1. ¹Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Ibaragi-city, Osaka, Japan
2. ²Pharmaceuticals and Medical Devices Agency, Tokyo, Japan
3. ³Graduate School of Pharmaceutical Sciences, Osaka University, Suita-city, Osaka, Japan

Abstract

Because of their capacity for self-renewal and multilineage differentiation into all types of mature blood cells, hematopoietic stem cells (HSCs) are highly attractive, not only as targets of gene therapy but also as cell sources for regeneration medicine. Hence, efficient transduction into HSCs holds promise for treating a number of diseases that result from abnormal blood cell function, and it would provide a powerful approach for understanding their basic biology and for manipulating their properties in vitro and in vivo. Numerous studies of transduction into HSCs have been performed using various types of gene transfer vectors. Retrovirus vectors and lentivirus vectors are often used for transduction into HSCs when stable transgene expression is desirable. In contrast, adenovirus (Ad) vectors are highly suitable when persistent gene expression followed by integrating viral vector-mediated gene transfer is not appropriate, for example, in ex vivo manipulation of HSCs and functional analysis of genes of interest in HSCs. We previously developed a novel type of Ad vector, composed of whole Ad serotype 35 (Ad35) belonging to subgroup B, and demonstrated that Ad35 vectors gave efficient gene transfer into human bone marrow CD34⁺ cells (Gene Ther. 2003, 10, 1041|[ndash]|1048; Gene Ther. 2005, 12, 1424|[ndash]|1433). However, rates of Ad35 vector-mediated transduction into mouse HSCs are relatively low because a receptor for Ad35, CD46, is not expressed in mouse HSCs. Because the cell fraction containing mouse HSCs is well characterized and the biology of the mouse HSC has been studied extensively in mouse models, efficient transduction with Ad vectors into mouse HSCs would be useful. Here, we performed Ad35 vector-mediated transduction into mouse HSCs isolated from human CD46-transgenic (CD46TG) mice, which express human CD46 in a pattern similar to that in humans. First, bone-marrow- derived C-kit(+)Lineage(|[minus]|) (KL) cells were recovered from wild- type mice and homozygous CD46TG mice as mouse HSCs, and human CD46 expression in KL cells was examined. More than 80% of KL cells from CD46TG mice expressed human CD46. Ad35 vector-mediated transduction demonstrated that KL cells from wild- type mice were refractory to Ad35 vectors; in contrast, KL cells from CD46TG mice showed high levels of transgene expression. The human elongation factor 1|[alpha]| promoter (EF1|[alpha]| promoter) worked most efficiently of all the promoters examined in KL cells. In addition, colony-forming assay revealed that Ad35 vector-mediated transgene-expressing KL cells from CD46TG mice differentiated into progenitor cells of all lineages. These results suggest that this model using Ad35 vector and KL cells from CD46TG mice would be a powerful tool for the study of gene function in mouse HSCs.