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Purging of chronic myelogenous leukemia cells by retrovirally expressed anti–bcr-abl ribozymes with specific cellular compartmentalization

Cancer Gene Therapy volume 9, pages 71–86 (2002)Cite this article

Abstract

In patients with chronic myelogenous leukemia (CML), abnormal expansion of myeloid cells is maintained by expression of the p210bcr-abl fusion protein. Thus, this protein and its mRNA represent primary targets to inhibit proliferation of these cells. Here we describe the properties of a ribozyme against the bcr-abl mRNA, expressed as a fusion transcript with the human U1 small nuclear RNA or the adenovirus VA1 RNA and delivered to the cells through retroviral vectors. These fusion ribozymes are specifically localized in the nucleus or in the cytoplasm, respectively. Transduction of 32D-LG7 myeloid cells, whose growth is IL-3 independent thanks to deregulated bcr-abl expression, imposed strong negative selective pressure on cell growth and induced restoration of an IL-3–dependent phenotype. Although expressed at a level similar to that of the U1-fusion ribozyme, the cytoplasmic VA1 ribozyme was a more powerful inhibitor of p210bcr-abl gene expression. In cells transduced with the vector expressing this ribozyme, the levels of the bcr-abl transcript were reduced up to 104-fold, the p210bcr-abl protein became undetectable, and the cells underwent massive apoptosis when cultured in the absence of IL-3. Transduction of primary hematopoietic cells obtained from bone marrow of patients with CML resulted in remarkable reduction of bcr-abl mRNA levels, starting a few days after transduction. These results show the feasibility and efficacy of vector-expressed anti–bcr-abl ribozymes for purging of CML cells.

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References

  1. Shtivelman E, Lifshitz B, Gale RP et al. Alternative splicing of RNAs transcribed from the human abl gene and from the bcr-abl fused gene Cell 1986 47: 277–284

    Article  CAS  PubMed  Google Scholar 

  2. Heisterkamp N, Stam K, Groffen J et al. Structural organization of the bcr gene and its role in the Ph′ translocation Nature 1985 315: 758–761

    Article  CAS  PubMed  Google Scholar 

  3. Kurzrock R, Gutterman JU, Talpaz M . The molecular genetics of Philadelphia chromosome-positive leukemias N Engl J Med 1988 319: 990–998

    Article  CAS  PubMed  Google Scholar 

  4. Litzow MR, Tefferi A . Bone marrow transplantation for chronic myelogenous leukemia Blood 1994 83: 2752–2753

    CAS  PubMed  Google Scholar 

  5. Reiffers J, Goldman J, Meloni G et al. Autologous transplantation in chronic myelogenous leukemia: European results: Chronic Leukemia Working Party of the EBMT Bone Marrow Transplant 1994 14: S51–S54

    PubMed  Google Scholar 

  6. Carella AM, Lerma E, Corsetti MT et al. Autografting with philadelphia chromosome-negative mobilized hematopoietic progenitor cells in chronic myelogenous leukemia Blood 1999 93: 1534–1539

    CAS  PubMed  Google Scholar 

  7. Khouri IF, Kantarjian HM, Talpaz M et al. Results with high-dose chemotherapy and unpurged autologous stem cell transplantation in 73 patients with chronic myelogenous leukemia: the MD Anderson experience Bone Marrow Transplant 1996 17: 775–779

    CAS  PubMed  Google Scholar 

  8. Kantarjian HM, Giles FJ, O'Brien S et al. Therapeutic choices in younger patients with chronic myelogenous leukemia Cancer 2000 89: 1647–1658

    Article  CAS  PubMed  Google Scholar 

  9. Bhatia R, Verfaillie CM, Miller JS et al. Autologous transplantation therapy for chronic myelogenous leukemia Blood 1997 89: 2623–2634

    CAS  PubMed  Google Scholar 

  10. Deisseroth AB, Zu Z, Claxton D et al. Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML Blood 1994 83: 3068–3076

    CAS  PubMed  Google Scholar 

  11. Degliantoni G, Mangoni L, Rizzoli V . In vitro restoration of polyclonal hematopoiesis in a chronic myelogenous leukemia after in vitro treatment with 4-hydroperoxycyclophosphamide Blood 1985 65: 753–757

    CAS  PubMed  Google Scholar 

  12. Carlo-Stella C, Mangoni L, Almici C et al. Autologous transplant for chronic myelogenous leukemia using marrow treated ex vivo with mafosfamide Bone Marrow Transplant 1994 14: 425–432

    CAS  PubMed  Google Scholar 

  13. Barnett MJ, Eaves CJ, Phillips GL et al. Autografting with cultured marrow in chronic myeloid leukemia: results of a pilot study Blood 1994 84: 724–732

    CAS  PubMed  Google Scholar 

  14. Szczylik C, Skorski T, Nicolaides NC et al. Selective inhibition of leukemia cell proliferation by Bcr-abl antisense oligodeoxynucleotides Science 1991 253: 562–565

    Article  CAS  PubMed  Google Scholar 

  15. Ledley FD . Nonviral gene therapy: the promise of genes as pharmaceutical products Hum Gene Ther 1995 6: 1129–1144

    Article  CAS  PubMed  Google Scholar 

  16. Wagner RW . Gene inhibition using antisense oligodeoxynucleotides Nature 1994 372: 333–335

    Article  CAS  PubMed  Google Scholar 

  17. Keating A, Wang XH, Laraya P . Variable transcription of Bcr-abl by Ph+ cells arising from hematopoietic progenitors in chronic myeloid leukemia Blood 1994 83: 1744–1749

    CAS  PubMed  Google Scholar 

  18. Leopold LH, Shore SK, Reddy EP . Multi-unit anti–Bcr-abl ribozyme therapy in chronic myelogenous leukemia Leuk Lymphoma 1996 22: 365–373

    Article  CAS  PubMed  Google Scholar 

  19. Snyder DS, Wu Y, McMahon R et al. Ribozyme-mediated inhibition of a Philadelphia chromosome-positive acute lymphoblastic leukemia cell line expressing the p190 bcr-abl oncogene Biol Blood Marrow Transplant 1997 3: 179–186

    CAS  PubMed  Google Scholar 

  20. Wright LA, Milliken S, Biggs JC et al. Ex vivo effects associated with the expression of a bcr-abl–specific ribozyme in a CML cell line Antisense Nucleic Acid Drug Dev 1998 8: 15–23

    Article  CAS  PubMed  Google Scholar 

  21. Kuwabara T, Warashina M, Tanabe T et al. A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo Mol Cell 1998 2: 617–627

    Article  CAS  PubMed  Google Scholar 

  22. Sullenger BA, Cech TR . Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA Science 1993 262: 1566–1569

    Article  CAS  PubMed  Google Scholar 

  23. Rossi JJ . Controlled, targeted, intracellular expression of ribozymes: progress and problems Trends Biotechnol 1995 13: 301–306

    Article  CAS  PubMed  Google Scholar 

  24. Snyder DS, Wu Y, Wang JL et al. Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line Blood 1993 82: 600–605

    CAS  PubMed  Google Scholar 

  25. James H, Mills K, Gibson I . Investigating and improving the specificity of ribozymes directed against the bcr-abl translocation Leukemia 1996 10: 1054–1064

    CAS  PubMed  Google Scholar 

  26. Prislei S, Buonomo SB, Michienzi A et al. Use of adenoviral VAI small RNA as a carrier for cytoplasmic delivery of ribozymes RNA 1997 3: 677–687

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Armentano D, Yu SF, Kantoff PW et al. Effect of internal viral sequences on the utility of retroviral vectors J Virol 1987 61: 1647–1650

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Tafuro S, Zentilin L, Falaschi A et al. Rapid retrovirus titration using competitive polymerase chain reaction Gene Ther 1996 3: 679–684

    CAS  PubMed  Google Scholar 

  29. Laneuville P, Heisterkamp N, Groffen J . Expression of the chronic myelogenous leukemia-associated p210bcr/abl oncoprotein in a murine IL-3 dependent myeloid cell line Oncogene 1991 6: 275–282

    CAS  PubMed  Google Scholar 

  30. Chomczynski P, Sacchi N . Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction Anal Biochem 1987 162: 156–159

    Article  CAS  PubMed  Google Scholar 

  31. Shifman MI, Stein DG . A reliable and sensitive method for non-radioactive Northern blot analysis of nerve growth factor mRNA from brain tissues J Neurosci Methods 1995 59: 205–208

    Article  CAS  PubMed  Google Scholar 

  32. Raap AK, van de Rijke FM, Dirks RW et al. Bicolor fluorescence in situ hybridization to intron and exon mRNA sequences Exp Cell Res 1991 197: 319–322

    Article  CAS  PubMed  Google Scholar 

  33. Celi FS, Zenilman ME, Shuldiner AR . A rapid and versatile method to synthesize internal standards for competitive PCR Nucleic Acids Res 1993 21: 1047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Grassi G, Zentilin L, Tafuro S et al. A rapid procedure for the quantitation of low abundance mRNAs by competitive RT-PCR Nucleic Acids Res 1994 22: 4547–4549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Diviacco S, Norio P, Zentilin L et al. A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates Gene 1992 122: 3013–3020

    Article  Google Scholar 

  36. Giacca M, Pelizon C, Falaschi A . Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction Methods 1997 13: 301–312

    Article  CAS  PubMed  Google Scholar 

  37. Sambrook J, Fritsch E, Maniatis T . Molecular Cloning — A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 1989

    Google Scholar 

  38. Kuwabara T, Warashina M, Nakayama A et al. Activities of tRNA-embedded dimeric minizymes Nucleic Acids Symp Ser 1997 37: 307–308

    CAS  Google Scholar 

  39. Ilves H, Barske C, Junker U et al. Retroviral vectors designed for targeted expression of RNA polymerase III–driven transcripts: a comparative study Gene 1996 171: 203–208

    Article  CAS  PubMed  Google Scholar 

  40. Wang FS, Kobayashi H, Liang KW et al. Retrovirus-mediated transfer of anti-MDR1 ribozymes fully restores chemosensitivity of P-glycoprotein–expressing human lymphoma cells Hum Gene Ther 1999 10: 1185–1195

    Article  CAS  PubMed  Google Scholar 

  41. Laneuville P, Sun G, Timm M et al. Clonal evolution in a myeloid cell line transformed to interleukin-3 independent growth by retroviral transduction and expression of p210bcr/abl Blood 1992 80: 1788–1797

    CAS  PubMed  Google Scholar 

  42. Lecoeur H, Ledru E, Prevost MC et al. Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods J Immunol Methods 1997 209: 111–123

    Article  CAS  PubMed  Google Scholar 

  43. Smetsers TF, Skorski T, van de Locht LT et al. Antisense Bcr-abl oligonucleotides induce apoptosis in the Philadelphia chromosome-positive cell line BV173 Leukemia 1994 8: 129–140

    CAS  PubMed  Google Scholar 

  44. Sullenger BA, Lee TC, Smith CA et al. Expression of chimeric tRNA–driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication Mol Cell Biol 1990 10: 6512–6523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhou C, Bahner IC, Larson GP et al. Inhibition of HIV-1 in human T-lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes Gene 1994 149: 33–39

    Article  CAS  PubMed  Google Scholar 

  46. Bertrand E, Castanotto D, Zhou C et al. The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization RNA 1997 3: 75–88

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Michienzi A, Prislei S, Bozzoni I . U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus Proc Natl Acad Sci USA 1996 93: 7219–7224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. De Young MB, Kincade-Denker J, Boehm CA et al. Functional characterization of ribozymes expressed using U1 and T7 vectors for the intracellular cleavage of ANF mRNA Biochemistry 1994 33: 12127–12138

    Article  CAS  PubMed  Google Scholar 

  49. Montgomery RA, Dietz HC . Inhibition of fibrillin 1 expression using U1 snRNA as a vehicle for the presentation of antisense targeting sequence Hum Mol Genet 1997 6: 519–525

    Article  CAS  PubMed  Google Scholar 

  50. Guthrie C, Patterson B . Spliceosomal snRNAs Annu Rev Genet 1988 22: 387–419

    Article  CAS  PubMed  Google Scholar 

  51. Carmo-Fonseca M, Tollervey D, Pepperkok R et al. Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery EMBO J 1991 10: 195–206

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Good PD, Krikos AJ, Li SX et al. Expression of small, therapeutic RNAs in human cell nuclei Gene Ther 1997 4: 45–54

    Article  CAS  PubMed  Google Scholar 

  53. Pe'ery T, Mellits KH, Mathews MB . Mutational analysis of the central domain of adenovirus virus–associated RNA mandates a revision of the proposed secondary structure J Virol 1993 67: 3534–3543

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Ma Y, Mathews MB . Secondary and tertiary structure in the central domain of adenovirus type 2 VA RNA I RNA 1996 2: 937–951

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Mellits KH, Mathews MB . Effects of mutations in stem and loop regions on the structure and function of adenovirus VA RNAI EMBO J 1988 7: 2849–2859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Furtado MR, Subramanian S, Bhat RA et al. Functional dissection of adenovirus VAI RNA J Virol 1989 63: 3423–3434

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Robbins PB, Yu XJ, Skelton DM et al. Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells J Virol 1997 71: 9466–9474

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Zentilin L, Qin G, Tafuro S et al. Variegation of retroviral gene expression in myeloid cells Gene Ther 2000 7: 153–166

    Article  CAS  PubMed  Google Scholar 

  59. Fu DJ, McLaughlin LW . Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme Proc Natl Acad Sci USA 1992 89: 3985–3989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Maria Elena Lopez for excellent assistance in tissue culture and Barbara Boziglav for skillful technical support. We are very grateful to Irene Bozzoni for the VA1 plasmid, to Maria Alessandra Santucci for the 32D and 32D-LG7 cell clones, and to Eli Gilboa for the N2A vector.

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Authors and Affiliations

  1. Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste, 34012, Italy

    Ramiro Mendoza-Maldonado, Lorena Zentilin & Mauro Giacca

  2. Cattedra di Ematologia, Universita' degli Studi di Udine. P.le S. Maria della Misericordia, Udine, 33100, Italy

    Renato Fanin

  3. Scuola Normale Superiore, Piazza dei Cavalieri 7, Pisa, 56126, Italy

    Mauro Giacca

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  1. Ramiro Mendoza-Maldonado
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Correspondence to Mauro Giacca.

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Mendoza-Maldonado, R., Zentilin, L., Fanin, R. et al. Purging of chronic myelogenous leukemia cells by retrovirally expressed anti–bcr-abl ribozymes with specific cellular compartmentalization. Cancer Gene Ther 9, 71–86 (2002). https://doi.org/10.1038/sj.cgt.7700410

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