Berkhout Lab - Ben Berkhout - HIV AIDS research Laboratory - Silencing of HIV-1 with shRNA interference

Silencing of HIV-1 with shRNA interference

Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). The double- stranded RNA can be expressed as a short hairpin molecule (shRNA) with a base-paired stem of 21-23 bp. Previously, we have shown that stable expression of a short hairpin targeting the Nef gene (shNEF) of HIV-1, via a retroviral vector, results in strong inhibition of HIV-1 replication J Virol. 2004 Mar;78(5):2601-5.

For the development of an anti-HIV-1 gene therapy we used lentiviral vectors that can efficiently transduce hematopoietic stem cells and T-cells. We introduced the shNEF pol-III expression cassette into a third generation lentiviral vector system containing the eGFP reporter gene (Zufferey et al., J Virol. 1998; Seppen et al., J Hepatol. 2002).

Donor blood derived T lymphocytes were transduced with the lentiviral vector containing the expression cassette. GFP+ and GFP- cells were sorted and both populations were infected with HIV-1 LAI. As a control, cells were infected with the R1 virus, which has a large deletion in the Nef gene and is therefore resistant to inhibition by shNEF (Das et al, 2003).

HIV-1 efficiently replicates in non-transduced cells, but not in shNEF-expressing transduced cells. The control R1 virus replicates equally well in both cells, confirming that the inhibition by shNEF is sequence specific.

These results show that HIV-1 can be strongly inhibited in human primary T cells by transduction with a lentiviral vector expressing shNEF. Currently we are in the process of establishing a human in vivo system based on the hu-SCID mouse model (Gimeno et al., Blood 2004). Additionally we have conducted a search for effective shRNAs targeting conserved regions of the HIV-1 genome. We have multiple shRNAs that are equally or even more effective than shNEF. This will allow us to design a combination shRNA therapy.

This shows that RNAi has great potential as an antiviral gene therapy approach and support our continuing effort in the development of this strategy for the treatment of HIV-1 infected individuals.

Westerhout EM, Ooms M, Vink M, Das AT, Berkhout B. HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res. 2005 Feb 01;33(2):796-804.

Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R, Berkhout B. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol. 2004 Mar;78(5):2601-5.