Inhibition of HIV-1 infection by lentiviral vectors expressing pol III-promoted anti-HIV RNAs

Abstract

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.

Gene therapy progress and prospects: Novel gene therapy approaches for AIDS

“…A single vector backbone harboring both CCR5RZ and U16TAR decoy transmitted a high degree of resistance against HIV-1 in both primary T cells and CD34(+)-derived monocytes. 48 This strategy has been extended to include siRNA. 49 Ribozymes against CCR5, Tat, rev, and env-coding mRNAs together with RNA decoy targeting rev and siRNA directed against a sequence common to rev and tat mRNAs were transduced into CD34 hematopoietic progenitor cells via retroviral or lentiviral vectors.…”

Effective Suppression of Human Immunodeficiency Virus Type 1 through a Combination of Short- or Long-Hairpin RNAs Targeting Essential Sequences for Retroviral Integration

“…Gene targeting in mammalian cells through the use of shorthairpin RNAs (shRNAs) has been advanced by the development of vector systems for efficient delivery and stable expression of shRNA sequences (4,7,22,30). Upon delivery into cells, shRNAs are converted into short double-stranded RNAs, termed small interfering RNAs (siRNAs), that mediate a sequence-specific RNA degradation process termed RNA interference (RNAi) (12,14,42).…”

Silencing of HIV-1 Gene Expression by Two Types of siRNA Expression Systems

“…(Naldini et al, 1996;Manganini et al, 2002) and deliver genes into dividing cells at high efficiency (Tavernarakis et al, 2000;Yu et al, 2002). (Follenzi et al, 2000;Li et al, 2003;Mautino & Morgan, 2002;Mukhtar et al, 2000;Pandya et al, 2001;Qin et al, 2003;Schroers et al, 2002 Figure 5A). Furthermore, siRNA and GFP expression was observed in the CS-env-shRNA plasmid-transfected SupT1 cells for 15 days (Figures 5B,C).…”