Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors

Abstract

The efficiency of gene transfer into human hematopoietic stem cells by oncoretroviral vectors is too low for effective gene therapy of most hematologic diseases. Retroviral vectors based on the nonpathogenic foamy viruses (FV) are an alternative gene-transfer system. In this study, human umbilical cord blood CD34 ؉ cells were transduced with FV vectors by a single 10-h exposure to vector stocks and then injected into sublethally irradiated nonobese diabetic-severe combined immunodeficiency (NOD͞SCID) mice. At 5-7 weeks after transplantation, high transgene expression rates were observed in engrafted human hematopoietic cells, including over 60% of clonogenic progenitors. Significant transgene silencing did not occur. We developed an approach for expanding human cell populations derived from transplanted mice to show that multiple SCID repopulating cells (SRCs) had been transduced, including some that were capable of both lymphoid and myeloid differentiation. These findings demonstrate for the first time that human pluripotent (lympho-myeloid) hematopoietic stem cells repopulate NOD͞SCID mice and can be efficiently transduced by FV vectors. S pumaviruses or foamy viruses (FV) are nonpathogenic retroviruses with a wide tissue tropism commonly found in mammals (1, 2), although humans are not a natural reservoir of infection (3, 4), and there is no evidence of human-to-human transfer (5, 6). FV vectors transduce nondividing cells more efficiently than oncoretroviral vectors, are not inactivated by human serum, and have a large packaging capacity (7). We previously developed methods for the production of high titer, helper-free FV vectors (8) and found that they could efficiently transduce murine hematopoietic stem cells (HSCs) (9). However, the high HSC transduction rates obtained in mice by other vector types have not been reproduced in primates, prompting us to test FV vectors in human cells.One model currently used to study human hematopoiesis is xenotransplantation of immunodeficient nonobese diabeticsevere combined immunodeficiency (NOD͞SCID) mice (10, 11). These mice support multilineage human hematopoiesis for several weeks, and the low transduction rates of SCID repopulating cells (SRCs) by oncoretroviral vectors indicate that it is an excellent preclinical gene therapy model of human repopulating cells (12). SRCs have proliferative capacities, repopulation kinetics, and cell-surface phenotypes that suggest they are true HSCs (12-14); however, multipotential differentiation has not been demonstrated conclusively by analysis of vector integration patterns in lineage-specific progeny cells. Here, we show that FV vectors efficiently transduce SRCs, and that clonal lymphomyeloid repopulation occurs with marked cells as expected for transduced pluripotent HSCs. Materials and MethodsHematopoietic Cell Culture and Transductions. Human CD34 ϩ cells were isolated from umbilical cord blood under an Institutional Review Board-approved protocol by using a CD34 ϩ cell isolation kit as per the manufacturer's instructions ...

Insulated Foamy Viral Vectors

“…20 Vectors developed from foamy virus (FV), the only member of the Spumaretrovirinae subfamily of retroviruses, have an integration profile that is potentially safer than other retroviral vectors. [20][21][22][23][24] FV vectors have a reduced preference for integration within CpG islands known to be DNA regulatory regions and near promoters compared with GV vectors, and a reduced likelihood to integrate within genes relative to lentiviral (LV) vectors. 20,21 In addition to a more desirable integration profile, FV vectors also have a lower propensity to dysregulate nearby genes when directly compared with GV and LV vectors.…”

In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMTP140K vector

“…The marking of human SCID-repopulating cells with FV vectors without in vivo selection or administration of cytokines has been demonstrated by our group and others [21][22][23]. High levels of FV marking in human cells may be due in part to the rapid expansion of the SCID-repopulating cells in the BM following homing and engraftment in the bone marrow of NOD/SCID mice.…”

“…Russell and colleagues demonstrated that murine stem cells residing in 5-fluorouracil (5FU)-treated bone marrow (BM) [24] and SCID-repopulating cells derived from umbilical cord blood and G-CSF-mobilized peripheral blood could be marked with FV vectors. [21,22] We previously demonstrated similar levels of gene marking using FV or lentiviral vectors to transduce SCID-repopulating cells derived from human umbilical cord blood CD34 + cells [23]. In addition, FV-mediated gene transfer of canine hematopoietic longterm repopulating cells has been recently reported [32].…”

“…Viral vector systems derived from lentivirus or FV are currently under investigation in small and large animal models [9,[19][20][21][22][23][24][25][26][27][28][29][30][31]. Russell and colleagues demonstrated that murine stem cells residing in 5-fluorouracil (5FU)-treated bone marrow (BM) [24] and SCID-repopulating cells derived from umbilical cord blood and G-CSF-mobilized peripheral blood could be marked with FV vectors.…”

Construction and characterization of efficient, stable and safe replication-deficient foamy virus vectors

“…2 Novel retroviral vectors based on primate-and nonprimate foamy viruses (FV) which constitute the Spumavirinae subfamily of the retroviruses have currently been established and intensively examined by us and others. [3][4][5][6][7][8][9][10][11][12][13] For instance, FV vectors based on the human/primate FV allow efficient, stable and long-term transduction of human haematopoietic cells without detectable side effects. 11,12 FVs are considered to be apathogenic in their natural animal hosts and after zoonotic transmission into humans.…”