Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants.

Abstract

Toward the goal of gene therapy, we have been attempting to establish model somatic cell systems with the potential of talned expression of the foreign gene. We report here that long-term expression of foreign genes in mouse embryo fibroblast implants can be achieved If a housekeeping gene promoter is used to drive transcription. Speitfcally, we have shown that in implants conining a f-galactosidase gene linked to either an immediate early promoter of cytomegalovirus or a dihydrofolate reductase (DHIFR) gene promoter, only the DHFR promoter allows long-term expression. We propose that choice of the promoter manife sgificant influence on the long-term expression of genes introduced in fibroblast implants by retroviral vectors.Current models for gene therapy involve the retrovirusmediated transfer of genetic material into cells derived from a variety of somatic host tissues, including cells of the hematopoietic system, fibroblasts, hepatocytes, endothelial cells, and myoblasts (1, 2). We have previously described an approach for gene product delivery by retroviral infection of mouse skin fibroblasts (3). The transduced genes used in our previous studies in fibroblasts were the human and dog factor IX cDNAs (3, 4). Although high levels of sustained expression could be achieved in tissue culture, when transplanted as allografts in rodents, these fibroblasts produced substantial amounts of factor IX for only a short period of time (3,5). This short duration of expression in vivo could theoretically be attributed to different factors: (i) an immune response of the host against the exogenous factor IX; (ii) destruction of the foreign cells after transplantation; and (iiM) a specific decrease in transcription of the transferred gene once the transduced cells are grafted to the animal. It has been shown (3, 5) that antibodies to human factor IX are present after implantation of modified fibroblasts, which could explain, at least in part, the short period of detection of factor IX.In this work, using different promoters to control the expression of 8-galactosidase, we demonstrate that, whereas in tissue culture long-term expression can be easily obtained, the type of promoter directing transcription of the gene of interest can be one of the key factors determining long-term expression in vivo. MATERIALS AND METHODSAnimal and Cell Culture Conditions. Adult male C57BL/6J mice (6-8 weeks old) and Nu/Nu athymic mice were obtained from The Jackson Laboratory. The retroviral packaging cell lines fCRE and *CRIP (6) and the cell lines NIH 3T3 and rat 208 F were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum. Primary fibroblasts were obtained from day 17 embryos of C57BL/6J mice and were grown in DMEM supplemented with 10% fetal calf serum. Infected cells were selected in medium containing 400 pkg of G418 per ml.Vector Construction. Retroviral vector LNCdF9L, which transduces canine factor IX, has been described (4). The vectors shown in Fig. 1

Effect of Bcr sequences on the cellular function of the Bcr-Abl oncoprotein

“…The C-terminal 34 amino acids of GCN4 which contain the leucine zipper domain were ®tted with a consensus eukaryotic translation initiation sequence and then fused in frame with the Bcr-Abl coding region (Figure 1, ZIP-Bcr/64-509-Abl). For expression in mammalian cells, the ZIP-Bcr-Abl gene was inserted into the retroviral vector pSLXCMV (Scharfmann et al, 1991).…”

A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line

“…The modified ␣1AT cDNA was cut with BglII and XhoI, and cloned into the BamHI-BglII site of pSLXCMV 30 to generate pSLXCMVm␣1AT (Figure 8). Orientations and sequences of retroviral vectors were confirmed by DNA sequencing.…”

Sustained gene expression in transplanted skin fibroblasts in rats

“…Vector inactivation could be the reason, at least in part, why viral promoters, such as SV40, Moloney LTR as well as CMV immediate-early promoter drive high levels of gene expression in cultured skin fibroblasts, but became inactive after cells were implanted in vivo. [26][27][28]36 Downregulation of retroviral vector sequences in transplanted keratinocytes has also been reported. 33 In mice, phosphoglycerate kinase promoter has been used to derive longterm expression of ␤-glucuronidase 30,38 and erythropoietin 32 in retrovirally transduced fibroblasts implanted into 'neo-organs' in the peritoneal cavity.…”

“…When fibroblasts are in a quiescent state the choice of promoter for the transduced gene was a major determinant for long-term in vivo gene expression. 36,37 When dihydrofolate reductase (DHFR) and CMV promoters were compared, long-term expression of ␤-galactosidase was only achieved by DHFR promoter. 36 This difference may reflect inactivation of the CMV viral promoter whereas DHFR, a house-keeping promoter, can still be active in quiescent cells.…”

“…36,37 When dihydrofolate reductase (DHFR) and CMV promoters were compared, long-term expression of ␤-galactosidase was only achieved by DHFR promoter. 36 This difference may reflect inactivation of the CMV viral promoter whereas DHFR, a house-keeping promoter, can still be active in quiescent cells. However, these studies used mouse embryo fibroblasts which may not correlate well with gene expression in non-embryonic cells, the only cells available for gene therapy protocols.…”