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...
Incorporation of a central polypurine tract (cPPT) and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression. We compared the effects of these elements individually and together on transduction efficiency and gene expression, using lentivirus vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) and encoding enhanced green fluorescent protein (GFP) and rat erythropoietin (EPO). The transduction efficiency was greater than 2-fold higher in the vector containing the PRE element, 3-fold higher in vector encoding the cPPT element, and 5-fold increased in the GFP virus containing both cPPT and PRE elements relative to the parent virus. In comparison with parent vector the mean fluorescence intensity (MFI) of GFP expression was 7-fold higher in cells transduced with virus containing PRE, 6-fold increased in cells transduced with virus containing cPPT, and 42-fold increased in GFP-virus containing both cPPT and PRE elements. EPO-virus containing a PRE element showed a nearly 5-fold increase in EPO secretion over the parent vector, and the vector encoding both PRE and cPPT showed a 65-fold increase. Thus, lentivirus vectors incorporating both PRE and cPPT showed expression levels significantly increased over the sum of the components alone, suggesting a synergistic effect.
Cultured vascular smooth muscle cells (SMCs) containing retrovirally introduced genes are a potential vehicle for gene replacement therapy. Because the cultured SMCs are selected for their ability to proliferate in vitro, it is possible that the SMCs might be permanently altered and lose their capacity to respond to growth-suppressing conditions after being seeded back into blood vessels. To investigate this possibility we measured SMC proliferation and intimal thickening in balloon-injured Fischer 344 rat carotid arteries seeded with SMCs stained with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and infected with replicationdefective retrovirus expressing human adenosine deaminase or human placental alkaline phosphatase. The majority of the seeded SMCs remained in the intima while a few of the cells appeared to migrate into the first layer of the media. Intimal SMC proliferation returned to background levels (< 0.1% thymidine labeling index) by 28 d. At late times (1 and 12 mo) the morphological appearance of the intima was the same for balloon-injured arteries with or without seeded SMC, except that the seeded arteries continued to express human adenosine deaminase or alkaline phosphatase. These results support the conclusion that cultured SMC infected with a replication-defective virus containing human adenosine deaminase or alkaline phosphatase are not phenotypically altered and do not become transformed. After seeding onto the surface of an injured artery, they stop replicating but continue to express the introduced human genes even over the long term. (J. Clin. Invest.
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