ALBERT

Beschreibung: Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.

Beschreibung: The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by immunodeficiency and severe thrombocytopenia in affected males, but no demonstrable clinical abnormalities in carrier females. Through analysis of the methylation patterns of X-linked genes that display restriction fragment length polymorphisms (RFLPs), we studied the pattern of X-chromosome inactivation in various cell populations from female relatives of patients with WAS. The peripheral blood T cells, granulocytes, and B cells of eight obligate WAS carriers were found to display specific patterns of X-chromosome inactivation clearly different from these of normal controls. Thus, carriers of WAS could be accurately identified using this analysis.

Beschreibung: We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long- lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.

Beschreibung: Two human melanoma cell lines were transduced with the human interleukin (IL)-7 and IL-2 genes using retroviral-mediated gene transfer. Stable, high-level cytokine expression was achieved. The in vitro growth of transduced tumors was unaltered. Neither of the IL-2- transduced melanoma cell lines grew in athymic mice, whereas one IL-7- transduced melanoma line showed retarded in vivo growth. This is consistent with animal studies suggesting a predominantly T-cell response to IL-7-transduced tumors and a more nonspecific response to IL-2-transduced tumors. Both IL-7- and IL-2-transduced melanoma cell lines could induce cytotoxic lymphocytes in mixed lymphocyte-tumor cultures. The expression of putative melanoma antigens (MAGE)-1 and MAGE-3 was unaltered by cytokine transduction. In one cell line, IL-7 transduction resulted in a marked inhibition of the immunosuppressive peptide transforming growth factor (TGF)beta 1. The results allow a comparison of immunobiologic properties of IL-7- and IL-2-transduced human melanoma cell lines in consideration of their use in genetically engineered tumor vaccines. IL-7 transduction results in stable cytokine expression and phenotypic alterations that appear to be favorable for enhanced immunogenicity and it deserves clinical testing.

Beschreibung: Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.

Beschreibung: Gaucher disease is an inherited lysosomal storage disease in which the loss in functional activity of glucocerebrosidase (GC) results in the storage of its lipid substrate in cells of the macrophage lineage. A gene therapy approach involving retroviral transduction of autologous bone marrow (BM) followed by transplantation has been recently approved for clinical trial. Amelioration of the disease symptoms may depend on the replacement of diseased macrophages with incoming cells expressing human GC; however, the processes of donor cell engraftment and vector gene expression have not been addressed at the cellular level in relevant tissues. Therefore, we undertook a comprehensive immunohistologic study of macrophage and microglia replacement after murine BM transplantation with retrovirus-marked BM. Serial quantitative PCR analyses were employed to provide an overview of the time course of engraftment of vector-marked cells in a panel of tissues. Following reconstitution of hematopoietic tissues with vector- marked donor cells at early stages, GC+ cells began to infiltrate the liver, lung, brain, and spinal cord by 3 months after transplant. Immunohistochemical analyses of PCR+ tissues using the 8E4 monoclonal antibody specific for human GC revealed that macrophages expressing human GC had partially reconstituted the Mac-1+ population in all tissues in a manner characteristic to each tissue type. In the brain, 20% of the total microglia had been replaced with donor cells expressing GC by 3 to 4 months after transplant. The finding that significant numbers of donor cells expressing a retroviral gene product immigrate to the central nervous system suggests that gene therapy for neuronopathic forms of lysosomal storage diseases as well as antiviral gene therapy for AIDS may be feasible.

Beschreibung: We have developed a novel cotransplantation system in which gene- transduced human CD34+ progenitor cells are transplanted into immunodeficient (bnx) mice together with primary human bone marrow (BM) stromal cells engineered to produce human interleukin-3 (IL-3). The IL- 3-secreting stroma produced sustained circulating levels of human IL-3 for at least 4 months in the mice. The IL-3-secreting stroma, but not control stroma, supported human hematopoiesis from the cotransplanted human BM CD34+ progenitors for up to 9 months, such that an average of 6% of the hematopoietic cells removed from the mice were of human origin (human CD45+). Human multilineage progenitors were readily detected as colony-forming units from the mouse marrow over this time period. Retroviral-mediated transfer of the neomycin phosphotransferase gene or a human glucocerebrosidase cDNA into the human CD34+ progenitor cells was performed in vitro before cotransplantation. Human multilineage progenitors were recovered from the marrow of the mice 4 to 9 months later and were shown to contain the transduced genes. Mature human blood cells marked by vector DNA circulated in the murine peripheral blood throughout this time period. This xenograft system will be useful in the study of gene transduction of human hematopoietic stem cells, by tracing the development of individually marked BM stem cells into mature blood cells of different lineages.