Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice

Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.     

Animal xenograft models for evaluation of gene transfer into human hematopoietic stem cells

Animal xenograft models of gene therapy have become increasingly popular to study the effects of various transduction strategies on human hematopoietic stem cells (HSC). Xenograft models provide an in vivo setting in which to monitor the duration and effects of vector expression in the progeny of the transduced stem and progenitor cells. Also, the ability of HSC to home to the bone marrow and differentiate into multilineage progeny following ex vivo manipulation can only be tested in a transplantation system. The current review will discuss the murine xenograft models that have been used recently to determine optimized methods for gene transfer into normal human hematopoietic stem cells.     

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34(+) cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.     

Efficient bone marrow transduction by gene transfer with allogeneic umbilical cord blood serum and plasma: an implication for clinical trials

Low in vivo transduction efficiency and safety concerns have been hurdles for effective hematopoietic stem cell (HSC) gene therapy. Here, we investigate whether the safety and efficiency of retroviral gene transfer into HSCs can be improved by using human allogeneic umbilical cord blood (UCB)-derived supplements instead of fetal bovine serum (FBS). When CD34(+) cells were cultured ex vivo in UCB-derived serum (CBS) or plasma (CBP), comparable or higher maintenance of HSCs was observed than in FBS and serum-free substitution medium (SFM) as assessed by the frequency of positive engraftment and the level of engraftment in NOD/SCID mice after transplantation of cultured cells. CBS and CBP also exhibited higher level stabilization of retroviral particles than SFM during in vitro culture of retrovirus pseudotyped with gibbon ape leukemia virus or vesicular stomatitis virus glycoprotein. Retroviral gene transfer into CD34(+) cells performed with CBS or CBP resulted in increased gene transfer into CD34(+) cells and increased transduction of reconstituted bone marrow cells compared to transfers with SFM or FBS. The increased transduction of bone marrow cells was associated with a larger number of transduced progenitors in the recipient mice. Significant oligoclonality in the transduced progenitors, as determined by ligation-mediated polymerase chain reaction, suggested efficient retroviral targeting of multiple HSCs in the CBS- or CBP-supplemented media. Combined, our results show that allogeneic UCB-derived serum or plasma is a safe and easily accessible serum supplement that can support efficient retroviral gene transfer into HSCs for the clinical-grade manipulation of HSCs.     

Efficient engraftment of in utero transplanted mice with retrovirally transduced hematopoietic stem cells

Using an experimental mouse model, we have investigated the kinetics of hematopoietic reconstitution of recipients transplanted during fetal development with fresh and transduced hematopoietic stem cells (HSCs). Total bone marrow (BM) and purified Lin(-)Sca-1(+) cells, either fresh or transduced ex vivo with enhanced green fluorescent protein (EGFP)-encoding retroviral vectors, were in utero transplanted (IUT) into fetal mice. Data obtained 2 months after transplantation showed a similar proportion of engrafted animals, regardless of the fact that samples were purified or not on HSCs, and subjected or not to ex vivo transduction with retroviral vectors. The transplantation of grafts enriched in HSCs, either fresh or transduced, always improved the levels of donor chimerism of IUT mice in comparison with results obtained in mice transplanted with unpurified BM grafts (6.8 and 7.3% versus 1.15% median values, respectively). Significantly, engrafted recipients that were transplanted with the transduced graft always contained transduced EGFP(+) cells in peripheral blood (around 5% of donor cells were EGFP(+) at 2 months post-transplantation). This proportion was essentially maintained at longer times post-transplantation, as well as in secondary recipients transplanted with the BM of IUT mice. Our study describes for the first time a significant and stable engraftment of unconditioned mice subjected to IUT with HSCs transduced with retroviral vectors.     

RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation

Hematopoietic stem cells (HSCs) sustain lifelong production of all blood cell types through finely balanced divisions leading to self-renewal and differentiation. Although several genes influencing HSC self-renewal have been identified, to date no gene has been described that, when activated, enhances HSC self-renewal and, when inactivated [corrected] promotes HSC differentiation. We observe that the retinoic acid receptor (RAR)gamma is selectively expressed in primitive hematopoietic precursors and that the bone marrow of RARgamma knockout mice exhibit markedly reduced numbers of HSCs associated with increased numbers of more mature progenitor cells compared with wild-type mice. In contrast, RARalpha is widely expressed in hematopoietic cells, but RARalpha knockout mice do not exhibit any HSC or progenitor abnormalities. Primitive hematopoietic precursors overexpressing RARalpha differentiate predominantly to granulocytes in short-term culture, whereas those overexpressing RARgamma exhibit a much more undifferentiated phenotype. Furthermore, loss of RARgamma abrogated the potentiating effects of all-trans retinoic acid on the maintenance of HSCs in ex vivo culture. Finally, pharmacological activation of RARgamma ex vivo promotes HSC self-renewal, as demonstrated by serial transplant studies. We conclude that the RARs have distinct roles in hematopoiesis and that RARgamma is a critical physiological and pharmacological regulator of the balance between HSC self-renewal and differentiation.     

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection

Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O6-methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O6-benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.     

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.     

Strategies to expand transduced hematopoietic stem cells in vivo.

Data in mice suggest that in vivo selection strategies will expand the numbers of transduced hematopoietic stem cells (HSC) to levels sufficient for clinical therapies, and it is argued that comparable strategies will benefit larger animals and humans. To test this assumption, we performed virtual gene therapy in mouse and cat, species in which the in vivo kinetics of HSC are defined. In the simulated experiments, 10% of HSC and 50% of short-term repopulating cells were transduced with a gene allowing a conditional replication or apoptosis advantage. After transplantation, differentiation proceeded stochastically and contributions of transduced cells were tracked for 2 years. Fifty independent transplantations were simulated per species for each analysis. When transduced HSC had a 2-fold increased chance of replication (self-renewal) extending for 4, 10, or 20 weeks after transplantation, or a 5-fold replication advantage extending for 4 weeks, results in mice were far better than in cat, a larger animal, with slower baseline HSC cell cycle kinetics. Similarly, when transduced HSC had a 2-, 4-, or 10-fold decreased chance of apoptosis, extending for 20 or more weeks after transplantation, the murine studies were poor predictors of feline results. Simulation may allow one to optimize and/or understand the limitations of a gene therapy strategy.     

New approaches to expand hematopoietic stem and progenitor cells

INTRODUCTION: Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and to differentiate into all blood cell lineages, and are currently the foundation of HSC transplantation therapy. A variety of methods have recently been explored to find a way to expand hematopoietic stem and progenitor cells (HSCs/PCs) ex vivo in order to improve the efficiency and outcome of HSC transplantation. AREAS COVERED: Recent studies of HSCs/PCs have led to the development of new ways to detect and purify HSCs/PCs and have also revealed several intrinsic and extrinsic factors that control the molecular signals fundamental to self-renewal and differentiation of HSCs. These findings have provided new approaches for expanding HSCs/PCs ex vivo utilizing protein factors and small-molecule compounds (SMCs) and have also demonstrated promising outcomes in clinical trials. EXPERT OPINION: Although further technical innovation is still needed, elucidation of the whole picture of signaling pathways critical to HSCs/PCs and manipulation of such pathways by SMCs could establish efficient, cost-effective, riskless and robust methods for ex vivo expansion of HSCs/PCs. With these efforts, more sophisticated HSC transplantation would be possible in the near future.     

Successful Treatment of Metachromatic Leukodystrophy Using Bone Marrow Transplantation of HoxB4 Overexpressing Cells

To evaluate the contribution of bone marrow (BM) cells to treat neurological disorders, we examined the effectiveness of BM cells expressing the homeobox B4 (HoxB4) gene to cure mice with metachromatic leukodystrophy (MLD) through transplantation. Increased number of donor cells was observed in brains of the MLD mice transplanted with HoxB4-transduced BM cells (B4MLD) in contrast to those transplanted with control green fluorescent protein (GFP)-transduced BM cells (MIGMLD). Immunohistochemical staining showed that most of the GFP⁺ cells were Iba1⁺ microglia. In addition, O4⁺ oligodendrocytes were identified only in the B4MLD brains but not in the MIGMLD brain. Alcian blue staining showed that accumulation of sulfatide was dramatically reduced in brain tissue from B4MLD mice, and there was a corresponding improvement in the animals'' ability to walk a balance beam 8 months after transplantation. Thus transplantation of BM cells overexpressing HoxB4 appears to effectively prevent the progression of MLD in this mouse model. These findings support the idea that hematopoietic stem cells (HSCs) transduced with a HoxB4 expression vector could be the useful carriers of therapeutic proteins into the brain for regeneration of oligodendrocytes to treat such demyelinating disorders as MLD, Krabbe disease, and multiple sclerosis.     

Transduction of long-term-engrafting human hematopoietic stem cells by retroviral vectors

Gene therapy using retroviral vectors to transfer functional exogenous genes into hematopoietic stem cells (HSCs) promises to provide a permanent cure for a wide array of both hematopoietic and nonhematopoietic disorders by virtue of the fact that retroviral vectors permanently integrate into the host cell genome and HSCs are able to self-renew and give rise to differentiated progeny throughout the life span of the patient. However, for transduction and genomic integration to occur, the target cells must undergo cell division and express the appropriate retroviral receptor, requirements that have thus far hindered attempts at inserting exogenous genes into human HSCs in vitro. In the present studies, we used the fetal sheep xenograft model of human hematopoiesis to evaluate whether human long-term engrafting HSCs could be transduced in vivo, within a fetal microenvironment. We transplanted adult human bone marrow-derived CD34(+)Lin(-) cells into preimmune fetal sheep recipients and subsequently (19 days later) administered clinical-grade murine retroviral vector supernatants to these fetal hematopoietic chimeras. Our results demonstrate that this approach successfully transduced adult human HSCs within all seven sheep that survived the procedure, and that these transduced HSCs had the ability to serially engraft primary, secondary, and tertiary fetal sheep recipients. Transgene expression persisted throughout the serial transplantation. The successful in vivo transduction of long-term engrafting human HSCs with the existing generation of murine retroviral vectors has significant implications for developing new approaches to pre- and postnatal gene therapy.     

Introduction of a Xenogeneic Gene via Hematopoietic Stem Cells Leads to Specific Tolerance in a Rhesus Monkey Model

Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.     

Hematopoietic stem cell transduction and amplification in large animal models

Progress in retroviral gene transfer to large animal hematopoietic stem cells (HSCs) has led to efficient, reproducible long-term marking in both canine and nonhuman primate models. Successes for HSC gene therapy have occurred in the severe combined immunodeficiency setting, in which transduced cells have a selective advantage. However, for most diseases, the therapeutic transgene does not confer a sufficient survival advantage, and increasing the percentage of gene-marked cells in vivo will be necessary to observe a therapeutic effect. In vivo amplification should expand the potential of HSC gene therapy, and progress in this area has benefited greatly from the use of large animal models where efficacy and toxicity have often not correlated with results in murine models. To date, the best results have been observed with O(6)-methylguanine-DNA methyltransferase (MGMT) selection, with which increases in gene-marked repopulating cells have been maintained long-term, likely because of the toxicity of 1,3-bis-(2-chloroethyl)-1-nitrosourea and temozolomide to quiescent HSCs. Using MGMT selection, long-term marking levels exceeding 50% can now be routinely attained with minimal toxicity. There is cause to be optimistic that HSC gene therapy with in vivo amplification will soon allow the treatment of several genetic and infectious diseases.     

Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.     

Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells

Reactivation of telomerase and maintenance of telomere length can lead to the prevention of replicative senescence in some human somatic cells grown in vitro. To investigate whether telomere shortening might also play a role in the limitation of hematopoietic stem cell (HSC) division capacity in vivo, we analyzed telomere length during serial transplantation of murine HSCs. Southern blot analysis of telomere length in donor bone marrow cells revealed extensive shortening ( approximately 7 kb) after just two rounds of HSC transplantation. The number of cycling HSCs increased after transplantation and remained elevated for at least 4 mo, while the frequency of HSCs in the bone marrow was completely regenerated by 2 mo after transplantation. Direct analysis of telomeres in HSCs by fluorescent in situ hybridization during serial transplantation also revealed a reduction in telomere size. Together, these data show that telomeres shorten during division of HSCs in vivo, and are consistent with the hypothesis that telomere shortening may limit the replicative capacity of HSCs.     

Efficient oncoretroviral transduction of extended long-term culture-initiating cells and NOD/SCID repopulating cells: enhanced reconstitution with gene-marked cells through an ex vivo expansion approach

Recent developments of surrogate assays for human hematopoietic stem cells (HSC) have facilitated efforts at improving HSC gene transfer efficiency. Through the use of xenograft transplantation models, such as nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, successful oncoretroviral gene transfer to transplantable hematopoietic cells has been achieved. However, because of the low frequency and/or homing efficiency of SCID repopulating cells (SRC) in bone marrow (BM), studies have primarily focused on cord blood (CB). The recently developed extended (> 60 days) long-term culture-initiating cell (ELTC-IC) assay detects an infrequent and highly quiescent candidate stem cell population in BM as well as CB of the CD34(+)CD38(-) phenotype. Although these characteristics suggest that ELTC-IC and SRC might be closely related, attempts to oncoretrovirally transduce ELTC-IC have been unsuccessful. Here, recently developed conditions (high concentrations of SCF + FL + Tpo in serum-free medium) supporting expansion of BM CD34(+)CD38(-) 12 week ELTC-IC promoted efficient oncoretroviral transduction of BM and CB ELTC-IC. Although SRC can be transduced with oncoretroviral vectors, this is frequently associated with loss of reconstituting activity, posing a problem for development of clinical HSC gene therapy. However, previous attempts at expanding transduced HSC posttransduction resulted in compromised rather than improved gene marking. Utilizing conditions promoting cell divisions and transduction of ELTC-IC we show that although 5 days of ex vivo culture is sufficient to obtain maximum gene transfer efficiency to SRC, extension of the expansion period to 12 days significantly enhances multilineage reconstitution activity of transduced SRC, supporting the feasibility of improving gene marking through ex vivo expansion.     

Microglia: a cellular vehicle for CNS gene therapy

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by deficiency of the enzyme arylsulfatase A (ARSA). MLD is characterized by progressive demyelination and neurological deficits. Treatment of MLD is still a challenge due to the fact that the blood-brain barrier is a major obstacle for most therapeutic substances. In this issue of the JCI, Biffi et al. report that genetically modified hematopoietic precursor cells transduced to overexpress ARSA and transplanted into mice with a targeted disruption of the murine Arsa gene (Arsa(-/-) mice) migrated into the CNS and cross-corrected brain ARSA deficiency (see the related article beginning on page 3070). Microglia served as a cellular vehicle to effectively deliver the enzyme to other brain cells while hepatocytes overexpressing ARSA increased plasma ARSA levels but failed to deliver ARSA into the CNS.     

Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency

Ex vivo culture of hematopoietic stem/progenitor cells could potentially improve the efficacy of human placental/umbilical cord blood (CB) in clinical hematopoietic stem cell (HSC) transplantation and allow gene transduction using conventional retroviral vectors. Therefore, we first examined the effects of a 7-day period of ex vivo culture on the hematopoietic capacity of CB CD34+ cells. Medium for the ex vivo cultures contained either serum and six recombinant human hematopoietic growth factors (GFs), including Flt-3 ligand (FL), Kit ligand (KL = stem cell factor), thrombopoietin (Tpo), interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6), or a serum-free medium containing only FL, KL, and Tpo. After culture under both ex vivo conditions, the total numbers of viable cells, CD34+ cells, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) were increased. In contrast, the severe combined immunodeficiency (SCID) mouse engrafting potential (SEP) of cultured cells was slightly decreased, as compared with fresh cells. Nevertheless, cultured human CB CD34+ cells were able to generate engraftment, shown to persist for up to 20 weeks after transplantation. We next tested the efficacy of retroviral transduction of cultured cells. Transduced cultured human cells were able to engraft in NOD/SCID mice, as tested 4 weeks after transplantation, and EGFP+CD34+ cells and EGFP+ CFCs were isolated from the chimeras. Thus, although additional improvements in ex vivo culture are still needed to expand the numbers and function of human HSCs, the current conditions appear to allow gene transduction into hematopoietic SCID engrafting cells, while at least qualitatively preserving their in vivo engraftment potential.     

Long-term tracking of murine hematopoietic cells transduced with a bicistronic retrovirus containing CD24 and EGFP genes

Hematopoietic stem cells (HSCs) are attractive targets for gene therapy, but current gene transfer methodologies are inadequate for efficient HSC transduction and perpetual transgene expression. To improve gene transfer vectors and transduction protocols, it is vital to establish a system to evaluate transgene expression and the long-term behavior of transduced cells in vivo. For this purpose, we constructed a bicistronic retrovirus encoding the human CD24 (as the first cistron) and the enhanced green fluorescent protein (EGFP; as the second cistron). Murine bone marrow cells were transduced with this vector and the transgene expression was monitored along with hematopoietic reconstitution. Stable expression of CD24 and EGFP was demonstrated in the long-term repopulating cells for at least 6 months, and multi-parameter flow cytometry illustrated expression of both markers in all the lymphohematopoietic lineages examined (B and T lymphoid, erythroid and myeloid). Sustained expression was also shown in the secondary transplants for 6 months, suggesting that self-renewing HSCs were transduced by this vector. Overall, EGFP-tagged bicistronic retroviruses would provide powerful tools for detailed in vivo analysis of transduced hematopoietic cells, such as transgene expression in conjunction with lineage differentiation. Gene Therapy (2000) 7, 1193-1199.