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作者：Cecilia Baresea, Nancy Pecha, Sara Dirscherla, Justin L. Meyersa, Anthony L. Sinna, Mervin C. Yodera,b,c, W. Scott Goebela,d, Mary C. Dinauera,d,e,f作者单位：aHerman B Wells Center for Pediatric Research, Department of Pediatrics, Sections of
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2 }8 x' H- W- O* t6 {* J6 y 【摘要】2 P- ~" |8 z2 m4 ~
The use of nonmyeloablative conditioning prior to bone marrow transplantation is an important component of transplantation-based therapies for nonmalignant blood diseases. In this study, treatment of recipient mice with granulocyte colony-stimulating factor (G-CSF) prior to low-dose total body irradiation (LD-TBI) enhanced long-term engraftment of freshly isolated congenic marrow 1.5- to 2-fold more than treatment with LD-TBI alone. This combined regimen was also evaluated in a mouse model of X-linked chronic granulomatous disease (X-CGD), where neutrophils have a defective NADPH oxidase due to genetic deletion of the gp91phox subunit. Long-term engraftment of male X-CGD bone marrow cells cultured ex vivo for retroviral transduction of gp91phox was enhanced by 40% when female X-CGD recipients were pretreated with G-CSF prior to 300 cGy. These data confirm that sequential treatment with G-CSF and LD-TBI prior to transplantation increases long-term engraftment of donor marrow, and they extend this approach to transplantation of murine donor marrow cultured ex vivo for gene transfer. Additional studies showed that the administration of G-CSF prior to LD-TBI did not alter early homing of donor marrow cells. However, the combined regimen significantly decreased the content of long-term repopulating cells in recipient marrow compared with LD-TBI alone, as assessed in competitive assays, which may contribute to the enhanced engraftment of donor marrow cells.
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e/ K( ?- Z8 J% |' ^' @Disclosure of potential conflicts of interest is found at the end of this article.
6 i9 ]! p5 t; Z3 V" J$ H; Z! T 【关键词】 Granulocyte colony-stimulating factor Irradiation Bone marrow transplant Retrovirus Gene therapy Neutrophil
+ S0 e7 a$ M7 p% P& c# K' ^" l INTRODUCTION
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Genetic modification of autologous hematopoietic stem cells (HSC) has the potential for effective treatment of a wide variety of inherited blood disorders, including inherited immunodeficiencies, hemoglobinopathies, and Fanconi anemia .4 P# f: y5 W( @% a7 q1 e: c
% s9 j3 x- ?7 XChronic granulomatous disease (CGD) is a congenital immune deficiency that could be treated by gene transfer into HSC . However, the limited availability of HLA-matched donors, immunosuppression, and other toxicities associated with transplant regimens and graft-versus-host disease have limited the application of this approach. Therefore, autologous transplantation with genetically corrected stem cells is a feasible alternative to allogenic transplantation for genetic disorders of hematopoietic cells. However, for disorders without an intrinsic selective advantage for hematopoietic stem cells following gene transfer, some form of marrow conditioning will be required to enable sufficient levels of long-term engraftment with genetically corrected cells following their transplantation.# i( r3 R: d" Z; o) R9 m
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Nonmyeloablative conditioning, such as low-dose total body irradiation (LD-TBI), has been successfully used in mice as a preparative regimen for HSC transplantation, resulting in moderate levels of donor engraftment . Thus, it remains controversial whether treatment with G-CSF prior to LD-TBI improves long-term engraftment of congenic marrow, and the mechanism(s) underlying the potential enhancement in donor cell chimerism are not understood.% {2 D' |9 c) G, ^5 B
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Because improved engraftment of transplanted marrow following nonmyeloablative conditioning is desirable, this study re-examined whether sequential G-CSF and LD-TBI increases engraftment in mice receiving congenic marrow and investigated the mechanistic basis for a beneficial effect of this combined regimen. Since this approach may be useful for transplantation of genetically modified HSC for inherited hematopoietic diseases, we also examined whether administration of G-CSF prior to LD-TBI enhances sustained long-term engraftment of retroviral vector-transduced HSC in a murine model of X model of X-linked chronic granulomatous disease (X-CGD)./ {0 S' b+ y' \ g
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MATERIALS AND METHODS
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X-CGD mice carry a null allele for gp91phox . Wild-type C57Bl/6J (C57; CD45.2 ) and C57 splenectomized mice were purchased from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). B6.SJL-PtrcaPep3b/BoyJ (BoyJ; CD45.1 ) and hemizygous transgenic C57-EGFP.Tg strains (C57-EGFP) from the Jackson Laboratory were maintained in a breeding colony at the Indiana University School of Medicine. All mice were housed under pathogen-free conditions and fed autoclaved food and acidified water ad libitum. Animal protocols were approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine.9 X$ V y, o4 x( E* |" L
8 W/ s! F5 [. q6 X2 VConditioning of Recipient Mice( a! C# R% B' i0 v( G- Z
+ {! C5 ] Y$ T, t) c& I" xG-CSF treatment and LD-TBI were performed as described by Mardiney and Malech . Prior to transplantation, recipient mice received twice-daily s.c. injections of either saline or 4 µg of recombinant human G-CSF (Amgen, Thousand Oaks, CA, http://www.amgen.com) in saline for 4 days. On the 5th day, mice received a single s.c. injection followed by 160 or 300 cGy (cesium 137 source) total body irradiation 2 hours later. Four hours after LD-TBI, recipients were transplanted with unfractionated fresh BM or BM cultured ex vivo for retroviral-mediated gene transfer. No G-CSF was administered post-transplantation." C4 ~( g/ z( v1 m6 L# H2 S
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Congenic Murine Models of Marrow Transplantation and Assessment of Donor Chimerism by Flow Cytometry0 ^ s8 e8 b# b+ Z" S
6 a5 V' o% k9 ?' y: a s" oTransplantation of fresh unfractionated BM into 8- to 10-week-old congenic recipients conditioned as above was performed as described . Thus, in experiments using C57-EGFP donors, the percentage of donor leukocytes was determined by multiplying the fraction of EGFP-positive peripheral blood leukocytes by 1.12 to correct for EGFP expression.: E2 D) ~' T' q! x% ?
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Table 1. Conditioning regimens for congenic or syngenic bone marrow transplants- }/ v3 r. X. u* d. W! o
* f7 _: ~+ X+ e+ A. |Early Homing of Donor Cells to Bone Marrow
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C57 mice were conditioned with saline or G-CSF as described above prior to 160 cGy and transplanted 4 hours after irradiation with 20 x 106 fresh unfractionated BM cells from BoyJ donors via tail vein. Recipients were sacrificed 20 hours after transplantation, and marrow was collected from each recipient and analyzed for CD45.1 (donor) cells by flow cytometry .
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Long-Term Repopulating Activity of Conditioned Marrow
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Stem cell function in C57 mice treated with G-CSF, LD-TBI, or G-CSF followed by LD-TBI was assessed using a competitive repopulation assay adapted from Harrison .: k3 G# {/ z) O
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Colony-Forming Units-Fibroblast Assay$ j5 n _& Z0 Z0 D
5 c, y, Z% r0 H' iColony-forming units-fibroblast (CFU-F) assays adapted from Phinney et al. were performed on BM harvested from C57 mice conditioned with saline or G-CSF prior to 300 cGy (two or three mice per group) or G-CSF alone. BM was harvested from each group (at 4 hours after 300 cGy, if applicable) and from a cohort of untreated control mice by flushing femurs and tibias with -minimum essential medium (MEM) (Gibco, Rockville, MD, http://www.invitrogen.com). Marrow cells were counted using a hemocytometer. In our hands, the numbers of cells obtained from each of these long bones are similar, and we normalized the CFU-F frequency to the mean number of cells obtained per long bone. Cells from individual mice were plated in six-well plates at 1.5 x 106 and 3 x 106 cells per well and cultured in 4 ml of Dulbecco's modified Eagle's medium-high glucose (Gibco) containing 10% fetal calf serum, 1% penicillin-streptomycin, 1% HEPES, and 1% L-glutamine. Plates were incubated under 5% CO2 at 37¡ãC for a total of 8 days. On day 3, adherent cells were washed once with phosphate-buffered saline (PBS), fresh medium was added, and cells were incubated for an additional 5 days. On day 8, adherent cells were stained with Coomassie Blue, and colonies 1 mm in diameter were counted and scored. Assays were performed in triplicate.; g$ ~7 u) e& _* ~3 y
3 s3 J$ g0 \- ?7 d6 ~$ \1 PRetroviral Transduction of Bone Marrow and Syngenic Sex-Mismatched Transplantation of X-CGD Mice
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Transduction of BM cells by coculture with a high titer MSCV-m91 Neo GP E86 ecotropic packaging line was performed as previously described .
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Fluorescent In Situ Hybridization Analysis for Y Chromosome% F/ }, c+ \4 o3 Q) c. b
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Fluorescence in situ hybridization assays were adapted from D'Hondt et al. with modifications. Female recipient mice were bled by the tail vein. Erythrocyte lysis was performed, and leukocytes were washed twice with a fixative solution containing 75% methanol/25% glacial acetic acid. Cytospin slides displaying total leukocytes were prepared and aged at least overnight at 4¡ãC. Slides were denatured at 74¡ãC for 4 minutes in a ThermoBrite slide processing system (StatSpin, Westwood, MA, http://www.statspin.com). Hybridization was performed using a concentrated FITC-labeled Y chromosome painting probe (Cambio, Cambridge, U.K., http://www.cambio.co.uk) at 37¡ãC overnight. The unbound probe was removed by two sequential washes with stringency wash solutions. Counterstaining and mounting was performed with blue DAPI II Antifade (Vysis, Downers Grove, IL, http://www.vysis.com). Hybridization signals were detected using standard epifluorescent filters for FITC and DAPI in a fluorescent microscope (Leica, Wetzlar, Germany, http://www.leica.com). At least 200 cells from three fields were scored per sample. A 100% positive male control and a 0% positive female control were included in every assay.
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) P* Q( s4 u H5 \) y4 o; n. uAnalysis of Respiratory Burst Oxidase Activity in Peripheral Blood Neutrophils# i; N6 G: p9 X$ o4 o0 E/ `/ ]
+ X! e- s% ^% ~& e2 UThe oxidative burst of PB neutrophils was assessed using dihydrorhodamine 123 (DHR 123) (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) as previously described , with minor modifications. Briefly, after erythrocyte lysis, PB leukocytes were pre-incubated for 15 minutes at 37¡ãC with DHR 123 (10¨C5 M) in PBS containing 0.9 mg/ml glucose and 1% gelatin, followed by stimulation with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) for 15 minutes at 37¡ãC. Fluorescence was measured by flow cytometry before and after PMA on a FACSCalibur instrument using CellQuest software. The photomultiplier tube for side scatter was operated using linear amplification and for FL1 (420 nm) using logarithmic amplification. Granulocytes were identified by forward and side scatter analysis and confirmed using Gr-1 monoclonal antibody (BD Pharmingen). In all samples, 5,000¨C10,000 events were collected in the granulocyte gate.
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Statistical Analysis2 Q/ _) g+ y. K7 O; f, l; j3 F" j
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All values were calculated as mean ¡À SD. Statistical significance was determined using unpaired Student's or Welch's t test (two-tailed). Values were considered significant when p
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8 w) x4 l% E: E4 URESULTS
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* t8 U% q1 e; x; C4 gEffect of Sequential G-CSF and LD-TBI on Engraftment of Freshly Isolated Donor Marrow
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) H. L$ U% o1 }: |" TCongenic and syngenic BM transplant protocols were designed to test whether host conditioning with G-CSF enhanced donor engraftment in LD-TBI-treated recipient mice. The details of each transplant protocol, including donor/recipient strain, transplant cell dose, G-CSF treatment, and TBI dose, are summarized in Table 1.
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In initial experiments, recipient mice were treated with G-CSF or saline for 5 days prior to irradiation with 160 or 300 cGy given in a single dose 4 hours before infusion with fresh unfractionated congenic BM cells. At 4 months post-transplant, donor chimerism in BoyJ recipients conditioned with saline and 160 cGy prior to transplantation of 20 x 106 C57 cells was 43.4% ¡À 3.5%, compared with 65.2% ¡À 1.0% when recipients were first pretreated with G-CSF prior to irradiation (Fig. 1A). Similar results were seen using C57-EGFP mice as donors and C57 mice as recipients. The addition of G-CSF pretreatment to 160 cGy conditioning of recipients increased long-term donor chimerism from 31.7% ¡À 13.1% to 71.5% ¡À 2.4% following transplantation of 20 x 106 C57-EGFP marrow cells into C57 mice (Fig. 1B). Using 300 cGy as conditioning for transplantation of 2.5 x 106 C57-EGFP marrow cells, an almost 10-fold smaller cell dose, pretreatment of C57 recipients with G-CSF increased donor chimerism from 28.7% ¡À 5.3% to 55.2% ¡À 4.6% (Fig. 1C). A 1.5- to 2-fold higher level of donor chimerism was evident in G-CSF-treated recipients even at 1 month post-transplant and was maintained for at least 6¨C8 months (Fig. 1B, 1C), suggesting a sustained effect of G-CSF on donor engraftment in LD-TBI-conditioned recipients.
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* L% B" @! X) A% z+ k- iFigure 1. Peripheral blood leukocyte chimerism following transplantation of syngenic bone marrow cells. All recipient mice received twice-daily subcutaneous injections of 4 µg of G-CSF in saline () or saline alone () for 4 days. On day 5, mice received a single injection of G-CSF followed by 160 cGy (A, B) or 300 cGy (C, D). Values are mean ¡À SD (n = 4 or 5). *, p .05, saline versus G-CSF. (A): BoyJ recipients transplanted with 20 x 106 C57 bone marrow (BM) cells. (B): C57 recipients transplanted with 20 x 106 C57-EGFP BM cells. (C): C57 recipients transplanted with 2.5 x 106 C57-EGFP BM cells. (D): Splenectomized C57 mouse recipients transplanted with 2.5 x 106 C57-EGFP BM cells.& G' F0 Y0 W# J
A/ g7 d# C* f+ _( g* I f6 h4 N! ?The spleen plays an active role in hematopoiesis in mice. Following treatment with G-CSF, the spleen is in equilibrium with circulating cells, including G-CSF-mobilized HSC and progenitors . To examine whether the effect on donor cell engraftment by G-CSF was influenced by the presence of the spleen, we performed similar C57-EGFP marrow transplants into splenectomized C57 mice. No donor cells were detected at either 4 or 8 weeks post-transplant in the peripheral blood of nonirradiated splenectomized recipients receiving only G-CSF prior to transplant (data not shown). In splenectomized mice conditioned with 300 cGy, those pretreated with G-CSF prior to 300 cGy exhibited a higher level of donor cell chimerism compared with those pretreated with saline, and this difference in the level of engraftment remained stable for at least 8 months (54.5% ¡À 2.5% versus 33.7% ¡À 10.3%, respectively; p - F- Z, S- \, ~: L+ v9 X, A/ h# R
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Homing of Transplanted Marrow Cells to Recipient Marrow Following Sequential G-CSF and LD-TBI+ B4 C* P# b$ N) i n& |( u8 [
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An effect on the early homing of transplanted donor cells to recipient BM was examined as a potential mechanism by which G-CSF administration prior to LD-TBI might increase marrow engraftment. C57 mice were treated with G-CSF or saline for 5 days and 160 cGy on the day of the transplant. Both groups received infusions of 20 x 10 6 BoyJ BM cells 4 hours after irradiation. Twenty hours after transplantation, the recipients were sacrificed, and the marrow was examined for content of BoyJ cells. As can be observed in Figure 2A, in two independent experiments, the fraction of donor origin BoyJ (CD45.1 ) cells was significantly lower in recipients treated with G-CSF 160 cGy than in recipients treated with 160 cGy alone (1.0% ¡À 0.1% versus 3.6% ¡À 0.9% in the first experiment and 3.4% ¡À 0.9% versus 9.5% ¡À 2.3% in the second experiment, respectively; p
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: U( m# K8 u8 B1 ]Figure 2. Effect of G-CSF and low-dose total body irradiation (LD-TBI) on donor cell homing and marrow LTRA of recipient mice. (A): C57 mice were treated with G-CSF and 160 cGy () or saline and 160 cGy () and transplanted with 20 x 106 BoyJ BM cells 4 hours after LD-TBI. Percentage of donor chimerism in marrow 20 hours post-transplant was analyzed by flow cytometry for CD45.1. Two independent experiments were performed (n = 4¨C5 per group), as shown. The mean of each group is shown as a horizontal bar. p 7 f6 u1 K- w: ?7 Y- ]) M/ |7 x0 q% @5 y: E
% p1 E4 c5 [7 g$ E3 W: N, R$ ]* KImpact of Sequential Administration of G-CSF and LD-TBI on Marrow Long-Term Repopulating Activity) O7 U# c6 z, f3 K: z$ ?
5 H# c `( S9 p' X# w& DStem cell competition between donor and host and the availability of HSC niches are believed to be major factors determining the level of donor engraftment in marrow transplantation. Therefore, we examined the impact of pretreatment with G-CSF prior to LD-TBI on host marrow HSC function using a competitive repopulation assay. The relative LTRA of marrow from G-CSF plus 160 cGy-treated mice was approximately threefold lower than for saline plus 160 cGy-treated mice (10.5% ¡À 4.9% versus 35.8% ¡À 6.1%; p 7 d6 C$ \, b' j1 G. @4 t
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Effect of G-CSF on Marrow Microenvironment
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Bone marrow stromal cells modulate donor engraftment during BM transplantation, but little is known about the effect of conditioning regimens on these cells. The CFU-F assay has been demonstrated to determine marrow stroma progenitor numbers and their proliferative ability . Thus, we examined the impact of G-CSF and LD-TBI on marrow CFU-F content. The results showed that CFU-F was substantially lower in mice treated with saline plus 300 cGy compared with untreated controls (32 ¡À 8 versus 165 ¡À 21.2; p 6 J( @( r/ g% G* X) n! R8 t
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Figure 3. Effect of conditioning regimens on marrow stromal cells. Recipient mice were treated with G-CSF, saline 300 cGy, or G-CSF 300 cGy, and marrow harvested 4 hours after 300 cGy was plated for CFU-F. Data were collected at two cell concentrations, and assays were plated in triplicate. Values are the mean ¡À SD (n = 3 mice per group). *, p = .02; **, p
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Sequential Treatment with G-CSF and 300 cGy as Conditioning Regimen for Transplantation of Retroviral-Transduced X-CGD Marrow& |% y8 S1 g, i6 k7 J
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We next examined whether sequential treatment with G-CSF and 300 cGy could enhance engraftment of RMGT-treated BM cells. We tested this conditioning regimen using X-CGD marrow transduced with MSCV-m91Neo . To enable analysis of donor chimerism, a sex-mismatched X-CGD murine transplant model was used, in which female X-CGD mice were conditioned with saline or G-CSF prior to 300 cGy irradiation and transplanted with 2.5¨C5 x 106 male RMGT-treated X-CGD BM cells (Table 1). Fluorescent in situ hybridization for the presence of the Y chromosome in peripheral blood leukocytes was used to identify male cells (i.e., donor cells) at various time points post-transplant, and neutrophil oxidase activity assayed in parallel.9 c6 Q# ?) h) z# {. g8 B6 \& C
+ X+ i' G$ }- ?6 q B' k/ sIn two independent experiments, female X-CGD recipients treated with sequential G-CSF and LD-TBI prior to transplantation with male RMGT-treated cells had significantly higher chimerism for male leukocytes than recipients treated with saline and 300 cGy (Fig. 4A, 4C). The higher levels of donor engraftment in recipients treated with G-CSF prior to 300 cGy was evident at all time points post-transplant in both experiments, persisting for at least 8 months. In the first experiment, the mean donor chimerism at 8 months for mice transplanted with 5 x 106 RMGT-treated cells following conditioning with G-CSF and 300 cGy was 65% ¡À 7.1% (n = 5), compared with 49.6% ¡À 9.2% (n = 5) for mice treated with saline and 300 cGy (p = .018). Similar results were obtained in a second experiment (Fig. 4C). Male leukocyte chimerism was 56.6% ¡À 5.7% (n = 5) at 8 months in female recipients conditioned with G-CSF plus 300 cGy prior to transplantation with either 2.5 x 106 (n = 2) or 5 x 106 RMGT-treated cells (n = 3); little difference in donor chimerism was seen at these two cell doses (50% or 61% compared with 64%, 57%, or 60%, respectively). Only two recipients conditioned with saline plus 300 cGy were available for study in the second experiment, and they exhibited 38% and 40% donor cells at 8 months following transplantation with 2.5 x 106 and 5 x 106 RMGT-treated cells, respectively. An additional cohort of animals in the second experiment was conditioned with ablative radiation (950 cGy) prior to transplantation with 2 x 106 RMGT-treated cells, and had 92% ¡À 4% (mean ¡À SD, n = 4) donor leukocytes in peripheral blood 8 months post-transplant (Fig. 4C).
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a3 H1 I3 e, e1 M" [; zFigure 4. Engraftment of retroviral-mediated gene transfer (RMGT)-treated X-linked chronic granulomatous disease (X-CGD) male bone marrow and reconstitution of neutrophil NADPH oxidase activity in female X-CGD recipients. Results from two independent experiments are shown ( and transplanted with 2 x 106 RMGT-treated cells () (n = 4) or with either G-CSF () (n = 5) or saline () (n = 2) pretreatment prior to 300 cGy and transplantation with 2.5¨C5 x 106 RMGT-treated male X-CGD BM cells. Mean values ¡À SD are shown. *, p 6 \/ Z g: e( z- N
% v* T @! d+ q- VNADPH oxidase activity was also measured in PB neutrophils of recipient X-CGD mice after transplantation with RMGT-treated marrow. X-CGD neutrophils are NADPH oxidase-negative, and thus oxidase activity is a good marker for functional expression of the MSCV-m91Neo provirus. The presence of NADPH oxidase-positive neutrophils after 4¨C6 months post-transplant, which were detected in all groups (Fig. 4B, 4D, 4E), indicates successful engraftment of genetically corrected long-term repopulating cells. In recipients conditioned with 950 cGy, the fraction of oxidase-positive neutrophils ranged from 20% to 30% in the first 6 months post-transplant (Fig. 4E), with similar levels of provirus marking in secondary CFU-S12 (not shown), a measure that reflects gene transfer efficiency (e.g., .). In recipients receiving LD-TBI, there was a trend for a higher fraction of oxidase-positive neutrophils using the combined regimen of G-CSF and 300 cGy compared with saline and 300 cGy (Fig. 4B, 4D), although this did not reach statistical significance. In the cohort conditioned with sequential G-CSF and 300 cGy, the fraction of the total peripheral blood neutrophils that were oxidase-positive generally ranged from 5% to 15%. In the second experiment, taking into account overall male donor chimerism in the mice receiving LD-TBI, the calculated fraction of male donor marrow-derived neutrophils that were oxidase-positive was similar to that of recipients receiving ablative irradiation.1 V4 n5 y% c- H G7 q* x6 R; f
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There was variability in the fraction of oxidase-positive neutrophils detected over time in individual recipients, similar to our previous studies using this vector . Another explanation for a decline in NADPH oxidase-positive neutrophils is an immune response to the gp91phox, the X-CGD protein; this seems less likely given that fraction of oxidase-positive cells was stable in the many recipients in both the 300-cGy- and 950-cGy-conditioned groups.
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$ m- Z5 D+ a# C3 {) mPotential effects of the MSCV-m91Neo-transduction on hematopoiesis were monitored by measuring the peripheral blood count and leukocyte differential at various time points after transplantation (Table 2). At 4 or 8 months post-transplant, the results were similar to those in previous experiments using cohorts of mice transplanted with MSCV-91Neo-transduced bone marrow, where blood counts were similar to wild-type controls 8¨C10 months post-transplant; n = 16) was similar to that of wild-type mice (100 ¡À 40 mg; n = 10; 2¨C6 months of age).
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Table 2. Peripheral blood cell counts in recipients of MSCV-m91Neo-transduced marrow, D' p1 b- `8 w6 H! N4 H
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DISCUSSION, K) J3 S, K4 u9 \' t
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In this study, we show that sequential G-CSF and LD-TBI as conditioning of recipient mice prior to transplantation of freshly isolated congenic marrow cells enhanced long-term chimerism compared with treatment with LD-TBI alone. This combined regimen also resulted in higher long-term chimerism for donor cells in experiments using X-CGD recipient mice transplanted with marrow transduced with a retroviral vector for expression of the X-CGD protein. This confirms and extends results previously reported in a study by Mardiney and Malech using Sca-1 -purified marrow, where mice were followed for only 4 months post-transplant . This may reflect an impact of dose and schedule of G-CSF, the main difference between this report and our study; we used a G-CSF dose that was 40% higher and given twice a day rather than as a continuous infusion, and for 5 days instead of 7 days.9 b: a+ \5 P3 j# R2 x! N% L
7 n% M) U9 S+ t2 j/ XExamination of marrow long-term-repopulating activity, when measured at completion of the conditioning regimen, indicated that conditioning of recipients with a combination of G-CSF/LD-TBI significantly decreased the content of functional HSCs compared with either G-CSF or LD-TBI alone. In contrast, the addition of G-CSF did not either increase early homing of donor cells or affect the content of marrow CFU-F compared with LD-TBI alone. Since the effect of the combined regimen on recipient long-term repopulating activity correlated with the enhancement of donor chimerism, we propose that this affords a competitive advantage to transplanted HSCs. This may be due to there being more available functional niches given that G-CSF is well-known to mobilize hematopoietic stem and progenitor cells from the marrow and that LD-TBI itself reduces marrow LTRA (Fig. 2; , suggest that having more HSC niches available at the time of transplantation can enhance long-term repopulation with donor marrow./ v0 g% a* m9 F+ I7 I* m* E3 k
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An important potential application of combined G-CSF and LD-TBI conditioning is to improve the outcome of autologous transplantation of genetically modified HSC for treatment of genetic defects in hematopoietic cells that do not have an intrinsic selective advantage following genetic correction. Approaches to enhance engraftment of RMGT-treated cells in the absence of marrow ablation are desirable, since BM cells cultured ex vivo for RMGT have impaired repopulation ability, leading to lower donor chimerism in congenic mouse models of autologous transplantation with nonablative conditioning .
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8 }2 Z+ _7 ~" U& HCONCLUSION; H( z8 i; `! l( j4 b( H9 V
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In conclusion, pretreatment with G-CSF prior to nonmyeloablative irradiation has attracted attention as a preparative regimen for HSC transplantation . Here, we show that sequential treatment with G-CSF and LD-TBI decreased the content of marrow HSC in mice as measured by competitive repopulating activity in vivo, which is associated with improved long-term engraftment of congenic or syngenic marrow and which also facilitated long-term chimerism with RMGT-treated BM cells. These results strengthen the possibility that similar approaches can be used in the clinic to augment nonmyeloablative conditioning in autologous transplantation for gene therapy of inherited blood cell disorders.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST ^ ]. |1 E# x+ i
7 K& Z# s( l1 K- tThe authors indicate no potential conflicts of interest.( c1 Q) {& G9 n a4 W2 u8 P! e5 P
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We thank Shari Upchurch for assistance with manuscript preparation and Natalie Stull for maintaining the mouse colony. This work was supported by NIH Grants P01-HL53586 (M.C.D.), K08-HL75253 (W.S.G.), American Cancer Society Grant IRG-84-002-19 (W.S.G.), Clarian Health Partners Values Fund for Research Grant VFR-145 (W.S.G.), and the Riley Children's Foundation.6 O8 P5 u# |8 }# c8 C9 g0 `
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