Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model.

Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.

Hematopoietic stem cell gene therapy: towards clinically significant gene transfer efficiency.

Early clinical gene therapy and gene marking trials using retroviral vectors to transduce hematopoietic stem cells (HSC) revealed two major shortcomings of this new treatment modality. One was insufficient expression or even silencing of the integrated vector sequences, and the second was the low gene transfer efficiency achieved to in vivo repopulating cells. It became clear that neither rodent models nor human in vitro surrogate assays for stem cells were predictive of in vivo transgene levels in human target cells. Using the rhesus monkey model we have focused on improving gene transfer efficiency into HSC. Immune rejection of trans duced cells has been shown to occur in mature peripheral target cells such as lymphocytes or myocytes. Our studies and those from other investiga tors suggest that transgenes introduced via HSC induces immunologic tolerance towards the foreign product. In vivo priming of target cells, i.e. mobilization of HSC into the circulation with granulocyte colony stimulating factor and stem cell factor, as well as optimization of the in vitro transduction conditions, now allow a stable in vivo gene transfer efficiency of up to 10-15% in both lymphoid and myeloid circulating cells in non-human primates, levels that would be adequate for many clinical applications.

Plasminogen activator expression in human atherosclerotic lesions.

The plasminogen activator (PA) system may participate in the pathogenesis of atherosclerosis by modulating the turnover of intimal fibrin and extracellular matrix deposits and by contributing to intimal cell migration. We present an analysis of tissue-type PA (tPA) and urokinase-type PA (uPA) expression at three levels: mRNA by in situ hybridization, antigen by immunohistochemistry, and enzymatic activity by histoenzymology and zymography. For PA colocalization with cellular or matrix components, we used double immunofluorescence labeling in conjunction with confocal microscopy. In normal arteries, tPA antigen and mRNA were detected in endothelial cells and smooth muscle cells (SMCs). In atherosclerotic arteries, tPA antigen and mRNA were increased in intimal SMCs and in macrophage-derived foam cells of fibro-fatty lesions. Part of the tPA was detected in the extracellular space and colocalized with fibrin deposits. uPA antigen and mRNA were detected in association with the intimal macrophages and SMCs. A particularly high uPA expression was noted on macrophages localized on the rims of the necrotic core. Moreover, using a novel histoenzymological assay as well as classic zymography, we revealed uPA-dependent lytic activity in the advanced lesions, whereas in normal arteries, only tPA-dependent activity was detected, mainly over the vasa vasorum. Also, strong tPA and uPA staining was detected in neomicrovessels of the plaques, suggesting that PAs may play a role in plaque angiogenesis. Our results suggest a local dynamic process of PA-dependent proteolysis in lesion areas that is associated with macrophages and SMCs. A better comprehension of these proteolytic mechanisms in advanced atherosclerotic plaques may provide the basis for therapeutic approaches for plaque stabilization.

Localization and production of plasminogen activator inhibitor-1 in human healthy and atherosclerotic arteries.

High plasma levels of plasminogen activator inhibitor type-1 (PAI-1), the principal inhibitor of the fibrinolytic system, have been associated with thrombotic and arterial disease. To study PAI-1 expression in healthy and atherosclerotic human arteries, a detailed analysis was made by light and electron microscopy immunocytochemistry and by in situ hybridization. In healthy arteries PAI-1 was found both at the level of endothelial cells and of smooth muscle cells (SMCs) of the arterial media. In early atherosclerotic lesions PAI-1 was also detected in intimal SMCs and in extracellular areas in association with vitronectin. Immunogold analysis by electron microscopy revealed PAI-1 in vesicular structures in endothelial cells and in SMCs with normal or foam cell characteristics. In advanced atheromatous plaques, PAI-1 mRNA expression in SMCs within the fibrous cap was increased compared with SMCs located in the adjacent media or in normal arterial tissue. PAI-1 mRNA was also detected in macrophages located at the periphery of the necrotic core. The increased synthesis of PAI-1 by cellular components of the atherosclerotic plaque and the extracellular accumulation of PAI-1 may contribute to the thrombotic complications associated with plaque rupture and possibly play a role in the accumulation of extracellular matrix deposits.