Antisense-mediated exon skipping to correct IL-12Rbeta1 deficiency in T cells.
Patients with Mendelian susceptibility to mycobacterial disease have severe, recurrent life-threatening infections with otherwise poorly pathogenic mycobacteria and salmonellae. The extreme susceptibility is the result of genetic defects in the interleukin-12/interferon-gamma (IL-12/IFN-gamma) pathway. The infections are difficult to treat, and therapeutic options are limited. We explored the feasibility of antisense-mediated exon skipping as therapy for Mendelian susceptibility to mycobacterial disease with cells from a complete IL-12Rbeta1(-/-) patient. Expression constructs were first studied to determine whether IL12RB1 lacking exon 2 encodes a functional protein. The IL-12Rbeta1 expression construct lacking exon 2 was expressed on T cells. On IL-12 or IL-23 stimulation, this construct phosphorylated similar amounts of STAT1, STAT3, and STAT4 and induced similar amounts of IFN-gamma compared with a normal IL-12Rbeta1 construct. Antisense oligonucleotides (AONs) directed at exon 2 resulted in transcripts lacking exon 2 in both controls' and patients' T cells. In IL-12Rbeta1(-/-) cells, skipping of exon 2 led to expression of IL-12Rbeta1 on the cell surface and responsiveness to IL-12. We showed that IL12RB1 lacking exon 2 encodes a functional IL-12Rbeta1. We demonstrated that T cells can be highly efficiently transduced with AONs and are amenable to antisense-mediated exon skipping. Furthermore, we showed that exon skipping (partly) corrects the IL-12Rbeta1 deficiency in patients' cells.
van de Vosse
Esther
E
Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. E.van_de_Vosse@LUMC.nl.
Verhard
Els M
EM
de Paus
Roelof A
RA
Platenburg
Gerard J
GJ
van Deutekom
Judith C T
JC
Aartsma-Rus
Annemieke
A
van Dissel
Jaap T
JT
D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005091:Exons / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018123:Receptors, Interleukin / Q000235:genetics / Q000378:metabolism; D053707:Receptors, Interleukin-12 / Q000037:antagonists & inhibitors / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D050795:STAT2 Transcription Factor / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*
C508438:IL12RB1 protein, human; C515500:IL23R protein, human; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12; D050794:STAT1 Transcription Factor; D050795:STAT2 Transcription Factor; C494084:STAT2 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D007371:Interferon-gamma