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Comparison of Retroviral Transduction Efficiency in CD34⁺ Cells Derived from Bone Marrow versus G-CSF–Mobilized or G-CSF Plus Stem Cell Factor–Mobilized Peripheral Blood in Nonhuman Primates

^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{at}intra.niddk.nih.gov

	ABSTRACT

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Hematopoietic stem cells (HSCs) are ideal targets for genetic manipulation in the treatment of several congenital and acquired disorders affecting the hematopoietic compartment. Although G-CSF–mobilized peripheral blood CD34⁺ cells are the favored source of hematopoietic stem cells in clinical transplantation, this source of stem cells does not provide meaningful engraftment levels of genetically modified cells compared with G-CSF + stem cell factor (SCF)–mobilized cells in nonhuman primates. Furthermore, the use of G-CSF mobilization can have disastrous consequences in patients with sickle cell disease, a long-held target disorder for HSC-based gene therapy approaches. We therefore conducted a study to compare the levels of genetically modified cells attainable after retroviral transduction of CD34⁺ cells collected from a bone marrow (BM) harvest with CD34⁺ cells collected from a leukapheresis product after mobilization with G-CSF (n = 3) or G-CSF in combination with SCF (n = 3) in the rhesus macaque autologous transplantation model. Transductions were performed using retroviral vector supernatant on fibronectin-coated plates for 96 hours in the presence of stimulatory cytokines. BM was equal to or better than G-CSF–mobilized peripheral blood as a source of HSCs for retroviral transduction. Although the highest marking observed was derived from G-SCF + SCF–mobilized peripheral blood in two animals, marking in the third originated only from the BM fraction. These results demonstrate that steady-state BM is at least equivalent to G-CSF–mobilized peripheral blood as a source of HSCs for retroviral gene transfer and the only currently available source for patients with sickle cell disease.

	INTRODUCTION

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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 [1–5]. 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 [6–9]. 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 [10]. 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 [11], 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 ß-globin gene along with key regulatory elements has also recently been achieved [12]. Furthermore, alternative anti-sickling genes have shown promise using similar vector systems [13–15]. 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 [16]. Thomasson et al. [17] 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 [18–20].

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

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Collection of PB HSCs from Rhesus Macaques
Young rhesus macaques (Macaca mulatta) used in these studies were housed and handled in accordance with the guidelines set by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHHS publication No. NIH 85-23), and the protocol was approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute. BM harvesting was performed on each animal as previously described [21]. Three days later, animals were started on either recombinant human (rhu) G-CSF (10 µg/kg; Amgen, Thousand Oaks, CA) alone or in combination with recombinant polyethylene glycol–conjugated human SCF (200 µg/kg; Amgen) as daily subcutaneous injections for 3 days and twice daily on the fourth day. Mobilized PB cells were collected by leukapheresis on day 5 (1 week after BM harvest) as described [21]. Mononuclear cells were isolated using density-gradient centrifugation over lymphocyte separation media (ICN Biomedicals/Cappel, Aurora, OH). CD34⁺ enrichment was performed using the 12.8 immunoglobulin M anti-CD34 biotinylated antibody and MACS streptavidin microbeads (Miltenyi Biotec, Auburn, CA). The degree of progenitor enrichment was determined by calculating the fold increase in colony-forming units (CFUs) achieved by column purification.

Vectors and Transduction Procedure
G1Na and LNL6 are amphotropic Moloney murine leukemia virus–derived retroviral vectors that carry an identical bacterial neomycin phosphotransferase (neo) gene. A 16-base pair polylinker insertion 5' of the neo gene allows quantitative assessment of marking from the two vectors within one polymerase chain reaction (PCR). The biologic titers of these vectors were assessed by making serial dilutions of the vectors and then monitoring the transfer of G418 resistance to HeLa cells. The biologic titers of both vectors were equivalent and were between 2 to 5 x 10⁵ biologically active vector particles per milliliter. For transduction, retro-viral supernatant was harvested from subconfluent producer cells cultured for 12–18 hours in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 4 mM L-glutamine, penicillin (50 mg/ml), and streptomycin (50 mg/ml) at 37°C in 5% CO₂. Fresh vector supernatant was passed through a 0.22-µm filter (Millipore, Bedford, MA) to remove cellular debris before transduction.

CD34-enriched cells were cultured at a starting concentration of 1 to 2 x 10 ⁵ cells/ml in filtered vector supernatant, supplemented with 100 ng/ml rhuSCF, 100 ng/ml rhuFlt-3 L, 20 ng/ml interleukin (IL)-3, and 50 ng/ml IL-6, in flasks previously coated with the CH-296 fragment of fibronectin (Retronectin, TaKara, Shiga, Japan) per manufacturer’s instructions. We have previously shown that this combination of cytokines produces in vivo marking results comparable to a cytokine combination of SCF, Flt3-L, and MGDF [7]. Every 24 hours, nonadherent cells were collected, spun down, and resuspended in fresh vector supernatant and cytokines and added back to the same fibronectin-coated flask. At the end of 96 hours, cultured cells were removed from the plates with trypsin, collected, and frozen viably in 50% autologous serum mixed with 40% DMEM and 10% dimethylsulfoxide, placed on dry ice, and then transferred to a liquid-nitrogen container.

One week later, the animals received 500 cGy x 2 total body irradiation, and both aliquots of transduced cells were thawed and reinfused via a central venous catheter. Twenty-four hours later, the animals were started on G-CSF at 5 µg/kg i.v. daily until the total white blood cell count reached 6,000/ml. Standard supportive care, including blood product transfusions, fluid and electrolyte management, and antibiotics, was administered as needed. Hematopoietic recovery was monitored by daily complete blood counts.

Sample Collection
PB samples were collected at the time of recovery, monthly through 6 months after transplantation, and then every 3 months. From each blood sample, mononuclear cells were isolated by density-gradient centrifugation, and granulocytes were obtained as previously described [7].

CFU Assays
The degree of progenitor enrichment was calculated from CFU assays performed before and after column purification. CFU assays were done using MethoCult H4230 methylcellulose media (StemCell Technologies, Vancouver, Canada) supplemented with 5 U/ml rhu erythropoietin (Amgen), 10 ng/ml rhuGM-CSF (Sandoz, East Hanover, NJ), 10 ng/ml rhuIL-3, (Sandoz), and 100 ng/ml rhuSCF (Amgen) at 37°C in 5% CO₂. Between days 10 and 14, colonies of more than 50 cells were counted, and 16 to 32 individual CFUs were plucked from the plates at each time point for PCR analysis. Colonies were plucked into 50 µl of distilled water, digested with 20 µg/ml proteinase K (Qiagen, Valencia, CA) at 55°C for 1 hour followed by 99°C for 10 minutes, and later assessed for the presence of vector sequences by nested PCR as described below.

PCR Analysis
Genomic DNA was extracted using the QIAamp DNA blood Midi kit (Qiagen). The primers and conditions used for neo PCR and ß-actin PCR have been previously described [22]. All neo and ß-actin PCR reactions were run under conditions optimized to yield linear results in the range of the intensity of the in vivo samples. DNA, 100–200 ng, was used for the outer reaction, and 18 to 20 cycles were performed for the inner reaction, based on the level of in vivo marking. For every PCR analysis, negative controls included DNA from normal rhesus PB samples extracted with the same methodology and a reagent control. Serial dilutions of G1Na DNA (containing two copies of integrated vector per cell) into normal rhesus PB DNA were used as positive controls for generating the control regression curve. Band intensity was quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Neo band intensity was normalized for amplifiable DNA content based on the ß-actin signal, and the overall contribution of each vector to in vivo marking was calculated by plotting the signal intensity of each band on a standard curve derived from known copy number controls amplified concurrently.

Statistical Analysis
Analysis of significance using the two-tailed Student’s t-test and regression analysis were carried out using SigmaPlot (SPSS Science, Chicago) and Excel software (Microsoft, Seattle).

	RESULTS

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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.

View larger version (28K): [in this window] [in a new window]   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.

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.

View larger version (38K): [in this window] [in a new window]   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. ß-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.

View larger version (21K): [in this window] [in a new window]   Figure 3. Summary of the percent positive granulocytes at different time points (in months [m]) 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

Top Abstract Introduction Materials and Methods Results Discussion References

Although clinical trials may be on the horizon for globin disorders, several issues, including stem cell source, need for marrow conditioning or in vivo selection, optimal vector design, and risk for insertional mutagenesis, remain unresolved and warrant additional investigation before proceeding to the clinic. The target cell population may have a significant impact on the efficiency of gene transfer in vivo; CD34⁺ cells collected after G-CSF + SCF administration were previously shown superior to CD34⁺ cells collected after G-CSF alone or G-CSF + Flt3-L administration [16]. The difficulty in transducing G-CSF–mobilized cells could in part explain the disappointing results obtained in some gene therapy clinical trials targeting G-CSF–mobilized HSCs. Because available growth factors cannot be used to mobilize stem cells in patients with sickle cell disease, we designed the current study to compare the transduction efficiency of CD34⁺ cells collected by BM harvest at steady state to CD34⁺ cells collected from the PB after growth factor mobilization.

Overall, our results suggest improved gene transduction efficiency using BM cells when compared with G-CSF–mobilized cells, the current preferred source of HSCs for transplantation and gene therapy applications. Additionally, our results again suggest an advantage for G-CSF + SCF–mobilized CD34⁺ cells as targets for retroviral gene transfer. A recent report using a canine model also describes poor gene transfer efficiency in G-CSF–mobilized CD34⁺ cells [17]. Interestingly, their results indicate that transduction of BM cells primed with G-CSF + SCF administration resulted in levels of marking higher than those obtained using G-CSF + SCF–mobilized CD34⁺ cells. However, SCF is no longer clinically available in the U.S., and for patients with SCA, severe sickle cell crises and even death have been reported after use of G-CSF for mobilization [18–20]. Although the results of our study are supportive of the use of steady-state BM as a source of HSCs for transduction using retroviral vectors under the conditions of this study, the variability of the outbred nonhuman primate model along with the high cost precludes larger comparative studies often required to reach statistical significance. Nonetheless, our results demonstrate that steady-state BM is a viable source of stem cells for disorders for which stem cell mobilization is problematic and presently represents the best clinically available source of HSCs for gene therapy applications in patients with SCA.

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