Construction And Analgesic Effect Of Immortalized Astrocytes Expressing Enkephalin Under Control Of Doxycycline

BackgroundChronic neuropathic pain is a common symptom in clinical practice and patients with chronic pain are subject to a greatly impaired quality of life. The treatment of chronic pain with traditional methods such as drug, acupuncture therapy or sympathetectomy has many unresolvable side effects and complications, especially the tolerance and dependence for long-period drug delivery that could cause the ineffectiveness of the treatment. An important issue of pain research is to find a novel method of analgesia that is safe and effective. Grafted genetically-modified cells secreting enkephalin have been considered an encouraging treatment for chronic pain. Importantly, the transplanted cell as a therapeutic agent should be reproducible, safe, and controllable. Astrocytes, as components of the brain, have served as grafted cells in gene therapy research because of their histocompatibility and lower immunogenicity. However, it is difficult to obtain primary astrocytes in large quantities and the life span of primary cells in culture is limited. Immortalized astrocytes are easy to manipulate and can be produced unlimitedly, which makes it possible for primary astrocytes to serve as the vehicle of therapeutic genes. Moreover, our previous work has shown that immortalized astrocytes are reproducible and nontumorigenic in vitro and in vivo.However, excessive and constant release of opioid peptides from implanted cells is not necessary and is potentially toxic. Therefore, it may be necessary to engineer a cell that enables tight regulation of the levels and duration of expression of transgene products. The recent development of tetracycline-regulated transactivation systems serve as gene switches to control gene expression promptly and appear to be well-suited for regulating long-term protein secretion.In this study, by taking advantage of both the tight regulation provided by the tetracycline-regulated ystem and the reproducibility of immortalized astrocyte cells, we aimed to develop an immortalized rat astrocyte cell whose enkephalin production can be controlled by doxycycline in a dose-responsive and reversible manner in vitro or in vivo. The human preproenkephalin (hPPE) gene encodes production of preproenkephalin which can be converted to bioactive enkephalin, which can ameliorate nociception by activating peripheral opioid receptors. Thus, we expected that the hPPE gene, introduced into modified cells and delivered into spinal cords, would be a good candidate to alleviate nociception. The regulatory analgesic effects of doxycycline on spared nerve injury (SNI) -induced nociceptive behavior in rats were investigated after these cells were transplanted into their subarachnoid space. The availability of regulatable grafted cells to treat chronic neuropathic pain was assessed.Methods and Results1. Retroviral vector constructs and virus productionMethods Human proenkephalin gene was amplificated by polymerase chain reaction (PCR) and was directionally cloned into the pRevTRE vector to obtain the recombinant vector pRevTRE/hPPE which was further verified by restriction endonuclease analysis and DNA sequencing. Then this recombinant plasmid and regulatory plasmid pRevTet-On were packed by packing cell line PT67 to yield two classes of viruses. The transfected PT67/Tet-On and PT67/TREhPPE cells were selected by the corresponding antibiotics and examined by using RT-PCR. Then selected cell were enriched to assay virus titer using NIH3T3 cell.Results Restriction endonuclease analysis, PCR analysis and DNA sequencing analysis confirmed that the recombinant RevTRE/hPPE retroviral vector was constructed successfully. The virus titer of recombinant was 3.2×105CFU/ml and the virus titer of pRevTet-On was 2.6×105CFU/ml.2. Construction of an immortalized rat astrocyte strain genetically modified with Tet-on geneMethods The RevTet-On virus supernatants was used to infect immortalized rat astrocyte strain (IAST). Forty-eight hours post-infection, cells were screened in medium containing 400μg/ml G418. Then the G418-resistant cell clones were rescreened by transient transfection with the reporter plasmid pRevTRE-Luc. One IAST/Tet-On clone with high inducibility and low background was selected by detecting Luciferase activity. Results A total of 48 IAST/Tet-on positive clones were assessed by luciferase assay. Finally, clone 6 was picked for its high induction of luciferase activity in response to doxycycline and low leakiness (activity in the absence of doxycycline); its inducible efficiency was 20.1-fold. The expression of luciferase was induced in dose-dependent manner by doxycycline at the concentrations between 100 and 2 000ng/ml .The expression of luciferase began 1 h after doxycycline administration (1 000ng/ml) and reached the maximum level 48 h later.3. Construction of an immortalized rat astrocyte strain with inducible enkephalin expressionMethods RevTRE/hPPE virus was used to infect the IAST/Tet-On strain, after 48 h, transfected cells were cultured for 21 days in the presence of G418 (500μg/ml) and hygromycin (400μg/ml). A single clone named IAST/Tet-On/hPPE was isolated. hPPE gene expression level of IAST/Tet-On/hPPE cells at different concentrations of doxycycline were detected by RT-PCR, immunocytochemistry and indirect immunofluorescence staining. The kinetics of Leu-enkephalin protein expression level secreted by IAST/Tet-On/hPPE cells under different concentrations of doxycycline administration and incubation for various times was assayed using a Leu-enkephalin radioimmunoassay.Results The regulatable expression of the hPPE gene by doxycycline in IAST/Tet-On/hPPE cells was confirmed by RT-PCR immunocytochemistry, indirect immunofluorescence staining and radioimmunoassay. Treatment of doxycycline at concentrations greater than 0.1μg/ml reliably induced enkephalin expression and its expression was increased following increasing concentrations of doxycycline in the culture medium. When doxycycline was added to the culture media (1μg/ml), enkephalin secretion increased gradually, then rapidly to 12-fold after 48 h, and returned to basal levels after doxycycline removal for at least 24 h. Comparable results were obtained during the second on-off cycle. These results suggest that the expression of the hPPE gene can be controlled by doxycycline in a dose-dependent manner.4. Analgesic effect of intrathecal transplantation of immortalizedenkephalin expressing astrocytes on chronic neuropathic pain in rats Methods 50 adult female Sprague-Dawley rats (150~200g) were randomly divided into five groups: Na?ve group, SNI group, SNI/control, SNI/hPPE/Dox(-)and SNI/hPPE/Dox(+). One week after right side spared nerve injury (SNI), IAST or IAST/Tet-On/hPPE cells were transplanted in the subarachnoid space of lumbar 4 to 6 of SNI rats and rats grafted with IAST/Tet-On/hPPE were administered saline or doxycycline in drinking water ad libitum for different predetermined times. One week before SNI and once a week for seven weeks following SNI and cell transplantation, all animals were retested on nociceptive tests. The spinal cord of L4-6 was removed eight weeks after cell transplantation, Leu-enkephalin expression using immunohistochemistry or real-time PCR, then the expression of hPPE was assessed in the dorsal horn, the content of Leu-enkephalin in the spinal cord was measured by radioimmunoassay.Results Allodynia-like behavior and the decrease of MWT and TWD levels were observed one week after SNI. The tactile allodynia induced by SNI was alleviated in IAST/Tet-On/hPPE group at the one week after cell transplantation. Administration of 2 mg/ml doxycycline in the drinking water increased the analgesia effect in animals grafted with IAST/Tet-On/hPPE cells (P<0.05). The expression of hPPE and the level of L-EK were higher in IAST/ hPPE /Dox(+) group than other groups (P<0.05) and the expression of Fos protein was lower in IAST/ hPPE /Dox(+) group than other groups (P<0.05).5. Statistical analysisAll of the analyses were performed by SPSS 11.0 software package. All data were expressed as the mean±standard deviation (SD). Group comparisons were made using repeated measures analysis of variance (behavioral testing data) or one-way analysis of variance (the level of enkephalin). A P-value of < 0.05 was considered statistically significant.ConclusionAn immortalized rat astrocyte strain secreting enkephalin under the control of doxycycline was established successfully in our study. Enkephalin production from IAST/Tet-On/hPPE cells can be controlled by doxycycline in a dose-responsive and reversible manner in vitro or in vivo. The tactile allodynia induced by SNI was alleviated in IAST/Tet-On/hPPE group and the analgesic effect was also able to be regulated by Dox. Our study provides a new strategy for regulatable gene therapy for chronic pain in the future.SummaryIn this study, Tet-on system and the reproducibility of immortalized astrocyte cells were in a novel combination to achieve the useful goal of regulated release of a transgene product in vivo to treat pain. An immortalized astrocyte cell line has been engineered which releases enkephalin in a doxycycline-dependent fashion. Both in vitro and in vivo data are presented to characterize regulation of the hPPE gene expression and these enkephalin-producing cells significantly suppressed nociceptive responses when grafted into the spinal cord of SNI rats. Although improvements in the Tet-on system are necessary, this study suggests transgenic hPPE gene cell transplantation regulated by a Tet-on system may provide an alternative approach for ex vivo cell transplantation to treat chronic pain.