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.

Figure 1

Human immunodeficiency virus type 1 (HIV-1) escape from short hairpin RNA (shRNA) therapy in different culture systems. In vitro panel: pure cultures of shRNA-expressing cells (blue) that are protected against HIV-1 infection. HIV-1 variants will be generated at a low rate and only a shRNA-resistant variant (red virus) will be able to spread. In vivo context: mixed cell cultures of protected (blue) and unprotected (pink) cells will allow virus replication in the unprotected cells, leading to the rapid generation of a viral quasispecies by spontaneously acquired mutations. This quasispecies may also contain one or more shRNA-resistant variants that are able to replicate in the protected cells. Test panel: direct genotyping means sequencing of proviral DNA target sequences when virus replication is observed in the primary virus cultures. Phenotyping: cell-free virus harvested in the primary culture is used to produce a replication curve on unprotected cells and shRNA-expressing cells. Standard genotyping of proviral DNA target sequences when virus replication is observed in the latter shRNA-expressing cells. WT, wild-type.

Figure 2

Impact of unprotected cells on the evolution of Pol47 resistance. A control vector transduced cell line was included (100% unprotected cells) and 5, 20, and 50% of unprotected cells was added to regular SupT1-Pol47 cultures to compare the evolution results with those obtained in pure cultures of Pol47-expressing cells. Six parallel cultures per experimental condition were challenged with human immunodeficiency virus type 1 LAI at a multiplicity of infection of 0.002. Virus replication was monitored by measuring CA-p24 for 55 days.

Figure 3

Phenotypic test of emerged LAI viruses. Cell-free virus at the peak of virus production in every scenario described (5, 20, and 50% unprotected cells and pure cultures of Pol47-expressing cells) was collected and passaged on (a) fresh unprotected cells and (b) SupT1-Pol47 cells (pure cultures of protected cells). Wild-type (WT) virus was included as control (virus that replicates exclusively on unprotected cells).

Figure 4

Impact of unprotected cells on the evolution of R3A resistance. A control vector transduced cell line was included (100% unprotected cells) and 5, 20, and 50% of unprotected cells were added to regular SupT1-R3A. Six parallel cultures per experimental condition were challenged with human immunodeficiency virus type 1 LAI at a multiplicity of infection of 0.002. Virus replication was monitored by measuring CA-p24 for 70 days.

Figure 5

Distribution of escape mutations within the 19-nucleotide target sequence (Pol47 shRNA escape). (a) Composite of mutations observed within the 19-nucleotide target in multiple independent viral escape cultures in SupT1 and (b) in PM1 T-cell line. (c) Composite of mutations observed in both T-cell lines (SupT1 + PM1). *Silent mutation. shRNA, short hairpin RNA.