US20050095225A1 - Compounds and methods for pharmico-gene therapy of epithelial sodium channel associated disorders - Google Patents

Compounds and methods for pharmico-gene therapy of epithelial sodium channel associated disorders Download PDF

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US20050095225A1
US20050095225A1 US10/815,557 US81555704A US2005095225A1 US 20050095225 A1 US20050095225 A1 US 20050095225A1 US 81555704 A US81555704 A US 81555704A US 2005095225 A1 US2005095225 A1 US 2005095225A1
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John Engelhardt
Liang Zhang
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University of Iowa Research Foundation UIRF
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Definitions

  • Cystic fibrosis is caused by a genetic mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), and is the most common genetic disorder in the Caucasian population.
  • CFTR is a chloride channel that localizes to the apical membrane of epithelial cells in many organs such as the lung. The channel is activated by cyclic AMP (cAMP) and regulated by PKA- and PKC-dependent phosphorylation.
  • cAMP cyclic AMP
  • CFTR has also been shown to regulate several other ion channels at the cell surface (Jiang et al., 1998), including the epithelial amiloride-sensitive sodium channel (ENaC) (Stults et al., 1997; Donaldson et al., 2002), outward rectifying chloride channel (ORCC) (Gabriel et al., 1993; Schwiebert et al., 1998), renal potassium channel (ROMK2) (Cahill et al., 2000), and the calcium activated chloride channel (Kunzelmann et al., 1997).
  • ENaC epithelial amiloride-sensitive sodium channel
  • ORCC outward rectifying chloride channel
  • ROMK2 renal potassium channel
  • Cahill et al., 2000 the calcium activated chloride channel
  • rAAV recombinant adeno-associated virus
  • rAAV-mediated gene delivery of CFTR includes the limited packaging capacity of this vector (about 5 kb) and the relatively large size of the CFTR cDNA (4.5 kb).
  • Several strategies have been used to fit the CFTR cDNA into rAAV vectors including the use of the ITR as a promoter (Flotte et al., 1993) and the deletion of regions of CFTR thought not to be necessary for in vivo function (Zhang et al., 1998; Ostedgaard et al., 2002). The first strategy is currently used in clinical trails with rAAV-2 based vectors.
  • the invention provides a method to identify an agent, or a combination of agents, that alters ENaC activity in a eukaryotic cell, e.g., a mammalian cell such as a mammalian lung, kidney or colon cell, or a population of eukaryotic cells, e.g., in tissues or organs.
  • the method comprises contacting the cell or population of cells with the one or more agents and determining whether the level or amount of ENaC is altered.
  • the cell or population of cells are epithelial cells such as airway epithelial cells.
  • the cell or cells are kidney tubule, e.g., distal nephron including distal convoluted tubule, connecting tubule, and cortical and medulary collecting duct, skin, liver, bladder, colon, sweat gland, mammary gland, salivary gland, placenta or uroepithelium cells.
  • Preferred cells include those of mammals, birds, fish, and reptiles, especially domesticated mammals and birds such as humans, non-human primates, cattle, sheep, pigs, horses, dogs, cats, mice, rats, rabbits, chickens, and turkeys.
  • polarized human airway epithelial cells grown at an air-liquid interface or human bronchial xenografts are useful to identify agents which inhibit or decrease the level or amount of ENaC.
  • the agents of the inventions may be contacted with any cell comprising native or recombinant ENaC, e.g., cell membrane bound ENaC. It is envisioned that agents identified as inhibiting the level or amount of ENaC may have variations in the degree of inhibition, time course and/or duration of inhibition, cell or tissue type specificities and/or the concentration employed for inhibition.
  • the invention provides a method to identify one or more agents that inhibit or decrease the level or amount of ENaC, e.g., agents including but not limited to those that inhibit transcription of one or more ENaC subunit genes, alter the level, amount or activity of a molecule that alters ENaC transcription, alter ENaC RNA stability, and/or alter the trafficking and processing of molecules, for instance, molecules of non-viral origin through intracellular compartments, including without limitation proteasomes, endosomes, and trans-golgi, and/or through the cytosol, e.g., via cytoskeletal components such as microtubules or microfilaments.
  • the agent is not an antagonist of ENaC.
  • the agent is not an agent that binds a cell membrane bound protein, e.g. ENaC or the receptor for hepatocyte growth factor.
  • the agent is not an agent that alters post-translational processing of ENaC.
  • the agent is not a gene of, or a gene product encoded by, a mammalian genome, e.g., a protein encoded by a mammalian cell, the complement of the gene, or a portion of the gene or its complement, e.g., an antisense oligonucleotide.
  • Agents to be tested may be selected from agents having desirable properties for a particular cell type, tissue type or disease type to be treated.
  • agents to be tested are selected from agents including those having desirable properties, e.g., therapeutic properties, for instance, agents in clinical trials or having FDA approval or functional and/or structural properties of agents identified as inhibiting or decreasing the level or amount of ENaC.
  • agents may be selected from agents that modulate the proteasome, e.g., agents including but not limited to those that bind to a proteasome, alter one or more activities of a proteasome, e.g., inhibit the proteolytic activity of the proteasome, alter subcellular positioning or trafficking of the proteasome, alter the interaction of one or more molecules with a proteasome, or stabilize the proteasome.
  • agents that modulate the proteasome e.g., agents including but not limited to those that bind to a proteasome, alter one or more activities of a proteasome, e.g., inhibit the proteolytic activity of the proteasome, alter subcellular positioning or trafficking of the proteasome, alter the interaction of one or more molecules with a proteasome, or stabilize the proteasome.
  • Proteasomes are the main proteolytic complex in the cytosol and nucleus, and can be transported between the cytoplasm and nucleus.
  • the 26S proteasome complex comprises a 19S regulatory unit and a 20S catalytic core which has chymotrypsin-like activity, i.e., cleavage after large hydrophobic residues, trypsin-like activity, i.e., cleavage after basic residues, post-glutamyl hydrolase activity, i.e., cleavage after acidic residues, branched amino acid cleavage activity and small neutral amino acid cleavage activity.
  • chymotrypsin-like activity i.e., cleavage after large hydrophobic residues
  • trypsin-like activity i.e., cleavage after basic residues
  • post-glutamyl hydrolase activity i.e., cleavage after acidic residues, branched amino acid cleavage activity and small neutral amino acid cleavage activity.
  • chemical libraries are selected based on chemical structures known to interact with the proteasome, or other intracellular processing pathways, e.g., endosomal compartments.
  • agents are selected from chemotherapeutics, antibiotics, lipid lowering agents or food additives.
  • Antibiotics include but are not limited to macrolides, penicillins, quinolones, sulfonamides and tetracyclines, e.g., cephalosporins, bacitracin, vancomycin, ristocetin, erythromycin, oleandomycin, carbomycin, spiramycin, lincomycin, clindamycin, chlortetracycline, minocycline, oxytetracycline, streptomycin, amikacin, gentamycin, kanamycin, neomycin, tobramycin, polymyxins, nystatin, amphotericin B, mitomycin, actinomycin, nalidixic acid, novobiocin, griseofulvin, rifampicins, and trimethoprim.
  • macrolides e.g., penicillins, quinolones, sulfonamides and trimethoprim.
  • Chemotherapeutics include but are not limited to anti-fungal agents, anti-bacterial agents, antiviral agents, e.g., nucleoside analogs, phosphonoacetate, phosphonoformate, amantadine, rimantadine, enviroxime, 4′, 6-dichloroflavan, chalcone Ro 09-0410, arildone, disoxaril, 3′-azidothymidine, suramin and HPA 23, and anticancer agents, e.g., alkylating agents, antimetabolites, plant alkaloids, antitumor antibiotics and steroid hormones such as cyclophosphamide, nitrosoureas, carmustine (BCNU), lomustine (CCNU), 6-mercaptopurine, 5-fluoroouracil (5FU), doxorubicin (adriamycin), mitomycin-C, bleomycin, vincristine, vinblastine, and tamoxifen
  • CFTR cDNA rAAV vectors were prepared. The vectors were packaged in either type 2 and 5 capsids, and following apical infection of polarized CF airway epithelia, analyzed for their ability to correct both Na hyperabsorption and Cl transport defects which accompany the CF phenotype, and for their efficiency of transduction in the presence or absence of proteasome modulating agents (LLnL/Doxorubicin).
  • the identical ITR-CFTR vector used in clinical trials for CF was compared to that of a vector harboring a minimal 83 bp promoter directing expression of the full-length CFTR cDNA (AVCF83).
  • the comparison included measurements of short circuit current, quantitative RS-PCR, and TaqMan DNA PCR, so as to quantify functional correction of CFTR chloride currents, vector-derived mRNA, and vector DNA, respectively.
  • the data demonstrated that rAAV-2 based vectors are more efficacious than rAAV-2/5 at expressing CFTR-derived mRNA and correcting CFTR chloride transport abnormalities in the presence of applied proteasome modulating agents.
  • proteasome modulating agents at the time of infection not only improved the functional conversion of rAAV genomes to expressible forms but also reduced the ENaC hyperabsorption CF phenotype in a manner independent of CFTR gene expression.
  • Quantitative RT-PCR demonstrated that the addition of proteasome modulating agents reduced ⁇ -ENaC subunit mRNA levels in polarized CF airway epithelia by 15-fold. The long-term (15 day) persistence of this effect on ENaC activity correlated with doxorubicin-dependent CpG methylation of the ⁇ -ENaC promoter.
  • proteasome modulating agents may have dual therapeutic potential for both enhancing rAAV transduction and ameliorating fluid transport defects in CF caused by dysregulated ENaC. For instance, agents which alter ENaC activity may be screened for their ability to alter fluid transport or absorption in polarized airway epithelial cells.
  • one or more agents and, optionally a dye, such as a fluorescent dye, in a small volume of liquid are contacted with polarized airway epithelial cells, and the presence or amount of the dye and/or the amount (depth) of extracellular liquid in the treated cells is detected or determined, e.g., using a confocal microscope, and compared to untreated cells.
  • agents which alter ENaC activity may be screened for their association with the methylation of other promoters, which may result in the identification of agents that are associated with the methylation of more than one promoter as well as agents that are associated with the methylation of only one promoter.
  • vectors harboring a short 83 bp minimal promoter improved functional correction and the transcriptional activity of vector genomes by 30% as compared to ITR promoter driven vectors.
  • the invention provides a method to identify one or more agents that decrease the level or amount of transcription of one or more subunits of ENaC in mammalian cells.
  • the method includes contacting mammalian cells which express ENaC with at least one agent that is a proteasome modulating agent, wherein the agent is not a gene or gene product encoded by the genome of the cells, the complement of the gene, or a portion of the gene or its complement, and identifying whether an agent decreases the level or amount of transcription from one or more subunits of ENaC in the mammalian cells.
  • the method includes contacting mammalian cells which express ENaC with the one or more agents and identifying one or more agents that decrease the level or amount of transcription from the ⁇ , ⁇ , and ⁇ subunits of ENaC in the mammalian cells.
  • the agent is not a gene or a gene product encoded by the genome of the cells, the complement thereof, or a portion thereof.
  • the method includes contacting mammalian cells which express ENaC with at least one agent that enhances viral transduction and identifying one or more agents that decrease the level or amount of transcription from one or more subunits of ENaC in the mammalian cells.
  • the agent is not a gene or gene product encoded by the genome of the cells, the complement thereof, or a portion thereof.
  • the method includes selecting one or more agents which inhibit or treat one or more symptoms of a disease which is associated with aberrant expression or activity of ENaC, contacting mammalian cells with the one or more agents and a gene therapy vector, and identifying an agent that enhances the efficacy of the gene therapy vector relative to mammalian cells contacted with the gene therapy vector but not contacted with the one or more agents.
  • the method includes selecting one or more agents that enhance the efficacy of a gene therapy vector in mammalian cells, contacting mammalian cells having aberrant expression or activity of ENaC with the one or more agents, and identifying an agent that alters ENaC expression or activity.
  • Agents of the invention may be used alone or in combination to produce additive or synergistic effects, e.g., to alter the level or amount of ENaC, to inhibit reabsorption of salts and water from mucous secretions in a tissue or organ, e.g., in the lung, to hydrate mucous secretions in a tissue or organ, to increase airway surface liquid volume, e.g., in the lung, to facilitate mucous clearance in a tissue or organ, to inhibit or treat conditions associated with aberrant ENaC activity, for instance, cystic fibrosis, Liddle's syndrome, hypertension, pain, and pulmonary edema, as well as chronic bronchitis, asthma, and acute lung injury.
  • ENaC activity for instance, cystic fibrosis, Liddle's syndrome, hypertension, pain, and pulmonary edema, as well as chronic bronchitis, asthma, and acute lung injury.
  • the agents of the invention may be employed with mineral corticoid receptor antagonists, glucocorticoid receptor antagonists, pyrazine diuretics, pyrazinoyl guanidine sodium channel blockers, amiloride, benzamil, phenamil, lanthione antibiotics, nucleotides or dinucleotides, as well as nucleic acids or oligonucleotides; viral gene transfer vectors (including adenovirus, adeno-associated virus, and retrovirus gene transfer vectors); enzymes; and hormone drugs or physiologically active proteins or peptides such as insulin, somatostatin, oxytocin, desmopressin, leutinizing hormone releasing hormone, nafarelin, leuprolide, adrenocorticotrophic hormone, secretin, glucagon, calcitonin, growth hormone releasing hormone, growth hormone, and the like.
  • mineral corticoid receptor antagonists such as insulin, somato
  • Enzyme drugs that may be used to carry out the present invention, include but are not limited to DNAse (for the treatment of, e.g., cystic fibrosis), ⁇ -antitrypsin (e.g., to inhibit elastase in the treatment of emphysema), etc.
  • Suitable anti-inflammatory agents including steroids, for use in the methods of the present invention include, but are not limited to, beclomethasone dipropionate, prednisone, flunisolone, dexamethasone, prednisolone, cortisone, theophylline, albuterol, cromolyn sodium, epinephrine, flunisolide, terbutaline sulfate, alpha-tocopherol (Vitamin E), dipalmitoylphosphatidylcholine, salmeterol and fluticasone dipropionate.
  • beclomethasone dipropionate prednisone, flunisolone, dexamethasone, prednisolone, cortisone, theophylline, albuterol, cromolyn sodium, epinephrine, flunisolide, terbutaline sulfate, alpha-tocopherol (Vitamin E), dipalmitoy
  • antibiotics examples include, but are not limited to tetracycline, choramphenicol, aminoglycosides, for example, tobramycin, beta-lactams, for example ampicillin, cephalosporins, erythromycin and derivatives thereof, clindamycin, and the like.
  • Suitable anti-viral agents include acyclovir, ribavirin, ganciclovir and foscamet.
  • Suitable anti-neoplastic agents include, but are not limited to, etoposid, taxol, and cisplatin.
  • Antihistamines include, but are not limited to, diphenhydramine and ranitadine.
  • Anti- Pneumocystis carinii pneumonia drugs such as pentamidine and analogs thereof may also be used.
  • Anti-tuberculosis drugs such as rifampin, erythromycin, chlorerythromycin, etc.
  • Chelators of divalent cations e.g., EGTA, EDTA), expectorants, and other agents useful in the loosening of mucous secretions (e.g., n-acetyl-L-cysteine) may also be administered as desired in the practice of the present invention.
  • Cells, tissues, organs or organisms may be contacted with one or more agents of the invention simultaneously or sequentially, at a single time point or at multiple time points.
  • agents of the invention may be contacted with Cells, tissues, organs or organisms simultaneously or sequentially, at a single time point or at multiple time points.
  • One of ordinary skill in the art will appreciate that the manner and timing of agent administration will be influenced by the duration and degree of inhibition of the agent, pharmaceutical properties of the agent, and underlying disease condition of the affected tissue, organ or organism.
  • Agents identified by the method of the invention may be also particularly useful in conjunction with or to potentiate gene therapy that employs nucleic acid-based vectors, e.g., viral vectors, to introduce and/or express a therapeutic peptide or polypeptide in cells of an animal, e.g., a mammal.
  • the agents are also useful in conjunction with nucleic acid-based vaccine vectors to introduce and/or express an immunogenic prophylactic polypeptide or peptide, such as one from a virus, fungus, bacterium, yeast or cancer cell, so as to induce an immune response to that polypeptide or peptide in an animal administered the nucleic acid-based vector.
  • cells may be contacted with one or more agents prior to nucleic acid-based therapy, concurrently with nucleic acid-based therapy, subsequent to nucleic acid-based therapy, or any combination thereof.
  • agents of the invention may be employed with gene therapy vectors, e.g., viral vectors such as adenovirus vectors, herpes virus vectors, lentivirus vectors, retroviral vectors and/or rAAV vectors.
  • the dual activities of certain agents of the invention may potentiate, or be employed in conjunction with, gene therapy vectors to decrease the dose or total number of molecules, e.g., viral particles, employed to achieve an efficacious result, increase the gene transfer, e.g., transduction, frequency, and/or for viral vectors, broaden the serotype infectivity pattern, and/or alter the in vivo microenvironment to allow increased availability of viral binding.
  • gene therapy vectors to decrease the dose or total number of molecules, e.g., viral particles, employed to achieve an efficacious result, increase the gene transfer, e.g., transduction, frequency, and/or for viral vectors, broaden the serotype infectivity pattern, and/or alter the in vivo microenvironment to allow increased availability of viral binding.
  • the use of agents of the invention to both treat primary pathophysiologic defects of a disease and potentiate gene therapy vectors is also referred to as pharmico-gene therapy.
  • the vector is not an rA
  • the gene being expressed in the vector can be either a DNA segment encoding a polypeptide, with whatever control elements (e.g., promoters, operators) are desired, or a non-coding DNA segment, the transcription of which produces all or part of some RNA-containing molecule (such as a transcription control element, +RNA, or anti-sense molecule).
  • therapeutic genes useful in such vectors include ones that encode a functional peptide or polypeptide.
  • a “functional” peptide or polypeptide is one which has substantially the same activity as a reference peptide or polypeptide, for example, a wild-type (full-length) peptide or polypeptide.
  • therapeutic genes useful in the vectors of the invention include but are not limited to the ⁇ -globin gene, the ⁇ -globin gene, Factor VIII gene, Factor IX gene, the erythropoietin gene, the cystic fibrosis transmembrane conductance regulator gene (CFTR), the dystrophin gene, the Fanconi anemia complementation group, a gene encoding a ribozyme, an antisense gene, a low density lipoprotein (LDL) gene, a tyrosine hydroxylase gene (Parkinson's disease), a glucocerebrosidase gene (Gaucher's disease), an arylsulfatase gene (metachromatic leukodystrophies), as well as genes encoding immunogenic polypeptides or peptide, such as those useful for vaccines, or genes encoding other polypeptides or proteins.
  • LDL low density lipoprotein
  • Parkinson's disease a glucocerebrosidas
  • Tissues, organs or organisms may be contacted with one or more agents of the invention and nucleic acid based vectors, simultaneously or sequentially, at a single time point or at multiple time points.
  • agents of the invention and nucleic acid based vectors, simultaneously or sequentially, at a single time point or at multiple time points.
  • agent administration will be influenced by the duration and degree of inhibition of the agents, pharmaceutical properties of the agent, and underlying disease condition of the affected tissue, organ or organism.
  • a method to inhibit or treat a condition associated with increased ENaC levels or increased ENaC activity includes contacting a mammal at risk of or having the condition with an effective amount of an agent that inhibits or decreases transcription of one or more ENaC subunit genes and/or alters the level, amount or activity of a molecule that alters transcription of one or more ENaC subunit genes, and enhances the efficacy of gene therapy vectors.
  • the method includes contacting a mammal at risk of or having the condition with an effective amount of an agent that inhibits or decreases transcription of one or more ENaC subunit genes and/or alters the level, amount or activity of a molecule that alters transcription of one or more ENaC subunit genes, wherein the agent is a proteasome modulating agent, and wherein the agent is not a gene or gene product encoded by the genome of the mammal, the complement of the gene, or a portion of the gene or its complement.
  • a method to inhibit or treat a condition associated with increased ENaC levels or increased ENaC activity in which a mammal at risk of or having the condition is contacted with an effective amount of an agent that inhibits or decreases transcription of the ⁇ , ⁇ , and ⁇ subunits of ENaC or alters the level, amount or activity of a molecule that alters transcription of the ⁇ , ⁇ , and ⁇ subunits of ENaC.
  • FIG. 2 In vivo enhancement of rAAV transduction with Doxil.
  • Male Balb/c mice intravenously administered Doxil were endotracheally instilled with 1 ⁇ 10 11 DRP AAV2FLAG-Luc (01:004).
  • FIG. 4 Dox and LLnL provide additive induction of rAV2 transduction.
  • FIG. 5 Combined administration of proteasome-modulating agents can synergistically induce rAAV transduction from the apical surface of polarized human airway epithelia.
  • A 1 ⁇ 10 9 particles of AV2Luc were applied to the apical surface of polarized human airway epithelia cultures in the absence and presence of various combinations of LLnL (40 ⁇ M) and/or Dox (5 ⁇ M). Luciferase expression was assayed at 3 and 17 days post-infection.
  • B-E Similar results were observed following apical infection with a self complementary (2.3 kb) scAV2eGFP vector at 15 days post-infection.
  • FIG. 8 Expression of transgene-derived and endogenous CFTR mRNA in CF airway epithelia.
  • FIG. 9 Quantification of vector DNA following rAAV infection of CF airway epithelia. The total DNA fraction (nuclear and cytoplasmic) remaining following mRNA isolation was quantified by TaqMan PCR for the number of vector genomes for the indicated conditions. Samples are identical to those analyzed in FIGS. 7 and 8 .
  • (A) Values represent the mean +/ ⁇ SEM (N 9) relative copies of rAAV CFTR vectors genomes for each sample.
  • FIG. 10 Proteasome modulation inhibits the function of amiloride-sensitive sodium channels in polarized CF airway epithelia.
  • the reduction in ENaC activity by LLnL/Dox is independent of the level of CFTR functional correction.
  • (C) Kinetics of doxorubicin inhibition of the amiloride-sensitive sodium channel in CF epithelia. Amiloride-sensitive Isc in polarized CuFi cells was measured at 1 day, 3 days, 1 week and 2 weeks after treatment with doxorubicin and compared untreated groups. Results depict the mean +/ ⁇ SEM of amiloride-sensitive sodium current for each of the indicated treatments (N 3 for each group).
  • FIG. 11 Doxorubicin treatment increases CpG methylation of the ⁇ -ENaC gene promoter.
  • B Schematic diagram of the ⁇ -ENaC gene promoter.
  • the transcription start site is labeled as +1; the position of the CpG island studied herein is shown in black rectangle; the position of restriction enzymes used to study CpG methylation are shown as vertical lines; and the position of primers used in the methylation sensitive PCR analysis are shown by arrows at ⁇ 3449 and ⁇ 3139 bp.
  • C Results from methylation-sensitive PCR analysis of the ⁇ 3449 to ⁇ 3139 bp region of the ⁇ -ENaC gene promoter. MboI digestion of genomic DNA prior to PCR analysis (Lane 9) served as a positive control and gave rise to two PCR products (more than one product is likely due to the GC rich content of the PCR fragment).
  • FIG. 13 Nasal trans epithelial potential differences (PD) obtained from 5 week old BLJ6 mice.
  • A) A continuous tracing beginning in 1) Herpes phosphate buffered Ringer's (HPBR), 2) HPBR +100 ⁇ M amiloride, 3) Cl-free HPBR+100 ⁇ M amiloride+10 ⁇ M forskolin.
  • B) A summary of the delta mV change following each buffer switch marked by arrows.
  • FIG. 14 Screening for anthracycline proteosome modulators.
  • A) Graph of luciferase activity versus concentration of tested agent.
  • FIG. 15 In vivo results for anthracycline proteosome modulators.
  • ENaC activity or “inhibit or decrease the level or amount of ENaC” as used herein include but are not limited to agents that inhibit ENaC activity of a cell, population of cells, tissue, or organ.
  • ENaC activity may be inhibited by inhibiting transcription of one or more ENaC subunit genes, altering the level, amount or activity of a molecule that alters ENaC transcription, altering ENaC RNA stability, and/or altering the trafficking and processing of molecules, for instance, molecules of non-viral origin, through intracellular compartments, including without limitation proteasomes, endosomes, and trans-golgi, and/or through the cytosol, e.g., via cytoskeletal components such as microtubules or microfilaments.
  • altering ENaC activity may include, for example, decreasing ENaC transcription via direct interaction with the promoter of one or more ENaC subunits, such as methylation of ENaC sequences, or may include affecting the binding of a negatively regulating protein to at least one of the ENaC subunit promoter sequences, e.g., a repressor of ENaC, or alternatively inhibiting binding of a positively regulating transcription factor, e.g., a transcription factor binding protein which binds to one or more of the ENaC promoters.
  • agents that alter ENaC activity or inhibit or decrease the level or amount of ENaC do not include agents that are antagonists of ENaC, bind a cell membrane bound protein, e.g., bind ENaC or the receptor for hepatocyte growth factor, alter post-translational processing of ENaC, and/or are genes of, or gene products encoded by, a mammalian cell, the complement thereof, or a portion thereof, e.g., an antisense oligonucleotide.
  • “Dual therapeutic activity,” “dual therapeutic action,” dual therapeutic,” “pharmico-gene therapy,” or “potentiate” as used herein to refer to agents of the invention refer to certain agents of the invention that are used to both treat primary pathophysiologic effects of a disease and enhance the efficiency of gene therapy vectors to treat the disease.
  • a “vector” as used herein refers to a macromolecule, e.g., a polynucleotide, or association of macromolecules that comprises or associates with a polynucleotide, and which can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo.
  • a vector may comprise a polynucleotide sequence of recombinant origin.
  • Illustrative vectors include, for example, plasmids, viral vectors, liposomes and other gene delivery vehicles.
  • the polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic or interest), a coding sequence of interest in vaccine development (such as a polynucleotide expressing a protein, polypeptide-or peptide suitable for eliciting an immune response in a mammal), and/or a selectable or detectable marker.
  • a coding sequence of interest in gene therapy such as a gene encoding a protein of therapeutic or interest
  • a coding sequence of interest in vaccine development such as a polynucleotide expressing a protein, polypeptide-or peptide suitable for eliciting an immune response in a mammal
  • a selectable or detectable marker such as a selectable or detectable marker.
  • AAV is adeno-associated virus, and may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.
  • serotype refers to an AAV which is identified by and distinguished from other AAVs based on capsid protein reactivity with defined antisera, e.g., there are eight serotypes of primate AAVs, AAV-1-AAV-8.
  • serotype AAV2 is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV 2 and a genome containing 5′ and 3′ ITR sequences from the same AAV2 serotype.
  • Pseudotyped AAV as refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5′-3′ ITRs of a second serotype.
  • Pseudotyped rAAV would be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype.
  • Pseudotyped rAAV are produced using standard techniques described in the art.
  • rAAV5 may be used to refer an AAV having both capsid proteins and 5′-3′ ITRs from the same serotype or it may refer to an AAV having capsid proteins from serotype 5 and 5′-3′ ITRs from a different AAV serotype, e.g., AAV serotype 2.
  • AAV serotype 2 e.g., AAV serotype 2.
  • the abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”).
  • Transduction are terms referring to a process for the introduction of an exogenous polynucleotide, e.g., a transgene in rAAV vector, into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell, and includes the use of recombinant virus to introduce the exogenous polynucleotide to the host cell, e.g., viral-mediated transfection is generally referred to as transduction.
  • an exogenous polynucleotide e.g., a transgene in rAAV vector
  • the process includes 1) endocytosis of the AAV after it has bound to a cell surface receptor, 2) escape from endosomes or other intracellular compartments in the cytosol of a cell, 3) trafficking of the viral particle or viral genome to the nucleus, 4) uncoating of the virus particles, and generation of expressible double stranded AAV genome forms, including circular intermediates.
  • the rAAV expressible double stranded form may persist as a nuclear episome or optionally may integrate into the host genome.
  • Transduction, transfection or transformation of a polynucleotide in a cell can be determined by methods well known to the art including, but not limited to, protein expression (including steady state levels), e.g., by ELISA, flow cytometry and Western blot, measurement of DNA and RNA by hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays.
  • Methods used for the introduction of the exogenous polynucleotide include well-known techniques such as chemical-mediated methods, e.g., Ca 2 +mediated methods and lipofection, viral infection, and electroporation, as well as non-viral gene delivery techniques.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Proteasome modulator refers to an agent or class of agents which interact with, bind to, or alter the function of, and/or alter the trafficking or location of the proteasome.
  • Proteasome modulators may have other cellular functions as described in the art, e.g., such as doxyrubicin, which is an antibiotic.
  • Gene delivery refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.
  • Gene transfer refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene.
  • Gene expression or “expression” refers to the process of gene transcription, translation, and post-translational modification.
  • a “detectable marker gene” is a gene that allows cells carrying the gene to be specifically detected (e.g., distinguished from cells which do not carry the marker gene). A large variety of such marker genes are known in the art.
  • a “selectable marker gene” is a gene that allows cells carrying the gene to be specifically selected for or against, in the presence of a corresponding selective agent.
  • an antibiotic resistance gene can be used as a positive selectable marker gene that allows a host cell to be positively selected for in the presence of the corresponding antibiotic.
  • positive and negative selectable markers are known in the art, some of which are described below.
  • viral vector refers to a viral vector comprising a polynucleotide sequence of recombinant origin, typically a sequence of interest for the genetic transformation of a cell.
  • the term viral vector encompasses both vector particles and vector plasmids.
  • a “viral vector vaccine” refers to a viral vector comprising a polynucleotide sequence not of viral origin (i.e., a polynucleotide heterologous to that virus), that encodes a peptide, polypeptide, or protein capable of eliciting an immune response in a host contacted with the vector. Expression of the polynucleotide may result in generation of a neutralizing antibody response and/or a cell mediated response, e.g., a cytotoxic T cell response.
  • infectious virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is trophic.
  • the term does not necessarily imply any replication capacity of the virus.
  • a “replication-competent” virus e.g., a replication-competent AAV, sometimes abbreviated as “RCA” refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (i.e., in the presence of a helper virus or helper virus functions).
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a “transcriptional regulatory sequence” or “TRS,” as used herein, refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked.
  • Transcriptional regulatory sequences of use in the present invention generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription.
  • Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared.
  • a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).
  • a TRS or promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous TRS or promoter.
  • Packaging refers to a series of subcellular events that results in the assembly and encapsidation of a viral vector. Thus, when a suitable vector is introduced into a packaging cell line under appropriate conditions, it can be assembled into a viral particle. Functions associated with packaging of viral vectors are described in the art.
  • a “terminator” refers to a polynucleotide sequence that tends to diminish or prevent read-through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator).
  • the degree to which transcription is disrupted is typically a function of the base sequence and/or the length of the terminator sequence.
  • transcriptional termination sequences are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and/or disengage from the DNA being transcribed.
  • sequence-specific terminators include polyadenylation (“polyA”) sequences, e.g., SV40 polyA.
  • polyA polyadenylation
  • insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed, and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated.
  • Terminators may thus prevent transcription from only one direction (“unidirectional” terminators) or from both directions (“bi-directional” terminators), and may be comprised of sequence-specific termination sequences or sequence-non-specific terminators or both.
  • sequence-specific termination sequences or sequence-non-specific terminators or both.
  • “Host cells,” “cell lines,” “cell cultures,” “packaging cell line” and other such terms denote higher eukaryotic cells, preferably mammalian cells, most preferably human cells, useful in the present invention. These cells can be used as recipients for recombinant vectors, viruses or other transfer polynucleotides, and include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.
  • a “therapeutic gene,” “prophylactic gene,” “target polynucleotide,” “transgene,” “gene of interest” and the like generally refer to a gene or genes to be transferred using a vector.
  • such genes are located within a viral vector thus can be replicated and encapsidated into viral particles.
  • Target polynucleotides can be used in this invention to generate vectors for a number of different applications.
  • polynucleotides include, but are not limited to: (i) polynucleotides encoding proteins useful in other forms of gene therapy to relieve deficiencies caused by missing, defective or sub-optimal levels of a structural protein or enzyme; (ii) polynucleotides that are transcribed into anti-sense molecules; (iii) polynucleotides that are transcribed into decoys that bind transcription or translation factors; (iv) polynucleotides that encode cellular modulators such as cytokines; (v) polynucleotides that can make recipient cells susceptible to specific drugs, such as the herpes virus thymidine kinase gene; and (vi) polynucleotides for cancer therapy, such as EIA tumor suppressor genes or p53 tumor suppressor genes for the treatment of various cancers.
  • cancer therapy such as EIA tumor suppressor genes or p53 tumor suppressor genes for the treatment of various cancers.
  • the transgene is preferably operably linked to a promoter, either its own or a heterologous promoter.
  • a promoter either its own or a heterologous promoter.
  • suitable promoters are known in the art, the choice of which depends on the desired level of expression of the target polynucleotide; whether one wants constitutive expression, inducible expression, cell-specific or tissue-specific expression, etc.
  • the vector may also contain a selectable marker.
  • control element or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3′ direction) from the promoter. Promoters include AAV promoters, e.g., P5, P19, P40 and AAV ITR promoters, as well as heterologous promoters.
  • An “expression vector” is a vector comprising a region which encodes a polypeptide of interest, and is used for effecting the expression of the protein in an intended target cell.
  • An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target.
  • the combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
  • Genetic alteration refers to a process wherein a genetic element is introduced into a cell other than by mitosis or meiosis.
  • the element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell.
  • Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex.
  • Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector.
  • the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and/or genotype of the cell and its progeny is included in this term.
  • a cell is said to be “stably” altered, transduced or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro.
  • such a cell is “inheritably” altered in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.
  • polypeptide and protein are used interchangeably herein to refer to polymers of amino acids of any length.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphonylation, lipidation, or conjugation with a labeling component.
  • Polypeptides such as “CFTR” and the like, when discussed in the context of gene therapy and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, that retains the desired biochemical function of the intact protein.
  • references to CFTR, and other such genes for use in gene therapy include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.
  • an “isolated” plasmid, virus, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from.
  • an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more preferred. Thus, for example, a 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, 1000-fold enrichment is even more preferred.
  • an “individual” or “subject” treated in accordance with this invention refers to vertebrates, particularly members of a mammalian species, and includes but is not limited to domestic animals, sports animals, and primates, including humans.
  • Treatment of an individual or a cell is any type of intervention in an attempt to alter the natural course of the individual or cell at the time the treatment is initiated, e.g., eliciting a prophylactic, curative or other beneficial effect in the individual.
  • treatment of an individual may be undertaken to decrease or limit the pathology caused by any pathological condition, including (but not limited to) an inherited or induced genetic deficiency, infection by a viral, bacterial, or parasitic organism, a neoplastic or aplastic condition, or an immune system dysfunction such as autoimmunity or immunosuppression.
  • Treatment includes (but is not limited to) administration of a composition, such as a pharmaceutical composition, and administration of compatible cells that have been treated with a composition. Treatment may be performed either prophylactically or therapeutically; that is, either prior or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
  • Agents useful to inhibit, treat or prevent conditions associated with aberrant ENaC levels, amount or activity include but are not limited those which inhibit or decrease the level or amount of ENaC, e.g., agents that alter the trafficking and processing of molecules through intracellular compartments, including without limitation proteasomes, endosomes, and trans-golgi, and/or cytosol e.g., via cytoskeletal components such as microtubules or microfilaments, inhibit transcription of one or more ENaC subunit genes and/or alter the level, amount or activity of a molecule that alters ENaC transcription.
  • agents that alter the trafficking and processing of molecules through intracellular compartments including without limitation proteasomes, endosomes, and trans-golgi
  • cytosol e.g., via cytoskeletal components such as microtubules or microfilaments, inhibit transcription of one or more ENaC subunit genes and/or alter the level, amount or activity of a molecule that alters ENaC transcription.
  • Classes of agents useful in the invention include but are not limited to antibiotics, chemotherapeutics, lipid lowering agents, and food additives, as well as proteasome modulators, e.g., such as tripeptidyl aldehydes, agents that inhibit calpains, cathepsins, cysteine proteases, and/or chymotrypsin-like protease activity of proteasomes (Wagner et al., 2002; Young et al., 2000; Seisenberger et al., 2001), and agents that modulate the proteasome and ubiquitin pathways, e.g., agents that bind to proteasomes and/or modulate the activity of proteasomes, ubiquitin, ubiquitin carrier protein, or ubiquitin ligase, but do not substantially alter the activity of the proteasome, e.g., the proteolytic activity of the proteasome or of ubiquitin, ubiquitin carrier protein, or ubiquitin liga
  • antibiotics e.g., epoxomicin
  • lipid lowering drugs e.g., simvastatin
  • food additives e.g., tannic acid
  • chemotherapeutics e.g., cisplatin
  • anthracyclines such as doxorubicin, epirubicin, daunorubicin and idarubicin, and camptothecin.
  • Cysteine protease inhibitors within the scope of the invention include the cystatins, e.g., cystatin B or cystatin C, antipain, leupeptin, E-64, E-64c, E-64d, KO2 (Wacher et al., 1998), LLnL, Z-LLL, CBZ-Val-Phe-H, cysteine protease inhibitors such as those disclosed in U.S. Pat. Nos. U.S. Pat. No.
  • cysteine protease inhibitors are peptides or analogs thereof.
  • peptide cysteine protease inhibitors within the scope of the invention comprise 2 to 20, more preferably 3 to 10, and even more preferably 3 to 8, amino acid residues.
  • Amino acid comprises the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g.
  • Peptide analogs are molecules which comprise at least one amino acid in D form and/or an unnatural amino acid, or other moiety which is not a natural amino acid.
  • Protease inhibitors include a compound of formula (I): R 1 -A-(B) n -C wherein R 1 is an N-terminal amino acid blocking group; each A and B is independently an amino acid; C is an amino acid wherein the terminal carboxy group has been replaced by a formyl (CHO) group; and n is 0, 1, 2, or 3; or a pharmaceutically acceptable salt thereof.
  • R 1 is (C 1 -C 10 )alkanoyl, acetyl or benzyloxycarbonyl.
  • each A and B is independently alanine, arginine, glycine, isoleucine, leucine, valine, nor-leucine or nor-valine, and more preferably each A and B is isoleucine.
  • C is alanine, arginine, glycine, isoleucine, leucine, valine, nor-leucine or nor-valine, wherein the terminal carboxy group has been replaced by a formyl (CHO) group, and more preferably, C is nor-leucine or nor-valine, wherein the terminal carboxy group has been replaced by a formyl (CHO) group.
  • R 1 is (C 1 -C 10 )alkanoyl. In another embodiment, R 1 is acetyl or benzyloxycarbonyl. In yet a further embodiment, R 1 is (C 1 -C 10 )alkanoyl or benzyloxycarbonyl; A and B are each isoleucine; C is nor-leucine or nor-valine, wherein the terminal carboxy group has been replaced by a formyl (CHO) group; and N is 1.
  • C is alanine, arginine, glycine, isoleucine, leucine, valine, nor-leucine or nor-valine, wherein the terminal carboxy group has been replaced by a CHO group, e.g., in one embodiment C is nor-leucine or nor-valine and the terminal carboxy group is replaced by a CHO group.
  • a and B are each independently alanine, arginine, glycine, isoleucine, leucine, valine, nor-leucine or nor-valine, e.g., in one embodiment A and B are each isoleucine.
  • R 1 is (C 1 -C 10 )alkanoyl or benzyloxycarbonyl; A and B are each isoleucine; C is nor-leucine or nor-valine, wherein the terminal carboxy group has been replaced by a formyl (CHO) group; and N is 1.
  • R 2 is an N-terminal amino acid blocking group
  • R 3 , R4, and R 5 are each independently hydrogen, (C 1 -C 10 )alkyl, aryl or aryl(C 1 -C 10 )alkyl;
  • R 6 , R 7 , and R 8 are each independently hydrogen, (C 1 -C 10 )alkyl, aryl or aryl(C 1 -C 10 )alkyl; or a pharmaceutically acceptable salt thereof.
  • R 2 is (C 1 -C 10 )alkanoyl, acetyl or benzyloxycarbonyl.
  • R 3 is hydrogen or (C 1 -C 10 )alkyl, e.g., 2-methylpropyl.
  • R 4 is hydrogen or (C 1 -C 10 )alkyl, e.g., 2-methylpropyl.
  • R 5 is hydrogen or (C 1 -C 10 )alkyl, for example, butyl or propyl.
  • R 2 is acetyl or benzyloxycarbonyl
  • R 3 and R 4 are each 2-methylpropyl
  • R 5 is butyl or propyl
  • R 6 , R 7 , and R 8 are each independently hydrogen.
  • R 2 may be (C 1 -C 10 )alkanoyl, e.g., acetyl or benzyloxycarbonyl; R 3 may be hydrogen or (C 1 -C 10 )alkyl, e.g., 2-methylpropyl.
  • R 5 may be hydrogen or (C 1 -C 10 )alkyl, e.g., butyl or propyl.
  • R 2 is acetyl or benzyloxycarbonyl; R 3 and R 4 are each 2-methylpropyl; R 5 is butyl or propyl; and R 6 , R 7 , and R 8 are each independently hydrogen.
  • R 1 is H, halogen, (C 1 -C 10 )alkyl, (C 1 -C 10 )alkenyl, (C 1 -C 10 )alkynyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, ( ⁇ O), ( ⁇ S), OH, SR, CN, NO 2 , trifluoromethyl or (C 1 -C 10 )alkoxy, wherein any alkyl, alkenyl, alkynyl, alkoxy or alkanoyl may optionally be substituted with one or more halogen, OH, SH, CN, NO 2 , trifluoromethyl, NRR or SR, wherein each R is independently H or (C 1 -C 10 )alkyl; R 2 is ( ⁇ O) or ( ⁇ S); R 3 is H, (C 1 -C 10 )alkyl, (C 1 -C 10 )alkenyl, (C 1 -
  • Another preferred agent useful in the methods of the invention is a compound of formula (III): wherein
  • R 1 is H, halogen, (C 1 -C 10 )alkyl, (C 1 -C 10 )alkenyl, (C 1 -C 10 )alkynyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, ( ⁇ O), ( ⁇ S), OH, SR, CN, NO 2 , trifluoromethyl or (C 1 -C 10 )alkoxy, wherein any alkyl, alkenyl, alkynyl, alkoxy or alkanoyl may optionally be substituted with one or more halogen, OH, SH, CN, NO 2 , trifluoromethyl, NRR or SR, wherein each R is independently H or (C 1 -C 10 )alkyl;
  • R 2 is ( ⁇ O) or ( ⁇ S);
  • R 3 is H, (C 1 -C 10 )alkyl, (C 1 -C 10 )alkenyl, (C 1 -C 10 )alkynyl, (C 1 -C 10 )alkoxy or (C 3 -C 8 )cycloalkyl, wherein any alkyl, alkenyl, alkynyl, alkoxy or cycloalkyl may optionally be substituted with one or more halogen, OH, CN, NO 2 , trifluoromethyl, SR, or NRR, wherein each R is independently H or (C 1 -C 10 )alkyl;
  • R 4 is H, (C 1 -C 10 )alkyl, (C 1 -C 10 )alkenyl, (C -C 1 o)alkynyl, (C 1 -C 10 )alkoxy or (C 3 -C 8 )cycloalkyl, wherein any alkyl, alkenyl, alkynyl, alkoxy or cycloalkyl may optionally be substituted with one or more halogen, OH, CN, NO 2 , trifluoromethyl, SR, or NRR, wherein each R is independently H or (C 1 -C 10 )alkyl;
  • R 5 is H, halogen, (C 1 -C 10 )alkyl, (C 1 -C 10 )alkenyl, (C 1 -C 10 )alkynyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, ( ⁇ O), ( ⁇ S), OH, SR, CN, N0 2 or trifluoromethyl, wherein any alkyl, alkenyl, alkynyl, alkoxy or alkanoyl may optionally be substituted with one or more halogen, OH, SH, CN, NO 2 , trifluoromethyl, NRR or SR, wherein each R is independently H or (C 1 -C 10 )alkyl; and
  • X is O, S or NR wherein R is H or (C 1 -C 10 )alkyl, or a pharmaceutically acceptable salt thereof.
  • R 1 is OH. It is also preferred that R 2 is ( ⁇ O); R 3 is H or (C 1 -C 10 )alkyl, and more preferably R 3 is methyl. Other preferred embodiments include R 4 is H or (C 1 -C 10 )alkyl, and more preferably, R 4 is H; R 5 is halogen, CN, NO 2 , trifluoromethyl or OH, and more preferably, R 5 is OH.
  • a compound of formula (III) includes X is O or S, preferably O; wherein both ----- are a single bond, wherein one ----- is a double bond, or wherein both ----- are a double bond.
  • R 1 is OH
  • R 2 is ( ⁇ O)
  • R 3 is methyl
  • R 4 is H
  • R 5 is OH
  • X is O, and both ----- are a double bond.
  • Yet another agent useful in the methods of the invention is a compound of formula (III):
  • R 1 is halogen, CN, NO 2 , trifluoromethyl or OH.
  • R 1 is OH.
  • R 2 is ( ⁇ O);
  • R 3 is H or (C 1 -C 10 )alkyl, and more preferably R 3 is methyl.
  • Other preferred embodiments include R 4 is H or (C 1 -C 10 )alkyl, and more preferably, R 4 is H;
  • R 5 is halogen, CN, NO 2 , trifluoromethyl or OH, and more preferably, R 5 is OH.
  • a compound of formula (IV) includes X is O or S, preferably O; wherein both ----- are a single bond, wherein one ----- is a double bond, or wherein both ----- are a double bond.
  • R 1 is OH
  • R 2 is ( ⁇ O)
  • R 3 is methyl
  • R 4 is H
  • R 5 is OH
  • X is O
  • both ----- are a double bond.
  • Another agent useful in the methods of the invention includes an agent that inhibits the activation of ubiquitin, the transfer of ubiquitin to the ubiquitin carrier protein, ubiquitin ligase, or a combination thereof.
  • Preferred ubiquitin ligase inhibitors include a compound of formula (IV): R-A-A 1 -R 1 wherein R is hydrogen, an amino acid, or a peptide, wherein the N-terminus amino acid can optionally be protected at the amino group with acetyl, acyl, trifluoroacetyl, or benzyloxycarbonyl;
  • A is an amino acid or a direct bond
  • a 1 is an amino acid
  • R 1 is hydroxy or an amino acid, wherein the C-terminus amino acid can optionally be protected at the carboxy group with (C 1 -C 6 )alkyl, phenyl, benzyl ester or amide (e.g., C( ⁇ O)NR 2 , wherein each R is independently hydrogen or (C 1 -C 6 )alkyl);
  • a specific value for R is hydrogen.
  • a specific value for A is an amino acid. Another specific value for A is Ile, Leu or His. Another specific value for A is Leu or His.
  • a specific value for A 1 is Ala or Gly. Another specific value for A 1 is Ala.
  • a specific value for R 1 is hydroxy.
  • the peptide can be a dipeptide (i.e., can comprise 2 amino acids).
  • the peptide can be H-Leu-Ala-OH, H-His-Ala-OH, H-Leu-Gly-OH, H-His-Gly-OH, H-Ile-Ala-OH, or H-Ile-Gly-OH. More specifically, the peptide can be H-Leu-Ala-OH or H-His-Ala-OH.
  • Alkyl denotes a straight or a branched group, but reference to an individual radical such as “propyl” embraces only the straight chain radical, a branched chain isomer such as “isopropyl” being specifically referred to.
  • Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic.
  • N-amino acid blocking groups are known to those skilled in the art (See, for example, T. W. Greene, Protecting Groups In Organic Synthesis; Wiley: N.Y., 1981, and references cited therein).
  • an agent may be employed in methods to inhibit reabsorption of salts and water from mucous secretions in a tissue or organ, e.g., in the lung, to hydrate mucous secretions in a tissue or organ, e.g., to increase airway surface liquid volume in the lung, to facilitate mucous clearance in a tissue or organ, to inhibit or treat conditions associated with aberrant ENaC activity, for instance, cystic fibrosis, Liddle syndrome, and pulmonary edema, as well as chronic bronchitis, asthma, and acute lung injury.
  • the identified agents may be administered alone, in combination with other agents, for instance, ENaC antagonists, and/or in combination with gene therapy vectors or vaccine vectors.
  • genetic material can be introduced into cells (such as mammalian “producer” cells for the production of viral vectors) using any of a variety of means to transform or transduce such cells.
  • such techniques include, for example, transfection with bacterial plasmids, infection with viral vectors, electroporation, calcium phosphate precipitation, and introduction using any of a variety of lipid-based compositions (a process often referred to as “lipofection”). Methods and compositions for performing these techniques have been described in the art and are widely available.
  • the polynucleotide sequences used to alter the cell may be introduced simultaneously with or operably linked to one or more detectable or selectable markers as is known in the art.
  • a drug-resistance gene as a selectable marker.
  • Drug-resistant cells can then be picked and grown, and then tested for expression of the desired sequence, i.e., a packaging gene product, or a product of the heterologous polynucleotide, as appropriate.
  • Testing for acquisition, localization and/or maintenance of an introduced polynucleotide can be performed using DNA hybridization-based techniques (such as Southern blotting and other procedures as is known in the art).
  • Testing for expression can be readily performed by Northern analysis of RNA extracted from the genetically altered cells, or by indirect immunofluorescence for the corresponding gene product. Testing and confirmation of packaging capabilities and efficiencies can be obtained by introducing to the cell the remaining functional components of the virus and a helper virus, to test for production of viral particles. Where a cell is inheritably altered with a plurality of polynucleotide constructs, it is generally more convenient (though not essential) to introduce them to the cell separately, and validate each step seriatim. References describing such techniques include those cited herein.
  • Viral vectors can be used for administration to an individual for purposes of gene or vaccine therapy.
  • Suitable diseases for gene or vaccine therapy include but are not limited to those induced by viral, bacterial, or parasitic infections, various malignancies and hyperproliferative conditions, autoimmune conditions, and congenital deficiencies.
  • Gene or vaccine therapy can be conducted to enhance the level of expression of a particular protein either within or secreted by the cell.
  • Vectors of this invention may be used to genetically alter cells either for gene marking, replacement of a missing or defective gene, or insertion of a therapeutic gene.
  • a polynucleotide may be provided to the cell that decreases the level of expression. This may be used for the suppression of an undesirable phenotype, such as the product of a gene amplified or overexpressed during the course of a malignancy, or a gene introduced or overexpressed during the course of a microbial infection.
  • Expression levels may be decreased by supplying a therapeutic polynucleotide comprising a sequence capable, for example, of forming a stable hybrid with either the target gene or RNA transcript (antisense therapy), capable of acting as a ribozyme to cleave the relevant mRNA or capable of acting as a decoy for a product of the target gene.
  • a therapeutic polynucleotide comprising a sequence capable, for example, of forming a stable hybrid with either the target gene or RNA transcript (antisense therapy), capable of acting as a ribozyme to cleave the relevant mRNA or capable of acting as a decoy for a product of the target gene.
  • the introduction of viral vectors by the methods of the present invention may involve use of any number of delivery techniques (both surgical and non-surgical) which are available and well known in the art.
  • delivery techniques include vascular catheterization, cannulization, injection, inhalation, inunction, topical, oral, percutaneous, intra-arterial, intravenous, and/or intraperitoneal administrations.
  • Vectors can also be introduced by way of bioprostheses, including, by way of illustration, vascular grafts (PTFE and dacron), heart valves, intravascular stents, intravascular paving as well as other non-vascular prostheses.
  • PTFE and dacron vascular grafts
  • Heart valves intravascular stents
  • intravascular paving as well as other non-vascular prostheses.
  • General techniques regarding delivery, frequency, composition and dosage ranges of vector solutions are within the skill of the art.
  • Vector means both a bare recombinant vector and vector DNA packaged into viral coat proteins, as is well known for virus administration. Simply dissolving a virus vector in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be coadministered with the vector (although compositions that degrade DNA should be avoided in the normal manner with vectors).
  • Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the invention.
  • the vectors can be used with any pharmaceutically acceptable carrier for ease of administration and handling.
  • solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions.
  • aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose.
  • Solutions of the viral vector as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • a dispersion of viral particles can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the viral vector in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • dilute sterile, aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, are prepared in containers suitable for incorporation into a transdermal patch, and can include known carriers, such as pharmaceutical grade dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • CFTR cystic fibrosis transmembrane conductance regulator
  • compositions of this invention may be used in vivo as well as ex vivo.
  • In vivo gene therapy comprises administering the vectors of this invention directly to a subject.
  • Pharmaceutical compositions can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to use.
  • a preferred mode of administration is by aerosol, using a composition that provides either a solid or liquid aerosol when used with an appropriate aerosolubilizer device.
  • Another preferred mode of administration into the respiratory tract is using a flexible fiberoptic bronchoscope to instill the vectors.
  • the viral vectors are in a pharmaceutically suitable pyrogen-free buffer such as Ringer's balanced salt solution (pH 7.4).
  • pharmaceutical compositions may optionally be supplied in unit dosage form suitable for administration of a precise amount.
  • an effective amount of virus is administered, depending on the objectives of treatment.
  • An effective amount may be given in single or divided doses.
  • the objective of treatment is generally to meet or exceed this level of transduction.
  • this level of transduction can be achieved by transduction of only about 1 to 5% of the target cells, but is more typically 20% of the cells of the desired tissue type, usually at least about 50%, preferably at least about 80%, more preferably at least about 95%, and even more preferably at least about 99% of the cells of the desired tissue type.
  • the number of vector particles present in a singl dose given by bronchoscopy will generally be at least about 1 ⁇ 10 8 , and is more typically 5 ⁇ 10 8 , 1 ⁇ 10 10 , and on some occasions 1 ⁇ 10 11 particles, including both DNAse-resistant and DNAse-susceptible particles.
  • the dose will generally be between 1 ⁇ 10 6 and 1 ⁇ 10 14 particles, more generally between about 1 ⁇ 10 8 and 1 ⁇ 10 12 particles.
  • the treatment can be repeated as often as every two or three weeks, as required, although treatment once in 180 days may be sufficient.
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence of a polypeptide expressed from a gene present in the vector, e.g., by immunological means (immunoprecipitations, immunoaffinity columns, ELISAs and Western blots) or by any other assay useful to identify the presence and/or expression of a particular nucleic acid molecule falling within the scope of the invention.
  • “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence of a polypeptide expressed from a gene present in the vector, e.g., by immunological means (immunoprecipitations, immunoaffinity columns, ELISAs and Western blots) or by any other assay useful to
  • RNA produced from introduced DNA segments may be employed.
  • PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA.
  • PCR techniques while useful, will not demonstrate integrity of the RNA product.
  • Further information about the nature of the RNA product may be obtained by Northern blotting. This technique demonstrates the presence of an RNA species and gives information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and only demonstrate the presence or absence of an RNA species.
  • Southern blotting and PCR may be used to detect the DNA segment in question, they do not provide information as to whether the DNA segment is being expressed. Expression may be evaluated by specifically identifying the polypeptide products of the introduced DNA sequences or evaluating the phenotypic changes brought about by the expression of the introduced DNA segment in the host cell.
  • the effectiveness of the genetic alteration can be monitored by several criteria. Samples removed by biopsy or surgical excision may be analyzed by in situ hybridization, PCR amplification using vector-specific probes, RNAse protection, immunohistology, or immunofluorescent cell counting. When the vector is administered by bronchoscopy, lung function tests may be performed, and bronchial lavage may be assessed for the presence of inflammatory cytokines. The treated subject may also be monitored for clinical features, and to determine whether the cells express the function intended to be conveyed by the therapeutic polynucleotide.
  • helper virus e.g., adenovirus
  • cellular proteins e.g., adenovirus
  • Administration of the agents identified in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • agents of the invention are amenable to chronic use, preferably by systemic administration.
  • the agents of the invention including a compound of formula (I), (II), (III), or (TV) including their salts, are preferably administered at dosages of about 0.01 ⁇ M to about 1 mM, more preferably about 0.1 ⁇ M to about 40 ⁇ M, and even more preferably, about 1 ⁇ M to 40 ⁇ M, although other dosages may provide a beneficial effect.
  • preferred dosages of LLnL include about 1 ⁇ M to 40 ⁇ M.
  • One or more suitable unit dosage forms comprising the agents of the invention can be administered by a variety of routes including oral, or parenteral, including by rectal, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrapulmonary and intranasal routes.
  • routes including oral, or parenteral, including by rectal, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrapulmonary and intranasal routes.
  • intravenous administration is preferred.
  • intrapulmonary administration intrapulmonary administration
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
  • the agents of the invention are prepared for oral administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • a pharmaceutically acceptable carrier diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • the total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.
  • pharmaceutically acceptable it is meant the carrier, diluent, excipient, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • the active ingredient for oral administration may be present as a powder or as granules; as a solution, a suspension or an emulsion; or in achievable base such as a synthetic resin for ingestion of the active ingredients from a chewing gum.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • compositions containing the agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients.
  • the agent can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like.
  • excipients, diluents, and carriers that are suitable for such formulations include the following fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose, HPMC and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.
  • fillers and extenders such as starch, sugars, mannitol, and silicic derivatives
  • binding agents such as carboxymethyl cellulose, HPMC and other cellulose derivatives
  • tablets or caplets containing the agents of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate.
  • Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, and zinc stearate, and the like.
  • Hard or soft gelatin capsules containing an agent of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil.
  • enteric coated caplets or tablets of an agent of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.
  • agents of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.
  • the pharmaceutical formulations of the agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.
  • the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative.
  • the active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • formulations can contain pharmaceutically acceptable vehicles and adjuvants which are well known in the prior art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name “Dowanol”, polyglycols and polyethylene glycols, C 1 -C 4 alkyl esters of short-chain acids, preferably ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name “Miglyol”, isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.
  • organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name “Dowanol”, poly
  • compositions according to the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They can also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.
  • an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes and colorings.
  • other active ingredients may be added, whether for the conditions described or some other condition.
  • t-butylhydroquinone t-butylhydroquinone
  • butylated hydroxyanisole butylated hydroxytoluene
  • ci-tocopherol and its derivatives may be mentioned.
  • the galenical forms chiefly conditioned for topical application take the form of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, or alternatively the form of aerosol formulations in spray or foam form or alternatively in the form of a cake of soap.
  • the agents are well suited to formulation as sustained release dosage forms and the like.
  • the formulations can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal or respiratory tract, possibly over a period of time.
  • the coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, and the like.
  • the agents of the invention can be delivered via patches for transdermal administration. See U.S. Pat. No. 5,560,922 for examples of patches suitable for transdermal delivery of an agent.
  • Patches for transdermal delivery can comprise a backing layer and a polymer matrix which has dispersed or dissolved therein an agent, along with one or more skin permeation enhancers.
  • the backing layer can be made of any suitable material which is impermeable to the agent.
  • the backing layer serves as a protective cover for the matrix layer and provides also a support function.
  • the backing can be formed so that it is essentially the same size layer as the polymer matrix or it can be of larger dimension so that it can extend beyond the side of the polymer matrix or overlay the side or sides of the polymer matrix and then can extend outwardly in a manner that the surface of the extension of the backing layer can be the base for an adhesive means.
  • the polymer matrix can contain, or be formulated of, an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer.
  • an adhesive polymer such as polyacrylate or acrylate/vinyl acetate copolymer.
  • Examples of materials suitable for making the backing layer are films of high and low density polyethylene, polypropylene, polyurethane, polyvinylchloride, polyesters such as poly(ethylene phthalate), metal foils, metal foil laminates of such suitable polymer films, and the like.
  • the materials used for the backing layer are laminates of such polymer films with a metal foil such as aluminum foil. In such laminates, a polymer film of the laminate will usually be in contact with the adhesive polymer matrix.
  • the backing layer can be any appropriate thickness which will provide the desired protective and support functions.
  • a suitable thickness will be from about 10 to about 200 microns.
  • those polymers used to form the biologically acceptable adhesive polymer layer are those capable of forming shaped bodies, thin walls or coatings through which agents can pass at a controlled rate.
  • Suitable polymers are biologically and pharmaceutically compatible, nonallergenic and insoluble in and compatible with body fluids or tissues with which the device is contacted. The use of soluble polymers is to be avoided since dissolution or erosion of the matrix by skin moisture would affect the release rate of the agents as well as the capability of the dosage unit to remain in place for convenience of removal.
  • Exemplary materials for fabricating the adhesive polymer layer include polyethylene, polypropylene, polyurethane, ethylene/propylene copolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetate copolymers, silicone elastomers, especially the medical-grade polydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates, chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, crosslinked polymethacrylate polymers (hydrogel), polyvinylidene chloride, poly(ethylene terephthalate), butyl rubber, epichlorohydrin rubbers, ethylene vinyl alcohol copolymers, ethylene-vinyloxyethanol copolymers; silicone copolymers, for example, polysiloxane-polycarbonate copolymers, polysiloxane-polyethylene oxide copolymers, polysiloxane-polymeth
  • a biologically acceptable adhesive polymer matrix should be selected from polymers with glass transition temperatures below room temperature.
  • the polymer may, but need not necessarily, have a degree of crystallinity at room temperature.
  • Cross-linking monomeric units or sites can be incorporated into such polymers.
  • cross-linking monomers can be incorporated into polyacrylate polymers, which provide sites for cross-linking the matrix after dispersing the agent into the polymer.
  • Known cross-linking monomers for polyacrylate polymers include polymethacrylic esters of polyols such as butylene diacrylate and dimethacrylate, trimethylol propane trimethacrylate and the like.
  • Other monomers which provide such sites include allyl acrylate, allyl methacrylate, diallyl maleate and the like.
  • a plasticizer and/or humectant is dispersed within the adhesive polymer matrix.
  • Water-soluble polyols are generally suitable for this purpose. Incorporation of a humectant in the formulation allows the dosage unit to absorb moisture on the surface of skin which in turn helps to reduce skin irritation and to prevent the adhesive polymer layer of the delivery system from failing.
  • transdermal drug delivery system must be capable of penetrating each layer of skin.
  • a transdermal drug delivery system must be able in particular to increase the permeability of the outermost layer of skin, the stratum corneum, which provides the most resistance to the penetration of molecules.
  • the fabrication of patches for transdermal delivery of agents is well known to the art.
  • the agents of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the composition may take the form of a dry powder, for example, a powder mix of the agent and a suitable powder base such as lactose or starch.
  • a powder mix of the agent and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatine or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.
  • the agent may be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler.
  • atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).
  • the local delivery of the agents of the invention can also be by a variety of techniques which administer the agent at or near the site of disease.
  • site-specific or targeted local delivery techniques are not intended to be limiting but to be illustrative of the techniques available.
  • local delivery catheters such as an infusion or indwelling catheter, e.g., a needle infusion catheter, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct applications.
  • the agents may be formulated as is known in the art for direct application to a target area.
  • Conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • the active ingredients can also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842.
  • the percent by weight of an agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1-25% by weight.
  • Drops such as eye drops or nose drops, may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents.
  • Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
  • the agent may further be formulated for topical administration in the mouth or throat.
  • the active ingredients may be formulated as a lozenge fuirther comprising a flavored base, usually sucrose and acacia or tragacanth;
  • pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier.
  • inert base such as gelatin and glycerin or sucrose and acacia
  • mouthwashes comprising the composition of the present invention in a suitable liquid carrier.
  • compositions and compositions described herein may also contain other ingredients such as antimicrobial agents, or preservatives.
  • active ingredients may also be used in combination with other agents,. for example, bronchodilators.
  • agents of this invention may be administered to a mammal alone or in combination with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers As noted above, the relative proportions of active ingredient and carrier are determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
  • the dosage of the present agents will vary with the form of administration, the particular compound chosen and the physiological characteristics of the particular patient under treatment. Generally, small dosages will be used initially and, if necessary, will be increased by small increments until the optimum effect under the circumstances is reached.
  • agents of the invention will be further described by, but is not limited to, the following examples.
  • agents of the invention are useful for all serotypes and pseudotypes of rAAV vectors.
  • nocodazole Sigma, St. Louis, Mo.; depolymerizes microtubules and causes lysosomal scattering
  • vinblastine sulfate Sigma, St. Louis, Mo.; depolymerizes microtubules, inhibits endocytosis by blocking intracellular endosomes and lysosomes movement
  • cytochalasin B Sigma, St. Louis, Mo.; depolymerizes microfilaments, i.e., actin, and blocks fusion of endosome with lysosome. Inhibits endocytosis by blocking intracellular endosome and lysosome movement
  • brefeldin A BFA, Sigma, St.
  • LLnL N-acetyl-L-Leucinyl-L-leucinal-L-norleucinal; Calbiochem-Novabiochem Corp., La Jolla, Calif.
  • Z-LLL N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal; Calbiochem-Novabiochem Corp., La Jolla, Calif.
  • tripeptides are structurally related to chloroquine but have different lipid solubility and specificity for cysteine proteases (Seglen, 1983). These molecules decrease endosomal degradation of molecules by a mechanism different than altering pH. They also have been shown to inhibit 26S ubiquitin and proteasome-dependent proteolytic pathways (Rock et al., 1994).
  • tripeptides LLnL and Z-LLL both significantly increased rAAV transduction. These tripeptides have been previously used to increase the transfection efficiency of plasmid DNA and are thought to inhibit the lysosomal degradation of DNA (Coonrod et al., 1997).
  • microtubule depolymerizing agents such as vinblastine did not inhibit rAAV-2 transduction are different than that previously reported for nocodazole inhibition of canine parvovirus (Vihinen-Tanta et al., 1998).
  • studies with tripeptide protease inhibitors demonstrated a significant augmentation in rAAV transduction.
  • endosomal degradation of virus and/or endosomal release may be an important rate-limiting step in rAAV transduction.
  • Endosomal Processing Inhibitors May Increase rAAV Transduction in Polarized Airway Cells
  • the culture medium used to feed only the basolateral side of the cells, contained 49% DMEM, 49% Ham's F12 and 2% Ultraser G (BioSepra, Cedex, France).
  • Dimethyl Sulphoxide (DMSO), camptothecin (Camp), etoposide (Etop), aphidicolin (Aphi), hydroxyurea (HU) and genistein (Geni) were purchased from Sigma (St. Louis, Mo.).
  • Tripeptidyl aldehyde proteasome inhibitors N-Acetyl-L-Leucyl-L-Leucyl-Norleucine (LLnL) and benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal (Z-LLL) were purchased from Calbiochem-Novabiochem Corporation (La Jolla, Calif.).
  • Ubiquitin ligase (E3) inhibitors were obtained from Bachem Bioscience Inc. (King of Prussia, Pa.).
  • Anti-AAV capsid monoclonal antibody Anti-AAV capsid monoclonal antibody (Anti-VP1,2 and 3) was purchased from American Research Products (Belmont, Mass.) and anti-ubiquitin antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.).
  • Recombinant AAV was produced by a CaPO 4 co-transfection protocol and purified through three rounds of isopycnic cesium chloride ultracentrifugation as described above in Example 1.
  • the proviral plasmid pCisAV.GFP3ori is described in Duan et al. (1998).
  • the proviral plasmid pCisRSV.Alkphos which encodes the alkaline phosphatase reporter gene under the transcriptional control of the RSV promoter and SV40 poly-adenylation signal, was used to generate AV.Alkphos (Yang et al., 1999).
  • the proviral plasmid pCisRSV.LacZ used for AV.LacZ production was generated by first inserting 3474 bp Not I digested ⁇ -galactosidase gene (from pCMVf ⁇ , Clontech) into the Not I site of the pRep4 (Invitrogene).
  • Millicell inserts were returned to the upright position, in the continued presence of the original viral inoculum plus an additional 450 ⁇ l of media.
  • rAAV containing media was removed after 24 hours and replaced with either fresh culture media (for the basal side) or exposed to air (for the apical side).
  • 1 ⁇ l of each solution was mixed with AAV prior to infection of airway epithelia. Agents were usually presented during the 24 hours AAV infection period unless indicated otherwise.
  • agents were dissolved in DMSO except for hydroxyurea (dissolved in phosphate buffered saline), H-Leu-Ala-OH (dissolved in 0.9% glacial acetic acid) and H-His-Ala-OH (dissolved in 50% methanol).
  • the working concentrations of the agents were as follows: 0.1 ILM camptothecin, 10 ⁇ M etoposide, 5 ⁇ g/ml aphidicolin, 40 mM hydroxyurea, 50 ⁇ M genistein, 40 ⁇ M LLnL and 4 ⁇ M Z-LLL.
  • Human primary fibroblast cells were maintained in 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, and 89% DMEM. Infection with AV.GFP3ori was performed with 80% confluent fibroblasts at an MOI of 1000 DNA particles/cell in 2% FBS DMEM for 24 hours.
  • L-methionine was added back to a final concentration of 30 mg/L, and cells were incubated for an additional 30 hours at 37° C.
  • Cell lysates were prepared and virus was purified by isopycnic cesium chloride ultracentrifugation as described above. Typical specific activities of labeled virus preparations were 5 ⁇ 10 ⁇ 6 cpm/particle, which is slightly higher than the 5.5 ⁇ 10 ⁇ 7 cpm/particle specific activity reported by other investigators (Bartlet et al., 1999).
  • the 1.6 kb single stranded viral DNA, the 2.7 kb double stranded circular intermediate, and the 4.7 kb double stranded replication from viral genome were detected with a transgene EGFP specific probe at 5 ⁇ 10 6 cpm/ml. Blots were washed at a stringency of 0.2 ⁇ SSC/0.1% SDS at 55° C. for 20 minutes twice. In studies aimed at evaluating viral internalization, virus attached to the cell surface was removed by trypsinization with 1 ml of buffer containing 0.5% trypsin, and 5.3 mM EDTA at 37° C.
  • AAV virus was determined by the intensity of the 1.6 kb viral genome band in Hirt DNA extracted from cells infected at 4° C. for 60 minutes.
  • the internalized virus was determined by the intensity of the 1.6 kb viral genome band in Hirt DNA extracted from trypsinized cells after infection at 37° C. for 4 and 24 hours. The dynamic changes in the molecular structure of the internalized virus were assayed at 2, 10, 30 and 50 days after virus was removed from culture medium.
  • ubiguitinated AAV capsid proteins by immunoprecipitation.
  • human primary fibroblasts were lysed at 6 hours post-viral infection in 1 ⁇ RIPA buffer. Cell lysates were then cleared with 30 ⁇ l Protein A-Agarose. The supernatant was incubated with 10 ⁇ l of monoclonal anti-VP1, 2, and 3 antibody (Clone B1, ARP) followed by the addition of 30 ⁇ l Protein A-Agarose. The pellets were washed 4 times with I ⁇ RIPA buffer and resolved on a 10% SDS-PAGE.
  • Viral infection controls in the absence of Z-LLL also contained a 1% final concentration of ethanol. Since studies in both primary cultured human airway cells and fibroblasts have demonstrated similar enhancement efficiency between 40 ⁇ M LLnL and 4 ⁇ M Z-LLL, and also due to the poor solubility of LLnL in ethanol (Example 7 employed a low dose of LLnL in DMSO which was administered to the trachea), only Z-LLL was tested in this particular mouse lung study. The animals were euthanized at 2, 10 and 150 days post infection and PBS (10 ml) was instilled into the right ventricle, followed by removal of the lungs and heart as an intact cassette.
  • the trachea was incubated and instilled at 10 cm of water pressure with the following solutions in order: PBS, 0.5% glutaraldehyde, 1 mM MgCl 2 /PBS, and finally X-gal staining reagent for an overnight incubation at room temperature.
  • the X-gal stained mouse lungs were then post fixed in 10% neutral buffered formalin for 48 hours at room temperature and cryopreserved in serial 10%, 20% and 30% sucrose/PBS solutions.
  • the diameter of the airway was recorded for classification (>360 ⁇ m, 260-350 ⁇ m, 160-250 ⁇ m, ⁇ 150 ⁇ m) of results following morphometric analysis. Greater than 150 airway cross-sections were quantified for each experimental condition.
  • Hirt DNA from the cultures was evaluated by Southern blot hybridization with 32 P-labeled EGFP probes.
  • a significant amount of apically applied rAAV was able to infect airway cells.
  • only single stranded viral genomes (ssDNA) were detected at this time point (50 days).
  • ssDNA single stranded viral genomes
  • rAAV can be endocytosed from the mucosal surface and that the endocytosed viral ssDNA was stably sequestered in some unknown subcellular compartment.
  • the majority of basolaterally applied rAAV was converted into double stranded forms that migrated at 2.8 kb and >12 kb in 1% non-denaturing agarose gels.
  • Proteasome modulators dramatically enhance rAAV infection in polarized airway epithelia. Given the fact that rAAV appears to remain latent within some cellular compartment(s) following apical infection in the airway, and that agents that alter the molecular conversion of the viral genome might enhance rAAV transduction in airway epithelia, several agents were tested in this regard, including DNA damaging agents (Alexander et al., 1994), DNA synthesis and topoisomerase inhibitors (Russell et al., 1995), and cellular tyrosine kinases inhibitors (Qing et al., 1997; Man et al., 1998).
  • DNA damaging agents Alexander et al., 1994
  • DNA synthesis and topoisomerase inhibitors Russell et al., 1995
  • cellular tyrosine kinases inhibitors Qing et al., 1997; Man et al., 1998).
  • camptothecin, etoposide, hydroxyurea, and genistein resulted a 10 to 60 fold enhancement in rAAV transduction from the basolateral surface.
  • none of these agents facilitated rAAV transduction from the apical surface (data not shown).
  • chemicals known to affect intra-nuclear events involved in rAAV transduction in other cell types did not enhance rAAV apical infection in the airway, other agents affecting endocytic processing, such as the ubiquitin-proteasome pathway, were tested.
  • Proteasome systems are known to modulate the intracellular processing of many foreign and endogenous molecules, including viruses such as HIV (Schwartz et al., 1998).
  • viruses such as HIV
  • Several specific, cell permeable, peptide aldehyde inhibitors of proteasome pathways have recently been discovered (Rock et al., 1994; Fenteany et al., 1995). These inhibitors bind to the active sites of proteolytic enzymes within the proteasome core and reversibly block their function (Rubin et al., 1995).
  • the tripeptidyl aldehyde proteasome inhibitor (a cysteine protease inhibitor) N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL, also called Calpain inhibitor I) was applied to polarized cultures of human bronchial epithelial cells at the time of rAAV infection. Surprisingly, a greater than 200 fold augmentation in transgene expression was obtained at 2 days post infection when 40 ⁇ M LLnL was applied to the serosal surface along with rAAV.
  • LLnL cysteine protease inhibitor
  • proteasome modulators augment rAAV transduction in airway epithelia in a polarized fashion.
  • proteasome modulators appear to significantly increase the efficacy of rAAV transduction from the serosal surface, the route most germane to clinical application of gene delivery in the airway is the mucosal surface.
  • a side-by-side kinetic comparison of rAAV transduction from both mucosal and serosal surfaces of airway epithelia following treatment with LLnL was performed.
  • Co-administration of LLnL and rAAV to the mucosal surface resulted a sustained augmentation in AAV transduction, which peaked at 22 days post-infection.
  • a rAAV vector encoding the alkaline phosphatase gene (Alkphos) was utilized. Transduced cell types were evaluated by standard histochemical staining for Alkphos to address this question. In the absence of LLnL, rAAV preferentially transduced basal cells at 3 days following serosal application of virus. Consistent with previous findings utilizing AV.GFP3ori virus, co-administration of LLnL resulted in a dramatic increase in AV.Alkphos transduction. Interestingly, ciliated cell transduction was most significantly increased by treatment with LLnL at the time of rAAV infection.
  • basal cells were the least responsive to LLNL treatment.
  • LLnL Viral binding and internalization are not affected by LLNL treatment.
  • the action of LLnL has been typically attributed to it selective and reversible inhibition of the proteasome system. However, it was important to rule out any possible effect on viral binding and/or endocytosis.
  • type 1 herpes simplex virus As has been found for type 1 herpes simplex virus (Everett et al., 1998), LLnL treatment had no significant effect on 4° C. rAAV binding. Similarly, the uptake of S 35 labeled rAAV for a 2 hour infection period at 37° C. was not altered by LLnL treatment. Given these results, LLnL acts at points distal to virus binding and entry.
  • LLnL enhances endosomal processing and nuclear trafficking of rAAV.
  • in situ localization of the S 35 -labeled rAAV particles following infection from the apical and basolateral surfaces was performed in the presence and absence of LLnL. Since loss of intact radiolabeled capsid proteins occurred at 24 hours post-infection, a 2 hour time point was chosen for this analysis.
  • photoemulsion overlay the subcellular distribution of S 35 -labeled rAAV particles was evaluated by blinded morphometric analysis. The majority of viral particles localized to the cytoplasm in the absence of LLnL.
  • LLnL augment rAAV transduction within a specific time frame after infection.
  • Evidence thus far has suggested that LLnL may act to increase intracellular routing of rAAV to the nucleus.
  • LLnL action is independent of the epithelial surface to which it is administered (i.e., serosal application of LLnL augments mucosal infection and vice versa). This indicates that LLnL need not be endocytosed with AAV particles to enhance transduction.
  • LLnL may act at a specific time following rAAV endocytosis but during endosomal processing.
  • a kinetic analysis of LLnL action at various times after infection from the basolateral surface was performed.
  • LLnL was added to the culture medium either at the time of AAV infection or at various time points after infection. Viral-mediated transgene expression was quantified at 24 hour intervals following infection. Augmentation was achieved regardless of whether LLnL was administrated at 0, 24, 48, and 72 hours after viral infection. However, addition of LLnL at 24 or 48 hours gave the strongest level of augmentation. The ability of LLnL to reduce AAV expression appeared to decline by 72 hour post-infection and was completely lost by 15 days after the initial AAV infection (data not shown). Taken together, it appears that after rAAV enters the cell, it may be targeted to an intracellular compartment that is sensitive to proteasome inhibitor-facilitated liberation. In addition, the loss of an LLnL augmentation effect at 15 days post-infection suggests that enhanced transcription, translation, and/or stability of the transgene products do not likely contribute to the mechanism responsible for this observation.
  • rAAV genomes in infected cells were analyzed by Southern blotting Hirt DNA. Consistent with studies using S 35 labeled virus, rAAV binding to either surface of polarized airway epithelia was not affected by LLnL treatment. Southern blotting also demonstrated 2 to 7 fold higher viral binding from the basal surface, which varied among different tissue samples (data not shown). The extent of virus internalization was compared after stripping surface bound virus with trypsin.
  • AAV capsid proteins were immunoprecipitated using anti-VP 1,2, 3 antibody from rAAV infected human polarized airway cells and confluent human fibroblasts at 6 hours post-viral infection. Subsequent Western analysis with anti-ubiquitin specific antibodies demonstrated a significant increase in the cellular level of ubiquitinated AAV capsid in fibroblasts following proteasome treatment.
  • AV.LacZ (5 ⁇ 10 10 particles) was delivered either alone or in the presence of 400 ⁇ M Z-LLL by intranasal administration.
  • Mouse lungs were harvested at 3, 10 and 150 days post-infection to evaluate short and long term effects.
  • Proteasome inhibitor treatment from basal surface, or in conjunction with EGTA from apical surface resulted in pronounced, immediate enhancement on rAAV transduction, however, X-gal staining of the lung tissues at 3 and 10 days post infection demonstrated no detectable transgene expression in either proteasome inhibitor treated or untreated groups. In contrast, significant transduction was achieved at 150 days in Z-LLL treated samples.
  • Targeted transgene expression was predominantly confined to the conducting airways, rather than in the parenchyma. Alveolar cells were rarely transduced. Although on average only about 5.88% of airway cells were transduced by AV.LacZ, and LacZ positive cells were observed throughout the entire conducting airway, a characteristic transduction profile was evident.
  • the transduction efficiency in larger bronchioles (>350 mm) reached a mean of 10.36 ⁇ 1.63% of the airway epithelium, while 1.37 ⁇ 0.41% of airways cells in the smaller bronchioles ( ⁇ 150 mm) expressed the ⁇ -galactosidase transgene.
  • the range of transgene expression in distal and proximal airways was 0 to 4% and 5 to 18%, respectively.
  • Endosomal processing barriers to rAAV transduction may not be limited to polarized epithelial cells.
  • impaired intracellular trafficking of viral particles to the nucleus has been observed in NIH 3T3 cells.
  • rAAV can remain in an inactive state for as long as 7 days in confluent primary fibroblast cells until rescued by UV irradiation to a functionally active state.
  • post-endocytic barriers to infection exist in multiple cell types.
  • proteasome is commonly know as a compartment for clearance of endogenous and foreign proteins.
  • proteasome inhibitors From the standpoint of gene delivery, proteasome inhibitors have been shown to protect transfected plasmid DNA from degradation (Coonrod et al., 1997).
  • the results described herein clearly demonstrate that rAAV mediated gene transfer to the airway epithelia is also significantly enhanced by proteasome inhibitors. Furthermore, this enhancement is correlated with proteasome inhibitor stimulated viral trafficking to the nucleus.
  • proteasome inhibitors increased long-term levels of AAV transduction form the apical surface, their effect on basolateral infection appeared predominantly to alter the rate, rather than the long-term levels, of transduction. These differences highlight fundamentally distinct pathways involved in rAAV transduction from apical and basolateral surfaces.
  • ubiquitination of virus following endocytosis may be a critical mechanism for sorting incoming AAV.
  • treatment of airway epithelia with proteasome inhibitors know to block ubiquitin-dependent degradation of proteins enhances rAAV gene transfer.
  • inhibition of ubiquitin E3 ligase activity in airway epithelia also enhances transduction.
  • rAAV capsid proteins are ubiquitinated following infection in confluent human fibroblasts, and that the extent of this ubiquitination is increased by inhibition of ubiquitin-proteasome degradative pathways.
  • the in vivo activity of rAAV in the presence or absence of an agent of the invention in the lung or liver may be tested using the LacZ gene.
  • Virus was directly instilled into C57Balb/c mice trachea with a 30 G needle in a total volume of 30 ⁇ l.
  • Ninety days after infection lungs were harvested intact and fixed in 4% paraformaldehyde followed by cryosection.
  • AAV-mediated transgene expression was evaluated by 10 ⁇ m tissue sections staining forLac Z.
  • Recombinant AV.LacZ (5 ⁇ 10 10 particles) was also administered to mouse liver either as virus alone in PBS, virus in combination with 40 ⁇ M Z-LLL in PBS, or virus in combination with 20 ⁇ M LLnL in PBS. Virus was directly instilled into portal vein of the C57B6 mice. AAV-mediated LacZ transgene expression was evaluated by histology staining at 2 and 4 weeks post infection in frozen tissue sections.
  • any number of cells can be used.
  • a range of concentrations of the agent to be tested can be determined based on, for instance desirable profiles of the agent, desirable toxicity profiles of the agent and/or concentration of the agents employed in vivo.
  • the usefulness of the cell type chosen for the screen can be confirmed by testing compounds, e.g., proteasome inhibitors such as LLnL and ZLL which are known to increase rAAV transduction.
  • proteasome inhibitors such as LLnL and ZLL which are known to increase rAAV transduction.
  • a AAV2 FLAG-Luc vector was employed to transduce HeLa, ferret fibroblasts, IB3 and Huh (liver) cells in the presence or absence of the proteasome inhibitor MG132.
  • MG132 was confirmed to enhance AAV transduction in all cell types tested: HeLa cell transduction was enhanced about 500-fold at 80 ⁇ M, and 200-fold at 40 ⁇ M, MG132; ferret fibroblast cell transduction was enhanced about 200-fold at 20 ⁇ M, and 17-fold at 4 ⁇ M, MG132; IB3-1 cell transduction was enhanced about 30 to 70-fold at 20 to 80 ⁇ M MG132; and Huh-7 cell transduction was enhanced about 15- fold at 20 to 80 ⁇ M MG132. There was no difference in rAAV transduction efficiency in HeLa cells when either DMSO or ETOH was used as a vehicle for MG132.
  • HeLa cells were selected to screen for additional agents that enhance rAAV transduction, although any cell strain or line; or primary cells, may be employed.
  • Agents were selected from various classes, such as anti-inflammatories (e.g., dexamethasone and cyclosporin A), NSAIDs (e.g., ibuprofen), ⁇ -adrenergics (e.g., albuterol), antibiotics (e.g., ciprofloxacin, colison, gentamycin, tobramycin, and epoxomycin), lipid lowering agents (e.g., lovastatin, simvastatin and eicosapentaenoic acid), food additives (e.g., tannic acid), viral protease inhibitors (e.g., Norvir, Kaletra, and Viracept), chemotherapeutics (e.g., aclacinomycin A, doxorubicin, doxil, camp
  • HeLa cells were infected for 2 hours with an MOI of 100 rAAV in the presence of agents, e.g., ritonavir (Norvir) (1, 10 and 100 ⁇ M), cyclosporin A (2.5 and 250 ⁇ g/ml), epoxomicin (1, 10 and 50 ⁇ M), alcacinomycin A (5, 50 or 500 ⁇ M), chymostatin (1, 10 and 100 ⁇ M), bestatin (1, 10 and 100 ⁇ M), doxorubicin (adriamycin) (0.1, 1 and 10 ⁇ M), camptothecin (camptosar) (1, 10 and 100 ⁇ M), eicosapentanoic acid (1, 10 and 100 ⁇ M), tannic acid (2, 20, 200 and 2000 ⁇ M), simvastatin, prodrug (2, 20 and 200 ⁇ M), cisplatin (0.2, 2 and 20 ⁇ g/mL), and chloroquine (4, 40 and 400 ⁇ M
  • rAAV transduction was measured by removing the media from the cell cultures, adding 100 ⁇ L reporter lysis buffer (RLB) and freezing. The supernatant was thawed and transferred to microfuge tubes, freeze thawed an additional 2 times, clarified by centrifugation for 10 minutes and then analyzed for reporter gene expression on the lumometer. Protein was determined by Bradford analysis and results were expressed as relative light units per mg protein (RLU/mg). Data is presented in FIGS. 1 A-E.
  • doxorubicin and epoxomicin increased transduction efficiency up to 169-fold and 120-fold, respectively
  • camptothecin increased transduction efficiency by 15-fold
  • tannic acid increased transduction efficiency by 17-fold
  • cisplatin increased transduction efficiency by 16-fold
  • simvastatin increased transduction efficiency by 4-fold.
  • agents which initially screened as statistically negative may be reflective of formulations that are not readily bioavailable to cell culture cells or may be reflective of the limited dose range or exposure time.
  • Epoxomicin a naturally occurring antibiotic isolated from Actinomycetes known to inhibit NF-KB-mediated signaling in vivo and in vitro, inhibits proteasomes by inhibiting a proteasome-specific chymotrypsin-like protease.
  • Doxorubicin an anti-tumor antibiotic which inhibits topoisomerase II and inhibits nucleic acid synthesis, is translocated by a 20S proteasome from the cytoplasm to the nucleus. Camptothecin, a reversible DNA topoisomerase inhibitor, down regulates topoisomerase via an ubiquitin/26S proteasome pathway.
  • Simvastatin is an agent that modulates proteasome activity, tannic acid inhibits chymotrypsin-like activity and is a cancer chemopreventative, and cisplatin is a chemotherapeutic which crosslinks DNA.
  • Av2RSVIuc 5 ⁇ 10 8 particle/ ⁇ l
  • Av2RSVlucCap5 also referred to as Av2/5 CMVLuc
  • Av2CMVluc 2 ⁇ 10 9 particle/ ⁇ l
  • Av2CMVluc 1.3 ⁇ 10 9 particle/ ⁇ l
  • Av2CMVlucCap5 1.1 ⁇ 10 9 particle/ ⁇ l.
  • LLnL was used at 40, 200 or 400 ⁇ M, Z-LLL at 4 ⁇ M and doxorubicin at 0.5 or 1 ⁇ M when employed alone.
  • LLnL was used at 4, 10, 20, 40, 200 or 400 ⁇ M and doxorubicin at 1 or 5 , ⁇ M.
  • the apical surface of polarized airway epithelia, HeLa cells or ferret fibroblast was contacted with the agents and rAAV (5 ⁇ 10 9 particles per well).
  • LLnL enhances transduction in HeLa, ferret fibroblast and polarized epithelial cells at 40 ⁇ M and A549 cells at 200 to 400 ⁇ M.
  • Doxorubicin enhanced transduction in HeLa and ferret fibroblast cells at 1 ⁇ M and A549 or polarized airway cells at 5 ⁇ M, and enhanced transduction about 100 fold when ferret fibroblasts were infected with lacZ splicing vectors.
  • Doxorubicin also enhanced AAV2 and AAV5 transduction to a greater extent than LLnL. Synergistic effects were noted when doxorubicin and LLnL were co-administered.
  • agents of the invention can enhance rAAV transduction, including in serotype, pseudotype and multiple vector strategies.
  • Liposomal formulations have desirable properties for in vivo use including their increased stability or circulation half life making them more bioavailable in vivo. Those same characteristics make liposomal formulations less desirable for in vitro screening as described above.
  • one skilled in the art can design formulation strategies for agents of the invention to tailor them to the desired application.
  • one skilled in the art can tailor routes of delivery in order to maximize rAAV transduction efficiencies.
  • a pseudotyped rAAV vector encoding FVIII was tested in male Rag-1 mice.
  • Rag-1 mice were used because as described in the art, normal mice produce inhibitors of human FVIII that can obscure protein detection in the serum.
  • Rag-1 mice are known to be deficient in the pathways necessary to produce these inhibitors and thus will either produce no inhibitors, lower levels of inhibitors or have extended time periods for development of inhibitors.
  • the rAAV vector was constructed containing serotype 5 capsid proteins and 5′-3′ ITRs of AAV-2 flanking a heterologous transgene comprised of the minimal liver specific element HNF3/EBP and a human B-domain deleted FVIII gene (a second construct was identical except it contained a B-domain deleted canine FVIII gene).
  • agents that interact with molecules in intracellular AAV trafficking pathways such as proteasomes or molecules in the ubiquitin pathway, by binding to those molecules and/or inhibiting their activity, are useful to enhance rAAV transduction.
  • Inhibition of the proteasome with small tripeptide inhibitors such as LLnL can significantly augment rAAV-2 transduction from the apical membrane of both polarized human airway epithelia in vitro and mouse lung in vivo (Duan et al., 2000).
  • AAV-5 has been reported to have higher tropism for, and alternate receptors on, the apical membrane of airway epithelia
  • increased transduction of airway epithelia from the apical membrane with rAAV-5 might be due to altered proteasome involvement.
  • Co-administration of a proteasome modulator and a proteasome inhibitor was found to augment transduction of both serotypes in a cell type dependent manner.
  • FIGS. 2 and 6 A proviral construct containing 5′ and 3′ ITRs from AAV-2 flanking a transgene was packaged into both AAV-2 and AAV-5 capsid to generate AV2.RSVluc and AV2.RSVlucCap5 viruses which express the luciferase transgene.
  • rAAV-2, but not rAVV-5 demonstrated a significant difference in transduction from the apical versus basolateral surface.
  • AV2.RSVlucCap5 transduced epithelia from the apical and basolateral membranes with similar efficiencies at both time points.
  • LLnL augments AV2.RSVluc transduction from the apical and basolateral surfaces.
  • application of LLnL selectively increased AV2.RSVlucCap5 transduction 12-fold only when virus was applied to the apical surface.
  • the proteasome inhibitor Z-LLL was found to induce long-term (5 month) transduction with rAAV-2 in mouse lung.
  • mice were infected with 6 ⁇ 10 10 particles of AV2.RSVlucCap5 by nasal aspiration alone (control) or in combination with 200 ⁇ M Z-LLL, 200 ⁇ M doxorubicin or 200 ⁇ M Z-LLL and 200 ⁇ M doxorubicin (12 mice per group).
  • Co-administration of Z-LLL induced whole lung luciferase expression 17.2- and 2.1-fold at 14 (2 weeks) and 42 (6 weeks) days post-infection, respectively.
  • luciferase expression was further reduced at 3 months post-infection ( FIG. 2 ).
  • doxorubicin Co-administration of doxorubicin induced whole lung luciferase expression at levels almost ten times higher than those for Z-LLL at 2 weeks. Doxorubicin also induced tracheal and bronchi luciferase expression at higher levels than Z-LLL at 2 weeks. At six weeks, a similar pattern was observed for Z-LLL and doxorubicin alone, however, luciferase levels were more than additive in trachea and bronchi in mice co-administered virus, Z-LLL and doxorubicin. By three months post-infection, the synergism was no longer observed.
  • agents and vectors that result in a steady increase in transgene expression in particular cells over time may be useful for certain disorders or conditions while agents and vectors that result in a high burst of transgene expression may be useful for metabolic disorders such as hemophilia.
  • Ubiquitination and proteasome activity can influence a myriad of intracellular processes that control protein degradation and intracellular trafficking.
  • the following examples are designed to identify the molecular mechanisms of rAAV transduction that are controlled by the ubiquitin/proteasome system. These studies may lead to a clearer understanding of pathways and/or molecules that influence rate-limiting steps in rAAV transduction and can also be used to identify further useful agents to enhance processing of rAAV (i.e., endosomal escape, trafficking to the nucleus, and uncoating) and hence transduction.
  • Proteasome inhibitors may modulate this aspect of the rAAV life cycle by either changing the rate of endosomal escape and/or the compartment from which rAAV enters into the cytoplasm.
  • Alexa Fluor system from Molecular Probes was chosen as a system for which multiple fluorochromes could be linked to the rAAV capsid at similar efficiencies.
  • Three dyes Alexa Fluor® 488 [green], Alexa Fluoa® 568 [Red] and Alexa Fluor® 647 [blue] were selected as useful in this regard.
  • dual labeling of rAAV does not change the infection pattern.
  • microinjection of quenching antibodies against Alexa-488 can shift fluorescence of dual-labeled rAAV.
  • the general approach to assess endosomal escape is to inject the cytoplasm of living cells with anti-Alexa-488 following infection with rAAV that is dual labeled with Alexa-488 and one of the other dyes. Alexa-488/568 dual-labeled rAAV, a shift in fluorescence of virus from yellow to red (i.e., quenching of the green fluorochrome) indicates movement of virus into the cytoplasm.
  • This approach is used in combination with GFP-tagged endosomal compartments and/or dominant negative Rabs to evaluate the compartment from which rAAV moves into the cytoplasm.
  • Alexa labeling of rAAV The monovalent Alexa succinimidyl ester reactive dye (Alexa-488 and/or Alexa-568) was dissolved in 50 ⁇ l of 1 M bicarbonate. 0.5 ⁇ 10 12 particles (determined by slot blot) of purified AV2Luc in 0.5 ml Hepes buffer was added to the reaction mixture and incubated for 2 hours. When dual labeling was performed, equal molar amounts of the two fluorochromes was used and the reaction time was extended to 3 hours. The labeled rAAV2 was separated from the free dye by exclusion chromatography. The fractions were tested for infectious titers on HeLa cells using luciferase assays. The 5 peak fractions were then combined and used for fluorescent imaging studies. Imaging studies were performed.
  • Proteasome-modulating agents act to increase rAAV transduction through one or more of the following mechanisms: 1) increasing the rate at which rAAV accumulates in the primary compartment through which it emerges to the cytoplasm without changing the pathway of intracellular trafficking; 2) altering the pathway of rAAV intracellular trafficking in a manner that leads to more efficient accumulation in a compartment through which it emerges to the cytoplasm; 3) increasing the efficiency at which rAAV breaks out of the endosomal compartment; and/or 4) enhancing the rate of nuclear trafficking of free rAAV in the cytoplasm.
  • proteasome inhibitors may act to enhance rAAV transduction by increasing the rate of viral transport to the nucleus (Duan et al., 2000) and/or enhancing viral processing of the capsid (Yan et al., 2002).
  • proteasome inhibitors such as the tripeptides LLnL and Z-LLL enhance transduction of both rAAV2 or rAAV5, viruses without enhancing 1) endocytosis of virus, 2) stability of viral DNA within the cell, or 3) promoter activity which drives transgene expression (Duan et al., 2000; Yan et al., 2002).
  • proteasome inhibitors can be added up to a week following infection of polarized human airway epithelia and still enhance transduction (i.e., gene expression).
  • viral capsids for type 2 and type 5 show enhanced ubiquitination in vivo in the presence of proteasome inhibitors, and purified virus can also be ubiquitinated in vitro (Yan et al., 2002).
  • Proteasome Inhibitors Increase Transport of rAAV2 and rAAV2/5 Cell to the Nucleus
  • proteasome inhibitors A large number of various classes of proteasome inhibitors were screened to identify those that had the largest effect. Two classes of compounds (the tripeptidyl aldehyde LLnL and an anthracyclin derivative doxorubicin), and their ability to induce rAAV2 and rAAV2/5 transduction in two airway cell lines (IB3 and A549) are described below.
  • LLnL and Z-LLL are two tripeptidyl aldehydes shown to inhibit calpains, cathepsins, cysteine proteases as well as the chymotrypsin-like protease activity of proteasomes (Wagner et al., 2002; Donkor, 2000; Sasaki et al., 1990). Doxorubicin has also been shown to inhibit chymotrypsin-like protease activity of proteasomes (Kiyomiya et al., 2002). Both classes of proteasome inhibitors bind tightly to the proteasome complex.
  • Dose response curves for these two proteasome-modulating agents were evaluated on E33, A549, Hela, and primary fibroblasts. The responses were consistent for a number of cell lines and for three different promoters driving luciferase expression.
  • CMV-driven luciferase constructs with an AAV2-based genome were employed that were packaged into AV2 or AV5 capsids.
  • Cells were infected at various doses of AV2Luc and AV2/5Luc (MOIs 100 to 1000 particles/cell). At the time of infection, cells were treated with various concentrations of LLnL or Doxorubicin and gene expression was assayed at 24 hours post-infection.
  • Hela, A549, IB3, and primary fetal fibroblasts were evaluated for AV2Luc and AV2/5Luc transduction in the presence of LLnL, Dox, or LLnL+Dox at various concentrations.
  • the data shown is from Hela and A549 cells at the most optimal dose combination that induces rAV2Luc transduction to a greater extent than each compound alone.
  • Hela cells appear to provide a greater additive effects of Dox and LLnL on rAAV transduction than A549 cells. Furthermore, it should be noted that in primary fetal fibroblasts, no additive effect on transduction is seen (data not shown). In this cell line, Dox most significantly enhances transduction of rAAV2 and rAAV5, and LLnL provides no additional induction despite the fact it induced transduction 10-fold by itself. These interesting cell-specific differences also imply that certain cellular processes that alter rAAV transduction may be uniquely controlled by LLnL and Dox interactions with the proteasome.
  • Airway epithelial cells isolated from bronchial tissue obtained from CF or non-CF patients were seeded onto collagen-coated, semi-permeable membranes (0.6 cm 2 Millicel-HA; Millipore, Bedford, Mass.). Methods to generate these air/liquid interface cultures and the medium used were as described in Zabner et al. (1998).
  • Four viral vectors, AV2CF83, AV2tgCF, AV2/5CF83, and AV2/5tgCF were used to infect polarized airway epithelial cells from the apical membrane.
  • AV2tgCF is the current clinically-used AAV2-based full-length CFTR vector in which expression of CFTR is driven off the ITR (Aitken et al., 2001; Wagner et al., 2002).
  • AV2/5tgCF virus has the identical proviral structure to AV2tgCF, but is pseudotyped into AAV5 capsid.
  • AV2CF83 and AV2/5CF83 viruses have an additional 83 bp minimal promoter (Lynch et al., 1999) inserted into the AV2tgCF proviral genome to increase gene expression, and were packaged into AAV2 and AAV5 capsids, respectively.
  • the airway epithelial cultures were infected with 75 ⁇ l of virus-containing medium applied to the apical surface of the epithelia at an MOI of 10 5 particles/cell in the presence or absence of 40 ⁇ M LLnL and 5 ⁇ M doxorubicin. The cells were incubated at 37° C.
  • Short-circuit current (Isc) measurement in polarized airway epithelia Transepithelial short-circuit currents were measured using an epithelial voltage clamp and a self-contained Ussing chamber as described in Zabner et al. (1998). The basolateral side of the chamber was filled with Ringer's buffer solution containing 135 mM NaCl, 1.2 mM CaCl 2 , 2.4 mM KH 2 PO 4 , 0.2 mM K 2 HPO 4 , 1.2 mM MgCl 2 , and 5 mM HEPES, pH 7.4.
  • the apical side of the chamber was filled with a low-chloride Ringer's containing 135 mM sodium gluconate, 1.2 mM CaCl 2 , 2.4 mM KH 2 PO 4 , 0.2 mM K 2 HPO 4 , 1.2 mM MgCl 2 , and 5 mM HEPES, pH 7.4.
  • the chamber was maintained at 37° C. and aerated with 100% O 2 .
  • Transepithelial voltage was clamped at zero, and the resulting Isc was measured and recorded following the sequential addition of the following channel antagonist and agonists: 1) 100 ⁇ M amiloride (apical), 2) 100 ⁇ M 4,4′-diisothiocyna-2,2′-disulfonic stilbene (DIDS) (apical), 3) 100 ⁇ M IBMX/10 ⁇ M forskolin (apical), 4) 100 ⁇ M bumetanide (basolateral). Voltage was referenced to the apical compartment. The series resistance of the Ringer's solution and transwell membrane was electrically compensated before starting experiments. All chemical agonists and antagonists were added to either the apical or the basolateral sides of the monolayer by direct injection and mixed by aerating the Ringer's solution. After the experiment, membranes were harvested and stored at ⁇ 80° C.
  • RNA Processing and RNA-Specific PCR Transgene-derived recombinant CFTR mRNA and endogenous CFTR mRNA were quantified using an RNA Specific real-time reverse transcriptase PCR (RS-PCR) method recently described and currently used in the Targeted Genetics Inc. clinical trails for CF (Gerard et al., 2003).
  • Total RNA was isolated from cells growing on Millicel-HA membranes using the RNeasy column purification method (Qiagen, Valencia Calif.). Specifically, 350 ⁇ L of RNeasy lysis buffer (RLT+ ⁇ -mercaptoethanol) was added directly to harvested membranes in a microfuge tube and samples were vortexed for 15 seconds.
  • DNA Processing and Real-Time DNA PCR for viral genomic DNA Cellular DNA was recovered by ethanol precipitation from a pool consisting of the RNeasy column load flow-through fraction and the first column rinse (from RNA processing). This allowed for a direct comparison of vector DNA and RNA for a given sample.
  • the recovered DNA was extracted once with phenol:chloroform:isoamyl alcohol (25:24:1), precipitated in 2.5 volumes of ethanol and quantitated by absorbance at 260 nm. Seventeen of the 90 DNA isolations were chosen at random and screened for matrix inhibition by evaluating DNA spike recovery; there was no evidence of matrix inhibition (data not shown).
  • test samples were analyzed in a real-time quantitative TaqMan PCR assay targeting AAV-CFTR (vector-specific) sequences.
  • Each 20 ⁇ L reaction contained 200 ng of genomic DNA and was run in triplicate in a 384-well format using an ABI Model 7900 Sequence Detection System (Applied Biosystems, Foster City Calif.).
  • Standards consisted of the plasmid ptgAAVCF (containing the AAV-CFTR sequence) diluted into a background of normal human lung DNA (Clontech/BD Biosciences, Palo Alto Calif.) and ranged from 8 ⁇ 10 6 to 8 ⁇ 10 1 copies per PCR.
  • AAV CFTR-specific PCR primers and Taqman probe were as follows: forward 5′-TTGCTGCTCTGAA AGAGGAGAC-3′ (SEQ ID NO: 1); reverse 5′-GATCGATGCATCTGAGCTCTTTAT-3′ (SEQ ID NO:2); probe 5′-(FAM)TGCTGCTCTCTAAAGCCTTGTATCTTGCACC(TAMRA)-3 (SEQ ID NO:3).
  • ⁇ -ENaC subunit forward 5′-CCTCAACTCGGACAAGCTCG-3′ (SEQ ID NO:4); reverse 5′-GAGAGTGGTGAAGGAGCTGTATTTG-3′ (SEQ ID NO:5); probe 5′-(FAM)ACCCTCAATCCCTACAGGTACCCGGAAATT(TAMRA)-3′ (SEQ ID NO:6).
  • ⁇ -ENaC subunit forward 5′-GGAACCACACACCCCTGG-3′ (SEQ ID NO:7); reverse 5′-CAAAGAGATCAAGGACCATGGG-3′ (SEQ ID NO:8); probe 5′-(FAM)CCTTATTGATGAACGGAACCCCCACC(TAMRA)-3′ (SEQ ID NO:9).
  • ⁇ -ENaC subunit forward 5′-GCTGGATTTTCCTGCAGTCAC-3′ (SEQ ID NO:10); reverse 5′ CAGGGCCTCTCTGGTCTCCT-3′ (SEQ ID NO:11); probe 5′-(FAM)AACATCAACCCCTACAAGTACAGCACCGTTC(TAMRA)-3′ (SEQ ID NO: 12). Copies of ENaC subunit mRNA were normalized to the number of ⁇ -actin mRNA copies in each sample using commercially available primer sets from Applied Biosystems (Foster City, Calif.).
  • ⁇ -ENaC promoter CpG Methylation The methylation status of a CpG island beginning at approximately ⁇ 1.8 kb of the ⁇ -ENaC promoter was analyzed using a previously described PCR method (Malik et al., 2001). Briefly, genomic DNA was isolated from A549 cells that had been treated with and without doxorubicin, and then digested overnight with MboI, MboI/MspI, or MboI/HpaII. PCR reactions were then performed using primers that flank the MspI/HpaII sites in this region. The relative positions of the CpG island, restriction sites, and primers are shown in FIG. 11B . Primers: forward 5′-TTGGAACCGAAAGGTGAGTT-3′ (SEQ ID NO: 13); reverse 5′-TGAACAGGCGCTGGGCGGAG-3′ (SEQ ID NO: 14).
  • Proteasome modulation agents enhance rAAV-mediated CFTR functional correction in polarized CF airway epithelia.
  • Proteasome inhibitors have been shown to dramatically increase the transduction efficiency of rAAV infection from the apical surface of polarized human airway epithelia (Duan et al., 2000; Ding et al., 2003). This enhancement by proteasome inhibitors appears to reflect an increased efficiency of intracellular processing of rAAV and accumulation in the nucleus (Duan et al., 2000) while not affecting processes that directly control the efficiency of second strand synthesis (Ding et al., 2003).
  • proteasome inhibitor action on rAAV transduction in polarized airway epithelia suggests that increased functional conversion of single-stranded genomes to expressible forms is facilitated by the increased bulk flow of rAAV into the nucleus.
  • AV2tgCF full-length CFTR rAAV vector
  • tgAAV2-CF full-length CFTR rAAV vector
  • rAAV2 or rAAV2/5 vectors utilizing the ITR or synthetic promoters were used to infect polarized CF airway epithelia from the apical surface in the presence or absence of a combined cocktail of LLnL and doxorubicin.
  • Fifteen days following infection CFTR-mediated cyclic AMP (cAMP)-sensitive short-circuit current (Isc) was assessed after stimulation by IBMX (100 ⁇ M) and forskolin (10 ⁇ M), and compared to normal human airway epithelia.
  • samples from 3 different CF donors (CFB-16, CFB-19, CFB-26) were infected and analyzed for CFTR correction. Results from these experiments are shown in FIG. 7 .
  • AV2CF83 minimal promoter vector of the type-2 serotype (0.76+/ ⁇ 0.16 ⁇ A) and no significant functional correction was seen with any of the other three viruses tested (AV2tgCF, AV2/5tgCF, or AV2/5CF83).
  • AV2CF83 restored CFTR-mediated chloride current upon IBMX/forskolin stimulation in the CF epithelia at the highest level (2.9+/ ⁇ 0.3 /1A), reaching approximately 80% of that seen in normal human airway epithelial (3.5+/ ⁇ 0.8 ⁇ A).
  • pseudotyped AV2/5CF83 with the same synthetic promoter gave significantly less correction of chloride currents in CF epithelia (1.0+/ ⁇ 0.2 ⁇ A), and was even lower than that seen with the ITR promoter CFTR vector AV2tgCF (1.9+/ ⁇ 0.2 ⁇ A).
  • the addition of the 83 bp synthetic promoter significantly increased (p ⁇ 0.03) IBMX/forskolin responsive Isc as compared to ITR-driven CFTR vectors.
  • Enhancement of vector derived mRNA/DNA ratios for the AV5 vector groups were also very large but could not be accurately calculated since mRNA levels in the absence of LLnL/Dox were at background levels.
  • RNA/DNA ratios were not significantly different for AV2CF83 as compared to AV2tgCF in the presence of LLNL and Dox, implying no significant augmentation in transcriptional activity of genomes containing the synthetic promoter.
  • RNA/DNA ratios for AV2/5CF83 were approximately 3-fold higher than that for AV2/5tgCF, suggesting that the synthetic promoter may have some beneficial effect on transcription, although not as great as in IB3 cells.
  • Proteasome modulating agents reduce the amiloride-sensitive sodium currents in CF airway epithelia by decreasing ENaC subunit mRNA levels.
  • ENaC is the major component of baseline short circuit current in CF airway epithelia and is greatly elevated due to a lack of functional CFTR. It has been previously suggested that as little as 6-10% transduction with a CFTR expressing vector can fully correct CFTR-mediated chloride currents in a polarized airway epithelia due to gap-junctional cell-cell coupling of Cl ⁇ ions in the epithelium (Johnson et al., 1992).
  • Doxorubicin treatment increases ⁇ -ENaC promoter methylation. Since it has been reported that doxorubicin treatment leads to CpG demethylation of MDR-1 gene promoter and a consequent increase in MDR expression (Kusaba et al., 1999), it was hypothesized that increased CpG methylation of the ⁇ -ENaC gene promoter might produce the opposite Dox-dependent effect. To test this hypothesis, an airway cell model system for which genomic DNA could be easily generated was developed. First, it was determined whether a non-CF airway cell line (A549) produced similar regulatory changes in ⁇ -ENaC mRNA expression following Dox treatment.
  • A549 non-CF airway cell line
  • This assay utilized primers flanking a 310 bp region in this CpG island that contained multiple MspI/HpaII sites. MspI and HpaII digest the same sequence in DNA, however, Hpall will not digest if CpG methylation is present. Results from this analysis demonstrated a significant Dox-dependent protection from HpaII digestion at this region of the CpG island ( FIG. 11 C ). These findings suggested that CpG methylation of the ⁇ -ENaC promoter indeed occurs in response to Dox treatment.
  • rAAV-2 vectors gave significantly higher levels of CFTR functional correction and mRNA expression than compared to rAAV-2/5. Additionally, the addition of the CF83 minimal promoter demonstrated only marginal improvement in functional correction and/or CFTR mRNA expression.
  • proteasome modulating agents may have dual therapeutic utility as pharmacologic agents to treat primary pathology and enhance gene therapy for CF lung disease.
  • the amiloride-sensitive epithelial sodium channel (ENaC) controls sodium transport across many types of epithelia, including airway, kidney and colon.
  • ENaC activity can be regulated either by altering the channel open probability or the number of functional ENaC molecules on cell surface. Previous studies have demonstrated a link between the ubiquitin-proteasome proteolytic system and regulation of ENaC turnover at the cell surface.
  • ENaC consists of three subunits ( ⁇ , ⁇ , and ⁇ ) each of which has a proline rich region (PPXY) at the C-terminal end.
  • the ubiquitin ligase Nedd4 interact with ENaC through this PPXY region and mutation of a group of lysine residues at the N-terminus of the ac and ⁇ subunits leading to inhibition of ubiquitination and increased ENaC activity (Staub et al., 1997).
  • doxorubicin has been shown to alter methylation of the MDR-1 promoter and increase transcriptional activity of the multi-drug resistance gene (MDR-1) (Kusaba et al., 1999; Nakayama et al., 1998; Ando et al., 2000). Although the net effect of doxorubicin on MDR-1 and ⁇ -ENaC promoter methylation are opposite, the processing controlling changes in CpG methylation may be similarly regulated.
  • MDR-1 multi-drug resistance gene
  • proteasome modulating agents alter baseline ENaC activity in CF airway epithelia may have practical therapeutic applications outside their combined ability to also enhance rAAV transduction.
  • ENaC hyperactivity is thought to dehydrate the surface airway fluid layer in the CF and decrease airway clearance of bacteria
  • inhaled proteasome modulating agents that inhibit ENaC activity could be applied as an aerosolized compound(s) to the lungs of CF patients to enhance airway clearance.
  • CFTR knockout mice In vivo based screening for function inhibition of ENaC using CF mice may also be employed. Electrophysiologic properties of CF mouse nasal epithelium are very similar to those found in the human nasal and lung airways (Grubb et al., 1994), and defective CFTR leads to hyperactivity of ENaC. To confirm the ability of agents identified in cell based screens to inhibit ENaC transcription and hence ENaC function, CFTR knockout mice may be employed. Agents to be tested are delivered to mice by the appropriate route including but not limited to i.v., i.p., endotracheal or nasal application at an interval (days to weeks) prior to functional analysis of nasal potential difference measurements.
  • a 200 ⁇ m internal diameter catheter was linked directly to a perfusion syringe pump.
  • the syringe was linked via a 1 M KCL agar salt bridge to a calomel electrode and a voltmeter.
  • the second calomel electrode was linked to a 1 M KCL agar-filled 21 gauge needle implanted subcutaneously in the mouse.
  • anthracyclines were tested for their relative in vitro and in vivo activities on AAV transduction.
  • HeLa cells were infected with 100 ppc AAV2FLAG-Luc for 2 hours in the presence of different anthracyclines, e.g., doxorubicin, daunarubicin (Cerubidine), epirubicin (EllenceTM), and idarubicin (Idamycin®), and cells harvested 48 hours later.
  • the anthracyclines were pharmaceutical grade, and prepared according to the manufacturer's instructions.
  • mice Six groups of ten, five-to-seven week-old, Balb/c mice (5 male and 5 female per group) were employed in a comparison of the relative in vivo potency and safety of different anthracycline derivatives at a single dose after intranasal delivery. Treatment was administered as shown in Table 3. Animals were followed for seven days post dose.
  • the dose of modulator was based on the Human Dose Equivalent (HDE) and is summarized below in Table 4.
  • HDE Human Dose Equivalent
  • the dose was held constant at 10% of the HDE.
  • Doxil intravenous positive control
  • a dose of 10 mg/kg (75%) of the HDE was used. This represented the lowest dose that gave a 10% increase in mean and median luciferase expression in earlier studies.
  • Safety endpoints included morbidity and mortality, clinical observations, body weights, gross necropsy observations and histopathology.
  • Transduction endpoints included luciferase and GFP analysis.
  • the left lung On the day of sacrifice, the left lung was clamped off at the level of the extrapulmonary bronchi, removed and frozen on dry ice. The left lung was homogenized and processed for luciferase expression using Promega's luciferase assay system (Madison, Wis.). Luminescence was measured using the Berthold AutoLumat LB953 instrument. Samples were normalized for total protein using Pierce's Coomassie Plus Protein Assay Reagent (Rockford, Ill.).

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