The CF Center Correction Core

About Objectives Personnel Contact

Core Director: Martina Gentzsch, PhD, Co-Director: Peter Bove, PhD, Co-Investigator: Barbara Grubb, PhD

About the Correction Core

Correction Core Figure 1

The Correction Core's Ussing Chamber Machine, used for measuring electrophysiological characteristics of tissue samples and cell cultures.

Cystic Fibrosis (CF) is a disease of defective ion and fluid transport. The identification and cloning of the gene altered in CF demonstrated that this protein codes for a cAMP-mediated Cl- channel, CFTR. The Correction Core and others have demonstrated that defects in CFTR lead to decreased hydration of the airway surface liquid (ASL), reduced mucus clearance, and chronic airways infection. In addition, 10-15% of neonatal human CF patients present with thick sticky intestinal mucus (meconium ileus) that result in intestinal blockage, often requiring surgical intervention.

Gene therapy of CF is a logical approach for treating both of these aspects of CF. The assessment of the efficacy of gene transfer requires routine and reliable assays to verify expression of the functional CFTR protein. The Correction Core uses a combination of in vivo and in vitro techniques to sensitively test for CFTR correction of the ion and fluid transport defect of 1) murine (native and cultured) airway epithelium; 2) CF human airway epithelium, cultured/freshly excised and 3) murine and human gut epithelia (native tissue).

Specifically, the Core uses transepithelial electrophysiological measurements of confluent cultured monolayers, as well as native tissue, mounted in Ussing chambers to determine the magnitude of both Cl- (CFTR) and Na+ (ENaC) currents. Fluid transport rates are measured in vitro by both the blue dextran technique and by confocal microscopy of fluorescently labeled ASL. In vivo approaches include nasal potential difference (PD) measurements (human and mouse) and murine nasopharyngeal PD measurements (a ciliated airway epithelium similar to the lower airways in humans). Because gene transfer to the neonate may be most effective for treating a number of genetic diseases, including CF, the Core has perfected techniques to study freshly excised epithelial sheets from neonatal murine airways and the GI tract to measure the effectiveness of in vivo vector dosing in neonatal CF mice. The studies performed are designed to provide gene transfer investigators routine access to high-quality, high-sensitivity, well-controlled, robust measures of CFTR correction.

Correction Core Objectives

  1. Analysis of efficacy of CFTR gene transfer to CF human and murine airways

    1. In Vitro

      1. Measure ion transport properties of “corrected” vs. control CF human and murine airway cultures in Ussing chambers.
      2. Measure fluid transport properties of “corrected” vs. control CF human and murine airway cultures by confocal microscopy.

      3. Measure fluid transport properties of “corrected” vs. control CF human and murine airway cultures by blue dextran optical absorption.

      4. Measure pH from the lumenal surface of well-differentiated “corrected” vs. control CF human and murine airway cultures.

      5. Measure mucus biophysical properties and mucus transport in corrected vs control CF airway cultures.

      6. Measure ion transport properties of freshly excised murine neonatal airways of mice corrected in

        in vivo vs control mice.

    2. In Vivo

      1. Measure electrical potential difference (PD) in murine nasal and nasopharyngeal epithelia in vivo.

      2. Measure nasal PD in humans.

  2. Analysis of efficacy of CFTR gene transfer to gastrointestinal epithelium of both mouse (neonatal and adult) and human

    1. Measure ion transport properties of freshly-excised murine neonatal and adult gut epithelia as well as transit times in CF corrected vs non-corrected gut epithelia.

    2. Measure ion transport properties of rectal biopsies from human CF patients.

  3. Analysis of efficacy of functional and biochemical correction of mutant CFTR by small-molecule compounds.

    1. Measure ion transport properties of "corrected" vs. control CF human airways cultures in Ussing chambers.

    2. Quantitate CFTR maturation by Western blotting and analyze CFTR cell surface localization by On-Cell Westerns.


    Correction Core Personnel

    Correction Core Gentzsch

    Martina Gentzsch, PhD, Core Director

    Correction Core _Bove

    Peter Bove, PhD Co-Director

    Correction Core_Grubb

    Barbara Grubb, PhD Co-Investigator

     

     

     

     

     

     

     

     

     

    Correction Core_Quinney

    Nancy Quinney, Research Specialist and Core Manager

    Correction Core_Mocny

    Jeffrey Mocny, Post-Doctoral Research Associate

    Correction Core_Rogers1

    Troy Rogers, Research Specialist

     

     

     

     

     

     

     

     

     

     

    Contact Information

    6013 Thurston-Bowles Bldg.
    Campus Box #7248
    The University of North Carolina at Chapel Hill
    Chapel Hill, NC 27599
    Phone: (919) 966-7059
    Fax:(919) 966-5178