Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.

Abstract

Genetically engineered fibroblasts have been successfully used to produce therapeutic proteins in animals, but sustained production of the proteins has not been achieved. This limits the potential of fibroblast-mediated gene therapy in humans. We have studied the phenomenon of decreased production in rats by using retroviral vectors carrying genes encoding human adenosine deaminase and neomycin phosphotransferase. While transplanted skin fibroblasts containing vector sequences persisted at constant levels for at least 8.5 mo, vector expression decreased by >1500-fold after 1 mo. Cellular or antibody-mediated immune responses were not detected in transplanted animals, and expression could not be restored in fibroblasts recultivated from the grafts. This phenomenon is reminiscent of sequence-specific gene inactivation observed in other cell types. Because genetic manipulation and expression of foreign proteins did not affect survival of the transplanted cells, effective long-term therapy may be possible with the use of alternative gene regulatory elements.Although many somatic cell types are potential gene therapy targets for treatment of genetic or acquired disease (1), skin fibroblasts are attractive because they are easy to obtain and transplant and can be rapidly grown to large numbers in culture. Immortalized fibroblasts make a variety of secretory products after genetic modification and transplantation into animals (2-7), but these are ultimately not appropriate models for gene therapy because these cell lines grow uncontrollably and often form tumors in recipient animals. Primary fibroblasts, however, could be used for human therapy. In animals, primary embryo or skin fibroblasts produce clotting factor IX at systemic levels that approach therapeutic utility (5, 6), but production of the protein for >1 mo has not been achieved. The gradual decrease in protein levels might be caused by immune response against the foreign protein, poor survival of the transplanted cells, or inactivation of the transferred genes.To address these issues, we have studied the transfer of a human adenosine deaminase (hADA)-encoding gene into skin fibroblasts of inbred rats. By using hADA in place of factor IX, we could avoid several complicating problems associated with production of a foreign secreted protein: hADA should not be immunogenic because it is an intracellular protein; hADA is localized at the site of the graft, and detection is not dependent on systemic distribution; and finally, sensitive assays make even low-level hADA readily detectable above endogenous rat adenosine deaminase (ADA). By careful examination of transplanted tissues, we show here that expression of hADA had no effect on cell survival, but that transplanted cells gradually inactivate retrovirally transferred genes.MATERIALS AND METHODS Primary Cell Culture. Primary skin fibroblasts were isolated by standard methods (8) from either human foreskin or forearm biopsies, or from inbred Fischer 344 rats. Cells were grown at 370C in a 10%o CO2 atmosph...

Cell type-specific activation of the cytomegalovirus promoter by dimethylsulfoxide and 5-Aza-2'-deoxycytidine

“…However, it becomes inactivated over time after insertion into the host genome (Cameron, Bachman, Myohanen, Herman, & Baylin, 1999;Choi et al, 2005;Knust et al, 1989;Palmer et al, 1991;Verma & Somia, 1997). Finding ways to activate the silenced CMV promoter should help overcome this limitation and facilitate the use of this promoter for enhancing the expression of the transgenes and production of recombinant proteins (Singh et al, 2004).…”

“…However, many transgenes under the control of viral promoters, including the CMV promoter, have been found to be gradually silenced over a period of months in culture (Choi, Basma, Singh, & Cheng, 2005;Knust, Bruggemann, & Doerfler, 1989;Krishnan et al, 2006;Palmer, Rosman, Osborne, & Miller, 1991;Verma & Somia, 1997). The gene silencing mechanism of these promoters is not clear, although DNA methylation, histone hypoacetylation, activation of signaling molecules and altered levels of transcriptional factors have been suggested (Brooks, Harkins, Wang, Qian, Liu, & Rubanyi, 2004;Swindle & Klug, 2002).…”

Gene transfer to the central nervous system by transplantation of cerebral endothelial cells

“…As noticed above for ␤-galactosidase-expressing cells, this decrease was probably due to progressive inactivation of the transgene since the grafted RBE/NGF cells were still present at that time. 27 These results demonstrate the efficacy of RBE/NGF cells in exerting specific neurotrophic and neurotropic effects through in situ delivery of biologically active ␤-NGF protein in the CNS parenchyma.…”

Transduction of axotomized retinal ganglion cells by adenoviral vector administration at the optic nerve stump: an in vivo model system for the inhibition of neuronal apoptotic cell death

“…It is more likely that silencing of the MCMC promoter occurred as it has been described for CMV and RSV viral promoters in organotypical cultures or cell culture. 41,42 In no case did we observe damages of the retinal structure following transduction of the nerve stump as seen after intraocular injection of a ␤-galactosidase coding vector. 43 Due to this detrimental effect of the Ad.lacZ vector it was not used as a control for the therapeutic Ad.p35 and Ad.crmA vectors in the present study.…”