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

Abstract

Agents and methods to alter epithelial sodium channel (ENaC) activity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of the filing date of U.S. application Ser. No. 60 / 459,323, filed Mar. 31, 2003, and U.S. application Ser. No. 60 / 512,347, filed Oct. 16, 2003, the disclosures of which are incorporated by reference herein.STATEMENT OF GOVERNMENT RIGHTS [0002] This invention was made at least in part with a grant from the Government of the United States of America (HL58340) from the National Institutes of Health). The Government may have certain rights in the invention.BACKGROUND OF THE INVENTION [0003] Cystic fibrosis (CF) 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. In ad...

AI Technical Summary

Benefits of technology

[0013] As described herein, to directly compare rAAV-2 or rAAV-2 / 5 vectors for their ability to correct the CFTR detect in polarized CF airway epithelia, several full-length 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). In particular, the identical ITR-CFTR vector used in clinical trials for CF (AVtgCF) 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.

[0014] Interestingly, the application of 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 γ-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 γ-ENaC promoter. These unexpected findings demonstrate for the first time the identification of a new class of dual therapeutic agents capable of both treating primary defects of a disease while enhancing gene therapy of the disorder. In particular, these findings suggest that in addition to improving rAAV transduction, modulation of the proteasome also significantly attenuates ENaC sodium hyperabsorption defects in CF airway epithelia. These studies, which provide the first demonstration of rAAV-mediated CFTR functional correction in CF polarized airway epithelial, suggest that 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. In one embodiment, 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. In addition, 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.

[0015] The identification of agents with dual therapeutic action may be extremely useful in clinical trials for CF lung disease as well as other diseases. Thus, one or more agents identified by the methods may provide clinically useful strategies for in vivo therapy, e.g., gene therapy of respiratory disorders such as cystic fibrosis and others associated with inflammation, e.g., due to infection with a pathogen, for instance, bacterial infection, and including conditions associated with aberrant, e.g., increased, ENaC activity. For instance, the amount of fluid absorption or transport in the lung may be linked to bacterial clearance, and aerosolized delivery of one or more agents of the invention, which alter ENaC activity to a mammal, may alter (e.g., modulate) the inflammatory response, resulting in enhanced clearance or decreased inflammation.

[0016] The data also showed that 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.

[0025] A method to inhibit or treat a condition associated with increased ENaC levels or increased ENaC activity is provided. 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, and enhances the efficacy of gene therapy vectors.

Problems solved by technology

Although recent trials with rAAV-2 have demonstrated an impressive safety profile and long-term persistence of vector genomes in the airway epithelia of the lung, successful transduction of airway cells as measured by vector derived CFTR mRNA have not been optimal possibly due to the relatively poor binding to the surface of polarized human airway cells (Zabner et al., 2000), and the fact that known rAAV-2 receptors do not reside on the apical surface of the human airway (Duan et al., 1998).

Additional limitations to rAAV-mediated gene delivery of CFTR include the limited packaging capacity of this vector (about 5 kb) and the relatively large size of the CFTR cDNA (4.5 kb).

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