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  • Viral Transfer Technology
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Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38− subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

Gene Therapy volume 7, pages 183–195 (2000)Cite this article

Abstract

Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and long-term gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/107particles) and adenovirus. Transduction of CD34+ cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated, quiescent CD34+ cells were resistant to transduction (less than 4%), but transduction increased to 23 ± 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor α (51 ± 4%) at a multiplicity of infection of 106. rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34+ and CD34+, CD38− subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 ± 7% of CD34+ progenitors and 21 ± 6% of CD34+, CD38− progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient.

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References

  1. Hawley RG, Fong AZ, Burns BF, Hawley TS . Transplantable myeloproliferative disease induced in mice by an interleukin 6 retrovirus J Exp Med 1992 176: 1149–1163

    Article  CAS  PubMed  Google Scholar 

  2. Persons DA et al. Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo Blood 1997 90: 1777–1786

    CAS  PubMed  Google Scholar 

  3. Brenner MK et al. Gene marking to determine whether autologous marrow infusion restores long-term haematopoiesis in cancer patients Lancet 1993 342: 1134–1137

    Article  CAS  PubMed  Google Scholar 

  4. Bodine D et al. Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells Blood 1993 82: 1975–1980

    CAS  PubMed  Google Scholar 

  5. Dunbar CE et al. Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation Blood 1995 85: 3057–3084

    Google Scholar 

  6. Orlic D et al. The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction Proc Natl Acad Sci USA 1996 93: 11097–11102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sorrentino BP, Nienhuis AW . The hematopoietic system as a target for gene therapy. In: Friedmann T (ed) The Development of Gene Therapy Cold Spring Harbor Laboratory Press: New York 1998 pp 351–354

    Google Scholar 

  8. Nienhuis AW et al. Gene transfer into hematopoietic cells Stem Cells 1997 15: 123–124

    Article  CAS  PubMed  Google Scholar 

  9. Fisher-Adams G et al. Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction Blood 1996 88: 492–504

    CAS  PubMed  Google Scholar 

  10. Goodman S et al. Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells Blood 1994 84: 1492–1500

    CAS  PubMed  Google Scholar 

  11. Walsh CE et al. Phenotypic correction of Fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vector J Clin Invest 1994 94: 1440–1448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Walsh CE et al. Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector Proc Natl Acad Sci USA 1992 89: 7257–7261

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Miller JL et al. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells Proc Natl Acad Sci USA 1994 91: 10183–10187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhou SZ et al. Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood J Exp Med 1994 179: 1867–1875

    Article  CAS  PubMed  Google Scholar 

  15. Alexander IE, Russell DW, Miller AD . Transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results Hum Gene Ther 1997 8: 1911–1920

    Article  CAS  PubMed  Google Scholar 

  16. Matsushita T et al. Adeno-associated virus vectors can be efficiently produced without helper virus Gene Therapy 1998 5: 938–945

    Article  CAS  PubMed  Google Scholar 

  17. Hargrove PW, Vanin EF, Kurtzman GJ, Nienhuis AW . High-level globin gene expression mediated by recombinant adeno-associated virus that contains the 3′ globin gene regulatory element and integrates as tandem copies in erythroid cells Blood 1997 89: 2167–2175

    CAS  PubMed  Google Scholar 

  18. Wang XS et al. Characterization of wild-type adeno-associated virus type 2-like particles generated during recombinant viral vector production and strategies for their elimination J Virol 1998 72: 5472–5480

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Snyder RO et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors Nat Genet 1997 16: 270–276

    Article  CAS  PubMed  Google Scholar 

  20. Zhou SZ et al. Adeno-associated virus 2-mediated gene transfer in murine hematopoietic progenitor cells Exp Hematol 1993 21: 928–933

    CAS  PubMed  Google Scholar 

  21. Bertran J et al. Recombinant adeno-associated virus-mediated high efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors J Virol 1996 70: 6759–6766

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Duan D et al. Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E4 ORF6 and E2a gene expression J Virol 1999 73: 161–169

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Snyder RO et al. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors Nature Med 1999 5: 64–70

    Article  CAS  PubMed  Google Scholar 

  24. Herzog RW et al. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector Nature Med 1999 5: 56–63

    Article  CAS  PubMed  Google Scholar 

  25. Kaplitt MG et al. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain Nat Genet 1994 8: 148–154

    Article  CAS  PubMed  Google Scholar 

  26. Malik P et al. Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line J Virol 1997 71: 1776–1783

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Schimmenti S et al. Long-term genetic modification of rhesus monkey hematopoietic cells following transplantation of adeno-associated virus vector-transduced CD34+ cells Hum Gene Ther 1998 9: 2727–2734

    Article  CAS  PubMed  Google Scholar 

  28. Chatterjee S et al. Transduction of primitive human marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors Blood 1999 93: 1882–1894

    CAS  PubMed  Google Scholar 

  29. Xiao X, Li J, Samulski RJ . Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus J Virol 1998 72: 2224–2232

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Grimm D, Kern A, Rittner K, Kleinschmidt JA . Novel tools for production and purification of recombinant adeno-associated virus vectors Hum Gene Ther 1998 9: 2745–2760

    Article  CAS  PubMed  Google Scholar 

  31. Salvetti A et al. Factors influencing recombinant adeno-associated virus production Hum Gene Ther 1998 9: 695–706

    Article  CAS  PubMed  Google Scholar 

  32. Allen JM, Debelak DJ, Reynolds TC, Miller AD . Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by nonhomologous recombination during AAV vector production J Virol 1997 71: 6816–6822

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Clark KR, Liu X, McGrath JP, Johnson PR . Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses Hum Gene Ther 1999 10: 1031–1039

    Article  CAS  PubMed  Google Scholar 

  34. Zolotukhin S et al. Utilization of AAV receptor affinity to develop an efficient and novel purification protocol for vector production Gene Therapy 1999 6: 973–985

    Article  CAS  PubMed  Google Scholar 

  35. Summerford C, Samulski RJ . Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions J Virol 1998 72: 1438–1445

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Ponnazhagan S et al. Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation J Virol 1997 71: 8262–8267

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Sitnicka E et al. Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells Blood 1996 88: 82–88

    CAS  PubMed  Google Scholar 

  38. Cashman JD, Eaves CJ, Sarris AH, Eaves AC . MCP-1, not MIP-1alpha, is the endogenous chemokine that cooperates with TGF-beta to inhibit the cycling of primitive normal but not leukemic (CML) progenitors in long-term human marrow cultures Blood 1998 92: 2338–2344

    CAS  PubMed  Google Scholar 

  39. Murphy M, Perussia B, Trinchieri G . Effects of recombinant tumor necrosis factor, lymphotoxin, and immune interferon on proliferation and differentiation of enriched hematopoietic precursor cells Exp Hematol 1988 16: 131–138

    CAS  PubMed  Google Scholar 

  40. Ratta M et al. Generation and functional characterization of human dendritic cells derived from CD34 cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells Br J Haematol 1998 101: 756–765

    Article  CAS  PubMed  Google Scholar 

  41. Muzyczka N . Use of adeno-associated virus as a general transduction vector for mammalian cells Curr Top Microbiol Immunol 1992 158: 97–129

    CAS  PubMed  Google Scholar 

  42. Halbert CL, Standaert TA, Wilson CB, Miller AD . Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure J Virol 1998 72: 9795–9805

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Manning WC et al. Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors Hum Gene Ther 1998 9: 477–485

    Article  CAS  PubMed  Google Scholar 

  44. Halbert CL et al. Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration J Virol 1997 71: 5932–5941

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Koeberl DD et al. Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors Proc Natl Acad Sci USA 1997 94: 1426–1431

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Fisher KJ et al. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis J Virol 1996 70: 520–532

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Russell DW, Alexander IE, Miller AD . DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors Proc Natl Acad Sci USA 1995 92: 5719–5723

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Summerford C, Bartlett JS, Samulski RJ . αVβ5 integrin: a co-receptor for adeno-associated virus type 2 infection Nature Med 1999 5: 78–82

    Article  CAS  PubMed  Google Scholar 

  49. Huang S, Endo RI, Nemerow GR . Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery J Virol 1995 69: 2257–2263

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Mah C et al. Adeno-associated virus type 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression J Virol 1998 72: 9835–9843

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Fietze E et al. Cytomegalovirus infection in transplant recipients. The role of tumor necrosis factor Transplantation 1994 58: 675–680

    Article  CAS  PubMed  Google Scholar 

  52. Laegreid A et al. Tumor necrosis factor induces lipopolysaccharide tolerance in a human adenocarcinoma cell line mainly through the TNF p55 receptor J Biol Chem 1995 270: 25418–25425

    Article  CAS  PubMed  Google Scholar 

  53. Civin CI et al. Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo Blood 1996 88: 4102–4109

    CAS  PubMed  Google Scholar 

  54. Bhatia M et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice Proc Natl Acad Sci USA 1997 94: 5320–5325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lubovy M et al. Stable transduction of recombinant adeno-associated virus into hematopoietic stem cells from normal and sickle cell patients Biol Blood Marrow Transplant 1996 2: 24–30

    CAS  PubMed  Google Scholar 

  56. Miao CH et al. The kinetics of rAAV integration in the liver Nat Genet 1998 19: 1913–1915

    Article  Google Scholar 

  57. Larochelle A et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy Nature Med 1996 2: 1329–1337

    Article  CAS  PubMed  Google Scholar 

  58. Persons DA et al. Use of the green fluorescent protein as a marker to identify and track genetically modified hematopoietic cells Nature Med 1998 4: 1201–1205

    Article  CAS  PubMed  Google Scholar 

  59. Samulski RJ, Chang LS, Shenk TE . A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication J Virol 1987 61: 3096–3101

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Albritton LM, Tseng L, Scadden D, Cunningham JM . A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection Cell 1989 57: 659–666

    Article  CAS  PubMed  Google Scholar 

  61. Samulski RJ, Chang LS, Shenk T . Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression J Virol 1989 63: 3822–3828

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Srivastava A, Lusby EW, Berns KI . Neucleotide sequence and organization of adeno-associated virus 2 genome J Virol 1983 45: 555–564

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We wish to thank Betty Ciullo for her outstanding assistance in preparation of the manuscript, Richard J Samulski for the gift of XX6 adeno-helper plasmid, and Dr Richard Ashmun for his help with the FACs analysis and figures. This work was supported by NHLBI Program Project Grant P01 HL 53749, the ASSISI Foundation of Memphis Grant 94–00, and American Lebanese Syrian Associated Charities (ALSAC). Amit C Nathwani was supported by a grant from the Wellcome Trust, number 049894/114.

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  1. Division of Experimental Hematology, Department of Hematology/Oncology, St Jude Children's Research Hospital, Memphis, TN, USA

    A C Nathwani, H Hanawa, J Vandergriff, P Kelly, E F Vanin & A W Nienhuis

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Nathwani, A., Hanawa, H., Vandergriff, J. et al. Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38− subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV. Gene Ther 7, 183–195 (2000). https://doi.org/10.1038/sj.gt.3301068

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