Ali Ramezani, Xue Zhong Ma, Reza Nazari, and Sadhna Joshi

Department of Medical Genetics and Microbiology, Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3E2, Canada

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 stable MT4 transductants
3.4. RT-PCR analysis of total RNA from stable MT4 transductants
3.5. In vitro cleavage activity of multimeric ribozymes amplified from pools of stable MT4 transductants
3.6. HIV-1 susceptibility of stable MT4 transductants

4. Results

4.1. MGIN-based retroviral vectors expressing multimeric ribozymes, Rz_1-9, Rz_10-14, or Rz_1-14
4.2. Development of pools of stable MT4 transductants expressing multimeric ribozymes
4.3. In vitro cleavage activity of multimeric ribozymes produced from the stable MT4 transductants
4.4. HIV-1 susceptibility of pools of stable MT4 transductants expressing multimeric ribozymes

5. Discussion

6. Acknowledgements

7. References

1. ABSTRACT

Rz_1-9 is a multimeric hammerhead ribozyme targeted against nine highly conserved sites within the env coding region of human immunodeficiency virus type-1 (HIV-1) RNA. This ribozyme was shown to inhibit HIV-1 replication (1, 2). However, Rz_1-9 target sites are only conserved within the clade B of HIV-1. In this study, we have designed another multimeric ribozyme, Rz_10-14. This multimeric ribozyme is targeted against five sites that are highly conserved amongst all major clades of HIV-1. A third multimeric ribozyme, Rz_1-14, was obtained by combining both Rz_1-9 and Rz_10-14. A mouse stem cell virus-based MGIN vector (3) was used to express these ribozymes in a human CD4+ T lymphoid cell line. Stable transductants expressed vector RNA containing ribozymes which were shown to be active. In HIV-1 challenge experiments, very little or no virus production could be detected in the pools of stable MT4 transductants expressing Rz_1-14 for 60 days tested. Inhibition of virus replication was most prominent with Rz_1-14, followed by Rz_1-9, and then Rz_10-14. Thus, the combined multimeric ribozyme, Rz_1-14, is more effective than Rz_1-9 or Rz_10-14. As Rz_1-14 is targeted against all major clades of HIV-1, it will be further pursued for use in HIV-1 gene therapy.