Skip to main content

Advertisement

SpringerLink
Log in

Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells

  • Original Paper
  • Published:
Journal of Cancer Research and Clinical Oncology Aims and scope Submit manuscript

Abstract

Purpose

Although gene transfer with retroviral vectors has shown distinct clinical success in defined settings, efficient genetic manipulation of hematopoietic progenitor cells remains a challenge. To address this issue we have evaluated different transduction protocols and retroviral constructs in the non-obese diabetes (NOD)/severe combined immunodeficiency disease (SCID) xenograft model.

Methods

An extended transduction protocol requiring 144 h of in vitro manipulation was compared to a more conventional protocol requiring 96 h only.

Result

While pretransplantation analysis of cells transduced with a retroviral vector, expressing the enhanced green fluorescent protein (EGFP) marker gene, demonstrated significantly higher overall transduction rates for the extended protocol (33.6 ± 2.3 vs. 22.1 ± 3.8%), EGFP expression in CD34+ cells before transplantation (4.0 ± 0.9 vs. 11.6 ± 2.5%), engraftment of human cells in NOD/SCID bone marrow 4 weeks after transplantation (4.5 ± 1.7 vs. 36.5 ± 9.4%) and EGFP expression in these cells (0 ± 0 vs. 11.3 ± 2.8%) were significantly impaired. When the 96 h protocol was used in combination with the spleen focus forming virus (SFFV)/murine embryonic stem cell (MESV) hybrid vector SFβ11-EGFP, high transduction rates for CD45+ (41.0 ± 5.3%) and CD34+ (38.5 ± 3.7%) cells prior to transplantation, as well as efficient human cell engraftment in NOD/SCID mice 4 weeks after transplantation (32.4 ± 3.5%), was detected. Transgene expression was observed in B-lymphoid (15.9 ± 2.0%), myeloid (36.5 ± 3.5%) and CD34+ cells (10.1 ± 1.5%).

Conclusion

Our data show that CD34+ cells maintained in cytokines for multiple days may differentiate and loose their capacity to contribute to the haematological reconstitution of NOD/SCID mice. In addition, the SFFV/MESV hybrid vector SFβ11-EGFP allows efficient transduction of and gene expression in haematopoietic progenitor cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Ahmed F, Ings SJ, Pizzey AR, Blundell MP, Thrasher AJ, Ye HT, Fahey A, Linch DC, Yong KL (2004) Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model. Blood 103:2079–2087

    Article  PubMed  CAS  Google Scholar 

  • Ballen K, Becker PS, Greiner D, Valinski H, Shearin D, Berrios V, Dooner G, Hsieh CC, Wuu J, Shultz L, Cerny J, Leif J, Stewart FM, Quesenberry P (2000) Effect of ex vivo cytokine treatment on human cord blood engraftment in NOD-scid mice. Br J Haematol 108:629–640

    Article  PubMed  CAS  Google Scholar 

  • Barquinero J, Segovia JC, Ramirez M, Limon A, Guenechea G, Puig T, Briones J, Garcia J, Bueren JA (2000) Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice. Blood 95:3085–3093

    PubMed  CAS  Google Scholar 

  • Baum C, Eckert HG, Stockschlader M, Just U, Hegewisch-Becker S, Hildinger M, Uhde A, John J, Ostertag W (1996) Improved retroviral vectors for hematopoietic stem cell protection and in vivo selection. J Hematother 5:323–329

    PubMed  CAS  Google Scholar 

  • Bjorgvinsdottir H, Bryder D, Sitnicka E, Ramsfjell V, De Jong I, Olsson K, Rusterholz C, Karlsson S, Jacobsen SE (2002) Efficient oncoretroviral transduction of extended long-term culture-initiating cells and NOD/SCID repopulating cells: enhanced reconstitution with gene-marked cells through an ex vivo expansion approach. Hum Gene Ther 13:1061–1073

    Article  PubMed  CAS  Google Scholar 

  • Bordignon C (1998) A new chance for SCID gene therapy. Nat Med 4:19–20

    Article  PubMed  CAS  Google Scholar 

  • Bordignon C, Roncarolo MG (2002) Therapeutic applications for hematopoietic stem cell gene transfer. Nat Immunol 3:318–321

    Article  PubMed  CAS  Google Scholar 

  • Carstanjen D, Dutt P, Moritz T (2001) Heparin inhibits retrovirus binding to fibronectin as well as retrovirus gene transfer on fibronectin fragments. J Virol 75:6218–6222

    Article  PubMed  CAS  Google Scholar 

  • Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A (2000) Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288:669–672

    Article  PubMed  CAS  Google Scholar 

  • Danet GH, Lee HW, Luongo JL, Simon MC, Bonnet DA (2001) Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34(+) cells after ex vivo expansion. Exp Hematol 29:1465–1473

    Article  PubMed  CAS  Google Scholar 

  • Dao MA, Hashino K, Kato I, Nolta JA (1998) Adhesion to fibronectin maintains regenerative capacity during ex vivo culture and transduction of human hematopoietic stem and progenitor cells. Blood 92:4612–4621

    PubMed  CAS  Google Scholar 

  • Demaison C, Brouns G, Blundell MP, Goldman JP, Levinsky RJ, Grez M, Kinnon C, Thrasher AJ (2000) A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector. Hum Gene Ther 11:91–100

    Article  PubMed  CAS  Google Scholar 

  • Denning-Kendall P, Singha S, Bradley B, Hows J (2003) Cytokine expansion culture of cord blood CD34+ cells induces marked and sustained changes in adhesion receptor and CXCR4 expressions. Stem Cells 21:61–70

    Article  PubMed  CAS  Google Scholar 

  • Dick JE, Kamel-Reid S, Murdoch B, Doedens M (1991) Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays. Blood 78:624–634

    PubMed  CAS  Google Scholar 

  • Donahue RE, Sorrentino BP, Hawley RG, An DS, Chen IS, Wersto RP (2001) Fibronectin fragment CH-296 inhibits apoptosis and enhances ex vivo gene transfer by murine retrovirus and human lentivirus vectors independent of viral tropism in nonhuman primate CD34+ cells. Mol Ther 3:359–367

    Article  PubMed  CAS  Google Scholar 

  • Dorrell C, Gan OI, Pereira DS, Hawley RG, Dick JE (2000) Expansion of human cord blood CD34(+)CD38(−) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function. Blood 95:102–110

    PubMed  CAS  Google Scholar 

  • Dunbar CE, Takatoku M, Donahue RE (2001) The impact of ex vivo cytokine stimulation on engraftment of primitive hematopoietic cells in a non-human primate model. Ann NY Acad Sci 938:236–244; discussion 244–245

    Google Scholar 

  • Flores-Guzman P, Gutierrez-Rodriguez M, Mayani H (2002) In vitro proliferation, expansion, and differentiation of a CD34+ cell-enriched hematopoietic cell population from human umbilical cord blood in response to recombinant cytokines. Arch Med Res 33:107–114

    Article  PubMed  CAS  Google Scholar 

  • Gatlin J, Melkus MW, Padgett A, Kelly PF, Garcia JV (2001) Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein. J Virol 75:9995–9999

    Article  PubMed  CAS  Google Scholar 

  • Gothot A, Giet O, Huygen S, Beguin Y (2003) Binding and migration across fibronectin and VCAM-1 of cycling hematopoietic progenitor cells. Leuk Lymphoma 44:1379–1383

    Article  PubMed  CAS  Google Scholar 

  • Guenechea G, Segovia JC, Albella B, Lamana M, Ramirez M, Regidor C, Fernandez MN, Bueren JA (1999) Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells. Blood 93:1097–1105

    PubMed  CAS  Google Scholar 

  • Hanenberg H, Xiao XL, Dilloo D, Hashino K, Kato I, Williams DA (1996) Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med 2:876–882

    Article  PubMed  CAS  Google Scholar 

  • Hanenberg H, Hashino K, Konishi H, Hock RA, Kato I, Williams DA (1997) Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells. Hum Gene Ther 8:2193–2206

    PubMed  CAS  Google Scholar 

  • Hanenberg H, Batish SD, Pollok KE, Vieten L, Verlander PC, Leurs C, Cooper RJ, Gottsche K, Haneline L, Clapp DW, Lobitz S, Williams DA, Auerbach AD (2002) Phenotypic correction of primary Fanconi anemia T cells with retroviral vectors as a diagnostic tool. Exp Hematol 30:410–420

    Article  PubMed  CAS  Google Scholar 

  • Hawley RG, Lieu FH, Fong AZ, Hawley TS (1994) Versatile retroviral vectors for potential use in gene therapy. Gene Ther 1:136–138

    PubMed  CAS  Google Scholar 

  • Hennemann B, Conneally E, Pawliuk R, Leboulch P, Rose-John S, Reid D, Chuo JY, Humphries RK, Eaves CJ (1999) Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables. Exp Hematol 27:817–825

    Article  PubMed  CAS  Google Scholar 

  • van Hennik PB, Verstegen MM, Bierhuizen MF, Limon A, Wognum AW, Cancelas JA, Barquinero J, Ploemacher RE, Wagemaker G (1998) Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice. Blood 92:4013–4022

    PubMed  Google Scholar 

  • Hildinger M, Abel KL, Ostertag W, Baum C (1999) Design of 5′ untranslated sequences in retroviral vectors developed for medical use. J Virol 73:4083–4089

    PubMed  CAS  Google Scholar 

  • Hogan CJ, Shpall EJ, Keller G (2002) Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD/SCID mice. Proc Natl Acad Sci USA 99:413–418

    Article  PubMed  CAS  Google Scholar 

  • Horn PA, Topp MS, Morris JC, Riddell SR, Kiem HP (2002) Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells. Blood 100:3960–3967

    Article  PubMed  CAS  Google Scholar 

  • Horn PA, Thomasson BM, Wood BL, Andrews RG, Morris JC, Kiem HP (2003) Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates. Blood 102:4329–4335

    Article  PubMed  CAS  Google Scholar 

  • Horwitz ME, Malech HL, Anderson SM, Girard LJ, Bodine DM, Orlic D (1999) Granulocyte colony-stimulating factor mobilized peripheral blood stem cells enter into G1 of the cell cycle and express higher levels of amphotropic retrovirus receptor mRNA. Exp Hematol 27:1160–1167

    Article  PubMed  CAS  Google Scholar 

  • Kelly PF, Carrington J, Nathwani A, Vanin EF (2001) RD114-pseudotyped oncoretroviral vectors. Biological and physical properties. Ann NY Acad Sci 938:262–276; discussion 276–277

    Google Scholar 

  • Kinsella TM, Nolan GP (1996) Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum Gene Ther 7:1405–1413

    PubMed  CAS  Google Scholar 

  • Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE (1996) Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 2:1329–1337

    Article  PubMed  CAS  Google Scholar 

  • Leurs C, Jansen M, Pollok KE, Heinkelein M, Schmidt M, Wissler M, Lindemann D, Von Kalle C, Rethwilm A, Williams DA, Hanenberg H (2003) Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells. Hum Gene Ther 14:509–519

    Article  PubMed  CAS  Google Scholar 

  • Lewis ID, Almeida-Porada G, Du J, Lemischka IR, Moore KA, Zanjani ED, Verfaillie CM (2001) Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 97:3441–3449

    Article  PubMed  CAS  Google Scholar 

  • Liu B, Buckley SM, Lewis ID, Goldman AI, Wagner JE, van der Loo JC (2003) Homing defect of cultured human hematopoietic cells in the NOD/SCID mouse is mediated by Fas/CD95. Exp Hematol 31:824–832

    Article  PubMed  CAS  Google Scholar 

  • Ma YP, Zhou P, Xiao J (2002) [Expression and function of CD95/fas antigen and Bcl-2 on cord blood hematopoietic progenitor cells]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 10:13–16

    PubMed  Google Scholar 

  • MacNeill EC, Hanenberg H, Pollok KE, van der Loo JC, Bierhuizen MF, Wagemaker G, Williams DA (1999) Simultaneous infection with retroviruses pseudotyped with different envelope proteins bypasses viral receptor interference associated with colocalization of gp70 and target cells on fibronectin CH-296. J Virol 73:3960–3967

    PubMed  CAS  Google Scholar 

  • Moritz T, Patel VP, Williams DA (1994) Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors. J Clin Invest 93:1451–1457

    Article  PubMed  CAS  Google Scholar 

  • Moritz T, Dutt P, Xiao X, Carstanjen D, Vik T, Hanenberg H, Williams DA (1996) Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments. Blood 88:855–862

    PubMed  CAS  Google Scholar 

  • Murray L, Luens K, Tushinski R, Jin L, Burton M, Chen J, Forestell S, Hill B (1999) Optimization of retroviral gene transduction of mobilized primitive hematopoietic progenitors by using thrombopoietin, Flt3, and Kit ligands and RetroNectin culture. Hum Gene Ther 10:1743–1752

    Article  PubMed  CAS  Google Scholar 

  • Ng YY, Bloem AC, van Kessel B, Lokhorst H, Logtenberg T, Staal FJ (2002) Selective in vitro expansion and efficient retroviral transduction of human CD34+ CD38- haematopoietic stem cells. Br J Haematol 117:226–237

    Article  PubMed  Google Scholar 

  • Novelli EM, Cheng L, Yang Y, Leung W, Ramirez M, Tanavde V, Enger C, Civin CI (1999) Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency. Hum Gene Ther 10:2927–2940

    Article  PubMed  CAS  Google Scholar 

  • Orlic D, Girard LJ, Anderson SM, Pyle LC, Yoder MC, Broxmeyer HE, Bodine DM (1998) Identification of human and mouse hematopoietic stem cell populations expressing high levels of mRNA encoding retrovirus receptors. Blood 91:3247–3254

    PubMed  CAS  Google Scholar 

  • Piacibello W, Bruno S, Sanavio F, Droetto S, Gunetti M, Ailles L, Santoni de Sio F, Viale A, Gammaitoni L, Lombardo A, Naldini L, Aglietta M (2002) Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice. Nonobese diabetic/severe combined immunodeficient. Blood 100:4391–4400

    Article  PubMed  CAS  Google Scholar 

  • Rice AM, Wood JA, Milross CG, Collins CJ, Case J, Vowels MR, Nordon RE (2001) Prolonged ex vivo culture of cord blood CD34(+) cells facilitates myeloid and megakaryocytic engraftment in the non-obese diabetic severe combined immunodeficient mouse model. Br J Haematol 114:433–443

    Article  PubMed  CAS  Google Scholar 

  • Sabatino DE, Do BQ, Pyle LC, Seidel NE, Girard LJ, Spratt SK, Orlic D, Bodine DM (1997) Amphotropic or gibbon ape leukemia virus retrovirus binding and transduction correlates with the level of receptor mRNA in human hematopoietic cell lines. Blood Cells Mol Dis 23:422–433

    Article  PubMed  CAS  Google Scholar 

  • Sellers SE, Tisdale JF, Agricola BA, Donahue RE, Dunbar CE (2004) The presence of the carboxy-terminal fragment of fibronectin allows maintenance of non-human primate long-term hematopoietic repopulating cells during extended ex vivo culture and transduction. Exp Hematol 32:163–170

    Article  PubMed  CAS  Google Scholar 

  • Takatoku M, Sellers S, Agricola BA, Metzger ME, Kato I, Donahue RE, Dunbar CE (2001) Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates. J Clin Invest 108:447–455

    Article  PubMed  CAS  Google Scholar 

  • Thomas CE, Ehrhardt A, Kay MA (2003) Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4:346–358

    Article  PubMed  CAS  Google Scholar 

  • Trarbach T, Greifenberg S, Bardenheuer W, Elmaagacli A, Hirche H, Flasshove M, Seeber S, Moritz T (2000) Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments. Cytotherapy 2:429–438

    Article  PubMed  CAS  Google Scholar 

  • Yokota T, Oritani K, Mitsui H, Aoyama K, Ishikawa J, Sugahara H, Matsumura I, Tsai S, Tomiyama Y, Kanakura Y, Matsuzawa Y (1998) Growth-supporting activities of fibronectin on hematopoietic stem/progenitor cells in vitro and in vivo: structural requirement for fibronectin activities of CS1 and cell-binding domains. Blood 91:3263–3272

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank A. Feldmann, M. Müller and M. Möllmann for excellent technical help. The SFβ11 backbones were kindly provided by C. Baum und W. Ostertag (Heinrich-Pette Institute, Hamburg, Germany). The work was supported by grant Fla 327/2 from Deutsche Forschungsgemeinschaft (DFG) to M.F. and T.M., the Benningsen Foerder-Preis of the Ministery of Research and Technology, North Rhine Westphalia to D.D. and T.M.

Author information

Authors and Affiliations

  1. Department of Internal Medicine (Cancer Research), University of Duisburg-Essen Medical School, Hufelandstraße 55, 45122, Essen, Germany

    Peter Ebeling, P. Bach, U. Sorg, A. Schneider, T. Trarbach, S. Seeber, T. Moritz & M. Flasshove

  2. Department of Paediatric Haematology, Oncology and Immunology, Children’s Hospital, Heinrich-Heine University, Dusseldorf, Germany

    D. Dilloo & H. Hanenberg

  3. Department of Gynaecology and Obstetrics, Elisabeth Krankenhaus Essen, Essen, Germany

    S. Niesert

Authors
  1. Peter Ebeling
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Bach
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. U. Sorg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T. Trarbach
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. D. Dilloo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. H. Hanenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S. Niesert
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. S. Seeber
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. T. Moritz
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. M. Flasshove
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter Ebeling.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ebeling, P., Bach, P., Sorg, U. et al. Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells. J Cancer Res Clin Oncol 133, 199–209 (2007). https://doi.org/10.1007/s00432-006-0158-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00432-006-0158-9

Keywords

Search

Navigation