[Frontiers in Bioscience 7, a15-28, February 1, 2002]

A Combination Anti-HIV-1 Gene Therapy Approach Using a Single Transcription Unit that Expresses Antisense, Decoy, and Sense RNAs, and Trans-Dominant NEGATIVE Mutant Gag and Env proteins

Shi-Fa Ding, Rocco Lombardi, 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 plasmid vectors allowing Tat- and Rev-inducible expression of anti-HIV-1 genes
3.2. Construction of bacterial expression vectors, pET-GE and pET-GmEm
3.3. Bacterial expression of WT and TDM gag and env genes
3.4. Construction of pTev-Puro expressing HIV-1 Tev
3.5. Stable transfection of HeLa cell line with pTev-Puro
3.6. Stable transfection of HeLa and HeLa-Tev cells
3.7. RT-PCR analysis of RNA extracted from the transfected HeLa and HeLa-Tev cells
3.8. Construction of retroviral vectors expressing anti-HIV-1 genes
3.9. Production of amphotropic retroviral vector particles
3.10. Production of stable MT4 transductants
3.11. HIV-1 susceptibility of stable MT4 transductants
3.12. RT-PCR analysis of RNA extracted from the uninfected and HIV-1 infected stable MT4 transductants, and from the progeny virus released from the HIV-1 infected stable MT4 transductants
3.13. Transduction of human PBLs
3.14. HIV-1 susceptibility of PBL transductants
4. Results
4.1. Assessment of the full-length gag and env open reading frames
4.1.1. Construction of pET-GE and pET-GmEm
4.1.2. Testing for expression of WT and TDM proteins
4.2. Assessment of Tat- and Rev-inducible gene expression
4.2.1. Construction of pBS-Ti-GE-Ri-Ter and pBS-Ti-GmEm-Ri-Ter
4.2.2. Testing for Tat-inducible expression of GE/GmEm mRNA and Tat- and Rev-inducible expression of WT/TDM Gag protein in HeLa and HeLa-Tev cells
4.3. Assessment of anti-HIV-1 potential of oncoretroviral vectors expressing the anti-HIV-1 genes <
4.3.1. Construction of MoTN-Ti-GE-Ri-Ter and MoTN-Ti-GmEm-Ri-Ter
4.3.2. Establishment of pools of stable MT4 transductants
4.3.3. HIV-1 susceptibility of stable MT4 transductants
4.3.4. HIV-1 susceptibility of human PBL transductants
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Y e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Y e signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.