Antitumor activity of systemically delivered ribozymes targeting murine telomerase RNA.
OBJECTIVE
To test ribozymes targeting mouse telomerase RNA (mTER) for suppression of the progression of B16-F10 murine melanoma metastases in vivo.


METHODS
Hammerhead ribozymes were designed to target mTER. The ribozyme sequences were cloned into a plasmid expression vector containing EBV genomic elements that substantially prolong expression of genes delivered in vivo. The activity of various antitelomerase ribozymes or control constructs was examined after i.v. injection of cationic liposome:DNA complexes containing control or ribozyme constructs. Expression of ribozymes and mTER at various time points were evaluated by quantitative real-time PCR. Telomerase activity was examined using the telomeric repeat amplification protocol.


RESULTS
Systemic administration of cationic liposome:DNA complexes containing a plasmid-expressed ribozyme specifically targeting a cleavage site at mTER nucleotide 180 significantly reduced the metastatic progression of B16-F10 murine melanoma. The antitumor activity of the anti-TER 180 ribozyme in mice was abolished by a single inactivating base mutation in the ribozyme catalytic core. The EBV-based expression plasmid produced sustained levels of ribozyme expression for the full duration of the antitumor studies. In addition to antitumor activity, cationic liposome:DNA complex-based ribozyme treatment also produced reductions in both TER levels and telomerase enzymatic activity in tumor-bearing mice.


CONCLUSIONS
Systemic, plasmid-based ribozymes specifically targeting TER can reduce both telomerase activity and metastatic progression in tumor-bearing hosts. The work reported here demonstrates the potential utility of plasmid-based anti-TER ribozymes in the therapy of melanoma metastasis.
Nosrati
Mehdi
M
Auerback Melanoma Research Laboratory, Cutaneous Oncology Program, Department of Dermatology, University of California San Francisco Cancer Center, San Francisco, California 94115, USA.
Li
Shang
S
Bagheri
Sepideh
S
Ginzinger
David
D
Blackburn
Elizabeth H
EH
Debs
Robert J
RJ
Kashani-Sabet
Mohammed
M
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D020134:Catalytic Domain; D002412:Cations; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D018450:Disease Progression; D005260:Female; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008168:Lung / Q000473:pathology; D008175:Lung Neoplasms / Q000473:pathology; D008546:Melanoma, Experimental / Q000628:therapy*; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D009154:Mutation; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000628:therapy*; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000737:chemistry* / Q000378:metabolism; D016615:Telomere / Q000648:ultrastructure; D013997:Time Factors
D000970:Antineoplastic Agents; D002412:Cations; D008081:Liposomes; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; C422952:telomerase RNA; D012313:RNA; D004247:DNA; D019098:Telomerase