Purging of chronic myelogenous leukemia cells by retrovirally expressed anti-bcr-abl ribozymes with specific cellular compartmentalization.
In patients with chronic myelogenous leukemia (CML), abnormal expansion of myeloid cells is maintained by expression of the p210(bcr-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 p210(bcr-abl) gene expression. In cells transduced with the vector expressing this ribozyme, the levels of the bcr-abl transcript were reduced up to 10(4)-fold, the p210(bcr-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.
Mendoza-Maldonado
Ramiro
R
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Zentilin
Lorena
L
Fanin
Renato
R
Giacca
Mauro
M
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001853:Bone Marrow / Q000378:metabolism; D002451:Cell Compartmentation / Q000502:physiology; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D015316:Genetic Therapy; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000628:therapy*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000166:cytology
D016337:RNA, Catalytic; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; C419304:VA1-ribozyme chimera; D016044:Fusion Proteins, bcr-abl