Clonal Analysis of Individual Marrow-Repopulating Cells after Experimental Peripheral Blood Progenitor Cell Transplantation
http://www.100md.com
《干细胞学杂志》
a German Cancer Research Center, Heidelberg, Germany;
b EUFETSAG, Idar-Oberstein, Germany;
c Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany
Key Words. Experimental PBPC transplantation ? SCID-repopulating cells ? Single cell analysis
Correspondence: Stefan Fruehauf, M.D., Department of Internal Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Telephone: 49-6221-562781; Fax: 49-6221-565722; e-mail: stefan_fruehauf@med.uni-hei-delberg.de
ABSTRACT
Recently, two cases of insertional mutagenesis have been described following retroviral gene transfer to human CD34+-selected bone marrow (BM) cells . Methods to monitor and quantify the contribution of individual retrovirally transduced marrow-repopulating cells to hematopoietic engraftment with high sensitivity and reproducibility would allow the recognition of aberrant proliferation at an early stage. For example, patients with leukemia having normal blood counts after high-dose therapy and stem cell transplantation are monitored for minimal residual disease. An increase in the clonal size of leukemic cells by one log, as detected with real-time polymerase chain reaction (PCR), can predict relapse . In the field of retrovirus-mediated gene therapy, quantitative and semiquantitative protocols to monitor all transduced cells have been applied. Additionally, a quantitative competitive PCR protocol to quantify individual transduced clones has been described, whereas highly accurate protocols using real-time PCR to quantify individual hematopoietic clones have not been reported yet.
We previously established a transplantation model for human retrovirally transduced peripheral blood progenitor cells (PBPCs) in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice . The retroviral vector used for transduction contains the human multidrug resistance 1 gene (MDR1) as a transgene. The retroviral vector—through its integration into the genomic DNA—serves as a unique tag of individually transduced cells and their progeny. We characterized retroviral integration sites in human marrow-repopulating cells .
To determine unique junctions between proviral and human genomic DNA, we used a ligation-mediated (LM)-PCR technique, cloned the LM-PCR products into plasmids, thus generating a plasmid library, and sequenced all the obtained proviral-genomic DNA junctions . The plasmids obtained in the cloning step were used later for quantitation of individual hematopoietic clones using real-time PCR technique .
Our results allow us to draw conclusions on the clone size with high sensitivity and specificity, suggesting that a plethora of individual hematopoietic cells are simultaneously contributing to human hematopoietic BM engraftment. The quantitative analysis of individual transduced cell clones also offers a means to analyze the clonality of an adverse event in preclinical or clinical stem cell gene therapy protocols.
MATERIALS AND METHODS
Our aim was to assess the contribution of individual progenitor cells to human hematopoietic engraftment in the BM of NOD/SCID mice (SCID-repopulating cells ). In our experiments, purified CD34+ PBPCs (CD34+ >87.9%) were retrovirally transduced and transplanted in a total of seven mice. The proportion of human CD45+ leukocytes in the chimeric NOD/SCID BM amounted to a median of 13% (range, 7%–42%). The proviral MDR1-DNA was found in a median of 23% (range, 13%–31%) of human cells, and a median of 10% (range, 4%–13%) of the human leukocytes expressed the MDR1 transgene (Table 1). The granularity of the leukocytes shows that both myeloid and lymphoid human cell populations harbored the transgene (Fig. 1). Individual hematopoietic clones were identified by their unique retroviral integration sites into the genomic DNA.
Table 1. Data of retrovirally transduced peripheral blood progenitor cells (PBPCs) in nonobese diabetic/severe combined immunodeficient mouse bone marrow
Figure 1. Flow cytometry analysis of retrovirally transduced chimeric mouse bone marrow (BM) (E17M19). (A): Six weeks after transplantation of retrovirally transduced CD34+ peripheral blood progenitor cells into nonobese diabetic/severe combined immunodeficient mouse, chimeric BM cells were obtained and analyzed for the presence of human leukocytes (R1). (B): Expression of the functional transcript—the plasma membrane P-glycoprotein—of the multidrug resistance 1 gene (MDR1) transgene in engrafted human CD45+ cells was measured. P-glycoprotein function in human leukocytes (R1) was proved by rhodamin-123 dye exclusion (R2). (C): MDR1 expression in the lymphoid and myeloid progeny of human transduced cells (R2) recovered from the chimeric mouse is demonstrated by the typical scatter profile . Expression of lymphoid and myeloid markers, respectively, in these two cell populations was confirmed by staining for CD3, CD7, CD10, CD11b, CD13, CD22, and CD64 (data not shown).
Detection of Retroviral Integration Sites
Chimeric BM DNA from seven mice was used to identify unique junctions between proviral and human genomic DNA sequence. Following an LM-PCR and cloning reaction (Fig. 2), we detected a median of 7 (range, 2–13) integration sites per chimeric BM (Table 1). Repeated LM-PCR analysis contributed to the total number of integration sites in sample E15M1 (Table 2). Six LM-PCR reactions did not seem to be enough to detect all integration sites, because only three reactions contained repeated junction fragments. Because of insufficient DNA from this mouse, we could not perform additional LM-PCR reactions. Identical integration site sequences were repeatedly found (Table 2, LM-PCR 1 and 2, E15M1EB9 and E15M1EB11). In some cases, PCR bands of different lengths contained the same integration site sequence (Fig. 2 and Table 2, E15M1EB9, PCR bands I and III, respectively), and one PCR band contained two integration site sequences (Fig. 2 and Table 2, LM-PCR 1, PCR band I). Because we cloned the PCR bands into plasmids, and several plasmids from the same cloning reaction were sequenced, it was possible to distinguish between the junction fragments with identical lengths but different sequences contained in the same PCR band. From the other six chimeric BM DNA samples, one LM-PCR analysis per sample was the basis for assembling the integration-site library. No integration sites were detected in mice transplanted with mocktransduced cells, confirming the specificity of our method (Fig. 2).
Figure 2. Reproducibility of the ligation-mediated polymerase chain reaction (LM-PCR) method. LM-PCR products were analyzed by agarose gel electrophoresis. Lanes 1, 2, and 3 show the PCR products of three separate LM-PCRs. Roman numbers denote PCR bands shown in Table 2. Identical integration site sequences were repeatedly found (LM-PCR 1 and 2, E15M1EB9, and E15M1EB11). In some cases, PCR bands of different lengths contained the same integration site sequence (E15M1EB9 and PCR bands I and III, respectively), and one PCR band contained two integration site sequences (LM-PCR 1 and PCR band I). The mock lane shows an LM-PCR of a control mouse that received transplants of untransduced (mock-transduced) human CD34+ cells. IB indicates internal band containing only vector sequences, thus representing an internal control of the LM-PCR method. Abbreviation: M, size marker.
Table 2. Reproducibility of the ligation-mediated polymerase chain reaction (LM-PCR) method
The chromosomal localization of the integration sites is defined by the unique accession numbers. The obtained accession numbers are listed in the order of integration sites, as follows: E15M1EB–E15M1EB13: Z83847 , Ac015823 Ac010746, Al445667, Al049569, Al355149, Ac005056, Al050317, Ac025471, Ac020904, Ac084854, Ac005632, Ac008533; E15M5K2–E15M5K8: Ac093759, Ap002907, Ap001437, Ac011979, Ac026703, Ac011501, Ac072052; E15M6K2–E15M6K6: AL157935 .28, AL391239 , Al137141, Al157877, Ac090117; E15M15K1–E15M15K7: Ac008760, Ac068134, Ac005208, Ac012100, Al138999, Al390961, Ac105245; E17M19K2–E17M19K5: AL162832 .6, AC009116 .8, AL136218 .26, AL160169 .12; E17M19K8–E17M19K10: AC130343 .7, AC003030 .1, AC026469 .8; E18M22K2–E18M22K4: AL445903 .3, BX005019 .10, AC011456 .2; E18M24K1–E18M24K2: AC036103 .8 and AC005208 .1. The integration sites not listed could not be mapped. The chromosomal localizations of the clones are given in Table 1.
Quantitation of Individual Clones
To quantify the clones detected by LM-PCR, we used a real-time quantitative PCR method. Unique primers specific for the flanking genomic DNA were designed for each integration site sequence, whereas the retroviral-specific primer and fluorogenic probe were identical for all integration sites. To test the specificity of the real-time quantitative PCR primer and probe set, we performed reactions with the corresponding plasmid and control plasmids. Reactions were only positive with the primer designed for a given clone (e.g., plasmid E15M1EB7 with primer for clone E15M1EB7) and were completely negative with primers designed for other clones (e.g., plasmid E15M1EB7 with primers for clones E15M1EB9 and E15M1EB11), thus demonstrating a very high specificity of the designed unique primers.
In a mixture of three plasmids each containing a different LM-PCR product, we were able to detect the proportion of an individual plasmid down to 10 copies in a total copy number of 105 (13 dilution steps; Fig. 3) using real-time quantitative PCR. To test the reproducibility of the results, three independent replicates were performed for each integration site sequence. The mean coefficient of variation was 0.16 (range, 0.0001–0.42).
Figure 3. Quantitation of an individual plasmid in a plasmid mixture. Three plasmids containing different ligation-mediated polymerase chain reaction product inserts were mixed, and the proportion of individual plasmids was studied. The error bars represent the standard deviation of three separate experiments. For example, plasmid A was detected with an accuracy of r = 0.99, p < .0001, as calculated by regression analysis.
Contribution of Individual Human Marrow-Repopulating Cells to Hematopoiesis
The amount of all transduced cells was determined as the copy number of the MDR1 transgene by real-time quantitative PCR and ranged between 263 and 6,807 copies per μg DNA (median, 1,380.5 copies per μg DNA; Table 3). A total of 17 integration site sequences from three different donors detected by LM-PCR were analyzed. Individual clone copy numbers ranged from 0.02–124 per μg DNA (median, 2.2 per μg DNA; Table 3). The ratio of the mean copy number of an individual clone and the mean copy number of the MDR1 transgene gives an estimate of the contribution of this clone to human transduced hematopoiesis. The analyzed individual marrow-repopulating cell clones contributed to 0.01%–1.82% of human retrovirally transduced hematopoiesis (median, 0.15%; Table 3). Considering the level of engraftment and the proportion of MDR1 transgene-marked cells, the transduced clones analyzed here amounted to 0.001%–0.4% of all human cells and to 0.0001%–0.14% (median, 0.03%) of all human and mouse cells in chimeric BM (Table 3). Interestingly, the clones, which were repeatedly found in separate LM-PCR analyses (Fig. 2 and Table 2), showed the highest engraftment (1.82% for E15M1EB11 and 1.23% for E15M1EB9).
Table 3. Proportion of individual clones among the transduced human severe combined immunodeficient mouse repopulating cells
DISCUSSION
The technical assistance of Bernhard Berkus, Hans-Jürgen Engel, Sigrid Heil (German Cancer Research Center), and Carmen Hoppstock (Department of Internal Medicine V, University of Heidelberg) and the support of the animal facility team of the German Cancer Research Center are gratefully acknowledged. We thank Birgit St?hle (EUFETS AG) for her help at the start of this project. This work was supported in part by grant 10-1294-Ze3 from the Deutsche Krebshilfe/Dr. Mildred-Scheel-Stiftung, grant M 20.4 from the H.W. & J. Hector-Stiftung, and by a grant from the AiP plus Forschung program of the University of Heidelberg.
K.Z.N. and S.L. contributed equally to these results.
REFERENCES
Hacein-Bey-Abina S, Von Kalle C, Schmidt M et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302:415–419.
Krauter J, Gorlich K, Ottmann O et al. Prognostic value of minimal residual disease quantification by real-time reverse transcriptase polymerase chain reaction in patients with core binding factor leukemias. J Clin Oncol 2003;21:4413–4422.
Schilz AJ, Kuhlcke K, Fauser AA et al. MDR1 gene expression in NOD/SCID repopulating cells after retro-viral gene transfer under clinically relevant conditions. Mol Ther 2000;2:609–618.
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.
Schmidt M, Carbonaro DA, Speckmann C et al. Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates. Nat Med 2003;9:463–468.
Schiedlmeier B, Kuhlcke K, Eckert HG et al. Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice. Blood 2000;95:1237–1248.
Laufs S, Gentner B, Nagy KZ et al. Retroviral vector integration occurs in preferred genomic targets of human bone marrow-repopulating cells. Blood 2003;101:2191–2198.
Heid CA, Stevens J, Livak KJ et al. Real time quantitative PCR. Genome Res 1996;6:986–994.
Alizadeh M, Bernard M, Danic B et al. Quantitative assessment of hematopoietic chimerism after bone-marrow transplantation by real-time quantitative polymerase chain reaction. Blood 2002;99:4618–4625.
Veldwijk MR, Topaly J, Laufs S et al. Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks. Mol Ther 2002;6:272–278.
Schiedlmeier B, Schilz AJ, Kuhlcke K et al. Multidrug resistance 1 gene transfer can confer chemoprotection to human peripheral blood progenitor cells engrafted in immunodeficient mice. Hum Gene Ther 2002;13:233–242.
Abonour R, Williams DA, Einhorn L et al. Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells. Nat Med 2000;6:652–658.
Fruehauf S, Veldwijk MR, Berlinghoff S et al. Gene therapy for sarcoma. Cells Tissues Organs 2002;172:133–144.
Eckert HG, Kuhlcke K, Schilz AJ et al. Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1). Bone Marrow Transplant 2000;25(suppl 2):114–117.
Hildinger M, Abel KL, Ostertag W et al. Design of 5' untranslated sequences in retroviral vectors developed for medical use. J Virol 1999;73:4083–4089.
Fruehauf S, Breems DA, Knaan-Shanzer S et al. Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area-forming cell assay and detection of vector-mediated P-glycoprotein expression by rhodamine-123. Hum Gene Ther 1996;7:1219–1231.
Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329–1337.
Cashman JD, Lapidot T, Wang JC et al. Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice. Blood 1997;89:4307–4316.
Gothot A, van der Loo JC, Clapp DW et al. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998;92:2641–2649.
Akashi K, Traver D, Miyamoto T et al. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 2000;404:193–197.
Dick JE, Guenechea G, Gan OI et al. In vivo dynamics of human stem cell repopulation in NOD/SCID mice. Ann N Y Acad Sci 2001;938:184–190.
Guenechea G, Gan OI, Dorrell C et al. Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2001;2:75–82.
Horn PA, Thomasson BM, Wood BL et al. Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates. Blood 2003;102:4329–4335.
Rosenthal A, Jones DS. Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucleic Acids Res 1990;18:3095–3096.
Schmidt M, Zickler P, Hoffmann G et al. Polyclonal long-term repopulating stem cell clones in a primate model. Blood 2002;100:2737–2743.
Dynabeads M-280 Streptavidin. Dynal, Oslo, Norway.
Nolta JA, Dao MA, Wells S et al. Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice. Proc Natl Acad Sci U S A 1996;93:2414–2419.
Bhatia M, Wang JC, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997;94:5320–5325.
Fruehauf S, Veldwijk MR, Kramer A et al. Delineation of cell cycle state and correlation to adhesion molecule expression of human CD34+ cells from steady-state bone marrow and peripheral blood mobilized following G-CSF-supported chemotherapy. STEM CELLS 1998;16:271–279.
Brunning et al. WHO Classification of Tumors: Pathology & Genetics. Tumors of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press, 2001:77.
Fruehauf S, Topaly J, Wilmes A, et al. Flow cytometric diagnostic of malignant h?matologic diseases. In: Zeller WJ, zur Hausen H, eds. Onkologie. Landsberg/Lech: Ecomed Verlag, 2000:1–32.(K. Zsuzsanna Nagya, Steph)
b EUFETSAG, Idar-Oberstein, Germany;
c Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany
Key Words. Experimental PBPC transplantation ? SCID-repopulating cells ? Single cell analysis
Correspondence: Stefan Fruehauf, M.D., Department of Internal Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Telephone: 49-6221-562781; Fax: 49-6221-565722; e-mail: stefan_fruehauf@med.uni-hei-delberg.de
ABSTRACT
Recently, two cases of insertional mutagenesis have been described following retroviral gene transfer to human CD34+-selected bone marrow (BM) cells . Methods to monitor and quantify the contribution of individual retrovirally transduced marrow-repopulating cells to hematopoietic engraftment with high sensitivity and reproducibility would allow the recognition of aberrant proliferation at an early stage. For example, patients with leukemia having normal blood counts after high-dose therapy and stem cell transplantation are monitored for minimal residual disease. An increase in the clonal size of leukemic cells by one log, as detected with real-time polymerase chain reaction (PCR), can predict relapse . In the field of retrovirus-mediated gene therapy, quantitative and semiquantitative protocols to monitor all transduced cells have been applied. Additionally, a quantitative competitive PCR protocol to quantify individual transduced clones has been described, whereas highly accurate protocols using real-time PCR to quantify individual hematopoietic clones have not been reported yet.
We previously established a transplantation model for human retrovirally transduced peripheral blood progenitor cells (PBPCs) in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice . The retroviral vector used for transduction contains the human multidrug resistance 1 gene (MDR1) as a transgene. The retroviral vector—through its integration into the genomic DNA—serves as a unique tag of individually transduced cells and their progeny. We characterized retroviral integration sites in human marrow-repopulating cells .
To determine unique junctions between proviral and human genomic DNA, we used a ligation-mediated (LM)-PCR technique, cloned the LM-PCR products into plasmids, thus generating a plasmid library, and sequenced all the obtained proviral-genomic DNA junctions . The plasmids obtained in the cloning step were used later for quantitation of individual hematopoietic clones using real-time PCR technique .
Our results allow us to draw conclusions on the clone size with high sensitivity and specificity, suggesting that a plethora of individual hematopoietic cells are simultaneously contributing to human hematopoietic BM engraftment. The quantitative analysis of individual transduced cell clones also offers a means to analyze the clonality of an adverse event in preclinical or clinical stem cell gene therapy protocols.
MATERIALS AND METHODS
Our aim was to assess the contribution of individual progenitor cells to human hematopoietic engraftment in the BM of NOD/SCID mice (SCID-repopulating cells ). In our experiments, purified CD34+ PBPCs (CD34+ >87.9%) were retrovirally transduced and transplanted in a total of seven mice. The proportion of human CD45+ leukocytes in the chimeric NOD/SCID BM amounted to a median of 13% (range, 7%–42%). The proviral MDR1-DNA was found in a median of 23% (range, 13%–31%) of human cells, and a median of 10% (range, 4%–13%) of the human leukocytes expressed the MDR1 transgene (Table 1). The granularity of the leukocytes shows that both myeloid and lymphoid human cell populations harbored the transgene (Fig. 1). Individual hematopoietic clones were identified by their unique retroviral integration sites into the genomic DNA.
Table 1. Data of retrovirally transduced peripheral blood progenitor cells (PBPCs) in nonobese diabetic/severe combined immunodeficient mouse bone marrow
Figure 1. Flow cytometry analysis of retrovirally transduced chimeric mouse bone marrow (BM) (E17M19). (A): Six weeks after transplantation of retrovirally transduced CD34+ peripheral blood progenitor cells into nonobese diabetic/severe combined immunodeficient mouse, chimeric BM cells were obtained and analyzed for the presence of human leukocytes (R1). (B): Expression of the functional transcript—the plasma membrane P-glycoprotein—of the multidrug resistance 1 gene (MDR1) transgene in engrafted human CD45+ cells was measured. P-glycoprotein function in human leukocytes (R1) was proved by rhodamin-123 dye exclusion (R2). (C): MDR1 expression in the lymphoid and myeloid progeny of human transduced cells (R2) recovered from the chimeric mouse is demonstrated by the typical scatter profile . Expression of lymphoid and myeloid markers, respectively, in these two cell populations was confirmed by staining for CD3, CD7, CD10, CD11b, CD13, CD22, and CD64 (data not shown).
Detection of Retroviral Integration Sites
Chimeric BM DNA from seven mice was used to identify unique junctions between proviral and human genomic DNA sequence. Following an LM-PCR and cloning reaction (Fig. 2), we detected a median of 7 (range, 2–13) integration sites per chimeric BM (Table 1). Repeated LM-PCR analysis contributed to the total number of integration sites in sample E15M1 (Table 2). Six LM-PCR reactions did not seem to be enough to detect all integration sites, because only three reactions contained repeated junction fragments. Because of insufficient DNA from this mouse, we could not perform additional LM-PCR reactions. Identical integration site sequences were repeatedly found (Table 2, LM-PCR 1 and 2, E15M1EB9 and E15M1EB11). In some cases, PCR bands of different lengths contained the same integration site sequence (Fig. 2 and Table 2, E15M1EB9, PCR bands I and III, respectively), and one PCR band contained two integration site sequences (Fig. 2 and Table 2, LM-PCR 1, PCR band I). Because we cloned the PCR bands into plasmids, and several plasmids from the same cloning reaction were sequenced, it was possible to distinguish between the junction fragments with identical lengths but different sequences contained in the same PCR band. From the other six chimeric BM DNA samples, one LM-PCR analysis per sample was the basis for assembling the integration-site library. No integration sites were detected in mice transplanted with mocktransduced cells, confirming the specificity of our method (Fig. 2).
Figure 2. Reproducibility of the ligation-mediated polymerase chain reaction (LM-PCR) method. LM-PCR products were analyzed by agarose gel electrophoresis. Lanes 1, 2, and 3 show the PCR products of three separate LM-PCRs. Roman numbers denote PCR bands shown in Table 2. Identical integration site sequences were repeatedly found (LM-PCR 1 and 2, E15M1EB9, and E15M1EB11). In some cases, PCR bands of different lengths contained the same integration site sequence (E15M1EB9 and PCR bands I and III, respectively), and one PCR band contained two integration site sequences (LM-PCR 1 and PCR band I). The mock lane shows an LM-PCR of a control mouse that received transplants of untransduced (mock-transduced) human CD34+ cells. IB indicates internal band containing only vector sequences, thus representing an internal control of the LM-PCR method. Abbreviation: M, size marker.
Table 2. Reproducibility of the ligation-mediated polymerase chain reaction (LM-PCR) method
The chromosomal localization of the integration sites is defined by the unique accession numbers. The obtained accession numbers are listed in the order of integration sites, as follows: E15M1EB–E15M1EB13: Z83847 , Ac015823 Ac010746, Al445667, Al049569, Al355149, Ac005056, Al050317, Ac025471, Ac020904, Ac084854, Ac005632, Ac008533; E15M5K2–E15M5K8: Ac093759, Ap002907, Ap001437, Ac011979, Ac026703, Ac011501, Ac072052; E15M6K2–E15M6K6: AL157935 .28, AL391239 , Al137141, Al157877, Ac090117; E15M15K1–E15M15K7: Ac008760, Ac068134, Ac005208, Ac012100, Al138999, Al390961, Ac105245; E17M19K2–E17M19K5: AL162832 .6, AC009116 .8, AL136218 .26, AL160169 .12; E17M19K8–E17M19K10: AC130343 .7, AC003030 .1, AC026469 .8; E18M22K2–E18M22K4: AL445903 .3, BX005019 .10, AC011456 .2; E18M24K1–E18M24K2: AC036103 .8 and AC005208 .1. The integration sites not listed could not be mapped. The chromosomal localizations of the clones are given in Table 1.
Quantitation of Individual Clones
To quantify the clones detected by LM-PCR, we used a real-time quantitative PCR method. Unique primers specific for the flanking genomic DNA were designed for each integration site sequence, whereas the retroviral-specific primer and fluorogenic probe were identical for all integration sites. To test the specificity of the real-time quantitative PCR primer and probe set, we performed reactions with the corresponding plasmid and control plasmids. Reactions were only positive with the primer designed for a given clone (e.g., plasmid E15M1EB7 with primer for clone E15M1EB7) and were completely negative with primers designed for other clones (e.g., plasmid E15M1EB7 with primers for clones E15M1EB9 and E15M1EB11), thus demonstrating a very high specificity of the designed unique primers.
In a mixture of three plasmids each containing a different LM-PCR product, we were able to detect the proportion of an individual plasmid down to 10 copies in a total copy number of 105 (13 dilution steps; Fig. 3) using real-time quantitative PCR. To test the reproducibility of the results, three independent replicates were performed for each integration site sequence. The mean coefficient of variation was 0.16 (range, 0.0001–0.42).
Figure 3. Quantitation of an individual plasmid in a plasmid mixture. Three plasmids containing different ligation-mediated polymerase chain reaction product inserts were mixed, and the proportion of individual plasmids was studied. The error bars represent the standard deviation of three separate experiments. For example, plasmid A was detected with an accuracy of r = 0.99, p < .0001, as calculated by regression analysis.
Contribution of Individual Human Marrow-Repopulating Cells to Hematopoiesis
The amount of all transduced cells was determined as the copy number of the MDR1 transgene by real-time quantitative PCR and ranged between 263 and 6,807 copies per μg DNA (median, 1,380.5 copies per μg DNA; Table 3). A total of 17 integration site sequences from three different donors detected by LM-PCR were analyzed. Individual clone copy numbers ranged from 0.02–124 per μg DNA (median, 2.2 per μg DNA; Table 3). The ratio of the mean copy number of an individual clone and the mean copy number of the MDR1 transgene gives an estimate of the contribution of this clone to human transduced hematopoiesis. The analyzed individual marrow-repopulating cell clones contributed to 0.01%–1.82% of human retrovirally transduced hematopoiesis (median, 0.15%; Table 3). Considering the level of engraftment and the proportion of MDR1 transgene-marked cells, the transduced clones analyzed here amounted to 0.001%–0.4% of all human cells and to 0.0001%–0.14% (median, 0.03%) of all human and mouse cells in chimeric BM (Table 3). Interestingly, the clones, which were repeatedly found in separate LM-PCR analyses (Fig. 2 and Table 2), showed the highest engraftment (1.82% for E15M1EB11 and 1.23% for E15M1EB9).
Table 3. Proportion of individual clones among the transduced human severe combined immunodeficient mouse repopulating cells
DISCUSSION
The technical assistance of Bernhard Berkus, Hans-Jürgen Engel, Sigrid Heil (German Cancer Research Center), and Carmen Hoppstock (Department of Internal Medicine V, University of Heidelberg) and the support of the animal facility team of the German Cancer Research Center are gratefully acknowledged. We thank Birgit St?hle (EUFETS AG) for her help at the start of this project. This work was supported in part by grant 10-1294-Ze3 from the Deutsche Krebshilfe/Dr. Mildred-Scheel-Stiftung, grant M 20.4 from the H.W. & J. Hector-Stiftung, and by a grant from the AiP plus Forschung program of the University of Heidelberg.
K.Z.N. and S.L. contributed equally to these results.
REFERENCES
Hacein-Bey-Abina S, Von Kalle C, Schmidt M et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302:415–419.
Krauter J, Gorlich K, Ottmann O et al. Prognostic value of minimal residual disease quantification by real-time reverse transcriptase polymerase chain reaction in patients with core binding factor leukemias. J Clin Oncol 2003;21:4413–4422.
Schilz AJ, Kuhlcke K, Fauser AA et al. MDR1 gene expression in NOD/SCID repopulating cells after retro-viral gene transfer under clinically relevant conditions. Mol Ther 2000;2:609–618.
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.
Schmidt M, Carbonaro DA, Speckmann C et al. Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates. Nat Med 2003;9:463–468.
Schiedlmeier B, Kuhlcke K, Eckert HG et al. Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice. Blood 2000;95:1237–1248.
Laufs S, Gentner B, Nagy KZ et al. Retroviral vector integration occurs in preferred genomic targets of human bone marrow-repopulating cells. Blood 2003;101:2191–2198.
Heid CA, Stevens J, Livak KJ et al. Real time quantitative PCR. Genome Res 1996;6:986–994.
Alizadeh M, Bernard M, Danic B et al. Quantitative assessment of hematopoietic chimerism after bone-marrow transplantation by real-time quantitative polymerase chain reaction. Blood 2002;99:4618–4625.
Veldwijk MR, Topaly J, Laufs S et al. Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks. Mol Ther 2002;6:272–278.
Schiedlmeier B, Schilz AJ, Kuhlcke K et al. Multidrug resistance 1 gene transfer can confer chemoprotection to human peripheral blood progenitor cells engrafted in immunodeficient mice. Hum Gene Ther 2002;13:233–242.
Abonour R, Williams DA, Einhorn L et al. Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells. Nat Med 2000;6:652–658.
Fruehauf S, Veldwijk MR, Berlinghoff S et al. Gene therapy for sarcoma. Cells Tissues Organs 2002;172:133–144.
Eckert HG, Kuhlcke K, Schilz AJ et al. Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1). Bone Marrow Transplant 2000;25(suppl 2):114–117.
Hildinger M, Abel KL, Ostertag W et al. Design of 5' untranslated sequences in retroviral vectors developed for medical use. J Virol 1999;73:4083–4089.
Fruehauf S, Breems DA, Knaan-Shanzer S et al. Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area-forming cell assay and detection of vector-mediated P-glycoprotein expression by rhodamine-123. Hum Gene Ther 1996;7:1219–1231.
Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329–1337.
Cashman JD, Lapidot T, Wang JC et al. Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice. Blood 1997;89:4307–4316.
Gothot A, van der Loo JC, Clapp DW et al. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998;92:2641–2649.
Akashi K, Traver D, Miyamoto T et al. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 2000;404:193–197.
Dick JE, Guenechea G, Gan OI et al. In vivo dynamics of human stem cell repopulation in NOD/SCID mice. Ann N Y Acad Sci 2001;938:184–190.
Guenechea G, Gan OI, Dorrell C et al. Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2001;2:75–82.
Horn PA, Thomasson BM, Wood BL et al. Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates. Blood 2003;102:4329–4335.
Rosenthal A, Jones DS. Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucleic Acids Res 1990;18:3095–3096.
Schmidt M, Zickler P, Hoffmann G et al. Polyclonal long-term repopulating stem cell clones in a primate model. Blood 2002;100:2737–2743.
Dynabeads M-280 Streptavidin. Dynal, Oslo, Norway.
Nolta JA, Dao MA, Wells S et al. Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice. Proc Natl Acad Sci U S A 1996;93:2414–2419.
Bhatia M, Wang JC, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997;94:5320–5325.
Fruehauf S, Veldwijk MR, Kramer A et al. Delineation of cell cycle state and correlation to adhesion molecule expression of human CD34+ cells from steady-state bone marrow and peripheral blood mobilized following G-CSF-supported chemotherapy. STEM CELLS 1998;16:271–279.
Brunning et al. WHO Classification of Tumors: Pathology & Genetics. Tumors of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press, 2001:77.
Fruehauf S, Topaly J, Wilmes A, et al. Flow cytometric diagnostic of malignant h?matologic diseases. In: Zeller WJ, zur Hausen H, eds. Onkologie. Landsberg/Lech: Ecomed Verlag, 2000:1–32.(K. Zsuzsanna Nagya, Steph)