Effects of altering dosing on cationic liposome-mediated gene transfer to the respiratory epithelium

Abstract

Liposome-mediated gene transfer is currently sub-optimal tering an equivalent DNA dose in aliquots over a 1-3 day with respect to both the extent and duration of transgene period resulted in significantly lower gene expression and expression. We investigated whether simple changes in did not increase the duration of expression. Administration DNA dosing could enhance either of these outcomes.at different times of the day (and hence wake/sleep cycles Increasing DNA doses produced highest transgene of the animals) did not alter gene expression. We conclude expression at an intermediate dose with toxicity observed that such simple changes in dosing regimes are unlikely to at higher doses, thereby likely limiting expression. Adminiscontribute to improvements in gene transfer efficiency.

Poly (D, L-lactide-co-glycolide)/DNA microspheres to facilitate prolonged transgene expression in airway epithelium in vitro, ex vivo and in vivo

“…All complexing reactions were carried out at a molar ratio of 1:4 (lipid:DNA) where the molarity of lipid #67 was 1.2 mM and the molarity of the DNA was calculated, based upon the assumption that the molecular weight of nucleotides in 330 g/mol. 33 The media were removed after 24 or 48 h. The cells were washed twice with PBS, and lysed in a solution containing 0.1% Triton X-100 and 250 mM Tris (pH 8, 500-ml per well) prior to undergoing three freeze/thaw cycles (À801C Â 30 min/371C Â 5 min). The cell lysate was assayed for b-gal using a luminescent reporter system (Clontech Laboratories Inc., Palo Alto, USA) according to the manufacturer's suggested protocol.…”

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor1α promoter

“…3 In animal models, nonviral gene transfer mediated either by naked plasmid DNA (pDNA) or by pDNA/cationic liposome complexes, showed transgene expression to be maximal 1-2 days following administration, declining to background levels after 2-4 weeks. [4][5][6] The loss of transgene expression may be explained in one of several ways: loss of the vector, transcriptional silencing of the transgene promoter, loss of the transfected cell through cell turnover, or the generation of an immune response to the transgene product or the transfected cell itself. The majority of pre-clinical and clinical airway gene transfer studies have used strong viral promoters in order to achieve high-level gene expression in the airways.…”

Use of ultrasound to enhance nonviral lung gene transfer in vivo

“…All procedures were approved by the Home Office under the Animals (Scientific Procedures) Act 1986. The pDNA doses used in the absence of Optison were: 20 mg/100 ml for PEI/pDNA, 42 80 mg/100 ml for GL67/ pDNA, 41 and 100 mg/100 ml for naked pDNA. 31 When Optison was added, the pDNA doses had to be reduced in some cases, in order to keep the final volume constant.…”

“…Similarly, equal volumes of GL67 (1.2 mM) and pDNA (4.8 mM, 1.6 mg/ml) were mixed at a molar ratio of 1:4 (GL67:pDNA) in sterilized water for injection and incubated at 301C, for 15 min. 41 In some cases, Optison (Amersham Health, Oslo, Norway), an ultrasound contrast agent, was mixed at a 1:1 volume: volume (v/v, DNA/Optison) ratio with the gene transfer agents described above, in a total volume of 100 ml.…”