Results from several experimental systems suggest that cells from one tissue type can form other tissue types after transplantation. This could be due to the presence of multipotential or several types of adult stem cells in donor tissues, or alternatively, to fusion of donor and recipient cells. In a model of tyrosinaemia type I, mice with mutations in the fumarylacetoacetate hydrolase gene (Fah-/-) regain normal liver function after transplantation of Fah+/+ bone marrow cells, and form regenerating liver nodules with normal histology that express Fah. Here we show that these hepatic nodules contain more mutant than wild-type Fah alleles, and that their hepatocytes express both donor and host genes, consistent with polyploid genome formation by fusion of host and donor cells. Using bone marrow cells marked with integrated foamy virus vectors that express green fluorescent protein, we identify common proviral junctions in hepatic nodules and haematopoietic cells. We also show that the haematopoietic donor genome adopts a more hepatocyte-specific expression profile after cell fusion, as the wild-type Fah gene was activated and the pan-haematopoietic CD45 marker was no longer expressed.

Foamy virus (FV) vectors show promise for gene therapy applications. However, existing FV vectors either retain a significant portion of the wild-type virus genome or are produced at low titers. We describe a transient cotransfection system that produces high-titer FV vectors with minimal cis-acting regions. These vector genomes have deletions in the gag, pol, env, and bel1-3 accessory genes, as well as the LTR U3 region, but retain an essential 2.5-kb cis-acting region. In addition, stop codons were introduced into the remaining gag sequences to prevent expression of viral peptides and to eliminate dominant-negative effects of a Gag-Pol fusion protein. Although these deleted foamy (deltaphi) vectors were produced at relatively low titers with our prior packaging construct, we designed separate helper plasmids for Gag, Pol, and Env expression that allowed us to routinely produce helper-free, unconcentrated vector stocks with titers of over 10(5) transducing units/ml by four-plasmid transient transfection. The deltaphi vector stocks were then concentrated by ultracentrifugation to titers over 10(7) transducing units/ml. A deltaphi vector containing a 9.2-kb transgene cassette was produced at unconcentrated titers of over 10(5) transducing units/ml, demonstrating the utility of these deleted vectors for large therapeutic genes.

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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 ...