These results indicated LV-mediated gene transfer and gene expression in HSC/primitive progenitors by iBM injection in mice without any preconditioning. Open in a separate window FIG. LAM-PCR exhibited that multiple transduced clones contributed to hematopoiesis in these animals. We also showed that GFP-expressing MSC retained multilineage differentiation potential, with 2.9 to 8.8% GFP-containing CFU-fibroblasts detected in both injected and BMT recipients. Our data provide evidence that adult stem cells in bone marrow can be efficiently transduced vector administration without preconditioning. This approach could lead to a novel application for treatment of human diseases. gene transfer, hematopoietic stem cells, lentiviral vectors, mesenchymal stem/progenitor cells, bone marrow transplantation, hematopoiesis Introduction HIV-based lentiviral vectors (LV) were proven to be capable of transducing a broad spectrum of nondividing cells in multiple mammalian species cIAP1 Ligand-Linker Conjugates 14 [1]. Using local injection, LV have been shown to mediate gene transfer and sustained gene expression in brain neuronal cells [2], retina cells [3], liver hepatocytes [4], rat cardiomyocytes [4], airway epithelium [5], and kidney tissue[6]. In contrast to adenoviral vectors, no pathology that could be specifically attributed to LV administration has been observed in any of these animal studies. Efficient transduction of purified hematopoietic stem cells (HSC) by LV has been shown by a number of investigators [7,8]; however, LV-mediated gene transfer into HSC has not been well studied. Following intravenous administration of a first-generation LV into adult mice, we found that bone marrow exhibited the highest levels of transgene among nine organs examined, with more than 10% green fluorescent protein-positive (GFP+) cells detected in peripheral blood leukocytes (PBL) in these mice [9]. It was also observed by others that a significant transgene signal was detected in the bone marrow (BM) by PCR analysis in adult mice of systemic administration of HIV-biased LV [10]. Recently, we exhibited detectable levels of transgene (up to 3.9%) in PBL of mice 4 months after secondary bone marrow transplantation (BMT) of HSC transduced by delivery of LV in newborn pups [11]. These data strongly suggest the possibility that delivery of LV may provide an alternative approach to transducing HSC. There is extensive clinical experience with the application of gene transfer targeting HSC, mostly with onco-retrovirus vectors (RV). However, the frequency of MUC16 gene transfer in multipotent HSC and the levels of long-term transgene expression have been variable and generally low. A few exceptions emerged when successful functional correction was exhibited in children with severe combined immunodeficiency (ADA-SCID and X-SCID) or chronic granulomatous disease following infusion of retrovirally transduced autologous CD34+ cells [12C14]. The success in these studies was related in part to the selective growth of genetically altered/corrected progenitors. This positive selection pressure may compensate for the relatively low numbers of transduced and successfully engrafted HSC [15]. While clinical efficacy has been achieved in these clinical trials using gene transfer, a more general clinical application of this approach is still impeded by several troubles. First, maintaining stem cell properties including repopulation potential during culture is usually a prerequisite of any successful gene transfer approach. Second, inadequate engraftment is usually another unsolved obstacle that affects the outcome of HSC gene therapy. Other issues associated with the approach include toxicity related to HSC enrichment procedures and cytokine stimulation [15], the potential for proliferation-induced genomic damage, and the contamination risk of multistep manipulations. As an alternative, gene transfer into HSC has the potential to overcome these problems. Bone marrow stem cells from adults have been viewed as the ideal target for gene- and cell-based therapy of genetic diseases, selected malignant diseases, and AIDS. In addition cIAP1 Ligand-Linker Conjugates 14 to HSC, bone marrow contains mesenchymal stem/progenitor cells (MSC), which can differentiate into mature cells of multiple mesenchymal tissues cIAP1 Ligand-Linker Conjugates 14 including fat, bone, and cartilage. Moreover, a populace of highly plastic BM cells (copurified initially with HSC from adult marrow of mice, rats, and humans) has also been shown to differentiate into hematopoietic cells, as well as epithelial cells, in liver, lung, and intestine to varying degrees after intravenous injection into immunodeficient mice [16] (see for review [17]). Although controversial in their interpretation as evidence for a common pluripotent precursor cell, the.