Deletion of individual exons and induction of soluble murine interleukin-5 receptor-alpha chain expression through antisense oligonucleotide-mediated redirection of pre-mRNA splicing.
Expression of the interleukin-5 receptor-alpha (IL-5Ralpha) chain is thought to play an important role in the pathogenesis of asthma and other eosinophilic diseases. With antisense oligonucleotides (ASOs) chemically modified to provide increased hybridization affinity for RNA but that do not support RNase H-mediated cleavage (2'-O-methoxyethyl-modified ASOs), we show that constitutive splicing of murine IL-5Ralpha mRNA can be modulated in cells such that individual exons may be selectively deleted from mature transcripts. Specific deletion of individual exons and redirection of alternative splicing of the IL-5Ralpha mRNA have been achieved with this approach, by targeting 3'-splice sites or exon sequences immediately downstream of an alternative splice site. ASO targeting with these strategies resulted in inhibition of mRNA and protein levels of the membrane IL-5Ralpha isoform capable of signaling IL-5-mediated growth and antiapoptotic signals to eosinophils. Membrane isoform IL-5Ralpha inhibition was coupled with an increase in expression of mRNA for the alternatively spliced soluble isoform, which binds IL-5 extracellularly and may block its function. These observations suggest the potential general therapeutic use of an antisense approach to increase expression of variant RNA transcripts and to thereby produce proteins devoid of specific functional domains that may impact disease processes, as well as its specific utility for modulating expression of a key cytokine receptor implicated in allergic inflammation.
Karras
J G
JG
Department of Molecular and Cellular Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008, USA. jkarras@isisph.com
McKay
R A
RA
Dean
N M
NM
Monia
B P
BP
D017398:Alternative Splicing / Q000187:drug effects*; D000818:Animals; D001483:Base Sequence; D005091:Exons / Q000235:genetics; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects*; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D020033:Protein Isoforms; D012322:RNA Precursors / Q000187:drug effects / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000235:genetics* / Q000378:metabolism; D053649:Receptors, Interleukin-5; D016914:Ribonuclease H / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D012322:RNA Precursors; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D016914:Ribonuclease H