Weiwen Liang

Date of Graduation


Document Type



In this study, we investigated the possibility of using antisense oligonucleotides directed against TNF{dollar}\\alpha{dollar} mRNA to specifically inhibit TNF{dollar}\\alpha{dollar} production in silica-exposed rats and to test whether this inhibition can provide any protection against silicosis. To optimize the length and sequence of antisense oligonucleotides against TNF{dollar}\\alpha{dollar} mRNA, the effect of oligonucleotide length and different target sites on TNF{dollar}\\alpha{dollar} mRNA on the antisense inhibitory activity of TNF{dollar}\\alpha{dollar} production was investigated. The antisense oligonucleotides targeted to the cap site, the initial codon and the coding region of TNF{dollar}\\alpha{dollar} mRNA was found equally effectively. The activity of antisense oligonucleotides against TNF{dollar}\\alpha{dollar} mRNA increased as the length of the oligonucleotides increased. To ensure that the inhibitory effect on TNF{dollar}\\alpha{dollar} production was an antisense effect, inverted and sense sequences were included as controls in antisense inhibition study. Neither sequence had any significant inhibition effect on TNF{dollar}\\alpha{dollar} production. TNF{dollar}\\alpha{dollar} mRNA level was also examined by RT-PCR to further confirm the antisense effect. Our results showed a significant reduction of mRNA level in cells treated with antisense oligonucleotides. In contrast, the inverted and sense sequences had no effect. A macrophage-specific carrier (MPL) which exploited endocytosis via the macrophage mannose receptor was developed in this study. We showed that MPL can significantly enhance the cellular uptake of oligonucleotides by macrophages. The mechanism of enhanced cellular uptake was through mannose-receptor mediated endocytosis. In vitro, the MPL was demonstrated to be capable of promoting the antisense inhibitory activity of oligonucleotides against macrophage production of TNF{dollar}\\alpha.{dollar} In vivo, we showed that antisense oligonucleotides can inhibit TNF{dollar}\\alpha{dollar} secretion and had a partial protection effect on lung inflammation and fibrosis, as demonstrated by our data on lung cell recruitment, lung weight, hydroxyproline content and histological examination. However, we failed to demonstrate the advantage of ON:MPL complex over free oligonucleotide. Nevertheless, these results support the hypothesis that antisense oligonucleotides can specifically inhibit TNF{dollar}\\alpha{dollar} production and this inhibition can slow down fibrosis development. However, further improvements in the drug delivery systems or administration methods, i.e., aerosolization instead of intratracheal instillation which was used in this study, may be required to fully take advantage of the promising antisense approach.