Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells.

Abstract

The human glucocerebrosidase (GC) gene has been transferred efficiently into spleen colony-forming unit (CFU-S) multipotential hematopoietic progenitor cells, and production of human GC RNA and protein has been achieved in transduced CFU-S colonies. High-titer retroviral vectors containing the human GC cDNA were constructed. Mouse bone marrow cells were stimulated with hematopoietic growth factors, infected by coculture with producer cells, and injected into lethally irradiated animals. Four vectors were compared with respect to gene-transfer efficiency into CFU-S progenitors. One vector (G vector) required high concentrations of interleukins 3 and 6 during stimulation and coculture for efficient transduction of CFU-S progenitors. The remaining three vectors (NTG, GTN, and GI vectors) transduced these progenitors at infection frequencies approaching 100% using low concentrations of hematopoietic growth factors to stimulate cell division prior to and during the infection. Vectors using the viral long terminal repeat enhancer/promoter to drive the human GC cDNA produced high levels of human GC RNA in the progeny of CFU-S progenitors after gene transfer. When an internal herpes simplex thymidine kinase promoter assisted by a mutant polyoma enhancer was used to drive the human GC cDNA (NTG vector), little or no human GC RNA was detected in transduced CFU-S colonies. All three vectors producing human GC RNA in CFU-S colonies can generate human GC as detected by immunochemical analysis of CFU-S colonies. NTG vector-infected bone marrow cells were transplanted into W/Wv recipients to generate long-term reconstituted mice. The capacity of the viral long terminal repeat and the internal thymidine kinase promoter to direct synthesis of RNA in transduced bone marrow and spleen cells 5 months after bone marrow transplantation reflected the performance of these promoters in NTG-transduced CFU-S colonies.

Expression of human alpha-globin and mouse/human hybrid beta-globin genes in murine hemopoietic stem cells transduced by recombinant retroviruses.

“…A similar observation has been reported in studies using W/WI mice (31). Recently, Bodine et al (32) and Correll et al (33) have shown that the combination of interleukins 3 and 6 in the culture medium increases the frequency of retroviral gene transfer by increasing the number of stem cells in the cycle. Alternatively, purification of stem cells prior to infection seems to increase the population of stem cells infected as shown recently (34).…”

Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease.

“…Progeny of these cells expressed human GCase mRNA and produced human GCase (immunologically cross-reacting material) protein (7). In addition, the vector used in the present report (NTG) efficiently transduced murine hematopoietic stem cells (7).…”

“…The three LTR vectors efficiently transduced murine colony-forming unit-spleen (CFU-S) multipotential progenitor cells (6,7). Progeny of these cells expressed human GCase mRNA and produced human GCase (immunologically cross-reacting material) protein (7).…”

“…Gaucher disease is a lysosomal storage disorder in which deficient glucocerebrosidase [glucosylceramidase (GCase); D-glucosyl-N-acylsphingosine glucohydrolase, EC (6,7). The three LTR vectors efficiently transduced murine colony-forming unit-spleen (CFU-S) multipotential progenitor cells (6,7).…”

“…Retroviral Vector. Construction of the NTG vector has been described (7). An amphotropic version of the N2 vector (15) (obtained from W. F. Anderson, National Heart, Lung and Blood Institute), which lacks the GCase cDNA, was used as a control.…”