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Peripheral blood stem cell and gene therapy
Baillière’s Clinical Haematology Vol. 12, Nos. 1/2, pp 129–138, 1999
10 Peripheral blood stem cell and gene therapy Marina Cavazzana-Calvo*
MD, PhD
Head of the Cell Therapy Lab E.T.S., INSERM U429 Hôpital Necker, 149 rue des Sevres 75015 Paris, France
Claude Bagnis
PhD
Senior Researcher Centre de Thérapie génique, Institu Paoli-Calmette, 232 Boulevard St Marguerite, 13009 Marseille, France
Patrice Mannoni
MD
Head of the Gene Therapy Lab Centre de Thérapie génique, Institu Paoli-Calmette, 232 Boulevard St Marguerite, 13009 Marseille, France
Alain Fischer
MD, PhD
Head INSERM U429 Hôpital Necker, 149 rue des Sevres 75015 Paris, France
Mobilized peripheral blood stem cells characterized by sustained re-populating ability could be optimal target cells for ex-vivo gene transfer. In spite of very attractive preliminary results obtained in the murine studies, theraputically efficient gene transfer and expression in human targeted cells must be proven. In recent years, effort has been spent on the identification of factors limiting gene transfer efficiency of haematopoietic stem cells. Increasing knowledge concerning haematopoiesis and gene transfer has helped in identifying a number of limiting factors. These factors as well as the strategies that showed increased retroviral infection of haematopoietic stem cells will be discussed. Finally, the results of the clinical trials will be reported. Key word: retrovirus; human stem cells; clinical trials of gene therapy; peripheral blood stem cells.
Gene transfer by viral vectors into human haematopoietic cells is a new approach to the treatment of a variety of genetic and acquired human diseases. Initially, the major obstacles to the clinical application of gene transfer into haematopoietic progenitor/stem cells were that few such cells were available and that they were poorly identified phenotypically and relatively refractory to infection by vectors. Haematopoietic cytokines have now been shown to mobilize large numbers of primitive haematopoietic cells (CD34+) into peripheral blood (PB) of mice, primates and humans.1,2 These cells have sustained re-populating ability in the murine model, and have been successfully used as a source of long-term re-populating cells in human autologous and allogeneic transplantation.3 These CD34+ cells can easily be purified * Author for correspondence. 1521–6926/99/010129 + 10 $12.00/00
© 1999, Harcourt Publishers Ltd.
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from PB using immunological methods and can be manipulated in vivo and/or ex vivo to improve gene transfer. The aim of this article is to review strategies used to increase gene transfer efficiency in peripheral blood stem cell (PBSC) and briefly describe the most widely used vectors. Pre-clinical and clinical studies based on the use of transduced PBSC are presented. The majority of these studies involved retrovirusmediated gene transfer and this technique will be considered in more detail. CHOICE OF VECTORS AND PRODUCING CELL LINES Gene delivery vehicles for gene therapy can be either viral or non-viral. Diverse viral delivery vectors are under investigation, all of which are naturally able to infect mammalian cells: retrovirus, adenovirus, adeno-associated virus, herpes-simplex virus, vaccinia virus, poliovirus, baculovirus and Sindbis virus. Some of them have been extensively developed so as to integrate into genomic DNA (i.e. adeno-associated virus and retrovirus). Adeno-associated virus (AAV) Efficient gene transfer into CD34+ cells using AAV has been reported.4,5 The mechanisms of integration, efficiency of gene transfer (and identification of an efficient packaging cell line), integration and expression in dividing and non-dividing haematopoietic cells remain to be clearly characterized.6 It will be especially interesting to learn whether the site-specific integration of AAV into the AAVS1 site on chromosome 19q13.3-qter will provide any advantage for gene transfer and gene therapy purposes.7 Despite the limitations in the size of transgene inserts that can be used (no more than 4 kilobases), and difficulties in preparing a high-titre supernatant devoid of contaminant adenovirus particles, it is likely that these vectors will be developed to allow transduction of haematopoietic stem cells. Transfer of the lacZ gene on AAV-derived vectors into haematopoietic cells has already been reported, but long-term expression of this gene in transduced haematopoietic cell remains undocumented.5,8,9 Adenoviral vectors Adenoviral vectors present some advantages over other vectors for gene delivery to haematopoietic precursor cells. In particular, target cells can be quiescent, their mechanism for cell entry is dependent on integrins and on the recently described common receptor for coxsackie B virus and adenovirus (CAR)10 expressed by CD34+ cells, and finally they pass efficiently from the cell surface to the nucleus. The major disadvantage is that gene expression mediated by adenoviral vectors is only transient because the epichromosome of the transgene is not duplicated during cell division. Moreover, the infection efficiency of adenoviral-vectors differs between various haematopoietic lineages and is high only for megakaryocytic and dendritic cells.11 As PBSC contain a large number of dendritic precursor cells this vector can be used to deliver tumoural antigens to ex vivo differentiated dendritic cells.12,13 Adenoviral gene transfer for therapeutic purposes may therefore be most effective if mature postmitotic precursor cells are targeted.14 The transient expression of therapeutic transgenes on adenoviruses in terminally differentiated haematopoietic cells may be used for alleviating complications of genetic disorders such as haemophilia.
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Retroviral vectors Murine retroviral vectors are the most widely used delivery systems and the most powerful tools for genomic integration of the transgene.15 Natural or artificial retroviral backbones are being developed that are more sophisticated than the Moloney murine leukaemia virus (Mo-Mu LV)-derived vectors and NIH-3T3-derived packaging cell lines currently in use. Few of these defective vectors, such as GN1a and LNL616, or MFG-derivatives17 have been used for gene marking or therapeutical clinical trials. Vectors derived from murine retroviral backbones other than Mo-MuLV are being tested for reporter gene expression in haematopoietic cells.18,19 Recently, retroviral vectors with improved cell transduction properties have been described. They include a chimeric viral backbone composed of retroviral elements of different origins, and pseudotyped particles expressing non-retroviral envelope proteins (VSV-G) or the gibbon ape leukaemia virus (GALV) envelope.20,21 Producing/packaging cell lines of both murine and human origin giving high virus titres are being developed.22 No clear comparative data have been reported about the relative ability of different producing cell lines to transduce haematopoietic cells, whereas such data are available for retroviral vectors.23 Factors limiting the use of murine retroviral vectors are that target cells must be actively cycling at the time of insertion and the variability of long-term expression of the transgene according to the differentiation and activation status of transduced cells. New vector designs, the modification of the hypermethylation status of the transgene or regulatory elements24, construction of inducible or specific promoters for these vectors25 and combined expression of selection and therapeutic genes may improve both long-term expression and control of the expression of the transgene. HIV-derived and lentivirus-derived vectors able to transduce quiescent cells are also promising tools for improving retroviral vector efficiency in non-dividing cells.26
IN VIVO AND EX VIVO MANIPULATION OF HUMAN STEM CELLS The most important reconstituting cell populations in the human haematopoietic system express the CD34 surface antigen but not lineage differentiated markers (lin–). Numerous in vitro and in vivo studies have described retroviral infection and transduction of immature haematopoietic progenitors and the long-term maintenance of provirus in cells arising from transduced immature cells. Stem cells transduction is a challenging problem as integration occurs only in cycling cells and most of the primitive cells are in the resting phase. Various strategies have recently been used to increase the transduction efficiency of HSC. In vivo manipulation of HSC prior to cell harvesting and transduction may improve retroviral gene transfer by promoting cell cycling or by increasing receptor density. This approach has been successfully used in the murine model by the administration of 5-fluorouracil (5-FU) to mice 2–4 days prior to harvesting of bone marrow.27,28 Gene transfer efficiencies similar to that obtained in the murine model have also been obtained in the rhesus model with or without the pre-harvest administration of 5-FU.29,30 Randall and Weissman31 analysed some of the physical and functional changes in the stem cell population after 5-FU treatment. 5-FU treatment itself causes a gradual loss
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of c-kit expression on the lineage negative, Sca-1-positive population concomitant with the induction of Mac-1 expression. These changes correlate with the induction of the cell cycle in this stem cell population in response to the loss of haematopoietic progenitors. The use of a similar protocol in 11 myeloma patients pre-treated with 5-FU at a clinically tolerable dose (i.e. 15 mg/kg/day for 3 days) did not, however, result in efficient gene transfer to re-populating cells.32,33 The patients were followed for at least 1 year after autologous transplantation of retrovirally-transduced BM and PB CD34enriched cells. The levels of gene marking were very low: only 1 : 1000 to 1 : 10 000 cells were positive for the neomycin resistance gene (Neo). Better results have been obtained in a paediatric study34 where autologous BM cells from children undergoing transplantation for acute leukaemia or neuroblastoma were transduced in vitro, prior to cryopreservation, by the same retroviral vector. An average of 5% of marrow progenitors and PB mononuclear cells were marked at 12–18 months post-transplantation. Three major factors could explain these divergent results: the chemotherapy regimen administered just before the harvest of BM cells, the age of patients and the differencies in the transduction protocol. In vivo pre-treatment of patients: chemotherapy or cytokines? The type of chemotherapy administered before collection and ex-vivo transduction of bone marrow cells greatly influences the proportion of marked progenitors. Brenner et al34 harvested BM cells after completion of intensive marrow ablative chemotherapy. Patients with acute myeloid leukaemia (AML, 12 patients) were treated with 2-chlorodeoxyadenosine and then three cycles of daunorubicine, cytosine arabinoside and etoposide. Patients with neuroblastoma received six cycles of chemotherapy that included cyclophosphamide, doxorubicin, etoposide and cisplatin. The bone marrow was harvested when peripheral blood neutrophil counts rose above 1000/ml. The low-dose chemotherapy used for mobilization is not sufficient to enrich the PBSC in very primitive haematopoietic cells and to improve the transduction rate. Nevertheless, high-dose chemotherapy cannot be safely used in patients affected by hereditary diseases, human immunodeficiency virus infection or other non-malignant diseases. For such cases stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) could be administered in vivo rather than chemotherapy. The combination of SCF and G-CSF increases the number of PBSC in murine, canine and primate models and in human clinical trials.35–38 Moreover, these two cytokines, administered together, were shown to increase significantly the number of primitive cells present in BM within 2–3 weeks. These in vivo primed haematopoietic stem cells seem to be a better target for retroviral gene transfer than steady-state BM cells.39 The higher amphotropic envelope receptor density on most primitive cells may explain these findings.40 Age of patients and transduction protocol Important ontogeny-associated changes in the cytokine responses of primitive human haematopoietic cells41,42 have been recently described and should be considered when defining ex-vivo transduction protocols. The use of cytokines in the transduction protocol can impair the long-term re-populating ability of the PBSC, and these changes are tightly linked with the source of haematopoietic stem cells (i.e. cord blood, steadystate bone marrow or mobilized PBSC from young children or adults).
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Two haematopoietic cytokines exert a key role in the in vitro expansion of longterm culture-initiating cells (LTC-IC): FLT3-L and thrombopoietin (TPO).43,44 FLT3-L preserves, at least in part, the ability of human CD34 progenitor cells to sustain longterm haematopoiesis, and TPO seems to exert an anti-apoptotic effect on activated HSC. It would be useful to test the effects of these cytokines in pre-clinical studies. There is a controversy on the usefulness of autologous or heterologous stroma for the infection of more immature progenitor cells. Although transduction of murine haematopoietic stem cells with retroviral vectors has been demonstrated by a number of laboratories using direct co-culture of the target bone marrow cells with the virus packaging cell line, this methodology may have significant drawbacks for use in human trials because of safety and quality considerations.45–48 The mechanism underlying this promoting effect of stroma on retroviral infection is unclear, but haematopoietic cell proliferation and differentiation is physiologically regulated when they are in direct contact with cells of the haematopoietic micro-environment.49,50 This environment includes cytokines, stromal cells and a complex extracellular matrix (ECM) made up, in particular, of a variety of adhesion molecules. ECM molecules such as laminin, collagen thrombospondin, proteoglycans, glycosamino-glycans and fibronectin constitute anchorage sites for both haematopoietic cells and growth factors.51,52 Fibronectin (FN) is abundantly expressed in the bone marrow micro-environment and has been studied as a prototype molecule in cell–cell as well as in cell–matrix interactions.53,54 Fibronectin participates in cell adhesion through at least three cell binding sites: the cell-binding domain (CBD) mediates adhesion via the integrin VLA-5; the heparin binding domain interacts via cell surface proteoglycan molecules, and the CS1 sequence mediates adhesion via the integrin VLA-4. These two integrins are both present on CD34+ cells; retroviral particles bind directly to the heparin-binding domain of FN. A number of groups has recently reported an improvement in gene transfer efficiency by the use of FN-coated plastic dishes due, at least in part, to the co-localization of retrovirus and target cells on the same molecule.55,56 Therefore, the use of FN for ex-vivo expansion of CD34+ cells57,58 and for gene transfer59 could improve the clinical results. PBSC seem to be more difficult to transduce than steady-state bone marrow cells and to require longer stimulation by growth factors. Efficient gene transfer into mobilized CD34+CD38– requires pre-stimulation for 40 hours in the presence of SCF, TPO and G-CSF followed by 8 hours’ infection. It is thus evident that each gene transfer protocol must be adapted according to a number of factors, particularly the source of stem cells, donor age, cytokine combinations and in vivo pre-treatment of patients. The reciprocal influence of these factors on the gene transfer efficiency has not been elucidated, and a knowledge of these interactions should help to improve future clinical results. Work on the possibilities of expanding CD34+/CD38– stem cells ex-vivo has been illuminating.60–62 CLINICAL APPLICATIONS Hereditary diseases Hereditary diseases caused by monogenic alterations have long been considered as a possible experimental field for gene therapy. Adenosine deaminase (ADA) deficiency was the first inherited disease for which human gene therapy was used.63–65 Four years
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after the treatment of three newborns with ADA deficiency by transduced autologous umbilical cord blood CD34+ cells, it is worth noting the continuing presence of peripheral blood leukocytes containing the transferred ADA gene, suggesting that stem cells were transduced and engrafted without any toxic effect. The selective increase of transduced T-cell frequency after PEG-ADA cessation shows that there is a selective advantage for this cell line, although a parallel reduction in the number of untransduced T-cells results in a lack of clinical benefit. We will consider clinical trials involving gene transfer into PBSC, that is, those for: chronic granulomatous disease (CGD), Fanconi anaemia and Gaucher disease. Bone marrow gene therapy for other primary immunodeficiences has recently been reviewed by Fischer66 and Kohn.64 Fanconi anaemia (FA) is a recessive inherited disease characterized by defective DNA repair. FA cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC) that cause chromosomal instability and cell death. The gene for some of the different types of FA, including type C, have been identified. Transfer of a normal FACC cDNA corrects the phenotypic defect resulting in normalized cell growth in the presence of MMC. FA haematopoietic stem cells rescued by gene transfer should have a selective growth advantage in the hypoplastic marrow environment. On the basis of pre-clinical results, Liu and Young67 have proposed to treat FA patients by retroviralmediated transfer of the FACC gene into peripheral blood progenitors (PBSC) from patients with defective alleles. No conditioning regimen is being used in order to avoid the toxicity of bone marrow conditioning regimens and to assess the potential selective advantage conferred to transduced cells. Patients are receiving up to four cycles of transduced PBSC every 2 months. The clinical results are pending. Chronic granulomatous diseases (CGD) are a group of four inherited disorders with a common phenotype characterized by a failure of blood phagocytic cells to produce superoxide. In pre-clinical studies; Malech et al68, by using MFGS based-retrovirus vectors, obtained significant correction of the functional oxidase defect in phagocytes developed in culture from transduced CD34 PBSC of patients with the p47 phoxdeficient, p22 phox-deficient, gp91 phox-deficient or p67 phox-deficient forms of CGD. In the first clinical trial, a transient detection of functional phagocytes, with a frequency of 1 out of 50 000 was achieved.68 The gene transfer approach appears still difficult to be successful in CGD due to the absence of any differentiation block. Consequently there is no selective advantage conferred to the transduced HSC. The use of a lineage-specific promotor might improve these results.69 Leucocyte adhesion deficiency type 1 (LAD) is characterized by defects in neutrophil adhesion due to a defect in the leukocyte integrin CD18 subunit. Bauer et al70 reported encouraging in vitro results with retrovirus CD18 gene transfer into PBSC CD34+ from a patient with LAD. In transduced LAD CD34+ cells, the adhesionrelated and CD18-dependent respiratory burst activities were reconstituted. Developing these findings into clinical apllication will, however, face the same type of problems as discussed for CGD. Acquired disorders Gene therapy may be of value for patients with the acquired immune deficiency syndrome (AIDS), although combinations of antiviral drugs are also making impressive progress. Specifically, gene therapy might be useful in cases such as resistance to therapy or in utero or neonatal infections. Several steps of the human immunodeficiency virus (HIV) replication cycle can be inhibited by transfer of anti-HIV genetic
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activity into stem cells before they differentiate into macrophages, dendritic cells, or T-lymphocytes, all cells susceptible to HIV infection. Genes of interest include those coding for antisense oligonucleotides, negative dominant-mutated viral proteins, protein fixation site decoys that compete with natural proteins encoded by the provirus, IFN-α and IFN-β, proteins required for the inhibition of viral replication, and ribozymes specifially able to cleave RNA viral genomes.71,72 In patients with leukaemia or cancer, two approaches appear fruitful. First, for autologous or allogenic stem cell transplantation gene marking can provide us with information on the fate of the engrafted cells and the involvement of the graft in disease relapse.34 Second, transfer of therapeutic genes, for example, the multidrug resistance gene (MDR-1), into normal progenitors, or the transfer of anti-oncogenes into leukaemic progenitors, may be an effective adjuvant therapy. However, one of the main worries about using this approach is the possibility of transfering the MDR gene into cancer cells or transfering anti-leukaemic genes into normal cells. Hesdorffer et al73 recently reported the clinical results of a phase I trial of retroviral-mediated transfer of human MDR1 gene. Despite the high level of pre-transplant MDR transduction, two post-tranplant marrow samples only were transiently positive for the exogenous MDR gene expression. These data are concordant with those previously reported by Hanania et al74 on a MDR-1 chemoprotection trial for cancer patients. Two different transduction protocols were tested, i.e. ‘suspension transduction’ and ‘stromal growth factor transduction’. Genetically modified cells were detected only in patients transplanted with cells transduced on a stromal cell line, although both groups of patients received post-transplantation taxol to induce a selective advantage for transduced cells. CONCLUSIONS Progress has been made in gene therapy protocols, but the clinical results are still disappointing when haematopoietic stem cells are used as targets. Improvements will come from basic research both in the field of vector design and that of the haematopoietic system. It is possible that new combination viral vectors with components from different viruses will provide properties to allow efficient gene transfer into PBSC. Advances in stem cell biology may lead to the development of in vitro expansion protocols leading to an increase in the number of dividing stem cells transducible by retroviral vectors. REFERENCES 1. Bensinger WI, Weaver CH, Appelbaum FR et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1995; 85: 1655–1658. 2. Begley CG, Basser R, Mansfield R et al. Enhanced levels and enhanced clonogenic capacity of blood progenitor cells following administration of stem cell factor plus granulocyte colony-stimulating factor to humans. Blood 1997; 90: 3378–3389. 3. Schmitz N, Linck DC, Dreger P et al. Randomized trial of filgrastim-mobilised peripheral blood progenitor cell transplantation in lymphoma patients. Lancet 1996; 347: 353–357. 4. Walsh CE, Nienhuis AW, Samulski RJ et al. Phenotypic correction of Fanconi Anemia in human hematopoietic cells with a recombinant adeno-associated virus vector. Journal of Clinical Investigation 1994; 94: 1440–1448.
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Williams DA, Lemischka IR, Nathan DG & Mulligan RC. Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse. Nature 1984; 310: 476–480. 46. Dick JE, Magli MC, Huszar D et al. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemapoietic system of W/Wv mice. Cell 1985; 42: 71–79. 47. Keller G, Paige C, Gilboa E & Wagner EF. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent hematopoietic precursors. Nature 1985; 318: 149–154. 48. Lemischka IR, Raulet DH & Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986; 45: 917–927. 49. Verfaillie C, Hurley R, Bhatia R & McCarthy JP. Role of bone marrow matrix in normal and abnormal hematopoiesis. Critical Reviews in Oncology and Hematology 1994; 16: 201–224. 50. Long MW, Briddell R, Walter AW et al. Human hematopoietic stem cell adherence to cytokines and matrix molecules. Journal of Clinical Investigation 1992; 90: 251–255. 51. Brunner G, Metz CN, Nguyen H et al. An endogenous glycosylphosphatidyl inositol-specific phospholipase D releases basic fibroblast growth factor-heparan sulfate proteoglycan complexes from human bone marrow cultures. Blood 1994; 83: 2115. 52. Gordon MY, Riley GP, Watt SM et al. Compartmentalization of a haematopoietic growth factor (GMCSF) by glycosaminoglycans in the bone marrow microenvironment. Nature 1987; 236: 403–405. 53. Verfaillie CM, McCarhy JB & McGlove PB. Differentiation of primitive human multipotent hematopoietic progenitor into single lineage clonogenic progenitors is accompanied by alterations in their interaction with fibronectin. Journal of Experimental Medicine 1991; 174: 693–703. 54. Williams DA, Rios M, Stephens C & Patel UP. Fibronectin and VLA4 in haemopoietic stem cell microenvironment interactions. Nature 1991; 352: 438–441. *55. Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nature Medicine 1996; 2: 876–882. 56. Moritz T, Patel VP & Williams DA. Bone marrow extracellular Matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors. Journal of Clinical Investigation 1994; 93: 1451–1457.
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57. Schofield KP, Humphries MJ, De Wynter E et al. The effect of α4β1 Integrin binding sequences of fibronectin on growth of cells from human hematopoietic progenitors. Blood 1998; 91: 3230–3238. 58. Yokota T, Oritani K, Mitsui H et al. Growth supporting activities of fibronectin on hematopoietic stem/progenitor cells in vitro and in vivo: structural requirement for fibronectin activities of CS1 and cell-binding domains. Blood 1998; 91: 3263–3272. 59. Hanenberg H, Haschino K, Konishi H et al. Optimization of fibronectin assisted retroviral gene transfer into human CD34+ hematopoietic cells. Human Gene Therapy 1997; 8: 2193–2206. 60. Petzer AL, Hogge DE, Landsdorp PM et al. Self rewewal of primitive human hematopoietic cells (longterm-culture-initiating cells) in vitro and their expansion in defined medium. Proceedings of the National Academy of Sciences of the USA 1996; 93: 1470–1474. *61. Zandstra PW, Conneally E, Petzer AL et al. Cytokine manipulation of primitive human hematopoietic cell self-renewal. Proceeding of the National Academy of Sciences of the USA 1997; 94: 4698–4703. 62. Piacilbello W, Sanario F, Garetto L et al. Extensive amplification and self renewal of human primitive hematopoietic stem cells from cord blood. Blood 1997; 89: 2644–2653. 63. Bordignon C, Notarangelo LD, Nobili N et al. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science 1995; 270: 470–474. 64. Kohn DB. Gene Therapy for haematopoietic and lymphoid disorders. Clinical and Experimental Immunology 1997; 107: 54–57. 65. Kohn D, Hershfield MS, Carbonaro D et al. T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates. Nature Medicine 1998; 4: 775–780. *66. Fischer A, De Saint-Basile G, Disanto J et al. Gene therapy of primary immunodeficiences. Advances in Nephrology 1997; 26: 107–120. 67. Liu JM & Young N. Clinical protocol: retroviral mediated gene transfer of the Fanconi Anemia complementation group C gene to hematopoietic progenitors of group C patients. Human Gene Therapy 1997; 8: 1715–1730. 68. Malech HL, Maples PB, Whiting-Theobald N et al. Prolonged production of NADPH oxidase corrected granulocytes after gene therapy of chronic ganulomatous disease. Proceedings of the National Academy of Sciences of the USA 1997; 94: 12 133–12 138. *69. Malik P, Krall WJ, Yu XJ et al. Retroviral-mediated gene expression in human myelomonocytic cells: a comparison of hematopoietic cell promoters to viral promoters. Blood 1995; 86: 2993–3005. 70. Bauer TR, Schwartz BR, Liles CW et al. Retroviral-mediated gene transfer of the leukocyte integrin CD18 into peripheral blood CD34+ cells derived from a patient with leukocyte adhesion deficiency type 1. Blood 1998; 91: 1520–1526. 71. Yu M, Poeschla E & Woug Staal F. Progress towards gene therapy for HIV infection. Gene Therapy 1994; 1: 13–26. 72. Junker U, Moon JJ, Kalfoglon CS et al. Hematopoietic potential and retroviral transduction of CD34+ Thy1+ peripheral blood stem cells from asymptomatic human immunodeficiency virus type 1 infected individuals mobilized with G-CSF. Blood 1997; 89: 4299–4306. 73. Hesdorffer C, Ayello J, Ward M et al. Phase 1 trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem cell transplantation. Journal of Clinical Oncology 1998; 16: 165–172. 74. Hanania EG, Giles RE, Kavanagh J et al. Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony forming unit cells do not contribute to post transplant hematopoietic recovery following intensive sytemic therapy. Proceedings of the National Academy of Sciences of the USA 1996; 93: 15 346–15 351.
10 Peripheral blood stem cell and gene therapy Marina Cavazzana-Calvo*
MD, PhD
Head of the Cell Therapy Lab E.T.S., INSERM U429 Hôpital Necker, 149 rue des Sevres 75015 Paris, France
Claude Bagnis
PhD
Senior Researcher Centre de Thérapie génique, Institu Paoli-Calmette, 232 Boulevard St Marguerite, 13009 Marseille, France
Patrice Mannoni
MD
Head of the Gene Therapy Lab Centre de Thérapie génique, Institu Paoli-Calmette, 232 Boulevard St Marguerite, 13009 Marseille, France
Alain Fischer
MD, PhD
Head INSERM U429 Hôpital Necker, 149 rue des Sevres 75015 Paris, France
Mobilized peripheral blood stem cells characterized by sustained re-populating ability could be optimal target cells for ex-vivo gene transfer. In spite of very attractive preliminary results obtained in the murine studies, theraputically efficient gene transfer and expression in human targeted cells must be proven. In recent years, effort has been spent on the identification of factors limiting gene transfer efficiency of haematopoietic stem cells. Increasing knowledge concerning haematopoiesis and gene transfer has helped in identifying a number of limiting factors. These factors as well as the strategies that showed increased retroviral infection of haematopoietic stem cells will be discussed. Finally, the results of the clinical trials will be reported. Key word: retrovirus; human stem cells; clinical trials of gene therapy; peripheral blood stem cells.
Gene transfer by viral vectors into human haematopoietic cells is a new approach to the treatment of a variety of genetic and acquired human diseases. Initially, the major obstacles to the clinical application of gene transfer into haematopoietic progenitor/stem cells were that few such cells were available and that they were poorly identified phenotypically and relatively refractory to infection by vectors. Haematopoietic cytokines have now been shown to mobilize large numbers of primitive haematopoietic cells (CD34+) into peripheral blood (PB) of mice, primates and humans.1,2 These cells have sustained re-populating ability in the murine model, and have been successfully used as a source of long-term re-populating cells in human autologous and allogeneic transplantation.3 These CD34+ cells can easily be purified * Author for correspondence. 1521–6926/99/010129 + 10 $12.00/00
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from PB using immunological methods and can be manipulated in vivo and/or ex vivo to improve gene transfer. The aim of this article is to review strategies used to increase gene transfer efficiency in peripheral blood stem cell (PBSC) and briefly describe the most widely used vectors. Pre-clinical and clinical studies based on the use of transduced PBSC are presented. The majority of these studies involved retrovirusmediated gene transfer and this technique will be considered in more detail. CHOICE OF VECTORS AND PRODUCING CELL LINES Gene delivery vehicles for gene therapy can be either viral or non-viral. Diverse viral delivery vectors are under investigation, all of which are naturally able to infect mammalian cells: retrovirus, adenovirus, adeno-associated virus, herpes-simplex virus, vaccinia virus, poliovirus, baculovirus and Sindbis virus. Some of them have been extensively developed so as to integrate into genomic DNA (i.e. adeno-associated virus and retrovirus). Adeno-associated virus (AAV) Efficient gene transfer into CD34+ cells using AAV has been reported.4,5 The mechanisms of integration, efficiency of gene transfer (and identification of an efficient packaging cell line), integration and expression in dividing and non-dividing haematopoietic cells remain to be clearly characterized.6 It will be especially interesting to learn whether the site-specific integration of AAV into the AAVS1 site on chromosome 19q13.3-qter will provide any advantage for gene transfer and gene therapy purposes.7 Despite the limitations in the size of transgene inserts that can be used (no more than 4 kilobases), and difficulties in preparing a high-titre supernatant devoid of contaminant adenovirus particles, it is likely that these vectors will be developed to allow transduction of haematopoietic stem cells. Transfer of the lacZ gene on AAV-derived vectors into haematopoietic cells has already been reported, but long-term expression of this gene in transduced haematopoietic cell remains undocumented.5,8,9 Adenoviral vectors Adenoviral vectors present some advantages over other vectors for gene delivery to haematopoietic precursor cells. In particular, target cells can be quiescent, their mechanism for cell entry is dependent on integrins and on the recently described common receptor for coxsackie B virus and adenovirus (CAR)10 expressed by CD34+ cells, and finally they pass efficiently from the cell surface to the nucleus. The major disadvantage is that gene expression mediated by adenoviral vectors is only transient because the epichromosome of the transgene is not duplicated during cell division. Moreover, the infection efficiency of adenoviral-vectors differs between various haematopoietic lineages and is high only for megakaryocytic and dendritic cells.11 As PBSC contain a large number of dendritic precursor cells this vector can be used to deliver tumoural antigens to ex vivo differentiated dendritic cells.12,13 Adenoviral gene transfer for therapeutic purposes may therefore be most effective if mature postmitotic precursor cells are targeted.14 The transient expression of therapeutic transgenes on adenoviruses in terminally differentiated haematopoietic cells may be used for alleviating complications of genetic disorders such as haemophilia.
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Retroviral vectors Murine retroviral vectors are the most widely used delivery systems and the most powerful tools for genomic integration of the transgene.15 Natural or artificial retroviral backbones are being developed that are more sophisticated than the Moloney murine leukaemia virus (Mo-Mu LV)-derived vectors and NIH-3T3-derived packaging cell lines currently in use. Few of these defective vectors, such as GN1a and LNL616, or MFG-derivatives17 have been used for gene marking or therapeutical clinical trials. Vectors derived from murine retroviral backbones other than Mo-MuLV are being tested for reporter gene expression in haematopoietic cells.18,19 Recently, retroviral vectors with improved cell transduction properties have been described. They include a chimeric viral backbone composed of retroviral elements of different origins, and pseudotyped particles expressing non-retroviral envelope proteins (VSV-G) or the gibbon ape leukaemia virus (GALV) envelope.20,21 Producing/packaging cell lines of both murine and human origin giving high virus titres are being developed.22 No clear comparative data have been reported about the relative ability of different producing cell lines to transduce haematopoietic cells, whereas such data are available for retroviral vectors.23 Factors limiting the use of murine retroviral vectors are that target cells must be actively cycling at the time of insertion and the variability of long-term expression of the transgene according to the differentiation and activation status of transduced cells. New vector designs, the modification of the hypermethylation status of the transgene or regulatory elements24, construction of inducible or specific promoters for these vectors25 and combined expression of selection and therapeutic genes may improve both long-term expression and control of the expression of the transgene. HIV-derived and lentivirus-derived vectors able to transduce quiescent cells are also promising tools for improving retroviral vector efficiency in non-dividing cells.26
IN VIVO AND EX VIVO MANIPULATION OF HUMAN STEM CELLS The most important reconstituting cell populations in the human haematopoietic system express the CD34 surface antigen but not lineage differentiated markers (lin–). Numerous in vitro and in vivo studies have described retroviral infection and transduction of immature haematopoietic progenitors and the long-term maintenance of provirus in cells arising from transduced immature cells. Stem cells transduction is a challenging problem as integration occurs only in cycling cells and most of the primitive cells are in the resting phase. Various strategies have recently been used to increase the transduction efficiency of HSC. In vivo manipulation of HSC prior to cell harvesting and transduction may improve retroviral gene transfer by promoting cell cycling or by increasing receptor density. This approach has been successfully used in the murine model by the administration of 5-fluorouracil (5-FU) to mice 2–4 days prior to harvesting of bone marrow.27,28 Gene transfer efficiencies similar to that obtained in the murine model have also been obtained in the rhesus model with or without the pre-harvest administration of 5-FU.29,30 Randall and Weissman31 analysed some of the physical and functional changes in the stem cell population after 5-FU treatment. 5-FU treatment itself causes a gradual loss
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of c-kit expression on the lineage negative, Sca-1-positive population concomitant with the induction of Mac-1 expression. These changes correlate with the induction of the cell cycle in this stem cell population in response to the loss of haematopoietic progenitors. The use of a similar protocol in 11 myeloma patients pre-treated with 5-FU at a clinically tolerable dose (i.e. 15 mg/kg/day for 3 days) did not, however, result in efficient gene transfer to re-populating cells.32,33 The patients were followed for at least 1 year after autologous transplantation of retrovirally-transduced BM and PB CD34enriched cells. The levels of gene marking were very low: only 1 : 1000 to 1 : 10 000 cells were positive for the neomycin resistance gene (Neo). Better results have been obtained in a paediatric study34 where autologous BM cells from children undergoing transplantation for acute leukaemia or neuroblastoma were transduced in vitro, prior to cryopreservation, by the same retroviral vector. An average of 5% of marrow progenitors and PB mononuclear cells were marked at 12–18 months post-transplantation. Three major factors could explain these divergent results: the chemotherapy regimen administered just before the harvest of BM cells, the age of patients and the differencies in the transduction protocol. In vivo pre-treatment of patients: chemotherapy or cytokines? The type of chemotherapy administered before collection and ex-vivo transduction of bone marrow cells greatly influences the proportion of marked progenitors. Brenner et al34 harvested BM cells after completion of intensive marrow ablative chemotherapy. Patients with acute myeloid leukaemia (AML, 12 patients) were treated with 2-chlorodeoxyadenosine and then three cycles of daunorubicine, cytosine arabinoside and etoposide. Patients with neuroblastoma received six cycles of chemotherapy that included cyclophosphamide, doxorubicin, etoposide and cisplatin. The bone marrow was harvested when peripheral blood neutrophil counts rose above 1000/ml. The low-dose chemotherapy used for mobilization is not sufficient to enrich the PBSC in very primitive haematopoietic cells and to improve the transduction rate. Nevertheless, high-dose chemotherapy cannot be safely used in patients affected by hereditary diseases, human immunodeficiency virus infection or other non-malignant diseases. For such cases stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) could be administered in vivo rather than chemotherapy. The combination of SCF and G-CSF increases the number of PBSC in murine, canine and primate models and in human clinical trials.35–38 Moreover, these two cytokines, administered together, were shown to increase significantly the number of primitive cells present in BM within 2–3 weeks. These in vivo primed haematopoietic stem cells seem to be a better target for retroviral gene transfer than steady-state BM cells.39 The higher amphotropic envelope receptor density on most primitive cells may explain these findings.40 Age of patients and transduction protocol Important ontogeny-associated changes in the cytokine responses of primitive human haematopoietic cells41,42 have been recently described and should be considered when defining ex-vivo transduction protocols. The use of cytokines in the transduction protocol can impair the long-term re-populating ability of the PBSC, and these changes are tightly linked with the source of haematopoietic stem cells (i.e. cord blood, steadystate bone marrow or mobilized PBSC from young children or adults).
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Two haematopoietic cytokines exert a key role in the in vitro expansion of longterm culture-initiating cells (LTC-IC): FLT3-L and thrombopoietin (TPO).43,44 FLT3-L preserves, at least in part, the ability of human CD34 progenitor cells to sustain longterm haematopoiesis, and TPO seems to exert an anti-apoptotic effect on activated HSC. It would be useful to test the effects of these cytokines in pre-clinical studies. There is a controversy on the usefulness of autologous or heterologous stroma for the infection of more immature progenitor cells. Although transduction of murine haematopoietic stem cells with retroviral vectors has been demonstrated by a number of laboratories using direct co-culture of the target bone marrow cells with the virus packaging cell line, this methodology may have significant drawbacks for use in human trials because of safety and quality considerations.45–48 The mechanism underlying this promoting effect of stroma on retroviral infection is unclear, but haematopoietic cell proliferation and differentiation is physiologically regulated when they are in direct contact with cells of the haematopoietic micro-environment.49,50 This environment includes cytokines, stromal cells and a complex extracellular matrix (ECM) made up, in particular, of a variety of adhesion molecules. ECM molecules such as laminin, collagen thrombospondin, proteoglycans, glycosamino-glycans and fibronectin constitute anchorage sites for both haematopoietic cells and growth factors.51,52 Fibronectin (FN) is abundantly expressed in the bone marrow micro-environment and has been studied as a prototype molecule in cell–cell as well as in cell–matrix interactions.53,54 Fibronectin participates in cell adhesion through at least three cell binding sites: the cell-binding domain (CBD) mediates adhesion via the integrin VLA-5; the heparin binding domain interacts via cell surface proteoglycan molecules, and the CS1 sequence mediates adhesion via the integrin VLA-4. These two integrins are both present on CD34+ cells; retroviral particles bind directly to the heparin-binding domain of FN. A number of groups has recently reported an improvement in gene transfer efficiency by the use of FN-coated plastic dishes due, at least in part, to the co-localization of retrovirus and target cells on the same molecule.55,56 Therefore, the use of FN for ex-vivo expansion of CD34+ cells57,58 and for gene transfer59 could improve the clinical results. PBSC seem to be more difficult to transduce than steady-state bone marrow cells and to require longer stimulation by growth factors. Efficient gene transfer into mobilized CD34+CD38– requires pre-stimulation for 40 hours in the presence of SCF, TPO and G-CSF followed by 8 hours’ infection. It is thus evident that each gene transfer protocol must be adapted according to a number of factors, particularly the source of stem cells, donor age, cytokine combinations and in vivo pre-treatment of patients. The reciprocal influence of these factors on the gene transfer efficiency has not been elucidated, and a knowledge of these interactions should help to improve future clinical results. Work on the possibilities of expanding CD34+/CD38– stem cells ex-vivo has been illuminating.60–62 CLINICAL APPLICATIONS Hereditary diseases Hereditary diseases caused by monogenic alterations have long been considered as a possible experimental field for gene therapy. Adenosine deaminase (ADA) deficiency was the first inherited disease for which human gene therapy was used.63–65 Four years
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after the treatment of three newborns with ADA deficiency by transduced autologous umbilical cord blood CD34+ cells, it is worth noting the continuing presence of peripheral blood leukocytes containing the transferred ADA gene, suggesting that stem cells were transduced and engrafted without any toxic effect. The selective increase of transduced T-cell frequency after PEG-ADA cessation shows that there is a selective advantage for this cell line, although a parallel reduction in the number of untransduced T-cells results in a lack of clinical benefit. We will consider clinical trials involving gene transfer into PBSC, that is, those for: chronic granulomatous disease (CGD), Fanconi anaemia and Gaucher disease. Bone marrow gene therapy for other primary immunodeficiences has recently been reviewed by Fischer66 and Kohn.64 Fanconi anaemia (FA) is a recessive inherited disease characterized by defective DNA repair. FA cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC) that cause chromosomal instability and cell death. The gene for some of the different types of FA, including type C, have been identified. Transfer of a normal FACC cDNA corrects the phenotypic defect resulting in normalized cell growth in the presence of MMC. FA haematopoietic stem cells rescued by gene transfer should have a selective growth advantage in the hypoplastic marrow environment. On the basis of pre-clinical results, Liu and Young67 have proposed to treat FA patients by retroviralmediated transfer of the FACC gene into peripheral blood progenitors (PBSC) from patients with defective alleles. No conditioning regimen is being used in order to avoid the toxicity of bone marrow conditioning regimens and to assess the potential selective advantage conferred to transduced cells. Patients are receiving up to four cycles of transduced PBSC every 2 months. The clinical results are pending. Chronic granulomatous diseases (CGD) are a group of four inherited disorders with a common phenotype characterized by a failure of blood phagocytic cells to produce superoxide. In pre-clinical studies; Malech et al68, by using MFGS based-retrovirus vectors, obtained significant correction of the functional oxidase defect in phagocytes developed in culture from transduced CD34 PBSC of patients with the p47 phoxdeficient, p22 phox-deficient, gp91 phox-deficient or p67 phox-deficient forms of CGD. In the first clinical trial, a transient detection of functional phagocytes, with a frequency of 1 out of 50 000 was achieved.68 The gene transfer approach appears still difficult to be successful in CGD due to the absence of any differentiation block. Consequently there is no selective advantage conferred to the transduced HSC. The use of a lineage-specific promotor might improve these results.69 Leucocyte adhesion deficiency type 1 (LAD) is characterized by defects in neutrophil adhesion due to a defect in the leukocyte integrin CD18 subunit. Bauer et al70 reported encouraging in vitro results with retrovirus CD18 gene transfer into PBSC CD34+ from a patient with LAD. In transduced LAD CD34+ cells, the adhesionrelated and CD18-dependent respiratory burst activities were reconstituted. Developing these findings into clinical apllication will, however, face the same type of problems as discussed for CGD. Acquired disorders Gene therapy may be of value for patients with the acquired immune deficiency syndrome (AIDS), although combinations of antiviral drugs are also making impressive progress. Specifically, gene therapy might be useful in cases such as resistance to therapy or in utero or neonatal infections. Several steps of the human immunodeficiency virus (HIV) replication cycle can be inhibited by transfer of anti-HIV genetic
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activity into stem cells before they differentiate into macrophages, dendritic cells, or T-lymphocytes, all cells susceptible to HIV infection. Genes of interest include those coding for antisense oligonucleotides, negative dominant-mutated viral proteins, protein fixation site decoys that compete with natural proteins encoded by the provirus, IFN-α and IFN-β, proteins required for the inhibition of viral replication, and ribozymes specifially able to cleave RNA viral genomes.71,72 In patients with leukaemia or cancer, two approaches appear fruitful. First, for autologous or allogenic stem cell transplantation gene marking can provide us with information on the fate of the engrafted cells and the involvement of the graft in disease relapse.34 Second, transfer of therapeutic genes, for example, the multidrug resistance gene (MDR-1), into normal progenitors, or the transfer of anti-oncogenes into leukaemic progenitors, may be an effective adjuvant therapy. However, one of the main worries about using this approach is the possibility of transfering the MDR gene into cancer cells or transfering anti-leukaemic genes into normal cells. Hesdorffer et al73 recently reported the clinical results of a phase I trial of retroviral-mediated transfer of human MDR1 gene. Despite the high level of pre-transplant MDR transduction, two post-tranplant marrow samples only were transiently positive for the exogenous MDR gene expression. These data are concordant with those previously reported by Hanania et al74 on a MDR-1 chemoprotection trial for cancer patients. Two different transduction protocols were tested, i.e. ‘suspension transduction’ and ‘stromal growth factor transduction’. Genetically modified cells were detected only in patients transplanted with cells transduced on a stromal cell line, although both groups of patients received post-transplantation taxol to induce a selective advantage for transduced cells. CONCLUSIONS Progress has been made in gene therapy protocols, but the clinical results are still disappointing when haematopoietic stem cells are used as targets. Improvements will come from basic research both in the field of vector design and that of the haematopoietic system. It is possible that new combination viral vectors with components from different viruses will provide properties to allow efficient gene transfer into PBSC. Advances in stem cell biology may lead to the development of in vitro expansion protocols leading to an increase in the number of dividing stem cells transducible by retroviral vectors. REFERENCES 1. Bensinger WI, Weaver CH, Appelbaum FR et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1995; 85: 1655–1658. 2. Begley CG, Basser R, Mansfield R et al. Enhanced levels and enhanced clonogenic capacity of blood progenitor cells following administration of stem cell factor plus granulocyte colony-stimulating factor to humans. Blood 1997; 90: 3378–3389. 3. Schmitz N, Linck DC, Dreger P et al. Randomized trial of filgrastim-mobilised peripheral blood progenitor cell transplantation in lymphoma patients. Lancet 1996; 347: 353–357. 4. Walsh CE, Nienhuis AW, Samulski RJ et al. Phenotypic correction of Fanconi Anemia in human hematopoietic cells with a recombinant adeno-associated virus vector. Journal of Clinical Investigation 1994; 94: 1440–1448.
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32. Dunbar CE, Cottler-Fox M, O’Shanghnessy JA et al. Retrovirally-marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation. Blood 1995; 85: 3048–3057. 33. Emmons RVB, Doren S, Zujewski J et al. Retroviral gene transduction of adult peripheral blood or Marrow-Derived 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. 34. Brenner MK, Rill KR, Moen RC et al. Gene marking to trace origin of relapse after autologous bone marrow transplantation. Lancet 1993; 341: 85–86. 35. Bodine DM, Seidel NE & Orlic D. Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow. Blood 1996; 88: 89–97. 36. Andrews RG, Bartelemez SH, Knitter GH et al. 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