1104. Imaging of sec-R Directed and PEG-Stabilized Gene Transfer Nanoparticles in CF Mice

Abstract

Compacted DNA targeted to the serpin enzyme complex receptor (sec-R) can deliver amounts of human CFTR DNA sufficient for partial correction of the chloride transport defect and NOS-2 downregulation in the noses of CF knock out mice. To examine the distribution of these compacted DNA particles in vivo, we used radioscintigraphy imaging. We modified our DNA compacting agent (a polymer of poly-L-lysine, chemically conjugated to the C105Y sec-R targeting ligand) with radiolabeled I-125. Prior to dosing, 12 S489X/FABP-hCFTR were imaged by X-ray for proper anatomical alignment with radiograph images. Following complex formulation, 9 mice were dosed intranasally (IN) with 25 |[mu]|g of sec-R targeted radiolabeled hCFTR DNA (specific activity 50 |[mu]|Ci each), while 3 control mice received administration of free I-125 (specific activity 50 |[mu]|Ci each). We used a small animal gamma scanner to analyze radio images at 2, 4, 24, and 48 hrs following nasal administration of the dose. At each time 3 animals were sacrificed, frozen and stored for subsequent sectioning. For each mouse, I-125 scintigraphic images, x-ray images, and autoradiographs were aligned and analyzed. Targeted delivery resulted in higher total retained activity than the carrier control. Both test and control groups begin to exhibit thyroid uptake by 2 hrs, while significantly higher activities were observed in the nasal and lower airways for animals that received the targeted complexes vs. controls. The highest observed activity was in thyroid, liver and bladder. Autoradiographic images correlated well with their corresponding in vivo scintigraphic images. For mice that received targeted complexes, strong signals were measured in both abdominal (stomach and liver) and bladder regions indicating possible ingestion during or aspiration following administration, as well as secretion into the bladder. This rapid clearance by 2 hrs was observed for all mice. Regional analysis showed significantly (p<0.05) higher activities for the targeted delivery in nose, lung and liver vs. controls, 2 and 4 hrs post administration. By 24 hrs, activities in the upper airways and lungs were back to the background level, while activity in the thyroid remained significant at 24 and 48 hrs. The data indicate a rapid clearance of sec-R targeted compacted DNA (by 2 hrs), followed by the appearance of at least the labeled component of the complex (the ligand) in the thyroid and bladder by 4 hrs. We also examined expression conferred by second generation PEG-stabilized compacted nanoparticles containing the Firefly luciferase gene using bioluminescent imaging. Expression was detected in lung 2 and 4 days following intra-tracheal (IT) but not IN administration. Lung homogenates from both methods of administration exhibited luciferase activity although levels in the IT group were significantly higher. These data suggest that a threshold level of expression, achieved only with IT administration, is necessary for successful bioluminescent detection . Development of these real-time imaging techniques has allowed us to better assess the localization and site of activity of our gene transfer complex formulations.