Dose-Dependent Doxycycline-Mediated Adrenocorticotropic Hormone Secretion from Encapsulated Tet-On Proopiomelanocortin Neuro2A Cells in the Subarachnoid Space

Abstract

We previously reported that polymer-encapsulated mouse neuroblastoma cells that are capable of secreting beta-endorphin may reduce pain sensitivity in rats after capsule implantation into the cerebrospinal fluid (CSF)-filled subarachnoid space of the spinal cord. The neuroblastoma cells carry the proopiomelanocortin (POMC) gene that encodes the precursor of adrenocorticotropic hormone (ACTH) and beta-endorphin. To control the expression of these hormones in the present study, a promoter that is inducible by administration of tetracycline derivatives such as doxycycline (Dox) was linked to the POMC gene. Encapsulated cells in the CSF space of rats stimulated by four intraperitoneal doses of Dox responded with ACTH expression as determined in a subsequence 36-hr in vitro incubation. The amount of ACTH released was dependent on the in vivo Dox dose. These findings indicate that gene expression in xenogeneic cells in the CSF space can be manipulated by injection of a relatively innocuous drug, and suggest that this system may be applicable to cell transplantation therapy in patients with central nervous system diseases that require temporary control of ligand delivery.

A novel doxycycline inducible autoregulatory plasmid which displays ‘on’/‘off’ regulation suited to gene therapy applications

“…In general the kinetics of induction for these clones is slightly slower than reported for the permanently integrated two plasmid 'on' system where maximal expression is achieved in less than 24 h. 7 The slower induction observed with our plasmids is probably explained by the autoregulated expression of rtTA resulting in a delay in optimal levels of rtTA expression. No previous in vitro data have been published on the kinetics of switching 'off' expression using the 'on' system, but our findings with these autoregulatory plasmids is comparable to observations in vivo with the two plasmid 'on' system 23 and demonstrates their suitability to gene therapy applications. Kinetics of gene regulation in the 'off' system has been reported for a selfcontained plasmid pSiaII, where Dox was shown to switch 'off' GM-CSF expression after 24 h, but following removal of Dox for 150 h the reversal in GM-CSF expression was only 50%, and maximal gene expression was only observed after more than 9 days.…”

Electroporation-mediated pain-killer gene therapy for mononeuropathic rats

“…With improved plasmid design, regulated long-term expression, specific promoter/enhancer elements to targeting gene expression within restricted tissue and/or groups of cells, or further refinement with variations in types of electrodes used (from a fine point to a long wire or plate), this in vivo approach may offer efficacy with just a single treatment. [21][22][23][24] This contrasts with intrathecal drug pumps which require ongoing maintenance and pose a continuous infection risk. Furthermore, multiple genes can also be simultaneously examined by co-electroporation of mixed expression vectors, 25 and blocking gene function or expression is also possible using dominant negative molecules.…”

Systemic and Localized Reversible Regulation of Transgene Expression by Tetracycline with tetR-Mediated Transcription Repression Switch

“…On the basis that nude NCr mice are immunodeficiency and unable to initiate all cell-mediated immune reactions, one could speculate that the decrease in overall hEGF expression after the tetracycline induction is likely because of one or combination of the following factors: poor survival of transplanted cells at the site of injection; potential loss of transgene in some of implanted cells; and intrinsic partial in vivo inactivation of the hCMV immediate-early promoter as observed previously [25,27]. The survival and replication capacity of implanted genetically modified non-autologous cells could potentially be enhanced and prolonged by enclosing cells in a permeable polymer capsule [35][36][37][38][39]. For example, Serguera et al [35] showed that implantation of encapsulated Epo expressing-NIH 3T3 cells can offer systemic delivery of Epo in immunocompetent mice over 6 months.…”