WO2007134906A1 - Thérapie génique à interféron bêta faisant appel à un système d'expression régulée amélioré - Google Patents

Thérapie génique à interféron bêta faisant appel à un système d'expression régulée amélioré Download PDF

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Publication number
WO2007134906A1
WO2007134906A1 PCT/EP2007/053308 EP2007053308W WO2007134906A1 WO 2007134906 A1 WO2007134906 A1 WO 2007134906A1 EP 2007053308 W EP2007053308 W EP 2007053308W WO 2007134906 A1 WO2007134906 A1 WO 2007134906A1
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Prior art keywords
expression
present
protein
molecule
vector
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PCT/EP2007/053308
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English (en)
Inventor
Maxine Bauzon
Richard N. Harkins
Terry Hermiston
Peter Kretschmer
Paul Szymanski
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Bayer Schering Pharma Aktiengesellschaft
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Priority claimed from PCT/EP2006/062431 external-priority patent/WO2006122972A1/fr
Application filed by Bayer Schering Pharma Aktiengesellschaft filed Critical Bayer Schering Pharma Aktiengesellschaft
Publication of WO2007134906A1 publication Critical patent/WO2007134906A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

  • the present invention relates to an improved expression system for the regulated expression of an encoded protein or nucleic acid therapeutic molecule, for use in the treatment of disease.
  • the present invention relates to an improved regulated gene expression system, and pharmaceutical compositions and uses thereof for treatment of disease.
  • TMs therapeutic molecules
  • TMs therapeutic molecules
  • the delivery of nucleic acids encoding a therapeutic protein, in gene therapy has the potential to provide significant advantages over conventional therapies requiring the administration of bolus protein.
  • These potential advantages include, e.g., the long-term and regulated expression of a TM in the cells of a patient resulting in maximum therapeutic efficacy and minimum side effects and, also, the avoidance of toxic and infectious impurities, and systemic impurities.
  • bolus protein for the treatment of disease is known to result in adverse side effects including, e.g., those related to infectious and toxic impurities, systemic toxicity, injection-site necrosis, influenza-like symptoms, chills, fever, fatigue, anorexia, and weight loss. In some cases these events are dose limiting and may lead to cessation of treatment altogether. Further, it is known that continuous exposure to some protein therapeutics may result in tolerance over time. Thus, there is a need for a regulated expression system that can provide a sustained or long-term, therapeutically efficacious level of a TM, with the additional feature of a means to rapidly reduce or modulate the level of TM within a dynamic therapeutic window.
  • a regulated expression system which has the capability to be turned off should the concentration of TM reach a level that is potentially toxic. Moreover, the ability to titrate the level of TM would allow dosing to be adjusted where there is a potential for an increase in tolerance to the TM over time.
  • a gene encoding a therapeutic protein that can be expressed in target patient cells to remedy a condition resulting in or caused by a disease, or to stop or slow the progression of a disease.
  • the etiologies of many disease states are the result of the expression of one or more defective gene products or the defective expression of one or more gene products, e.g., the expression of a mutated protein, or the over or under expression of a protein, respectively.
  • conventional treatment methods include the administration of recombinant proteins to correct such defective protein expression or expression of a defective protein.
  • the administration of protein therapeutics to a patient is known to result in the generation of antibodies against the protein and its rejection by the patient immune system as foreign.
  • MS multiple sclerosis
  • IFN- ⁇ protein therapeutic a chronic inflammatory autoimmune disorder of the central nervous system that affects approximately 400,000 patients in North America and approximately one million people worldwide.
  • MS is a disease that affects more women than men, with onset typically between 20 and 40 years of age. Further, the disease is progressive, and in the early stages is characterized by a relapsing and remitting phase that is characterized by "attacks” or "relapses" of neurological dysfunction that are sub-acute over hours to days followed by periods of improvement that may last months (B. M. Keegan et al. (2002) Annu. Rev. Med. 53: 258-302; J. Noseworthy (2000) 343: 938-52).
  • the symptoms include, for example, disruption of coordinated movement of the eyes, limbs, and axial muscles leading to paralysis.
  • the course of the disease may evolve over several years with neurological symptoms that worsen until the patient becomes severely disabled.
  • the symptoms and signs of MS can reflect demyelination of neuronal axons in the brain resulting in impaired conductance of neural impulses along the axon.
  • the pathology of MS can manifest itself as acute focal inflammatory demyelination and axonal loss that eventually results in, e.g., chronic multifocal sclerotic plaques from which the disease gets its name (A. Compston and A. Coles (2002) Lancet 359: 1221-31 ; L. Steinman (1996) Cell 85: 299-302).
  • Betaseron® has been approved for secondary progressive MS in EU, Canada, and Europe.
  • IFN- ⁇ products are purified recombinant protein preparations.
  • Betaseron®/Betaferon® IFN- ⁇ 1 b
  • the recombinant protein may be purified from a bacterial cell culture (e.g., E. coli) that expresses the protein.
  • the recombinant protein is purified from a mammalian cell culture that expresses the protein.
  • IFN- ⁇ products for MS can be administered by subcutaneous (s.c.) or intramuscular (i.m.) injection of a bolus protein solution at a frequency ranging from once a week to every other day.
  • Type I interferons e.g., IFN- ⁇
  • IFN protein therapeutics can cause dose-dependent side effects, e.g., flu-like symptoms, nausea, and leukopenia in patients (E. U. Walther (1999) Neurology 53: 1622-27). These side effects can result in an intolerance to further IFN therapy.
  • s.c. subcutaneous
  • i.m. intramuscular
  • nucleic acid delivery systems are not suitable for clinical use and do not afford regulated or long-term expression in cells. Only a few known nucleic acid delivery systems are reported to have an ability to regulate transgene expression under laboratory conditions, but the suitability and workability of these delivery systems for clinical use are not known (see e.g., M. Gossen and H. Bujard Science 268: 1766-69; D. No et al. (1996) Proc. Natl.
  • the present invention provides an improved expression system for the regulated expression of an encoded protein or nucleic acid therapeutic molecule (TM) for use in the treatment of disease, wherein therapeutic efficacy of the TM can be maximized and side effects minimized.
  • the present invention provides an improved regulated gene expression system, and pharmaceutical compositions and methods thereof for treatment of disease.
  • the encoded TM can be a nucleic acid or protein that provides a therapeutic benefit to a subject having, or susceptible to, a disease.
  • therapeutic benefit or activity includes, but is not limited to, the amelioration, modulation, diminution, stabilization, or prevention of a disease or a symptom of a disease.
  • the present invention provides an improved regulated expression system comprising at least a first expression cassette having a nucleic acid sequence encoding a TM, such that, when delivered to cells of a subject, the encoded TM is expressed, and the expression and/or activity of the TM is regulated in the presence of a regulator molecule (RM).
  • a regulator molecule RM
  • Examples of such regulation include, but are not limited to, the induction, repression, increase, or decrease of TM expression and/or activity in the presence of an RM.
  • the expression and/or activity of the TM is regulated in a dose-responsive or dose-dependent manner, e.g., according to the amount of a RM present in the cells of the subject or administered to the subject.
  • the expression and/or activity of the TM is regulated in a dose-responsive or dose-dependent manner, e.g., according to the amount of an activator molecule (AM) or inactivator molecule (IM) present in the cells of the subject or administered to the subject.
  • AM activator molecule
  • IM inactivator molecule
  • the expression and/or activity of the TM is orientation-dependent.
  • the expression and/or activity of the TM in cells is modulated with respect to the 5' to 3' orientation of the expression cassette encoding the TM, or with respect to the 5' to 3' orientation of the transcription or translation of the encoded TM. Consequently, TM expression and/or activity can be modulated by selection of a particular orientation of the expression cassette encoding the TM or the orientation of transcription or translation of the TM.
  • the regulated expression system of the present invention further comprises a second expression cassette encoding an RM, such that, when delivered to cells of a subject, the encoded RM is expressed and the presence thereof regulates the expression and/or activity of the TM.
  • a first expression cassette encoding a TM and a second expression cassette encoding an RM of the present invention are present in a single vector.
  • the single vector is pGT23, pGT24, pGT25, pGT26, pGT27, pGT28, pGT29, or pGT30.
  • the single vector is pGT54, pGT57, pGT713, pGT15, or pGT16.
  • a TM of the present invention can be an isolated DNA, RNA, or protein, or variant thereof, encoded by a nucleic acid sequence and having a therapeutic activity. More particularly, a TM of the present invention can be a modified, synthetic, or recombinant DNA, RNA or protein.
  • the encoded TM is a nucleic acid, e.g., a DNA or RNA, having a therapeutic activity.
  • the encoded TM is an RNA e.g., an siRNA or shRNA.
  • the encoded TM is a protein having a therapeutic activity and, preferably, a human protein or variant thereof.
  • the encoded TM is a monoclonal antibody having a therapeutic activity.
  • the encoded TM is the monoclonal antibody, CAMPATH®.
  • the nucleic acid sequence encoding such a protein is a gene or gene fragment.
  • the encoded TM is a granulocyte macrophage colony stimulating factor (GMCSF) or variant of GMCSF (e.g., Leukine ® ).
  • the encoded TM is an interferon, e.g., interferon-alpha (IFN- ⁇ ) or interferon-beta (IFN- ⁇ ), and more particularly, is IFN- ⁇ -1a.
  • an RM of the present invention can be a naturally-occurring molecule or variant thereof, or an isolated molecule.
  • an RM of the present invention is a synthetic or recombinant molecule.
  • an RM of the present invention is a chemical compound, DNA, RNA, or protein.
  • an RM of the present invention is a modified molecule.
  • the RM is a humanized protein.
  • the RM is a human protein or variant thereof.
  • the RM is a transcriptional activator, e.g., a steroid receptor and, more particularly, a progesterone receptor.
  • the RM comprises a transactivation domain (e.g., a VP16 or p65 transactivation domain).
  • the RM comprises a ligand-binding domain (LBD).
  • LBD ligand-binding domain
  • an AM binds to the LBD of the RM, thereby activating the RM such that the presence of the activated RM regulates TM expression and/or activity.
  • the RM comprises a DBD, e.g., a GAL-4 DBD.
  • the RM comprises a DBD that binds to a functional sequence (e.g., a promoter sequence) operably linked to a nucleic acid encoding a TM, thereby regulating TM expression (e.g., inducing TM expression).
  • a functional sequence e.g., a promoter sequence
  • TM expression e.g., inducing TM expression
  • an RM of the present invention is activated and thereby TM expression and/or activity is regulated in the presence of the activated RM.
  • an RM of the present invention is expressed or present in cells of a subject in an inactivated form, and is activated in the presence of an AM, thereby, TM expression and/or activity is regulated by the activated RM.
  • the AM is a biomarker.
  • the AM is a biomarker for a disease or condition and, more particularly, is a biomarker for a disease state or condition, or symptom thereof.
  • the AM activates the RM by promoting or inhibiting conformational change, enzymatic processing or modification, specific binding, or dimerization of the RM. In a preferred aspect, the AM activates the RM by promoting homodimerization of the RM.
  • an AM of the present invention can be a naturally-occurring molecule or variant thereof, or an isolated molecule.
  • the AM of the present invention is a synthetic or recombinant molecule.
  • the AM of the present invention is a chemical compound, DNA, RNA, or protein.
  • the AM of the present invention is a modified molecule.
  • the AM is a humanized protein.
  • the AM is a human protein or variant thereof.
  • the AM is a chemical compound, e.g., an antiprogestin.
  • the AM is mifepristone.
  • an RM of the present invention is inactivated and thereby TM expression and/or activity is regulated in the presence of an inactivated RM.
  • an RM of the present invention is expressed or present in cells of a subject in an activated form, and is inactivated in the presence of an IM, thereby, TM expression and/or activity is regulated by the inactivated RM.
  • the IM is a biomarker.
  • the IM is a biomarker for a disease or condition and, more particularly, is a biomarker for a disease state or condition, or symptom thereof.
  • the IM inactivates the RM by promoting or inhibiting conformational change, enzymatic processing, specific binding, or dimerization of the RM. In a preferred aspect, the IM inactivates the RM by inhibiting homodimerization of the RM.
  • an IM of the present invention can be a naturally-occurring molecule or variant thereof, or an isolated molecule.
  • the IM of the present invention is a synthetic or recombinant molecule.
  • the IM of the present invention is a chemical compound, DNA, RNA, or protein.
  • the IM of the present invention is a modified molecule.
  • the IM is a humanized protein.
  • the IM is a human protein or variant thereof.
  • the IM is a chemical compound.
  • TM, RM, AM, or IM of the present invention can be consitutive or transient.
  • expression of a TM, RM, AM, or IM is regulated or tissue-specific (e.g. muscle-specific).
  • tissue-specific e.g. muscle-specific
  • a regulated RM include, but are not limited to, an RM that is activated by an AM or inactivated by an IM.
  • the expression of a TM, RM, AM, or IM of the present invention is driven by a regulated promoter or a tissue-specific promoter.
  • the regulated or tissue-specific promoter is regulated in the presence of an RM and, more particularly, by the binding of the RM to the promoter.
  • an RM of the present invention binds to a promoter operably linked to a nucleic acid sequence encoding a TM and thereby, regulates the expression of the encoded TM as described herein, in the cells of a subject.
  • the promoter that is operably linked to a nucleic acid encoding the TM comprises at least one GAL-4 DNA-binding site (DBS), and preferably comprises 3-18 GAL-4 DBS.
  • the promoter is a Pol Il or Pol III promoter.
  • the promoter is the Pol Il promoter U6H1.
  • the promoter is a Pol Il promoter selected from a group consisting of: a muscle creatine kinase promoter (MCK), a promoter comprising hypoxia responsive element (HRE promoter), endothelial leukocyte adhesion molecule (ELAM) promoter, chimeric promoter (e.g., CMV/actin chimeric promoter), cyclin A promoter, and cdc6 promoter.
  • MCK muscle creatine kinase promoter
  • HRE promoter hypoxia responsive element
  • ELAM endothelial leukocyte adhesion molecule
  • chimeric promoter e.g., CMV/actin chimeric promoter
  • cyclin A promoter cyclin A promoter
  • cdc6 promoter a Pol Il promoter selected from a group consisting of: a muscle creatine kinase promoter (MCK), a promoter comprising hypoxia responsive element (HRE promoter), endothelial leukocyte
  • the present invention also provides pharmaceutical compositions and methods for treatment of disease or condition comprising the improved regulated expression system of the present invention as described herein.
  • the present invention provides pharmaceutical compositions and methods for treating a disease or condition; regulating the expression of a TM; adminstering a TM; 4) delivering a TM; or expressing a TM in cells of a subject, where the methods comprise contacting the cells with a regulated expression system of the present invention, such that the encoded TM is expressed in the cells, and such TM expression is regulated in the presence of an RM.
  • the present invention provides pharmaceutical compositions and methods for treatment of leukemia, melanoma, hepatitis, and cardiomyopathy.
  • the encoded TM of the regulated expression system of the present invention is an IFN, e.g., an IFN- ⁇ or an IFN- ⁇ , for treatment of leukemia, melanoma, hepatitis, or cardiomyopathy.
  • an IFN e.g., an IFN- ⁇ or an IFN- ⁇
  • compositions of the present invention comprise at least one of the expression systems described herein, particularly, at least one of the TM and RM of the present invention, more particularly, at least one of the vectors of the present invention (e.g., pGT23, pGT24, pGT25, pGT26, pGT27, pGT28, pGT29, pGT30, pGT54, pGT57, pGT713, pGT715, pGT716, pTR-m IFN- ⁇ , or pTR-hlFN- ⁇ ).
  • the pharmaceutical compositions of the present invention comprise at least one AM or IM of the present invention.
  • a pharmaceutical composition of the present invention comprises one or more vectors encoding at least one TM and/or RM.
  • the TM, RM, AM, and IM of the present invention can be administered to a subject separately or together and ex vivo or in vivo, using any suitable means of administration described herein or known in the art. Examples of such suitable means of administration include, but are not limited to injection (e.g., subcutaneous injection), oral administration, and electroporation.
  • a TM and RM of the present invention are present in a single vector, and separately administered from an AM that activates the RM (and thereby, the presence of the activated RM regulates TM expression and/or activity).
  • the AM is a compound (e.g., mifepristone) administered orally
  • the single vector encoding a TM and RM is a single vector administered by injection or electroporation to cells of a subject (e.g., skeletal muscle cells).
  • the present invention further provides vectors and kits comprising the improved regulated expression system of the present invention.
  • the improved regulated expression system of the present invention comprises one or more vectors, and each vector comprises one or more expression cassettes.
  • the improved regulated expression system of the present invention comprises a single vector having at least one expression cassette and, more preferably, at least two expression cassettes.
  • the improved regulated expression system of the present invention comprises a single vector comprising a first expression cassette having at least one cloning site for insertion of a first nucleic acid sequence encoding a TM, and a second expression cassette having at least one cloning site for insertion of a second nucleic acid sequence encoding an RM.
  • the vector is a vector that is used for producing virus, e.g., an adeno-associated virus (AAV) shuttle plasmid and, more particularly, an AAV-1 shuttle plasmid.
  • the vector of the present invention is a nonviral vector (i.e., a vector that does not produce virus), e.g., a plasmid vector that does not produce virus.
  • the vector is a plasmid vector of the present invention comprising a cloning site for insertion of a nucleic acid sequence comprising a sequence encoding a TM.
  • plasmid vectors of the present invention include, but are not limited to, pGT1 , pGT2, pGT3, pGT4, pGT11 , pGT12, pGT13, or pGT14.
  • the expression cassettes of the present invention comprise functional sequences for expression of an encoded molecule of the present invention, e.g., a TM, RM, AM, or IM.
  • the expression cassette comprises at least one functional sequence operably linked to a nucleic acid sequence encoding a molecule of the present invention.
  • Examples of a functional sequence are, but not limited to, a 5' or 3' untranslated region (e.g., UT12), intron (e.g., IVS8), poly(A) site (e.g, SV40 or hGH poly(A) site), or a DNA-binding site (DBS) (e.g., GAL-4 DBS).
  • the functional sequence comprises at least one GAL-4 DBS and preferably comprises multimers of a GAL-4 DBS (e.g., 3-18 GAL-4 DBS).
  • Such functional sequences also include, for example, sequences encoding a regulated promoter or tissue- specific promoter that promotes the regulated or tissue-specific expression, respectively, of a molecule encoded by a nucleic acid sequence operably linked to such functional sequences in an expression cassette of the present invention.
  • the expression cassettes of the present invention comprise at least one cloning site and, more preferably, a multiple cloning site (MCS), for the insertion of a nucleic acid sequence encoding a molecule of the present invention, e.g., a TM, RM, AM, or IM.
  • a first expression cassette of the present invention comprises an MCS for insertion of a first nucleic acid sequence encoding a TM, an inducible promoter comprising at least one DBS (e.g., 3-18 GAL-4 DBS), 5' untranslated region (e.g., UT12), an intron (e.g., IVS8), and hGH poly(A) site, such that when the first nucleic acid sequence is inserted at the MCS, these functional sequences are operably linked to this sequence.
  • DBS e.g., 3-18 GAL-4 DBS
  • 5' untranslated region e.g., UT12
  • an intron e.g., IVS8
  • hGH poly(A) site e.g., hGH poly(A) site
  • a second expression cassette of the present invention comprises an MCS for insertion of a second nucleic acid sequence encoding a regulated RM and SV40 poly(A) site, such that when the second nucleic acid sequence is inserted at the MCS, these functional sequences are operably linked to this sequence.
  • the first and second expression cassettes are present in a single vector.
  • the kits of the present invention comprise at least one of the expression systems of the present invention described herein and, more particularly, at least one of the pharmaceutical compositions, vectors, or molecules (e.g., TM, RM, AM, or IM) of the present invention.
  • the present invention is a method of treating an anti-inflammatory disease or condition.
  • the method comprises administering a regulated gene expression system comprising at least a vector comprising a first gene expression cassette and a second gene expression casette.
  • the first gene expression cassette comprises: i) a first nucleic acid sequence encoding a therapeutic molecule (TM) having a therapeutic activity, and ii) a first promoter and a first poly(A) site operably linked to the first nucleic acid sequence, wherein the TM is expressed in cells of a subject in a therapeutically effective amount and is an interferon molecule (IFNM) or a variant thereof, and the TM expression or activity is induced in the presence of an activated regulator molecule (RM).
  • TM therapeutic molecule
  • IFNM interferon molecule
  • RM activated regulator molecule
  • the second gene expression cassette comprises: i) a second nucleic acid sequence encoding the RM, and ii) a second promoter and a second poly(A) site operably linked to the second nucleic acid sequence, wherein the RM is expressed in the cells and activated in the presence of an activator molecule (AM), thereby inducing the TM expression or activity in a dose dependent manner.
  • the anti-inflammatory disease is multiple sclerosis.
  • the vector and/or AM is administered one or more times.
  • the vector and/or AM is administered daily, every other day, three times per week, twice per week, weekly, biweekly, monthly, bimonthly, every three months, quarterly, semiannually, annually, or some combination thereof.
  • the lag time between administration of the vector encoding the TM and the induction of the expression of the TM by an AM is for example, 5 days, 12 days, 20 days or 55 days.
  • the AM is formulated as an oral dosage or an injectable dosage.
  • the AM is formulated as an immediate release dosage or a controlled release dosage. In a further aspect, the AM is formulated as a controlled release dosage. In another aspect, the AM is mifepristone. In a further aspect, the mifepristone is administered at a dosage from about 0.01 mg/kg to about 10 mg/kg, from about 0.05 mg/kg to about 5 mg/kg or about 0.01 mg/kg.
  • Figure 1 illustrates unlimiting examples of a regulated expression system of the present invention.
  • Figure 1A illustrates an unlimiting example of a regulated expression system of the present invention comprising: 1 ) a first expression cassette comprising a first nucleic acid sequence encoding a therapeutic molecule (TM) and a first promoter sequence encoding a DNA-binding site (DBS) and TATA sequence operably linked to the first nucleic acid sequence; 2) a second expression cassette comprising a second nucleic acid sequence encoding a regulator molecule (RM) and a second promoter sequence operably linked to the second nucleic acid sequence; 3) the expressed RM that is a fusion or chimeric protein comprising a DNA-binding domain (DBD), ligand-binding domain (LBD), and regulatory domain (RD); and 4) an activator or inactivator molecule (NM) that activates the RM or inactivates the RM, respectively.
  • TM therapeutic molecule
  • DBS DNA-binding site
  • RM
  • an activator molecule binds to the RM and activates the RM and, thereby, the activated RM binds to the DBS of the promoter sequence operably linked to the TM sequence, resulting in the induction of TM expression in cells (e.g., mammalian cells).
  • the first and second expression cassettes are present in a single vector.
  • Figure 1 B illustrates an unlimiting example of a regulated expression system of the present invention comprising: 1 ) a first expression cassette comprising a first nucleic acid sequence encoding a TM and a first promoter sequence encoding a DBS and TATA sequence operably linked to the first nucleic acid sequence; 2) a second expression cassette comprising a second nucleic acid sequence encoding a regulator molecule (RM) and a second promoter sequence operably linked to the second nucleic acid sequence; 3) the expressed RM that is a fusion or chimeric protein comprising a DBD, LBD, and activation domain (AD); and 4) an activator or inactivator molecule (NM).
  • a first expression cassette comprising a first nucleic acid sequence encoding a TM and a first promoter sequence encoding a DBS and TATA sequence operably linked to the first nucleic acid sequence
  • RM regulator molecule
  • NM activator or inactivator molecule
  • an activator molecule binds to the RM and activates the RM, and thereby, the activated RM forms a homodimer that binds to the DBS of the promoter operably linked to the TM sequence, resulting in the induction of TM expression, in cells (e.g., mammalian cells).
  • the first and second expression cassettes are present in a single vector.
  • Figure 2 illustrates murine IFN- ⁇ and human IFN- ⁇ plasmid vectors for generation of recombinant protein.
  • Figures 2 A and B illustrate murine IFN- ⁇ expression vectors for generation of recombinant protein (A, pGER90 (pCEP4/mlFN) and for gene-based delivery studies (B, pGER101 (pgWiz/mlFN).
  • the CMV promoter and enhancer present in pGER90 extends from -831 bp to +1 bp relative to the transcription start site, with no 5' UTR or intron.
  • the CMV sequences present in pGER101 include the promoter, enhancer, 5' UTR, and natural Intron A from -674 bp to +942 bp.
  • Figure 2 C and D illustrate human IFN- ⁇ expression vectors for generation of recombinant protein (C, pGER123 (pCEP4/hlFN) and for gene-based delivery studies (D, pGER125 (pgWiz/hlFN).
  • Figure 3 illustrates the pharmacokinetic profile following injection of human IFN- ⁇ 1a protein in mice.
  • C57BL/6 mice were administered either 25 ng (Low Dose) or 250 ng (High Dose) of recombinant hlFN- ⁇ 1a protein by either i.v. or i.m. injection.
  • Figure 4 illustrates the pharmacokinetic profile following intramuscular injection of AAV-1-hlFN- ⁇ in mice.
  • Figure 5 illustrates Mx1 RNA induction in vitro (in L929 cells) by mIFN- ⁇ .
  • L929 cells were seeded at 5x10 5 cells in 6 well plates and stimulated with increasing amounts of purified recombinant mIFN- ⁇ protein.
  • Mx1 RNA expression is plotted as the fold increase relative to GAPDH RNA.
  • Figure 6 illustrates Mx1 RNA induction following i.v. (A) or i.m. injection (B) of mlFN- ⁇ protein.
  • RNA was isolated from PBMCs.
  • Mx1 RNA was measured by quantitative RT-PCR. The fold increase in Mx1 RNA is expressed relative to GAPDH values measured in the same samples.
  • the controls include naive mice (N), and mice injected with the vehicle buffer only followed by Mx1 analysis at 2 hours (V2h) or 4h (V4h) post-injection. Each column represents the mean value +/- standard deviation.
  • Figure 7 illustrates induction levels of IP-10 (A) and JE (B) following i.v. or i.m. injection of murine IFN- ⁇ protein.
  • Figure 8 illustrates the induction of IP-10 following intramuscular injection of AAV-1- mIFN- ⁇ DNA or mIFN- ⁇ plasmid DNA with electroporation (EP) in mice.
  • Figure 9 illustrates the induction of Mx1 mRNA following intramuscular injection of mIFN- ⁇ plasmid DNA.
  • FIG 10 illustrates the induction of Mx1 mRNA following intramuscular injection of AAV-1-mlFN- ⁇ virus or mIFN- ⁇ plasmid DNA with electroporation in mice.
  • Normal mice C57BL/6 were injected i.m. with either AAV-1-mlFN- ⁇ (5x10 10 viral particles), or mIFN- ⁇ plasmid DNA (150 ug) with electroporation.
  • Controls included PBS injected mice (i.m. control), and mice injected with SEAP plasmid (pSEAP) or AAV- 1 expressing SEAP (AAV- SEAP). Mice were bled at the indicated time points and Mx1 RNA levels were determined by quantitative RT-PCR in RNA isolated from PBMCs.
  • Figure 14 illustrates plasmid vectors pGT1 , pGT2, pGT3, and pGT4 (A, B, C, D, respectively), which are unlimiting examples of one-plasmid regulated expression vectors of the present invention.
  • the regulated expression vectors of the present invention contain, in a single plasmid vector: 1 ) a first expression cassette with a multiple cloning site (MCS) for insertion of a nucleic acid encoding a therapeutic molecule (TM); and 2) a second expression cassette with a cloning site for insertion of a nucleic acid encoding a regulator molecule (RM).
  • MCS multiple cloning site
  • TM therapeutic molecule
  • RM regulator molecule
  • the skeletal muscle promoter (sk actin pro), untranslated region 12 (UT12), intervening sequence 8 (IV8) from the plasmid pLC1674 are located upstream of the MCS and human growth hormone poly (A) site (hGH polyA).
  • a nucleic acid comprising a therapeutic molecule (TM) of interest, e.g., a transgene, can be inserted at the MCS.
  • Figure 15 illustrates unlimiting examples of regulated expression plasmid vectors of the present invention for gene-based delivery of murine IFN- ⁇ (pGT23, pGT24, pGT25, and pGT26) (A), or human IFN- ⁇ (pGT27, pGT28, pGT29, and pGT30) (B).
  • murine IFN- ⁇ pGT23, pGT24, pGT25, and pGT26
  • human IFN- ⁇ pGT27, pGT28, pGT29, and pGT30
  • the regulated expression vectors of the present invention contain, in a single plasmid vector: 1 ) a first expression cassette with a multiple cloning site (MCS) and a nucleic acid inserted at the MCS encoding either a human IFN- ⁇ gene or a murine IFN- ⁇ gene; and 2) a second expression cassette with a cloning site and a nucleic acid inserted at the site encoding a regulator molecule (RM) that contains the modified LBD of the progesterone receptor (e.g., comprising the amino acid sequence of SEQ ID NO: 22 or encoded by the nucleic acid sequence of SEQ ID NO: 21 ).
  • MCS multiple cloning site
  • RM regulator molecule
  • Figure 16 illustrates the in vitro validation of hlFN- ⁇ regulated expression plasmid vectors of the present invention in murine skeletal muscle cells as fully described in Example 6, subsection C.
  • Constitutive (pGER125) and inducible (pGT27, pGT28, pGT29, and pGT30) hlFN- ⁇ plasmid vectors were transfected into mouse muscle C2C12 cells, treated with MFP (10 nM), and media collected. Media was assayed for hlFN- ⁇ by ELISA. The average of two independent transfections are shown.
  • Plasmid vectors pGS1694 + pGER129 is a two- plasmid system of Valentis in which the present inventors inserted the hlFN- ⁇ gene.
  • the regulated expression vectors of the present invention were constructed with the hlFN- ⁇ gene in either the forward (hlFN, — >) or reverse (hlFNr, ⁇ — ) direction, either upstream or downstream of the RM
  • Figure 17 illustrates the in vitro validation of mIFN- ⁇ regulated expression plasmid vectors of the present invention in murine skeletal muscle cells as fully described in Example 6, subsection C.
  • Constitutive (pGER101 ) and inducible (pGT23, pGT24, pGT25, and pGT26) mIFN- ⁇ expression plasmids were transfected into mouse muscle C2C12 cells.
  • Media was collected 24 hr later and assayed for mIFN- ⁇ by a reporter gene assay.
  • the chart shows the average of three independent transfections.
  • pGS1694 + pGER127 is a two-plasmid system of Valentis in which the present inventors inserted the mIFN- ⁇ gene.
  • the regulated expression vectors of the present invention were constructed with the mIFN- ⁇ gene in either the forward (mlFN, — >) or reverse (mlFNr, ⁇ — ) direction, either upstream or downstream of the RM cassette. .
  • FIG. 18 illustrates Mx1 RNA induction in vivo using a pBRES mIFN- ⁇ regulated expression system of the present invention.
  • Constitutive (pGER101 ) and inducible regulated expression (pGT26) mIFN- ⁇ plasmid vectors were injected and electroporated into the tibialis and gastrocnemius muscles of mice (150 ug per animal). Blood was collected at 7 days after injection. Mice were treated with MFP (0.33 mg/kg) by oral gavage once per day 7-10 days after injection. Blood was collected at 11 and 18 days after injection.
  • PBMCs were isolated from the blood and RNA was prepared from PBMCs and assayed by RT-PCR to determine the level of Mx1 RNA.
  • FIG 19 illustrates IP-10 and JE induction with a pBRES mIFN- ⁇ regulated expression system of the present invention.
  • Constitutive (pGER101 ) and inducible pBRES (pGT26) mlFN expression plasmids were injected and electroporated into hind limb muscles of C57BL/6 mice. Animals were bled and the plasma was assayed for the chemokines IP-10 and JE by ELISA on day 7 (absence of MFP), day 11 (following four consecutive days of oral administration of MFP, and day 18.
  • Figure 20 illustrates plasmid vectors pbSER189 (A) and pgWIZ (B) used in the construction of plasmid vector pGER (pgWiz/mlFN) (C), as fully described in the Materials and Methods, subsection F.
  • Figure 21 illustrates the plasmid vector pGER125 (pgWiz/hlFN) as fully described in the Materials and Methods, subsection F.
  • Figure 22 illustrates the plasmid vector pGene/V5-HisA as fully described in the Materials and Methods, subsection F.
  • Figure 23 illustrates the plasmid vector pGene-mlFN (pGER127) as fully described in the Materials and Methods, subsection F.
  • Figure 24 illustrates the plasmid vector pGene-hlFN (pGER129) as fully described in the Materials and Methods, subsection F.
  • Figure 25 illustrates the plasmid vector pSwitch (Invitrogen) as fully described in the Materials and Methods, subsection F.
  • Figure 26 illustrates the plasmid vector pGS1694 as fully described in the Materials and Methods, subsection F.
  • Figure 27 illustrates the plasmid vector pLC1674 as fully described in the Materials and Methods, subsection F.
  • Figure 28 illustrates the pGT-hGMCSF and pGT-mGMCSF shuttle plasmids and construction thereof, as fully described in the Materials and Methods, subsection F.
  • Figure 29 illustrates the pZac2.1-RM-hGMCSF and pZac2.1-RM-mGMCSF (A) and pZac2.1-CMV-hGMCSF (pGT713) and pZac2.1-CMV-mGMCSF (pGT714) (B) shuttle plasmids and construction thereof, as fully described in the Materials and Methods, subsection F.
  • Figure 30 illustrates the pORF-hGMCSF and pORF9-mGMCSF used in the construction of pZac2.1-RM-hGMCSF and pZac2.1-RM-mGMCSF, respectively, as fully described in the Materials and Methods, subsection F.
  • Figure 31 illustrates the pGT715 (A) and pGT716 (B) shuttle plasmids, as fully described in the Materials and Methods, subsection F.
  • Figure 32 illustrates IP-10 induction in vivo with mIFN- ⁇ regulated expression plasmid vectors of the present invention.
  • Figure 33 illustrates hlFN induction in vivo with hlFN- ⁇ regulated expression plasmid vectors of the present invention.
  • Figure 34A illustrates hEPO induction in vivo with hEPO regulated expression plasmid vectors of the present invention.
  • Inducible two-plasmid pGS1694 + pEP1666
  • one-plasmid pBRES pGT27, pGT28, pGT29, and pGT30
  • hEPO expression plasmids were injected and electroporated into hind limb muscles of C57BL/6 mice.
  • Figure 34B illustrates induction of hematocrit count in vivo with hEPO regulated expression plasmid vectors of the present invention.
  • Inducible two-plasmid pGS1694 + pEP1666
  • one-plasmid pBRES pGT27, pGT28, pGT29, and pGT30
  • hEPO expression plasmids were injected and electroporated into hind limb muscles of C57BL/6 mice and animals were treated with MFP or left untreated and bled as above.
  • Figure 35 illustrates long-term, persistent, multiple hlFN inductions in vivo with a hlFN- ⁇ regulated expression AAV vector of the present invention.
  • Figure 36 illustrates long-term, persistent, multiple IP-10 inductions in response to increasing dosages of MFP in vivo with repeated administrations of a mIFN- ⁇ regulated expression plasmid vector of the present invention.
  • Figure 37A illustrates the kinetics of hlFN induction in vivo with a hlFN- ⁇ regulated expression AAV vector of the present invention.
  • Figure 37B illustrates the kinetics of hlFN de-induction in vivo with a hlFN- ⁇ regulated expression AAV vector of the present invention.
  • Figure 37C illustrates the kinetics of mlFN induction and de-induction, response to pulsatile or chronic MFP treatment, and the persistence of gene expression over several months with a mIFN- ⁇ regulated expression plasmid vector of the present invention.
  • Figure 38 illustrates Mx-1 induction in vivo with a mIFN- ⁇ regulated expression plasmid vector of the present invention.
  • the inducible mIFN- ⁇ expression plasmid pBRES mlFN (pGT26) or pBRES NuII-MFP (control) plasmid was injected and electroporated into hind limb muscles of SJL mice with acute EAE. Mice were treated with MFP (0.33 mg/kg) by i.p. injection once per day (d) or every third day (etd) after plasmid injection. Blood was collected at day 5 after injection. PBMCs were isolated from the blood and RNA was prepared from and assayed by RT-PCR to determine the level of Mx1 RNA. Mx1 expression levels were normalized to GAPDH. The results are shown as the mean +/- the standard deviation.
  • Figure 39A illustrates the effect of treatment of mice with induced EAE neuroinflammation with mIFN- ⁇ protein by SC injection every other day for 25 days.
  • SJL mice were immunized on day 1 with proteolipid peptide (PLP) amino acids 139-151/pertussis according to the protocol described in Material and Methods.
  • Figure 39B illustrates the delay in the onset of disease and decrease in the severity of disease following administration of pBRES mIFN- ⁇ as compared to the null vector control group.
  • One group of mice were treated daily with Prednisolone by IP injection and served as the positive control for the study.
  • Plasmid treated mice were administered one mifepristone (MFP) containing slow release pellets by SC injection on day -3.
  • MFP mifepristone
  • AAV adeno-associated virus
  • AAV-1 (adeno-associated virus, serotype 1 )
  • AAV-2 (adeno-associated virus, serotype 2)
  • AM activator molecule
  • AMP ampicillin
  • bp base pairs
  • BGH bovine growth hormone
  • CMV cytomegalovirus
  • DBD DNA-binding domain
  • DAI Dynamic Activity Index
  • E1 b TATA Addenovirus E1 b gene promoter TATA box
  • EBNA-1 Epstein-Barr virus Nuclear Antigen
  • EDTA ethylene diamine tetraacetic acid
  • ELAM endothelial leukocyte adhesion molecule
  • GAL-4 yeast GAL-4 protein
  • GAPDH glycosylcholine dehydrogenase
  • GMCSF granulocyte macrophage stimulating factor
  • hGMCSF human granulocyte macrophage colony-stimulating factor
  • hlFN human interferon
  • hlFN- ⁇ human interferon- ⁇
  • HR hormone receptor
  • HRE hyperoxia responsive element
  • hGH human growth hormone
  • hPR human progesterone receptor
  • HTLV human T-cell lymphotropic virus
  • HSV herpes simplex virus
  • IFN ⁇ i b interferon- ⁇ 1b
  • IFNM IFN molecule
  • IFN sig seq interferon signal sequence
  • IgK immunoglobulin kappa
  • IM intramuscular inj.
  • INR inr
  • IP-10 or IP-10 (interferon-alpha inducible protein 10)
  • ITR inverted terminal repeats
  • IP intraperitoneal
  • IVS8 intervening sequence or intron 8
  • IV intravenous
  • JE murine analog of MCP-1
  • kDA kilodalton
  • kan kanamycin
  • KanR KanR (Kanamycin resistence gene)
  • LBD ligand-binding domain
  • MCP-1 monocyte chemoattractant protein
  • MFP (mifepristone) mg (milligram)
  • mGMCSF mae granulocyte macrophage colony-stimulating factor
  • mlFN murine interferon
  • mIFN- ⁇ murine interferon-beta
  • MCK muscle creatine kinase
  • ORF open reading frame
  • Ori oil of replication
  • OriP replication origin of Epstein Barr Virus
  • pBRES plasmid Berlex Regulated Expression System
  • p65 transcription regulatory domain of NFkappaB p65 protein
  • PBS phosphate buffered saline
  • PEG polyethylene glycol
  • PLP proteolipid peptide
  • PLP139-151 proteolipid peptide amino acids 139-151
  • polyA poly(A) (polyadenylation site)
  • PTK promoter of the Herpes Simplex Virus thymidine kinase gene
  • pUC ori replication origin of pUC plasmids
  • r reverse
  • RNA ribonucleic acid
  • rpm repetitions per minute
  • RT room temperature
  • SC subcutaneous
  • SHR steroid hormone receptor
  • shRNA short hairpin RNA
  • siRNA small interfering RNA
  • sk actin pro skeletal muscle promoter
  • SV40 simian virus 40
  • TK thymidine kinase
  • TKpA thymidine kinase poly A
  • UbiB Ubiquitin B
  • ug microgram
  • 5'UTR 5' untranslated region
  • the improved regulated, expression system of the present invention is a highly innovative technology which provides for nucleic acids that encode a therapeutic molecule
  • TM therapeutic molecule
  • TM therapeutic molecule
  • a further advantage of the present invention is that it provides for the expression and/or activity of a TM, in the cells of a subject, in a dose-dependent or orientation-dependent manner (as described herein), e.g., depending on the amount of a regulator molecule (RM) present in or administered to a subject, or the orientation of a nucleic acid encoding a TM, respectively. Consequently, another advantage of the compositions and methods of the present invention is that it can be used to optimize therapy in a manner specific to a disease or disease state of a subject.
  • a further advantage of the present expression system is that it can comprise a single nucleic acid vector, which can be administered to a subject via a single injection. Thus, the present expression system provides significant advantages over known nucleic acid-based therapy or bolus protein-based therapy.
  • the expression system of the present invention provides for the regulated, long-term expression of a TM (e.g., a protein or nucleic acid) in the cells of a subject, resulting in therapeutic efficacy while minimizing dose-limiting side effects.
  • gene therapy using the expression system of the present invention, can provide regulated, long-term expression of a protein and thereby minimize dose-limiting side effects and maximize therapeutic efficacy of the protein for the treatment of disease in a subject.
  • Interferon beta IFN- ⁇
  • MS multiple sclerosis
  • IFN- ⁇ is known to have a short half-life in circulation. Further, frequent, local administration of the protein may cause dose-dependent side effects.
  • a nucleic acid encoding an IFN- ⁇ (e.g., IFN- ⁇ -1a) can be administered to the cells of a subject, and the expression of the encoded IFN- ⁇ in the cells can be regulated long-term, and optimized, to achieve maximum therapeutic efficacy and minimum dose-limiting side effects of the IFN- ⁇ drug, for treatment of MS.
  • an AM that is a small molecule activator, in the form of an orally available pill, controls promoter induction and subsequent expression of a TM encoded by a nucleic acid sequence of the regulated, expression system of the present invention.
  • the level of the expressed TM (e.g., a protein or nucleic acid) in circulation in a subject can be tightly regulated in an on/off manner and/or in a dose-dependent manner.
  • An AM of the present invention can directly or indirectly control expression of a TM.
  • the AM activates an RM, and the presence of the activated RM thereby modulates (e.g., induces) expression of the TM in the cells of a subject.
  • another advantage of the regulated expression system of the present invention is that it allows for the option for continuous versus pulsatile therapy of a TM expressed in the cells of a subject (e.g., a protein or nucleic acid), and the modulation of expression levels of the TM, in order to optimize therapeutic efficacy of the TM while minimizing any side effects thereof.
  • the regulated expression system of the present invention allows for the first time the option for continuous and durable, versus pulsatile, IFN- ⁇ protein therapy in MS subjects.
  • another advantage of the present invention is that it can provide renewable expression of a TM in the cells of a subject, by repeated administration of a nucleic acid vector encoding the TM.
  • the present regulated expression system allows for the subject- specific or disease-specific therapy, by modulating and optimizing the expression level of a TM in the cells of a subject, to achieve maximum therapeutic efficacy and minimum side effects.
  • subject-specific or disease-specific therapy refers to treatment that is specific to a subject having a specific disease, stage of disease, or disease condition or symptom.
  • the level of IFN- ⁇ expressed in the cells of a subject having MS can be modulated and optimized to achieve maximum therapeutic efficacy and minimum side effects, for treatment of a specific condition, symptom, or stage of MS (e.g., relapsing remitting, primary progressive, or secondary progressive); or according to a subject's response or tolerance to IFN- ⁇ .
  • the present invention provides an improved regulated gene expression system, and pharmaceutical compositions and methods thereof for treatment of disease.
  • the encoded TM can be a nucleic acid or protein that provides a therapeutic benefit to a subject having, or susceptible to, a disease.
  • therapeutic benefit or “therapeutic activity” includes, but is not limited to, the amelioration, modulation, diminution, repression, stabilization, or prevention, delay, or slowing of the onset or progression of a disease or symptom or condition of a disease.
  • subject refers to a mammal (e.g., a human), and more particularly, refers to a mammal in need of treatment for a disease.
  • Treatment refers to providing a therapeutic benefit to a subject for a disease, including a stage, symptom or condition of a disease.
  • Disease as used herein encompasses a stage, symptom, condition, or pathology of a disease, or genetic predisposition for a disease. Such diseases can be autoimmune or inflammatory diseases.
  • the disease is a cancer.
  • the disease is e.g., multiple sclerosis, leukemia, melanoma, hepatitis, or cardiomyopathy.
  • the improved regulated expression system of the present invention provides a novel approach for engineering changes in an animal genome (e.g., a murine genome) so that gene function in an animal model can be accurately analyzed and credible animal models (e.g., murine models) of human diseases can be generated.
  • the improved regulated expression system of the present invention provides an invaluable tool for biomedical research because using the present system, expression of a target molecule e.g., a target gene in an animal genome (or other molecule of the present invention) can be regulated temporally and in a spacial-specific manner.
  • the improved regulated expression system of the present invention provides a novel approach for the selective or unique expression of target shRNA both in vitro and in vivo.
  • a polymerase Il (POL II) based expression system can be modified to generate a target shRNA selectively or uniquely.
  • the present regulated, expression system can be modified and used to generate the shRNA by operably linking a POL Il promoter to an intron-containing gene, and the resulting spliced intron processed by the inclusion of MIR sequences to express the target shRNA.
  • the RM protein-targeted GAL-4 binding sites of the present vectors and expression cassettes described herein could be inserted upstream of a U6 promoter to create an RM-reponsive system, with the additional potential modification of exchanging the p65 transactivator with a polymerase III (POL III) activator (e.g., Oct-2 Q ).
  • POL III polymerase III
  • the present invention provides an improved regulated expression system comprising at least a first expression cassette having a nucleic acid sequence encoding a TM, such that, when delivered to cells of a subject, the encoded TM is expressed, and the expression and/or activity of the TM is regulated in the presence of a regulator molecule (RM).
  • a regulator molecule RM
  • Regulation of the activity and/or expression of a molecule of the present invention (e.g., a TM) as used herein refers to the modulation of the expression and/or an activity of the molecule resulting in e.g., the induction, repression, increase, or decrease of an activity and/or the expression of such a molecule.
  • TM a molecule of the present invention
  • TM a molecule of the present invention
  • examples of other molecules that are suitable for regulation in the present expression system include, but are not limited to an RM, activator molecule (AM), and inactivator molecule (IM) as described herein.
  • the expression and/or activity of the TM is regulated in a dose-responsive or dose-dependent manner, e.g., according to the amount of a RM present in the cells of the subject or administered to the subject.
  • the expression and/or activity of the TM is regulated in a dose-responsive or dose-dependent manner, e.g., according to the amount of an activator molecule (AM), or inactivator molecule (IM) present in the cells of the subject or administered to the subject.
  • the expression and/or activty of the TM is regulated in a dose-responsive or dose-dependent manner according to the amount of the same TM or different TM present in the cells of the subject or administered to the subject.
  • dose-responsive refers to the correlation of the expression and/or activity of a molecule of the present invention (e.g. a TM), with the presence in, or administration to, the cells of a subject, a particular dose or amount of a second molecule.
  • a second molecule include, but are not limited to, an RM, AM, IM, or TM.
  • a second molecule include a cellular molecule e.g., a biomarker (e.g., a biomarker associated with a disease).
  • cells of a subject refer to autologous cells from a subject, or heterologous cells (or donor cells) that are not from a subject but are delivered or administered to a subject as described herein.
  • the autologous cells are present in a subject, and the heterologous cells are delivered to and present in a subject.
  • a composition of the present invention e.g., a vector encoding a TM and/or RM, is delivered in vivo to autologous cells of a subject, such that the encoded molecule is expressed in cells present in the subject.
  • a composition of the present invention e.g., a vector encoding a TM and/or RM, is delivered ex vivo to autologous or heterologous cells of a subject and then the treated cells are delivered to the subject, such that the encoded molecule is expressed in cells present in the subject.
  • the expression and/or activity of the TM is orientation-dependent.
  • orientation-dependent refers to the the 5' to 3' orientation of an expression cassette encoding a TM of the present invention, or the 5' to 3' direction of transcription or translation of an encoded TM of the present invention, and in some embodiments the orientation is: with respect to a vector comprising the expression cassette or encoding the TM; with respect to the orientation of another expression cassette on the same vector; or with respect to the orientation of the expression of another molecule encoded by the same vector.
  • the expression and/or activity of the TM in cells is modulated with respect to the 5' to 3' orientation of the expression cassette encoding the TM, or with respect to the 5' to 3' orientation of the transcription or translation of the encoded TM. Consequently, TM expression and/or activity can be modulated by selection of a particular orientation of the expression cassette encoding the TM or the orientation of transcription or translation of the TM.
  • the regulated expression system of the present invention comprises at least one expression cassette encoding a TM and can comprise additional expression cassettes encoding one or more of the molecules of the present invention, e.g., a TM, RM, AM, or IM. Further, one or more expression cassettes can be present in a single vector, or in more than one vector. Further, the present invention is not limited to a single TM, RM, AM, or IM and encompasses embodiments having one or more or multiples of a TM, RM, AM, or IM of the present invention, which can be present alone or together in a single vector or in more than one vector.
  • vector refers to a nucleic acid suitable for inserting and expressing in cells a nucleic acid sequence encoding one or more molecules of the present invention, e.g., a TM, RM, AM, or IM.
  • Expression cassette refers to a nucleic acid encoding the requisite components or functional sequences for the expression in cells of a molecule of the present invention (e.g., a protein or nucleic acid TM, RM, AM, or IM), where the molecule is encoded by a nucleic acid sequence operably inserted into the expression cassette (e.g., at a cloning site in the expression cassette) and operably linked to the functional sequences of the expression cassette.
  • operably linked or “operably inserted” sequence or sequences, as used herein, refers to a sequence or sequences fused, joined, attached or otherwise brought together with another sequence such that the respective sequences function as intended, known, and/or to achieve a particular outcome (e.g., a promoter sequence operably linked to a gene sequence to promote transcription of the encoded gene).
  • Figures 1A and 1 B illustrate unlimiting examples of a regulated expression system of the present invention comprising: 1 ) a first expression cassette comprising a first nucleic acid sequence encoding a therapeutic molecule (TM) and a first promoter sequence encoding a DNA-binding site (DBS) and TATA sequence operably linked to the first nucleic acid sequence; 2) a second expression cassette comprising a second nucleic acid sequence encoding a regulator molecule (RM) and a second promoter sequence operably linked to the second nucleic acid sequence, wherein the RM comprises a DNA-binding domain (DBD), ligand-binding domain (LBD), and regulatory domain (RD) and more specifically, in Figure 1 B, an activation domain (AD); and 4) an activator or inactivator molecule (NM) that activates the RM or inactivates the RM, respectively, such that the presence of the activated or inactivated RM regulates the expression and/or activity of the TM.
  • TM therapeutic molecule
  • a first expression cassette comprises the following operably linked functional sequences: 6x GAL-4 DBS, E1 b TATA, and transcription start site (e.g., SEQ ID NO: 1 ), 5' untranslated region UT12 (e.g., SEQ ID NO: 2), synthetic intron IVS8 (e.g, SEQ ID NO: 3), multiple cloning site (e.g, SEQ ID NO: 4 or SEQ ID NO: 5), human growth hormone (hGH) polyadenylation (poly(A)) site (e.g., SEQ ID NO: 6); and a second expression cassette comprises the following operably linked functional sequences: chicken skeletal muscle alpha-actin promoter (e.g., SEQ ID NO: 7) and SV40 poly(A) site (e.g., SEQ ID NO: 8).
  • the first and second expression cassettes are present in a single vector (e.g., as schematically illustrated in Figures 1A-B).
  • the vector is a single plasmid vector e.g., pGT1 (comprising the sequence of SEQ ID NO: 9 and SEQ ID NO: 4), pGT2 (comprising the sequence of SEQ ID NO: 10 and SEQ ID NO: 4), pGT3 (comprising the sequence of SEQ ID NO: 11 and SEQ ID NO: 4), or pGT4 (SEQ ID NO: 12),wherein each vector comprises a multiple cloning site (SEQ ID NO: 4) located 3' of the IVS8 and 5' of the hGH poly(A) site (e.g., as schematically depicted in Figures 14A-D), or pGT11 (comprising the sequence of SEQ ID NO: 9 and SEQ ID NO: 5), pGT12 (comprising the sequence of SEQ ID NO:
  • the TM is an IFN- ⁇ or GMCSF.
  • the first nucleic acid sequence encodes a TM that is human IFN- ⁇ 1a comprising the amino acid sequence of SEQ ID NO: 13, and is encoded by the nucleic acid sequence of SEQ ID NO: 14.
  • the first nucleic acid sequence encodes a TM that is a mouse IFN- ⁇ comprising the amino acid sequence of SEQ ID NO:15, and is encoded by the nucleic acid sequence of SEQ ID NO: 16.
  • the first nucleic acid sequence encodes a TM that is a human GMCSF comprising the amino acid sequence of SEQ ID NO: 17, and is encoded by the nucleic acid sequence of SEQ ID NO: 18.
  • the first nucleic acid sequence encodes a TM that is a mouse GMCSF comprising the amino acid sequence of SEQ ID NO: 19, and is encoded by the nucleic acid sequence of SEQ ID NO: 20.
  • the RM is a variant of a wild-type or naturally-occurring progesterone receptor (PR).
  • the second nucleic acid sequence encodes an RM that is a mutated PR comprising the amino acid sequence of SEQ ID NO: 22, and is encoded by the nucleic acid sequence of SEQ ID NO: 21.
  • the nucleic acid sequence encoding a TM is inserted or cloned into the Spe I and Not I restriction enzyme sites of an MCS of a first expression cassette, of a single plasmid vector.
  • the single plasmid vector comprising a nucleic acid sequence encoding a TM is e.g., pGT23 (SEQ ID NO: 23), pGT24 (SEQ ID NO: 24), pGT25 (SEQ ID NO: 25), or pGT26 (SEQ ID NO: 26), where the encoded TM is a mouse IFN- ⁇ (e.g., comprising the amino acid sequence of SEQ ID NO: 15 and/or encoded by a nucleic acid sequence comprising SEQ ID NO: 16), and the 5'-3' orientation of transcription of the encoded TM and of the inserted nucleic acid sequence is schematically illustrated in Figure 15A (see arrow).
  • the single plasmid vector comprising a nucleic acid sequence encoding a TM is e.g., pGT27 (SEQ ID NO: 27), pGT28 (SEQ ID NO: 28), pGT29 (SEQ ID NO: 29), or pGT30 (SEQ ID NO: 30), where the encoded TM is a human IFN- ⁇ (e.g., comprising the amino acid sequence of SEQ ID NO: 13 and/or encoded by a nucleic acid sequence comprising SEQ ID NO: 14), and the orientation of trancription of the encoded TM and of the inserted nucleic acid sequence is schematically illustrated in Figure 15B (see arrow).
  • pGT27 SEQ ID NO: 27
  • pGT28 SEQ ID NO: 28
  • pGT29 SEQ ID NO: 29
  • pGT30 SEQ ID NO: 30
  • the encoded TM is a human IFN- ⁇ (e.g., comprising the amino acid sequence of SEQ ID NO: 13
  • the single plasmid vector comprises the nucleic acid sequence of a vector backbone (e.g., SEQ ID NO: 12), MCS (e.g., SEQ ID NO: 31 ), and Spel-Notl fragment (SEQ ID NO: 31 ), wherein the fragment encodes a TM that is a mouse IFN- ⁇ , has an Spel sequence at the 5' end and Notl sequence at the 3' end compatible for insertion of the fragment at the Spel-Notl site in the MCS, and is inserted at the Spel-Notl site of the MCS.
  • a vector backbone e.g., SEQ ID NO: 12
  • MCS e.g., SEQ ID NO: 31
  • Spel-Notl fragment SEQ ID NO: 31
  • the single plasmid vector comprises the nucleic acid sequence of a vector backbone (e.g., SEQ ID NO: 12), MCS (e.g., SEQ ID NO: 32), and Spel-Notl fragment (SEQ ID NO: 31 ), wherein the fragment encodes a TM that is a human IFN- ⁇ , has an Spel sequence at the 5' end and Notl sequence at the 3' end compatible for insertion of the fragment at the Spe I-Notl site in the MCS, and is inserted at the Spel-Notl site of the MCS.
  • a vector backbone e.g., SEQ ID NO: 12
  • MCS e.g., SEQ ID NO: 32
  • Spel-Notl fragment SEQ ID NO: 31
  • an AM binds to the RM and activates the RM, thereby, the activated RM binds to the DBS of the promoter sequence operably linked to the TM sequence, resulting in the induction of TM expression and/or activity, in cells (e.g., mammalian cells).
  • an inactivator molecule binds to the RM and inactivates the RM, thereby, the inactivated RM does not bind to the DBS of the TM promoter, resulting in the repression or in the lack of induction of TM expression and/or activity.
  • an activator molecule binds to the LBD of the RM and activates the RM, thereby, the activated RM forms a homodimer that binds to the DBS of the promoter operably linked to the TM sequence, resulting in the induction of TM expression and/or activity, in cells (e.g., mammalian cells).
  • the first and second expression cassettes are present in a single vector.
  • a first expression cassette encoding a TM and a second expression cassette encoding an RM of the present invention are present in a single vector (e.g., pGT23 (SEQ ID NO: 23), pGT24 (SEQ ID NO: 24), pGT25 (SEQ ID NO: 25), pGT26 (SEQ ID NO: 26), pGT27 (SEQ ID NO: 27), pGT28 (SEQ ID NO: 28), pGT29 (SEQ ID NO: 29), or pGT30 (SEQ ID NO: 30)).
  • pGT23 SEQ ID NO: 23
  • pGT24 SEQ ID NO: 24
  • pGT25 SEQ ID NO: 25
  • pGT26 SEQ ID NO: 26
  • pGT27 SEQ ID NO: 27
  • pGT28 SEQ ID NO: 28
  • pGT29 SEQ ID NO: 29
  • pGT30 SEQ ID NO: 30
  • a “therapeutic molecule” or “TM” as used herein refers to a molecule having a therapeutic activity or providing a therapeutic benefit.
  • a TM of the present invention can be an isolated DNA, RNA, or protein, or variant thereof, encoded by a nucleic acid sequence and having a therapeutic activity.
  • "Variants” as used herein include muteins, e.g., muteins of an isolated DNA, RNA, protein, or chemical compound. More particularly, a TM of the present invention can be a modified, synthetic, or recombinant DNA, RNA or protein.
  • Modified encompasses molecules modified chemically, synthetically, or by recombinant technology, including e.g., mutated, fusion, or chimeric molecules.
  • the encoded TM is a protein that is expressed and cleaved or processed in the cells of a subject and thereby results in multiple TMs, or an activated TM, or a TM that differs from the expressed and uncleaved or unprocessed TM.
  • the encoded TM is a nucleic acid (e.g., an RNA) having a therapeutic activity.
  • the encoded RNA encodes multiple splice sites that are multiply or differentially spliced in the cells of a subject.
  • the multiply- or differentially-spliced RNAs encode for different or variant proteins, or comprise different or variant RNAs, having a similar or separate therapeutic activity.
  • the multiply- or differentially-spliced RNAs are spliced in response to the presence of a specific factor, disease, condition, or tissue.
  • the encoded TM is a protein having a therapeutic activity and, preferably, a human protein or variant thereof.
  • the nucleic acid sequence encoding such a protein is of a gene or gene fragment.
  • the TM is a granulocyte macrophage colony stimulating factor (GMCSF).
  • the TM is an interferon, e.g., interferon-beta (IFN- ⁇ ), and more particularly, is IFN- ⁇ 1a or IFN- ⁇ 1 b.
  • the encoded TM is an antibody, and preferably a monoclonal antibody (e.g., CAMPATH).
  • Suitable sequences encoding a monoclonal antibody can be identified and made using methods known in the art, and inserted into a vector of the regulated expression system of the present invention as described herein.
  • the therapeutic activity of monoclonal antibodies has been reported (see e.g., Gatto, B. (2004) 4:411-414; Groner et al. (2004) 4:539-547).
  • "Regulator molecule” or “RM” as used herein refers to a molecule that regulates the expression and/or activity of a TM of the present invention.
  • Examples of such regulation by an RM of the present invention include, but are not limited to, the modulation of TM expression and/or activity, and more particularly, an increase, decrease, activation (or induction), or inactivation (or repression) of TM expression and/or activity, by an RM of the present invention. Further such modulation of TM expression and/or activity by an RM of the present invention can be direct (e.g., by direct contact of an RM with a TM) or indirect (e.g., where the RM effects a molecule in a signal transduction pathway that results in the modulation of TM expression and/or activity).
  • RMs suitable for use in the regulated, expression system of the present invention include, but are not limited to, molecules that effect cellular expression, activity, or processing of a TM of the present invention.
  • suitable RMs include, but are not limited to, transcriptional regulatory molecules (e.g., that activate, inactivate, decrease, or increase transcription of an RNA of an expressed TM); RNA processing molecules (e.g., molecules that activate, inactivate, decrease, or increase RNA processing such as RNA splicing, polyadenylation, or cleavage of an RNA of an expressed TM); or molecules that effect protein translation or post- translational processing of a protein (e.g., enzymes that activate, inactivate, decrease, or increase the phosphorylation, cleavage, or formation of a particular conformation or multimeric form of a protein of an expressed TM).
  • transcriptional regulatory molecules e.g., that activate, inactivate, decrease, or increase transcription of an RNA of an expressed TM
  • RNA processing molecules
  • an RM of the present invention can be a naturally-occurring or isolated molecule, or variant thereof.
  • an RM of the present invention is a synthetic or recombinant molecule.
  • an RM of the present invention is a chemical compound, DNA, RNA, or protein.
  • an RM of the present invention is a modified molecule.
  • the RM is a humanized protein.
  • the RM is a human protein or variant thereof.
  • the RM is a transcriptional activator e.g., a steroid receptor and, more particularly, a progesterone receptor.
  • the RM comprises a transactivation domain (e.g., a VP16 or p65 transactivation domain, see e.g., Schmitz et al. (1991 ) EMBO J 10:3805-3817; Moore et al. (1993) Molec and Cell Biol 13:1666; Blair et al. (1994) Molec and Cell Biol 14:7226-7234), and/or other functional domain (e.g., a basal factor interaction domain) of a co-activator (e.g., p300/CBP), a basal transcription factor (e.g. TFIIB), or a histone acetyltransferase (e.g.
  • a transactivation domain e.g., a VP16 or p65 transactivation domain, see e.g., Schmitz et al. (1991 ) EMBO J 10:3805-3817; Moore et al. (1993) Molec and Cell Bio
  • the RM comprises a ligand-binding domain (LBD).
  • LBD ligand-binding domain
  • an AM binds to the LBD of the RM, thereby activating the RM such that the presence of the activated RM regulates TM expression and/or activity.
  • the RM comprises a DBD, e.g., a GAL-4 DBD.
  • the RM comprises a DBD that binds to a functional sequence (e.g., a promoter sequence) operably linked to a nucleic acid encoding a TM, thereby regulating TM expression (e.g., inducing TM expression).
  • a functional sequence e.g., a promoter sequence
  • an RM of the present invention is activated by an activator molecule (AM) and, thereby, TM expression and/or activity is regulated in the presence of the activated RM.
  • A activator molecule
  • Activator molecule refers to a molecule that induces or increases the expression and/or activity of an RM of the present invention.
  • Examples of such activation by an AM include, but are not limited to the induction or increase in expression and/or activity of an RM of the present invention. Further such activation in RM expression and/or activity by an AM of the present invention can be direct (e.g., by direct contact of an AM with a RM) or indirect (e.g., where the AM affects a molecule in a signal transduction pathway that results in the modulation of RM expression and/or activity). Further examples of AMs suitable for use in the regulated expression system of the present invention include, but are not limited to, molecules that effect cellular processing of an RM of the present invention (examples of such cellular processing are decribed herein, e.g., above).
  • the AM is a biomarker. In a further embodiment, the AM is a biomarker for a disease or condition and, more particularly, is a biomarker for a disease state or condition, or symptom thereof. In one embodiment, the AM activates the RM by promoting or inhibiting conformational change, enzymatic processing or modification, specific binding, or dimerization of the RM. In a preferred embodiment, the AM activates the RM by promoting homodimerization of the RM. In one embodiment, the AM activates the RM by binding to the RM and, more particularly, to a functional domain of the RM, e.g., an AD of the RM.
  • an AM of the present invention can be a naturally-occurring or isolated molecule, or variant thereof.
  • the AM of the present invention is a synthetic or recombinant molecule.
  • the AM of the present invention is a chemical compound, DNA, RNA, or protein.
  • the AM of the present invention is a modified molecule.
  • the AM is a humanized protein.
  • the AM is a human protein or variant thereof.
  • the AM is a chemical compound, e.g., an antiprogestin.
  • the AM is mifepristone.
  • the regulated expression system of the present invention comprises: 1 ) a first expression cassette having a first nucleic acid sequence encoding a TM, and at least one GAL-4 DNA-binding site (DBS) and, more particularly, six GAL-4 DBS (6XGAL-4 DBS), located upstream and operably linked to the first nucleic acid sequence; 2) a second expression cassette having a second nucleic acid sequence encoding an RM that is a modified progesterone receptor comprising a VP-16 AD or p65 AD (e.g., a p65 AD comprising the nucleic acid sequence of SEQ ID NO: 39 or amino acid sequence of SEQ ID NO: 40), progesterone (PR) LBD, and GAL-4 DBD, and an actin promoter sequence located upstream and operably linked to the second nucleic acid sequence; and 3) an AM that is a small molecule inducer, e.g., mifepristone (MFP) that when orally administered to a subject,
  • the RM of the present invention is a transcriptional regulator and more particularly, a mutated steroid receptor.
  • the RM is a mutated human PR (hPR) and comprises a mutated hPR receptor LBD, (e.g., having a C-terminal deletion of about 19-66 amino acids), wherein the RM is activated in the presence of an AM that is an antagonist of the wild-type PR from which the mutant PR was derived.
  • the RM of the present invention comprises a regulatory domain (RD), e.g., an activation domain (AD), and more particularly, a transactivation domain (TD). Examples of suitable regulatory domains for use in the RM of the present invention, include, but are not limited to, those known in the art or described herein (e.g., TAF-1 , TAF-2, TAU-1 , and TAU- 2).
  • an RM of the present invention is inactivated and thereby TM expression and/or activity is regulated in the presence of an inactivated RM.
  • "Inactivator molecule” or “IM” as used herein refers to a molecule that inactivates the expression and/or activity of an RM of the present invention. Examples of such inactivation by an IM include, but are not limited to the repression or decrease in expression and/or activity of an RM of the present invention.
  • an IM of the present invention can be direct (e.g., by direct contact of an IM with a RM) or indirect (e.g., where the IM affects a molecule in a signal transduction pathway that results in the inactivation of RM expression and/or activity).
  • IMs suitable for use in the regulated, expression system of the present invention include, but are not limited to, molecules that effect cellular processing of an RM of the present invention (examples of such cellular processing are decribed herein, e.g., above).
  • an RM of the present invention is expressed or present in cells of a subject in an activated form, and is inactivated in the presence of an inactivator molecule (IM), thereby, TM expression and/or activity is regulated by the inactivated RM.
  • IM is a biomarker.
  • the IM is a biomarker for a disease or condition and, more particularly, is a biomarker for a disease state or condition, or symptom thereof.
  • the IM inactivates the RM by promoting or inhibiting conformational change, enzymatic processing, specific binding, or dimerization of the RM.
  • the IM inactivates the RM by inhibiting homodimerization of the RM.
  • the IM inactivates the RM by binding to the RM and, more particularly, to a functional domain of the RM, e.g., an AD of the RM.
  • an IM of the present invention can be a naturally-occurring or isolated molecule, or variant thereof.
  • the IM of the present invention is a synthetic or recombinant molecule.
  • the IM of the present invention is a chemical compound, DNA, RNA, or protein.
  • the IM of the present invention is a modified molecule.
  • the IM is a humanized protein.
  • the IM is a human protein or variant thereof.
  • the IM is a chemical compound.
  • the expression of a TM, RM, AM, or IM of the present invention can be constitutive or transient.
  • expression of a TM, RM, AM, or IM is regulated or tissue- specific (e.g. muscle-specific).
  • a regulated RM include, but are not limited to, an RM that is activated by an AM or inactivated by an IM.
  • the expression of a TM, RM, AM, or IM of the present invention is driven by a regulated promoter or a tissue- specific promoter.
  • the regulated or tissue-specific promoter is regulated in the presence of an RM and, more particularly, by the binding of the RM to the promoter.
  • the tissue-specific promoter is a muscle-specific promoter and, more particularly, an actin promoter.
  • an RM of the present invention binds to a promoter operably linked to a nucleic acid sequence encoding a TM and thereby regulates the expression of the encoded TM as described herein, in the cells of a subject.
  • TM, RM, AM, or IM of the present invention can be isolated, produced, and modified using known methods and assays for nucleic acids, proteins, and chemical compounds, as described herein, e.g., below.
  • the present invention also provides pharmaceutical compositions and methods for treatment of a variety of diseases comprising the improved regulated expression system of the present invention as described herein.
  • the present invention provides pharmaceutical compositions and methods for treating a disease or condition; regulating the expression of a TM; administering a TM; delivering a TM; or expressing a TM in cells of a subject, where the methods comprise contacting the cells of a subject with a regulated expression system of the present invention, such that the encoded TM is expressed in the cells of the subject, and such TM expression is regulated in the presence of an RM.
  • the pharmaceutical compositions of the present invention comprise at least one TM,
  • the pharmaceutical compositions of the present invention can comprise more than one of each TM, RM, AM, or IM, and more than one kind thereof (e.g., a first and second TM, RM, AM, and/or IM). More particularly, the pharmaceutical compositions of the present invention can comprise nucleic acid sequences encoding more than one of each TM, RM, AM, or IM, and more than one kind thereof.
  • a pharmaceutical composition of the present invention comprises at least one of the vectors of the present invention (e.g., pGT23 (SEQ ID NO: 23), pGT24 (SEQ ID NO: 24), pGT25 (SEQ ID NO: 25), pGT26 (SEQ ID NO: 26), pGT27 (SEQ ID NO: 27), pGT28 (SEQ ID NO: 28), pGT29 (SEQ ID NO: 29), or pGT30 (SEQ ID NO: 30)).
  • the vectors of the present invention e.g., pGT23 (SEQ ID NO: 23), pGT24 (SEQ ID NO: 24), pGT25 (SEQ ID NO: 25), pGT26 (SEQ ID NO: 26), pGT27 (SEQ ID NO: 27), pGT28 (SEQ ID NO: 28), pGT29 (SEQ ID NO: 29), or pGT30 (SEQ ID NO: 30)).
  • a pharmaceutical composition of the present invention comprises at least one AM or IM of the present invention.
  • a pharmaceutical composition of the present invention comprises one or more vectors encoding at least one TM and/or RM.
  • the TM, RM, AM, and IM of the present invention can be administered to a subject separately or together, and ex vivo or in vivo, using any suitable means of administration described herein or known in the art. Examples of such suitable means of administration include, but are not limited to injection (e.g., intramuscular or subcutaneous injection), oral administration, and electroporation.
  • a TM and RM of the present invention are present in a single vector, and separately administered from an AM that activates the RM (and thereby, the presence of the activated RM regulates TM expression and/or activity).
  • the AM is a compound (e.g., mifepristone) administered orally to a subject
  • the single vector encoding a TM and RM is a single vector administered by injection or by electroporation to cells of a subject (e.g., skeletal muscle cells).
  • a suitable means for administering a composition of the present inventions include the ex vivo delivery of the composition, e.g., a nucleic acid vector encoding a TM and/or RM, to cells of a subject and then the delivery of the treated cells to the subject, such that the encoded molecule is expressed in cells in the subject (see e.g., Studeny et al. (2004) J Natl Cancer Inst 96(21 ): 1593-1603; Studeny et al. (2002) Cancer Res 62(13):3603-3608).
  • the composition e.g., a nucleic acid vector encoding a TM and/or RM
  • the regulated expression system of the present invention comprises a nucleic acid sequence encoding a therapeutic gene (e.g., IFN- ⁇ gene) that is administered to a subject by injection.
  • a therapeutic gene e.g., IFN- ⁇ gene
  • therapeutic gene refers to a gene encoding a TM, e.g., a protein having a therapeutic activity (e.g., which may be an interferon molecule (IFNM) such as IFN- ⁇ 1a or 1 b).
  • IFNM interferon molecule
  • the gene is IFN- ⁇ 1a and is administered as a single intramuscular injection periodically, e.g., 3 to 6 months, using a vector of the present invention.
  • a therapeutic gene is administered every 1-3 months, 3 to 6 months, 6 to 9 months, or 9 to 12 months.
  • the regulation of the circulating levels of the expressed protein is achieved by controlled induction of a promoter driving expression of the encoded protein in the target subject cells or tissue.
  • the RM is a small molecule activator, in the form of an orally available pill, that controls promoter induction and subsequent expression of a TM and, more particularly, a therapeutic gene.
  • a TM e.g., a protein or nucleic acid
  • the regulated, expression system of the present invention allows for the first time the option for continuous versus pulsatile therapy of a TM (e.g., a protein or nucleic acid), and the modulation of expression levels of a TM, in order to optimize therapeutic efficacy of a TM while minimizing any side effects thereof.
  • the regulated expression system of the present invention allows for the first time the option for continuous versus pulsatile TM therapy in subjects and, more particularly, allows for subject- specific therapy by modulating and optimizing expression levels of a TM in cells of the subject to achieve maximum therapeutic efficacy and minimum side effects, for treatment of a disease.
  • nucleic acids encoding a TM e.g., a protein or nucleic acid
  • nucleic acids encoding a TM can be delivered to target cells of a subject, for treatment of disease.
  • nucleic acids encoding a TM can be delivered to target cells of a subject, such that the expressed TM is provided in a therapeutically effective dose or amount.
  • a "therapeutically effective dose” or “therapeutically effective amount” of a TM of the present invention is a dose or amount that, when present in the cells of a subject in need of treatment of a disease, results in a therapeutic benefit to the subject (i.e., results in treatment of the disease).
  • a suitable dose or amount of an RM or a nucleic acid encoding an RM, administered to a subject is a dose or amount that regulates (e.g., induces) the expression and/or activity of a TM in the cells of a subject such that a therapeutically effective dose is achieved.
  • Factors influencing the amount of TM that constitutes a therapeutically effective dose include, but are not limited to, the severity and history of the disease to be treated, and the age, health, and physical condition of the subject undergoing therapy.
  • a therapeutically effective dose of a TM of the present invention can also depend upon the dosing frequency and severity of the disease in the subject undergoing treatment.
  • the dosing regimen of a TM of the present invention can be continued for as long as is required to achieve the desired effect, i.e., for example, prevention and/or amelioration of the disease, symptoms associated with the disease, disease severity, and/or periodicity of the recurrence of the disease, as described herein.
  • the dosing regimen is continued for a period of up to one year to indefinitely, such as for one month to 30 years, about three months to about 20 years, about 6 months to about 10 years.
  • the TM may be administered daily, every other day, three times per week, twice per week, weekly, biweekly, monthly, bimonthly, every three months, quarterly, semiannually, annually, or any combination thereof.
  • suitable nucleic acids for use in the regulated expression system of the present invention include, but are not limited to, those nucleic acids encoding a gene for a hormone, growth factor, enzyme, cytokine, receptor, or MHC molecule having a therapeutic activity.
  • suitable genes for use in the compositions and methods of the present invention include nucleic acid sequences that are exogenous or endogenous to cells into which the nucleic acid encoding the gene of interest can be introduced.
  • nucleic acid sequences that are exogenous or endogenous to cells into which the nucleic acid encoding the gene of interest can be introduced.
  • genes encoding a polypeptide that is either absent, produced in diminished quantities, or produced in a mutant form in those subjects having or are susceptible to a genetic disease.
  • genetic diseases include, but are not limited to, retinoblastoma, Wilms tumor, adenosine deaminase deficiency (ADA), thalassemias, cystic fibrosis, Sickle cell disease, Huntington's disease, Duchenne's muscular dystrophy, Phenylketonuria, Lesch-Nyhan syndrome, Gaucher's disease, and Tay-Sach's disease.
  • nucleic acids encoding a tumor suppressor gene.
  • Suitable tumor suppressor genes include, but are not limited to, retinoblastoma, GM-CSF, G-CSF, M-CSF, human growth hormone (HGH), TNF, TGF- ⁇ , TGF- ⁇ , hemoglobin, interleukins, co-stimulatory factor B7, insulin, factor VIII, factor IX, PDGF, EGF, NGF, EPO, and ⁇ -globin, as well as biologically or therapeutically active muteins of the proteins encoded by such genes.
  • Suitable genes for delivery to target cells can be from any species, but preferably a mammalian species, and more preferably a human. Further, preferred species, as sources of suitable genes, are those species into which the gene of interest is to be delivered using the methods and compositions of the present invention, e.g., a mammalian species and preferably a human.
  • suitable nucleic acids include, but are not limited to, those encoding a granulocyte macrophage stimulating colony factor (GMCSF) or variant thereof (e.g., Leukine ® or human GMCSFLeu 23 Asp 27 Glu 39) ), having an anticancer activity (see e.g., the GMCSF mutants of US Patent No.s 5,032,676; 5,391 ,485; and 5,393,870).
  • GMCSF granulocyte macrophage stimulating colony factor
  • suitable nucleic acids include, but are not limited to, those encoding an interferon having an antiinflammatory or antiviral activity, e.g., an inteferon, particularly IFN- ⁇ , and more particularly, an IFN- ⁇ 1a or IFN- ⁇ 1 b.
  • compositions and methods of the present invention are used to treat MS by delivering to a subject in need of treatment, a nucleic acid encoding a TM that is an IFN- ⁇ and, more particularly, is IFN- ⁇ 1a, such that the IFN- ⁇ is expressed in the cells of the subject and the expression and/or activity of the IFN- ⁇ is regulated by an RM, as described herein.
  • MS is a chronic and severe disease characterized by focal inflammation in the central nervous system (CNS) (see e.g., Hemmer ef a/. (2002) Neuroscience 3: 291-301 ; Keegan ef a/. (2002) Ann. Rev. Med. 53: 285-302; Young, V.
  • MS subject populations there are two major types of MS subject populations at the onset of the disease: those subjects with relapsing-remitting MS and those subjects with primary progressive MS.
  • Relapsing-remitting MS is characterized by episodes (the so called relapses or exacerbation) where new neurologic deficits emerge or preexisting neurologic deficits worsen and periods of remission where the clinical symptoms are stabilized or diminished, whereas, primary progressive MS subjects suffer from progressive neurological deterioration without exacerbations.
  • a large proportion of subjects with relapsing-remitting MS also experience during the course of their disease a worsening of neurologic symptoms independent of relapses, with or without superimposed relapses. Once this stage of the disease is reached, it is called secondary progressive MS.
  • MS The clinical symptoms of MS are thought to result from a focal breakdown in the blood-brain barrier (BBB) which permits the entry of inflammatory infiltrates into the brain and spinal cord. Further, these infiltrates are thought to consist of various lymphocytes and macrophages that lead to demyelination, axonal degeneration and scar tissue formation, and the degeneration of oligodendrocytes imperative to CNS myelin production (see e.g., Martin (1997) J. Neural Transmission (Suppl) 49:53-67). Consequently, the nerve-insulating myelin and the ability of oligodendroglial cells to repair damaged myelin are seriously compromised (see e.g., Scientific American 269(1993):106-114). These symptoms of MS include pain and tingling in the arms and legs, localized and generalized numbness, muscle spasm and weakness, difficulty with balance when standing or walking, difficulty with speech and swallowing, cognitive deficits, fatigue, and bowel and bladder dysfunction.
  • interferons are important cytokines characterized by antiviral, antiproliferative, and immunomodulatory activities. These activities form a basis for the clinical benefits that have been observed in the treatment of subjects with multiple sclerosis.
  • the interferons are divided into the type I and type Il classes. IFN- ⁇ belongs to the class of type I interferons, which also includes interferons alpha, tau and omega, whereas interferon gamma is the only known member of the distinct type Il class.
  • Human IFN- ⁇ is a regulatory polypeptide with a molecular weight of 22 kDa consisting of 166 amino acid residues.
  • the polypeptide can be produced by most cells in the body, in particular fibroblasts, in response to viral infection or exposure to other biologies.
  • IFN- ⁇ binds to a multimeric cell surface receptor, and productive receptor binding results in a cascade of intracellular events leading to the expression of IFNB inducible genes which in turn produces effects which can be classified as antiviral, antiproliferative and immunomodulatory.
  • Human IFN- ⁇ is a well-characterized polypeptide.
  • the amino acid sequence of human IFN- ⁇ is known (see e.g., Gene 10:11-15,1980, and in EP 83069, EP 41313 and U.S. Pat. No. 4,686,191 ).
  • crystal structures have been reported for human and murine IFN- ⁇ , respectively (see e.g., Proc. Natl. Acad. Sci. USA 94:11813-11818, 1997. J. MoI. Biol. 253:187-207, 1995; reviewed in Cell MoI. Life Sci. 54:1203-1206, 1998).
  • IFN- ⁇ fusion proteins are reported, e.g., in WO 00/23472.
  • Betaseron® also termed Betaferon® or IFN- ⁇ 1 b ser17 , which is non-glycosylated, produced using recombinant bacterial cells, has a deletion of the N-terminal methionine residue and the C17S mutation
  • Avonex® and Rebif® also termed IFN- ⁇ 1a, which is glycosylated, produced using recombinant mammalian cells.
  • IFN- ⁇ is the first therapeutic intervention shown to delay the progression of MS.
  • the approved dose of IFN- ⁇ has been shown to be effective in reducing the exacerbation rate of MS, and more subjects remain exacerbation-free for prolonged periods of time as compared with placebo-treated subjects.
  • the accumulation rate of disability is reduced (see e.g., Neurol. 51 :682-689, 1998).
  • IFN- ⁇ has inhibitory effects on the proliferation of leukocytes and antigen presentation. Furthermore, IFN- ⁇ may modulate the profile of cytokine production towards an anti-inflammatory phenotype. Finally, IFN- ⁇ can reduce T-cell migration by inhibiting the activity of T-cell matrix metalloproteases. Such IFN- ⁇ activities are likely to act in concert to account for the beneficial effect of IFN- ⁇ in the treatment of subjects with MS (see e.g., Neurol. 51 :682-689, 1998).
  • compositions and methods of the present invention are for use in the treatment of subjects suffering from various clinically recognized forms of MS, including but not limited to, relapsing-remitting MS, different types of progressive MS (including, but not limited to, e.g., primary and secondary progressive MS, progressive- relapsing MS) and, also, clinically isolated syndromes suggestive of MS.
  • MS including but not limited to, relapsing-remitting MS, different types of progressive MS (including, but not limited to, e.g., primary and secondary progressive MS, progressive- relapsing MS) and, also, clinically isolated syndromes suggestive of MS.
  • Relapsing-remitting MS is a clinical course of MS that is characterized by clearly defined, sporadic exacerbations or relapses, during which existing symptoms become more severe and/or new symptoms appear. Such exacerbations or relapses, may be followed by partial recovery, or full recovery and remission. The length of time between these sporadic exacerbations or relapses may be months or years, during which time inflammatory lesions, demyelination, axonal loss, and scar formation may still proceed. Relapsing-remitting MS is the most common beginning phase of MS, and it has been reported that about 50% of the cases have progression within 10 to 15 years, and another 40% within 25 years of onset.
  • primary-progressive MS is a clinical course of MS that is characterized from the beginning by progressive disease, with no plateaus or remissions, or an occasional plateau and very short-lived, minor improvements. As the disease progresses, the subject may experience difficulty in walking, the steadily decline in motor skills, and an increase in disabilities over many months and years, generally, in the absence of those distinct inflammatory attacks characteristic of relapsing-remitting MS.
  • secondary-progressive MS is a clinical course of MS that initially is relapsing-remitting and then becomes progressive at a variable rate independent of relapses. Although subjects experiencing this type of MS may continue to experience inflammatory attacks or exacerbations, eventually the exacerbations and periods of remission may diminish, with the disease taking on the characteristic decline observed with primary- progressive MS.
  • MS progressive-relapsing
  • MS is a clinical course of MS that may show permanent neurological deterioration from the onset of the disease, but with clear, acute exacerbations or relapses that look like relapsing-remitting MS. For these subjects, lost functions may never return. It has been reported that this type of MS has a high mortality rate if untreated.
  • Clinically isolated syndromes suggestive of MS include, but are not limited to, early onset multiple sclerosis and monosymptomatic MS.
  • multiple sclerosis is intended to encompass each of these clinical manifestations of the disease and clinically isolated syndromes suggestive of MS unless otherwise specified.
  • a subject having MS or symptoms associated with MS is a subject in need of treatment of MS or associated symptoms of MS.
  • treatment when a subject suffering from MS undergoes treatment in accordance with the pharmaceutical compositions and methods of the present invention, treatment can result in the prevention and/or amelioration of MS disease symptoms, disease severity, and/or periodicity of recurrence of the disease, i.e., treatment of MS using the compositions and methods of the present invention can result in lengthening the time period between episodes in which symptoms flare, and/or can suppress the ongoing immune or autoimmune response associated with the disease, which, left untreated, can enhance disease progression and disability.
  • a subject can be pre-treated with a pharmaceutical composition or can be a naive subject who has not been pre-treated with a pharmaceutical composition, prior to treatment using a pharmaceutical composition or method of the present invention.
  • a pre-treated subject can be one who has been pretreated with an IFN- ⁇ protein drug (e.g., IFN- ⁇ 1a) or IFN- ⁇ variant (e.g., IFN- ⁇ 1 b), prior to treatment with the compositions or methods of the present invention.
  • an approved dose of Betaseron®, Avonex®, or Rebif® can be used to pre-treat subjects.
  • the pharmaceutical compositions and methods of the present invention are suitable for use in the treatment of pre-treated and naive subjects.
  • compositions and methods of the present invention can also be used to block or reduce the physiological and pathogenic deterioration associated with a disease, e.g., inflammatory response in the brain and other regions of the nervous system, breakdown or disruption of the blood-brain barrier, appearance of lesions in the brain, tissue destruction, demyelination, autoimmune inflammatory response, acute or chronic inflammatory response, neuronal death, and/or neuroglial death.
  • a disease e.g., inflammatory response in the brain and other regions of the nervous system, breakdown or disruption of the blood-brain barrier, appearance of lesions in the brain, tissue destruction, demyelination, autoimmune inflammatory response, acute or chronic inflammatory response, neuronal death, and/or neuroglial death.
  • Beneficial effects of the pharmaceutical compositions and methods of the present invention include, but are not limited to, preventing the disease, slowing the onset of an established disease, ameliorating symptoms of a disease, reducing an exacerbation rate, slowing the progression of the disease, and postponing or preventing disability including cognitive decline, loss of employment, hospitalization, and finally death.
  • the episodic recurrence of a particular type of disease e.g.
  • MS can be treated, e.g., by decreasing the severity of the symptoms (such as the symptoms described above) associated with the episode, or by lengthening the time period between the occurrence of episodes, e.g., by days, weeks, months, or years, where the episodes can be characterized by the flare-up and exacerbation of disease symptoms, or preventing or slowing the appearance of brain inflammatory lesions e.g. in MS (see, e.g., Adams (1993) Principles of Neurology, page 777, for a description of a neurological inflammatory lesion).
  • suitable nucleic acids for use in the compositions and methods of the present invention can encode a TM that is a fusion or chimeric protein, or a fusion or chimeric nucleic acid (e.g., RNA).
  • a TM of the present invention can regulate expression of a gene product or block one or more steps in a biological pathway (e.g., a sepsis pathway) and, thereby, provide a therapeutic benefit.
  • the nucleic acid can encode a toxin fused to a TM (e.g., a receptor ligand gene or an antibody that directs the fused toxin to a target such as a tumor cell or a virus) and, thereby, have a therapeutic effect.
  • Adverse effects due to some disease treatment regimens are known in the art (see, e.g., Munschauer ef al. (1997) Clinical Therapeutics 19(5): 883-893; Walther ef al. (1999) Neurology 53: 1622-1627; Lublin ef al. (1996) 46: 12-18; Bayas ef al. (2000) 2: 149-159; Ree ef al. (2002) 8: 15-18; Walther ef a/. (1998) 5(2): 65-70).
  • some of the adverse effects due to treatment of MS include, but are not limited, e.g., flu-like symptoms; increased spasticity or deterioration of neurological symptoms; menstrual disorders; laboratory abnormalities (e.g., abnormal blood count/value for hemoglobin, leukocytes, granulocytes, lymphocytes, or thrombocytes); abnormal laboratory value for liver enzymes (e.g. bilirubin, transaminases, or alkaline phosphatases); injection site reactions, (e.g., inflammation, pain, or erythema); cutaneous or subcutaneous necroses; and depression.
  • laboratory abnormalities e.g., abnormal blood count/value for hemoglobin, leukocytes, granulocytes, lymphocytes, or thrombocytes
  • abnormal laboratory value for liver enzymes e.g. bilirubin, transaminases, or alkaline phosphatases
  • injection site reactions e.g., inflammation, pain, or erythema
  • Suitable co-medications and the use of these co-medications, in conjunction with the compositions and methods of the present invention, for treating adverse effects due to treatment of a disease can be determined according to co-medications generally known in the art for treatment of such effects (see, e.g., Munschauer ef al. (1997) Clinical Therapeutics 19(5): 883-893; Walther ef al. (1999) Neurology 53: 1622-1627; Lublin ef al. (1996) 46: 12-18; Bayas ef a/. (2000) 2: 149-159; Ree ef a/. (2002) 8: 15-18; Walther ef a/. (1998) 5(2): 65-70).
  • a disease e.g., MS
  • co-medications are well known in the art and may include, but are not limited to, e.g., those that help alleviate or mitigate adverse effects due to a disease or due to treatment of a disease.
  • co-medications include, but are not limited to, analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), and steroids.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • co-medications also include, but are not limited to, e.g., ibuprofen, acetaminophen, acetylsalicyclic acid, prednisone, pentoxifylline, baclofen, steroids, antibacterial agents, and antidepressants (see e.g., Walther ef al. (1999) Neurology 52: 1622-1627).
  • flu-like symptoms can be treated with NSAIDs (e.g., ibuprofen or acetylsalicylic acid) or with paracetamol or with pentoxifylline; increased spasticity or deterioration of neurological symptoms can also be treated with NSAIDs and/or muscle relaxants (e.g., baclofen); menstrual disorders can be treated with oral contraceptives; injection site reactions can be treated with systemic NSAIDs and/or steroids (e.g., hydrocotisone); cutaneous or subcutaneous necrosis can be treated with antibacterial agents and depression can be treated with antidepressants (see e.g., Walther ef al. (1999) Neurology 53: 1622-1627).
  • NSAIDs e.g., ibuprofen or acetylsalicylic acid
  • paracetamol or with pentoxifylline e.g., acetylsalicylic acid
  • pentoxifylline e.g.
  • Combination therapies with other drugs which are effective in the treatment of a particular disease and have a different adverse event profile may increase the treatment effect and level out the adverse event profile.
  • examples of combination therapies include, but are not limited to, e.g., glatiramer acetate (Copaxone), mitoxantrone, cyclophosphamide, cydosporine A, cladribine, monoclonal antibodies (e.g., Campath-H1® or Antegren®/Natazulimab®), and statins.
  • Effective treatment of disease in a subject using the methods of the invention can be examined in several alternative ways including, for example, EDSS (extended disability status scale) score, Functional Composite Score, cognitive testing, appearance of exacerbations, or MRI e.g., for the treatment of MS.
  • the EDSS is a means to grade clinical impairment due to MS (see e.g., Kurtzke (1983) Neurology 33:1444).
  • Eight functional systems, the walking range, the ability to walk, and the ability to maintain self-care functions are evaluated for the type and severity of neurologic impairment. For example, prior to treatment, impairment in the following systems is evaluated: pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual, cerebral, and other.
  • the grade scale may range, e.g., from 0 (normal) to 10 (death due to MS).
  • An increase of one full step (or a one-half step at the higher baseline EDSS scores) may define disease progression (see e.g., Kurtzke (1994) Ann. Neural. 36:573-79, Goodkin (1991 ) Neurology. 41 :332.).
  • exacerbations can be defined as the appearance of a new symptom that is attributable to MS and accompanied by an appropriate new neurologic abnormality (see e.g., IFN- ⁇ MS Study Group). Exacerbations typically last at least 24 hours, and are preceded by stability or improvement for at least 30 days or a separation of at least 30 days from onset of the last event. Standard neurological examinations may result in the exacerbations being classified as either mild, moderate, or severe according to changes in a Neurological Rating Scale (see e.g., Sipe et al. (1984) Neurology 34:1368), and/or changes in EDSS score or evaluating physician opinion.
  • a Neurological Rating Scale see e.g., Sipe et al. (1984) Neurology 34:1368
  • An annual exacerbation rate (or other measures for the frequency of relapses, like e.g., a hazard ratio for recurrent relapses), the proportion of exacerbation-free subjects, and other relapse-based measures for disease activity are then determined, and the effectiveness of therapy is assessed between the treated group and the placebo group, for any of these measurements.
  • suitable vectors for use in the compositions and methods of the present invention for the treatment of disease are those having minimal immunological toxicity, e.g., plasmid or AAV vectors.
  • plasmid vectors encoding either TGF- ⁇ or IL-4, under control of a CMV promoter reportedly protect mice from myelin basic protein (MBP) induced EAE with minimal immunolgical toxicity (see e.g., CA. Piccirillo and G.J. Prud'ans (1999) Human Gene Therapy 10: 1915-22)).
  • MBP myelin basic protein
  • cytokines and target tissues are reportedly suitable for use for expressing cytokines in an EAE model, including a non- replicative herpes simplex (HSV) type-1 vector expressing IL-4, IL-10, or IL-1 antagonist following intrathecal administration (see e.g., G. Martino et al. (2000) J. Neuroimmunol 107: 184-90).
  • HSV herpes simplex
  • MRI magnetic resonance imaging
  • MRI can be used to measure active lesions using, e.g., gadolinium-DTPA-enhanced T1-weighted imaging (see e.g., McDonald ef a/. (2001 ) Ann. Neurol. 50: 121-127) or the location and extent of lesions using T2-weighted and T1-weighted techniques.
  • Baseline MRIs can be obtained and thereafter, the same imaging plane and subject position can be used for each subsequent study.
  • areas of lesions can be outlined and summed slice by slice for total lesion area, and various criteria may be examined, e.g.: 1 ) evidence of new lesions; 2) rate of appearance of active or new lesions; and 3) change in lesion area or lesion volume (see e.g., Paty ef al. (1993)
  • improvement due to therapy may then be established, e.g., when there is a statistically significant improvement in an individual subject compared to baseline or in a treated group versus a placebo group.
  • the nucleic acid compositions of the present invention are formulated for administration or delivery to the cells of a subject.
  • the nucleic acid compositions of the present invention are formulated with non-ionic and/or anionic polymers. Such polymers can enhance transfection efficiency and expression of molecules encoded by the nucleic acid, and protect the nucleic acid from degradation. Thus, in some embodiments where transfection efficiency or expression is enhanced using formulated nucleic acid compositions of the present invention, lower amounts of the nucleic acid composition (e.g., a vector encoding a molecule of the present invention, e.g., a TM and/or RM of the present invention) can be used.
  • biodegradable polymers refers to polymers that can be metabolized or cleared in vivo by a subject and having no or minimal toxic effects or side effects on the subject.
  • anionic polymers refers to polymers having a repeating subunit which includes, for example, an ionized carboxyl, phosphate or sulfate group having a net negative charge at neutral pH.
  • anionic polymers suitable for use in the present invention include, but are not limited to, poly-amino acids (e.g., poly-glutamic acid, poly- aspartic acid and combinations thereof), poly-nucleic acids, poly-acrylic acid, poly- galacturonic acid, and poly-vinyl sulfate.
  • the polymer is a polymeric acid
  • the polymer is utilized as a salt form.
  • poly-L-glutamic acid is used interchangeably herein with “poly-L-glutamic acid, sodium salt”, “sodium poly-L-glutamate” and “poly-L-glutamate.”
  • Poly- L-glutamate refers to a sodium salt of poly-L- glutamic acid.
  • the L stereoisomer of polyglutamic acid is used in the compositions of the present invention, but, in other embodiments, other stereoisomer or racemic mixtures of isomers are suitable for use in the compositions of the present invention.
  • other salts of anionic amino acid polymers are suitable for use in the compositions of the present invention.
  • anionic amino acid polymers refers to polymeric forms of a given anionic amino acid, for example, a poly-glutamic acid or poly-aspartic acid.
  • polymers formed of a mixture of anionic amino acids for example glutamic acid and aspartic acid, may be equally suitable for use in compositions of the present invention.
  • a pharmaceutically acceptable carrier and other components may be used in the pharmaceutical compositions and methods of the present invention.
  • pharmaceutically acceptable carrier is a carrier or diluent that is conventionally used in the art to facilitate the storage, administration, and/or the desired effect of the therapeutic ingredients of the pharmaceutical composition.
  • a carrier may also reduce undesirable side effects of administering or delivering to a subject a pharmaceutical composition of the present invention.
  • a suitable carrier is preferably stable, e.g., incapable of reacting with other ingredients in the formulation. Further, a suitable carrier preferably does not produce significant local or systemic adverse effect in a subject at the doses and concentrations employed for therapy. Such carriers are generally known in the art.
  • Suitable pharmaceutically acceptable carriers are, e.g., solvents, dispersion media, antibacterial and antifungal agents, microcapsules, liposomes, cationic lipid carriers, isotonic and absorption delaying agents and the like which are not incompatible components of the pharmaceutical compositions of the present invention.
  • the use of such media and agents for therapeutically effective or active substances is well known in the art.
  • Supplementary active ingredients may also be incorporated into the pharmaceutical compositions of the present invention and used in the methods of the present invention.
  • compositions of the present invention are large stable macromolecules such as albumin, gelatin, collagen, polysaccharide, monosaccharides, polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polymeric amino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose, lactose, mannose, dextrose, dextran, cellulose, mannitol, sorbitol, polyethylene glycol (PEG), heparin alginate, and the like.
  • Slow-release carriers such as hyaluronic acid, may also be suitable.
  • Stabilizing agents such as human serum albumin (HSA), mannitol, dextrose, trehalose, thioglycerol, and dithiothreitol (DTT), may also be added to the pharmaceutical compositions of the present invention to enhance their stability.
  • HSA human serum albumin
  • mannitol mannitol
  • dextrose trehalose
  • thioglycerol trehalose
  • DTT dithiothreitol
  • Suitable stabilizing agents include but are not limited to ethylenediaminetetracetic acid (EDTA) or one of its salts such as disodium EDTA; polyoxyethylene sorbitol esters e.g., polysorbate 80 (TWEEN 80), polysorbate 20 (TWEEN 20); polyoxypropylene-polyoxyethylene esters e.g., Pluronic F68 and Pluronic F127; polyoxethylene alcohols e.g., Brij 35; semethicone; polyethylene glycol e.g., PEG400; lysophosphatidylcholine; and polyoxyethylene-p-t-octyphenol e.g., Triton X-100. Stabilization of pharmaceutical compositions by surfactants is generally known in the art (see e.g., Levine et al. (1991 ) J. Parenteral Sci. Technol. 45(3): 160-165).
  • compositions of the present invention may include, but are not limited to, buffers that enhance isotonicity such as water, saline, phosphate, citrate, succinate, acetic acid, aspartate, and other organic acids or their salts.
  • buffers that enhance isotonicity such as water, saline, phosphate, citrate, succinate, acetic acid, aspartate, and other organic acids or their salts.
  • pharmaceutical compositions of the present invention comprise a non-ionic tonicifying agent in an amount sufficient to render the compositions isotonic with body fluids.
  • compositions of the present invention can be made isotonic with a number of non-ionic tonicity modifying agents generally known to those in the art, e.g., carbohydrates of various classifications (see, e.g., Voet and Voet (1990) Biochemistry (John Wiley & Sons, New York); monosaccharides classified as aldoses (e.g., glucose, mannose, arabinose), and ribose, as well as those classified as ketoses (e.g., fructose, sorbose, and xylulose); disaccharides (e.g., sucrose, maltose, trehalose, and lactose); and alditols (acyclic polyhydroxy alcohols) e.g., glycerol, mannitol, xylitol, and sorbitol.
  • non-ionic tonicifying agents are trehalose, sucrose, and mannitol, or
  • the non-ionic tonicifying agent is added in an amount sufficient to render the formulation isotonic with body fluids.
  • a pharmaceutical composition of the present invention including, e.g., an HSA-free pharmaceutical composition
  • the non-ionic tonicifying agent is present at a concentration of about 1 % to about 10 %, depending upon the agent used (see e.g., U.S. Application No.s 10/190,838, 10/035,397; and PCT International Application No.s PCT/US02/21464 and PCT/US01/51074).
  • compositions of the present invention may incorporate buffers having reduced local pain and irritation resulting from injection, or improve solubility or stability of a component of the pharmaceutical compositions of the present invention (e.g., comprising and/or encoding a TM, RM, AM, and/or IM).
  • buffers include, but are not limited to, e.g., low-phosphate, aspartate, and succinate buffers.
  • the pharmaceutical compositions of the present invention may additionally comprise a solubilizing compound or formulation that is capable of enhancing the solubility of the components of the compositions. Suitable solubilizing compounds include, e.g., compounds containing a guanidinium group, preferably arginine.
  • Suitable solubilizing compounds include, but are not limited to, e.g., the amino acid arginine, or amino acid analogues of arginine that retain the ability to enhance the solubility of an IFN- ⁇ mutein of the present invention.
  • amino acid analogues include but are not limited to, e.g., dipeptides and tripeptides that contain arginine.
  • suitable solubilizing compounds are discussed in, e.g., U.S. Patent No.s 4,816,440; 4,894,330; 5,005,605; 5,183,746; 5,643,566; and in Wang et al. (1980) J. Parenteral Drug Assoc. 34: 452-462).
  • the pharmaceutical compositions of the present invention are formulated in a unit dosage and in an injectable form such as a solution, suspension, or emulsion, or in the form of lyophilized powder, which can be converted into solution, suspension, or emulsion prior to administration.
  • an injectable form such as a solution, suspension, or emulsion, or in the form of lyophilized powder, which can be converted into solution, suspension, or emulsion prior to administration.
  • the pharmaceutical compositions of the present invention may be sterilized by membrane filtration, which also removes aggregates, and stored in unit-dose or multi-dose containers such as sealed vials, ampules or syringes.
  • an AM or IM of the present invention is formulated for oral administration.
  • the nucleic acids encoding a TM and/or RM of the present invention are formulated for administration by injection or electroporation
  • an AM and/or IM of the present invention is formulated for oral administration.
  • the regulated expression system of the present invention comprises a single vector encoding at least a TM and an RM formulated for delivery by injection or electroporation to the cells of a subject, and an AM that is formulated for oral administration to the subject, such that the presence of the AM in the cell of the subject activates the RM and thereby the RM induces expression of the TM in the cells of the subject.
  • Liquid, lyophilized, or spray-dried pharmaceutical compositions of the present invention may be prepared as known in the art, e.g., as an aqueous or nonaqueous solution or suspension for subsequent administration to a subject in accordance with the methods of the present invention.
  • Each of these pharmaceutical compositions may comprise a therapeutically or prophylactically effective or active component.
  • a therapeutically or prophylactically “effective” or “active” component is an amount of a molecule of the present invention (e.g., comprising and/or encoding a TM, RM, AM, and/or IM) that is included in the pharmaceutical composition of the present invention to bring about a desired therapeutic or prophylactic response with regard to treatment, prevention, or diagnosis of a disease or condition in a subject in need of treatment, using the pharmaceutical compositions and methods of the present invention.
  • the pharmaceutical compositions of the present invention comprise appropriate stabilizing agents, bulking agents, or both to minimize problems associated with loss of biological or therapeutic activity during preparation and storage.
  • Formulation of the pharmaceutical compositions of the present invention are preferably stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. Methods of preventing microorganism contamination are well known, and can be achieved e.g., through the addition of various antibacterial and antifungal agents.
  • Suitable forms of the pharmaceutical composition of the present invention may include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Suitable forms are preferably sterile and fluid to the extent that they can easily be taken up and injected via a syringe.
  • Typical carriers may include a solvent or dispersion medium containing, for example, water buffered aqueous solutions (i.e., biocompatible buffers), ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants, or vegetable oils. Sterilization can be accomplished by any art-recognized technique, including but not limited to filtration or addition of antibacterial or antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.
  • water buffered aqueous solutions i.e., biocompatible buffers
  • polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants, or vegetable oils.
  • Sterilization can be accomplished by any art-recognized technique, including but not limited to filtration or
  • sterile injectable solutions containing a pharmaceutical composition of the present invention may be accomplished by incorporating the composition in the desired amount, in an appropriate formulation with various ingredients (e.g., those enumerated herein) as desired, and followed by sterilization.
  • the above solutions can be vacuum-dried or freeze-dried as necessary.
  • compositions of the present invention can thus be compounded for convenient and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in a therapeutically effective dose.
  • a suitable pharmaceutically acceptable carrier in a therapeutically effective dose.
  • the precise therapeutically effective amount of the compositions and methods of the present invention for application to humans can be determined by the skilled artisan with consideration of individual differences in age, weight, extent of cellular infiltration by inflammatory cells and condition of the MS subject.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the principal active ingredients may be compounded for convenient and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as described herein.
  • the co-medications are contained in a unit dosage form in amounts generally known in the art.
  • the dosages may be determined, e.g., by reference to the known dose and manner of administration of the ingredients.
  • compositions of the present invention may be administered in a manner compatible with the dosage formulation and in such an amount as will be therapeutically effective. Further, the pharmaceutical compositions of the present invention may be administered in any way which is medically acceptable and which may depend on a specific type or stage of disease or associated symptoms being treated. Possible administration routes include injections, by parenteral routes such as intravascular, intravenous, intra-arterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural or others, as well as oral, nasal, ophthalmic, rectal, topical, or by inhalation. In a preferred embodiment, the administration route is intramuscular. In a preferred embodiment, the pharmaceutical composition of the present invention is administered intramuscularly, every 3-12 months.
  • the intramuscular administration is via automated or manual injection (e.g., using a syringe) of the pharmaceutical composition.
  • Sustained release administration is also contemplated, e.g., using erodible implants.
  • the nucleic acid pharmaceutical compositions of the present invention can be delivered to cells of a subject using any means described herein or known in the art, including e.g., by injection or other suitable means.
  • known methods of delivery of nucleic acids to cells by physical means are suitable for use and include, but are not limited to, electroporation, sonoporation, and pressure.
  • delivery of a nucleic acid composition of the present invention is by electroporation and comprises the application of a pulsed electric field to create transient pores in the cellular membrane and, thereby, an exogenous molecule, e.g., a nucleic acid composition of the present invention, is delivered to the cell.
  • an exogenous molecule e.g., a nucleic acid composition of the present invention
  • nucleic acid molecules can find their way through passageways or pores in the cell that are created during such a procedure (see e.g., U. S. Patent 5, 704,908, U.S. Patent 5,704,908).
  • pulse voltage device or “pulse voltage injection device” refers to an apparatus that is capable of causing or causes uptake of nucleic acid molecules into the cells of a subject by emitting a localized pulse of electricity to the cells, thereby, causing the cell membrane to destabilize and result in the formation of passageways or pores in the cell membrane.
  • Conventional devices of this type are suitable for use for the delivery of a nucleic acid composition of the present invention.
  • the device is calibrated to allow one of ordinary skill in the art to select and/or adjust the desired voltage amplitude and/or the duration of pulsed voltage and therefore.
  • a pulse voltage nucleic acid delivery device can include, for example, an electroporetic apparatus as described e.g. in U.S. Patent No. 5,439,440, U.S. Patent No. 5,704,908, U.S. Patent No. 5,702,384, PCT No.
  • Packaging material used to contain the active ingredient of a pharmaceutical composition of the present invention can comprise glass, plastic, metal or any other suitable inert material and, preferably, is packaging material that does not chemically react with any of the ingredients contained therein.
  • the pharmaceutical composition is packaged in a clear glass, single-use via[; and a separate yja[ containing diluent is included for each vial of drug.
  • the diluent is provided in a syringe (i.e., the syringe is pre-filled with the diluent).
  • the pharmaceutical composition of the present invention is provided in solution in a syringe (i.e., the syringe is pre-filled with the pharmaceutical composition in solution) and is ready for use.
  • the pharmaceutical composition of the present invention can be stored under refrigeration, between 2° to 8 0 C (36° to 46 0 F). In a preferred embodiment, the pharmaceutical composition is stored at room temperature.
  • the present invention further provides vectors and kits comprising the improved regulated expression system of the present invention for treatment of disease.
  • the improved regulated expression system of the present invention comprises one or more vectors, and each vector comprises one or more expression cassettes.
  • the improved regulated expression system of the present invention comprises a single vector having at least one expression cassette and, more preferably at least two expression cassettes.
  • Suitable vectors for use in the regulated, expression system of the present invention include, but are not limited to, those that are capable of expressing an encoded TM, and/or other encoded molecule of the present invention (e.g., RM, AM, or IM), when administered to the cells of a subject.
  • suitable vectors include, but are not limited to, those described herein and those known in the art, including vectors for producing virus and nonviral vectors (vectors that do not produce virus).
  • one class of suitable vectors utilize DNA elements which provide autonomously replicating extra-chromosomal plasmids, derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus.
  • vectors for producing virus may be modified and adapted for use in the regulated expression system of the present invention.
  • the vectors of the present invention can be modified to include additional functional and operably linked sequences for optimal expression of an encoded molecule.
  • suitable functional sequences include, but are not limited to, splicing, polyadenylation and other types of RNA processing sequences; and transcriptional promoter, enhancer, and termination sequences.
  • Suitable cDNA expression vectors incorporating such functional sequences include those described by Okayama, H., MoI. Cell. Biol.
  • Plasmid refers to a composition comprising extrachromosomal genetic material, usually of a circular duplex of DNA, that can replicate independently of chromosomal DNA. Plasmids may be used as vectors, as described herein.
  • Vector refers to a composition (e.g., a nucleic acid construct) comprising genetic material designed to direct transformation or transfection of a targeted cell. Further, a vector may contain multiple functional sequences positionally and sequentially oriented with respect to other sequences of the vector such that an encoded molecule of the present invention can be transcribed and when necessary translated in the transfected or transformed cells.
  • a vector or expression cassette encodes a molecule of the present invention (e.g., a TM, RM, AM or IM), it comprises the essential components (e.g., promoter, poly(A) site, transcription start and stop sites) for expression of the encoded molecule in a heterologous cell (e.g., cells of a subject) according to the regulated expression system of the present invention, described herein.
  • essential components e.g., promoter, poly(A) site, transcription start and stop sites
  • the improved regulated expression system of the present invention comprises a single vector comprising a first expression cassette having at least one cloning site for insertion of a first nucleic acid sequence encoding a TM, and a second expression cassette having at least one cloning site for insertion of second nucleic acid sequence encoding an RM.
  • the vector is a vector for producing virus encoding a molecule of the present invention (e.g., a TM and/or RM) for delivery to cells of a subject as described herein, e.g., a shuttle plasmid and more particularly, an AAV-1 shuttle plasmid (see e.g., Table 8).
  • the vector is a single plasmid vector e.g., pGT1 (comprising the sequence of SEQ ID NO: 9 and SEQ ID NO: 4), pGT2 (comprising the sequence of SEQ ID NO: 10 and SEQ ID NO: 4), pGT3 (comprising the sequence of SEQ ID NO: 11 and SEQ ID NO: 4), or pGT4 (SEQ ID NO: 12), wherein each vector comprises a multiple cloning site (SEQ ID NO: 4) located 3' of the IVS8 and 5' of the hGH poly(A) site (e.g., as schematically depicted in Figures 14A-D), or pGT11 (comprising the sequence of SEQ ID NO: 9 and SEQ ID NO: 5), pGT12 (comprising the sequence of SEQ ID NO: 10 and SEQ ID NO: 5), pGT13 (comprising the sequence of SEQ ID NO: 11 and SEQ ID NO: 5), or
  • the expression cassettes of the present invention comprise functional sequences for expression of an encoded molecule of the present invention, e.g., a TM, RM, AM, or IM.
  • the expression cassette comprises at least one functional sequence operably linked to a nucleic acid sequence encoding a molecule of the present invention.
  • functional sequence refers to a nucleic acid or amino acid sequence having a function or activity in a cell, e.g., a function or activity relating to the cellular expression, processing, or cloning of a molecule, or to the biological or cellular activity or function of a molecule.
  • Examples of a functional sequence include a sequence encoding a molecule of the present invention (e.g., a TM, RM, AM, or IM), promoter, protein or nucleic acid binding site, splice site, transcription stop site, regulatory domain (e.g., activation domain), transcription start site, protein or nucleic acid stabilization site, intervening sequence, restriction enzyme site or cloning site, viral packaging signal, or other cellular, protein, or nucleic acid processing or regulatory signal (e.g., a signal transduction sequence or tissue-specific sequence).
  • a encoding a molecule of the present invention e.g., a TM, RM, AM, or IM
  • promoter e.g., protein or nucleic acid binding site, splice site, transcription stop site, regulatory domain (e.g., activation domain), transcription start site, protein or nucleic acid stabilization site, intervening sequence, restriction enzyme site or cloning site, viral packaging signal, or other cellular,
  • a functional sequence are, but not limited to, a 5' or 3' untranslated region (e.g., UT12, SEQ ID NO: 2), intron (e.g., IVS8, SEQ ID NO: 3), polyadenylation (PoIy(A)) site (e.g, SV40, SEQ ID NO: 8 or hGH poly(A) site, SEQ ID NO: 6), or a DNA- binding site (DBS) (e.g., SEQ ID NO: 49).
  • a 5' or 3' untranslated region e.g., UT12, SEQ ID NO: 2
  • intron e.g., IVS8, SEQ ID NO: 3
  • PoIy(A)) site e.g, SV40, SEQ ID NO: 8 or hGH poly(A) site, SEQ ID NO: 6
  • DBS DNA- binding site
  • Such functional sequences also include, for example, sequences encoding a regulated promoter or tissue-specific promoter that promotes the regulated or tissue-specific expression, respectively, of a molecule encoded by a nucleic acid sequence operably linked to such functional sequences in an expression cassette of the present invention.
  • suitable promoters include, but are not limited to, a CMV promoter, muscle-specific promoter (e.g., actin promoter or muscle creatine kinase (MCK) promoter), condition-specific (e.g., hypoxia or inflammation) promoter or element (e.g.
  • the promoter is a physiologically responsive promoter, e.g., a promoter responsive to inflammation and, preferably responsive to the presence of cytokines or chemokines or other cellular or biological molecules indicative of the onset or presence of a disease or condition. Further examples of suitable promoters are provided in Table 1 below. Table 1
  • the regulated expression system of the present invention comprises one or more vectors comprising at least one expression cassette having a CMV promoter sequence that is operably linked to a nucleic acid sequence encoding a molecule of the present invention that is a fusion or chimeric protein, and the promoter drives the expression of the encoded molecule (e.g., TM, RM, AM, or IM).
  • a suitable promoter would be one that would provide a durable level of expression of the encoded molecule in the cells of a subject.
  • a nucleic acid vector encodes a molecule of the present invention (e.g., TM, RM, AM, or IM) that is operably linked to a promoter that provides durable expression in the cells of a subject, and the nucleic acid vector is administered to the cells of the subject via electroporation, such that the molecule is expressed in the cells, preferably, at the site of administration.
  • the encoded molecule is a TM.
  • the regulated expression system of the present invention comprises one or more vectors comprising a first expression cassette having a promoter sequence comprising at least one GAL-4 DBS, operably linked to a nucleic acid sequence encoding a TM of the present invention.
  • the promoter sequence comprises multimers of a GAL-4 DBS, e.g., 3-18 GAL-4 DBS.
  • the expression cassettes of the present invention can be suitably modified to comprise cloning sites for the insertion of a desired nucleic sequence.
  • the expression cassettes of the present invention comprise at least one cloning site and, more preferably a multiple cloning site (MCS), for the insertion of a nucleic acid sequence encoding a molecule of the present invention, e.g., a TM, RM, AM, or IM.
  • MCS multiple cloning site
  • cloning site refers to an enzyme site or other site in a nucleic acid wherein a nucleic acid sequence can be inserted, operably linked, or otherwise attached using conventional methods known in the art e.g., such that the sequence functions for its intended purpose.
  • a first expression cassette of the present invention comprises an MCS for insertion of a first nucleic acid sequence encoding a TM, an inducible promoter comprising at least one DBS (e.g., 3-18 GAL-4 DBS), 5' untranslated region (e.g., UT12, SEQ ID NO: 2), an intron (e.g., IVS8, SEQ ID NO: 3), and hGH poly(A) site (e.g., SEQ ID NO: 6), such that when the first nucleic acid sequence is inserted at the MCS (e.g., SEQ ID NO: 4, or SEQ ID NO: 5), these functional sequences are operably linked to the first nucleic acid sequence.
  • DBS e.g., 3-18 GAL-4 DBS
  • 5' untranslated region e.g., UT12, SEQ ID NO: 2
  • an intron e.g., IVS8, SEQ ID NO: 3
  • hGH poly(A) site e.g.
  • a second expression cassette of the present invention comprises an MCS for insertion of a second nucleic acid sequence encoding a regulated RM and SV40 poly(A) site (e.g., SEQ ID NO: 8), such that when the second nucleic acid sequence is inserted at the MCS, these functional sequences are operably linked to the second nucleic acid sequence.
  • the first and second expression cassettes are present in a single vector.
  • the kits of the present invention comprise at least one of the expression systems of the present invention described herein and, more particularly, at least one of the pharmaceutical compositions, vectors, or molecules (e.g., TM, RM, AM, or IM) of the present invention.
  • compositions of the present invention include, for example, chemical compounds, proteins, and nucleic acids (e.g., DNA or RNA molecules), particularly, nucleic acids encoding a protein or RNA.
  • the chemical, nucleic acid, and protein compositions of the present invention can be isolated, constructed, and/or tested using conventional methods and assays, as described herein or in the art.
  • Protein or “amino acid molecule” as used herein refers to a peptide, full-length protein, or fragment or portion of a full-length protein.
  • a protein of the present invention can be a fused, chimeric, modified, isolated, synthetic, or recombinant amino acid molecule.
  • proteins suitable for use in the compositions and methods of the present invention include, but are not limited to, a wild-type, full-length protein (including a secreted form thereof), or an analog, derivative, or variant thereof having a biological or therapeutic activity.
  • protein variants of the present invention can be muteins i.e., comprising a mutation e.g., a single or multiple amino acid substitution, deletion, or addition such that the variant retains or has a biological or therapeutic activity.
  • Sequences encoding a protein may include, e.g., codon-optimized versions of wild-type protein sequences, or humanized sequences. Optimal codon usage in humans can be identified from codon usage frequencies for expressed human genes and may be determined by methods known in the art e.g., program "Human High.codN" from the Wisconsin Sequence Analysis Package, Version 8.1 , Genetics Computer Group, Madison, Wl. For example, codons that are most frequently used in highly expressed human genes may be optimal codons for expression in the cells of a human subject, and, thus, can be used as a basis for constructing a synthetic coding sequence.
  • Nucleic acid refers to a nucleic acid molecule, e.g., a DNA or RNA, or fused, chimeric, modified, isolated, synthetic, or recombinant form thereof.
  • nucleic acids suitable for use in the compositions and methods of the present invention include, but are not limited to, a wild-type, full-length DNA or RNA (e.g., mRNA) encoding a protein, or other nucleic acid molecule having a biological or therapeutic activity (e.g., shRNA, siRNA, ribozyme, antisense RNA or DNA, RNA or DNA oligonucleotide), or an analog, derivative, or variant thereof.
  • nucleic acid variants of the present invention can be muteins i.e., comprising a mutation, e.g., a single or multiple nucleic acid substitution, deletion, or addition such that the variant retains or has a biological or therapeutic activity.
  • modifications of the regulated, expression system of the present invention can be carried out and tested using conventional methods and assays, as described herein or in the art.
  • Modified refers to any reaction or manipulation resulting in a change or alteration of a reference nucleic acid, amino acid, or chemical molecule to arrive at a desired composition or molecule of the present invention (e.g., mutation of a wild-type protein or nucleic acid to arrive at a desired variant thereof having a specific biological and/or therapeutic activity; mutation of a protein or nucleic acid sequence to arrive at a desired humanized sequence; or mutation of a chemical compound to arrive at a desired chemical structure, and/or biological and/or therapeutic activity).
  • the functional domains or functional sequences of a molecule of the present invention can be modified to arrive at a desired composition or molecule of the present invention.
  • the regulated expression system of the present invention can be modified or optimized to achieve a particular specificity (e.g., specific to a particular tissue, condition, disease, or biomarker or other molecule), stringency, or amount of regulation, expression, and/or activity for use in the treatment of disease, as described herein.
  • a particular specificity e.g., specific to a particular tissue, condition, disease, or biomarker or other molecule
  • stringency e.g., specific to a particular tissue, condition, disease, or biomarker or other molecule
  • amount of regulation, expression, and/or activity for use in the treatment of disease as described herein.
  • the regulated, expression system of the present invention can be modified or optimized to achieve such objectives by isolating or constructing: 1 ) novel or variant AMs and IMs having a desired binding specificity for the LBD of an RM; 2) RMs having a novel or variant AD, LBD (e.g., that binds a novel or variant AM or IM), and/or DBD (e.g., that binds a novel or variant promoter sequence); 3) promoters having activity that is highly specific and responsive to the presence of a particular RM (e.g., that are specifically activated or inactivated in the presence of, e.g., by the binding of, a particular RM); 4) fully humanized sequences (e.g., modifying sequences encoding a GAL-4 DBD and GAL-4 DBS such that the sequences are fully humanized); 5) an expression cassette for expression of an RNA (e.g., shRNA, siRNA, ribozyme, or antisense RNA),
  • the regulated expression system of the present invention is modified such that the basal expression of a TM is significantly reduced in order to increase reliance on administration of an RM and, thereby, provide an increased margin of safety by virtue of extrinsically controlled TM expression rather than through dependence on the dose of plasmid administrated.
  • a promoter sequence operably linked to a nucleic acid coding sequence encoding a TM may be modified and optimized for the number of copies of a GAL-4 DBS, such that the responsiveness of the promoter (and resulting TM expression) can be modulated by the presence of (e.g., binding of) an RM having a GAL-4 DBD.
  • a minimal promoter can be constructed and modified using standard methods and operably linked to a TM to reduce the basal expression of the TM.
  • the TM is encoded by a nucleic acid sequence that when delivered to and/or is present in the cells of a subject, the TM is expressed at a low level.
  • the TM is encoded by a nucleic acid sequence that is operably linked to an inducible promoter sequence (e.g., SEQ ID NO: 1 ) having 6X GAL-4 DBS operably linked to a TATA box sequence.
  • an inducible promoter sequence e.g., SEQ ID NO: 1
  • 6X GAL-4 DBS operably linked to a TATA box sequence.
  • the sequence from -33 to -22, which contains the TATA box from the EIb region of Adenovirus type 2 (residues 1665-1677 of NCBI accession no. J01917) is suitable for use in such an embodiment of the present invention.
  • the promoter sequence comprises 6X GAL-4 DBS operably linked to an Ad EIb TATA box sequence and a CMV sequence that contains the putative initiator (inr) region of the CMV promoter (Macias et al., Journ. of Virol. 70(6):3628 (1996)), such that these functional sequences are operably linked to a nucleic acid encoding a TM.
  • the TM is encoded by a nucleic acid sequence that is operably linked to multiple copies of a GAL-4 DBS comprising a 17 nucleotide sequence ⁇ '-TGGAGTACTGTCCTCCG-S' or 5'-CGGAGTACTGTCCTCCG-3' (e.g., of the consensus GAL-4 DBS of SEQ ID NO: 49).
  • the TM is encoded by a nucleic acid sequence that is operably linked to 4 copies of a GAL-4 DBS each comprising the 17 nucleotide sequence ⁇ '-CGGAGTACTGTCCTCCG-S' separated by a 10 nucleotide spacer having the nucleotide sequence 5-AGTTTAAAAG-3' as in e.g., SEQ ID NO: 50.
  • the TM is encoded by a nucleic acid sequence that is operably linked to 6 copies of a GAL-4 DBS arranged in two groups with 3 copies each of a GAL-4 DBS, and wherein: 1 ) in each group (containing 3 copies of a GAL-4 DBS) the second copy of the GAL- 4 DBS comprises the 17 nucleotide sequence ⁇ '-TGGAGTACTGTCCTCCG-S' and the first and third copy of the GAL-4 DBS each comprise the 17 nucleotide sequence 5'- CGGAGTACTGTCCTCCG-3; 2) each copy within the group of 3 copies is separated by two nucleotides 5'-AG-3'; and 3) between the two groups of 3 copies there is a longer spacer sequence ⁇ '-AGTCGAGGGTCGAAG-S' (e.g., the sequence of SEQ ID NO: 51 comprising 6 copies of a GAL-4 DBS as described).
  • the regulated expression system of the present invention may be modified to comprise tissue- specific promoters.
  • tissue-specific promoters e.g., an actin promoter sequence.
  • Tissue-specific promoters e.g., muscle-specific promoters
  • tissue-specific promoters may provide the advantage of reduced expression in dendritic and other antigen- presenting cells, thus avoiding immune responses to the expressed TM (e.g., protein or nucleic acid).
  • the lag time between administration of the vector encoding the TM and the time in which an AM is imposed is for example, 5 days, 12 days, 20 days or 55 days.
  • the AM may be administered one or more times over a period of time of one day to more than one year.
  • the AM may be administered daily, every other day, three times per week, twice per week, weekly, biweekly, monthly, bimonthly, every three months, quarterly, semiannually, annually, or any combination thereof.
  • the regulated expression system is modified such that the specificity, selectivity, precise timing, and/or level of TM expression and/or activity is modulated in the presence of an RM.
  • the RM has a rapid clearance in a subject administered an RM of the present invention.
  • the RM is a protein and is modified such that it is activated in the presence of a specific or cognate ligand and, thereby, the presence of the activated RM regulates the expression and/or activity of a TM.
  • the specificity and stringency of activation of the RM can be optimized by mutation of the GAL-4 DBD of the RM to minimize any propensity to form dimers in the absence of an AM.
  • the RM can be modified by mutation of the GAL-4 domain by deleting or otherwise mutating the C-terminal portion of the GAL-4 DBD (e.g., 20 C-terminal residues) and, thereby, reducing the length of a coiled-coil structure that is predicted to contribute to GAL-4 homodimer formation.
  • the GAL-4 DNA Binding Domain (“GAL-4 DBD”) comprises a portion or fragment of amino acids 1-93 of the N-terminal DBD of GAL-4 (where e.g., the sequence of amino acids 2-93 is SEQ ID NO: 37 and amino acid 1 is a methionine).
  • the GAL-4 DBD comprises amino acids 2-93 of the N- terminal DBD of GAL-4 (e.g., comprising the amino acid sequence of SEQ ID NO: 38, or encoded by the nucleic acid sequence of SEQ ID NO: 37).
  • the GAL-4 DBD comprises amino acids 2-93 of the N-terminal DBD of GAL-4 and an operably linked N- terminal peptide sequence e.g.
  • the GAL-4 DBD comprises amino acids 2-74 of the N-terminal DNA binding domain of GAL-4 (e.g., comprising the amino acid sequence of SEQ ID NO: 48, or encoded by the nucleic acid sequence of SEQ ID NO: 47).
  • a suitable GAL-4 DBD has a modification in a nucleic acid sequence or amino acid sequence that results in a mutation of the GAL-4 DBD such that it retains the ability to bind to a canonical 17-mer binding site, CGGAAGACTCTCCTCCG, but has a reduced ability to form a helical tertiary structure needed for autodimerization.
  • mutations or deletions are made to the region spanning amino acids 75 to 93 and/or 54 to 74 of the GAL-4 DBD sequence.
  • a deletion is made of the amino acids 54 to 64 or 65 to 75 of the GAL-4 DBD sequence, such that autodimerization is minimized through the coiled-coil region of an RM comprising the mutated GAL-4 DBD.
  • the nucleic acid sequence of the RM is modified to encode a fusion or chimeric protein comprising one or more functional domains, e.g., a DNA binding domain (DBD), ligand-binding domain (LBD), and/or regulatory domain (RD) (e.g, an activation domain).
  • suitable functional domains for use in the fusion or chimeric proteins of the present invention include, but are not limited to the GAL-4 DBD, human progesterone receptor (hPR) LBD, and NFKappaB p65 AD.
  • Suitable regulatory domains for use in an RM of the present invention, include, but are not limited to, NFkappaBp65, VP-16, TAF- 1 , TAF-2, TAU-1 , TAU- 2, ORF-10, TEF-1 , and any other nucleic acid or amino acid sequences having a regulatory function (e.g., regulates the expression and/or activity of a molecule of the present invention) and, more particularly, a transcriptional regulatory function (see e.g., Pham et al. (1992) 6(7) :1043-50 MoI. Endocrinol.; Dahlman-Wright et al. (1994) Proc. Natl. Acad. Sci. U.S.A.
  • a regulatory function e.g., regulates the expression and/or activity of a molecule of the present invention
  • a transcriptional regulatory function see e.g., Pham et al. (1992) 6(7) :1043-50 MoI. Endocrinol
  • the preferred RD is a human transactivation domain (e.g., NFkappaB p65).
  • the RM particularly a functional domain of the RM (e.g., an RD), is humanized.
  • the LBD of an RM of the present invention is derived from an amino acid sequence correlating to a wild-typed LBD of a receptor in the steroid-receptor family, e.g., a progesterone receptor (PR) and more particularly, a human progesterone receptor (hPR).
  • the RM is a steroid receptor, and the amino acid sequence of the LBD of the steroid receptor (e.g., a PR, and more particularly an hPR) is mutated to result in a mutated steroid-receptor LBD (e.g., a mutated hPR LBD) that selectively binds to an AM that is an antiprogestin instead of progestin.
  • an RM that is a mutated steroid-receptor LBD can be selectively activated by an AM that is an antiprogestin, instead of a naturally-occurring progestin.
  • the antiprogestin binds to a natural PR, but acts as an antagonist.
  • the progestin binds to a wild-type PR and acts as an agonist, and does not bind to a truncated or mutated PR.
  • a mutated PR retains the ability to bind antiprogestins, but responds to them as agonists.
  • the antiprogestin when the antiprogestin binds to a mutated PR that is an RM, the mutated PR protein is activated and forms a dimer.
  • the dimer-antiprogestin complex then binds to the DBS of a promoter sequence and, thereby, induces transcription of a nucleic acid sequence encoding a TM, where the nucleic acid sequence is operably linked to the promoter.
  • the presence of the anti-progestin MFP (RU486), the chimeric RM binds to a 17-mer GAL-4 DBS operably linked to a nucleic acid sequence encoding a TM, and results in an efficient ligand-inducible transactivation of TM expression.
  • the modified steroid-hormone LBD of the RM may also be modified by deletion of carboxy terminal amino acids, preferably, from about 1 to 120 carboxy terminal amino acids. The extent of deletion desired can be obtained using standard molecular biological techniques to achieve both selectivity for the desired ligand and high inducibility when the ligand is administered.
  • the mutated steroid hormone receptor LBD is mutated by deletion of about 1 to about 60 carboxy terminal amino acids. In another embodiment 42 carboxy terminal amino acids are deleted. In yet another embodiment, having both high selectively and high inducibility, 19 carboxy terminal amino acids are deleted.
  • the nucleic acid sequence of an RM comprises a sequence encoding a truncated GAL-4 DBD, a mutated progesterone receptor having a C-terminal deletion of 19 amino acids, and a p65 transactivation domain (e.g., SEQ ID NO: 39).
  • the nucleic acid sequence of an RM comprises a sequence encoding a chimeric receptor having a mutated progesterone-receptor ligand-binding domain, a truncated GAL-4 DNA binding domain, and a VP16 or p65 transregulatory domain, where the p65 transregulatory domain is part of the activation domain of the human p65 protein; a component of the NFkappaB complex.
  • a DBD of an RM of the present invention is not limited to a modified GAL-4 DBD as described herein.
  • a suitable DBD is one that has been modified to remove sequences that are not essential for recognition of binding sites but may be predicted to contribute to autodimerization by virtue of their secondary structure.
  • DBDs that may be so modified and suitable, include e.g., the known DBD of a member of the steroid-receptor family (e.g., glucocorticoid receptor, progesterone receptor, retinoic acid receptor, thyroid receptor, androgen receptor, ecdysone receptor) or other cellular DNA- binding proteins such as the cAMP Response Element Binding protein (CREB) or zinc finger DNA binding proteins, such as SP1.
  • a member of the steroid-receptor family e.g., glucocorticoid receptor, progesterone receptor, retinoic acid receptor, thyroid receptor, androgen receptor, ecdysone receptor
  • CREB cAMP Response Element Binding protein
  • SP1 zinc finger DNA binding proteins
  • the steroid-receptor family of gene regulatory proteins is also suitable for the construction of an RM of the present invention.
  • Steroid receptors are ligand-activated transcription factors whose ligands can range from steroids to retinoids, fatty acids, vitamins, thyroid hormones, and other presently unidentified small molecules. These compounds bind to receptors and either up-regulate or down-regulate the expression of steroid-regulated genes. The compounds are reportedly cleared from the body by existing mechanisms and are usually non-toxic.
  • a ligand of a steroid receptor may be any compound or molecule that activates the steroid receptor e.g., by binding to, or otherwise interacting with, the LBD of the steroid receptor.
  • steroid-hormone receptor refers to steroid-hormone receptors in the superfamily of steroid receptors.
  • Representative examples of the steroid- hormone receptor family include, but are not limited to, the estrogen, progesterone, glucocorticoid, mineralocorticoid, androgen, thyroid hormone, retinoic acid, retinoid X, Vitamin D, COUP-TF, ecdysone, Nurr-1 and orphan receptors.
  • the receptors for hormones in the steroid/thyroid/retinoid supergene family for example, are transcription factors that bind to target sequences in the regulatory regions of hormone-sensitive genes to enhance or suppress their transcription. These receptors have evolutionarily conserved similarities in a series of discrete structural domains, including a ligand binding domain (LBD), a DNA binding domain (DBD), a dimerization domain, and one or more transactivation domain(s).
  • LBD ligand binding domain
  • DBD DNA binding domain
  • mutated steroid receptor is capable of preferentially binding to a non-natural or non-native ligand rather than binding to the wild-type or naturally-occurring hormone receptor ligand.
  • a mutated hormone receptor is generated by deletion of amino acids at the carboxy terminal end of a reference hormone receptor (e.g., a wild-type or naturally-occuring hormone receptor) e.g., a deletion of from about 1 to about 120 amino acids from the carboxy terminal end of the reference hormone receptor.
  • a mutated progesterone receptor of the present invention comprises a carboxy terminal deletion of from about 1 to about 60 amino acids of a reference progesterone receptor (e.g., a wild-type or naturally-occuring hormone receptor). In another embodiment, a mutated progesterone receptor comprises a carboxy terminal deletion of 19 amino acids of a reference progesterone receptor (e.g., a wild-type or naturally-occuring hormone receptor).
  • modified or mutated steroid-hormone receptors for modification and/or use in the compositions and methods of the present invention are described in, for example: (1 ) "Adenoviral Vector-Mediated Delivery of Modified Steroid Hormone Receptors and Related Products and Methods" International Patent Publication No. WO0031286 (PCTAJS99/26802); (2) “Modified Glucocorticoid Receptors, Glucocorticoid Receptor/ Progesterone Receptor Hybrids" International Patent Publication No. WO9818925 (PCTAJS97/19607); (3) “Modified Steroid Hormones for Gene Therapy and Methods for Their Use” International Patent Publication No.
  • a mutated steroid-hormone receptor LBD may be selected based on the ability of an antagonist of a wild-type steroid-hormone receptor to activate the mutant receptor even in the presence of an agonist for the wild-type receptor.
  • progesterone is the normal ligand for the progesterone receptor and functions as a strong agonist for the receptor.
  • the anti-progestin, MFP (RU486), is a non-natural or non-native ligand for the progesterone receptor. MFP is considered an "anti-progestin" because, although it is able to exert an agonist effect on the wild-type progesterone receptor, MFP inhibits the agonistic effects of progesterone.
  • MFP may be considered an "antagonist" for the wild-type progesterone receptor when in the presence of the normal agonist, i.e., when both MFP and progesterone are together in the presence of the wild-type progesterone receptor.
  • the mutated progesterone steroid-hormone receptor is not activated by progesterone (agonist for the wild type receptor) but is activated in the presence of MFP ("antagonist" for the wild type receptor).
  • progesterone does not block the activation of the mutated steroid-hormone receptor by MFP.
  • the mutated receptor may be characterized as activated when bound to an antagonist (e.g, MFP) for the wild-type receptor even in the presence of an agonist (e.g., progesterone) for the wild-type progesterone receptor.
  • the mutated steroid receptor activates the transcription of a desired TM in the presence of an antagonist for a wild-type steroid hormone receptor.
  • the antagonist is a non-naturally-occuring or non-wild-type ligand that acts as an antagonist of a wild-type steroid receptor (e.g., a wild-type steroid hormone receptor).
  • an antagonist of a wild-type steroid hormone receptor is a molecule that interacts with or binds to the wild-type steroid hormone receptor and blocks the activity of an agonist of the receptor.
  • an agonist of a wild-type steroid hormone receptor is a molecule that interacts with the wild-type steroid hormone receptor to regulate the expression and/or activity of a TM in the cells of a subject.
  • agonists include, but are not limited to, progesterone or progestin for the progesterone receptor 10, where progesterone binds to a wild-type progesterone receptor to activate the transcription of progesterone-regulated genes.
  • suitable progesterone receptor agonists are chemical compounds that mimic progesterone.
  • MFP Mifepristone
  • RU486 is a non-natural ligand that also binds to the wild-type progesterone receptor and competes with progesterone for binding.
  • MFP exerts an antagonistic effect on the receptor by blocking the activation of the receptor by progesterone.
  • the therapeutic dose of MFP for use as an abortifacient in humans is 600 mg, or approximately 10 mg/kg.
  • the present MFP dose response studies in animals have shown that the EC 50 for induction of transgene expression using the pBRES regulated expression system in vivo is approximately 0.50 mg/kg, two orders of magnitude below the abortifacient dose. This value has been reported by other laboratories using the two plasmid GeneSwitch (Abruzzesse et al. (2002) Methods Mo. Med. 69:109-122).
  • a large safety database exists demonstrating the safety of low doses of MFP administered over several months in humans (Sitruk-Ware and Spitz. (2003) Contraception 68:409-420).
  • MFP at low doses does have a pharmacological effect as a contraceptive in women.
  • this effect would not pose a problem with its use as an AM of the present invention in female MS patients because women patients receiving IFN- ⁇ protein therapy are advised not to bear children during IFN- ⁇ treatment.
  • the progesterone receptor may be modified, e.g. in the LBD of the progesterone receptor, such that it only binds to MFP and not to progesterone.
  • mutation of the LBD of the progesterone receptor may be such that binding of the MFP activates the progesterone receptor.
  • the mutated PR LBD, or more generally any other mutated steroid receptor LBD is fused with a particular DBD (e.g., the GAL-4 DBD), such that binding of MFP selectively activates the RM to transactivate TM expression and/or activity that is driven by a promoter recognized by the DBD of the PR.
  • the mutated steroid receptor of the present invention is not activated in the presence of agonists for the wild-type steroid receptor, but instead the mutated steroid receptor is activated in the presence of non-natural ligands.
  • non-natural ligands or “non-native ligands” refers to compounds which are non-wild-type or not naturally-occurring ligands that bind to the ligand binding domain of a receptor.
  • non-natural ligands are Selective Progesterone Receptor Modulators (SPRMs) or mesoprogestins (see e.g., Chwalisz et al. (2002) Ann NY Acad Sci 955:373-388; Elger et al. (2000) Steroids 65(10-11 )713-723; Chwalisz et al. (2004) Semin Reprod Med 22(2):113-119; DeManno et al.
  • SPRMs Selective Progesterone Receptor Modulators
  • mesoprogestins see e.g., Chwalisz et al. (2002) Ann NY Acad Sci 955:373-388; Elger et al. (2000) Steroids 65(10-11
  • non-natural ligands and non-native ligands are anti-hormones that may include the following: 11-(4-dimethylaminophenyl)-17-hydroxy-17c- propynyl-4, 9- estradiene- 3-one (RU38486 or Mifepristone); 11-(4-dimethylaminophenyl)-17o-hydroxy-17-(3- hydroxypropyl)-13-methyl-4, 9-gonadiene- 3-one (ZK98299 or Onapristone); 11-(4- acetylphenyl)-17-hydroxy-17c-(1- propynyl)- 4,9-estradiene-3-one (ZK112993); 11-(4- dimethylaminophenyl)- 17-hydroxy-17(z-(3- hydroxy- 1 (Z)-propenyl-estra-4,9-diene-3-one (ZK98734); (7, 11 , 17)-11-(4- dimethylaminophen
  • ligands are non-steroidal progesterone receptor-binding ligands e.g., that act as an inducer of an RM of the present invention.
  • a protein domain e.g., AD, LBD, DBD
  • functional nucleic acid sequence e.g., sequence encoding a protein, RNA, promoter, splice site, intron, DNA binding site, or poly(A) site
  • a TM, RM, AM, IM or nucleic acid encoding a TM, RM, AM, or IM
  • TM, RM, AM, IM or nucleic acid encoding a TM, RM, AM, or IM
  • a molecule of the present invention e.g., a TM, RM, AM, IM, or nucleic acid encoding a TM, RM, AM, or IM
  • a molecule of the present invention can be modified such that the expression and/or activity of the molecule is transient or constitutive, and/or is regulated by the presence of a particular condition, disease, biomarker or other molecule, or is self-regulated.
  • the primary, secondary, or tertiary structure of a nucleic acid or amino acid molecule, or chemical compound, of the present invention can be modified to achieve a particular stringency, specificity, or amount of binding, activation, inactivation, or conformation (e.g., to form a homo- or hetero-dimer or other multimer, or bind a specific or cognate ligand or site).
  • a transcribed portion of an expression cassette of the present invention can be modified to include post-transcriptional elements (e.g., a UTR, splice site, intron, and/or poly(A) signal) that optimize or improve the specificity, level and fidelity of expression and/or activity of an operably linked, encoded, and expressed molecule (e.g., a TM, RM, AM, or IM).
  • post-transcriptional elements e.g., a UTR, splice site, intron, and/or poly(A) signal
  • an operably linked, encoded, and expressed molecule e.g., a TM, RM, AM, or IM
  • the promoter sequence of an operably linked sequence encoding a molecule of the present invention can be modified such that the expression of the encoded molecule is, e.g., transient or constitutive, inducible or repressible, and/or modulated or otherwise regulated by the presence of a specific condition or molecule.
  • an expression cassette of the present invention can be modified to optimize for the expression and/or activity of an encoded molecule (e.g., TM, RM, AM, or IM).
  • an encoded molecule e.g., TM, RM, AM, or IM
  • intron sequences can be modified to optimize for the highly efficient and accurate splicing of RNA transcripts from such sequences. Thereby, cryptic splicing can be minimized and expression can be maximized of the desired molecule (e.g., TM, RM, AM, or IM) encoded by a nucleic acid of the present invention.
  • suitable synthetic introns for use in the compositions of the present invention include, but are not limited to, consensus sequences for a 5' splice site, 3' splice site, and/or branch point.
  • the 5' splice site is reported to pair with U1 snRNA.
  • a suitable 5' splice site consensus sequence is one that is optimized to minimize the free energy of helix formation between U1 RNA and the synthetic 5' splice site e.g., 5' splice site sequence comprising 5'-CAGGUAAGU-3'.
  • the branch point (BP) sequence except for a single bulged A residue, is reported to pair with U2 snRNA.
  • BP branch point
  • a branch point sequence can be optimized to minimize the free energy of helix formation between U2 RNA and the sequence.
  • the BP is typically located 18-38 nts upstream of the 3' splice site.
  • the BP sequence of the synthetic intron is located 24 nts upstream from the 3' splice site and is e.g., BP sequence comprising 5'
  • the polypyrimidine tract of the consensus sequence for 3' splice sites can be optimized for 3' splice site function. For example, it has been reported that at least 5 consecutive uracil residues are optimal for 3' splice site function, and thus, in some embodiments the polypyrimidine tract of a synthetic intron of the present invention, has 7 consecutive uracil residues.
  • a synthetic intron is IVS8 (e.g., SEQ ID NO: 3) and comprises restriction enzyme sites, Bbsl and Earl (located within the synthetic intron), and Pstl and Nhel.
  • the restriction enzyme Bbsl may be used to cleave the DNA near the 5' splice site, and Earl may be used to cleave the DNA precisely at the 3' splice site.
  • a synthetic intron may be inserted at multiple locations of a nucleic acid sequence encoding a molecule of the present invention.
  • a nucleic acid sequence encoding a molecule of the present invention is modified to comprise multiple introns.
  • an expression cassette of the present invention in addition to the synthetic intron, IVS8 (e.g., SEQ ID NO: 3) an expression cassette of the present invention is modified to comprise a nucleic acid sequence encoding a CMV 5' UTR termed UT12, an expression control element (e.g., SEQ ID NO: 2).
  • an expression cassette of the present invention is modified to comprise a nucleic acid sequence encoding a SV40 poly(A) signal (e.g., SEQ ID NO: 8).
  • an expression cassette of the present invention is modified to comprise a nucleic acid sequence encoding a human growth hormone ("hGH") poly (A) signal (e.g., SEQ ID NO: 6).
  • hGH human growth hormone
  • an encoded molecule e.g., TM, RM, AM, IM
  • a nucleic acid sequence of the present invention e.g., encoded by a nucleic acid sequence of an expression cassette, or vector
  • intron refers to a sequence encoded in a DNA sequence that is transcribed into an RNA molecule by RNA polymerase but is removed by splicing to form the mature messenger RNA.
  • a "synthetic intron” refers to a sequence that is not initially replicated from a naturally-occurring intron sequence and generally will not have a naturally- occurring sequence, but will be removed from an RNA transcript during normal post- transcriptional processing. Such synthetic introns can be designed to have a variety of different characteristics, in particular such introns can be designed to have a desired strength of splice site and a desired length.
  • both the molecular switch expression cassette and the therapeutic gene expression cassette include a synthetic intron.
  • the synthetic intron includes consensus sequences for the 5' splice site, 3' splice site, and branch point. When incorporated into eukaryotic vectors designed to express therapeutic genes, the synthetic intron will direct the splicing of RNA transcripts in a highly efficient and accurate manner, thereby minimizing cryptic splicing and maximizing production of the desired gene product.
  • a functional sequence encoding a domain of a protein can be modified to optimize for the activity of the protein.
  • a truncation mutant can be designed such that there is lower dimerization potential while retaining sequence-specific DNA binding activity of a protein having a GAL-4 DBD.
  • the GAL-4 DBD is reported to bind as a dimer to the palindromic 17-mer GAL-4 DBS (CGGAAGACTCTCCTCCG) and such dimer binding reportedly results in the activation of an inducible promoter having the GAL-4 DBS.
  • nucleic acid sequence encoding a protein having a GAL-4 DBD can be modified such that the tertiary structure of the GAL-4 DBD is optimized to reduce any unregulated (e.g., AM-independent) or undesired dimerization, resulting in activation of an inducible promoter.
  • unregulated e.g., AM-independent
  • undesired dimerization e.g., AM-independent
  • the cysteine (C) may be involved in chelating zinc; the coiled-coil structures that form the dimerization elements comprise residues 54-74 and 86-93; the generally hydrophobic amino acids are reportedly at the first and fourth positions of each heptad repeat sequence; residue Ser 47 and Arg 51 are reported to form a hydrogen bond between the protein chains forming the dimer; and residues 8-40 reportedly form the Zn binding domain or the DNA recognition unit.
  • This DNA recognition unit has two alpha helical domains that form a compact globular structure and in the presence of Zn resulting in a structure that reportedly is a binudear metal ion cluster rather than a zinc finger, i.e., the cysteine-rich amino-acid sequence (CysXa-Xaa2-Cys14-Xaaa-CysZ1-Xaa6-CysZS-Xaa2- Cys31-Xaaa-Cys38) binds two Zn(II) ions (Pan and Coleman (1990) PNAS 87: 2077-81 ).
  • the Zn cluster may be responsible for making contact with the major groove of the 3 bp at extreme ends of the 17-met binding site; and a proline at 26 (cis proline) reportedly forms the loop that joins the two alpha-helical domains of the zinc cluster domain and is also critical for this function. Further, residues 41-49 reportedly join the DNA recognition unit and the dimerization elements, residues 54-74 and 86-93.
  • residues 47-51 of the dimer can also interact with phosphates of the DNA target.
  • Residues 50-64 may be involved in weak dimerization.
  • the dimers consist of a short coiled-coil that forms an amphipathic alpha-helix and wherein two alpha-helices are packed into a parallel coiled-coil similar to a leucine zipper.
  • Residues 65-93 may form a strong dimerization domain.
  • residues 65-71 is a continuation of the coiled-coil structure for one heptad repeat.
  • Residues 72-78 contain a proline and therefore disrupt the amphipathic helix.
  • Residues 79- 99 contain three more potentially alpha-helical heptad sequences (Marmorstein et al (1992) Nature 356: 408-414).
  • the Kd for binding of GAL-4 residues 1-100 is reported to be 3 nM (Reece and Ptashne (1993) Science 261 : 909-911 ).
  • the GAL-4 regions are modified to optimize for the elimination or reduction of any basal expression and retention of sequence-specific DNA binding. More particularly, in some embodiments, the length of the region that contains the interacting coiled-coil sequences of the GAL-4 DBD (e.g., residues 54-74 and residues 86-93) can be shortened by deletion e.g., by deleting amino acid sequence 54-64, 65-74, 54-74, or 86-93. Also, GAL-4 mutants with only one coiled-coil region can be constructed by deleting one of the coiled-coil regions.
  • mutant or artificial sequences may be inserted into the GAL-4 domain using unique restriction sites positioned at, e.g., the junctions of each of the alpha-helical heptad sequences.
  • modified versions of the GAL-4 domain can be produced that have progressively reduced alpha-helical heptad sequences.
  • the native GAL-4 sequence is modified to remove the N- terminal methionine and additional amino acids are added to the N-terminal end of the sequence.
  • the modifications to the N-terminal amino acids of the native GAL-4 sequence are not of consequence as long as they do not affect the tertiary structure of residues 8-40 of the Zn binding domain.
  • the specific binding of a small molecule AM, e.g. MFP, to a mutated hPR LBD of a protein having a GAL-4 DBD e.g., an RM
  • a mutated hPR LBD of a protein having a GAL-4 DBD e.g., an RM
  • an expression cassette of the present invention comprises a nucleic acid sequence encoding residues 2-93 of the GAL-4 DBD sequence of SEQ ID NO: 37.
  • the DNA recognition sequence of the GAL-4 DBD comprises residues 9-40 of the GAL-4 DBD sequence of SEQ ID NO: 37.
  • the GAL-4 domain is truncated by deletion of 19 amino acids at the C- terminal portion of the GAL-4 DBD and comprises residues 75-93 of the GAL-4 DBD sequence of SEQ ID NO: 37.
  • an RM of the present invention is a chimeric protein comprising a mutated progesterone receptor comprising residues 2-74 of the GAL-4 DBD sequence of SEQ ID NO: 37 and a mutated progesterone receptor LBD that is specifically activated in the presence of an AM. Further, in the absence of the AM there is little or no RM activation and resulting induction or activation of transcription of a nucleic acid sequence operably linked to a promoter having a GAL-4 DBS.
  • nucleic acids encoding variants of a native molecule are also suitable for use in the compositions and methods of the present invention.
  • a variant of IFN- ⁇ e.g., IFN- ⁇ 1 b
  • the IFN- ⁇ variant is a variant of a native human IFN- ⁇ .
  • Variants of native human IFN- ⁇ which may be naturally-occurring (e.g., allelic variants that occur at the IFN- ⁇ locus) or recombinantly or synthetically produced, have amino acid sequences that are similar to, or substantially similar to a mature native IFN- ⁇ sequence.
  • Nucleic acids encoding a native human IFN- ⁇ e.g., comprising the amino acid sequence of SEQ ID NO: 13
  • IFN- ⁇ 1a e.g., SEQ ID NO: 14
  • nucleic acids encoding a human IFN- ⁇ variant are suitable for use in the compositions and methods of the present invention e.g., IFN- ⁇ 1 b (see e.g., US Patent Ser. No. s 4,588,585, 4,737,462, and 4,959,314).
  • Variants also encompass nucleic acids encoding fragments or truncated forms of a native molecule (e.g., protein or nucleic acid) that retain a biological or therapeutic activity.
  • nucleic acids encoding these biologically active fragments or truncated forms of a native protein may be glycosylated or not glycosylated.
  • suitable protein or nucleic acid variants for use in the compositions and methods of the present invention can be variants of a native or wild-type protein or nucleic acid, respectively, of any mammalian species including, but not limited to, avian, canine, bovine, porcine, equine, and human.
  • IFN- ⁇ variants encompassed by the present invention e.g., encoded by a nucleic acid , e.g., Nagata ef a/. (1980) Nature 284:316-320; Goeddel ef a/. (1980) Nature 287:411-416; Yelverton et al. (1981 ) Nucleic Acids Res.
  • Changes or modifications of expressed proteins and nucleic acids (e.g., RNA) of the present invention can be introduced by mutation into the nucleotide sequences encoding them, thereby leading to changes in the amino acid sequence of the expressed protein or nucleic acid sequence without altering the biological or therapeutic activity of the expressed molecule.
  • an isolated nucleic acid molecule encoding a variant protein having a sequence that differs from the amino acid sequence for a reference or starting protein can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence (for IFN- ⁇ variants, see, e.g., U.S. Pat. No. 5,588,585, U.S. Pat. No.
  • nucleic acid sequences encoding a protein can be modified to encode conservative amino acid substitutions at one or more predicted, preferably nonessential amino acid residues.
  • a "nonessential" amino acid residue is a residue that can be altered from a reference sequence of a protein without altering its biological or therapeutic activity, whereas an "essential” amino acid residue is required for such activity.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • such substitutions are not made for conserved amino acid residues, or for amino acid residues residing within a conserved motif.
  • nucleotide sequences of a variant molecule can be made by introducing mutations randomly along all or part of the coding sequence of a reference molecule, such as by saturation mutagenesis, and the resultant mutants can be screened for biological or therapeutic activity.
  • the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques described herein or known in the art.
  • biologically or therapeutically active protein variants have at least 80 %, more preferably about 90 % to about 95 % or more, and most preferably about 96 % to about 99 % or more amino acid sequence identity to the amino acid sequence of a reference protein, which serves as the basis for comparison or reference.
  • sequence identity is the same amino acid residues that are found within a variant protein and a protein molecule that serves as a reference when a specified, contiguous segment of the amino acid sequence of the variant is aligned and compared to the amino acid sequence of the reference molecule.
  • the contiguous segment of the amino acid sequence of the variant may have additional amino acid residues or deleted amino acid residues with respect to the amino acid sequence of the reference molecule.
  • the contiguous segment used for comparison to the reference amino acid sequence will comprise at least 20 contiguous amino acid residues. Corrections for increased sequence identity associated with inclusion of gaps in the amino acid sequence of the variant can be made by assigning gap penalties. Methods of sequence alignment are well known in the art.
  • the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm utilized for the comparison of sequences is e.g., the algorithm of Myers and Miller (1988) Comput. Appl. Biosci. 4:11-7. Such an algorithm is utilized in the ALIGN program (version 2.0), which is part of the GCG alignment software package. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.
  • Another preferred, non- limiting example of a mathematical algorithm for use in comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
  • gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • PSI-BLAST can be used to perform an integrated search that detects distant relationships between molecules (see e.g., Altschul et al. (1997) supra.).
  • the default parameters can be used (see e.g., www.ncbi.nlm.nih.gov).
  • ALIGN program Dayhoff (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3, National Biomedical Research Foundation, Washington, D. C.
  • programs in the Wisconsin Sequence Analysis Package, Version 8 available from Genetics Computer Group, Madison, Wis.
  • percent sequence identity may be adjusted upwards to account for the similarity in conservatively substituted amino acids. Such adjustments are well known in the art (see, e.g., Myers and Miller (1988) Comput. Appl. Biosci. 4:11-17).
  • the protein can be covalently linked with, e.g., polyethylene glycol (PEG) or albumin.
  • PEG polyethylene glycol
  • albumin covalent hybrid molecules
  • Methods for creating PEG-IFN adducts involve chemical modification of monomethoxypolyethylene glycol to create an activated compound that will react with a protein of the present invention.
  • Methods for making and using PEG-linked proteins are reported, e.g., in Delgado ef a/. (1992) Crit. Rev. Ther. Drug. Carrier Syst. 9:249- 304 (and as described herein in the Background).
  • Methods for creating albumin fusion proteins involve fusion of the coding sequences for the protein of interest and albumin and are reported, e.g., in U.S. Pat. No. 5,876,969.
  • Biologically or therapeutically active protein or nucleic acid variants encompassed by the invention preferably retain or have a biological or therapeutic activity.
  • the variant retains at least about 25 %, about 50 %, about 75 %, about 85 %, about 90 %, about 95 %, about 98 %, about 99 % or more of the biologically or therapeutic activity of the reference molecule (e.g., protein or nucleic acid).
  • Variants whose activity is increased in comparison with the activity of the reference molecule e.g., protein or nucleic acid
  • the biological or therapeutic activity of variants can be measured by any method known in the art (see e.g., assays described in Fellous ef a/.
  • the nucleic acid, protein and chemical compositions of the present invention can be produced or synthesized using methods known in the art.
  • proteins can be produced by culturing a host cell transformed with an expression vector comprising a nucleotide sequence that encodes a protein or nucleic acid of the present invention.
  • the host cell is one that can transcribe the nucleotide sequence and produce the desired protein or nucleic acid, and can be prokaryotic (see, e.g., E. coli) or eukaryotic (e.g., a yeast, insect, or mammalian cell).
  • IFN- ⁇ examples include suitable expression vectors, are provided in, e.g., Mantei et al. (1982) Nature 297:128; Ohno ef a/. (1982) Nucleic Acids Res. 10:967; Smith ef a/. (1983) MoI. Cell. Biol. 3:2156, and U.S. Pat. No. 4,462,940, 5,702,699, and 5,814,485; U.S. Pat. No. 5,795,779).
  • genes have been cloned using recombinant DNA (“rDNA”) technology and can be produced and tested in e.g., animal or plant cells, or transgenic animals (see e.g., Nagola et al. (1980) Nature 284:316; Goeddel et al. (1980) Nature 287:411 ; Yelverton et al. (1981 ) Nuc. Acid Res. 9:731 ; Streuli et al. (1981 ) Proc. Natl. Acad. Sci. U.S.A. 78:2848).
  • rDNA recombinant DNA
  • Proteins may also be produced with rDNA technology, e.g., by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded cDNA using the mRNA as a template, introducing the cDNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the microorganisms, and extracting the protein therefrom (see, e.g., European Patent Application No.s 28033
  • proteins can be synthesized chemically and tested, by any of several techniques that are known to those skilled in the peptide art (see e.g., Li ef a/. (1983) Proc. Natl. Acad. Sci. USA 80:2216-2220, Steward and Young (1984) Solid Phase Peptide
  • a protein of the present invention can also be chemically prepared e.g., by the method of simultaneous multiple peptide synthesis.
  • compositions of the present invention for the treatment of disease using accepted and appropriate animal models and methods known in the art.
  • gene delivery systems can be used to deliver cytokines in several animal autoimmune disease models, e.g., including experimental allergic encephalomyelitis (EAE), arthritis, lupus, and NOD diabetes models (see e.g., G. C. Tsokos and GT. Nepom (2000) Clin. Invest. 106: 181-83; G.J.
  • EAE is a model of central nervous system inflammation that ensues after immunization with certain CNS auto-antigens, for example brain derived proteolipid or myelin basic protein. Its course and clinical manifestations are similar to multiple sclerosis (MS) in humans and it has become an accepted model to study MS.
  • MS multiple sclerosis
  • Type I IFN's delivered as a protein (15- 20) and by a vector (see e.g., K. Triantaphyllopoulos et al. (1998) Gene Therapy 5: 253-63; J. L. Croxford et al. (1998) J. Immunol.
  • mice administered mIFN- ⁇ protein at the time of EAE disease induction, exhibit delayed progression to disability (as measured by clinical score), delayed onset of relapse, and a decrease in exacerbation frequency compared to normal mice (see e.g., M. Yu et al. (1996) J. Immunol. 64: 91-100). This result closely resembles the human results with IFN- ⁇ treatment.
  • Plasmid based vectors were used by Triantaphyllopoulos et al. in a gene therapy-based approach to deliver IFN- ⁇ to the CNS under the control of a neuron specific promoter (see e.g., K.
  • mIFN- ⁇ mIFN- ⁇ expression vectors were constructed, assays developed to measure IFN- ⁇ directly in serum, and biomarkers were identified that correlate with IFN- ⁇ expression in vivo. Further, pharmacokinetic studies were performed in normal mice comparing gene-based delivery of IFN- ⁇ with bolus protein delivery and a superior pharmacokinetic profile was demonstrated using intramuscular injection of non-viral plasmid DNA or an adeno-associated viral (AAV) vector encoding IFN- ⁇ . In addition, long term, stable expression of hlFN- ⁇ was observed for nearly 6 months with an AAV-1 vector expressing hlFN- ⁇ .
  • AAV adeno-associated viral
  • plasmid DNA encoding mIFN- ⁇ was shown to be efficacious in a murine model of experimental allergic encephalomyelitis (EAE), and equally as effective as an every-other-day injection of mIFN- ⁇ protein.
  • EAE allergic encephalomyelitis
  • examples of the regulated expression system of the present invention were constructed and tested.
  • regulated expression of IFN- ⁇ was demonstrated in normal mice using a regulated expression system of the present invention, where a TM and RM are contained in a single plasmid vector.
  • the delivery vector is a single plasmid vector comprising a first and second expression cassette encoding a TM (e.g., IFN- ⁇ ) and RM, respectively which provides persistent (e.g., greater than 3 months) and renewable expression through oral administration of an AM e.g., a small molecule inducer (e.g., MFP) and, further, the vector is capable of repeat administration by intramuscular injection.
  • TM e.g., IFN- ⁇
  • RM e.g., a small molecule inducer
  • EXAMPLE 1 Construction of Vectors for Use in IFN- ⁇ or GMCSF Gene Therapy
  • Plasmid Vectors The murine IFN- ⁇ (mIFN- ⁇ ) gene from the bacterial expression vector pbSER189 was PCR amplified, with immunoglobulin kappa (IgK) (for protein purification) or mIFN- ⁇ (for gene therapy) signal sequence added on the 5' primer.
  • the PCR products were inserted downstream of the cytomegalovirus (CMV) promoter in the expression vectors pCEP4/WPRE, to generate pGER90 ( Figure 2A) for recombinant protein expression and purification, and pgWiz, to generate pGER101 ( Figure 2B) for gene therapy.
  • CMV cytomegalovirus
  • the human IFN- ⁇ gene from the bacterial expression vector pbSER178 was PCR amplified by the same procedure as the mIFN- ⁇ gene (except with the hlFN signal sequence for the gene therapy vector) and inserted into pCEP4/WPRE to generate pGER123 ( Figure 2C) for recombinant protein expression and purification, and pgWiz to generate pGER125 ( Figure 2D) for gene therapy.
  • AAV-1 Vectors An AAV-1-hlFN- ⁇ shuttle plasmid encoding hlFN- ⁇ was constructed by inserting the blunted Hincll/Notl fragment of pGWIZ/h IFN- ⁇ encoding hlFN- ⁇ into the blunted Agel-Sall site of the AAV-1 vector, pTReGFP. The resulting shuttle plasmid was named pGT62 (SEQ ID NO: 44) and used to produce AAV-1 virus encoding hlFN- ⁇ .
  • Two batches of the AAV-1 virus were prepared as described herein using standard methods and used in pharmacokinetic studies ( Figure 4). The expression levels of h IFN- ⁇ in these two viral batches were validated by ELISA.
  • AAV-1-GMCSF shuttle plasmids pGT714 and pGT713 ( Figure 30B), encoding mGMCSF or hGMCSF, were constructed by inserting a fragment encoding mGMCSF or hGMCSF into the vector pGENE/V5HisA (Invitrogen).
  • the resulting vectors were named pGT723-GENE/hGMCSF and pGT724-GENE/mGMCSF.
  • a fragment encoding mGM-CSF was then excised from pGT724-GENE/mGMCSF by digesting the vector with Kpnl-Xbal.
  • a fragment encoding hGM-CSF was excised from pGT723-GENE/hGMCSF by digesting the vector with Kpnl-Xbal.
  • the vector pZac2.1 was digested with Kpnl-Xbal and treated with calf intestinal phosphatase (CIP) and then the excised fragment encoding either mGMCSF or hGMCSF was inserted into pZac2.1 at the Kpnl-Xbal site.
  • CIP calf intestinal phosphatase
  • the resulting shuttle plasmids were named pGT713 (pZac2.1-CMV- hGMCSF) and pGT714 (pZac2.1-CMV-mGMCSF) ( Figure 30B).
  • LOD limit of detection
  • the hlFN- ⁇ 1a serum level reached a maximum level at 2 hours post-injection and then decreased by approximately 10-fold by 6 hours.
  • the amount of hlFN- ⁇ 1a remaining in the serum at 6 hours was higher with the i.m. injection compared to the i.v. injection. With both the i.v. and i.m.
  • AAV-1-CMV hlFN- ⁇ 1a An AAV-1 vector was constructed to constitutively express human IFN- ⁇ (AAV-1-
  • Biomarkers for mIFN- ⁇ activity were identified in mice after injection of mIFN- ⁇ protein or mIFN- ⁇ encoded gene therapy vectors. Biomarkers can be used to follow human IFN- ⁇ activity in clinical samples from patients treated with Betaseron (IFN- ⁇ 1 b) (see e.g., Arnason, BG (1996) CHn Immunol lmmunopathol 81 : 1-11 ; Deisenhammer, F et al. (2000) Neurology 54: 2055-60; Knobler, RL et al.
  • MxA MxA
  • PBMCs peripheral blood mononuclear cells
  • RNA for the Mx1 assay was purified using the "RNAeasy" mini extraction kit from Qiagen. The RNA was stored in H 2 O at -80 0 C.
  • Mx1 RT-PCR was performed using TaqMan ® chemistry and analysis was done on the Applied Biosystems (ABI) PRISM ® 7700 Sequence Detection instrument.
  • the "One-step TaqMan ® RNA" kit from ABI was used for reverse transcription of the RNA and amplification of cDNA.
  • RNA was reverse transcribed for 30 minutes at 48 0 C and the amplification was done in 40 cycles with a denaturation step at 95 0 C for 30 seconds, and an annealing/elongation step at 60 0 C for 1 minute.
  • the samples were analyzed with an Mx1 -specific probe/primer combination.
  • Mx1 expression was normalized to GAPDH expression measured in parallel using a standard assay from ABI.
  • the assay was validated in vitro by examining Mx1 RNA induction in murine L929 cells treated with purified recombinant mIFN- ⁇ protein (Figure 5).
  • a dose dependent increase in the expression of Mx1 RNA was observed with an EC50 of approximately 50 pg/ml mIFN- ⁇ resulting in a 100-fold increase in the level of Mx1 RNA.
  • Mx1 RNA expression in the i.v. injected mice also peaked at approximately 2 hours post-injection and dropped rapidly thereafter.
  • Mx1 RNA is expressed constitutively in mouse PBMCs at a low level and can be strongly upregulated by mIFN- ⁇ treatment.
  • the upregulation is short term and rapidly drops from high expression levels to background within 12-24 hours.
  • the rapid kinetics correspond with the short half-life time reported for type I interferons in humans (see e.g., Salmon, P et al. (1996) J Interferon Cytokine Res 16: 759-64; Buchwalder, P-A et al.
  • IP-10 is known as a biological marker for IFN- ⁇ by virtue of the interferon responsive element (ISRE) in the promoter region (see e.g., Luster, AD et al. (1985) Nature 315: 672-76), it is has not previously been shown to be a specific biomarker for mIFN- ⁇ in mice.
  • ISRE interferon responsive element
  • Plasmid Delivery of mIFN- ⁇ Gene For plasmid delivery different doses of plasmid DNA encoding mIFN- ⁇ were injected i.m. into mice followed by electroporation of the injected muscle. Mx1 expression was measured from PBMCs isolated from each individual animal and expressed as the fold increase over background levels of the control group ( Figure 9). There was a strong up-regulation of Mx1 RNA (40- to 130-fold induction) in all four groups receiving mIFN- ⁇ plasmid DNA at day 2 post-injection. Mx1 expression in all four groups was significantly above background (p ⁇ 0.002). The Mx1 expression data showed that there is a dose response with 250 ⁇ g as the optimal DNA concentration.
  • AAV-1 Delivery of mIFN- ⁇ Gene C57BL/6 mice were injected with the DNA of pGT61 encoding mIFN- ⁇ , or with the virus produced from pGT61 encoding mIFN ⁇ , or the DNA of pGER75 encoding SEAP (see Materials and Methods, subsection G).
  • mice were bled at days 2, 10, 14 and 17 post-injection. Mice that received the the pGT61 DNA showed an approximately 15-fold induction of Mx1 RNA over background at day 2 (Figure 10). Mx1 expression continued to increase to greater than 100-fold over background by day 10. By day 17 Mx1 RNA expression level was about 180-fold above background. No increased Mx1 expression was observed in the control group that received the pGER75 DNA. The Mx1 RNA expression levels in the mice injected with the virus produced from pGT61 were about 5-fold higher on day 10, 14 and 17 than in mice that received the pGT61 DNA. This was supported by IFN- ⁇ RNA RT-PCR analysis performed on the injected muscles.
  • the plasma samples were also analyzed for IP-10 and JE ( Figures 7 and 8). The results obtained were very similar to that obtained for Mx1 RNA induction.
  • the mice that received the DNA by electroporation showed higher IP-10 plasma level compared to the mice that were injected with the AAV-1-mlFN- ⁇ expressed virus.
  • the IP-10 levels in the mice injected with AAV-1-mlFN- ⁇ showed a strong increase and averaged approximately 5- to 10- fold greater than plasmid m IFN- ⁇ injected mice.
  • mIFN- ⁇ biomarkers Three mIFN- ⁇ biomarkers were identified and validated to perform pharmacokinetic studies using murine IFN-B protein or gene delivery.
  • a highly sensitive quantitative RT-PCR assay was developed to measure the induction of Mx1 RNA isolated from PBMCs of mice administered mIFN- ⁇ .
  • Two murine chemokines, IP-10 and JE, were also identified as sensitive IFN- ⁇ biomarkers and commercial ELISA's allowed the means to rapidly quantitate and support the results obtained with the Mx1 TaqMan assay.
  • Two different types of gene delivery vectors were tested, plasmid DNA (plus electroporation) and AAV-1.
  • Administration of bolus mIFN- ⁇ protein either i.v. or i.m.
  • Biomarker levels rapidly dropped to background within 12 to 24 hours post-injection.
  • a dose response was observed for Mx1 , IP-10 and JE when mIFN- ⁇ was injected i.m.
  • the rapid drop in the biomarker levels directly reflects the rapid systemic clearance of IFN- ⁇ following bolus protein administration.
  • Gene-based delivery of mIFN- ⁇ using plasmid plus electroporation or an AAV-1 vector resulted in biomarker responses that were greater than those observed when mIFN- ⁇ protein was injected. With plasmid DNA a biomarker response was measured out to 49 days and was down to background levels at day 63.
  • the level of IFN- ⁇ expressed is equal to or greater than the levels achieved with protein delivery, as measured directly in the serum or as reflected by the induction of IFN- ⁇ biomarkers.
  • the duration of IFN- ⁇ expression from a single injection of an IFN- ⁇ vector is far longer (stable expression for weeks to months) compared to the transient kinetics observed with protein administration (hours).
  • EXAMPLE 5 Efficacy Studies using Gene-based Delivery of mIFN- ⁇ These studies demonstrate that gene-based delivery is efficacious in an animal model of MS.
  • the rodent EAE model is an accepted model of MS and there are several reports in which IFN- ⁇ has been shown to be active in these models (Yu, M et al. (1996) J lmm 64: 91-100).
  • gene-based delivery of IFN-B is efficacious in some of these models (see e.g., Triantaphyllopoulos, K et al., (1998) Gene Therapy 5: 253-63).
  • the gene encoding mIFN- ⁇ was cloned into a pCEP4 expression vector (Invitrogen).
  • the expression plasmid encoding mIFN- ⁇ was transiently transfected into 293E cells (Edge Biosystems) using X-tremeGene Ro-1539 Transfection Reagent (Roche).
  • Murine IFN- ⁇ protein was purified from the medium by ion-exchange chromatography and by hydrophobic-interaction chromatography. The product was sialyzed and concentrated against dilution buffer (50 mM sodium acetate, pH 5.5, 150 mM sodium chloride, and 5% polypropylene glycol) and sterile filtered.
  • dilution buffer 50 mM sodium acetate, pH 5.5, 150 mM sodium chloride, and 5% polypropylene glycol
  • mIFN- ⁇ purified mIFN- ⁇ was diluted to 100 ug/mL in dilution buffer. Immediately prior to injection of the animals, the mIFN- ⁇ stock solution was diluted to the desired concentration of mIFN- ⁇ . The vehicle control used in these studies was the dilution buffer minus mIFN- ⁇ .
  • mice received an intramuscular injection on day 2 of the study with either PBS, null plasmid DNA (pNull) with electroporation (EP), mIFN- ⁇ plasmid DNA (plus EP), or mIFN- ⁇ plasmid DNA formulated with a polymer formulation called "PINC" (Mumper, RJ et al (1998) J Controlled Release 52: 191-203).
  • PINC polymer formulation
  • the regulated expression systems of the present invention has advantages over known expression systems.
  • the system of the present invention solves several development and manufacturing issues by having in a single vector a first expression cassette encoding a therapeutic molecule of interest (TM) (e.g., an IFN- ⁇ transgene) and a second expression cassette encoding a regulator molecule (RM) that regulates the expression of the TM.
  • TM therapeutic molecule of interest
  • RM regulator molecule
  • the present inventors provide a new and improved regulated expression system.
  • the expression cassettes of the regulated, expression system of the present invention are present in a single plasmid vector called pBRES.
  • the pBRES single vector has a number of versatile features incorporated into its design, including multiple cloning sites (MCS) for the insertion of different transgenes as well as different promoters to drive expression of the regulatory protein.
  • MCS multiple cloning sites
  • the size of the pBRES expression cassettes is compatible with many different delivery vectors, including plasmid and AAV vectors.
  • the resulting plasmids have either the expression cassette encoding the murine or human IFN- ⁇ gene and the expression cassette encoding the RM gene in four different orientations relative to each other ( Figure 15A-B).
  • the resulting pBRES plasmids encoding the mIFN- ⁇ were designated as pGT23, pGT24, pGT25, and pGT26 ( Figure 15A), and the resulting pBRES plasmids encoding the hlFN- ⁇ were designated as pGT27, pGT28, pGT29, and pGT30 ( Figure 15B). See the Materials and Methods, subsection F for a complete description of the construction of these plasmids.
  • the plasmid vector pGT26 was used to test whether the expression of mIFN- ⁇ could be regulated in an off/on/off pulsatile manner through oral administration of the inducer, MFP.
  • Constitutive and inducible pBRES mIFN- ⁇ expression plasmids were injected with electroporation into the hind limb muscles of mice. Mice were treated with MFP for four consecutive days, beginning on day 7 after plasmid injection. Blood was collected at days 11 and 18 post-injection, PBMCs were isolated and Mx1 RNA levels were determined by RT- PCR. Plasma samples were also assayed for the chemokines IP-10 and JE.
  • FIG. 39a shows the validation of the murine active EAE model in SJL mice in which the animals were injected with PLP/pertussis on day 1 to initiate disease and then treated with mIFN- ⁇ protein (100,000 or 300,000 units) by SC injection every other day throughout the course of the 25 day study. Based upon the clinical scores, both concentrations of mIFN- ⁇ protein significantly reduced the severity of the disease compared to vehicle treated animals.
  • pBRES plasmid (either Null vector or IFN- ⁇ ) was administered by IM injection to the hind limbs of the animals on day -7 and slow release MFP pellets were implanted on day -3 of the study.
  • Fig. 39b shows the results of the study demonstrating that the pBRES mIFN- ⁇ plasmid treated animals displayed a significant delay in the onset of disease as well as a significant decrease in the overall severity of disease scores compared to the null vector control group.
  • mRNA analysis of the plasmid injected muscles confirmed highly elevated levels of mIFN- ⁇ RNA in the pBRES mIFN- ⁇ treated animals (data not shown).
  • mice Eight-week old female SJL mice (Jackson Labs, Bar Harbor, ME) were immunized on day 1 of the study with a 0.1 ml subcutaneous (SC) injection containing 150 ug Proteolipid Protein (PLP) 139-151 in Complete Freund's Adjuvant (CFA) supplemented with 200 ug M. tuberculosis H37Ra.
  • SC subcutaneous
  • PLP Proteolipid Protein
  • CFA Complete Freund's Adjuvant
  • mice received a 0.1 ml IP injection of pertussis toxin.
  • mice Two days after immunization, all mice received a second IP injection of pertussis toxin.
  • Mice were housed under standard conditions, with food and water available ad libitum and a schedule of 12 hours of light and 12 hours of darkness. The mice were handled according to protocols and experimental design approved by the Berlex Biosciences Institutional Animal Care and Use Committee.
  • the positive control used for this study was Predn
  • mice were given SC injections of purified recombinant mlFN- ⁇ protein every other day, starting on day 1 of the study (100,000 or 300,000 IU per animal per injection in 100 ul vehicle buffer).
  • Recombinant mIFN- ⁇ protein was purified according to Schaefer et al. ((2006) J. Interferon Cytokine Res., 26:449-454) and stored at at -8O 0 C in vehicle buffer (50 mM sodium acetate, pH 5.5, 150 mM sodium chloride, and 5% propylene glycol) prior to use.
  • vehicle buffer 50 mM sodium acetate, pH 5.5, 150 mM sodium chloride, and 5% propylene glycol
  • the specific activity of the purified protein was 2x10 8 international units (IU) per mg.
  • mice received an IM injection on day -7 of either pBRES null vector plasmid DNA with electroporation (EP), or pBRES mIFN- ⁇ vector plasmid DNA with EP.
  • EP electroporation
  • Each animal was injected bilaterally with PBS or a total of 200 ⁇ g plasmid DNA (100 ug per limb) in 120 ⁇ l PBS (20 ⁇ l into the tibialis muscle and 40 ⁇ l into the gastrocnemius muscle of each hind limb).
  • mice Immediately following injection the muscles were electroporated with a caliper electrode (8 pulses at 200 V/cm, 1 Hz, 20 msec/pulse) using a BTX ECM 830 EP instrument supplied by Genetronics (San Diego, CA). On day -3 the null vector and mFNb plasmid treated mice were each administered a single MFP pellet (2.5 mg/ 60 day release pellet, Innovative Research of America, Sarasota, FL) by SC injection.
  • a caliper electrode 8 pulses at 200 V/cm, 1 Hz, 20 msec/pulse
  • BTX ECM 830 EP instrument supplied by Genetronics (San Diego, CA).
  • the null vector and mFNb plasmid treated mice were each administered a single MFP pellet (2.5 mg/ 60 day release pellet, Innovative Research of America, Sarasota, FL) by SC injection.
  • the present inventors have identified two vectors, a non-viral plasmid DNA and an adeno-associated virus type 1 (AAV-1 ), that are suitable for delivery of a therapeutic molecule (TM), e.g. an IFN- ⁇ gene, to treat a chronic disease e.g., MS. Further, the present inventors have shown that both vectors can be delivered to skeletal muscle by intramuscular injection and generate IFN- ⁇ expression levels that are measurable and persistent in murine animal models.
  • TM therapeutic molecule
  • MS chronic disease
  • plasmid DNA In the case of plasmid DNA the present inventors have demonstrated that a single injection of a mIFN- ⁇ encoded plasmid (with electroporation) is efficacious and as effective as an every other day injection of mIFN- ⁇ protein in an animal model of MS- The present inventors developed biomarkers of mIFN- ⁇ to show that plasmid encoded mIFN- ⁇ expression persists for at least 45 days following a single plasmid injection. In the case of the AAV-1 vector the present inventors have shown that a single intramuscular injection of a human IFN- ⁇ encoded AAV-1 vector results in high serum levels of the human IFN- ⁇ protein that persists for 6 months.
  • Both plasmid and AAV-1 vectors have been shown to be compatible with the BRES regulated expression system of the present invention.
  • the present inventors demonstrated the regulated expression of IFN- ⁇ in mice using a single-plasmid vector BRES regulated expression system of the present invention.
  • the expression cassettes are present in a single vector, e.g., a single plasmid vector.
  • the pBRES single plasmid vector contains the expression cassettes for both the Regulator molecule (RM) (e.g., a transcriptional activator such as a modified steroid hormone receptor) and the therapeutic molecule (TM) (e.g., human or murine IFN- ⁇ ) on a single shuttle plasmid expression vector.
  • RM Regulator molecule
  • TM therapeutic molecule
  • the single vector of the BRES regulated expression system contains multiple cloning sites (MCS) to simplify the insertion or replacement of promoters, regulatory elements and transgenes into the plasmid backbone.
  • pBRES mIFN- ⁇ and pBRES hlFN- ⁇ single-plasmid vectors were constructed and tested in vitro, and shown to have low background activity in the absence of the activator molecule (AM), the small molecule inducer MFP, and showed high inducibility (comparable to a two-plasmid system) in the presence of MFP.
  • AM activator molecule
  • an outcome of these studies and the compositions and methods of the present invention is a gene-based delivery system for IFN- ⁇ that will provide long-term, regulated expression of IFN- ⁇ for the treatment of a disease or condition, e.g., an anti-inflammatory disease or condition, and more preferably MS.
  • the gene therapy vectors of the present invention can incorporate one or more expression cassettes for delivery of a therapeutic molecule (TM) of interest (e.g., IFN- ⁇ ) for treatment of a disease or condition.
  • TM therapeutic molecule
  • the regulated expression system as described herein can provide long-term, renewable expression through oral administration of the small molecule inducer, MFP.
  • the single-vector pBRES system is capable of repeat administration, e.g., by intramuscular injection, and will allow the testing of continuous versus pulsatile IFN- ⁇ therapy in the clinic.
  • pBRES Orientation The in vivo studies performed utilized one of the four pBRES orientations that were constructed as described in Figure 15. As described herein, this was based upon in vitro data that showed that the construct, pGT26, had the highest level of transgene expression in the presence of MFP. These four pBRES orientations can be tested in vivo using the protocols described herein to determine which one provides the best "window" of transgene expression (e.g., the lowest basal expression level minus MFP, and highest induced expression level plus MFP). i.
  • pBRES plasmids A study was performed in na ⁇ ve C57BL/6 mice using the mIFN- ⁇ pBRES plasmid vectors pGT23, 24, 25, and 26, to determine the level of mlFN expression as assayed by the level of the chemokine IP-10, which serves as a biomarker for mlFN expression.
  • pGT23, pGT24, pGT25, and pGT26 were injected with electroporation into the hind limb muscles of mice, with 15 animals per group. Five mice from each group were bled at day 7 in the absence of MFP.
  • mice in each group were treated with MFP for four consecutive days, beginning on day 7 after plasmid injection. Blood was collected at days 11 and 18 post-injection, and plasma samples were assayed for IP-10 (See “Experimental Design” for details).
  • the results show that pGT26 offered the best combination of low expression - MFP and high expression + MFP, consistent with the in vitro results ( Figure 32).
  • pGT24 had the highest induction of IP-10 expression, but the IP-10 levels in the absence of MFP were higher than the other orientations.
  • pGT25 had lower IP-10 levels both - and + MFP, and pGT23 had IP-10 levels + MFP about the same as pGT26.
  • the IP-10 levels with pGT24 - MFP were considerably higher than for pGT26.
  • mice In vivo transfection of pBRES/mlFN plasmids and comparison of the four orientations of pBRES/mlFN was performed as follows. Normal C57BL/6 mice were injected and electroporated with single-vector pBRES mouse IFN expression plasmids. mlFN expression was monitored by biomarker response and mlFN RNA analysis. The mice went through one off/on/off cycle of MFP treatment.
  • DNA delivery Adult male C57BL/6 mice were injected bilaterally on day 0 with 250 ug plasmid DNA in 150 ul PBS. The DNA solution was injected 25 ul into the tibialis muscle and 50 ul into the gastrocnemius muscle of each hind leg, followed by electroporation with a caliper (8 pulses at 200 V/cm, 1 Hz, 20 msec/pulse).
  • MFP treatment Groups 1-4 (all injected mice) were administered MFP by oral gavage at 0.33 mg/kg (100 ul of 0.083 mg/ml in sesame oil, made fresh) as indicated in the table below.
  • Plasmid DNA solutions Group 2-6 mice (plasmid) received 250 ug DNA per mouse in a volume of 150 ul. Table 5
  • DNA delivery For Groups 2-6 (plasmid), 25 ul of the DNA solution was injected into the tibialis muscle and 50 ul into the gastrocnemius muscle of each hind leg, followed by electroporation with a caliper (8 pulses at 200 V/cm, 1 Hz, 20 msec/pulse).
  • MFP treatment Groups 2-5 were administered MFP by i.p. injection at 0.33 mg/kg (100 ul of 0.083 mg/ml in sesame oil, made fresh) on day 7-10, as indicated in the table below.
  • pBRES/hEPO plasmids were also tested in a similar manner as above. Inducible one-plasmid pBRES/hEPO (pGT15, pGT16, pGT17, and pGT18) or two-plasmid (pGS1694 + pEP1666) vectors were injected with electroporation into the hind limb muscles of naive C57BL/6 mice, with 10 animals per group. Five mice from each group were treated with MFP for four consecutive days, beginning on day 7 after plasmid injection, and five mice from each group did not receive MFP.
  • mice were injected with MFP days 7-10 in the morning, and tail bled on day 10 in the afternoon, about 6 hrs after the last MFP injection (induced samples). The remaining five mice in each group were not induced with MFP, and were terminally bled at day 11 (terminal bleeds were necessary for accurate uninduced levels). Group 6 was terminally bled at day 11.
  • the pBRES and two plasmid systems were compared as to their MFP- induced and uninduced levels, and also compared was their constantly-induced levels over time.
  • Plasmid DNA solutions Group 1 mice received 100 ug each plasmid per mouse in a volume of 150 ul. Group 2-5 mice received 200 ug DNA per mouse in a volume of 15O uI.
  • DNA delivery 25 ul was injected into the tibialis muscle and 50 ul was injected into the gastrocnemius muscle of each hind leg, followed by electroporation with a caliper (8 pulses at 200 V/cm, 1 Hz, 20 msec/pulse).
  • MFP treatment Mice were administered MFP by i.p. injection of 100 ul of MFP solution (0.083 mg/ml in sesame oil). Table 8
  • Hematocrit Approximately 10 ul blood was collected (aspirated) directly from a tail nick in a capillary tube, sealed with clay, and centrifuged ⁇ 5 min at -10,000 g within 10 minutes after collection. The blood was separated in the capillary tube into 3 layers, i.e.: RBCs at the bottom (40-50% total volume), a small “buffy layer” (WBC and platelets) and the remainder plasma. A sliding gauge was used to read the hematocrit (percentage of RBC to total blood).
  • B. pBRES Backbone Any of the plasmid backbone modifications of the pBRES vectors of the present invention, as described herein, that demonstrate a significant increase in the level and/or duration of transgene expression (as determined by the methods described herein) can be incorporated in the pBRES vectors of the present invention. Additional modifications to the pBRES vectors of the present invention may include the use of a stronger promoter. This type of modification is relatively easy to test due to the modular design of the pBRES system.
  • C. pBRES/Vector Testing Pharmacokinetics and efficacy studies by the present inventors have employed IFN- ⁇ expression cassettes utilizing the CMV promoter enhancer.
  • the BRES expression system of the present invention can be tailored to suit a particular therapeutic need as described herein. For example, changes or modifications of the vectors or expression cassettes of the present invention can be tested in C57BL/6 na ⁇ ve mice.
  • A vector and activator molecule
  • TM therapuetic molecule
  • IFN- ⁇ therapuetic molecule
  • the selection of the type of vector can be determined by specific studies. For example, for plasmid DNA it can be determined whether electroporation is a desirable component of intramuscular injection to obtain a therapeutic level of the therapeutic molecule (TM), e.g., a therapeutic level of IFN- ⁇ . If administration by electroporation of the gene therapy vector of the present invention is desirable, then as described herein and additionally from what is known in the art, a device and protocol that can be clinically feasible and acceptable can be designed. For example, for AAV-1 vectors of the present invention, it can be determined whether repeat administration of the vector is desirable based on potential immunogenic properties reported for AAV vectors (see e.g., Chirmule, N et al. (2000) J Virol 74: 2420-25).
  • Plasmid DNA i is a desirable vector for gene-based delivery because it is simple, non- immunogenic, and easy to produce and manufacture.
  • Several different methods have been developed to enhance skeletal muscle (SkM) transfection efficiency of plasmid DNA by intramuscular injection. These methods include the use of enzymes such as hyaluronidase to treat the muscle and surrounding extracellular matrix prior to delivery (see e.g., Mennuni, C et al. (2002) Hum Gene Ther 13: 355-65), various polymer formulations (see e.g., Mumper, RJ et al. (1998) J Controlled Release 52: 191-203; Nicol, F et al.
  • Electroporation devices for clinical use in the delivery of IFN- ⁇ plasmid DNA can be evaluated and determined by testing in rabbits and other larger animals using methods described herein or known in the art. Electroporation devices that may be suitable for such testing and use may include those developed by Inovio AS, Ichor Medical Systems, Genetronics, Inc. Genetronics has reported testing of a device in humans (unpublished presentation at Gordon Research Conference on Bioelectrochemistry, July 25-30, NH). Inovio has also reported the results of testing electroporation technology in human volunteers (see e.g., Kjelen, R et al. (2004) MoI Ther 9: Supp1 , S60). Ichor Medical Systems has recently reported the development of an electroporation device suitable for the delivery of therapeutic DNA (see e.g., Evans, CF et al. (2004) MoI Ther 9: Supp 1 , S56).
  • a plasmid encoding a LacZ reporter gene the present inventors have demonstrated high transfection efficiency to rat hind limb skeletal muscle using intra-arterial delivery of a plasmid solution.
  • Mirus has recently reported an intra-venous delivery method for delivery of plasmid DNA with decreased volume under decreased pressure which can be tested for plasmid delivery to SkM using methods described herein or known in the art (see e.g., Hagstrom, JE et al. (2004) Mole Ther 10: 386-98).
  • Methods other than electroporation or intravascular delivery to enhance the uptake of plasmid DNA to SkM and the subsequent expression of the transgene can be tested and their suitability for delivery of plasmid DNA determined using methods described herein or known in the art.
  • certain chemical agents that have been reported to enhance vector uptake to SkM can be tested and may be suitable for use in the delivery of the plasmid vectors of the present invention, including polymer formulations and antennopedia peptides (AP).
  • AP antennopedia peptides
  • "F68" is a poloxamer formulation that can be used to formulate and deliver plasmid DNA and has been reported to enhance the delivery of plasmid DNA to SkM by approximately 10-fold (see e.g., Mumper, RJ et al.
  • a pBRES plasmid vector encoding IFN- ⁇ is modified to increase and prolong the level of transgene expression.
  • expression of the IFN- ⁇ transgene in the regulated expression system of the present invention was driven by the strong cytomegalovirus (CMV) promoter to constitutively express IFN- ⁇ .
  • CMV cytomegalovirus
  • the results from the in vivo study by the present inventors using the pBRES gene therapy plasmid vector pGT26- mIFN- ⁇ showed higher IFN- ⁇ levels using the pBRES expression cassette than the levels achieved using the CMV driven expression cassette (see e.g., Example 6).
  • the IFN- ⁇ pBRES expression system of the present invention may generate significantly higher and more persistent expression levels than what has thus far been observed using CMV driven plasmid DNA expression cassettes and therefore it is suitable for examining the delivery of the pBRES plasmid vector without electroporation.
  • the method of administration is by intramuscular injection of an IFN- ⁇ plasmid solution in the absence of electroporation. Detectable levels of IFN- ⁇ in serum can be tested by administering plasmid vector by intramuscular injection to naive mice. A complete characterization of the expression level and persistence of the pBRES expression cassette can be performed and compared with the CMV vectors previously used.
  • Plasmid formulations including F68 poloxamer and Antennapedia peptides can be tested for their ability to enhance plasmid transfection of SkM and subsequent IFN- ⁇ transgene expression.
  • modifications of the plasmid vector backbone e.g., removal of bacterial sequences
  • transgene expression can be explored as a means to increase and prolong transgene expression.
  • AAV-1 Vectors Adeno-associated virus (AAV) is a single stranded DNA virus (parvovirus) that was initially isolated as a contaminant in adenoviral isolates from humans. AAV has a number of features that make it particularly attractive as a gene therapy vector. In addition to its non-pathogenic and replication deficient nature in the absence of a helper virus it contains a very simple genome consisting of only two genes, rep and cap. These genes are replaced in recombinant AAV vectors with the desired transgene flanked by characteristic 5' and 3' inverted terminal repeats (ITR's) of approximately 135 base pairs each. The ITR's are the only remaining components of AAV derived DNA required for vector delivery.
  • ITR's characteristic 5' and 3' inverted terminal repeats
  • AAV vectors without the rep gene do not integrate in vivo but rather form large concatameric structures that remain episomal in non-dividing cells (see e.g., Duan, D et al. (1998) J Virol 72: 8568-77; Vincent-Lacaze, N et al. (1999) J Virol 73: 1949-55; Schnepp, BC et al. (2003) J Virol 77: 3495-3504).
  • AAV has a relatively small capacity for DNA, approximately 4.5 kb, but this is usually sufficient to accommodate all but the largest therapeutic transgenes.
  • AAV2 has been tested in human gene therapy trials and has shown to provide long term expression and minimal inflammation (see e.g., Silwell, JL and Samulski, RJ (2003) BioTechniques 34: 148-59). Recently, alternative AAV serotypes have been shown to have excellent transfection efficiency to SkM in addition to long term expression characteristic of this vector system (see e.g., Grimm, D and Kay, MA (2003) Curr Gene Ther 3: 281-304). The studies by the present inventors using an AAV-1 vector expressing luciferase under a CMV promoter have shown high expression persisting beyond 12 months following intramuscular injection into the hind limb of mice (see e.g., Qian, HS et al.
  • AAV-1 IFN- ⁇ expressing vectors (constitutive expression using the CMV promoter/enhancer) to demonstrate robust levels of hlFN- ⁇ protein as well as mIFN- ⁇ biomarker responses following intramuscular injection into the hind limbs of C57BL/6 mice.
  • the regulated expression systems of the present invention can be tested, using methods as described herein or as known in the art, for their ability to demonstrate not only long term expression of the therapeutic transgene but also the ability to re-administer the gene (see e.g., Chirmule, N et al. (2000) J Virol 74: 2420-25).
  • studies can be performed e.g., using the candidate vector, AAV-1 , and AAV2, the serotype that is believed to be the most prevalent in the human population.
  • Such an approach can be used to determine: 1 ) whether pre-existing antibodies to AAV2 or AAV-1 will affect the ability to deliver and express genes encoded in an AAV-1 vector, and 2) whether an AAV-1 vector can be re-administered at a dose sufficient to maintain therapeutic levels of IFN- ⁇ in mice.
  • Vectors expressing either the reporter gene luciferase or the therapeutic gene, murine IFN- ⁇ , in either AAV-1 or AAV2 vectors can be administered i.m. at different doses and transgene expression monitored. After four weeks the vector can be re-administered and again transgene expression can be monitored,
  • Neutralizing antibodies can be measured ex vivo by the ability of immunized mouse serum to inhibit viral uptake in a cell-based assay.
  • In vivo activity of a pBRES-hlFN AAV vector A study was performed in na ⁇ ve C57BL/6 mice using the hlFN- ⁇ pBRES AAV vector AAV-1 GT58 injected into the hind limb muscles of mice. Mice were bled at day 4 in the absence of MFP, then treated with MFP for four consecutive days, beginning on day 7 after plasmid injection. Blood was collected at days 11 and 18 post-injection.
  • mice Normal adult C57BL/6 mice were injected with an AAV vector carrying the pBRES/hlFN expression cassettes, as follows. Human IFN expression was assayed through multiple off/on/off cycles of MFP treatment.
  • Viral solution Group 1 mice (AAV) received 5 x 10 10 viral particles (vp) per mouse in a volume of 75 ul.
  • MFP treatment Group 1 was administered MFP by i.p. injection at 0.33 mg/kg (100 ul of 0.083 mg/ml in sesame oil, made fresh) on day 7-10, as indicated in the tables below.
  • Example 9 Gene Therapy Using a Regulated Expression System A. Dose/Response and Kinetics Studies: Dose/response studies can be performed to determine the amount of gene therapy vector of the present invention for delivery, whether delivered by plasmid or by AAV-1 , that is necessary to achieve therapeutic levels of the therapeutic molecule (TM) (e.g., IFN- ⁇ ) in mice (or other suitable animals).
  • the therapeutic level is defined as the induced level of the therapeutic molecule (TM), e.g. a transgene encoding a therapeutic protein, achieved systemically by the vector that is equivalent to the level of therapeutic protein achieved by a therapeutic amount of bolus of the therapeutic protein given in humans on a mg/kg basis.
  • the therapeutic level is defined as the induced level of IFN- ⁇ achieved systemically by a gene therapy vector of the present invention that is equivalent to the level of IFN- ⁇ achieved by a therapeutic amount of bolus IFN- ⁇ protein given in humans on a mg/kg basis.
  • the current single dose of IFN- ⁇ 1a is either 30 ug or 44 ug (Rebif).
  • a complete pharmacokinetic profile of the expression of the therapeutic molecule can be performed using an activator molecule (AM) to determine the AM dose/response, as well as the kinetics of induction of TM expression following AM administration.
  • AM activator molecule
  • a complete pharmacokinetic profile of IFN- ⁇ expression can be performed with the inducer MFP to determine the MFP dose/response, as well as the kinetics of IFN- ⁇ induction following MFP administration.
  • MFP dose/response and plasmid re-injection with a pBRES/mlFN plasmid in vivo A study was performed in na ⁇ ve C57BL/6 mice using the mIFN- ⁇ pBRES plasmid vector pGT26 to determine the level of mlFN expression in response to various doses of MFP, as assayed by the level of the chemokine IP-10. pGT26 was injected with electroporation into the hind limb muscles of mice, and the animals were treated with MFP at 0.0033 mg/kg to 1.0 mg/kg on day 7-10 after plasmid injection.
  • a complete characterization of the rate at which the activator molecule (AM) and TM expression can be turned “off” following withdrawal of the AM can be performed.
  • the frequency of AM dosing necessary to achieve steady state levels of the TM can be evaluated.
  • a complete characterization of the rate at which MFP and IFN- ⁇ expression can be turned "off' following withdrawal of the inducer can be evaluated. Further, the frequency of MFP dosing necessary to achieve steady state levels of IFN- ⁇ can be evaluated.
  • mice Normal C57BL/6 mice were injected and electroporated with a single-vector pBRES murine IFN expression plasmid and treated with various doses of MFP, and mlFN expression was assayed by biomarker response, as follows. Table 12
  • DNA delivery Adult male C57BL/6 mice were injected bilaterally on day 0 with 250 ug plasmid DNA per mouse in 150 ul PBS. The DNA solution was injected 25 ul into the tibialis muscle and 50 ul into the gastrocnemius muscle of each hind leg, followed by electroporation with a caliper (8 pulses at 200 V/cm, 1 Hz, 20 msec/pulse).
  • MFP treatment As indicated in the tables above and below, Groups 1-7 received 100 ul sesame oil alone or with MFP at various concentrations by i.p. injection on days 7-10. Table 15: Cycle 1
  • Cycle 2 Same MFP concentrations as Cycle 1
  • Each mouse received 250 ug of plasmid DNA in 150 ul PBS.
  • Group 9 were new control mice, where the age was matched as closely as possible.
  • Group 1 was also injected with plasmid.
  • Cycle 8 Same MFP concentrations as Cycle 7. Groups 3-7 were terminally harvested, and RNA and DNA prepared from muscle.
  • mice received 75 ul PBS and Groups 2-6 mice received 5 x 10 10 viral particles (vp) per mouse in a volume of 75 ul PBS in one hind leg (right leg). 25 ul was injected into the tibialis muscle and 50 ul injected into the gastrocnemius muscle.
  • Blood collection and Endpoint Analysis/Assay Procedure Mice were bled by cardiac punture (terminal bleed) at the time points indicated in the Table 27. Blood was collected in Microtainer tubes (no anti-coagulant) and the serum was separated from the blood, collected and assayed for hlFN by ELISA.
  • the presence of neutralizing antibodies against the BRES vector, expressed therapeutic molecule (e.g., a transgene), regulator molecule (RM) or therapeutic protein, can be monitored.
  • expressed therapeutic molecule e.g., a transgene
  • regulator molecule RM
  • therapeutic protein e.g., a transgene
  • the activator molecule can be administered chronically as well as in a pulsatile manner to evaluate the ability to maintain expression levels of the therapeutic molecule (TM) over time as well as provide renewable expression levels in an on/off manner with AM dosing.
  • the IFN- ⁇ BRES vector of the present invention can be tested in C57BL/6 mice (or other suitable animal) for repeat biomarker endpoints in response to MFP administration or administation of IFN- ⁇ .
  • the presence of neutralizing antibodies against the vector, IFN molecule (IFNM) e.g., a IFN- ⁇ transgene), regulator molecule (RM), or IFN- ⁇ protein, can be monitored.
  • IFN molecule e.g., a IFN- ⁇ transgene
  • RM regulator molecule
  • IFN- ⁇ protein can be monitored.
  • MFP can be administered chronically as well as in a pulsatile manner to evaluate the ability to maintain IFN- ⁇ expression levels over time as well as provide renewable expression levels in an on/off manner with MFP dosing.
  • mice that received pGT26 were injected with MFP i.p. every day from day 35-50, and five mice from each group were bled during this period on day 35 (before MFP treatment), 39, 42, 45, and 51.
  • mice were bled on day 105, then treated with MFP on day 105-108, and bled on day 109 and 116. Serum samples were assayed for IP-10 as a biomarker for mlFN expression.
  • Figure 37C show that induction of IFN expression from the pBRES plasmid upon MFP treatment occurred within 24 hr, and expression decreased to baseline 24-48 hr following peak induction. Expression of mlFN from the pBRES system was higher than that driven by the CMV promoter. All three cycles of mlFN expression over the course of two to three months were at high levels. Continuous MFP treatment resulted in sustained high-level expression of mlFN over two weeks.
  • IP-10 levels after 3.5 months were about one-half to two-thirds less than in earlier MFP cycles, but was still well above the background IP-10 levels and also considerably higher than that generated by expression of mlFN from the CMV promoter, which also decreased over time with about the same kinetics (two-thirds of expression lost after 3.5 months).
  • this experiment demonstrates the capacity of the pBRES system for continuous, high-level target gene expression over several months, with the ability to be rapidly turned off and on again multiple times.
  • mice Normal C57BL/6 mice were injected and electroporated with single-vector pBRES and CMV promoter mouse IFN expression plasmids, as follows. IFN expression was monitored for at least several months, using IP-10 as the endpoint biomarker for mlFN activity. MFP treatment and bleeds are designed to determine the kinetics of the "on" and "off' responses.
  • MFP treatment Group 2 was administered MFP by i.p. injection at 0.33 mg/kg (100 ul of 0.083 mg/ml in sesame oil, made fresh) as indicated in the table below.
  • Cycle 1 (day 7-18): Determine the "off” kinetics.
  • Cycle 2 (day 21- 25): Determine the "on” kinetics.
  • Bioequivalence Studies can be conducted in normal mice and in one other species, preferably non-human primates, using the candidate vector and pBRES expression system, under delivery conditions, e.g., as established in the studies described above.
  • "Bioequivalence” can be, but is not limited to, e.g., to demonstrate that the present regulated expression system of the present invention provides a superior pharmacokinetic profile for IFN- ⁇ gene-based delivery over IFN- ⁇ protein delivery in both animal models as defined, but not limited to, e.g., in the table below.
  • Table 31 Non-Limiting Examples of Bioequivalence Criteria
  • therapeutic level in an animal model is defined as the systemic level of IFN- ⁇ (as determined, e.g., by ELISA and/or biomarker induction) that is equivalent to the level achieved by a therapeutic amount of bolus IFN- ⁇ 1a protein administered in humans on a mg/kg basis.
  • AAV-1 is selected as the candidate vector biodistribution studies following i.m. administration of the candidate vector can be performed.
  • the endpoints will include vector DNA and expressed IFN- ⁇ RNA and protein distribution to target tissue (muscle), blood, lymph, heart, liver, kidney, lungs, male and female gonads (testis, ovary).
  • E. Gene-Based Delivery for Treatment of a Disease or Condition An outcome from these studies is a gene-based delivery system for delivery of a therapeutic molecule (TM), e.g. a transgene encoding a therapeutic protein, for treatment of a disease or condition.
  • TM therapeutic molecule
  • the present invention provides a regulated expression system of delivery of an IFN- ⁇ transgene that will provide long term, regulated expression of IFN- ⁇ for the treatment of MS.
  • an outcome of these studies is that the pBRES vector of the present invention can provide persistent, renewable expression (e.g., greater than 3 months) through the oral administration of the small molecule inducer, MFP; and is capable of repeat administration by intramuscular injection.
  • mice were treated with MFP (0.33 mg/kg) by i.p. injection once per day (d) or every third day (etd) after plasmid injection. Blood was collected at day 5 after injection. PBMCs were isolated from the blood and RNA was prepared from and assayed by RT-PCR to determine the level of Mx1 RNA (See “Experimental Design” below for details). The results show about an 8-fold increase in Mx1 RNA levels with pBRES/mlFN plus daily MFP injections, and no significant increase over a null vector in the absence of MFP (Figure 38). This demonstrates a biological response in an animal disease model similar to that which has been shown to be efficacious in this model.
  • CMV plasmid plus Electroporation Two bilateral intramuscular (im) injections of either Null (pgWiz) or mlFN (pGER101 ) CMV plasmid DNA into the tibialis (20 ul) and gastrocnemius muscles (40 ul), followed immediately by EP on Day 2, as administered.
  • BRES1 Plasmid plus EP Two bilateral intramuscular (im) injections of either pBRES Null (pGT4) or pBRES mlFN (pGT26) plasmid DNA into the tibialis (20 ul) and gastrocnemius muscles (40 ul), followed by EP one week (Day -7) prior to initiation of disease, was administered.
  • MFP (0.33 mg/kg) was administered daily or every third day (etd) by ip injection beginning on Day 1. The animals receiving MFP "etd" were dosed on Days 1 , 4, 7, 10, 13, 16, 19, and 22.
  • mice A ⁇ ent Dose Volume/dose i I I U Inl ItLilO KSa CltLot?H U I O M INrUjnI IpC
  • Buffer control 10 buffer 100 ul/inj.* sc
  • the process for production of a gene therapy vector of the present invention comprising a pBRES hlFN- ⁇ expression cassette can be suitable for cGMP- manufacturing.
  • the pBRES vectors of the present invention can be made of sufficient purity, potency, and stability to perform preclinical development studies.
  • the gene therapy vectors of the present invention, and preferably the pBRES vectors of the present invention can be fully characterized with respect to the plasmid backbone, capsid (in the case of AAV as a delivery vector), transgene expression product (IFN- ⁇ ), and inducer (MFP), using methods described herein or known in the art.
  • A inducer or activator molecule
  • Repeat administration and persistence of transgene expression can be fully characterized, lmmunogenicity studies can be conducted with the candidate vector.
  • Pharmacokinetics/safety/toxicology Pharmacokinetic studies of the expressed IFN- ⁇ transgene can be conducted using direct detection of the expressed transgene as well as measurement of IFN- ⁇ biomarkers. If AAV-1 is the selected candidate vector, biodistribution studies can be performed to examine the fate of the vector DNA and its expression products.
  • EAE Seventy 8-week old female SJL mice from Jackson Labs were immunized with a 0.1 ml SC (divided between base of tail and upper back) injection containing 150 ug Proteolipid Protein (PLP)139-151 in Incomplete Freund's Adjuvant (IFA) supplemented with 200 ug M. tuberculosis H37Ra.
  • PLP Proteolipid Protein
  • IFA Incomplete Freund's Adjuvant
  • mice Immediately after immunization, all mice received a 0.1 ml IP injection of pertussis toxin. Two days after immunization (day 3 of study), all mice received a second IP injection of pertussis toxin.
  • mice treated with IFN- ⁇ protein or its vehicle (20 nM NaAc, pH 5.5, 150 mM NaCI, 5% propylene glycol) were dosed with 0.1 ml, SC once every other day beginning on the day of immunization until the end of the study.
  • the positive controls used for this study were 9 mg/kg Mesopram (ZK-117137) and 2.5 mg/kg Prednisolone. Both controls use a dose volume of 0.1 ml/injection and are administered IP, twice daily, beginning on the morning of immunizations until the end of the study.
  • Experimental Groups (n 10): 1. Vehicle
  • mice in the plasmid + PINC groups received the same treatment as on day 2.
  • mice in the plasmid + PINC groups received the same treatment as on day 2.
  • mice treated with IFN- ⁇ protein or its vehicle (20 nM NaAc, pH 5.5, 150 mM NaCI, 5% propylene glycol) were dosed with 0.1 ml, sc once every other day beginning on the day of immunization until the end of the study.
  • the positive controls used for this study were 9 mg/kg Mesopram (ZK-117137) and 2.5 mg/kg Prednisolone. Both controls use a dose volume of 0.1 ml/injection and both controls are administered IP, twice daily, beginning on the morning of immunizations until the end of the study.
  • mice There were a total of 10 groups in this study. Each group had 13 mice. The last 3 mice in each group were bled via tail nick on day 6 of the study for Mx 1 RNA analysis. The same 3 animals that were bled on day 6 were bled via cardiac puncture on day 13 of the study for Mx1 RNA analysis from PBMCs, and injected muscles were collected for analysis.
  • IFN15-GS5 In vivo transfection of pBRES/IFN plasmids: Mifepristone- regulated mIFN- ⁇ expression was demonstrated from a pBRES/mlFN- ⁇ plasmid electroporated into mouse muscle.
  • mice Normal C57BL/6 mice can be injected and electroporated with a pBRES single vector of the present invention and control plasmid DNAs as described in Tables 34 and 35 below. Table 34
  • mIFN- ⁇ expression can be assayed by biomarkers and RNA levels in muscle at 3 time points.
  • Groups 1 , 3, and 4 can be terminally harvested and Group 2 can be tail bled to determine uninduced background mIFN- ⁇ expression and biomarker activity in mice receiving GS/mlFN - MFP (Group 3) in comparison to uninjected mice (Group 1 ) and mice receiving empty pBRES vector (Group 2).
  • CMV/mlFN (Group 4) serves as a positive control.
  • Group 12 can be tail bled to determine uninduced background levels of SEAP expression.
  • mice in Groups 2, 6, 9, 1 1 , and 12 can be treated with MFP on days 7-10 after DNA injection.
  • Groups 5-7 can be terminally harvested to determine induced mlFN expression and biomarker activity in mice receiving RM/mlFN + MFP (Group 6) in comparison to mice receiving CMV/mlFN (Group 7). Uninduced levels in mice receiving RM/mlFN - MFP (Group 5) will also be assayed.
  • Group 2 empty pBRES vector
  • Groups 8- 10 can be tail bled to determine if biomarker activity is detectable from small volumes of blood and to provide an induced time point in the same mice with which to compare the uninduced levels on day 18.
  • Group 11 (CMV/mlFN + MFP) can be terminally harvested to determine whether MFP affects mlFN expression or inhibits the biomarker response.
  • Group 12 can be tail bled to determine induced levels of SEAP expression.
  • Group 13 will provide a negative control for SEAP expression.
  • Groups 8-10 can be terminally harvested to determine if the mIFN- ⁇ RNA levels and biomarker activity in the RM/mlFN - /+/- MFP group (Group 9) have returned to baseline in comparison to RM/mlFN mice that never received MFP (Group 8).
  • CMV/mlFN (Group 10) again serves as a positive control.
  • Group 12 can be terminally bled to determine if SEAP expression has returned to baseline.
  • Each mouse in Groups 2-11 can receive 250 ug of plasmid DNA in 150 ul PBS.
  • Each mouse in Group 12 can receive 25 ug of plasmid DNA in 150 ul PBS (see Table 36 below).
  • Table 36 Prepare DNA solutions for 5 mice/group plus extra
  • DNA delivery (Groups 2-12): Adult male C57BL/6 mice (5 per group) can be injected bilaterally on day 0 with 250 ug (Groups 2-11 ) or 25 ug (Group 12) plasmid DNA per mouse in 150 ul PBS. The DNA solution can be injected 25 ul into the tibialis muscle and 50 ul into the gastrocnemius muscle of each hind leg, followed by electroporation with a caliper (8 pulses at 200 V/cm, 1 Hz, 20 msec/pulse).
  • MFP treatment Groups 2, 6, 9, 11, and 12
  • Mice in Groups 2, 6, 9, 11 , and 12 can be administered MFP by oral gavage at 0.33 mg/kg (100 ul of 0.083 mg/ml in sesame oil, made fresh) on days 7 through 10 post-injection as indicated in Table 37 below.
  • Group 12 mice can be bled prior to MFP treatment on day 7.
  • *pGT31 was constructed by digestion of pGER75 (CMV/SEAP) with Nhe I and Not I, and insertion of the resulting fragment carrying the SEAP gene between the Spe I and Not I sites of pGT1.
  • mice can be tail bled or terminally bled. When mice are terminally bled, the injected muscles can be collected.
  • Blood can be collected into Microtainer tubes (containing EDTA) at RT and then PBMCs can be separated and collected. The leftover plasma can be stored at -20 0 C for cytokine assays.
  • Muscle The injected muscles of both legs can be harvested, pooled together, and cut into pieces no larger than 5 mm on one side. Approximately one-fourth of the chopped muscle can be placed into 1.5 ml of RNA-Later solution in a 2 ml tube. The remainder of the muscle can be stored at - 70 0 C. The DNA and RNA can be extracted from the muscle samples in RNA-Later solution. The samples can be stored at 4 0 C for at least 24 h and then transferred to -20 0 C if they can be stored for more than 5 days.
  • Blood (Groups 12 and 13): Mice in Group 12 can be tail bled on day 7 and day 11 and terminally bled on day 18 into yellow Microtainer tubes (no anti-coagulant). The mice can be bled prior to MFP treatment on day 7. Mice in Group 13 can be terminally bled on day 11 into yellow Microtainer tubes (no anti-coagulant).
  • JE and IP-10 protein from plasma The plasma can be assayed for JE and IP-10 cytokines by ELISA .
  • mIFN- ⁇ RNA from muscle RNA can be prepared from the injected muscles and assayed for mIFN- ⁇ RNA by TaqMan.
  • Plasmid DNA from muscle DNA can be prepared from the injected muscles and assayed for plasmid DNA by TaqMan.
  • Primers and probe specific for the CMV promoter can be used for DNA from Groups 4, 7, 10, and 11.
  • Primers and probe specific for the GAL-4 DNA binding domain of the regulator protein can be used for Groups 2, 3, 5, 6, 8, and 9.
  • SEAP protein from serum The serum can be assayed for SEAP expression by the chemiluminescent activity assay, using the serum from the Group 13 mice as a diluent.
  • Plasmid Vectors pGER101 (pgWiz/mlFN): The mouse IFN- ⁇ (mIFN- ⁇ ) gene was amplified by PCR from the plasmid vector pbSER189 ( Figure 20A) with the mlFN signal sequence placed on the 5' primer and Sal I and Not I restriction enzyme sites added at the 5' and 3' ends. The fragment was digested with Sal I and Not I and inserted into the Sal I and Not I sites of plasmid vector pgWIZ ( Figure 20B) resulting in plasmid vector pGER101 ( Figure 20C).
  • pGER125 (pgWiz/hlFN): The human IFN- ⁇ (hlFN- ⁇ ) gene was amplified by PCR from plasmid vector pbSER178 with the hlFN signal sequence replaced on the 5' primer and Sal I and Not I restriction enzyme sites added at the 5' and 3' ends. The fragment was digested with Sal I and Not I and inserted into the Sal I and Not I sites of plasmid vector pgWIZ resulting in plasmid vector pGER125 ( Figure 21).
  • Plasmid vector was purchased from Invitrogen and contains 6 GAL-4 binding sites upstream of a minimal promoter (E1 b TATA), a 5' untranslated region (UTR) that is UT12 derived from CMV, a synthetic intron 8 (IVS8), a multiple cloning site (MCS) and the bovine growth hormone (bGH) poly(A) site.
  • E1 b TATA minimal promoter
  • UTR 5' untranslated region
  • IVS8 synthetic intron 8
  • MCS multiple cloning site
  • bGH bovine growth hormone
  • Genes inserted at the MCS can be regulated by a regulator molecule (RM).
  • RM regulator molecule
  • a gene inserted at the MCS can be induced by the activated form of the modified progesterone receptor (e.g.
  • Plasmid vector pGER101 was digested with Sal I, filled in with Klenow, ligated to Hind III linkers, and digested with Hind III and Not I. The mIFN- ⁇ gene fragment was inserted into the Hind III and Not I sites of plasmid vector pGene/V5-HisA resulting in plasmid vector pGene-mlFN ( Figure 23).
  • pGene-hlFN Plasmid vector pGER125 was digested with Sal I, filled in with Klenow, ligated to Hind III linkers, and digested with Hind III and Not I. The mIFN- ⁇ gene fragment was inserted into the Hind III and Not I sites of plasmid vector pGene/V5-HisA resulting in plasmid vector pGene-hlFN (pGER129) ( Figure 24).
  • pSwitch This plasmid vector was purchased from Invitrogen and encodes the modified progesterone receptor (e.g.
  • Plasmid vector pGS1694 was provided by Valentis and contains the chicken skeletal muscle actin promoter (sk actin pro), 5' untranslated region 12 (UT12) and synthetic intron 8 (IVS8) driving expression of the gene encoding the modified progesterone receptor (e.g.
  • Plasmid vector pLC1674 was provided by Valentis and contains a "RM- responsive" promoter (i.e., a promoter responsive to the activated form of the modified progesterone receptor (e.g. comprising the amino acid sequence of SEQ ID NO: 22 or encoded by the nucleic acid sequence of SEQ ID NO: 21 ), 5' untranslated region 12 (UT12) and synthetic intron 8 (IVS8) driving expression of the gene encoding the firefly luciferase gene (luc) ( Figure 27).
  • RM- responsive promoter i.e., a promoter responsive to the activated form of the modified progesterone receptor (e.g. comprising the amino acid sequence of SEQ ID NO: 22 or encoded by the nucleic acid sequence of SEQ ID NO: 21 ), 5' untranslated region 12 (UT12) and synthetic intron 8 (IVS8) driving expression of the gene encoding the firefly luciferase gene (luc) ( Figure 27).
  • Vectors for producing virus e.g., shuttle plasmids
  • methods of producing virus e.g., AAV-1 virus
  • the viruses of the present invention are produced from shuttle plasmids (e.g., see Table 38) and used for the delivery and expression of a molecule of the present invention (e.g., a TM and/or RM encoded by a sequence contained in the vector) in the cells of a subject, for treatment of disease.
  • pGT2/mGMCSF and pGT/hGMCSF Shuttle plasmids pGT2/mGMCSF and pGT/hGMCSF were constructed as follows ( Figure 28).
  • a fragment encoding mouse GMCSF (mGM-CSF) was excised from pORF9-mGMCSF ( Figure 30) by digesting the vector plasmid with Agel and Nhel. This fragment was then blunted.
  • a fragment encoding human (hGM-CSF) was excised from pORF-hGMCSF ( Figure 30) by digesting with the vector plasmid with SgrAI and Nhel. This fragment was then blunted.
  • the excised and blunted fragment encoding either mGMCSF or hGMCSF was inserted into the EcoRV site of pGT2 vector plasmid. The orientation of the insert was then checked by restriction digest mapping.
  • pGT2/mGMCSF encoding mouse GMCSF
  • pGT2/hGMCSF encoding human GMCSF
  • Figure 28 pZac2.1-RM-hGMCSF
  • pZac2.1-RM-mGMCSF Shuttle plasmids pZac2.1-RM- hGMCSF and pZac2.1-RM-mGMCSF were constructed as follows ( Figure 29A).
  • a fragment encoding a mouse GMCSF was excised from the plasmid pORF9-mGMCSF ( Figure 30) by digesting the plasmid with Agel and Nhel and blunted, and the resulting blunted fragment inserted into the EcoRV site of the plasmid pGT2, resulting in the plasmid pGT2/mGMCSF.
  • the vector plasmids pGT2/hGMCSF and pGT2/mGMCSF were then each digested with Fsel and Srfl. These pBRES-GMCSF fragments were then blunted.
  • the plasmid vector pZac2.1 was digested with Bgl2 and CIaI, and blunted. The blunted pBRES-GMCSF fragments were each ligated to a blunted pZac2.1 vector. Positive clones were verified by restriction digests.
  • pZac2.1-RM-hGMCSF encoding human GMCSF
  • pZac2.1-RM-mGMCSF encoding mouse GMCSF
  • Figure 29A pZac2.1-CMV-mGMCSF
  • pZac2.1-CMV-hGMCSF Shuttle plasmids pZac2.1- CMV-mGMCSF and pZac2.1-CMV-hGMCSF were constructed as follows ( Figure 29B).
  • a fragment encoding a human GMCSF and a fragment encoding a mouse GMCSF were each separately cloned into the plasmid vector pGENE/V5HisA (Invitrogen) resulting, respectively, in the plasmids pGT723-GENE/hGMCSF (encoding human GMCSF) and pGT724-
  • GENE/mGMCSF (encoding mouse GMCSF).
  • a fragment encoding mouse GMCSF was excised from pGT724/mGMCSF by digesting the plasmid with Kpnl and Xbal, and a fragment encoding human GMCSF was excised from pGT723/hGMCSF by digesting the plasmid with Kpnl and Xbal.
  • the resulting fragments were each separately inserted into the Kpnl/Xbal site of the vector pZac2.1 that had been digested with Kpnl and Xbal, and treated with calf alkaline phosphatase (CIP).
  • CIP calf alkaline phosphatase
  • the resulting shuttle plasmids were named pGT713 (or pZac2.1- CMV-hGMCSF) encoding human GMCSF and pGT714 (or pZac2.1-CMV-mGMCSF) encoding mouse GMCSF ( Figure 29B).
  • the pZAC2.1 shuttle plasmid was modified at the MCS resulting in shuttle plasmid pGT53, in order to enable the insertion of the fragment containing the BRES sequence into the vector.
  • the appropriate pGT plasmid (as described above in Table 8) was digested with restriction enzymes that resulted in a fragment containing the entire BRES sequence encoding the respective IFN, and this fragment was inserted into a compatible site of pGT53.
  • the resulting AAV-1 shuttle plasmids were used to make AAV-1 virus preps as described herein using standard methods for producing AAV-1 virus.
  • a fragment encoding the respective IFN gene was isolated from the appropriate plasmid vector via restriction enzyme digestion and inserted into the pZAC2.1 plasmid vector at (a) compatible restriction site(s) as described in Table 8 above.
  • mice and human GMCSF genes from the respective pORF9 plasmids were cloned through the plasmid pGENE/V5HisA plasmid (Invitrogen) so that they could each be excised with Kpnl/Xbal and cloned into pZAC2.1.
  • compositions and methods of the present invention are well adapted to carry out the objects and obtain the ends and advantages described herein, as well as those inherent in the present invention. Changes to the compositions and methods of the present invention, and other uses, will occur to those skilled in the art and such changes are contemplated and encompassed herein as described and as claimed.

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Abstract

La présente invention se rapporte à un système d'expression amélioré assurant l'expression régulée d'une molécule thérapeutique codée à protéine ou à acide nucléique dans les cellules d'un sujet, aux fins d'utilisation pour le traitement d'une maladie. En particulier, l'invention concerne un système d'expression génétique régulée amélioré, des compositions pharmaceutiques et des procédés d'utilisation associés destinés à traiter une maladie.
PCT/EP2007/053308 2006-05-18 2007-04-04 Thérapie génique à interféron bêta faisant appel à un système d'expression régulée amélioré WO2007134906A1 (fr)

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EP3235516A1 (fr) * 2016-04-22 2017-10-25 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts Universitätsmedizin Vecteur de virus adéno-associés (aav) régulable

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WO2006122972A1 (fr) * 2005-05-19 2006-11-23 Bayer Schering Pharma Aktiengesellschaft Therapie genique a base d'interferons beta utilisant un systeme d'expression regulee, ameliore

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WO2006122972A1 (fr) * 2005-05-19 2006-11-23 Bayer Schering Pharma Aktiengesellschaft Therapie genique a base d'interferons beta utilisant un systeme d'expression regulee, ameliore

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