[Frontiers in Bioscience 11, 2940 -2948, September 1, 2006]

Assessment of an anti-HIV-1 combination gene therapy strategy using the antisense RNA and multimeric hammerhead ribozymes

Ali Ramezani 1,2, Xue Zhong Ma 1, Masoud Ameli 1, Alka Arora1, and Sadhna Joshi 1

1 Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, 150 College Street #212, Toronto, Ontario M5S 3E2, Canada; 2 Present address, Department of Anatomy and Cell Biology, The George Washington University Medical Center, Washington, DC 20037

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and Methods
3.1. Construction of retroviral vectors
3.2. Transduction and selection of stable MT4 transductants
3.3. PCR analysis of genomic DNA from MT4 transductants
3.4. RT-PCR analysis of total RNA from MT4 transductants
3.5. In vitro cleavage activity of multimeric ribozymes amplified from the pools of MT4 transductants
3.6. HIV-1 susceptibility of MT4 transductants
4. Results
4.1. MGIN-based vectors expressing the multimeric ribozymes, the antisense RNA, or the multimeric ribozymes and the antisense RNA
4.2. Development of pools of stable MT4 transductants expressing the interfering RNAs
4.3. HIV-1 susceptibility of MT4 transductants expressing the interfering RNAs
5. Discussion
6. Acknowledgements
7. References

1. Abstract

A combination gene therapy strategy using an ASPsi-gag antisense RNA (targeted against the packaging signal and the gag-coding region) and a multimeric hammerhead ribozyme Rz1-9 (targeted against nine sites within the env-coding region) or Rz1-14 (targeted against 14 sites within the 5' leader and the pro-, pol-, vif- and env-coding regions) was assessed for inhibiting HIV-1 replication. A murine stem cell virus (MSCV)-based MGIN vector was used to express Rz1-9, Rz1-14, ASPsi-gag, Rz1-9ASPsi-gag, or Rz1-14ASPsi-gag RNA in a CD4+ T lymphoid cell line. Stable transductants were shown to express similar levels of interfering RNA. HIV-1 replication was inhibited in cells expressing Rz1-9 and Rz1-14. Little inhibition of HIV-1 replication was observed in cells expressing ASPsi-gag RNA. Thus, the multimeric hammerhead ribozymes inhibit HIV-1 replication better than the antisense RNA. Inhibition of HIV-1 replication in cells expressing Rz1-9ASPsi-gag or Rz1-14ASPsi-gag RNA was worse than that obtained with the multimeric ribozymes alone. This result suggests that co-expression of antisense RNA decreases the anti-HIV potential of ribozymes. The multimeric ribozymes and the antisense RNA were designed to target different sites within the HIV-1 RNA. They are not expected to interact with each other. Neither are they expected to compete with each other for binding to the HIV-1 RNA. Instead, the antisense RNA binding to its (1553 nt-long) target site may have resulted in a decreased ribozyme turn over. Furthermore, since the antisense RNA/HIV-1 RNA hybrids are degraded by the cells, the co-expressed antisense RNA may have led to ribozyme degradation.