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Researchers: HIV-1 Proves Elusive Target for RNAi Drugs; Multiference May Be the Answer

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For those looking to harness RNA interference for drug therapies, HIV has become one of the most promising targets. But, according to Daniel Boden and colleagues from Brown Medical Brown Medical School’s laboratory of retrovirology, the virus’ ability to rapidly mutate could pose as much a problem for RNAi-based drugs as it does for traditional small molecule therapies.

Boden and colleagues published a paper in the November issue of the Journal of Virology in which they described how they set out to characterize the potency and durability of HIV-1-specific RNAi compounds using a synthetic siRNA molecule that targets exon 1 of the HIV-1 tat gene, which encodes a transactivator protein essential for viral replication. What they found is worrisome — but maybe not unexpected — for anyone who hopes to quickly turn RNAi into an HIV therapeutic.

“Our results demonstrate a critical obstacle to using RNAi as an antiretro- viral agent,” Boden and colleagues wrote in their paper. “The rapid replication kinetics of HIV-1 and the error-prone nature of viral reverse transcriptase led to the emergence and selection of viral quasispecies that were genotypically distinct in the tat shRNA region, thereby eliminating the antiviral effect.”

According to the paper, the researchers created a recombinant AAV DNA vector that contained an H1 promoter-driven short hairpin RNA expression cassette with tat-specific sequences. The H1 promoter was chosen, they wrote, because recent evidence suggests it can effectively drive the expression of shRNA, dsRNA that are processed into siRNA by Dicer, with retroviral vectors.

Boden et al. cotransfected the vector, along with a control vector lacking tat sequences in the shRNA expression cassette, with an infectious molecule clone of HIV-1 in 293T cells. After 48 hours, HIV-1 p24 antigen levels were quantified, and the researchers noted a 97 percent drop in antigen level associated with the cells expressing tat shRNA versus the mock construct.

They then attempted to create a cell line that stably expresses shRNAs by introducing the two vectors into H9 cells, which are derived from human cutaneous T-cell lymphoma and are permissive for HIV-1 replication. These cells were then infected with the HIV-1 clone.

Over a two-month period, Boden and his colleagues wrote, the p24 antigen levels were checked weekly. For the first three weeks, HIV-1 replication was cut by 95 percent in cultures of cells expressing tat shRNA. By day 25, however, p24 levels began to rise, indicating a loss of shRNA-related antiviral activity.

“That brought us to the idea to first check if there’s anything wrong with the expression of these short hairpin RNAs … if maybe the construct doesn’t contain the expression cassette anymore, or if the siRNAs aren’t processed,” Boden told RNAi News. “We did a Northern [blot analysis] to see if the expression is still ongoing, and it was, in fact. Then we though … maybe it’s a resistance occurring there.

“Then we found out that there was, indeed, a mutation in the middle of the target sequence,” he added. Specifically, the nonsynonymous mutation was at nucleotide position 9 of the targeted sequence, the researchers wrote, “leading to an amino acid change from threonine to serine.”

“It looked pretty clear to us [that the mutation caused the drop in antiviral activity], but the next step was to come up with a mutant clone where we artificially included that mutation,” Boden said.

They did so, and found that infection of a tat shRNA-expressing H9 cell line with the mutant version of the HIV-1 clone resulted in zero inhibition of viral replication. Inhibition of HIV-1 replication, however, was seen by day 10 when the same cell line was challenged with the original HIV-1 clone.

According to Boden, the outcome of these experiments didn’t come as much of a surprise, given that viral replication was never totally knocked out. “Since there was still residual replication, you would expect that, at some point, the virus would escape that selective pressure [of RNAi].”

Boden and colleagues suggested in the paper that “more potent shRNAs need to be designed which target highly conserved regions of the viral genome (e.g. gag and pol) essential for the viral life cycle” in order to more durably suppress HIV-1 replication. “Alternatively, similar to present antiviral drug therapy paradigms, RNAi constructs coexpressing multiple shRNAs need to be developed that simultaneously target different regions of the viral genome, thereby reducing the probability of gener- ating shRNA escape mutants.”

But in practice, Boden is exploring a combination of these two potential solutions.

“We tried to come up with strategies … like the HAART ther- apy,” he said. “So, we try to target multiple gene regions within HIV which are highly conserved. The tat region we selected was kind of conserved — more conserved, of course, than [HIV] envelope — but there are even higher conserved regions … in the active center of protease and the gag region, which we’ve tested. We now have a panel of short hairpin RNAs [for these targets] and we’ve created already cell lines expressing different targets from different promoters within one molecular construct,” Boden said.

“We call it multiference … because it’s RNA interference targeting multiple genes,” Boden said. “It increases the potency of your construct in a synergistic way … and it delays emergence of resistance, maybe [indefinitely]. We’re testing that right now.”

He added that these tests have been going for about two weeks, and that it may be “a long time before we see anything.”

—DM

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