The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS

Abstract

RNA interference (RNAi) is highly effective in inhibiting human immunodeficiency virus type 1 (HIV-1) replication by the expression of antiviral short hairpin RNA (shRNA) in stably transduced T-cell lines. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against highly conserved regions of the HIV-1 RNA genome. The future in vivo application of such a gene therapy protocol will reach only a fraction of the T cells, such that HIV-1 replication will continue in the unmodified T cells, thereby possibly frustrating the therapy by generation of HIV-1 variants that escape from the inhibition imposed by the protected cells. We studied virus inhibition and evolution in pure cultures of shRNA-expressing cells versus mixed cell cultures of protected and unprotected T cells. The addition of the unprotected T cells indeed seems to accelerate HIV-1 evolution and escape from a single shRNA inhibitor. However, expression of three antiviral shRNAs from a single lentiviral vector prevents virus escape even in the presence of unprotected cells. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models.

Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

“…In any case, CCR5 knockdown should be done in concert with another strategy to constrain HIV (i.e., including another anti-HIV moiety, combining with efficient antiretrovirals, or boosting the immune response in parallel to transplantation). Indeed, the solidness of successful gene engineering by the expression of more than one antiviral moiety may prevent HIV evolution (51). Gene engineering could be combined with conventional ART: combining two treatment modalities was efficient in cell lines (52), as induction therapy (53) or with anti-PD1 antibodies that decrease viral load and increase the level of CD4 ϩ T cells in HIV-infected mice (54).…”

Gene Therapy Strategies to Block HIV-1 Replication by RNA Interference

“…Modifi ed from ref. [ 106 ] but this obviously does not result in 100 % coverage due to the considerable genetic variability among HIV-1 strains [ 30 ]. The survey of clinical HIV-1 isolates should include drug-resistant HIV-1 variants, but we tried to avoid targets that correspond to protein domains in which drug-resistance mutations are located [ 16 ].…”

“…7 ). We studied virus inhibition and evolution in pure cultures of shRNAexpressing cells versus mixed cell cultures of protected and unprotected T cells [ 106 ]. Transcripts start with the HIV-1 R and U5 regions, the packaging signal (ψ).…”

Gene Therapy for HIV-1 Infection

“…The finding that tRNA promoters may generate artificial antiviral RNAi activators was an important development. Corroboration of the merits of the approach came from demonstration that expression of three antiviral shRNA sequences in a stably transduced T cell line effectively prevented viral escape when replication occurred in mixed populations comprising protected and naïve cells [170]. Corroboration of the merits of the approach came from demonstration that expression of three antiviral shRNA sequences in a stably transduced T cell line effectively prevented viral escape when replication occurred in mixed populations comprising protected and naïve cells [170].…”