Transfection efficiency and toxicity following delivery of naked plasmid DNA and cationic lipid–DNA complexes to ovine lung segments

Abstract

We defined, using a novel large animal model system, the acute pathologic response to localized pulmonary administration of either naked plasmid DNA (pDNA) or cationic lipid-pDNA complexes (pDNA:GL67) and related such responses to concomitant indicators of transfection efficiency, namely levels of chloramphenicol acetyl transferase (CAT) protein and mRNA in specific lung tissue compartments. We instilled doses of 0.2, 1, and 5 mg pDNA to spatially distinct lung segments in six anesthetized sheep and doses of 0.2, 1, and 5 mg pDNA:GL67 to a further six sheep. Twenty-four hours after gene delivery the sheep were euthanized and necropsy examination with sampling of relevant tissues was carried out. Levels of plasmid-derived CAT-specific mRNA and CAT protein in samples derived from segments treated with either pDNA or pDNA:GL67 increased in relation to the administered dose. Levels of mRNA and protein expression were greater for pDNA:GL67 than for pDNA alone. A significant correlation was observed between mRNA and protein expression in samples derived from airways treated with pDNA:GL67. Histopathological changes following administration of both pDNA and pDNA:GL67 were characterized by a neutrophilic inflammation predominantly oriented on airways. The severity of the inflammatory response appeared to correlate with the administered dose of DNA and was generally more severe for pDNA:GL67.

Use of ultrasound to enhance nonviral lung gene transfer in vivo

“…Sheep, in particular, are an attractive option, as proof of principle for nonviral gene transfer to the ovine lung has already been established. 29 Also, their lung is similar to humans both in structure and size. Sonoporation studies in this model could assess how US is applied, for instance endobronchially or via the chest wall, and optimize US conditions for lung gene transfer while assessing any US-induced side effects.…”

“…28 Nonetheless, we assessed whether the energy that is able to penetrate the lung tissue was sufficient to enhance the gene delivery of cationic lipid 67 (GL67)/ pDNA or polyethylenimine (PEI)/pDNA complexes, and naked pDNA, all previously used for lung gene transfer. [29][30][31] …”

Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung

“…A further group received pCIKLux complexed with PEI (n¼8) to act as a control group for CFTR protein detection; PEI was chosen for the control group for the pragmatic reasons of availability and cost of material. Aerosol delivery was performed using the nebulisation and ventilation system described previously 8,9 and was well tolerated in all sheep with no adverse effects apparent beyond those normally associated with general anaesthesia in this species. The nebuliser charge volume was held constant at 20 ml, as a similar volume has been safely delivered to patients within an acceptable administration time, therefore different amounts of pDNA were delivered owing to constraints on GTA formulation.…”

“…Although large animal CF models continue to be developed [5][6][7] these are not yet widely available; thus, we have successfully used wild-type sheep to evaluate the safety and efficacy of lung-directed gene therapy in a clinically relevant way. [8][9][10] Our choice of species is based on anatomical and functional similarities to human lungs and the observation that similar mechanisms are initiated in response to lung inflammation that follow a familiar pattern of repair and regeneration. Indeed, the sheep model is proving valuable for assessing gene delivery and efficacy, the localisation of transgene expression and the safety of the gene transfer protocol, all crucial end point measurements in human trials.…”

Gene therapy for cystic fibrosis: an example for lung gene therapy

“…This, combined with the fact that the nasal epithelium can easily be exposed to GTAs, makes the CF mouse nose an ideal organ for assessing and optimizing gene transfer. In addition, non-CF primates, 58,59 pigs 44 and most recently sheep 60 have been used to optimize airway gene transfer and allowed clinically relevant delivery methods such as nebulization to be assessed. More recently, first attempts have been made at generating CF ferret and sheep based on targeting of the CFTR locus in somatic cells coupled with nuclear transfer 61 (and Jim McWhir, Roslin Institute, personal communication).…”