Specific transgene expression in human and mouse CD4+ cells using lentiviral vectors with regulatory sequences from the CD4 gene

Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4(+) cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from the CD4 gene--a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 10(7) infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2-deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4 gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4(+) T cells in reconstituted mice and in ex vivo-transduced human T cells. Non-T CD4(+) dendritic and macrophage cells derived from human CD34(+) cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4(+) T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4(+) cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.     

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to moloney murine leukemia virus-based vectors

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34(+) cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin(-) bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.     

Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors

The efficiency of gene transfer into human hematopoietic stem cells by oncoretroviral vectors is too low for effective gene therapy of most hematologic diseases. Retroviral vectors based on the nonpathogenic foamy viruses (FV) are an alternative gene-transfer system. In this study, human umbilical cord blood CD34(+) cells were transduced with FV vectors by a single 10-h exposure to vector stocks and then injected into sublethally irradiated nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. At 5-7 weeks after transplantation, high transgene expression rates were observed in engrafted human hematopoietic cells, including over 60% of clonogenic progenitors. Significant transgene silencing did not occur. We developed an approach for expanding human cell populations derived from transplanted mice to show that multiple SCID repopulating cells (SRCs) had been transduced, including some that were capable of both lymphoid and myeloid differentiation. These findings demonstrate for the first time that human pluripotent (lympho-myeloid) hematopoietic stem cells repopulate NOD/SCID mice and can be efficiently transduced by FV vectors.     

The AFT024 stromal cell line improves MDR1 gene transfer to CD34+ cells derived from human umbilical cord blood

Human hematopoietic stem cells (HSCs) are difficult to transfect with retroviral vectors because of their quiescent nature. Based on the theory that the murine fetal stromal cell line AFT024 can recruit significant numbers of HSC into cell cycle without loss of their primitive function, we transduced human umbilical cord blood cells (UCB) derived CD34+ cells with a retroviral vector pHaMDR1/A containing the human multidrug resistant 1 gene (MDR1) during co-culture with the AFT024 feeders. We found that the presence of the AFT024 cells increased the proportion of Rh-123dull cells up to 35.5%+/-11.4% and transduced colony-forming cells (CFCs) up to 15.2%. Six weeks after transplantation of 5x10(4) day 0 uncultured CD34+ HSCs or their equivalents expanded in the presence or absence of the AFT024 cells for 21 days into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, we found that CD34+ cells expanded in the presence of the AFT024 cells engrafted in each receptor mouse and the percentage of CD45+ cells reached 18.8%+/-9.5%, of which 18.1%+/-6.0% were Rh-123dull cells. These results suggest that the AFT024 stromal cells can significantly improve MDR1 gene transfer efficiency and maintain the engrafting ability of the CD34+ HSCs derived from UCB.     

Differential regulation of the human and murine CD34 genes in hematopoietic stem cells

Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34(-/lo) express the hCD34 transgene. These data strongly support the notion that hCD34(+) human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.     

Transduction of hematopoietic cells by foamy virus vectors

Foamy viruses are retroviruses of the spumavirus family that are often isolated from primary cultures of primate cells. We previously constructed vectors based on human foamy virus (HFV) and found that they were able to transduce a wide variety of vertebrate cells by integration of the vector genome. Here we show that several types of hematopoietic cells are efficiently transduced by an HFV vector that encodes alkaline phosphatase (AP). These cell types include transformed cell lines and primary hematopoietic progenitors from mice, baboons, and humans. The transduction rates of HFV vectors compare favorably with those obtained by murine leukemia virus vectors, which suggests that HFV vectors may be effective in the treatment of hematologic diseases by gene therapy.     

Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary,secondary, and tertiary multilineage repopulation in NOD/SCID mice. Nonobese diabetic/severe combined immunodeficient

The ability of advanced-generation lentiviral vectors to transfer the green fluorescent protein (GFP) gene into human hematopoietic stem cells (HSCs) was studied in culture conditions that allowed expansion of transplantable human HSCs. Following 96 hours' exposure to flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), and interleukin-6 (IL-6) and overnight incubation with vector particles, cord blood (CB) CD34(+) cells were further cultured for up to 4 weeks. CD34(+) cell expansion was similar for both transduced and control cells. Transduction efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) was assessed by transplants into NOD/SCID mice. Mice that received transplants of transduced week 1 and week 4 expanded cells showed higher levels of human engraftment than mice receiving transplants of transduced nonexpanded cells (with transplants of 1 x 10(5) CD34(+) cells, the percentages of CD45(+) cells were 20.5 +/- 4.5 [week 1, expanded] and 27.2 +/- 8.2 [week 4, expanded] vs 11.7 +/- 2.5 [nonexpanded]; n = 5). The GFP(+)/CD45(+) cell fraction was similar in all cases (12.5% +/- 2.9% and 12.2% +/- 2.7% vs 12.7% +/- 2.1%). Engraftment was multilineage, with GFP(+)/lineage(+) cells. Clonality analysis performed on the bone marrow of mice receiving transduced and week 4 expanded cells suggested that more than one integrant likely contributed to the engraftment of GFP-expressing cells. Serial transplantations were performed with transduced week 4 expanded CB cells. Secondary engraftment levels were 10.7% +/- 4.3% (n = 12); 19.7% +/- 6.2% of human cells were GFP(+). In tertiary transplants the percentage of CD45(+) cells was lower (4.3% +/- 1.7%; n = 10); 14.8% +/- 5.9% of human cells were GFP(+), and human engraftment was multilineage. These results show that lentiviral vectors efficiently transduce HSCs, which can undergo expansion and maintain proliferation and self-renewal ability.     

CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages

The CD34 antigen serves as an important marker for primitive hematopoietic cells in therapeutic transplantation of hematopoietic stem cells (HSC) and gene therapy, but it has remained an open question as to whether or not most HSC express CD34. Using a competitive long-term reconstitution assay, the results of this study confirm developmental changes in CD34 expression on murine HSC. In fetuses and neonates, CD34 was expressed on Lin(-)c-Kit(+) long-term repopulating HSC of bone marrow (BM), liver, and spleen. However, CD34 expression on HSC decreased with aging, and in mice older than 10 weeks, HSC were most enriched in the Lin(-)c-Kit(+)CD34(-) marrow cell fraction. A second transplantation was performed from primary recipients who were transplanted with neonatal Lin(-)c-Kit(+) CD34(high) HSC marrow. Although donor-type HSC resided in CD34-expressing cell fraction in BM cells of the first recipients 4 weeks after the first transplantation, the stem cell activity had shifted to Lin(-)c-Kit(+)CD34(-) cells after 16 weeks, indicating that adult Lin(-)c-Kit(+)CD34(-) HSC are the progeny of neonatal CD34-expresssing HSC. Assays for colony-forming cells showed that hematopoietic progenitor cells, unlike HSC, continue to express CD34 throughout murine development. The present findings are important because the clinical application of HSC can be extended, in particular as related to CD34-enriched HSC and umbilical cord blood HSC.     

Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.     

Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells

Vector-containing medium harvested from murine packaging cell lines has been shown to contain factors that can negatively influence the transduction and maintenance of hematopoietic stem cells. Thus, we generated a human packaging cell line with a gibbon ape leukemia virus pseudotype (Phoenix-GALV), and we evaluated vectors produced by Phoenix-GALV for their ability to transduce hematopoietic progenitor/stem cells. In 3 baboons, we used a competitive repopulation assay to directly compare GALV-pseudotype retrovirus vectors produced by either Phoenix-GALV or by the NIH 3T3-derived packaging cell line, PG13. In 3 additional baboons we compared Phoenix-GALV-derived vectors to more recently developed lentiviral vectors. Gene transfer efficiency into hematopoietic repopulating cells was assessed by evaluating the number of genetically modified peripheral blood and marrow cells using flow cytometry and real-time polymerase chain reaction. Transduction efficiency of hematopoietic repopulating cells was significantly higher using the Phoenix-GALV-derived vector as compared with the PG13-derived vectors or lentiviral vectors, with stable transduction levels up to 25%. We followed 2 animals for more than one year. Flow cytometric analysis of hematopoietic subpopulations in these animals revealed transgene expression in CD13(+) granulocytes, CD20(+) B lymphocytes, CD3(+) T lymphocytes, CD61(+) platelets, as well as red blood cells, indicating multilineage engraftment of cells transduced by Phoenix-GALV-pseudotype vectors. In addition, transduction of human CD34(+) cells was significantly more efficient than transduction of baboon CD34(+) cells, suggesting that Phoenix-GALV-derived oncoretroviral vectors may be even more efficient in human stem cell gene therapy applications.     

Optimization of multidrug resistance 1 gene transfer confers chemoprotection to human hematopoietic cells engrafted in immunodeficient mice

OBJECTIVE: To investigate whether an optimization of MDR1 gene transfer protocol would result in stable hematopoietic stem cell (HSC) engraftment and myeloprotection in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice after paclitaxel chemotherapy. METHODS: We transplanted freshly isolated CD34+ cells or MDR1-transduced CD34+ cells derived from human umbilical cord blood (UCB) into sublethally irradiated NOD/SCID mice. Twenty-eight days after transplantation, mice received paclitaxel chemotherapy and peripheral blood (PB) was collected for analysis of WBC, RBC and PLT counts once every week. RESULTS: We found that MDR1-transduced human hematopoietic cells could facilitate hematopoietic recovery and completely reconstitute hematopoiesis in mice as well as freshly isolated CD34+ cells. Mice transplanted with MDR1-transduced human hematopoietic cells were protected from paclitaxel chemotherapy with higher survival rate and higher level of WBC counts and RBC counts compared with mice transplanted with untransduced HSCs. We also demonstrated that hematopoietic cells transduced with MDR1 gene were enriched in vivo after paclitaxel chemotherapy determined by the higher percentage of human Rh-123(dull) CD45+ cells in bone marrow of mice. CONCLUSION: Our results demonstrated successful chemoprotection against myelosuppression in mice by MDR1-transduced repopulating human hematopoietic cells with an optimized transduction protocol.     

Soluble factors elaborated by human brain endothelial cells induce the concomitant expansion of purified human BM CD34+CD38- cells and SCID-repopulating cells

The CD34(+)CD38- phenotype identifies a population in the bone marrow that is enriched in the steady state for hematopoietic stem cells (HSCs). Following ex vivo culture of CD34(+) cells, HSC content is difficult to measure since committed CD34(+)CD38+ progenitors down-regulate CD38 surface expression during culture. In this study, we sought to define the phenotype of human HSCs following ex vivo culture under conditions that support the expansion of human cells capable of repopulating non-obese diabetic/severe combined immunodeficiency (SCID)-repopulating cells (SRCs). Contact coculture of fluorescence-activated cell sorter (FACS)-sorted bone marrow (BM) CD34(+)CD38- cells with human brain endothelial cells (HUBECs) supported a 4.4-fold increase in CD34(+)CD38- cells with a concordant 3.6-fold increase in SRCs over 7 days. Noncontact HUBEC cultures and the addition of thrombopoietin, stem cell factor (SCF), and macrophage colony stimulating factor I receptor (Fms)-like tyrosine kinase 3 (Flt-3) ligand supported further increases in CD34(+)CD38- cells (6.4-fold and 13.1-fold), which correlated with significant increases in SRC activity. Moreover, cell-sorting studies performed on HUBEC-cultured populations demonstrated that SRCs were significantly enriched within the CD34(+)CD38- subset compared with the CD34(-)CD38- population after culture. These results indicate that human HSCs can be identified and characterized by phenotype following expansion culture. These studies also demonstrate that HUBEC-elaborated soluble factors mediate a unique and potent expansion of human HSCs.     

Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein

Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector-mediated gene transfer. Human hematopoietic cell lines and cord blood-derived CD34(+) and CD34(+), CD38(-) cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34(+) cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive for enhanced green fluorescent protein represented as much as 90% of the graft. The use of RD114-pseudotyped vectors may be advantageous for therapeutic gene transfer into hematopoietic stem cells. (Blood. 2000;96:1206-1214)     

Hot spots of retroviral integration in human CD34+ hematopoietic cells

Insertional oncogenesis is a possible consequence of the integration of gamma-retroviral (RV) or lentiviral (LV) vectors into the human genome. RV common insertion sites (CISs) have been identified in hematopoietic malignancies and in the nonmalignant progeny of transduced hematopoietic stem/progenitor cells (HSCs), possibly as a consequence of clonal selection in vivo. We have mapped a large number of RV and LV integrations in human CD34(+) HSCs, transduced in vitro and analyzed without selection. Recurrent insertion sites (hot spots) account for more than 21% of the RV integration events, while they are significantly less frequent in the case of LV vectors. RV but not LV hot spots are highly enriched in proto-oncogenes, cancer-associated CISs, and growth-controlling genes, indicating that at least part of the biases observed in the HSC progeny in vivo are characteristics of RV integration, already present in nontransplanted cells. Genes involved in hematopoietic and immune system development are targeted at high frequency and enriched in hot spots, suggesting that the CD34(+) gene expression program is instrumental in directing RV integration. The lower propensity of LV vectors for integrating in potentially dangerous regions of the human genome may be a factor determining a better safety profile for gene therapy applications.     

Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells.

We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34(+) thy-1(+) cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34(+) thy-1(+) cells on AC6.21 stroma, a murine bone marrow-derived stromal cell line, caused expansion of cells with CD34(+) thy-1(+) phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34(+) thy-1(+) phenotype. The ex vivo-expanded CD34(+) thy-1(+) cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34(+) thy-1(+) cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34(+) CD38(-) cells, shows a similar pattern of proliferative response. This suggests that ex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34(+) thy-1(+) cells.     

Hematopoietic capability of CD34+ cord blood cells: a comparison with CD34+ adult bone marrow cells

The characteristics of hematopoietic progenitor and stem cell (HPC/HSC) populations in mammals vary according to their ontogenic stage. In humans, HPC/HSCs from umbilical cord blood (CB) are increasingly used as an alternative to HPC/HSCs from adult bone marrow (BM) for the treatment of various hematologic disorders. How the hematopoietic activity of progenitor and stem cells in CB differs from that in adult BM remains unclear, however. We compared CD34+ cells, a hematopoietic cell population, in CB with those in adult BM using phenotypic subpopulations analyzed by flow cytometry, the colony-forming activity in methylcellulose clonal cultures, and the repopulating ability of these cells in NOD/Shi-scid (NOD/SCID) mice. Although the proportion of CD34+ cells was higher in adult BM than in CB mononuclear cells, the more immature subpopulations, CD34+ CD33- and CD34+ CD38- cells, were present in higher proportions in CD34+ CB cells. Clonal culture assay showed that more multipotential progenitors were present in CD34+ CB cells. When transplanted into NOD/SCID mice. CD34+ adult BM cells could not reconstitute human hematopoiesis in recipient BM, but CD34+ CB cells achieved a high level of engraftment, indicating that CD34+ CB cells possess a greater repopulating ability. These results demonstrated that human hematopoiesis changes with development from fetus to adult. Furthermore, CD34+ CB cells contained a greater number of primitive hematopoietic cells, including HSCs, than did adult BM, suggesting the usefulness of CD34+ CB cells not only as a graft for therapeutic HSC transplantation but also as a target cell population for ex vivo expansion of transplantable HSCs and for gene transfer in gene therapy.     

Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.