Expression of human adenosine deaminase in mice transplanted with hemopoietic stem cells infected with amphotropic retroviruses.

Abstract

SummaryAmphotropic recombinant retroviruses were generated carrying sequences encoding human adenosine deaminase (ADA) . Transcription of the human ADA gene was under control of a hybrid long terminal repeat in which the enhancer from the Molony murine leukemia virus was replaced by an enhancer from the 17101 host-range mutant of polyoma virus. Hemopoietic stem cells in murine bone marrow were infected with this virus under defined culture conditions. As a result, 59% ofday-12 colony forming unit spleen (CFU-S) stem cells became infected without any in vitro selection. Infected CFU-S were shown to express human ADA before transplantation and this expression sustained upon in vivo maturation. Mice transplanted with infected bone marrow exhibited human ADA expression in lymphoid, myeloid, and erythroid cell types. Moreover, human ADA expression persisted in secondary and tertiary transplanted recipients showing that human ADAexpressing cells were derived from pluripotent stem cells. These characteristics of our amphotropic viruses make them promising tools in gene therapy protocols for the treatment of severe combined immunodeficiency caused by ADA deficiency. In this respect it is also relevant that the viral vector that served as backbone for the ADA vector was previously shown to be nonleukemogenic .

Expression pattern of CD2 locus control region containing retroviral vectors in hemopoietic cells in vitro and in vivo

“…To study the ADA expression levels of our basic vector LgAL(⌬Mo+PyF101) 9 in T cells we transduced human ADA − SCID T cells by cocultivation with the amphotropic recombinant retrovirus vector producing cell line POAM-P1. 31 Without applying any selection, we isolated four different T cell clones containing the retroviral construct, each exhibiting a unique insertion pattern.…”

“…5 When the fragment encompassing this mutant enhancer was used to replace the Moloney murine leukemia virus (MoMLV) enhancer in a recombinant retroviral construct, expression was obtained in EC cells and in the mouse hemopoietic system. 8,9 The discovery of locus control region (LCR) sequences has raised the expectation that well controlled expression levels of retrovirally introduced sequences will become feasible. LCR sequences are defined as regulatory units that confer a cell type-specific, copy number-dependent and integration site-independent expression pattern on a linked gene in transgenic animals.…”

“…9 It contains the human adenosine deaminase (ADA) cDNA driven by the retroviral promoter. Furthermore, the wild-type enhancer in the MoMLV long terminal repeat (LTR) has been replaced by the enhancer of the PyF101 mutant of polyoma virus.…”

Ganciclovir-mediated in vivo elimination of myeloid leukemic cells expressing the HSVtk gene induces HSVtk loss variants

“…The LTR is a hybrid in which the MLV enhancer is replaced by the PyF101 enhancer that is active in both primitive and mature cells in vitro and in vivo. 31,32 The pCD-2 retroviral vector (kindly provided by Dr CA Mullen, MDACC, Houston, TX, USA) contains the E. coli cytosine deaminase (CD) gene under control of the MMTV LTR promotor and the Neo R gene under control of the SV40 early promotor.…”

Methotrexate selectable retroviral vectors for Gaucher disease

“…Cocultivation transduction was performed as described previously. 34 Before addition of the murine BMC, producer cells were irradiated (20 Gy, 0.70 Gy/min, 137 Cs gammarays). After 72 h, BMC were harvested by trypsinisation and seeded in equal numbers in DMEM supplemented with mIL-3 and hIL-1␣ and either normal or dialysed FCS.…”