Retrovirus‐Mediated Expression of an Artificial β‐Endorphin Precursor in Primary Fibroblasts

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.

Amelioration of pain and histopathologic joint abnormalities in the Col1‐IL‐1βXAT mouse model of arthritis by intraarticular induction of μ‐opioid receptor into the temporomandibular joint

“…First, HuMOR transduction of the afferent sensory nerve fibers innervating the TMJ, and the subsequent overexpression of MOR along the peripheral and central nerve axons and in the cell body, may be linked to the observed amelioration of joint pain. Previous studies have shown similar results following the injection of viral vectors encoding HuMOR directly into nerve trunks or sensory ganglia in combination with the exogenous administration of opioids (26)(27)(28)(29)(30)(31)(32)(33). However, in our experiments, there was no need for such opioid administration, since injection in the TMJ resulted in the induction of antinociception, presumably due to an interaction of the MOR with endogenous opioid ligands at the brainstem or within the joint.…”

“…Previous studies have shown that opioid precursor genes, such as enkephalins and ␤-endorphin or its receptors, can be successfully transferred to afferent sensory neurons via intrathecal administration as well as via direct injections into dorsal root ganglia or the sciatic nerve (26)(27)(28)(29)(30)(31)(32)(33). Those studies have demonstrated enhanced production of opioid peptides or receptors and a reduction of hyperalgesia in various nociceptive models.…”

Antinociceptive effect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion

“…The mechanism of adenoviral gene transfer is more like cell transplantation studies which have been reported. For example, retrovirus vectors have been used to transfect and express ␤-endorphin in fibroblasts, 27 and spinal transplantation of adrenal medullary cells which naturally release opioid peptides reduces nocifensive behaviors (tail flick and paw pinch response) 28 and have been reported to decrease experienced pain in humans with pain and terminal cancer; 29 similar results have been demonstrated using the AtT-20 pituitary tumor-derived cell line modified to constitutively secrete enkephalin. 30,31 However, these approaches all rely on release of peptides into CSF, rather than localized release of enkephalin in the dorsal horn.…”

Gene-gun particle with pro-opiomelanocortin cDNA produces analgesia against formalin-induced pain in rats

“…Beutler et al 13 demonstrated that addition of pre-pro-sequence of mouse nerve growth factor to beta-endorphin in primary fibroblast was necessary for the secretion of beta-endorphin. They showed that the pre-pro-sequence is cleaved from pre-pro-sequence/beta-endorphin construct before secretion, resulting in bona fide beta-endorphin.…”