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Journal of Virology, July 2004, p. 7079-7088, Vol. 78, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.13.7079-7088.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Antisense-Mediated Inhibition of Human Immunodeficiency Virus (HIV) Replication by Use of an HIV Type 1-Based Vector Results in Severely Attenuated Mutants Incapable of Developing Resistance

Xiaobin Lu,^1, Qiao Yu,^1, Gwendolyn K. Binder,¹ Ziping Chen,¹ Tatiana Slepushkina,¹ John Rossi,² and Boro Dropulic^1,3*

VIRxSYS Corporation, Gaithersburg, Maryland 20877,¹ Division of Biology and Department of Hematology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, California 91010 ,² Sydney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231³

Received 15 October 2003/ Accepted 9 March 2004

We have constructed a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector expressing a 937-base antisense sequence against the HIV-1 envelope gene. Transduction of CD4⁺ T lymphocytes with this vector results in expression of the therapeutic antisense sequence and subsequent inhibition of productive HIV-1 replication. In this report, we examined the effect of antisense-mediated suppression on the potential development of virus escape mutants using a permissive T-cell line cultured under conditions that over serial passages specifically allowed for generation and amplification of mutants selected for by antisense pressure. In the resulting virus clones, we found a significant increase in the number of deletions at the envelope target region (91% compared to 27.5% in wild-type HIV). Deletions were most often greater than 1 kb in length. These data demonstrate for the first time that during antisense-mediated suppression of HIV, mutants develop as a direct result of selective pressure on the HIV genomic RNA. Interestingly, in clones where deletions were not observed, there was a high rate of A-G transitions in mutants at the antisense target region but not outside this region, which is consistent with those mutations that are predicted as a result of antisense-mediated modification of double-stranded RNA by the enzyme double-stranded RNA-specific adenosine deaminase. These clones were not found to be escape mutants, as their replicative ability was severely attenuated, and they did not replicate in the presence of vector.

X.L. and Q.Y. contributed equally to this work.

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