Granulocyte-macrophage colony-stimulating factor mRNA stabilization enhances transgenic expression in normal cells and tissues.
To increase transgenic production of granulocyte-macrophage colony-stimulating factor (GM-CSF), we mutated the mRNA's 3'-untranslated region, AUUUA instability elements. Expression vectors containing human or murine GM-CSF cDNAs coding for wild-type (GM-AUUUA) or mutant versions with reiterated AUGUA repeats (GM-AUGUA) were transfected into cells in culture or animals using particle-mediated gene-transfer technology. Normal peripheral blood mononuclear cells accumulated 20-fold greater levels of GM-CSF mRNA and secreted comparably greater amounts of cytokine after transfection with hGM-AUGUA expression vectors versus hGM-AUUUA. hGM-AUGUA mRNA was fivefold more stable (t 1/2 = 95 minutes) than hGM-AUUUA mRNA (t 1/2 = 20 minutes), accounting for elevated steady-state levels. Transfection site extracts and serum samples obtained 24 hours after gene transfer of hGM-AUGUA cDNA into mouse skin contained greater than 32 ng/mL and 650 pg/mL of GM-CSF protein, respectively, compared with 0.33 ng/mL and less than 8 pg/mL for hGM-AUUUA cDNA. GM-CSF produced from mGM-AUGUA cDNA transfected into rat abdominal epidermis induced a profound neutrophil infiltrate. These data suggest a novel strategy for enhanced production of biologically active cytokines by normal cells after in vivo gene transfer.
Rajagopalan
L E
LE
Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison 53792-2472, USA.
Burkholder
J K
JK
Turner
J
J
Culp
J
J
Yang
N S
NS
Malter
J S
JS
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004355:Drug Stability; D004817:Epidermis / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins; D012091:Repetitive Sequences, Nucleic Acid; D012867:Skin / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor