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Home , Cell potency, Progenitor cell, Stem cell

(2000) 7, 451–457  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt MILLENNIUM REVIEW Stem therapy and gene transfer for

T Asahara, C Kalka and JM Isner Cardiovascular Research and Medicine, St Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, MA, USA

The committed stem and progenitor cells have been recently In this review, we discuss the promising appli- isolated from various adult tissues, including hematopoietic cation of adult stem and progenitor cells in terms of mod- , , and ifying stem , altering organ property, accelerating endothelial . These adult stem cells have sev- regeneration and forming expressional organization. Gene eral advantages as compared with embryonic stem cells as Therapy (2000) 7, 451–457. their practical therapeutic application for tissue regeneration.

Keywords: stem cell; gene therapy; regeneration; progenitor cell; differentiation

Introduction poietic stem cells to cells. The determined stem cells differentiate into ‘committed progenitor cells’, which The availability of embryonic stem (ES) cell lines in mam- retain a limited capacity to replicate and phenotypic fate. malian species has greatly advanced the field of biologi- In the past decade, researchers have defined such com- cal research by enhancing our ability to manipulate the mitted stem or progenitor cells from various tissues, and by providing model systems to examine including marrow, peripheral blood, , . ES cells, which are derived from and reproductive organs, in both adult animals and the inner mass of or primordial germ cells, humans (Figure 1). While most cells in adult organs are can be maintained continuously in an undifferentiated composed of differentiated cells, which express a variety pluripotent state. They have the potential to contribute of specific phenotypic adapted to each organ’s to a variety of differentiated cell lineages in the , environment, quiescent stem or progenitor cells are main- including germ cells.1,2 Furthermore, targeted mutations in ES cells have been introduced into the mouse germ tained locally or in the systemic circulation and are acti- line to elucidate target gene function in vivo and create vated by environmental stimuli for physiological and mouse models of human genetic diseases and abnormali- pathological tissue regeneration. ties.3,4 ES cells have also been used to study the events Tissue replacement in the body takes place by two regulating the differentiation of various cell types and mechanisms. One is the replacement of differentiated tissues , for example, into multiple hematopoietic cells by newly generated populations derived from lineages, endothelial cells, neural cells and mesenchymal residual cycling stem cells. Blood cells are a typical cell lineages. The transfer of ES-derived cells into fetal example of this kind of regeneration. Whole hematopo- and adult mice has demonstrated their functional ietic lineage cells are derived from a few self-renewal integration into organs of recipient animals.5–10 stem cells by regulated differentiation under the influ- In 1998, Thomson’s group at the University of Wiscon- ence of appropriate cytokines and/or growth factors. The sin and Gearhart’s group at the Johns Hopkins Hospital second mechanism is the self-repair of differentiated isolated human cells with the pluripotent property of ES functioning cells preserving their proliferative activity. cells.11,12 This exciting achievement opened the way to the , endothelial cells, smooth muscle cells, kera- clinical application of stem cell therapy: the replacement tinocytes and fibroblasts are considered to possess this of diseased or degenerating cell populations, tissues ability. Following physiological stimulation or injury, fac- and organs. tors secreted from surrounding tissues stimulate cell rep- lication and replacement. In contrast, those cells which Stem cell in adult species are more fully differentiated are limited in terms of their proliferative potential by , and by their ES cells are not the only candidates for stem cell gener- inability to incorporate into remote target sites. ation. During , the pluripotency Thus, quiescence stem or progenitor cells in most adult of ES cell is narrowed to ‘determined stem cells’ which tissues are mobilized in response to environmental stim- 13 give rise to cells of a particular tissue. Thus neural stem uli when an emergent regenerative process is required, cells give rise to cells of the and hemato- while during a minor event, neighboring differentiated cells are relied upon.

Correspondence: JM Isner, St Elizabeth’s Medical Center, 736 Cambridge Street, Boston, MA 02135, USA Millennium review T Asahara et al 452

Figure 1 Cell stages of embryonic and adult stem/progenitor cell during lineage differentiation.

Stem cell therapy Thus, while ES cells represent a promising cell source for gene therapy in the future, this review will focus Recently, the regenerative potential of stem cells has been primarily on gene-modified cell therapy using adult under intense investigation. In vitro, stem and progenitor stem cells. cells possess the capability of self-renewal and differen- tiation into organ-specific cell types. In vivo, transplan- tation of these cells may reconstitute organ systems, as Gene-modified stem cell therapy shown in animal models of diseases.14–18 In contrast, dif- ferentiated cells do not exhibit such characteristics. Considering their regenerative ability, gene modification Human endothelial progenitor cells (EPCs), for example, of stem cells has several advantages over conventional have been isolated from the peripheral blood of adult gene therapy. Ex vivo gene of stem cells may individuals, expanded in vitro and committed into an avoid administration of vectors and vehicles into the endothelial lineage in culture.15 The transplantation of recipient . Possible targets of gene-modified human EPCs has been shown to facilitate successful stem cells include the following (Figure 2). salvage of limb vasculature and perfusion in athymic nude mice with severe hindlimb ischemia, while Modification of stem cell potency/stem cell target differentiated endothelial cells (human microvascular Certain properties of stem cells can be modified by gene endothelial cells, HUMEC) failed to accomplish limb-saving transfer. Stem cells isolated from adults may exhibit age- neovascularization.19 related, genetic, or acquired disease-related impairment The committed stem and progenitor cells isolated from of their regenerative ability. Transcriptional or enzymatic the adult species have several advantages as compared gene modification may constitute effective means to with ES cell in terms of their possible therapeutic appli- maintain, enhance or inhibit their capacity to proliferate cation for tissue regeneration. First, committed stem and or differentiate. progenitor cells can be transplanted autologously with- out immunologic consequences. As adult stem and pro- genitor cells are already determined or committed to cell Modification of organ property/progeny target types for targeted organs, they spontaneously differen- Genes introduced into stem cells are inherited by their tiate under controlled conditions in vitro into specific lin- progeny through differentiation cascades. Specifically, eage cells, and have the ability to reconstitute target expression of the inserted gene may persist through the organs in vivo. In contrast, ES cells are still under investi- life of the organ system which is reconstructed by the gation for direct induction of specific cell differentiation gene-modified stem cells. Genetically disordered organs, for therapeutic application. Furthermore, ethical issues especially in the case of hematopoietic pathology, could about the isolation and the use of human ES are currently be replaced by this strategy following quite controversial. transplantation.20–24

Gene Therapy Millennium review T Asahara et al 453

Figure 2 Targets of stem cell gene modification for therapeutic regeneration.

Acceleration of regeneration/process target Stem cell gene transfer and associated Regeneration of injured tissues is in part or primarily target disorders achieved via stem cell expansion and differentiation. Examples include endothelial progenitor cells for neovas- Hematopoietic stem cells cularization, neural stem cells for , and hem- atopoietic stem cells (HSCs) and mesenchymal stem cells A is a determined multipotent (MSCs) for bone marrow reconstruction. Natural repara- stem cell, which is able to differentiate along a number tory processes are often too impaired from unexpectedly of pathways and thereby generate erythrocytes, granulo- severe injury, basic diseases, or aging to accomplish cytes, , mast cells, and megakary- regeneration. Gene modification of stem cells to sup- ocytes. These stem cells are limited in number, occurring with a frequency of one stem cell per 104 bone marrow plement mitogens25 or to deliver inhibitory factors for negative control might accelerate retarded regenerative cells. By virtue of their capacity for self-renewal, stem processes following stem , incorporation, cells are maintained at homeostatic levels throughout and in situ. adult life. Due to their low frequency and the difficulties associated with maintaining stem cells in culture, little is known about the regulation of HSC proliferation and Expressional organization/systemic target differentiation. To supply target molecules for therapeutic use, trans- While the true of the human HSC has not plantation of genetically modified stem cells might gener- been fully characterized, the CD34+CD38− population is ate expressional tissues or pseudo-organs. The estab- known to be enriched for cells with high repopulation lished tissues or organs will continuously express a capacity and is essential for engraftment in animal mod- certain amount of molecules locally or generally for the els.14 More recently, KDR expression has been shown to short or long term by means of vector selection and con- be essential for the CD34+ subset of hematopoietic stem ditioning. This strategy could represent an alternative cells.26 Several organ systems have been evaluated for approach for therapeutic delivery in lieu of standard isolation of HSCs. Bone marrow serves as a source of drug distribution and uptake. HSCs as does the peripheral blood, where HSCs circulate,

Gene Therapy Millennium review T Asahara et al 454 although in much lower frequencies than they are found Neural stem cells in bone marrow. To increase the number of circulating Several groups have identified adult neural stem cells HSCs, patients have been treated with cytokines such as (NSCs) that are able to differentiate to , granulocyte colony-stimulating factor (G-SCF), granulo- and . The potential of treating neuro- cyte–macrophage colony-stimulating factor (GM-CSF) degenerative disorders such as Parkinson’s disease or and stem cell factor, all of which are known to mobilize multiple sclerosis reveals a promising therapeutic poten- bone marrow-derived progenitors into the peripheral cir- tial of NSCs.17,18 Genetically engineered NSCs have been culation. The relative abundance of HSCs, characterized shown to differentiate selectively into oligodendrocytes, by their CD34+CD38− phenotype makes human umbilical which, after in vitro expansion, were used to remyelinate an ideal source for gene therapy protocols.27 the vast majority of axons after transplantation in a In addition, highly purified CD34+CD38− hematopoietic demyelinated area.39 Furthermore, recombinant adenovi- progenitors have been isolated from human fetal liver.28 ral vectors can successfully transfect human neural pro- The unique ability of hematopoietic stem cells to genitor cells with exogenous genes allowing options of engraft in a recipient and establish long-term repopu- ex vivo gene therapy.40 In fact, transplantation of NSCs lation of the hematopoietic system makes them ideal tar- genetically modified to secrete (NGF) gets for gene therapy vectors designed to correct was able to ameliorate the death of striatal projection inherited or acquired diseases affecting the hematopoietic neurons caused by transient focal ischemia in the adult and immune systems. Theoretically, many single human rat.41 The clinical potential of genetically modified neural gene disorders (eg hemoglobinopathies, immunodefi- precursors for the treatment of neurodegenerative dis- ciencies, lipid storage disorders and platelet glycoprotein eases is promising, but development of successful ther- deficiencies) which affect the function of mature blood apies will depend on better characterization of the origin cells of a specific lineage20 could be treated by the intro- and differentiation factors affecting NSCs.42 duction of the defective or missing gene into autologous hematopoietic stem cells. Indeed, gene therapy of hema- Mesenchymal stem cells topoietic cells has already been applied to patients with Bone marrow stroma provides the microenvironment for adenosine deaminase (ADA) deficiency,29 Gaucher hematopoiesis by supporting proliferation and differen- disease,21 HIV infection22 and .23,24 tiation of hematopoietic stem cells and is also the source of non-hematopoietic, mesenchymal stem cells (MSCs).43 Endothelial progenitor cells MSCs share certain characteristics of stem cells, including Conventionally, physiological (reproduction system) and the capability to differentiate in culture into , pathological (ischemia, tumor, ) vascular , adipocytes, fibroblasts, myoblasts and car- developments have been considered to be established by diomyoblasts.44 Characteristically quiescent and non-cyc- sprout formation, ie proliferation of pre-existing differen- ling with low cell turnover, MSCs have been explored as tiated ECs.30,31 Transplantation of differentiated ECs, vehicles for gene therapy based on their ability to engraft however, reportedly fails to augment impaired neovascu- in marrow, ie to deliver certain secreted to a local larization in severely ischemic tissues.32 Recently, EPCs site, and to provide progeny for the repopulation of other have been isolated from peripheral blood and bone mar- tissues. Genetic-engineered MSCs have been shown to row, and shown to be incorporated into sites of physio- serve as an effective vehicle for the replacement of genes logical and pathological neovascularization in vivo.15,33,34 responsible for deficiencies in circulating . There In contrast to differentiated ECs, transplantation of EPCs are, for example, reports of a therapeutic approach for successfully enhanced vascular development by in situ hemophilia B or severe osteogenesis imperfecta by trans- differentiation and proliferation within ischemic organs.16 fecting human MSCs with a gene for factor IX45,46 or for This beneficial property of EPC is attractive for I collagen.47 The ability to induce MSC differen- therapy as well as gene therapy applications. tiation in specific tissues by using gene transfer tech- As cells already committed to an endothelial lineage, niques reveals the therapeutic possibility of treating a cells isolated at the present time were termed progenitors variety of human diseases. This potential has been used in our initial report.15 During embryogenesis, - for example by adenovirus-mediated bone - derived hemangioblasts are common stem cells for both etic protein-2 gene transfer into MSCs, which could angioblasts (EPCs) and HSCs.35 Although EPCs and potentially direct differentiation into cells having an HSCs of adult species express in common certain epi- phenotype in vitro and bone formation in topes (flk-1, tie-2, CD34, Sca-1, c-Kit etc),36 a bipotent vivo.48 While cultured murine stromal cells have been stem cell for both lineages has not been defined. EPCs shown to engraft into murine recipients following intra- are so few in number in peripheral blood and bone mar- venous infusion,49 engraftment of transplanted stromal row that mobilization and/or ex vivo expansion will be cells in humans remains to be documented. required for clinical applications.37,38 Ex vivo cultured EPCs may be genetically transduced Gene transfer vectors by recombinant adenoviral vectors encoding VEGF, and demonstrate significantly enhanced neovascularization A number of requirements for gene transfer to stem cells even when 10 times fewer cells were injected.25 This strat- determine the choice of the appropriate vector or gene egy could be applied to peripheral vascular disease and transfer vehicle. Sufficient integration of the transferred myocardial ischemia to augment impaired collateral gene into the host genome of the target stem cell is neces- development. Genetic modification of EPCs by genes sary to achieve subsequent transmission to all progeny designed to growth and development of neoplasms will cells and to obtain durable effects. Other applications – be achieved in situ via incorporation of EPCs into such as targeting regenerative processes – require only tumor vasculature. temporary cell engineering.

Gene Therapy Millennium review T Asahara et al 455 Retroviral vectors Adenovirus vectors Most reported attempts to transduce HSCs for gene ther- Adenovirus vectors have been successfully used for tran- apy protocol have used retroviral vectors, such as the sient gene expression in many systems, although stan- murine leukemia virus (MLV). These vectors, which can dard adenovirus does not usually allowed stable inte- accept up to 8 kilobases (kb) of exogenous DNA, require gration.59 It does not require cycling of the target cell for in order to integrate in their genome.50 gene transfer, appears to utilize an integrin-dependent Therefore, most ex vivo retroviral gene transfer protocols mechanism for cell entry, and has evolved an efficient include the addition of a variety of stimulatory cytokines mechanism for gene delivery from the cell surface to the to induce cycling in the HSC population before sequential nucleus. It is utilized to achieve transient genetic engin- addition of vector-containing supernatants. Current eering of stem cells especially for acceleration of regener- investigations differ in the number of cycles of infection ative responses, such as cytokine or growth factor gene and the length of exposure of stem cells to retroviral vec- transduction of EPCs for , MSCs for forma- tors, the liability being progressive differentiation and the tion of bone, or muscle development, and NSCs loss of repopulation ability. However, progress in the for neurogenesis. field of genetic transfer or modification into human long- term repopulating stem cells mediated by retroviral vec- Factors limiting gene transfer into stem cells tors has been blocked by the fact that levels of transfec- One important issue which potentially limits gene trans- tion are too low for any likely therapeutical benefit.51,52 fer of retroviral vectors into HSCs is the quiescent nature Several reports have demonstrated that an important rea- and reduced expression of primate HSCs. Lenti- for the low levels of transduction might involve cer- virus-based vectors overcome this limitation and may tain incompatible features of the vectors used and the therefore represent an alternative to retroviruses. While stem cells that they target. rAAVs achieve sufficient short-term transduction, long- term expression with this vector system is reported to be limited to less than 6 months. Lentiviral vectors Successful gene therapy applications require optimized There is increasing evidence that lentivirus-based sys- strategies to increase gene transfer efficiency and expan- tems might be ideal vectors for the transduction of sion in balance with maintenance of the immature state of human HSCs. Lentiviruses constitute another retrovirus HSCs. Today, important experimental variables include subgroup and include the human immunodeficiency multiplicity of infection, length of time or viral incu- virus (HIV) type 1 envelope. While retrovirus systems are bation, medium used for viral incubation, the viral con- inefficient at transducing nondividing or slowly dividing struct (including promoter and gene), and the source of cells, lentivirus-based vectors when pseudotyped with the stem cells. The determination of factors controlling vesicular stomatitis virus glycoprotein G (VSV-G)53 or 54 HSC proliferation, differentiation and expression of the gibbon ape leukemia virus (GALV) have the capability after each cycle of transduction are important to mediate genome integration into nondividing human 55 issues which are currently under investigation. HSCs. Pseudotyping implicates incorporation of protein One final issue concerns the selection of successfully envelopes from other viruses into the HIV-1-based vec- transduced stem cells. Ex vivo selection involves cell sur- tors and is necessary for these vectors, because they nat- + face antigen expression and autofluorescent (GFP) mark- urally infect only CD4 T lymphocytes and macrophage- ing while an in vivo selection method is based on separat- lineage cells. There is evidence that lentiviral vectors ing transduced cells by conferring drug resistance could transduce more primitive, quiescent progenitors (dihydrofolate reductase) or by selective amplifier genes than MLV with stable transgene integration 60 days 60,61 56 (chimeric receptor). The in vivo selection by drug after infection. treatment after transplant also offers the possibility to In comparison with retrovirus vectors, lentiviral sys- selectively increase the number of primitive and mature tems allow the immediate transduction after transduced stem cells.62 without any prior expansion or growth factor stimulation with only short exposure times. Transduction efficiencies of around 60% HSCs in cord blood have been reported.14 References Further investigations analyzing gene transfer into long- 1 Evans MJ, Kaufman MH. Establishment in culture of pluripotent term repopulating cells of large animals and the use of cells from mouse . Nature 1981; 292: 154–156. improved vector systems will be needed to establish the 2 Martin GR. Isolation of a pluripotent cell line from early mouse superiority of lentiviral vectors. embryos cultured in medium conditioned by teratocarcinoma cells. Proc Natl Acad Sci USA 1981; 78: 7634–7638. 3 Smithies O et al. Insertion of DNA sequences into the human Adeno-associated viral vectors chromosomal beta-globin locus by homologous recombination. Recombinant adenovirus-associated viruses (rAAV), Nature 1985; 317: 230–234. which can take up to 4.8 kb of exogenous DNA are also 4 Cawpecchi MR. Altering the genome by homologous recombi- being explored as potential vectors for introduction of nation. 1989; 244: 1288–1292. genes into HSCs.57 This encapsulated single-stranded, 5 Kennedy M et al. A common precursor for primitive erythropo- nonpathogenic DNA parvovirus has been shown to infect iesis and definitive . Nature 1997; 386: 488–493. primate hematopoietic progenitor cells with reasonable 6 Hole N, Graham GJ, Menzel U, Ansell JD. A limited temporal window for the derivation of multilineage repopulating hemato- efficiency; even so its use involves coinfection with a poietic progenitors during embryonal stem cell differentiation helper virus such as adenovirus or herpes simplex virus. in vitro. Blood 1996; 88: 1266–1276. Use of rAAV will require the development of optimized 7 Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cotransfection and helper strategies for pro- cardiomyocytes from differentiating embronic stem cells from ductive infection.58 stable intracardiac grafts. J Clin Invest 1996; 98: 216–224.

Gene Therapy Millennium review T Asahara et al 456 8 Deacon T et al. Blastula-stage stem cells can differentiate into cells responsible for postnatal in physiological dopaminergic and serotonergic neurons after transplantation. and pathological neovascularization. Circ Res 1999; 85: 221–228. Exp Neurol 1998; 149: 28–41. 34 Shi Q et al. Evidence for circulating bone marrow-derived endo- 9 Brustle O et al. In vitro-generated neural precursors participate thelial cells. Blood 1998; 92: 362–367. in mammalian brain development. Proc Natl Acad Sci USA 1997; 35 Risau W, Flamme I. Vasculogenesis. Ann Rev Cell Dev Biol 1995; 94: 14809–14814. 11: 73–91. 10 Potocnik AJ, Kohler H, Eichmann K. Hemato-lymphoid in vivo 36 Hatzopoulos AK et al. Isolation and characterization of endo- reconstitution potential of subpopulations derived from in vitro thelial progenitor cells from mouse embyros. Development 1998; differentiated embryonic stem cells. Proc Natl Acad Sci USA 1997; 125: 1457–1468. 94: 10295–10300. 37 Takahashi T et al. Ischemia- and cytokine-induced mobilization 11 Thomas JA et al. lines derived from human of bone marrow-derived endothelial progenitor cells for neovas- blastocysts. Science 1998; 282: 1145–1147. cularization. Nature Med 1999; 5: 434–438. 12 Shamblott MJ et al. Derivation of pluripotent stem cells from 38 Asahara T et al. VEGF contributes to postnatal neovasculariz- cultured human primordial germ cells. Proc Natl Acad Sci USA ation by mobilizing bone marrow-derived endothelial progeni- 1998; 95: 13726–13731. tor cells. EMBO J 1999; 18: 3964–3972. 13 Gage FH. Cell therapy. Nature 1998; 392: 18–24. 39 Groves AK et al. Repair of demyelinated lesions by transplantation 14 Evans JT, Kelly PF, O’Neill E, Garcia JV. Human cord blood of purified 0–2A progenitor cells. Nature 1993; 362: 453–455. CD34+CD38− cell transduction via lentivirus-based gene trans- 40 Sabate O et al. Transplantation to the rat brain of human neural fer vectors. Hum Gene Ther 1999; 10: 1479–1489. progenitors that were genetically modified using adenoviruses. 15 Asahara T et al. Isolation of putative progenitor endothelial cells Nat Genet 1995; 9: 256–260. for angiogenesis. Science 1997; 275: 965–967. 41 Andsberg G et al. Amelioration of ischaemia-induced neuronal 16 Kalka C et al. Administration of culture-expanded endothelial death in the rat striatum by NGF-secreting neural stem cells. progenitor cells (EPC) augments therapeutic neovascularization. Eur J Neurosci 1998; 10: 2026–2036. Circulation 1998; 98: I-455 (Abstr.). 42 Whittemore SR, Eaton MJ, Onifer SM. Gene therapy and the use 17 Flax JD et al. Engraftable human neural stem cells respond to of stem cells for regeneration. Adv Neurol developmental cues, replace neurons, and express foreign 1997; 72: 113–119. genes. Nat Biotech 1998; 16: 1033–1039. 43 Clark BR, Keating A. of bone marrow stroma. Ann NY 18 Lindvall O et al. Grafts of fetal dopamine neurons survive and Acad Sci 1995; 770: 70–78. improve motor function in Parkinson’s disease. Science 1990; 44 Prockop DJ. Marrow stromal cells as stem cells for nonhemato- 247: 574–577. poietic tissues. Science 1997; 276: 71–74. 19 Schuler JJ et al. Efficacy of prostaglandin E1 in the treatment of 45 Hurwitz DR et al. Systemic delivery of human growth hormone lower extremity ischemic ulcers secondary to peripheral vascu- or human factor IX in dogs by reintroduced genetically modified lar occlusive disease. J Vasc Surg 1984; 1: 160–170. autologous bone marrow stromal cells. Hum Gene Ther 1997; 8: 20 Karlsson S. Treatment of genetic defects in hematopoietic cell 137–156. function by gene transfer. Blood 1991; 78: 2481–2492. 46 Cherington V et al. Retroviral vector-modified bone marrow 21 Dunbar CE et al. Retroviral transfer of the glucocerebrosidase stromal cells secrete biologically active factor IX in vitro and gene into CD34+ cells from patients with Gaucher disease: in transiently deliver therapeutic levels of human factor IX to the plasma of dogs after reinfusion. Hum Gene Ther 1998; 9: 1397– vivo detection of transduced cells without myeloablation. Hum 1407. Gene Ther 1998; 9: 2629–2640. 47 Horwitz EM et al. Transplantability and therapeutic effects of 22 Wong-Staal F, Poeschla EM, Looney DJ. A controlled phase 1 bone marrow-derived mesenchymal cells in children with osteo- clinical trial to evaluate the safety and effects in HIV-1 infected genesis imperfecta. Nature Med 1999; 5: 309–313. humans of autologous lymphocytes transduced with a ribozyme 48 Lou J, Xu F, Merkel K, Manske P. Gene therapy: adenovirus- that cleaves HIV-1 RNA. Hum Gene Ther 1998; 9: 2407–2425. mediated human bone morphogenetic protein-2 gene transfer 23 Reisner Y, Segall H. Hematopoietic stem cell transplantation for induces mesenchymal progenitor cell proliferation and differen- Curr Opin Immunol 7 cancer therapy. 1995; : 687–693. tiation in vitro and bone formation in vivo. J Orthop Res 1999; 17: 24 Cowan KH et al. Paclitaxel after autologous stem- 43–50. cell transplantation and engraftment of hematopoietic cells 49 Anklesaria P et al. Engraftment of a clonal bone marrow stromal transduced with a retrovirus containing the multidrug resist- cell line in vivo stimulates hematopoietic recovery from total ance complementary DNA (MDR1) in metastatic breast cancer body irradiation. Proc Natl Acad Sci USA 1987; 84: 7681–7685. patients. Clin Cancer Res 1999; 5: 1619–1628. 50 Miller DG, Adam MA, Miller AD. Gene transfer by retroviral 25 Asahara T et al. Gene therapy of endothelial progenitor cell for vectors occurs only in cells that are actively replicating at the vascular development in severe ischemic disease. Circulation time of infection. Mol Cell Biol 1990; 10: 4239–4242. 1999; 100: I-480 (Abstr.). 51 Havenga M, Hoogerbrugge P, Valerio D, van Es HH. Retroviral 26 Ziegler BL et al. KDR receptor: a key marker defining hematopo- stem cell gene therapy. Stem Cells 1997; 15: 162–179. ietic stem cells. Science 1999; 285: 1553–1558. 52 Chu P, Lutzko C, Stewart AK, Dube ID. Retrovirus-mediated + − 27 Hao QL et al. A functional comparison of CD34 CD38 cells gene transfer into human hematopoietic stem cells. J Mol Med in cord blood and bone marrow. Blood 1995; 86: 3745–3753. 1998; 76: 184–192. 28 Humeau L et al. Successful reconstitution of human hematopo- 53 Emi N, Friedmann T, Yee JK. Pseudotype formation of murine iesis in the SCID-hu mouse by genetically modified, highly leukemia virus with the G protein of vesicular stomatitis virus. enriched progenitors isolated from fetal liver. Blood 1997; 90: J Virol 1991; 65: 1201–1207. 3496–3506. 54 Kavanaugh MP et al. Cell-surface receptors for gibbon ape leu- 29 Blaese RM et al. T -directed gene therapy for ADA- kemia virus and amphotropic muring retrovirus are inducible SCID: initial trial results after 4 years. Science 1995; 270: 475–480. sodium-dependent phosphate symporters. Proc Natl Acad Sci 30 Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992; 267: USA 1994; 91: 7071–7075. 10931–10934. 55 Naldini L et al. In vivo gene delivery and stable transduction of 31 Risau W. Mechanisms of angiogenesis. Nature 1997; 386: 671–674. nondividing cells by a lentiviral vector. Science 1996; 272: 263– 32 Ojeifo JO et al. Angiogenesis-directed implantation of genetically 267. modified endothelial cells in mice. Cancer Res 1995; 55: 2240– 56 Case SS et al. Stable transduction of quiescent CD34(+)CD38(−) 2244. human hematopoietic cells by HIV-1-based lentiviral vectors. 33 Asahara T et al. Bone marrow origin of endothelial progenitor Proc Natl Acad Sci USA 1999; 96: 2988–2993.

Gene Therapy Millennium review T Asahara et al 457 57 Goodman S et al. Recombinant adeno-associated virus-mediated 60 Persons DA et al. Use of the green fluorescent protein as a gene transfer into hematopoietic progenitor cells. Blood 1994; 84: marker to identify and track genetically modified hematopoietic 1492–1500. cells. Nature Med 1998; 4: 1201–1205. 58 Chatterjee S et al. Transduction of primitive human marrow and 61 Sorrentino BP et al. Selection of drug-resistant bone marrow cells cord blood-derived hematopoietic progenitor cells with adeno- in vivo after retroviral transfer of human MDR1. Science 1992; associated virus vectors. Blood 1999; 93: 1882–1894. 257: 99–103. 59 Watanabe T et al. Gene transfer into human bone marrow hema- 62 Allay JA et al. In vivo selection of retrovirally transduced hema- topoietic cells mediated by adenovirus vectors. Blood 1996; 87: topoietic stem cells. Nature Med 1998; 4: 1136–1143. 5032–5039.

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