1. ¹Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
2. ²Division of Virology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
3. ³Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
4. ⁴Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA

Correspondence: John J. Rossi, E-mail: jrossi@coh.org

Received 7 April 2005; Revised 11 July 2005; Accepted 11 July 2005.

Abstract

Combinatorial therapies for the treatment of HIV-1 infection have proven to be effective in reducing patient viral loads and slowing the progression to AIDS. We have developed a series of RNA-based inhibitors for use in a gene therapy-based treatment for HIV-1 infection. The transcriptional units have been inserted into the backbone of a replication-defective lentiviral vector capable of transducing a wide array of cell types, including CD34⁺ hematopoietic progenitor cells. The combinatorial therapeutic RNA vector harbors a U6 Pol III promoter-driven short hairpin RNA (shRNA) targeting the rev and tat mRNAs of HIV-1, a U6 transcribed nucleolar-localizing TAR RNA decoy, and a VA1-derived Pol III cassette that expresses an anti-CCR5 ribozyme. Each of these therapeutic RNAs targets a different gene product and blocks HIV infection by a distinct mechanism. Our results demonstrate that the combinatorial vector suppresses HIV replication long term in a more-than-additive fashion relative to the single shRNA or double shRNA/ribozyme or decoy combinations. Our data demonstrate the validity and efficacy of a combinatorial RNA-based gene therapy for the treatment of HIV-1 infection.