Date of Award

2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Isabel Virella-Lowell

Second Advisor

Michael Bowman

Third Advisor

David T. Kurtz

Fourth Advisor

Lucille London

Fifth Advisor

Gabriel Virella

Abstract

Cystic fibrosis (CF), the most common lethal autosomal recessive disease in Caucasians resulting in a median life span of approximately 35 years, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The primary cause of morbidity and mortality is respiratory failure caused by progressive obstructive lung disease. CF airway disease is characterized by the abundance of pro inflammatory cells, chemokines and cytokines with a deficiency of regulatory/anti-inflammatory cytokine interleukin 10 (lL10). We hypothesized that CF airway obstruction originates from excessive inflammation and gene transfer of IL-I0 would decrease inflammation in the airways of mice infected with Pseudomonas aeruginosa, a common pathogen in CF. IL-10 gene transfer was accomplished using an adeno-associated viral vector (AAV) selected for the inherent benefits of long-term gene expression, a proven safety profile, and the ability to elicit a minimal inflammatory response in comparison to other gene transfer agents. Using intratracheal injection, we administered AAV5.β-mIL10 to IL-10 deficient mice, chronically infected the mice with P. aeruginosa, and observed high levels of IL-10, decreased lung pathology, and IL-β and IL-8 (MIP-1α and KC in mice), cytokines frequently seen at increased levels in patients with CF. To characterize IL-10 gene transfer in CFTR knockout mice, we modified a noninvasive procedure, aspiration challenge, to include surfactant phospholipids as a vehicle for transfer of P. aeruginosa and AAV-based vectors. This was important because CFTR knockout mice are sensitive to manipulations such as intratracheal injection, resulting in mortality from the surgical procedure. We demonstrated aspiration challenge with surfactant phospholipids was similar to intratracheal injection for P. aeruginosa and AAV delivery, as measured by percentage weight loss, proinflammatory cytokine production, inflammation associated histopathology, and bacterial burden for P. aeruginosa and vector distribution and transgene expression for AAV. Then, we characterized AAV5.Cβ-mIL-10 in CFTR knockout mice chronically infected with P. aeruginosa, observing decreased percentage weight loss, lung pathology, pro-inflammatory cytokines (IL-lβ, IL-6, TNF-α, KC, MIP-1α), histopathology, and neutrophil migration with no change in bacterial burden. These studies support that inflammation is excessive to bacterial infection in CF and demonstrate AAV -based IL-10 gene transfer is a promising anti-inflammatory therapeutic for CF lung disease.

Rights

All rights reserved. Copyright is held by the author.

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