Comparison of Retroviral Transduction Efficiency in CD34+ Cells Derived from Bone Marrow versus G-CSF–Mobilized or G-CSF Plus Stem Cell Fact
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《干细胞学杂志》
a Hematology Branch, NHLBI, National Institutes of Health, and
b Molecular and Clinical Hematology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland, USA
Key Words. Stem cell ? Gene tranfer ? retrovirus ? bone marrow
Correspondence: John F. Tisdale, M.D., Molecular and Clinical Hematology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 9N116, 9000 Rockville Pike, Bethesda, MD 20892, USA. Telephone: 301-402-6497; Fax: 301-480-1373; e-mail: Johntis@intra.niddk.nih.gov
ABSTRACT
Nonhuman primates are valuable as a preclinical model for the evaluation of both the safety and potential efficacy of promising gene therapy protocols before their implementation in human studies. Indeed, the model has proven useful in predicting and evaluating the major toxicities observed in human gene therapy trials to date . Furthermore, significant advances in hematopoietic stem cell (HSC) gene transfer technology have also been made, with in vivo gene transfer levels of 5%–10% or higher now achievable . Recently, the first definitive evidence for efficacy of any gene therapy trial was reported in children with severe combined immunodeficiency who received bone marrow (BM) cells transduced with a standard retroviral vector carrying a corrective gene under optimized conditions . These results have established the therapeutic potential of HSC-based gene transfer methods, yet the subsequent development of leukemia in two children has raised new safety concerns , placing a higher priority on preclinical models.
Concurrent with the progress attained in achieving significant rates of gene transfer to repopulating cells of the hematopoietic system in large animals and humans, significant progress using lentiviral vectors that faithfully deliver the human ?-globin gene along with key regulatory elements has also recently been achieved . Furthermore, alternative anti-sickling genes have shown promise using similar vector systems . These achievements have renewed enthusiasm for moving toward clinical application of gene therapy in monogenic disorders of globin synthesis such as sickle cell anemia (SCA) and thalassemia.
The optimal source of HSCs for transduction has not yet been established, and the ability to transduce primitive HSCs varies depending on the source of HSCs. The use of G-CSF and stem cell factor (SCF)–mobilized peripheral blood (PB) CD34+ cells resulted in significantly higher in vivo marking levels compared with G-CSF alone or G-CSF + Flt3-L–mobilized cells in the rhesus macaque competitive repopulation model . Thomasson et al. have also recently shown in a canine model that BM cells harvested 14 days after G-SCF + SCF administration were superior to G-CSF + SCF–mobilized cells or unprimed BM cells. Nevertheless, none of these cytokine regimens can be used in patients with SCA, because SCF is no longer clinically available in the U.S. due to anaphylactic reactions after its use and, more important, severe sickle cell crisis and even death have been reported following the use of G-CSF for mobilization in patients with SCA .
In the current study, we compared the in vivo levels of genetically modified cells attainable after transduction of CD34+ cells collected from steady-state BM (clinically applicable to patients with SCA) versus G-CSF–mobilized PB (the commonest clinically used regimen for collection of HSCs in humans) or G-CSF + SCF–mobilized PB (the regimen that has resulted in the highest marking level in our large animals) in the rhesus macaque competitive repopulation model.
MATERIALS AND METHODS
The experimental design for comparison of BM and either G-CSF alone or G-CSF + SCF–mobilized CD34+ cells as targets for retroviral transduction is shown in Figure 1. Three animals were used to compare BM to G-CSF alone, and three animals were used to compare BM to G-CSF + SCF. Table 1 summarizes the retroviral vector used to transduce each population of CD34+ cells and the characteristics of each trans-duction procedure.
Figure 1. Experimental design. For each animal, bone marrow was initially harvested. Bone marrow CD34+ cells were selected and transduced with either G1Na or LNL6 vectors for 96 hours in the presence of SCF, FLT, IL-3, IL-6, and FN. One week later, each animal was mobilized with five doses of either G-CSF alone or G-CSF + SCF before leukapheresis. Mobilized CD34+ cells were selected and transduced with the alternate vector (G1Na or LNL6) using the same transduction conditions. Both transduced aliquots were frozen at the end of transduction and subsequently thawed and reinfused to the monkey after 500 cGy x 2 total body irradiation. Abbreviations: FLT, Flt-3 ligand; FN, fibronectin; IL, interleukin; SCF, stem cell factor.
Table 1. Summary of CD34+ enrichment, CFU transduction efficiency, cell expansion, and engraftment kinetics of animals
With the exception of animal RC803, there were no significant differences in the efficiency of transduction of committed progenitors, as defined using the in vitro CFU assay, between CD34+ cells collected by BM harvest or G-CSF + SCF–induced or G-CSF–induced PB mobilization and apheresis. There was no difference in results observed based on the vectors used (G1Na versus LNL6) (Table 1). In animal RC803, there was a marked difference between the transduction efficiency in the BM experiment (22 of 24 or 92% transduced colonies) compared with the G-CSF + SCF experiment (6 of 27 or 22% transduced colonies, p < .001).
The average number of CD34+ cells collected was similar in the experimental group comparing BM harvest to G-CSF–mobilized PB (3.1 x 107 versus 2.8 x 107, respectively; p = .83). There was a trend toward a greater number of CD34+ cells collected by apheresis after G-CSF + SCF administration compared with the BM harvest (5 x 107 versus 3.2 x 107), but the difference did not reach statistical significance (p = .2). The fold expansion of cells after 4 days of transduction was similar within groups. In the first group, an expansion of 4.5 was noted in BM-harvested cells compared with 3.1 in G-CSF–mobilized cells. In the second group, a 3.8-fold expansion was seen in BM-harvested cells versus 3.5 in G-CSF + SCF mobilized cells. When comparing the average number of cells collected and infused at the end of transduction, the first group showed twice as many cells in the BM-harvested fraction compared with the G-CSF–mobilized fraction (15.1 x 107 versus 7.4 x 107, respectively). However, in paired two-tailed t-test, the difference did not reach statistical significance (p = .105). In contrast, in the second group, the average number of cells infused from the G-CSF + SCF fraction was two–fold higher compared with the BM-harvested fraction (17.3 x 107 versus 9.6 x 107, respectively), but, again, the difference was not statistically significant (p = .15). All animals recovered their PB counts without significant morbidity and reached an absolute neutrophil count of >500/μl between 6 and 12 days after infusion.
After transplantation, semiquantitative PCR analysis of PB samples allowed comparison of the relative contribution of marked cells derived from CD34+ target cells collected from steady-state BM or after mobilization. We assayed granulocytes, because these cells have a short half life and are better representative of cells produced by transduced HSCs. A representative gel is shown in Figure 2. Figure 3 summarizes the marking levels in the two groups of animals. In group one, animal RQ2223 had very low to undetectable marking and was thus not informative. In the other two animals in this group (RQ2800 and RC904), the gene marking predominantly originated from the BM fraction rather than the G-CSF–mobilized cells. In the second group comparing BM cells and G-CSF + SCF–mobilized cells, the first animal showed low-level marking from the BM fraction and none from G-CSF + SCF fraction. However, the very low gene transfer efficiency in CFU obtained at the end of 4 days of transduction suggests an overall poor transduction procedure in the G-CSF + SCF fraction in this animal. In the other two animals in this group, in vivo gene marking originated predominantly from G-CSF + SCF–mobilized cells.
Figure 2. Representative polymerase chain reaction gel from peripheral blood granulocytes of animals RQ2800 and RC904. Control dilutions of G1Na in normal rhesus DNA are shown along with an LNL6 control. ?-actin controls are shown below and were used to correct for DNA amount. In animal RQ2800, only marking from the BM-derived fraction (LNL6) is detectable up to 9 months after transplantation; no marking is detectable from the G-CSF–mobilized fraction (G1Na). In RC904, better marking is observed from the BM-derived fraction (G1Na) compared with the G-CSF–mobilized fraction (LNL6) at 1 and 9 months after transplantation. Abbreviation: BM, bone marrow.
Figure 3. Summary of the percent positive granulocytes at different time points (in months ) after transplantation. Upper panel: comparison of BM and G-CSF–mobilized cells. Lower panel: comparison of BM and G-CSF + stem cell factor–mobilized cells. Abbreviations: BM, bone marrow; PB, peripheral blood.
DISCUSSION
Donahue RE, Kessler SW, Bodine D et al. Helper virus induced T cell lymphoma in nonhuman primates after retro-viral mediated gene transfer. J Exp Med 1992;176:1125–1135.
Lozier JN, Csako G, Mondoro TH et al. Toxicity of a first-generation adenoviral vector in rhesus macaques. Hum Gene Ther 2002;13:113–124.
Lozier JN, Metzger ME, Donahue RE et al. Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity. Blood 1999;94:3968–3975.
Hu J, Dunbar CE. Update on hematopoietic stem cell gene transfer using non-human primate models. Curr Opin Mol Ther 2002;4:482–490.
Kiem H-P, Sellers S, Thomasson B et al. Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: no progression to clonal hematopoiesis or leukemia. Mol Ther 2004;9:389–395.
Tisdale JF, Hanazono Y, Sellers SE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 1998;92:1131–1141.
Wu T, Kim HJ, Sellers S et al. Prolonged high-level detection of retrovirally marked hematopoietic cells in nonhuman primates after transduction of CD34+ progenitors using clinically feasible methods. Mol Ther 2000;1:285–293.
Kiem HP, Andrews RG, Morris J et al. Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor. Blood 1998;92:1878–1886.
Goerner M, Bruno B, McSweeney PA et al. The use of granulocyte colony-stimulating factor during retroviral transduction on fibronectin fragment CH-296 enhances gene transfer into hematopoietic repopulating cells in dogs. Blood 1999;94:2287–2292.
Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease . Science 2000;288:669–672.
Hacein-Bey-Abina S, von Kalle C, Schmidt M et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255–256.
May C, Rivella S, Callegari J et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 2000;406:82–86.
Pawliuk R, Westerman KA, Fabry ME et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001;294:2368–2371.
Persons DA, Allay ER, Sawai N et al. Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells. Blood 2003;102:506–513.
Levasseur DN, Ryan TM, Pawlik KM et al. Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells. Blood 2003;102:4312–4319.
Hematti P, Sellers SE, Agricola BA et al. Retroviral transduction efficiency of G-CSF+SCF-mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L-mobilized cells in nonhuman primates. Blood 2003;101:2199–2205.
Thomasson B, Peterson L, Thompson J et al. Direct comparison of steady-state marrow, primed marrow, and mobilized peripheral blood for transduction of hematopoietic stem cells in dogs. Hum Gene Ther 2003;14:1683–1686.
Adler BK, Salzman DE, Carabasi MH et al. Fatal sickle cell crisis after granulocyte colony-stimulating factor administration. Blood 2001;97:3313–3314.
Abboud M, Laver J, Blau CA. Granulocytosis causing sickle-cell crisis . Lancet 1998;351:959.
Grigg AP. Granulocyte colony-stimulating factor-induced sickle cell crisis and multiorgan dysfunction in a patient with compound heterozygous sickle cell/beta+ thalassemia. Blood 2001;97:3998–3999.
Donahue RE, Kirby MR, Metzger ME et al. Peripheral blood CD34+ cells differ from bone marrow CD34+ cells in Thy-1 expression and cell cycle status in nonhuman primates mobilized or not mobilized with granulocyte colony-stimulating factor and/or stem cell factor. Blood 1996;87:1644–1653.
Dunbar CE, Cottler-Fox M, O’Shaughnessy 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.
Pauling L, Itano HA, Singer SJ et al. Sickle cell anemia, a molecular disease. Science 1949;110:543.
Tisdale JF, Sadelain M. Toward gene therapy for disorders of globin synthesis. Semin Hematol 2001;38:382–392.
Rivella S, May C, Chadburn A et al. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer. Blood 2003;101:2932–2939.(Peiman Hemattia, Sascha T)
b Molecular and Clinical Hematology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland, USA
Key Words. Stem cell ? Gene tranfer ? retrovirus ? bone marrow
Correspondence: John F. Tisdale, M.D., Molecular and Clinical Hematology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 9N116, 9000 Rockville Pike, Bethesda, MD 20892, USA. Telephone: 301-402-6497; Fax: 301-480-1373; e-mail: Johntis@intra.niddk.nih.gov
ABSTRACT
Nonhuman primates are valuable as a preclinical model for the evaluation of both the safety and potential efficacy of promising gene therapy protocols before their implementation in human studies. Indeed, the model has proven useful in predicting and evaluating the major toxicities observed in human gene therapy trials to date . Furthermore, significant advances in hematopoietic stem cell (HSC) gene transfer technology have also been made, with in vivo gene transfer levels of 5%–10% or higher now achievable . Recently, the first definitive evidence for efficacy of any gene therapy trial was reported in children with severe combined immunodeficiency who received bone marrow (BM) cells transduced with a standard retroviral vector carrying a corrective gene under optimized conditions . These results have established the therapeutic potential of HSC-based gene transfer methods, yet the subsequent development of leukemia in two children has raised new safety concerns , placing a higher priority on preclinical models.
Concurrent with the progress attained in achieving significant rates of gene transfer to repopulating cells of the hematopoietic system in large animals and humans, significant progress using lentiviral vectors that faithfully deliver the human ?-globin gene along with key regulatory elements has also recently been achieved . Furthermore, alternative anti-sickling genes have shown promise using similar vector systems . These achievements have renewed enthusiasm for moving toward clinical application of gene therapy in monogenic disorders of globin synthesis such as sickle cell anemia (SCA) and thalassemia.
The optimal source of HSCs for transduction has not yet been established, and the ability to transduce primitive HSCs varies depending on the source of HSCs. The use of G-CSF and stem cell factor (SCF)–mobilized peripheral blood (PB) CD34+ cells resulted in significantly higher in vivo marking levels compared with G-CSF alone or G-CSF + Flt3-L–mobilized cells in the rhesus macaque competitive repopulation model . Thomasson et al. have also recently shown in a canine model that BM cells harvested 14 days after G-SCF + SCF administration were superior to G-CSF + SCF–mobilized cells or unprimed BM cells. Nevertheless, none of these cytokine regimens can be used in patients with SCA, because SCF is no longer clinically available in the U.S. due to anaphylactic reactions after its use and, more important, severe sickle cell crisis and even death have been reported following the use of G-CSF for mobilization in patients with SCA .
In the current study, we compared the in vivo levels of genetically modified cells attainable after transduction of CD34+ cells collected from steady-state BM (clinically applicable to patients with SCA) versus G-CSF–mobilized PB (the commonest clinically used regimen for collection of HSCs in humans) or G-CSF + SCF–mobilized PB (the regimen that has resulted in the highest marking level in our large animals) in the rhesus macaque competitive repopulation model.
MATERIALS AND METHODS
The experimental design for comparison of BM and either G-CSF alone or G-CSF + SCF–mobilized CD34+ cells as targets for retroviral transduction is shown in Figure 1. Three animals were used to compare BM to G-CSF alone, and three animals were used to compare BM to G-CSF + SCF. Table 1 summarizes the retroviral vector used to transduce each population of CD34+ cells and the characteristics of each trans-duction procedure.
Figure 1. Experimental design. For each animal, bone marrow was initially harvested. Bone marrow CD34+ cells were selected and transduced with either G1Na or LNL6 vectors for 96 hours in the presence of SCF, FLT, IL-3, IL-6, and FN. One week later, each animal was mobilized with five doses of either G-CSF alone or G-CSF + SCF before leukapheresis. Mobilized CD34+ cells were selected and transduced with the alternate vector (G1Na or LNL6) using the same transduction conditions. Both transduced aliquots were frozen at the end of transduction and subsequently thawed and reinfused to the monkey after 500 cGy x 2 total body irradiation. Abbreviations: FLT, Flt-3 ligand; FN, fibronectin; IL, interleukin; SCF, stem cell factor.
Table 1. Summary of CD34+ enrichment, CFU transduction efficiency, cell expansion, and engraftment kinetics of animals
With the exception of animal RC803, there were no significant differences in the efficiency of transduction of committed progenitors, as defined using the in vitro CFU assay, between CD34+ cells collected by BM harvest or G-CSF + SCF–induced or G-CSF–induced PB mobilization and apheresis. There was no difference in results observed based on the vectors used (G1Na versus LNL6) (Table 1). In animal RC803, there was a marked difference between the transduction efficiency in the BM experiment (22 of 24 or 92% transduced colonies) compared with the G-CSF + SCF experiment (6 of 27 or 22% transduced colonies, p < .001).
The average number of CD34+ cells collected was similar in the experimental group comparing BM harvest to G-CSF–mobilized PB (3.1 x 107 versus 2.8 x 107, respectively; p = .83). There was a trend toward a greater number of CD34+ cells collected by apheresis after G-CSF + SCF administration compared with the BM harvest (5 x 107 versus 3.2 x 107), but the difference did not reach statistical significance (p = .2). The fold expansion of cells after 4 days of transduction was similar within groups. In the first group, an expansion of 4.5 was noted in BM-harvested cells compared with 3.1 in G-CSF–mobilized cells. In the second group, a 3.8-fold expansion was seen in BM-harvested cells versus 3.5 in G-CSF + SCF mobilized cells. When comparing the average number of cells collected and infused at the end of transduction, the first group showed twice as many cells in the BM-harvested fraction compared with the G-CSF–mobilized fraction (15.1 x 107 versus 7.4 x 107, respectively). However, in paired two-tailed t-test, the difference did not reach statistical significance (p = .105). In contrast, in the second group, the average number of cells infused from the G-CSF + SCF fraction was two–fold higher compared with the BM-harvested fraction (17.3 x 107 versus 9.6 x 107, respectively), but, again, the difference was not statistically significant (p = .15). All animals recovered their PB counts without significant morbidity and reached an absolute neutrophil count of >500/μl between 6 and 12 days after infusion.
After transplantation, semiquantitative PCR analysis of PB samples allowed comparison of the relative contribution of marked cells derived from CD34+ target cells collected from steady-state BM or after mobilization. We assayed granulocytes, because these cells have a short half life and are better representative of cells produced by transduced HSCs. A representative gel is shown in Figure 2. Figure 3 summarizes the marking levels in the two groups of animals. In group one, animal RQ2223 had very low to undetectable marking and was thus not informative. In the other two animals in this group (RQ2800 and RC904), the gene marking predominantly originated from the BM fraction rather than the G-CSF–mobilized cells. In the second group comparing BM cells and G-CSF + SCF–mobilized cells, the first animal showed low-level marking from the BM fraction and none from G-CSF + SCF fraction. However, the very low gene transfer efficiency in CFU obtained at the end of 4 days of transduction suggests an overall poor transduction procedure in the G-CSF + SCF fraction in this animal. In the other two animals in this group, in vivo gene marking originated predominantly from G-CSF + SCF–mobilized cells.
Figure 2. Representative polymerase chain reaction gel from peripheral blood granulocytes of animals RQ2800 and RC904. Control dilutions of G1Na in normal rhesus DNA are shown along with an LNL6 control. ?-actin controls are shown below and were used to correct for DNA amount. In animal RQ2800, only marking from the BM-derived fraction (LNL6) is detectable up to 9 months after transplantation; no marking is detectable from the G-CSF–mobilized fraction (G1Na). In RC904, better marking is observed from the BM-derived fraction (G1Na) compared with the G-CSF–mobilized fraction (LNL6) at 1 and 9 months after transplantation. Abbreviation: BM, bone marrow.
Figure 3. Summary of the percent positive granulocytes at different time points (in months ) after transplantation. Upper panel: comparison of BM and G-CSF–mobilized cells. Lower panel: comparison of BM and G-CSF + stem cell factor–mobilized cells. Abbreviations: BM, bone marrow; PB, peripheral blood.
DISCUSSION
Donahue RE, Kessler SW, Bodine D et al. Helper virus induced T cell lymphoma in nonhuman primates after retro-viral mediated gene transfer. J Exp Med 1992;176:1125–1135.
Lozier JN, Csako G, Mondoro TH et al. Toxicity of a first-generation adenoviral vector in rhesus macaques. Hum Gene Ther 2002;13:113–124.
Lozier JN, Metzger ME, Donahue RE et al. Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity. Blood 1999;94:3968–3975.
Hu J, Dunbar CE. Update on hematopoietic stem cell gene transfer using non-human primate models. Curr Opin Mol Ther 2002;4:482–490.
Kiem H-P, Sellers S, Thomasson B et al. Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: no progression to clonal hematopoiesis or leukemia. Mol Ther 2004;9:389–395.
Tisdale JF, Hanazono Y, Sellers SE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 1998;92:1131–1141.
Wu T, Kim HJ, Sellers S et al. Prolonged high-level detection of retrovirally marked hematopoietic cells in nonhuman primates after transduction of CD34+ progenitors using clinically feasible methods. Mol Ther 2000;1:285–293.
Kiem HP, Andrews RG, Morris J et al. Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor. Blood 1998;92:1878–1886.
Goerner M, Bruno B, McSweeney PA et al. The use of granulocyte colony-stimulating factor during retroviral transduction on fibronectin fragment CH-296 enhances gene transfer into hematopoietic repopulating cells in dogs. Blood 1999;94:2287–2292.
Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease . Science 2000;288:669–672.
Hacein-Bey-Abina S, von Kalle C, Schmidt M et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255–256.
May C, Rivella S, Callegari J et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 2000;406:82–86.
Pawliuk R, Westerman KA, Fabry ME et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001;294:2368–2371.
Persons DA, Allay ER, Sawai N et al. Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells. Blood 2003;102:506–513.
Levasseur DN, Ryan TM, Pawlik KM et al. Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells. Blood 2003;102:4312–4319.
Hematti P, Sellers SE, Agricola BA et al. Retroviral transduction efficiency of G-CSF+SCF-mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L-mobilized cells in nonhuman primates. Blood 2003;101:2199–2205.
Thomasson B, Peterson L, Thompson J et al. Direct comparison of steady-state marrow, primed marrow, and mobilized peripheral blood for transduction of hematopoietic stem cells in dogs. Hum Gene Ther 2003;14:1683–1686.
Adler BK, Salzman DE, Carabasi MH et al. Fatal sickle cell crisis after granulocyte colony-stimulating factor administration. Blood 2001;97:3313–3314.
Abboud M, Laver J, Blau CA. Granulocytosis causing sickle-cell crisis . Lancet 1998;351:959.
Grigg AP. Granulocyte colony-stimulating factor-induced sickle cell crisis and multiorgan dysfunction in a patient with compound heterozygous sickle cell/beta+ thalassemia. Blood 2001;97:3998–3999.
Donahue RE, Kirby MR, Metzger ME et al. Peripheral blood CD34+ cells differ from bone marrow CD34+ cells in Thy-1 expression and cell cycle status in nonhuman primates mobilized or not mobilized with granulocyte colony-stimulating factor and/or stem cell factor. Blood 1996;87:1644–1653.
Dunbar CE, Cottler-Fox M, O’Shaughnessy 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.
Pauling L, Itano HA, Singer SJ et al. Sickle cell anemia, a molecular disease. Science 1949;110:543.
Tisdale JF, Sadelain M. Toward gene therapy for disorders of globin synthesis. Semin Hematol 2001;38:382–392.
Rivella S, May C, Chadburn A et al. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer. Blood 2003;101:2932–2939.(Peiman Hemattia, Sascha T)