Neutrophil-selective CD18 silencing using RNA interference in vivo

Abstract

Tissue-specific silencing of genes may be used for genetic engineering in mice and has possible therapeutic applications in humans. Current strategies in mice rely on Cre/loxP technology requiring the generation of multiple transgenic lines and breeding strategies. Here, we describe the selective silencing of CD18, a leukocyte-specific integrin in neutrophils using a micro RNA (miRNA) strategy that requires the generation of one transgenic line. CD18-specific miRNA hairpin driven by the myeloid specific human MRP8 promoter resulted in the generation of transgenic lines with 75% to 95% reduction in CD18 protein levels in neutrophils and monocytes. Minimal decreases in T cells and a partial diminution in macrophages were observed. Neutrophil CD18 silencing resulted in neutrophilia, splenomegaly, and significant defects in neutrophil trafficking with the degree of alterations correlating with the extent of CD18 silencing. Thus, our data demonstrate the utility of using miRNA approaches to silence genes in neutrophils, which are terminally differentiated cells with a short half-life that largely precludes their genetic manipulation in vitro. Furthermore, the mouse models provide a valuable tool to examine the contribution of CD18 on neutrophils to leukocyte adhesion deficiency type I (LAD-I), a complex inherited disorder in which reduced or absent CD18 expression in multiple leukocyte subsets leads to impaired innate and adaptive immune responses. IntroductionHomologous recombination combined with the Cre/loxP system has been used to achieve conditional or tissue-specific targeting of gene expression in mice. This approach requires the generation of homologous targeted embryonic stem cells, and multiple transgenic lines and breeding strategies. 1 Recent advances in RNA interference (RNAi) have provided an attractive alternative to silencing genes in vivo that is more rapid and also has possible therapeutic applications. RNAi is a posttranscriptional genesilencing mechanism that is induced by double-stranded RNA. Introduction of chemically synthesized small interfering RNAs (siRNAs) and/or transfections of DNA-based vector systems driving the expression of short hairpin RNAs (shRNAs) by RNA pol III promoters (U6 and H1) have been used as powerful tools to examine the function of specific genes. Endogenous micro RNAs (miRNA) are small single-stranded RNAs that regulate genes required for a wide range of cellular functions. miRNAs are produced by the processing of primary transcripts (pri-mRNA) by the RNAse III type enzyme Drosha into pre-miRNAs (approximately 70 nucleotide stem-loop RNAs), which are exported to the cytoplasm and further cleaved by the cytoplasmic RNase III Dicer into miRNA-containing RNA duplexes. In the case of near-perfect complementarity to the targets, the outcome is endonucleolytic cleavage of the target mRNA, 2-5 while binding to partially complementary sites, predominantly in the 3ЈUTR, causes mRNA degradation or repression of translation. [6][7][8] The expression of artificial miRNAs by RNA P...

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