Comparison of Retroviral Transduction Efficiency in CD34 Cells Derived from Bon - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:36 |只看该作者 |倒序浏览 |打印

a Hematology Branch, NHLBI, National Institutes of Health, and" u) O# G9 G7 O7 J5 U, c

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# Q8 T+ A( J5 N! k. @& b7 D5 x

ABSTRACT1 R/ W2 q2 e2 [2 ^+ G; x
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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.9 P) w& b/ f( P& k1 S
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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.: T4 m/ l$ k+ u3 g9 q0 g* E

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
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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 1 j0 H+ d$ x) n/ ]  v
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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.: M* a  X  D% t) W! _+ F: V- ^# P
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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.
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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., q  S: U4 y. w. B) v# a8 @0 _7 F  A

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.. B+ ^5 w$ D  f
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