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

a technology pharmico-gene therapy, which is applied in the field of compound and method for pharmico-gene therapy of epithelial sodium channel associated disorders, can solve the problems of limited packaging capacity of this vector, mediated gene delivery of cftr, and inability to achieve optimal transduction of airway cells as measured by vector derived cftr mrna, etc., to achieve the effect of improving the functional conversion

Inactive Publication Date: 2005-05-05
UNIV OF IOWA RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

Proteasome modulating agents significantly enhance rAAV-mediated CFTR functional correction and reduce ENaC hyperabsorption defects, demonstrating dual therapeutic potential for improving gene therapy and treating cystic fibrosis by increasing transduction efficiency and correcting fluid transport abnormalities.

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).

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

example 1

Endosomal Processing Limits AAV Transduction

[0166] Based on the finding that basolateral membranes have higher endocytic rates and UV irradiation enhances endosomal uptake and rAAV transduction from the apical membrane, it is possible that endosomal pathways influencing viral uptake and transport to the nucleus may be limiting from the apical membrane. In contrast, these pathways may be active at maximal levels from the basolateral membrane of airway epithelial cells. To further investigate the importance of endosomal processing, the effect(s) of several chemical agents known to alter endosomal processing was evaluated.

Methods

[0167] Initial studies were performed in confluent primary human fibroblasts since dose titrations and toxicity could be quickly assessed. Selected agents were used to treat fibroblast monolayers prior to rAAV infection. rAAV transduction was assessed at 96 hours post-infection by FACS analysis, and the percentage of dead cells was simultaneously assessed b...

example 2

Endosomal Processing Inhibitors May Increase rAAV Transduction in Polarized Airway Cells

Materials and Methods

[0172] Primary culture of human bronchial epithelia and reagents utilized. Primary human airway epithelial cells were collected by enzymatic digestion of bronchial samples from lung transplants, as previously described (Kondo et al., 1991; Zabner et al., 1996). Isolated primary airway cells were seeded at a density of 5×105 cells / cm2 onto collagen-coated Millicell-HA culture inserts (Millipore Corp., Bedford, Mass.). Primary cultures were grown at the air-liquid interface for more than 2 weeks, by which time differentiation into a mucociliary epithelium occurs. 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...

example 3

Expression of the LacZ Gene in Lung

Airway Epithelium and Liver In vivo

[0201] 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. A rAAV vector containing the LacZ gene, recombinant AV.LacZ (5×1010 particles), was administered to mouse lung either as virus alone in PBS or virus in combination with 40 μM LLnL in PBS. Virus was directly instilled into C57Balb / c mice trachea with a 30 G needle in a total volume of 30 μl. To insure the spread of the virus in mouse lung, 50 μl air was pumped into lung through the same syringe immediately after virus was administrated. Ninety days after infection, lungs were harvested intact and fixed in 4% paraformaldehyde followed by cryosection. AAV-mediated transgene expression was evaluated by 10 μm tissue sections staining forLac Z.

[0202] Recombinant AV.LacZ (5×1010 particles) was also administered to mouse liver either as virus alone in PBS, virus in combinat...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N15/86C12N15/861C12N15/864C12Q1/68C12Q1/70G01N33/50G01N33/68
CPCA61K48/00C12N15/86C12N2750/14143G01N33/6893G01N33/502G01N33/6872G01N33/5008A61P3/06A61P3/12A61P7/04A61P11/00A61P25/00A61P25/16A61P31/04A61P35/00A61P43/00
Inventor ENGELHARDT, JOHNZHANG, LIANG
Owner UNIV OF IOWA RES FOUND
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