Granulocyte-macrophage colony-stimulating factor mRNA stabilization enhances transgenic expression in normal cells and tissues

• Published in: Blood Vol. 86; no. 7; pp. 2551 - 2558
• Main Authors: RAJAGOPALAN, L. E, BURKHOLDER, J. K, TURNER, J, CULP, J, NING-SUN YANG, MALTER, J. S
• Format: Journal Article
• Language: English
• Published: Washington, DC The Americain Society of Hematology 01.10.1995
• Subjects: Animals; Antineoplastic drugs. Cytokines; Base Sequence; Biological and medical sciences; Biotechnology; Cells, Cultured; DNA, Complementary - genetics; Drug Stability; Epidermis - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor - genetics; Health. Pharmaceutical industry; Humans; Industrial applications and implications. Economical aspects; Leukocytes, Mononuclear; Mice; Molecular Sequence Data; Mutagenesis; Production of active biomolecules; Rats; Recombinant Proteins; Repetitive Sequences, Nucleic Acid; RNA, Messenger - metabolism; Skin - metabolism; Transfection; Human; Stabilization; Cytokine; Rodentia; Biosynthesis; Genetic transfer; Vertebrata; Mammalia; Mononuclear cell; Messenger RNA; Transfection; Mouse; Polypeptide; Animal; Skin; Granulocyte macrophage colony stimulating factor
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.v86.7.2551.2551

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.