Retrovirus-mediated expression of an artificial beta-endorphin precursor in primary fibroblasts.
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 beta-endorphin. To overcome this problem, addition of the pre-pro-sequence of mouse nerve growth factor to beta-endorphin was tested. Retrovirus-mediated expression of a hybrid construct of the pre-pro-sequence of nerve growth factor and human beta-endorphin in primary fibroblasts resulted in the secretion of beta-endorphin immunoreactivity at a rate of 620 pg/h/10(6) cells. Analysis of the secreted beta-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 mu-opioid receptors suggests that the pre-pro-sequence is cleaved off from the pre-pro-sequence/beta-endorphin construct prior to secretion, resulting in bona fide beta-endorphin. Transplantation of beta-endorphin-secreting cells into brain or spinal cord may provide a gene therapy approach for the treatment of chronic, opioid-sensitive pain states.
Beutler
A S
AS
Department of Neurosciences, University of California, San Diego, La Jolla 92093-0818.
Banck
M S
MS
Bach
F W
FW
Gage
F H
FH
Porreca
F
F
Bilsky
E J
EJ
Yaksh
T L
TL
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism* / Q000821:virology; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000235:genetics; D011498:Protein Precursors / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D012192:Retroviridae Infections / Q000378:metabolism; D001615:beta-Endorphin / Q000378:metabolism*
D015335:Molecular Probes; D009414:Nerve Growth Factors; D011498:Protein Precursors; D001615:beta-Endorphin