Retrovirus-Mediated Expression of an Artificial β-Endorphin Precursor in Primary Fibroblasts |
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Authors: | Andreas S Beutler Michaela S Banck †Flemming W Bach Fred H Gage ‡Frank Porreca ‡Edward J Bilsky † Tony L Yaksh |
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Institution: | Department of Neurosciences and; Department of Anesthesiology, University of California, San Diego, La Jolla, California,; Molecular Biology and Virology Laboratory, Salk Institute, La Jolla, California, and; Department of Pharmacology, University of Arizona, Tucson, Arizona, U.S.A. |
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Abstract: | Abstract: Peptides are of potential interest in the field of gene therapy but require modification by genetic engineering to facilitate their secretion. Amino terminal addition of a signal peptide is not always sufficient to achieve this goal, as found in this study for β-endorphin. To overcome this problem, addition of the pre-pro-sequence of mouse nerve growth factor to β-endorphin was tested. Retrovirus-mediated expression of a hybrid construct of the pre-pro-sequence of nerve growth factor and human β-endorphin in primary fibroblasts resulted in the secretion of β-endorphin immunoreactivity at a rate of 620 pg/h/106 cells. Analysis of the secreted β-endorphin immunoreactivity with reverse-phase HPLC, immunoassays using three different antibodies, and an assay for the specific displacement of 3H] d -Ala2, N -MePhe4,Gly-ol5]enkephalin from μ-opioid receptors suggests that the pre-pro-sequence is cleaved off from the pre-pro-sequence/β-endorphin construct prior to secretion, resulting in bona fide β-endorphin. Transplantation of β-endorphin-secreting cells into brain or spinal cord may provide a gene therapy approach for the treatment of chronic, opioid-sensitive pain states. |
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Keywords: | Gene therapy Pain Opioid peptides Pre-pro-sequence Nerve growth factor Furin |
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