Retroviral-mediated gene expression in human myelomonocytic cells: a comparison of hematopoietic cell promoters to viral promoters

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.     

Effect of the calcium channel blocker verapamil on human immunodeficiency virus type 1 replication in lymphoid cells.

Cell signaling events are known to affect human immunodeficiency virus type 1 (HIV-1) replication. Treatment of lymphoid CEM cells with the calcium channel blocker verapamil (25-75 microM) enhanced HIV-1 expression in acute, whole virus infection experiments, despite lowering intracellular calcium levels, ablating the acute rise in intracellular calcium normally seen with infection, and lengthening the doubling time of cell replication. Verapamil had no effect on cell surface CD4 expression. Transfection of CEM cells with plasmids containing the HIV-1 long terminal repeat linked to the chloramphenicol acetyltransferase reporter gene showed that verapamil enhanced expression of the HIV-1 long terminal repeat in a dose-dependent fashion. This effect was abolished by mutations in the binding sites for nuclear factor kappa-B. Electrophoretic mobility shift assays confirmed that verapamil induced nuclear factor kappa-B activity in CEM cells. Thus, verapamil, in high concentrations, can potentiate HIV-1 replication in lymphoid cells, and this effect may be mediated by induction of nuclear factor kappa-B.     

Selective transduction of HIV-1-infected cells by the combination of HIV and MMLV vectors

Human immunodeficiency virus 1 (HIV-1)-infected cells are important targets of gene therapy for acquired immune deficiency syndrome. We have developed a novel strategy for targeted gene transfer into HIV-1-infected cells based on 2-step gene transfer. The first step involves the stable introduction of the HIV vector containing the ecotropic Moloney murine leukemia virus (MMLV) receptor gene (EcoRec) into human CD4+ T cells as a molecular switch. Because the HIV-long terminal repeat (HIV-LTR) is Tat inducible, it is expected that EcoRec is expressed only after HIV-1 infection. Northern blot analysis and a retrovirus binding assay confirmed that the HIV-LTR of the integrated vector was silent in transduced cells but strongly transactivated in HIV-1 infection. High levels of EcoRec expression were observed only in HIV-1-infected cells. These cells became highly susceptible to ecotropic MMLV infection and, therefore, in the second step, HIV-1-infected cells were selectively transduced with ecotropic MMLV vectors. More than 70% of HIV-1-infected cells were transduced by this strategy. These findings indicate that this 2-step method can be used for selective and stable gene transfer into HIV-1-infected cells.     

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.     

Performance- and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator

Retroviral vectors are the most efficient means of stable gene delivery to hematopoietic stem cells (HSCs). However, transgene expression from retroviral vectors is frequently subject to the negative influence of chromosomal sequences flanking the site of integration. Toward the development of autonomous transgene expression cassettes, we inserted the human interferon-beta scaffold attachment region (IFN-SAR) and the chicken beta-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator both separately and together into a series of self-inactivating (SIN) lentiviral vector backbones. Transduced cells of the human CD34+ hematopoietic progenitor line KG1a-pooled populations as well as individual clones harboring single integrants--were analyzed for reporter expression during culture periods of up to 4 months. Vectors carrying both the 5'HS4 insulator and the IFN-SAR consistently outperformed control vectors without inserts as well as vectors carrying either element alone. The performance of a set of vectors containing the murine stem cell virus long terminal repeat as an internal promoter was subsequently assessed during in vitro monocytic differentiation of transduced primary human CD34+ cord blood cells. Similar to what was observed in the KG1a hematopoietic progenitor cell model, optimal reporter expression in primary monocytes was obtained with the vector bearing both regulatory elements. These findings indicate that the 5'HS4/IFN-SAR combination is particularly effective at maintaining open chromatin domains permissive for high-level transgene expression at early and late stages of hematopoietic development, and thus could be of utility in HSC-directed retroviral vector-mediated gene transfer applications.     

Cytomegalovirus infection and trans-activation of HIV-1 and HIV-2 LTRs in human astrocytoma cells

Susceptibility of a human astrocytoma cell line to human cytomegalovirus (HCMV) infection was investigated. Infection of U-373MG astrocytoma cells with two strains of HCMV resulted in both production of extracellular, infectious virus and expression of immediate early and early antigens within 18 hours and late antigens after 72 hours of infection. The kinetics of infection in U-373MG cells were the same as in human diploid fibroblasts (MRC-5). Since HCMV and human immunodeficiency virus (HIV) have reportedly been found in astrocytic cells in vivo, we studied the possible interaction between HCMV and HIV long terminal repeat (LTR) elements in this cellular environment. HCMV infection transactivated the LTR of HIV-1 and HIV-2 to similar levels. Interestingly, transfection of these cells with infectious HIV-1 provirus did not result in expression of gag, env, or F proteins detectable by immunofluorescence. However, provirus gene expression was not completely silent, since it transactivated HIV-1 LTR. The level of this transactivation was similar to that seen following cotransfection with a tat expression vector. These results suggest that opportunistic infection with HCMV may reactivate latent HIV genomes in glial cells.     

rpt-1, an intracellular protein from helper/inducer T cells that regulates gene expression of interleukin 2 receptor and human immunodeficiency virus type 1.

The Rpt-1 (for regulatory protein, T-lymphocyte, 1) gene, selectively expressed by resting but not by activated CD4+ inducer T cells, encodes an intracellular protein (rpt-1, Mr 41,000) that down-regulates gene expression directed by the promoter region of the gene encoding interleukin 2 receptor alpha chain and by the long terminal repeat of human immunodeficiency virus type 1. The data reported here suggest that rpt-1 levels may be inversely correlated with activation of CD4+ T cells and human immunodeficiency virus replication leading to clinical symptoms of the acquired immunodeficiency syndrome.     

Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice

A recombinant AAV2 (rAAV2) vector encoding antisense RNA to HIV-1 transactivating region (TAR) was evaluated for transduction of human cord blood CD34+CD38- hematopoietic stem cells (HSC) capable of serial engraftment in nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Results revealed long-term multilineage marking in primary and secondary recipients, and significantly, an enrichment of transduced cells in secondary hosts, indicating efficient transduction of multipotential self-renewing HSC. These results were confirmed by the persistence of rAAV marking of clonogenic progenitors in serial analyses of recipient marrow. Upon HIV-1 challenge, the macrophage progeny of transduced CD34+ cells expressed antisense RNA and exhibited sustained and significant inhibition of virus replication as compared with controls in every donor tested, without selective pressure. This study represents a clear in vivo demonstration of efficient rAAV2 transduction of human HSC.