High-level β-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells

Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and β-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling β^A-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo–regenerated cells produced high levels of β^A-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 ± 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroid-specific production of therapeutically relevant levels of β-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.

Sickle-cell disease (SCD) and β-thalassemia are life-threatening inherited disorders of red cell malfunction of major worldwide importance. Both are caused by alterations to the β-globin gene, which is required for normal hemoglobin production in adults. Currently, allogeneic bone marrow transplantation is the only therapy that can produce permanent cures in patients with these disorders (1, 2). However, this approach is limited by lack of suitably matched donors and the significant morbidity and mortality associated with the treatment.

Transplantation of genetically corrected autologous hematopoietic stem cells (HSCs) could circumvent both of these drawbacks, and the development of this approach has therefore been a goal of basic and preclinical studies for many years. These investigations have led to a number of important advances including the identification of the locus control region (LCR) of the β-globin gene cluster (3–6) and the demonstration of the importance of specific elements within the LCR in promoting high-level, erythroid-specific, and position-independent expression of the β-globin gene (7–10).

Efforts to incorporate these LCR elements into oncoretroviral vectors have yielded some promising results in both mouse (11–14) and human targets (15, 16). However, the generation of high titers of intact oncoretroviral vectors encoding sufficient portions of the LCR to achieve maximal β-globin transgene expression in primary cells has proven difficult (11, 15–20). In addition, obtaining clinically relevant yields of transduced HSCs remains a challenge with these vectors. This is due to the decline in HSC activity that occurs during the several days of growth factor stimulation in culture required for their efficient transduction (21–23).

Lentiviral vectors can also infect primitive primary human hematopoietic cells at high efficiencies (50% and more) but with much shorter in vitro–transduction protocols (24–32). Also, the rev-responsive element in the vector facilitates the export of unspliced transcripts into the cytoplasm, thereby promoting higher protein expression (33). Lentiviral vectors may also be less prone to silencing (34, 35). Thus, a number of groups have been evaluating the potential of lentiviral vectors as potentially superior vehicles for globin gene transfer applications (36–41). We have focused on the development of a lentiviral vector containing an anti-sickling human β^A-T87Q-globin expression cassette that includes a 2.7-kb region of the LCR (comprising the DNase I–hypersensitive 2, 3, and 4 elements) (37, 38). Evaluations of the therapeutic utility of this vector in murine models of SCD (37) and β-thalassemia (38) have, in both cases, demonstrated pancellular expression of the transduced β-globin gene with nearly complete correction of the disease phenotype in recipients of transduced HSCs containing an average of 3 proviral integrations per cell.

The present study was undertaken to test the ability of this vector to transduce HSCs of human origin and to measure the level of β-globin transgene expression achieved in their erythroid progeny. The results show efficient transduction of HSCs in normal cord blood as detected by their ability to repopulate sublethally irradiated immunodeficient (NOD/LtSz-scid/scid, or NOD/SCID) mice for at least 6 months. In addition, production of clinically relevant levels of β-globin protein in the erythroid progeny of the transduced HSCs has been demonstrated. Additional evidence of the preferred intragenic integration of such vectors has also been obtained including insertions into genes implicated in human leukemia.

Efficient lentiviral transfer of a β^A-T87Q-globin transgene into primitive human cord blood cells. As an initial test of the ability of our safety-modified β^A-T87Q-globin lentivirus (Figure 1) to transduce primitive human hematopoietic cells, the proportion of vector-containing hematopoietic colonies was determined on cells plated immediately after a 16-hour exposure of a CD34⁺ cell–enriched cord blood cell suspension to virus following a prior 48-hour period of stimulation in vitro with the following growth factors alone: Flt-3 ligand (FL), Steel factor (SF), thrombopoietin (Tpo), and hyper–IL-6 (hIL-6). PCR analysis of individual colonies (both granulopoietic and erythroid) using primer pairs specific for the β^A-T87Q-globin transgene and for GAPDH showed that the DNA from 15 of 40 GAPDH⁺ colonies analyzed (38%) also contained the β^A-T87Q-globin transgene. To determine the efficiency of transducing more primitive cells with multiple-lineage in vivo–repopulating ability, 26 NOD/SCID and 3 NOD/LtSz-scid/scid nu/nu (NOD/SCID-nu/nu) mice were transplanted with infected cord blood cells. In these experiments, CD34⁺ cell–enriched suspensions (n = 4; including experiments 1 and 2) or CD34⁺CD38^– cells isolated by FACS (n = 1) were first stimulated with growth factors for 18 hours, exposed to β^A-T87Q-globin lentivirus for the next 6 hours, and then injected into the mice. FACS analysis of serial femoral marrow aspirates obtained from these mice confirmed that the transplants had regenerated persistent populations of hematopoietic (CD45/71⁺) cells. Figure 2A shows representative data from two experiments. The variation in the number of human cells regenerated in these two experiments is probably due to differences in the number of CD34⁺ cells transplanted per mouse (1.65 × 10⁵ versus 4.0 × 10⁵) and also to differences in frequency of repopulating cells in the two pools of cord blood used. As expected (42), by 16–20 weeks after the transplant, the regenerated human cells were predominantly B-lymphoid (CD19/20⁺), with a minority of mature granulopoietic (CD15⁺) elements, in almost all (15/19) of the mice. Overall, from five experiments, 19 of 29 recipients were monitored for a minimum of 18 and up to 24 weeks, and 75% of these maintained significant levels of human lymphoid and myeloid cells (35% ± 9% total human cells; 23% ± 6% human B-lymphoid cells, and 7% ± 3% mature human granulopoietic cells).

Figure 1

Schematic diagram of the self-inactivating β^A-T87Q-globin lentiviral vector used in this study. Features include the HIV LTR; the packaging signal (ψ⁺); the central polypurine tract (cPPT); the rev-responsive element (RRE); the β-globin promoter, from SnaBI to the cap site (βp); the 3′ β-globin enhancer (to the downstream AvrII site); the 372-bp IVS2 deletion (indicated by the triangle); the β^A-T87Q mutation (ACA Thr [β⁸⁷ Thr] to CAG Gln [γ⁸⁷ Gln]); and the DNase I_hypersensitive sites, SmaI to XbaI (HS2), SacI to PvuII (HS3), and StuI to SpeI (HS4) of the β-globin LCR; 2 stop codons in the ψ⁺ packaging signal; the 400-bp deletion in U3 of the right-hand HIV LTR; and the rabbit β-globin polyA signal. The locations of the β^A-T87Q-globin transgene-specific primers in exons 2 and 3 (E2, E3) are indicated by arrowheads.

Figure 2

Assessment of 16 NOD/SCID mice and 3 NOD/SCID-nu/nu mice transplanted with β^A-T87Q-globin lentivirus-infected cord blood cells. (A) Time course studies of the total number of human hematopoietic (CD45/71⁺) cells generated in two representative experiments. Values are the mean ± SEM for 8 mice in experiment 1 (filled squares) and 11 mice in experiment 2 (filled circles). Of all cells present in the marrow in experiment 1, 20 weeks after transplant, 4% ± 2% were B-lymphoid (CD19/20⁺) cells and 0.7% ± 0.4% were mature granulopoietic (CD15⁺) cells; of all cells present in the marrow in experiment 2, 16 weeks after transplant, 63% ± 4% were B-lymphoid cells and 7% ± 1% were mature granulopoietic cells. (B) PCR analysis to detect provirus-positive hematopoietic colonies produced by human CFCs isolated from the marrow of individual mice (n = 18) at different time points after transplant in a total of five experiments. Values determined at 3 weeks after transplant are not connected to the values determined at later time points because the cells present at these two different times are thought to be derived from different types of repopulating cells (43). The overall proportion of colonies with the β^A-T87Q-globin transgene in all 17 recipients analyzed 11_24 weeks after transplant was 45% ± 6% (range 12%_91%).

Figure 2B shows the results of the PCR analysis of the regenerated human CFCs isolated from 15 NOD/SCID and 3 NOD/SCID-nu/nu mice at different time points after the transplant (5–40 colonies per mouse per time point indicated). In all but 1 of the 15 positive NOD/SCID mice evaluated between 3 and 6 months after the transplant and also in the single positive NOD/SCID-nu/nu recipient followed for evaluation until 13 months after transplant, more than 20% of the CFCs were found to contain the transgene. In 2 mice, as many as 80–90% of the regenerated human CFCs were positive. Based on the data for all of the CFCs analyzed (obtained at least 11 weeks after the transplant from 17 mice in five experiments), the overall average transduction efficiency of long-term NOD/SCID mouse–repopulating cells was 45%.

High levels of β^A-T87Q-globin protein are produced in the erythroid progeny of transduced human cord blood cells. To investigate the levels of β^A-T87Q-globin protein produced by erythroid cells derived from transduced human cord blood cells, we transferred aliquots of cord blood cells immediately after virus exposure to culture conditions designed to promote erythroblast generation and differentiation over a period of 14 days (see Methods). In the two such experiments performed, more than 85% of the cells at the time of harvest had phenotypic features of maturing glycophorin-A⁺ erythroblasts (Figure 3, A and B). HPLC analysis of lysates of these cells showed that β^A-T87Q-globin protein composed 48% and 59% of all β-globin chains (and 24% and 41% of all β-like globin chains) in the two experiments (Figure 3C).

Figure 3

Lineage-specific expression of human β^A-T87Q -globin protein in human erythroid cells generated in vitro. (A) Photomicrograph of Wright-Giemsa_stained slide preparation of cells from erythroid suspension cultures initiated 14 days previously with cells obtained immediately after exposure to lentivirus. (B) FACS analysis of the cells shown in A after staining with antibodies specific to human glycophorin-A. (C) Representative HPLC profile of a cell lysate obtained from a 14-day culture of cord blood cells set up immediately after infection. Peaks representing differently eluting β-globin proteins are indicated. (D) Isolation by FACS of human CD45/71⁺ cells from bone marrow (BM) aspirates of transplanted mice to initiate CFC or suspension cultures that support terminal human erythroid cell differentiation. (E) Representative HPLC profile of a cell lysate from a 14-day suspension culture of human erythroid cells initiated with cells obtained from a mouse that received a transplant of transduced cord blood cells 11 weeks previously. (F) RT-PCR analysis of RNA extracted from cells shown in A (right panels) and human CD19/20⁺ B-lymphoid cells isolated by FACS (left panels) from a marrow sample of a repopulated mouse 19 weeks after transplant. The β^A-T87Q-globin transcript was detected in erythroblasts even when diluted 32 times (top right panel), whereas no signal was seen in the extract of B-lymphoid cells (top left panel). Triangles indicate decreasing concentration.

The ability of transduced HSCs to generate erythroid progeny that would also produce this level of β^A-T87Q-globin protein was then investigated. Direct assessment of in vivo–generated human erythroblasts is, unfortunately, not possible in NOD/SCID mice repopulated with human hematopoietic cells because of the failure of these hosts to support terminal human erythropoiesis beyond 3–4 weeks after irradiation (43). Therefore, we used the FACS to isolate the human (CD45/71⁺) cells present in marrow samples obtained from engrafted mice and then stimulated these to differentiate into erythroid cells in culture, either in CFC assays or in suspension cultures (Figure 3D). HPLC analysis of lysates from the cells harvested from the suspension cultures showed β^A-T87Q-globin protein to represent 35% of all β-globin chains and 19% of all β-like globin chains, with values as high as 59% and 31%, respectively (Table 1). Figure 3E shows a representative HPLC profile resulting from a culture initiated with cells present in a mouse receiving a transplant of transduced cord blood cells 11 weeks before. Similar levels of β^A-T87Q-globin protein (37%) were also detected in lysates of single or 5–10 pooled erythroid colonies produced from regenerated burst forming unit–erythroids (BFU-Es; Table 1).

Table 1

Expression of β^A-T87Q-globin protein in human erythroid cells produced ex vivo from the in vivo progeny of transduced cord blood cells transplanted into NOD/SCID mice

RT-PCR analysis of RNA from erythroblasts generated in vitro from transduced cord blood cells confirmed the presence of β^A-T87Q-globin transcripts in these cells. A strong signal could be detected when the cDNA prepared from these extracts was diluted up to 32 times (Figure 3F, top right panel). In contrast, similar analysis of RNA extracts prepared from human B-lymphoid cells isolated by FACS from 2 mice repopulated with transduced cells showed no evidence of β^A-T87Q-globin transcripts in the undiluted cDNA (Figure 3F, top left panel). This demonstrates the erythroid specificity of expression of the β^A-T87Q-globin transgene achieved in human cells transduced with this vector, as previously observed in murine cells (37).

Polyclonal reconstitution of NOD/SCID mice transplanted withβ^A-T87Q-globin lentiviral vector–infected cord blood cells. To investigate the number and stability of transduced clones obtained in NOD/SCID mice transplanted with β^A-T87Q-globin lentiviral vector–infected cord blood cells, and to obtain an estimate of the number of copies present in each repopulating cell, we used “bubble” linker-mediated PCR (LM-PCR). A total of 121 human CFC–derived colonies from 11 mice that had been confirmed to contain the β^A-T87Q-globin transgene were analyzed individually. These included CFCs from 2 mice that were assessed at different time points after receiving transplants. Agarose gel electrophoresis of the LM-PCR products showed that the cells in each colony contained, on average (± SEM), 2.1 ± 0.1 copies of provirus. Multiple unique integration patterns were frequently detected in the different CFCs harvested from a single mouse, indicating the presence of multiple clones transduced by the β^A-T87Q-globin lentivirus contributing to the regenerated human cell population present (Figure 4, A–C). Analysis of colonies generated from sequential marrow aspirates indicated dynamic changes in clonal prevalence during the first 6 months after the transplant in both mice followed in this way. Representative data from one of these mice is shown in Figure 4, D and E.

Figure 4

Bubble LM-PCR analysis of the number and dynamics of β^A-T87Q -globin lentivirus-positive clones in mice transplanted with transduced cord blood cells. (A_C) Analysis of individual CFC-derived colonies from 3 recipients (A, 1 recipient from experiment 4 analyzed 24 weeks after transplant; B and C, 2 mice from experiment 2, described in Figure 2A, analyzed 19 weeks after transplant). DNA from a transgene-negative colony was used as a negative control for the PCR, and DNA from a mouse spleen colony with a single integration confirmed by Southern blotting was used as a positive control. Note that the expected 750-bp 3′ LTR_related fragment (IB) could not be detected in all of the colonies. The presence of identical faint bands in the analyses of several colonies is probably caused by cross contamination that occurred while plucking these colonies from the methylcellulose cultures. (D and E) Analysis of CFC-derived colonies from a third mouse from experiment 2 described in Figure 2A analyzed 11 and 19 weeks after transplant. Bands with asterisks were gel purified and sequenced. Asterisks with the same color indicate colonies that showed the same integration site upon sequencing.

Preferential intragenic integration ofβ^A-T87Q-globin lentiviral vector into human HSCs. The products of the LM-PCR include genomic sequences flanking the integrated provirus, thus allowing the site of proviral integration to be ascertained by DNA sequencing. Using this approach, we analyzed the sequences of 48 gel-purified fragments that contained the expected vector-related long terminal repeat (LTR) sequence linked to a flanking genomic sequence. Of these 48 fragments, 35 were unique and were obtained from 33 individual colonies generated in vitro from human CFCs produced in the mice. Of 48 fragments, 13 (27%) were repeats; that is, the same sequence was observed in at least 2 colonies generated from the same mouse. For 7 of the 35 inserts (20%), no match was found in the human genome database. Of the remaining 28 inserts, 24 mapped within identified genes (86%) and 4 were repetitive elements that could not be mapped. The chromosomal distribution and names of the mapped genes are shown in Figure 5 and in Table 2, respectively. Notably, several integrations occurred in known oncogenes (e.g., MLL and NUP214, both implicated in acute myeloid leukemias), genes involved in signal transduction pathways (e.g., SCAP2), or genes with tumor suppressor activity (e.g., CGBP). Interestingly, the majority of intragenic integration events occurred in introns (21/24 or 88%).

Figure 5

Chromosomal distribution of the sites of β^A-T87Q-globin lentiviral vector integration. Asterisks indicate sites of integration within predicted genes.

Table 2

Identity of intragenic β^A-T87Q-globin lentiviral transgene integration sites

Historically, the development of gene therapy approaches to SCD and β-thalassemia has been plagued by problems inherent in the design of stable vectors encoding human β-globin cassettes sufficient to direct the permanently very high levels of expression needed for the correction of these diseases. More recently, the risks of insertional mutagenesis have highlighted the need to achieve these goals with minimal copies of proviral integrants per cell (44). Here, we present the first evidence that a recently described self-inactivating lentiviral vector can be used to efficiently engineer the sustained production of anticipated therapeutic levels of an anti-sickling β^A-T87Q-globin protein in the erythroid progeny of primary human hematopoietic cells with in vivo–repopulating ability. We focused on normal cord blood as a target population for these initial studies because of the high frequency of NOD/SCID mouse–repopulating cells in cord blood (45, 46) and their reported ease of transduction after brief in vitro exposure to virus (25, 28, 29, 32, 47–49). The demonstrated levels of anti-sickling β^A-T87Q-globin protein in these cells constitute a significant advance and now underscore the need to extend these studies to adult HSCs.

Many groups have now shown that human HSCs with NOD/SCID mouse–repopulating activity can be efficiently transduced after very brief exposure to lentiviral vectors in vitro without affecting their subsequent hematopoietic potential or ability to express the acquired transgene (25, 31, 32, 48, 50–52). However, most of these findings have been limited to vectors carrying a ubiquitously expressed reporter or drug resistance gene. It remained to be demonstrated that such models could be extended effectively to vectors encoding a therapeutic gene and any additional sequences required for regulating their level and cell-specific expression. The findings presented here are the first to document this achievement with a vector carrying a β-globin gene. At least 20% of the CFCs regenerated in mice that had been repopulated with multiple clones of human cord blood cells exposed to concentrated preparations of this virus (0.5 × 10⁹ to 1 × 10⁹ infectious units/ml) had the vector transgene, and values of more than 50% were noted in several mice. These findings indicate that the β^A-T87Q-globin lentivirus can be used to transduce human cord blood cells with long-term NOD/SCID mouse–repopulating activity with the same efficiency as has been reported for simpler constructs (25, 27, 28, 31, 32, 48, 53).

Because we had found that prior overnight exposure to growth factors can increase the proportion of NOD/SCID mouse–repopulating cells that are ultimately transduced (data not shown), we adopted this approach. No perturbation of the normal pattern of differentiation was observed in mice repopulated with the transduced cells. Also, multiple-lineage hematopoiesis was stable for as long as 4–6 months after transplant in the NOD/SCID hosts and for at least 13 months after transplant in the one NOD/SCID-nu/nu mouse that could be followed. Taken together, these findings indicate that the transduction protocol used had no significant or consistent biological effect on cord blood HSCs.

We did not attempt to document any changes in NOD/SCID mouse–repopulating cell activity that may have been incurred by the use of an overnight infection protocol, nor did we try to determine how these might have affected an ultimate net gain or loss in the absolute yield of transduced HSCs (32, 54). Clearly such data would be important to design a clinical protocol. The latter would also require re-examination of the timing and growth factor treatment needed to optimize the transduction of NOD/SCID mouse–repopulating cells in adult sources of human HSCs, since these are present at much lower frequencies than in cord blood (45, 55). Adult sources of HSCs are also known to be qualitatively as well as quantitatively different from cord blood HSCs in terms of the rate at which they can be activated into G₁ and the rate at which HSC function may be lost in vitro (16, 42, 56–59).

We took advantage of the high gene transfer to human HSCs obtained in many experiments and the resultant high degree of repopulation of some mice with transduced human cells to investigate the clonal dynamics of this process. To identify different clones by the detection of unique proviral integration events, we analyzed hematopoietic colonies produced in vitro from in vivo–regenerated progenitors using a bubble LM-PCR method. Bubble LM-PCR was chosen because it is simpler to implement than other methods for detecting proviral inserts in small numbers of cells (fewer than 10³). However, we do not yet know the sensitivity of this protocol, particularly where multiple integrations may be encountered and hence the number of proviral copies per cell may be underestimated. Nevertheless, this analysis allowed multiple clones (CFCs with different inserts) to be identified in individual animals, providing strong evidence of their polyclonal reconstitution by transduced human HSCs. CFCs with different patterns of integration were also seen in a single repopulated mouse examined over a 2-month interval (i.e., 3 and 5 months after transplant). This is similar to what has been observed in large animals (60).

The present studies are also important in their demonstration of the high lineage-specific levels of β^A-T87Q-globin produced in the erythroblast progeny of transduced HSCs. In many examples, these levels were above the estimated values of 20% of total β-globin per erythroid cell required to achieve a therapeutic effect in SCD and β-thalassemia (41, 61), even when all the β-like globin chains present in the cells were taken into consideration. Although the transgene copy number was estimated as 2 copies per cell, no meaningful calculation of the amount of β^A-T87Q-globin protein produced per transgene copy was possible because the extracts analyzed were obtained from a highly heterogeneous mixture of erythroblasts at different stages of differentiation. Nevertheless, it is clear that the results obtained here represent a significant improvement over the levels of β-globin transgene–derived transcripts and protein recently documented in erythroblasts derived from human cord blood cells transduced with an oncoretroviral vector containing a similar β-globin cassette with a smaller LCR fragment (16).

We found that the majority (24/28, 86%) of the proviral integrations occurred within genes identified in RefSeq, and these included a number of genes that possess cell signaling or regulatory functions or that have been associated with leukemia. This proportion is significantly higher than would be expected (P < 0.0001) from the current estimates that genes occupy approximately 35% of the genome (62, 63). Thus our findings support the concept of preferred intragenic integration of lentiviral vectors into human DNA, as noted by others who reported 67% and 58% intragenic HIV-1 integrations in human lymphoid (64) and HeLa cells (65), respectively. We also observed preferential integration into introns (21/24 = 88% of inserts characterized), as reported by others (64), perhaps reflecting the greater relative size of introns as compared with exons (62, 63). Taken together, these findings refocus attention on the possibility that such integration could result in activation or ablation of the expression of endogenous genes following the transduction of human HSCs, with functional consequences. As such, they point to the need for improvements in vector design aimed at both insulating integration sites from the effects of the provirus and minimizing the integrated proviral copy number required for therapeutic levels of expression.

This work was supported by grants from the NIH (Program Project grant HL 55435 to C.J. Eaves, M.E. Fabry, R.K. Humphries, P. Leboulch, and R.L. Nagel; HL38055 and HL68962 to R.L. Nagel), the Stem Cell Network (to C.J. Eaves and R.K. Humphries), and the International Cooley’s Anemia Foundation (to R.L. Nagel). P. Tang received a Research Studentship from the Stem Cell Network. The authors thank Lars Palmquist for integration site analysis; Sandy Suzuka, members of the Stem Cell Assay Service of the Terry Fox Laboratory, Sanja Sekulovic, Margaret Hale, and Michael Schertzer for technical help; StemCell Technologies Inc., Immunex Corporation, Genentech, P. Lansdorp, and S. Rose-John for gifts of reagents; and Colleen MacKinnon for assistance in manuscript preparation.

See the related Commentary beginning on page 889.

Connie J. Eaves and R. Keith Humphries contributed equally to this work.

Nonstandard abbreviations used: BFU-E, burst forming unit–erythroid; Epo, erythropoietin; FL, Flt-3 ligand; HSC, hematopoietic stem cell; LCR, locus control region; LM-PCR, linker-mediated PCR; LTR, long terminal repeat; NOD/SCID, NOD/LtSz-scid/scid; NOD/SCID-nu/nu, NOD/LtSz-scid/scid nu/nu; SCD, sickle-cell disease; SF, Steel factor; TAE, Tris-acetate EDTA; Tpo, thrombopoietin.

Conflict of interest: C.J. Eaves and R.K. Humphries are paid consultants of StemCell Technologies Inc., suppliers of some of the culture reagents used in this study; R. Pawliuk, K.A. Westerman, and P. Leboulch are officers of Genetix Pharmaceutical Inc.

1. Olivieri, NF. The beta-thalassemias. N. Engl. J. Med. 1999. 341:99-109.
   View this article via: PubMed CrossRef Google Scholar
2. Walters, MC, et al. Barriers to bone marrow transplantation for sickle cell anemia. Biol. Blood Marrow Transplant. 1996. 2:100-104.
   View this article via: PubMed Google Scholar
3. Tuan, D, London, IM. Mapping of DNase I-hypersensitive sites in the upstream DNA of human embryonic epsilon-globin gene in K562 leukemia cells. Proc. Natl. Acad. Sci. U. S. A. 1984. 81:2718-2722.
   View this article via: PubMed CrossRef Google Scholar
4. Tuan, D, Solomon, W, Li, Q, London, IM. The “beta-like-globin” gene domain in human erythroid cells. Proc. Natl. Acad. Sci. U. S. A. 1985. 82:6384-6388.
   View this article via: PubMed CrossRef Google Scholar
5. Wright, S, Rosenthal, A, Flavell, R, Grosveld, F. DNA sequences required for regulated expression of beta-globin genes in murine erythroleukemia cells. Cell. 1984. 38:265-273.
   View this article via: PubMed CrossRef Google Scholar
6. Forrester, WC, Takegawa, S, Papayannopoulou, T, Stamatoyannopoulos, G, Groudine, M. Evidence for a locus activation region: the formation of developmentally stable hypersensitive sites in globin-expressing hybrids. Nucleic Acids Res. 1987. 15:10159-10177.
   View this article via: PubMed CrossRef Google Scholar
7. Grosveld, F, et al. The regulation of expression of human beta-globin genes. Prog. Clin. Biol. Res. 1987. 251:133-144.
   View this article via: PubMed Google Scholar
8. Blom van Assendelft, G, Hanscombe, O, Grosveld, F, Greaves, DR. The beta-globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner. Cell. 1989. 56:969-977.
   View this article via: PubMed CrossRef Google Scholar
9. Talbot, D, et al. A dominant control region from the human beta-globin locus conferring integration site-independent gene expression. Nature. 1989. 338:352-355.
   View this article via: PubMed CrossRef Google Scholar
10. Fraser, P, Hurst, J, Collis, P, Grosveld, F. DNaseI hypersensitive sites 1, 2 and 3 of the human beta-globin dominant control region direct position-independent expression. Nucleic Acids Res. 1990. 18:3503-3508.
    View this article via: PubMed CrossRef Google Scholar
11. Leboulch, P, et al. Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure. EMBO J. 1994. 13:3065-3076.
    View this article via: PubMed Google Scholar
12. Sadelain, M, Wang, CH, Antoniou, M, Grosveld, F, Mulligan, RC. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene. Proc. Natl. Acad. Sci. U. S. A. 1995. 92:6728-6732.
    View this article via: PubMed CrossRef Google Scholar
13. Raftopoulos, H, Ward, M, Leboulch, P, Bank, A. Long-term transfer and expression of the human beta-globin gene in a mouse transplant model. Blood. 1997. 90:3414-3422.
    View this article via: PubMed Google Scholar
14. Kalberer, CP, et al. Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta-globin in engrafted mice. Proc. Natl. Acad. Sci. U. S. A. 2000. 97:5411-5415.
    View this article via: PubMed CrossRef Google Scholar
15. Nicolini, FE, et al. Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells. Blood. 2002. 100:1257-1264.
    View this article via: PubMed CrossRef Google Scholar
16. Oh, IH, et al. Expression of an anti-sickling beta-globin in human erythroblasts derived from retrovirally transduced primitive normal and sickle cell disease hematopoietic cells. Exp. Hematol. 2004. 32:461-469.
    View this article via: PubMed CrossRef Google Scholar
17. Dzierzak, EA, Papayannopoulou, T, Mulligan, RC. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature. 1988. 331:35-41.
    View this article via: PubMed CrossRef Google Scholar
18. Bender, MA, Gelinas, RE, Miller, AD. A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells. Mol. Cell. Biol. 1989. 9:1426-1434.
    View this article via: PubMed Google Scholar
19. Plavec, I, Papayannopoulou, T, Maury, C, Meyer, F. A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells. Blood. 1993. 81:1384-1392.
    View this article via: PubMed Google Scholar
20. Rivella, S, Sadelain, M. Genetic treatment of severe hemoglobinopathies: the combat against transgene variegation and transgene silencing. Semin. Hematol. 1998. 35:112-125.
    View this article via: PubMed Google Scholar
21. Rebel, VI, et al. One-day ex vivo culture allows effective gene transfer into human nonobese diabetic/severe combined immune-deficient repopulating cells using high-titer vesicular stomatitis virus G protein pseudotyped retrovirus. Blood. 1999. 93:2217-2224.
    View this article via: PubMed Google Scholar
22. Hennemann, B, et al. Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin. Blood. 2000. 96:2432-2439.
    View this article via: PubMed Google Scholar
23. Dorrell, C, Gan, OI, Pereira, DS, Hawley, RG, Dick, JE. Expansion of human cord blood CD34(+)CD38(–) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function. Blood. 2000. 95:102-110.
    View this article via: PubMed Google Scholar
24. Uchida, N, et al. HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc. Natl. Acad. Sci. U. S. A. 1998. 95:11939-11944.
    View this article via: PubMed CrossRef Google Scholar
25. Miyoshi, H, Smith, KA, Mosier, DE, Verma, IM, Torbett, BE. Transduction of human CD34⁺ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science. 1999. 283:682-686.
    View this article via: PubMed CrossRef Google Scholar
26. Case, SS, et al. Stable transduction of quiescent CD34(+)CD38(–) human hematopoietic cells by HIV-1-based lentiviral vectors. Proc. Natl. Acad. Sci. U. S. A. 1999. 96:2988-2993.
    View this article via: PubMed CrossRef Google Scholar
27. Follenzi, A, Ailles, LE, Bakovic, S, Geuna, M, Naldini, L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 2000. 25:217-222.
    View this article via: PubMed CrossRef Google Scholar
28. Guenechea, G, et al. Transduction of human CD34⁺ CD38^– bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors. Mol. Ther. 2000. 1:566-573.
    View this article via: PubMed CrossRef Google Scholar
29. Woods, NB, et al. Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells. Blood. 2000. 96:3725-3733.
    View this article via: PubMed Google Scholar
30. Scherr, M, et al. Lentiviral gene transfer into peripheral blood-derived CD34⁺ NOD/SCID-repopulating cells. Blood. 2002. 99:709-712.
    View this article via: PubMed CrossRef Google Scholar
31. Ailles, L, et al. Molecular evidence of lentiviral vector-mediated gene transfer into human self-renewing, multi-potent, long-term NOD/SCID repopulating hematopoietic cells. Mol. Ther. 2002. 6:615-626.
    View this article via: PubMed CrossRef Google Scholar
32. Mazurier, F, Gan, OI, McKenzie, JL, Doedens, M, Dick, JE. Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood. 2004. 103:545-552.
    View this article via: PubMed CrossRef Google Scholar
33. Naldini, L, Verma, IM. Lentiviral vectors. Adv. Virus Res. 2000. 55:599-609.
    View this article via: PubMed CrossRef Google Scholar
34. Lois, C, Hong, EJ, Pease, S, Brown, EJ, Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002. 295:868-872.
    View this article via: PubMed CrossRef Google Scholar
35. Pfeifer, A, Verma, IM. Gene therapy: promises and problems. Annu. Rev. Genomics Hum. Genet. 2001. 2:177-211.
    View this article via: PubMed CrossRef Google Scholar
36. May, C, et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature. 2000. 406:82-86.
    View this article via: PubMed CrossRef Google Scholar
37. Pawliuk, R, et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science. 2001. 294:2368-2371.
    View this article via: PubMed CrossRef Google Scholar
38. Imren, S, et al. Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells. Proc. Natl. Acad. Sci. U. S. A. 2002. 99:14380-14385.
    View this article via: PubMed CrossRef Google Scholar
39. May, C, Rivella, S, Chadburn, A, Sadelain, M. Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene. Blood. 2002. 99:1902-1908.
    View this article via: PubMed CrossRef Google Scholar
40. Persons, DA, Hargrove, PW, Allay, ER, Hanawa, H, Nienhuis, AW. The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number. Blood. 2003. 101:2175-2183.
    View this article via: PubMed CrossRef Google Scholar
41. Levasseur, DN, Ryan, TM, Pawlik, KM, Townes, TM. Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells. Blood. 2003. 102:4312-4319.
    View this article via: PubMed CrossRef Google Scholar
42. Holyoake, TL, Nicolini, FE, Eaves, CJ. Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Exp. Hematol. 1999. 27:1418-1427.
    View this article via: PubMed CrossRef Google Scholar
43. Glimm, H, et al. Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice. J. Clin. Invest. 2001. 107:199-206.
    View this article via: JCI PubMed CrossRef Google Scholar
44. Hacein-Bey-Abina, S, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003. 302:415-419.
    View this article via: PubMed CrossRef Google Scholar
45. Wang, JC, Doedens, M, Dick, JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood. 1997. 89:3919-3924.
    View this article via: PubMed Google Scholar
46. Conneally, E, Cashman, J, Petzer, A, Eaves, C. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc. Natl. Acad. Sci. U. S. A. 1997. 94:9836-9841.
    View this article via: PubMed CrossRef Google Scholar
47. Ailles, LE, Naldini, L. HIV-1-derived lentiviral vectors. Curr. Top. Microbiol. Immunol. 2002. 261:31-52.
    View this article via: PubMed Google Scholar
48. Amsellem, S, Ravet, E, Fichelson, S, Pflumio, F, Dubart-Kupperschmitt, A. Maximal lentivirus-mediated gene transfer and sustained transgene expression in human hematopoietic primitive cells and their progeny. Mol. Ther. 2002. 6:673-677.
    View this article via: PubMed CrossRef Google Scholar
49. Zielske, SP, Gerson, SL. Cytokines, including stem cell factor alone, enhance lentiviral transduction in nondividing human LTCIC and NOD/SCID repopulating cells. Mol. Ther. 2003. 7:325-333.
    View this article via: PubMed CrossRef Google Scholar
50. Woods, NB, Ooka, A, Karlsson, S. Development of gene therapy for hematopoietic stem cells using lentiviral vectors. Leukemia. 2002. 16:563-569.
    View this article via: PubMed CrossRef Google Scholar
51. Scherr, M, Eder, M. Gene transfer into hematopoietic stem cells using lentiviral vectors. Curr. Gene Ther. 2002. 2:45-55.
    View this article via: PubMed CrossRef Google Scholar
52. Zielske, SP, Reese, JS, Lingas, KT, Donze, JR, Gerson, SL. In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning. J. Clin. Invest. 2003. 112:1561-1570. doi:10.1172/JCI200317922.
    View this article via: JCI PubMed Google Scholar
53. Piacibello, W, et al. 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. Blood. 2002. 100:4391-4400.
    View this article via: PubMed CrossRef Google Scholar
54. Takatoku, M, et al. Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates. J. Clin. Invest. 2001. 108:447-455. doi:10.1172/JCI200112593.
    View this article via: JCI PubMed Google Scholar
55. van der Loo, JC, et al. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse as a model system to study the engraftment and mobilization of human peripheral blood stem cells. Blood. 1998. 92:2556-2570.
    View this article via: PubMed Google Scholar
56. Hennemann, B, et al. Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables. Exp. Hematol. 1999. 27:817-825.
    View this article via: PubMed CrossRef Google Scholar
57. Cashman, JD, Clark-Lewis, I, Eaves, AC, Eaves, CJ. Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model. Blood. 1999. 94:3722-3729.
    View this article via: PubMed Google Scholar
58. Glimm, H, Oh, IH, Eaves, CJ. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0). Blood. 2000. 96:4185-4193.
    View this article via: PubMed Google Scholar
59. Oh, IH, Lau, A, Eaves, CJ. During ontogeny primitive (CD34(+)CD38(–)) hematopoietic cells show altered expression of a subset of genes associated with early cytokine and differentiation responses of their adult counterparts. Blood. 2000. 96:4160-4168.
    View this article via: PubMed Google Scholar
60. Kuramoto, K, et al. The impact of chronic low-dose busulfan on clonal dynamics in nonhuman primates. Blood. 2004. 104:1273-1280.
    View this article via: PubMed CrossRef Google Scholar
61. Persons, DA, et al. Functional requirements for phenotypic correction of murine beta-thalassemia: implications for human gene therapy. Blood. 2001. 97:3275-3282.
    View this article via: PubMed CrossRef Google Scholar
62. Lander, ES, et al. Initial sequencing and analysis of the human genome. Nature. 2001. 409:860-921.
    View this article via: PubMed CrossRef Google Scholar
63. Venter, JC, et al. The sequence of the human genome. Science. 2001. 291:1304-1351.
    View this article via: PubMed CrossRef Google Scholar
64. Schroder, AR, et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002. 110:521-529.
    View this article via: PubMed CrossRef Google Scholar
65. Wu, X, Li, Y, Crise, B, Burgess, SM. Transcription start regions in the human genome are favored targets for MLV integration. Science. 2003. 300:1749-1751.
    View this article via: PubMed CrossRef Google Scholar
66. Westerman, KA, Morales, T, Pawliuk, R, Cohen, E, LeBoulch, P. A high titer, “supersplit” packaging system in combination with self-inactivating vector for the prevention of replication competent lentivirus (RCL) contamination [abstract]. Mol. Ther. 2003. 7:6A.
67. Pawliuk, R, Kay, R, Lansdorp, P, Humphries, RK. Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer. Blood. 1994. 84:2868-2877.
    View this article via: PubMed Google Scholar
68. Sutherland, HJ, Eaves, CJ, Eaves, AC, Dragowska, W, Lansdorp, PM. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood. 1989. 74:1563-1570.
    View this article via: PubMed Google Scholar
69. Meyerrose, TE, Herrbrich, P, Hess, DA, Nolta, JA. Immune-deficient mouse models for analysis of human stem cells. Biotechniques. 2003. 35:1262-1272.
    View this article via: PubMed Google Scholar
70. Verlinden, SF, van Es, HH, van Bekkum, DW. Serial bone marrow sampling for long-term follow up of human hematopoiesis in NOD/SCID mice. Exp. Hematol. 1998. 26:627-630.
    View this article via: PubMed Google Scholar
71. Eaves, C, et al. Hematopoietic stem cells: inferences from in vivo assays. Stem Cells. 1997. 15(Suppl. 1):1-5.
    View this article via: PubMed Google Scholar
72. Fabry, ME, et al. A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity. Blood. 1995. 86:2419-2428.
    View this article via: PubMed Google Scholar
73. Riley, J, et al. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res. 1990. 18:2887-2890.
    View this article via: PubMed CrossRef Google Scholar