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/Wv 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.

Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme

“…[6][7][8][9][10][11][12] The presence of the selectable marker sequence, bacterial neomycin resistance gene (neo), has produced inconsistent effects on retroviral vector expression. In some experiments neo appears to inhibit expression, 13,14 whereas in other experiments it does not. 12,15 In the Moloney murine leukemia virus (Mo-MuLV), a number of regulatory elements have been found to influence transcriptional down-regulation of viral genes from the LTR.…”

Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors.

“…Successful gene transfer into the most primitive hematopoietic cells, long-term repopulating stem cells, may lead to a lifelong cure for a variety of diseases manifested in the progeny of these cells. Although gene transfer and longterm gene expression in repopulating stem cells have been achieved in murine models by a number ofinvestigators (3)(4)(5)(6)(7)(8)(9), in vivo experiments in larger animals such as dogs and primates have met with limited success, largely because ofthe low efficiency of infection of primitive hematopoietic stem cells (10)(11)(12)(13).…”

Transcriptional Silencing of Retroviral Vectors

“…It has been found that identical proviruses inserted at differ multidrug resistance (MDR-1) [56] and glucocerebrosidase (GC) [16,19,20,79] into murine hematopoietic stem cells.…”