Keith Munson

Distinct Classes of Proteasome-Modulating Agents Cooperatively Augment Recombinant Adeno-Associated Virus Type 2 and Type 5-Mediated Transduction from the Apical Surfaces of Human Airway Epithelia

ABSTRACT Tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus type 2 (rAAV-2) and rAAV-5 serotypes. In the present study we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their ability to induce rAAV transduction. The anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with the proteasome through a mechanism distinct from that of tripeptidyl aldehydes. Our studies demonstrated that doxorubicin and aclarubicin also significantly augmented rAAV transduction in airway cell lines, polarized human airway epithelia, and mouse lungs. Both tripeptidyl aldehyde and anthracycline proteasome-modulating agents similarly augmented nuclear accumulation of rAAV in A549 and IB3 airway cell lines. However, these two cell types demonstrated cell specificity in the ability of N-acetyl-l-leucyl-l-leucyl-l-norleucine (LLnL) or doxorubicin to augment rAAV transduction. Interestingly, the combined administration of LLnL and doxorubicin resulted in substantially increased transduction (>2,000-fold) following apical infection of human polarized epithelia with either rAAV-2 or rAAV-5. In summary, the cell type specificity of LLnL and doxorubicin to induce rAAV transduction, together with the ability of these compounds to synergistically enhance rAAV transduction in polarized airway epithelial induction, suggests that these two classes of compounds likely modulate different proteasome functions that affect rAAV transduction. Findings from this study provide new insights into how modulation of proteasome function can be effectively used to augment rAAV transduction in airway epithelia for gene therapy of cystic fibrosis.

Calculating expected lung deposition of aerosolized administration of AAV vector in human clinical studies

Cystic fibrosis is an autosomal recessive disease affecting approximately 1 in 2500 live births. Introducing the cDNA that codes for normal cystic fibrosis transmembrane conductance regulator (CFTR) to the small airways of the lung could result in restoring the CFTR function. A number of vectors for lung gene therapy have been tried and adeno‐associated virus (AAV) vectors offer promise. The vector is delivered to the lung using a breath‐actuated jet nebulizer. The purpose of this project was to determine the aerosolized AAV (tgAAVCF) particle size distribution (PSD) in order to calculate target doses for lung delivery.

756. Distinct Classes of Proteasome-Modulating Agents Cooperatively Augment Recombinant Adeno-Associated Virus Type 2 and Type 5-Mediated Transduction from the Apical Surface of Human Airway Epithelia|[ast]|

Top of pageAbstract Tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus (rAAV) type 2 and 5 serotypes. In the present study, we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their ability to induce rAAV transduction. The anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with the proteasome through a mechanism distinct from that of tripeptidyl aldehydes. Our studies demonstrated that doxorubicin and aclarubicin also significantly augmented rAAV transduction in airway cell lines and polarized human airway epithelia, human bronchial xenografts, and mice lungs. Both tripeptidyl aldehyde and anthracycline proteasome-modulating agents similarly augmented nuclear accumulation of rAAV in A549 and IB3 airway cell lines but did not directly enhance the efficiency of second- strand synthesis for AAV genome conversion. However, these two cell types demonstrated cell specificity in terms of the ability of LLnL or doxorubicin to augment rAAV transduction. Interestingly, the combined administration of LLnL and doxorubicin resulted in substantially increased transduction (>2000 fold) following apical infection of human polarized epithelia with either rAAV-2 or rAAV-5. In summary, the cell-type specificity of LLnL and doxorubicin to induce rAAV transduction, together with the ability of these compounds to synergistically enhance rAAV transduction in polarized airway epithelial induction, suggest that these two classes of compounds likely modulate different proteasome functions that affect rAAV transduction. Findings from this study provide new insights into how modulation of the proteasome function can be used to augment rAAV transduction in airway epithelia for gene therapy of cystic fibrosis.

489. Comparison of AAV2 and AAV2/5 CFTR vectors in the lungs of cynomolgus macaques

Cystic fibrosis is the most common autosomal recessive disorder in North America, leading to significant morbidity and early mortality. Cystic fibrosis is caused by a defect in the cystic fibrosis transmembrane regulator (CFTR) gene which encodes a chloride channel. The defect in chloride transport in epithelial cells affects a number of organs. The defect in airway epithelium in the lung leads to progressive loss of pulmonary function which is the primary cause of death. rAAV vectors have shown potential utility for treatment of cystic fibrosis. The primary objective of this study was to assess transduction efficiency of several full-length CFTR cDNA rAAV vectors. Two CFTR vectors, one with the AAV2 ITR as a promoter and the other with a small, synthetic 83 base promoter, were evaluated in either AAV type 2 or 5 capsids. Secondary objectives were to collect safety data and to assess the cell types transduced using GFP as a reporter gene. Safety parameters included clinical chemistry, hematology, gross necropsy, histopathology and AAV neutralizing antibody titers.