the serum, with a subsequent return to background levels.
  • Example P4C In vivo induction of a hormone in non-human primates
  • Examples P4A and P4B are repeated with flavone as the inducer.
  • Materials and methods are as described in Examples P4A and P4B, where the artemisinin is replaced with flavone.
  • Levels of the hormone at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the hormone from NHPs that had no AAV injected into them are used as background or pre-induction level of the hormone. Pre-induction levels of the hormone can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • Example P4D In vivo induction of a hormone in non-human primates
  • Examples P4A and P4B are repeated with each of the following inducers: artesunate, arteether, dihydroartemisinin, and artemether.
  • Materials and methods are as described in Examples P4A and P4B, where the artemisinin is replaced with one of the above inducers. Levels of the hormone at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the hormone from NHPs that had no AAV injected into them are used as background or pre-induction level of the hormone. Pre-induction levels of the hormone can be equivalent to the no AAV NHP suggesting that the promoters have no background leak. However, treatment with the inducer leads to a significant increase in the hormone, e.g., in the serum, with a subsequent return to background levels.
  • Example P5A In vivo induction of tumor necrosis factor-alpha in non-human primates
  • artemisinin is used as the inducer to induce the in vivo expression in NHPs of tumor necrosis factor-alpha (TNF- ⁇ ), from AAV containing 3xhPB-MinTK driving the expression of a transgene encoding the TNF- ⁇ to assess the following attributes: • Induction with via IP injection • Induction via oral ingestion • Differences in male vs female for induction • Long-term expression and induction • Dose response curves via induction with different concentrations of inducer • Kinetics of induction, to obtain response times of the promoter to the inducer Materials and methods: [0068] A vector as described herein, e.g., 3xhPB-MinTK and encoding TNF- ⁇ is packaged into AAV9.
  • the AAV9 containing 3xhPB-MinTK driving the expression of the TNF- ⁇ is produced as described in Greiger et al.2015.
  • AAV9 containing LSP driving the expression of the TNF- ⁇ is used a control.
  • Male and/or female NHPs Macaca fascicularis
  • Male and/or female NHPs are pre-screened for neutralizing Abs against AAV9 and injected with 1e13vg/kg of AAV9 containing either 3xhPB-MinTK or LSP with each promoter driving the expression of the TNF- ⁇ .
  • the levels of the TNF- ⁇ are measured weekly, e.g., by ELISA or similar assay.
  • the animals are administered the inducer as follows: • Day 30: 80mg/kg via IP injection • Day 74: 80mg/kg via IP injection • Day 123: 40mg/kg via IP injection • Day 131: 20mg/kg via IP injection • Day 160: 10mg/kg via IP injection • Day 180: 1mg/kg via IP injection • Day 200: 6mg/kg via oral gavage [0070] Levels of the TNF- ⁇ at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured. Levels of the TNF- ⁇ from NHPs that had no AAV injected into them are used as background or pre-induction level of the TNF- ⁇ .
  • Pre-induction levels of the TNF- ⁇ can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the TNF- ⁇ , e.g., in serum with a subsequent return to background levels.
  • Example P5B In vivo induction of a growth factor in non-human primates [0071]
  • Example P5A is repeated with each of the following growth factors: adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), colony-stimulating factor (e.g., macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), or granulocyte macrophage colony-stimulating factor (GM-CSF)), epidermal growth factor (EGF), ephrin (e.g., ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, or ephrin B
  • Example P5A Materials and methods are as described in Example P5A, where the TNF- ⁇ is replaced by one of the above growth factors.
  • Levels of the growth factor at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the growth factor from NHPs that had no AAV injected into them are used as background or pre- induction level of the growth factor.
  • Pre-induction levels of the growth factor can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • treatment with the inducer leads to a significant increase in the growth factor, e.g., in serum with a subsequent return to background levels.
  • Example P5C In vivo induction of a growth factor in non-human primates
  • Examples P5A and P5B are repeated with flavone as the inducer.
  • Materials and methods are as described in Examples P5A and P5B, where the artemisinin is replaced with flavone.
  • Levels of the growth factor at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.
  • Levels of the growth factor from NHPs that had no AAV injected into them are used as background or pre- induction level of the growth factor. Pre-induction levels of the growth factor can be equivalent to the no AAV NHP suggesting that the promoters have no background leak.
  • Example P5D In vivo induction of a growth factor in non-human primates
  • Examples P5A and P5B are repeated with each of the following inducers: artesunate, arteether, dihydroartemisinin, and artemether.
  • Materials and methods are as described in Examples P5A and P5B, where the artemisinin is replaced with one of the above inducers. Levels of the growth factor at 12 hours, 24 hours, 48 hours, 72 hours, and/or 1 weeks after each administration of the inducer are measured.

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Abstract

La technologie décrite dans la présente invention concerne de manière générale des procédés et des compositions pour l'expression régulable et contrôlable d'un transgène chez un sujet, par exemple, un sujet primate.
EP24793343.5A 2023-04-17 2024-04-17 Expression régulable et contrôlable d'un transgène chez des primates Pending EP4698662A2 (fr)

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