Safety of Adenovirus-Mediated Transfer of the Human Cystic Fibrosis Transmembrane Conductance Regulator cDNA to the Lungs of Nonhuman Primates

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Abstract
To define the toxicity of cystic fibrosis transmembrane conductance regulator gene (CFTR) gene therapy with a replication-deficient recombinant adenovirus (Av1Cf2) in a nonhuman primate model, 1010 plaque forming units (pfu) were instilled directly through a bronchoscope into the right lung of 5 macaques, and a lower dose of 4×106 pfu was administered to the right lung of 1 macaque. One sham-treated control received phosphate-buffered saline (PBS). The macaques were evaluated sequentially by clinical examination, vital signs, weight, hematology, blood chemistry, chest radiography, pulse oximetry, and bronchoalveolar lavage (BAL) at baseline and 3–28 days post-treatment. After the period of observation, macaques were sacrificed for autopsy and histological examination. The macaques tolerated the experimental therapy clinically with no changes in body temperature, oxygen saturation, heart rate, body weight, or blood pressure. However, 1 macaque with visible evidence of aspiration at the time of initial bronchoscopy developed tachypnea with right lower lobe (RLL) pneumonia on chest radiograph and by histology. There were no changes in Hgb, Wbc, BUN, plasma electrolytes, bilirubin, or hepatic transaminases. In the macaques that received 1010 pfu, there was a progressive increase in the number of CD8+ lymphocytes in BAL that was maximal at 28 days. Histological examination of the treated lungs of the high-dose macaques at 3 days showed marked peribronchial and perivascular cuffing by inflammatory cells and alveolar accumulation of neutrophils and macrophages. The alveolitis appeared to be resolving at 28 days, although the perivascular and peribronchial aggregates of mononuclear cells were still present. In the high-dose macaques, BAL interleukin-8 (IL-8) was increased at all time points (256–388 pg/ml versus 1–84 pg/ml at baseline and in control), whereas IL-1β was increased only at days 21 and 28 (341–852 pg/ml versus 30–92 pg/ml at baseline and in control). There were no increases in BAL cell counts, IL-1β or IL-8, and histological changes were mild in the macaque that received 4 × 106 pfu. Evaluation for Av1Cf2-derived human CFTR expression using RS-PCR demonstrated expression at 3, 10, and 21, but not 28 days in macaques treated with 1010 pfu of Av1Cf2. In situ hybridization analysis demonstrated human CFTR mRNA in the alveolar regions of the lobes that received the vector at 10 and 21 days. There was no evidence of expression after treatment with 4 × 106 pfu. This study showed that high-dose adenoviral vector administration to the lung achieved CFTR gene transfer and expression but was associated with increased concentrations of cytokines in BAL and alveolar inflammation. A low dose, equivalent to the maximum clinical dose currently proposed for phase I trials in human subjects, was not associated with cellular or cytokine evidence of inflammation, and histological abnormalities were mild. Transfer of the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the human airway using an E1-deleted, E3-deleted adenoviral vector, Av1Cf2, has been proposed for correction of the epithelial cell secretion defect of CF. The present study presents data that demonstrates the biological efficacy and the clinical safety of Av1Cf2-mediated gene transfer in vivo in nonhuman primates.

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