Genetic modification of hematopoietic stem cells: recent advances in the gene therapy of inherited diseases

Archives of Medical Research 34 (2003) 589–599

REVIEW ARTICLE

Genetic Modification of Hematopoietic Stem Cells: Recent Advances in the Gene Therapy of Inherited Diseases Juan A. Bueren, Guillermo Guenechea, Jose´ A. Casado, Marı´a Luisa Lamana and Jose´ C. Segovia Hematopoietic Gene Therapy Program, Comisio´n Interministerial de Ciencia y Tecnologı´a (CIEMAT)/Fundacio´n Marcelino Botı´n, Madrid, Spain Received for publication September 3, 2003; accepted September 5, 2003 (03/147).

Hematopoietic stem cells constitute a rare population of precursor cells with remarkable properties for being used as targets in gene therapy protocols. The last years have been particularly productive both in the fields of gene therapy and stem cell biology. Results from ongoing clinical trials have shown the first unquestionable clinical benefits of immunodeficient patients transplanted with genetically modified autologous stem cells. On the other hand, severe side effects in a few patients treated with gene therapy have also been reported, indicating the usefulness of further improving the vectors currently used in gene therapy clinical trials. In the field of stem cell biology, evidence showing the plastic potential of adult hematopoietic stem cells and data indicating the multipotency of adult mesenchymal precursor cells have been presented. Also, the generation of embryonic stem cells by means of nuclear transfer techniques has appeared as a new methodology with direct implications in gene therapy. 쑖 2004 IMSS. Published by Elsevier Inc. Key Words: Gene marking, Gene therapy, Stem cells, Inherited diseases, Retroviral vectors, Immunodeficiencies.

Properties of Hematopoietic Stem Cells as Targets in the Gene Therapy of Inherited Hematopoietic Diseases Transplantation of allogeneic hematopoietic stem cells (HSCs) from healthy donors constitutes a good therapeutic option for patients suffering from a number of inherited diseases, including severe immunodeficiencies, hemoglobinopathies, metabolic diseases, and some genetic instability disorders (1–3). Unfortunately, the proportion of patients having an HLA identical related donor is low, and the success of transplants from other donors is limited (4). Even in cases in which allogeneic transplantation can improve the clinical status of these patients, the potential side effects associated with this transplantation modality must be carefully considered. In this scenario, the treatment of genetic

Address reprint requests to: Juan A. Bueren, CIEMAT, Hematopoietic Gene Therapy Program, Av. Complutense #22, Madrid, 28040, Spain. Phone: (⫹34) (91) 346-6518; FAX: (⫹34) (91) 346-6484; E-mail: juan. [email protected]

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hematopoietic diseases by means of gene transfer strategies is considered a new therapeutic option that may overcome the main problems associated with allogeneic transplantation. As will be described throughout this review, the very primitive HSCs constitute a very good target population for the gene therapy of inherited diseases. One of the advantages of HSCs derives from the fact that this population can be readily harvested from accessible hematopoietic tissues and then concentrated using procedures routinely used in the clinical setting (5). However, the main advantage of using HSCs as a target of a gene therapy treatment relies on the fact that this population is capable of undergoing self-renewing divisions and also of differentiating into mature cells of different lineages. Transducing the true HSCs, therefore, facilitates the generation of genetically modified peripheral blood (PB) T and B-lymphocytes, natural killer cells, monocytes, granulocytes, eosinophils, basophils, megakaryocytes, and macrophages, including hepatic and alveolar macrophages, dermal Langerhans cells (6), microglial cells of the brain (7), and osteoclasts (8). All these cell types could also participate as secreting pumps for the delivery of therapeutic

쑖 2004 IMSS. Published by Elsevier Inc.

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products that may facilitate the correction of non-hematological disorders. A number of advances have recently facilitated the efficient transduction of HSCs from experimental animals and most probably also the true human HSCs. In this respect, new oncoretroviral vectors with optimized long-terminal repeats and leader sequences have been generated (9), improving the in vivo expression of therapeutic transgenes both in immature precursors as well as in mature lymphoid and myeloid cells (10). New packaging cell lines capable of producing high titers of retroviral vectors with more stable envelopes have been developed (11). Also, new lentiviral vectors capable of transducing HSCs not subjected to active stimulation have been engineered (12–15).

approach, HSC multipotentiality was inferred from the observation of the same proviral insert in purified lymphoid and myeloid cells. In addition, the evolution of the different repopulating clones in a transplanted recipient provided a unique opportunity for investigating the dynamics of individual repopulating clones after transplantation (see Figure 1). These studies showed that transplanted hematopoiesis is highly dynamic during the early stages post-transplantation, while it is much more stable in the long term. Different models of clonal succession and selection of the clonal repertoire have been reported in the mouse hematopoietic system. Significantly, these studies also showed that a single primitive HSC can be sufficient to sustain a normal and longterm hematopoietic function (16–20).

First Evidence of Gene Transfer in Mouse HSCs The efficient transduction of the true HSCs is one of the prerequisites to ensure that a therapeutic gene can be expressed throughout the life span of a patient. Early studies demonstrated that recombinant retroviruses can stably transduce the mouse HSCs and showed the efficacy of these vectors as genetic markers of individually transduced HSCs (16–19). Moreover, the extensive application of this technology in mouse experimental models allowed investigators to elucidate the hallmark properties of the HSCs (17–21). In these studies, the presence of an HSC clone was inferred by detection of unique proviral inserts on lymphoid and myeloid populations obtained from a recipient animal that was transplanted with the transduced graft. Southern blots capable of revealing the integration sites of proviruses in the genomic DNA have been routinely used for investigating the properties of the HSCs, defined as hematopoietic repopulating cells (HRCs) in these assays (Figure 1). Using this gene marking

Gene Marking Studies in Human HSCs Once the possibility of transducing mouse HSCs and human hematopoietic progenitors assessed by in vitro cultures was demonstrated, the first clinical trials aimed at gene marking and gene therapy of human diseases were proposed. Initial trials of gene marking aimed to investigate the origin of relapse of cancer patients subjected to autologous bone marrow (BM) transplantation. In these trials, a fraction of the human BM of patients with hematological or solid tumors was transduced with retroviral vectors used as genetic markers. After transplantation, the researchers followed the marker gene both in the normal hematopoietic cells that repopulated the patients (22), as well as in tumor cells in relapse cases (23–25). These studies demonstrated the presence of malignant cells bearing the genetic marker, indicating that remission BM contained cells that contributed to the relapse (23–25). In addition, these authors showed that

Figure 1. Retroviral marking of hematopoietic stem cells and Southern blot analysis of provirus integration sites. HSCs are transduced ex vivo with retroviral vectors and transplanted into recipient mice. Genomic DNA extracted from recipient hematopoietic tissues is digested with restriction enzymes and hybridized with a probe of the transgene. Each band corresponds to a unique retroviral integration site, indicating the clonal descendants of one repopulating cell.

HSC Therapy of Inherited Diseases

transduced BM was capable of reconstituting the hematopoiesis of recipients, indicating the possibility of transducing the HRCs of human patients (22,24). In most studies, however, the efficiency for transducing human HRCs was low (24,26–28). One issue that hindered the development of protocols aimed at targeting of the human HSCs was the absence of an experimental assay capable of assessing the functionality of these rare cells. Several xenotransplantation models are now available for evaluating the in vivo repopulating properties of human hematopoietic grafts (see Figure 2). The experimental model most widely used for these purposes relies upon the non-obese, diabeticsevere combined immunodeficiency (NOD/SCID) mouse (29,30). The hematopoiesis of these animals can be largely replaced by human hematopoietic cells (predominantly myeloid and B cells) after irradiation and transplantation of a human graft. Transplantation of limiting numbers of human hematopoietic cells into these animals facilitated the definition of a human HSC candidate, known as the SCID-repopulating cell (SRC) (30). Criteria to define this precursor cell were based on its capability to give rise to human lymphoid and myeloid cells in the BM of engrafted mice. Significantly, the studies of Larochelle et al. showed that the experimental conditions capable of transducing efficiently CFC and LTCIC progenitors only transduced a minority of the SRCs (30), data that were very similar to those obtained in a number

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of the early human clinical trials. Other immunodeficient mouse models (31,32) and models based on the transplantation of preimmune sheep (33) have rendered invaluable information with regard to the functionality of human HSCs (34–36). Several factors likely contributed to the inefficient gene transfer of the human HRCs. Given that most HSCs are quiescent under physiological stationary conditions and therefore refractory to transduction by oncoretroviral vectors, efforts were focused on identifying the cytokine combinations that may activate the HSC cell cycle without compromising their functionality (37–40). Another important advance in the development of more efficient gene transfer protocols was based on the production of molecules from the extracellular matrix capable of co-localizing the hematopoietic target cell and the retroviral particle (30,41,42). Improvements in retroviral transduction technologies allowed investigators to develop more efficient procedures for the gene transfer of the human HRCs and to investigate the clonal dynamics of these precursors. Nolta et al. (34) showed the presence of T-cell and myeloid clones containing identical proviral integration sites, demonstrating the transduction of human HRCs capable of differentiating into both lineages in bnx mice. Using an inverse PCR approach, these authors observed an oligoclonal repopulation pattern of human engrafted cells.

Figure 2. Evolution of the experimental assays used for characterization of human hematopoietic stem cells (HSCs). Initial assays were based on evaluation of the in vitro clonogenic potential of human hematopoietic samples. Non-clonogenic assays based on the long-term culture of human samples were used subsequently as more reliable indicators of the human HSCs in vitro. In vivo repopulation assays based on transplantation of immunodeficient mice or preimmune sheep are used at present as experimental assays that more closely reflect the functionality of human HSCs.

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In an attempt to develop efficient procedures of gene transfer in human HSCs, we purified and transduced CD34⫹ cord blood cells using optimized conditions of hematopoietic growth factors. Transduced samples were transplanted into NOD/SCID mice and BM from transplanted recipients was periodically aspirated to evaluate the repopulating potential of transduced grafts. Periodic analyses of recipient BM showed a sustained engraftment of human hematopoietic cells expressing the marker transgene (40). The high and stable engraftments achieved in our NOD/SCID mice allowed us to confirm by Southern blotting the predominant oligoclonal reconstitution pattern of human hematopoiesis in a xenogenic transplantation model (40). The sequential analysis of the provirus insertion sites in large series of transplanted NOD/SCID mice showed that some human HRCs only appeared at early times after the transplant, while others only appeared at late times and others persisted from the early stages to the late stages posttransplantation. These studies thus indicated the heterogeneous nature of the human repopulating clones that engrafted the NOD/SCID mice (43,44). While all these data documented the existence of short-term SRCs in the human hematopoietic tissue, designation and identification of long-term SRCs is limited by the relatively brief period of observation permitted by the NOD/SCID system. The tentative designation of persistent clones, detected after 3 months post-transplant as long-term HRCs, is based on a murine experimental model where clonal stability often occurs after 3–4 months. However, extrapolation of the SRC to the human HSC must be cautious because the overall hematopoietic demand in a small host should be largely reduced compared to that which takes place in a human recipient. Also of significance was the fact that in most of these mice, primitive CD34⫹ cells as well as lymphoid and myeloid cells expressed the transgene for the life span of these transplanted mice (3–4 months). The percentage of marked cells within each lineage was approximately equal, providing strong evidence that individual marked SRCs have multilineage repopulation capacity (43,44). Very recently, the efficiency of transduction of a baboon SRC has been compared to that observed in baboons subjected to autologous transplantation. Gene-marking levels observed in the baboons long term after transplantation were significantly lower compared to values obtained in NOD/ SCID mice transplanted with the same grafts (45). These authors thus proposed that a distinct, more committed, and more easily transducible population is responsible for the NOD/SCID repopulation, compared to the one that reconstituted the baboons in the long term. These findings emphasize that caution must be taken when experimental assays are used to predict the functionality of the human HSC. Studies conducted in non-human primate models have offered conflicting results regarding the pattern of clonal repopulation in these animals. Using a highly sensitive inverse PCR technique, Kim et al. detected 48 unique insertion sequences long term after transplantation. In addition,

these authors showed that multiple clones contributed to hematopoiesis at two or more time points (46). In a more recent work, the clonal composition of retrovirus-marked PB leukocytes was analyzed by a novel direct genomic sequencing technique that allowed identification of vector insertion sites. More than 80 contributing long-term hematopoietic clones were identified in individual rhesus macaque PB transplant recipients and ⬎25 different clones in a baboon marrow transplant (47). This study provided direct molecular evidence for a polyclonal, multilineage, and sustained contribution of individual stem cells to primate hematopoiesis. In contrast to these observations, it has been recently reported in rhesus recipients exhibiting high levels of reconstitution by genetically transduced cells that a single repopulating clone accounted for the majority of the hematopoietic reconstitution of the animal (48). In humans, a clinical trial based on the transfer of the adenosine deaminase (ADA) gene into umbilical cord CD34⫹ cells showed that ADA⫹ PB T lymphocytes persisted in patients in the long term (49,50). In a recent paper, the authors used linear amplification-mediated PCR (LAM-PCR) to identify clonal vector proviral integrants (51). In one patient, a single vector integrant was predominant in T lymphocytes during most of the 8-year period analyzed. T-cell clones with the predominant integrant showed multiple patterns of T-cell receptor gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time.

Treatment of Inherited Diseases Using Genetically Modified HSCs Diseases that can be ameliorated as a result of an allogeneic transplant are of particular interest in the context of HSC gene therapy. Among these, life-threatening recessive diseases not having an efficient conventional therapy have been selected for the development of the first gene therapy trials. Considering the technical limitations of current tools of gene transfer, diseases in which a limited number of genetically corrected cells could result in clinical benefit for the patient and diseases in which a regulated expression of the transgene is not required are of particular interest. The possibility that genetically corrected HSCs develop a proliferation advantage over the non-transduced HSCs has been considered a critical issue for the success of gene therapy trials. Whether or not this process can be expected as a result of a gene therapy approach can be inferred from the natural evolution of the disease. In this respect, a phenomenon known as somatic mosaicism has been observed in a number of recessive hereditary disorders, including SCID-Xl (52), ADA deficiency (53,54), Bloom’s syndrome (55), Wiskott-Aldrich (56), and Fanconi anemia (57–59), some of which have been efficiently treated with gene therapy. If a gene mutation causes a defect in cell survival

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or proliferation, repopulating cells that develop a spontaneous correction of the mutation could progressively predominate over the background of mutated cells. In the case of Fanconi anemia, a significant proportion of patients showed evidence of somatic mosaicism in PB (57). This natural form of gene therapy is associated with a remarkable expansion of the corrected cells, most frequently in T- and B-lymphocytes. In some patients, a spontaneous recovery of both the lymphoid and myeloid lineages has been observed, suggesting a natural genetic correction in a HSC clone (57– 59). On the basis of these observations, it is believed that at least in the case of diseases with evidence of somatic mosaicism, gene therapy protocols capable of complementing the genetic defect in a relatively reduced number of HSCs may result in the restoration of the hematological defect. Using a mouse model of Fanconi anemia, we showed an in vitro proliferation advantage of genetically complemented cells over Fanconi anemia-deficient cells (60). Other authors confirmed this advantage in in vivo models (61), supporting the idea that genetically corrected cells would have a selective advantage in the patient. The relevance of selective advantage in clinical gene therapy has been clearly documented in two immunodeficiencies successfully treated by gene therapy, SCID-X1 and ADA. In the case of SCID-X1 patients, the selective advantage phenomenon is supported by observations showing the spontaneous genetic correction of T cells that provide protection from infections in vivo (62).

Advances in the Gene Therapy of Inherited Immunodeficiencies Hematopoietic gene therapy applied to the treatment of immunodeficiency diseases has offered important new results during recent years. Both the first unquestionable clinical benefits and also severe side effects have been recently described. Regarding ADA immunodeficiency, ADA⫺ patients lacking an appropriate donor are conventionally treated by enzyme replacement strategies with pegylated ADA (PEGADA). This is an expensive treatment that needs to be continued throughout the life span of the patient. Unfortunately, PEG-ADA is not efficient for restoring permanently and efficiently the immunity of all these patients, who therefore require an alternative therapeutic option. The genetic treatment of ADA⫺ patients constituted the first attempt to cure an inherited disease by means of a gene therapy approach. Blaese and colleagues began a clinical trial for the treatment of ADA immunodeficiency in 1990 using autologous T cells that were transduced ex vivo with a retroviral vector encoding the human ADA minigene (63). The first two patients enrolled in the protocol were infused with 1011 ADA-transduced PB T lymphocytes that were administered in 11 and 12 infusions, respectively, during a period of 2

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years. Concomitant with these infusions, administration of PEG-ADA was reduced to approximately one half. The persistence of the integrated vector as well as the expression of the ADA gene was observed from the first infusion of transduced lymphocytes, resulting in a significant improvement in their immune functions. Twelve years after the gene therapy treatment, both patients still required a low dose of PEG-ADA. In one patient, ca. 20% of the lymphocytes still expressed the exogenous ADA gene, while in the remaining patient a very low proportion of transduced lymphocytes (⬍0.1%) contained the transgene (64). This difference was mainly attributed to differences in the efficiencies of transduction and total number of infused T cells, as well as in the development of antibodies against components of the retroviral envelope and the fetal calf serum used for the ex vivo manipulation of the lymphocytes. Longterm follow-up of these two genetically treated patients provides invaluable information regarding the longevity and functionality of PB T-lymphocytes transduced with therapeutic retroviral vectors. Additionally, this study also provides very useful information concerning the safety of the gene therapy protocol. Transduced BM cells have also been used for the gene therapy of ADA patients either as a sole source of transduced cells or in combination with transduced T lymphocytes. In 1995, Bordignon and colleagues published the results of a trial based on the combined infusion of transduced BM and PB T lymphocytes (65). Two different retroviral vectors encoding the human ADA were used for the transduction of these populations, aiming to trace their progeny in the infused recipient. A total of nine and five infusions were administered in 24 and 10 months, respectively, to the patients. This therapy resulted in the normalization of the immune repertoire and restoration of the cellular immunity of the patients. Two years after initiation of the gene therapy treatment, the presence of transduced T lymphocytes, BM cells, and granulocytes was observed. In addition, the expression of the ADA transgene was demonstrated in these cells. Notably, during the period of administration of the transduced populations, ADA⫹ T cells of the patients consistently contained the marker provirus that was inserted into the infused T-cell population. However, the discontinuation of infusions led to a predominance of ADA⫹ T cells derived from the transduced BM population. The absolute clinical impact of this gene therapy trial was difficult to assess because of the concomitant administration of PEG-ADA to the patients. However, in a series of six ADA⫺ patients receiving transduced T lymphocytes, one patient showed compelling conditions for the discontinuation of the enzyme therapy (66). During 3 months, the dose of PEG-ADA given to this patient was lowered until complete discontinuation. At the same time, the patient received an intensified infusion of transduced lymphocytes. This treatment led to a selective growth advantage of transduced lymphocytes expressing the ADA gene, reaching nearly 100% of lymphocytes in

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PB. In addition, an increase in total CD3⫹ counts and intracellular ADA activity of PB lymphocytes was observed. In addition, full restoration of T-cell functions was observed, accompanied by normal proliferative responses and a polyclonal repertoire of T cells. In 1993, a trial of gene transfer of ADA into autologous CD34⫹ cells was begun by the group of Kohn (50). The results observed in this trial also indicated a long-term engraftment of transduced HRCs. However, in this case the cessation of PEG-ADA administration led to a decline in the immune function of the patients. Differences in the regulatory sequences of vectors used in the protocols of Kohn and Bordignon as well as in the target population and total number of infused cells could account for the discrepancies between both studies. A further protocol showed the relevance of nonmyeloablative conditioning as a procedure to facilitate the engraftment of transduced BM CD34⫹ cells in two patients for whom enzyme replacement therapy was not available (67). In these two patients, a sustained engraftment of transduced HRCs was observed. In both patients, increased leukocyte counts as well as improved immune functions and decreased toxic metabolites were observed, showing the efficacy of ADA gene therapy based on the transplantation of transduced CD34⫹ cells in patients subjected to submyeloablative conditioning. A particular impact in the gene therapy of inherited diseases was obtained by the trial developed in the Necker

Hospital of Paris for the treatment of SCID-X1, because it offered the first unquestionable evidence of clinical benefit as a result of a gene transfer protocol. Fisher, Cavazzana-Calvo, Hacein-Bey, and co-workers cured patients with this SCID disorder that is generally fatal in the first year of life with the use of gene therapy. SCID-X1 is caused by mutations in the X-linked gene IL2RG that encodes the common γchain (γc) of the lymphocyte receptors for interleukin-2 and other cytokines (68). Initially, this group transduced autologous BM CD34⫹ cells from two SCID-X1 patients aged 11 and 8 months, respectively. After transduction, a total of 19 and 17 million CD34⫹ cells, respectively, were infused into the patients in the absence of any conditioning (Figure 3). As early as 15 days after infusion, cells carrying the γc transgene were detectable by PCR. The transgene persisted in the long term and T, B, and NK counts of the patients became comparable to those observed in age-matched controls (69). The observations of this study were confirmed in a larger series of patients analyzed over a long-term period after the gene transfer. Of the first five children (70) and the six included later and treated with the gene therapy protocol, nine have a repaired immune system and have been living at home for 3.5 years. Safety Issues in Gene Therapy Although gene therapy has evolved as a therapeutic alternative for a number of inherited diseases, the risks associated

Figure 3. Scheme of the clinical trial used for the gene therapy of immunodeficient SCID-X1 patients. Bone marrow cells from the patients were harvested and CD34⫹ stimulated cells were transduced with retroviral vectors encoding the γc chain in the presence of fibronectin and protamine sulfate. Transduced samples were then infused into the patients in the absence of previous conditioning (see Reference 69).

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with use of integrative retroviral vectors have also been shown during these years. In experimental models, the group of Baum described the generation of a leukemia as a result of a retroviral transduction of mouse BM cells that were subjected to two series of transplantation in irradiated recipients (71). Although primary recipients did well for 28 weeks, secondary recipients developed a hematopoietic disorder within 22 weeks. The development of the disease was associated with an insertional mutagenesis event, given that the integrated provirus was inserted into the vicinity of, and deregulated the expression of, the Evi1 (ecotropic viral integration site-1) gene. The authors also suggested a role of the marker transgene—a mutant of the nerve growth factor receptor lacking the intracellular domain of the protein, ∆LNGFR—in leukemia development. This marker transgene, however, has been used in other experimental and clinical studies without evidence of side effects (72). In the SCID-X1 clinical trial (69), two lymphocytic leukemias have been observed in 2 of 11 patients subjected to the gene therapy protocol, both associated with an insertional mutagenesis event (73,74). Surprisingly, in both patients the vector inserted itself near the same gene, LMO2 (73–75), whose deregulated expression has been previously associated with T-cell leukemia (76). A third case of retroviral insertion in the same LMO2 gene has been detected in a third patient, although this patient is healthy at the present time (77). As soon as the first malignancy was detected, French investigators halted their clinical trial and reported the observed adverse side effect to National and International Regulatory Agencies. These events caused the U.S. National Institutes of Health’s Recombinant DNA Advisory Committee (RAC) and the Food and Drug Administration (FDA) to urge approximately 90 other retroviral-targeting blood cell studies to consider a pause. After a period of discussion, the RAC concluded that resuming other trials could be justified if changes were made in the informed consent forms and monitoring plans. Moreover, the RAC suggested that SCID-X1 trial might continue for patients who had previously failed a standard transplant therapy (77). The observations of the French trial may support the idea that integration of retroviruses is much less random than predicted. Using the available information of the whole sequences of the human genome, it has been possible to define a pattern of integration sites to integrative vectors, transcriptionally active genes being the most susceptible to viral insertion (78,79). Despite these results, the observation that the same oncogene is involved in the two leukemias of the French trial strongly suggests a cooperative mechanism between the product of the LMO2 gene and the therapeutic γc gene. If this cooperation actually takes place, a very rare cell in which both the γc and the LMO2 genes are overexpressed may progressively expand in the recipient and finally result in a leukemic clone. Supporting the cooperative effect between the γc and LMO2 gene products is the observation that only in the SCID-X1 protocol was a leukemogenic effect produced as a result of the provirus insertion.

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In this respect, ADA⫺ patients treated with gene therapy up to 12 years ago are healthy, with PB lymphocytes carrying stable integrations of the transgene (50,51,63,64,66,67,80). Also, in the clinical trial developed for the control of the graft-vs.-host disease transduction of PB lymphocytes with retroviral vectors did not produce any serious adverse effect in the patients (72,81). Regardless of the mechanism accounting for the leukemogenesis of SCID-X1 patients, new improvements in current integrative vectors are required. Until gene replacement by means of homologous recombination (82–84) becomes clinically efficient, vectors including safety elements should be developed. In this respect, the introduction of a suicide gene such as HSV-tk—already shown to be efficient for the in vivo eradication of transduced cells (81)—constitutes an interesting procedure to control a potential leukemogenic event associated with gene transfer. Also, insertion of DNA sequences capable of isolating the expression of the transgene (85) and use of promoters only active in a restricted population of differentiated cells (86) may reduce the risks associated with insertional mutagenesis (87). Finally, according to the evolution of hematopoietic gene therapy strategies, it is clear that new preclinical studies not only aiming at demonstration of efficacy, but also of safety, are required. As occurred in other therapeutic options such as radiotherapy or chemotherapy, serious side effects have now been proven in the gene therapy of inherited diseases. As observed with these other therapeutic modalities, it seems that the balance between risks and benefits associated with gene therapy will be also highly positive. Future Perspectives of Stem Cells in Gene Therapy Recent years have been extremely active in the research of stem cell biology. In this respect, provocative studies showing the plastic potential of adult HSCs have been published (88–93). Although some data suggest that cell fusion may account for results originally interpreted as HSC transdifferentiation (94,95), data showing evident changes of differentiation have been observed in BM cells not subjected to cell fusion (96). This new capacity of the HSCs opens the possibility of transducing these cells with the aim of treating non-hematopoietic damaged tissues (97). Apart from adult HSCs, mesenchymal stem cells present in adult tissues may evolve as new critical targets of gene therapy. In this respect, the group of Verfaille has shown that a population known as mesenchymal adult progenitor cells (MAPCs) not only generates cells of the endodermal, mesodermal, and ectodermal tissues, but also integrates in the blastocyst and contributes to regeneration of the different tissues of the adult animal (98). As soon as the biology and manipulation of the MAPCs is better characterized, new applications based on the transduction of these cells with therapeutic genes will probably appear as a new approach for the gene therapy of inherited diseases.

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Figure 4. Scheme of a hypothetical gene therapy protocol involving the use of nuclear transfer technologies, generation of embryonic stem cells, and gene transfer. In the scheme, cells from a patient biopsy are obtained and their nuclei transferred into enucleated oocytes. In a second step, embryonic stem cells are generated and expanded stem cells are subjected to genetic correction. Finally, genetically corrected cells are differentiated toward the tissue of interest and then transplanted into the patient.

Finally, on the basis of advances in the knowledge of embryonic stem cells and of nuclear transfer technology these cells have also been considered as potential new targets for future gene therapy strategies. Studies showing the generation of multipotential embryonic stem cells as a result of the transfer of a mature nucleus into an enucleated oocyte have opened new perspectives in the field of gene therapy (99). By means of these new approaches, nuclei from mature cells of a patient would be transferred to recipient oocytes. The embryonic stem cells generated by this procedure could be genetically corrected either by gene insertion or gene replacement strategies (99) (Figure 4). Although several practical concerns currently limit the potentiality of this approach (i.e., risks associated with uncontrolled cell proliferation), many laboratories are at present working on this strategy, and most probably new developments based on the genetic modification of embryonic stem cells will soon appear in the scientific literature.

Acknowledgments Gene therapy studies conducted at the CIEMAT have received grant support from the Commission of the European Communities, the Comisio´n Interministerial de Ciencia y Tecnologı´a, the Direccio´n General de Investigacio´n de la Comunidad de Madrid, and the Fundacio´n Marcelino Botı´n.

References 1. Parkman R. The application of bone marrow transplantation to the treatment of genetic diseases. Science 1986;232:1373–1378. 2. Krivit W, Peters C, Shapiro EG. Bone marrow transplantation as effective treatment of central nervous system disease in globoid cell leukodystrophy, metachromatic leukodystrophy, adrenoleukodystrophy, mannosidosis, fucosidosis, aspartylglucosaminuria, Hurler, MaroteauxLamy, and Sly syndromes, and Gaucher disease type III. Curr Opin Neurol 1999;12:167–176. 3. Chapel H, Geha R, Rosen F. Primary immunodeficiency diseases: an update. Clin Exp Immunol 2003;132:9–15. 4. Guardiola P, Pasquini R, Dokal I, Ortega JJ, van Weel-Sipman M, Marsh JC, Ball SE, Locatelli F, Vermylen C, Skinner R, Ljungman P, Miniero R, Shaw PJ, Souillet G, Michallet M, Bekassy AN, Krivan G, Di Bartolomeo P, Heilmann C, Zanesco L, Cahn JY, Arcese W, Bacigalupo A, Gluckman E. Outcome of 69 allogeneic stem cell transplantations for Fanconi anemia using HLA-matched unrelated donors: a study on behalf of the European Group for Blood and Marrow Transplantation. Blood 2000;95:422–429. 5. Gaipa G, Dassi M, Perseghin P, Venturi N, Corti P, Bonanomi S, Balduzzi A, Longoni D, Uderzo C, Biondi A, Masera G, Parini R, Bertagnolio B, Uziel G, Peters C, Rovelli A. Allogeneic bone marrow stem cell transplantation following CD34⫹ immunomagnetic enrichment in patients with inherited metabolic storage diseases. Bone Marrow Transplant 2003;31:857–860. 6. Mollah ZU, Aiba S, Manome H, Yoshino Y, Tagami H. Cord blood CD34⫹ cells differentiate into dermal dendritic cells in co-culture with cutaneous fibroblasts or stromal cells. J Invest Dermatol 2002;118: 450–460. 7. Krivit W, Sung JH, Shapiro EG, Lockman LA. Microglia: the effector cell for reconstitution of the central nervous system following bone marrow transplantation for lysosomal and peroxisomal storage diseases. Cell Transplant 1995;4:385–392.

HSC Therapy of Inherited Diseases 8. Matayoshi A, Brown C, DiPersio JF, Haug J, Abu-Amer Y, Liapis H, Kuestner R, Pacifici R. Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells. Proc Natl Acad Sci USA 1996;93:10785–10790. 9. Hildinger M, Abel KL, Ostertag W, Baum C. Design of 5′ untranslated sequences in retroviral vectors developed for medical use. J Virol 1999; 73:4083–4089. 10. Wahlers A, Kustikova O, Zipfel PF, Itoh K, Koester M, Heberlein C, Li Z, Schiedlmeier B, Skerka C, Fehse B, Baum C. Upstream conserved sequences of mouse leukemia viruses are important for high transgene expression in lymphoid and hematopoietic cells. Mol Ther 2002;6:313–320. 11. Porter CD, Collins MK, Tailor CS, Parkar MH, Cosset FL, Weiss RA, Takeuchi Y. Comparison of efficiency of infection of human gene therapy target cells via four different retroviral receptors. Hum Gene Ther 1996;7:913–919. 12. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998;72:8463–8471. 13. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998;72:9873–9880. 14. 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. 15. Guenechea G, Gan OI, Inamitsu T, Dorrell C, Pereira DS, Kelly M, Naldini L, Dick JE. Transduction of human CD34⫹ CD38⫺ bone marrow and cord blood-derived SCID-repopulating cells with thirdgeneration lentiviral vectors. Mol Ther 2000;1:566–573. 16. Dick JE, Magli MC, Huszar D, Phillips RA, Bernstein A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell 1985; 42:71–79. 17. Lemischka IR, Raulet DH, Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986;45:917–927. 18. Jordan CT, Lemischka IR. Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes Dev 1990;4:220–232. 19. Bernad A, Varas F, Gallego JM, Almendral JM, Bueren JA. Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene-transfer to murine lympho-haemopoietic stem cells. Br J Haematol 1994;87:6–17. 20. Varas F, Bernad A, Almendral JM, Bueren JA. Relevance of myeloablative conditioning in the engraftment of limiting numbers of normal and genetically marked lympho-hematopoietic stem cells. Bone Marrow Transplant 1996;18:981–989. 21. Varas F, Bernad A, Bueren JA. Granulocyte colony-stimulating factor mobilizes into peripheral blood the complete clonal repertoire of hematopoietic precursors residing in the bone marrow of mice. Blood 1996;88:2495–2501. 22. Brenner MK, Rill DR, Holladay MS, Heslop HE, Moen RC, Buschle M, Krance RA, Santana VM, Anderson WF, Ihle JN. Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients. Lancet 1993;342:1134–1137. 23. Brenner MK, Rill DR, Moen RC, Krance RA, Mirro J Jr, Anderson WF, Ihle JN. Gene-marking to trace origin of relapse after autologous bone-marrow transplantation. Lancet 1993;341:85–86. 24. Deisseroth AB, Zu Z, Claxton D, Hanania EG, Fu S, Ellerson D, Goldberg L, Thomas M, Janicek K, Anderson WF, Hester J, Korbling M, Durett A, Moen R, Berenson R, Heimfeld S, Hamer J, Calvert L, Tibbits P, Talpaz M, Kantarjian H, Champlin R, Reading C. Genetic marking shows that Ph⫹ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML. Blood 1994;83:3068–3076. 25. Rill DR, Santana VM, Roberts WM, Nilson T, Bowman LC, Krance RA, Heslop HE, Moen RC, Ihle JN, Brenner MK. Direct demonstration

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36. 37.

38.

39.

40.

597

that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 1994;84:380–383. Dunbar CE, Cottler-Fox M, O’Shaughnessy JA, Doren S, Carter C, Berenson R, Brown S, Moen RC, Greenblatt J, Stewart FM. Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation. Blood 1995;85:3048–3057. Cornetta K, Srour EF, Moore A, Davidson A, Broun ER, Hromas R, Moen RC, Morgan RA, Rubin L, Anderson WF, Hoffman R, Tricot G. Retroviral gene transfer in autologous bone marrow transplantation for adult acute leukemia. Hum Gene Ther 1996;7:1323–1329. Emmons RV, Doren S, Zujewski J, Cottler-Fox M, Carter CS, Hines K, O’Shaughnessy JA, Leitman SF, Greenblatt JJ, Cowan K, Dunbar CE. Retroviral gene transduction of adult peripheral blood or marrowderived CD34⫹ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo. Blood 1997;89:4040–4046. Shultz LD, Schweitzer PA, Christianson SW, Gott B, Birdsall-Maller I, Tennent B, McKenna S, Mobraaten L, Rajan TV, Greiner DL, Leitter EH. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol 1995;154:180–191. Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329–1337. Dao MA, Shah AJ, Crooks GM, Nolta JA. Engraftment and retroviral marking of CD34⫹ and CD34⫹CD38⫺ human hematopoietic progenitors assessed in immune-deficient mice. Blood 1998;91:1243–1255. Nolta JA, Hanley MB, Kohn DB. Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: analysis of gene transduction of long-lived progenitors. Blood 1994;83:3041–3051. Zanjani ED, Pallavicini MG, Ascensao JL, Flake AW, Langlois RG, Reitsma M, MacKintosh FR, Stutes D, Harrison MR, Tavassoli M. Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero. J Clin Invest 1992;89:1178–1188. Nolta JA, Dao MA, Wells S, Smogorzewska EM, Kohn DB. Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice. Proc Natl Acad Sci USA 1996;93:2414–2419. Kawashima I, Zanjani ED, Almaida-Porada G, Flake AW, Zeng H, Ogawa M. CD34⫹ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells. Blood 1996;87:4136–4142. Almeida-Porada G, Porada C, Zanjani ED. Adult stem cell plasticity and methods of detection. Rev Clin Exp Hematol 2001;5:26–41. Crooks GM, Kohn DB. Growth factors increase amphotropic retrovirus binding to human CD34⫹ bone marrow progenitor cells. Blood 1993; 82:3290–3297. van Beusechem VW, Bart-Baumeister JA, Hoogerbrugge PM, Valerio D. Influence of interleukin-3, interleukin-6, and stem cell factor on retroviral transduction of rhesus monkey CD34⫹ hematopoietic progenitor cells measured in vitro and in vivo. Gene Ther 1995;2:245–255. Guenechea G, Segovia JC, Albella B, Lamana M, Ramı´rez M, Regidor C, Ferna´ndez MN, Bueren JA. Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(⫹) cord blood cells. Blood 1999; 93:1097–1105. Barquinero J, Segovia JC, Ramı´rez M, Limo´n A, Guenechea G, Puig T, Briones J, Garcı´a J, Bueren JA. Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice. Blood 2000;95: 3085–3093.

598

Bueren et al. / Archives of Medical Research 34 (2003) 589–599

41. Moritz T, Patel VP, Williams DA. Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors. J Clin Invest 1994;93:1451–1457. 42. Hanenberg H, Xiao XL, Dilloo D, Hashino K, Kato I, Williams DA. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med 1996;2:876–882. 43. Guenechea G, Gan OI, Dorrell C, Dick JE. Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2001;2:75–82. 44. Dick JE, Guenechea G, Gan OI, Dorrell C. In vivo dynamics of human stem cell repopulation in NOD/SCID mice. Ann N Y Acad Sci 2001; 938:184–190. 45. Horn PA, Thomasson BM, Wood BL, Andrews RG, Morris JC, Kiem HP. Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice versus nonhuman primates. Blood 2003 (Epub, ahead of print). 46. Kim HJ, Tisdale JF, Wu T, Takatoku M, Sellers SE, Zickler P, Metzger ME, Agricola BA, Malley JD, Kato I, Donahue RE, Brown KE, Dunbar CE. Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates. Blood 2000; 96:1–8. 47. Schmidt M, Zickler P, Hoffmann G, Haas S, Wissler M, Muessig A, Tisdale JF, Kuramoto K, Andrews RG, Wu T, Kiem HP, Dunbar CE, Von Kalle C. Polyclonal long-term repopulating stem cell clones in a primate model. Blood 2002;100:2737–2743. 48. Kelly PF, Donahue RE, Vandergriff JA, Takatoku M, Bonifacino AC, Agricola BA, Metzger ME, Dunbar CE, Nienhuis AW, Vanin EF. Prolonged multilineage clonal hematopoiesis in a rhesus recipient of CD34 positive cells marked with a RD114 pseudotyped oncoretroviral vector. Blood Cells Mol Dis 2003;30:132–143. 49. Kohn DB, Weinberg KI, Nolta JA, Heiss LN, Lenarsky C, Crooks GM, Hanley ME, Annett G, Brooks JS, el-Khoureiy A. Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat Med 1995;1:1017–1023. 50. Kohn DB, Hershfield MS, Carbonaro D, Shigeoka A, Brooks J, Smogorzewska EM, Barsky LW, Chan R, Burotto F, Annett G, Nolta JA, Crooks G, Kapoor N, Elder M, Wara D, Bowen T, Madsen E, Snyder FF, Bastian J, Muul L, Blaese RM, Weinberg K, Parkman R. T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34⫹ cells in ADAdeficient SCID neonates. Nat Med 1998;4:775–780. 51. Schmidt M, Carbonaro DA, Speckmann C, Wissler M, Bohnsack J, Elder M, Aronow BJ, Nolta JA, Kohn DB, von Kalle C. Clonality analysis after retroviral-mediated gene transfer to CD34⫹ cells from the cord blood of ADA-deficient SCID neonates. Nat Med 2003;9:463–468. 52. Stephan V, Wahn V, Le Deist F, Dirksen U, Broker B, Muller-Fleckenstein I, Horneff G, Schroten H, Fischer A, de Saint Basile G. Atypical X-linked severe combined immunodeficiency due to possible spontaneous reversion of the genetic defect in T cells. N Engl J Med 1996; 335:1563–1567. 53. Arredondo-Vega FX, Santisteban I, Richard E, Bali P, Koleilat M, Loubser M, Al-Ghonaium A, Al-Helali M, Hershfield MS. Adenosine deaminase deficiency with mosaicism for a “second-site suppressor” of a splicing mutation: decline in revertant T lymphocytes during enzyme replacement therapy. Blood 2002;99:1005–1013. 54. Hirschhorn R, Yang DR, Puck JM, Huie ML, Jiang CK, Kurlandsky LE. Spontaneous in vivo reversion to normal of an inherited mutation in a patient with adenosine deaminase deficiency. Nat Genet 1996; 13:290–295. 55. Ellis NA, Ciocci S, German J. Back mutation can produce phenotype reversion in Bloom syndrome somatic cells. Hum Genet 2001;108: 167–173. 56. Wada T, Schurman SH, Otsu M, Garabedian EK, Ochs HD, Nelson DL, Candotti F. Somatic mosaicism in Wiskott-Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism. Proc Natl Acad Sci USA 2001;98:8697–8702.

57. Lo Ten Foe JR, Kwee ML, Rooimans MA, Oostra AB, Veerman AJ, van Weel M, Pauli RM, Shahidi NT, Dokal I, Roberts I, Altay C, Gluckman E, Gibson RA, Mathew CG, Arwert F, Joenje H. Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance. Eur J Hum Genet 1997;5:137–148. 58. Gregory JJ Jr, Wagner JE, Verlander PC, Levran O, Batish SD, Eide CR, Steffenhagen A, Hirsch B, Auerbach AD. Somatic mosaicism in Fanconi anemia: evidence of genotypic reversion in lymphohematopoietic stem cells. Proc Natl Acad Sci USA 2001;98:2532–2537. 59. Gross M, Hanenberg H, Lobitz S, Friedl R, Herterich S, Dietrich R, Gruhn B, Schindler D, Hoehn H. Reverse mosaicism in Fanconi anemia: natural gene therapy via molecular self-correction. Cytogenet Genome Res 2002;98:126–135. 60. Rio P, Segovia JC, Hanenberg H, Casado JA, Martı´nez J, Gottsche K, Cheng NC, Van de Vrugt HJ, Arwert F, Joenje H, Bueren JA. In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice. Blood 2002;100:2032–2039. 61. Galimi F, Noll M, Kanazawa Y, Lax T, Chen C, Grompe M, Verma IM. Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors. Blood 2002;100:2732–2736. 62. Bousso P, Wahn V, Douagi I, Horneff G, Pannetier C, Le Deist F, Zepp F, Niehues T, Kourilsky P, Fischer A, de Saint Basile G. Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor. Proc Natl Acad Sci USA 2000;97: 274–278. 63. Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, Anderson WF. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years. Science 1995;270:475–480. 64. Muul LM, Tuschong LM, Soenen SL, Jagadeesh GJ, Ramsey WJ, Long Z, Carter CS, Garabedian EK, Alleyne M, Brown M, Bernstein W, Schurman SH, Fleisher TA, Leitman SF, Dunbar CE, Blaese RM, Candotti F. Persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial. Blood 2003; 101:2563–2569. 65. Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, Mazzolari E, Maggioni D, Rossi C, Servida P, Ugazio AG, Mavilio F. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science 1995;270: 470–475. 66. Aiuti A, Vai S, Mortellaro A, Casorati G, Ficara F, Andolfi G, Ferrari G, Tabucchi A, Carlucci F, Ochs HD, Notarangelo LD, Roncarolo MG, Bordignon C. Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement. Nat Med 2002;8:423–425. 67. Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, Morecki S, Andolfi G, Tabucchi A, Carlucci F, Marinello E, Cattaneo F, Vai S, Servida P, Miniero R, Roncarolo MG, Bordignon C. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002;296:2410–2413. 68. Noguchi M, Nakamura Y, Russell SM, Ziegler SF, Tsang M, Cao X, Leonard WJ. Interleukin-2 receptor gamma chain: a functional component of the interleukin-7 receptor. Science 1993;262:1877–1880. 69. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000;288:669–672. 70. Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay J-P, Thrasher AJ, Wulffraat N, Sorensen R, Dupuis-Girod S, Fischer A, Davies EG, Kuis W, Leiva L, Cavazzana-Calvo M. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002;346:1185–1193. 71. Li Z, Dullmann J, Schiedlmeier B, Schmidt M, von Kalle C, Meyer J, Forster M, Stocking C, Wahlers A, Frank O, Ostertag W, Kuhlcke

HSC Therapy of Inherited Diseases

72.

73.

74. 75. 76.

77. 78.

79.

80.

81.

82. 83. 84.

85.

K, Eckert HG, Fehse B, Baum C. Murine leukemia induced by retroviral gene marking. Science 2002;296:497. Bonini C, Grez M, Traversari C, Ciceri F, Marktel S, Ferrari G, Dinauer M, Sadat M, Aiuti A, Deola S, Radrizzani M, Hagenbeek A, Apperley J, Ebeling S, Martens A, Kolb HJ, Weber M, Lotti F, Grande A, Weissinger E, Bueren JA, Lamana M, Falkenburg JHF, Heemskerk MHM, Austin T, Kornblau S, Marini F, Benati C, Magnani Z, Cazzaniga S, Toma S, Gallo-Stampino C, Introna M, Slavin S, Greenberg PD, Bregni M, Mavilio F, Bordignon C. Safety of retroviral gene marking with a truncated NGF receptor. Nat Med 2003;9:367–369. Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, Radford I, Villeval JL, Fraser CC, CavazzanaCalvo M, Fischer A. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255–256. Marshall E. Gene therapy. Second child in French trial is found to have leukemia. Science 2003;299:320. Marshall E. Gene therapy. What to do when clear success comes with an unclear risk? Science 2002;298:510–511. Larson RC, Lavenir I, Larson TA, Baer R, Warren AJ, Wadman I, Nottage K, Rabbitts TH. Protein dimerization between Lmo2 (Rbtn2) and Tal1 alters thymocyte. EMBO J 1996;15:1021–1027. Kaiser J. Gene therapy. RAC hears a plea for resuming trials, despite cancer risk. Science 2003;299:991. Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV1 integration in the human genome favors active genes and local. Cell 2002;110:521–529. 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. Bordignon C, Bonini C, Verzeletti S, Nobili N, Maggioni D, Traversari C, Giavazzi R, Servida P, Zappone E, Benazzi E. Transfer of the HSVtk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. Hum Gene Ther 1995;6:813–819. Bonini C, Ferrari G, Verzeletti S, Servida P, Zappone E, Ruggieri L, Ponzoni M, Rossini S, Mavilio F, Traversari C, Bordignon C. HSVTK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 1997;276:1719–1724. Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science 2003;300:764. Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science 2003;300:763. Ya´n˜ez RJ, Porter AC. Differential effects of Rad52p overexpression on gene targeting and extrachromosomal homologous recombination in a human cell line. Nucleic Acids Res 2002;30:740–748. Ramezani A, Hawley TS, Hawley RG. Performance- and safety-enhanced lentiviral vectors containing the human interferon-{beta} scaffold attachment region and the chicken {beta}-globin insulator. Blood 2003;101:4717–4724.

599

86. Follenzi A, Sabatino G, Lombardo A, Boccaccio C, Naldini L. Efficient gene delivery and targeted expression to hepatocytes in vivo by. Hum Gene Ther 2002;13:243–260. 87. Baum C, Dullmann J, Li Z, Fehse B, Meyer J, Williams DA, von Kalle C. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 2003;101:2099–2114. 88. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410:701–705. 89. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F. Muscle regeneration by bone marrowderived myogenic progenitors. Science 1998;279:1528–1530. 90. Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782. 91. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, Grompe M. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234. 92. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377. 93. Bjornson CR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999;283:534–537. 94. Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545. 95. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, Grompe M. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897– 901. 96. LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002;111:589–601. 97. Grove JE, Lutzko C, Priller J, Henegariu O, Theise ND, Kohn DB, Krause DS. Marrow-derived cells as vehicles for delivery of gene therapy to pulmonary epithelium. Am J Respir Cell Mol Biol 2002;27:645–651. 98. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, OrtizGonza´lez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418:41–49. 99. Rideout WM 3rd, Hochedlinger K, Kyba M, Daley GQ, Jaenisch R. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 2002;109:17–27.