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We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.

A persistent obstacle in the use of vector systems for gene therapy has been the inability to attain high-level expression of the target gene in primary cells in vivo. The MFG retroviral vector was designed to yield improved expression over the widely used N2 or LN vectors; however, the molecular basis for this effect has not been examined. Using the human glucocerebrosidase (GC) enzyme as a reporter, we compared expression from the MFG and N2 vector backbones in transduced murine hematopoietic cells after syngeneic bone marrow transplantation. Reporter enzyme activities in primary spleen colonies of transplanted mice were seven-fold higher per vector copy in cells transduced with the (MFG-based) MGC vector than in cells bearing the (N2-based) G2 vector. In spleen colonies harboring the MGC vector, the ratio of spliced to unspliced vector RNA was increased four-fold relative to the G2 vector transcripts in Northern blot analyses. Further analyses indicated that MGC-transduced cells contained five-fold higher levels of spliced RNA per vector copy. Since translation of spliced RNA species (in which the complex secondary structure of the packaging signal has been excised) is likely to proceed with enhanced efficiency, the augmented levels of spliced RNA produced by MFG may represent the key element of increased protein expression from this vector. These findings suggest that the MFG retroviral vector may provide higher level expression of target genes used in human gene therapy.

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.

A major obstacle in the study of B lymphocyte development subsequent to immunization has been the paucity of distinctive phenotypic markers which might permit the separation and scrutiny of a mature antigen-experienced or memory cell population. To address this issue, we have introduced a recombinant non-replicating retrovirus into antigen-reactive lymph nodes in vivo using the expression of a reporter gene to monitor the trafficking, phenotype and persistence of retrovirus-marked B cells. Marked cells are first observed in the medullary cords where they proliferate for approximately 10 days prior to migration to the spleen. Up to half of the marked cells bear antigen-specific immunoglobulin receptors, but otherwise phenotypically resemble conventional B cells. The abundance and persistence of the marked cells suggest an association with the memory compartment. These experiments demonstrate a feasible approach for the identification and lineage analysis of antigen-responsive B cells, and suggest that the lymph node medulla supports a transient phase of post-antigenic B cell development associated with a long-lived antigen-specific B cell subset.