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    • For some genes the difference in transgene expression

    2018-10-26

    For some genes, the difference in transgene expression in BM/blood and other target organs can be both quantitative and qualitative. For example, the activities of introduced TAK-875 α-glucosidase, which is deficient in a lysosomal glycogen storage disorder, Pompe disease, were different in blood, BM and spleen 8m post lentivirus-transduced HSC transplant (van Til et al., 2010). The activities of lentivirus-encoded α-l-iduronidase, which is deficient in another lysosomal storage disorder, Type I mucopolysaccharidosis, were also different in PBMC compared to other target organs, including the brain (Visigalli et al., 2010). Accordingly, in a recent trial of human gene therapy mediated by lentivirus, the introduced gene function was detected directly in the target organ using cerebrospinal fluid, in addition to blood (Biffi et al., 2013). In our studies, introduced Ii had more enhancing effect on I-Ag7 levels in secondary immune organs, such as spleen and PLN than it did in BM and blood (Figs. 5 and 6). The professional APC (i.e. B cells, macrophages and DC) we studied all originate in BM and are released into circulation, followed by maturation in secondary immune organs, such as spleen and lymph nodes (Gordon and Taylor, 2005; LeBien and Tedder, 2008). Ii is required for B cell maturation (Matza et al., 2002), and Ii level increases during the development of B cells (data not shown, https://gexc.stanford.edu/ (Seita et al., 2012)). GM-CSF (granulocyte-macrophage colony-stimulating factor), the main cytokine promoting the transition of monocytes to mature macrophages, increases Ii and not class II expression in monocytes, but not in macrophages (Klagge et al., 1997). Ii deficiency affects the maturation of DC carrying certain murine class II alleles, i.e. H2b (Rovere et al., 1998). As Ii is involved in the maturation of all three professional APC cell types, it is perhaps not surprising that we observed Ii function at sites of mature cells (i.e. secondary immune organs), compared to BM and blood. The incomplete function of introduced Ii in APC from BM and blood may be due to higher levels of cellular cystatin C than in APC from spleen or PLN. Cystatin C is an inhibitor of cathepsin S, the main protease in charge of Ii processing in tissues other than thymus, and is expressed at higher levels in immature vs. mature DC (Pierre and Mellman, 1998). Our results suggest that during the maturation of APC, Ii function is enhanced, in addition to the reported increase in its synthesis (Engering et al., 1998). The phenomenon we observed likely is not broadly generalizable, but may extend to other immune molecules that, like Ii, interact with other developmentally regulated proteins. When normalized for expression differences, M98A mutant Ii had a modestly increased ability to enhance I-Ag7 levels compared to wt Ii, confirming our in vitro data (Rinderknecht et al., 2010). However, in these in vivo experiments, overcoming the reduced affinity between Ii and I-Ag7 by increased wt expression alone is also an efficient way to stabilize I-Ag7 and may have therapeutic utility.
    Conclusions We report that two transgenes (invariant chain and GFP as a reporter gene) were both expressed using a dual-promoter lentivirus, with the EF1a promotor driving Ii expression. After transplantation of transduced hematopoietic stem cells (HSC), GFP expression correlated with Ii expression in certain myeloid cells, but not other HSC-derived cells. Ii levels in APC in bone marrow did not reflect Ii expression in APC in secondary immune organs, specifically spleen and pancreatic lymph nodes (PLN); a contributor to this result may be differences in the composition of B220+ cells in these locations. Blood Ii levels paralleled Ii levels in PLN only in DC. Interestingly, Ii expression had more robust functional consequences in the more mature immune environments, such as spleen and PLN, compared to BM and blood cells, potentially due to other interacting proteins expressed in these cells. Our results highlight the phenomena of importance for the analysis of gene expression after transplantation of genetically-modified HSC, particularly evaluation of transgene expression in relevant target organs.