Expression of Human Glucocerebrosidase in Long-Term Reconstituted Mice Following Retroviral-Mediated Gene Transfer into Hematopoietic Stem Cells

Abstract

A retroviral vector (GTN) in which the glucocerebrosidase (GCase) cDNA is driven by the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) was tested for transfer efficiency and expression of the GCase gene in long-term reconstituted mice. Eleven W/W^v mice were transplanted with unselected GTN-infected bone marrow cells and 10 of these mice were analyzed 3 months later. Seven of these 10 mice (70%) contained the intact proviral genome in bone marrow, spleen, and thymus. Of these 7,3 mice contained a high-copy number of the provirus in all the hematopoietic tissues tested. The mice contained anywhere from one to four proviral integration sites that were the same in all three tissues, indicating that these mice have been repopulated by one or more transduced multipotential hematopoietic stem cells. Five months after transplantation, bone marrow from the eleventh mouse was transplanted into secondary recipient animals. The secondary recipients contained the intact proviral genome in the bone marrow, spleen, thymus, and macrophages 4 months after the secondary transplantation. This further supports the conclusion that hematopoietic stem cells have indeed been targeted. Human GCase RNA was detected in all 7 mice containing the proviral DNA. These results demonstrate expression of the human GCase gene in the progeny of repopulating hematopoietic stem cells of mice following gene transfer. Gaucher disease, an inherited disorder caused by a deficiency of the enzyme glucocerebrosidase, is a leading candidate for human gene therapy. Correll et al., demonstrate efficient retroviral-mediated gene transfer of the human glucocerebrosidase cDNA into murine repopulating hematopoietic stem cells. The Moloney murine leukemia virus long terminal repeat directed synthesis of glucocerebrosidase mRNA in the progeny of these cells.