Concocting a Knock-Out Punch for HIV-1

INITIAL RESISTANCE

At first glance, the outcome of these experiments seems obvious: RNA interference is an RNA silencing mechanism; HIV-1 needs RNA to replicate. EMBO Journal rejected a related study submitted earlier by Bryan Cullen and colleagues at Duke University Medical School, Cullen says, because the reviewers called the results predictable. The work was subsequently published in the Journal of Virology.⁵

The Hot Paper authors similarly weren't astonished by the result. "At that time, it was becoming clear that the RNA-interference machinery could be educated to attack any RNA, whether it was viral or cellular. So it wasn't really a surprise that we could do this," says Stevenson. The ease, efficiency, and potency of the technique did amaze, though, he says.

But researchers recognized a critical limitation in viral resistance: Long-term therapy targeting viral genes alone would likely be impossible. "A single point mutation is enough to make the virus resistant to RNA interference," says Cullen.

VIRAL RESISTANCE

Courtesy Carl D. Novina, Helen Cargill and Philip A. Sharp

People possessing a mutant form of CCR5 that is deficient in HIV binding have normal immune function but are resistant to HIV infection. Due to a high HIV mutation rate and concerns about generating virus escape mutants, cellular targets are preferable to viral targets. Thus, reducing CCR5 expression by RNA interference is a potential application for therapeutic gene silencing.

Indeed, Ben Berkhout's group at the University of Amsterdam demonstrated HIV escape using retroviral transduction, which stably introduces siRNAs targeting the HIV-1 Nef gene in T cells.⁶ They discovered several mutants that resisted RNAi, all of which contained Nef mutations. "The escape actually demonstrates that the initial pressure that was put on the virus was really potent ... it really is a perfect demonstration of the specificity of the RNA interference," says Berkhout. If multiple targets are used, escape would be more difficult, he adds, making an effective HIV therapy "doable."

Other laboratories followed up on CD4 targeting by going after the corecep-tor CCR5; humans who are homozygous for a defect in this gene are naturally resistant to HIV infection.⁷⁸ Sharp speculates that if the technology will work as a therapy, it will do so by silencing cellular genes. "Not CD4, but more likely CCR5, whose expression may not be critical for normal immune function."

But this may not be the answer, either. "You can certainly imagine that HIV-1 can mutate and utilize different coreceptors within a host," says virologist Irvin Chen, director of the AIDS Institute at the University of California, Los Angeles. Chen adds, however, that such mutations would not likely impede the effectiveness of siRNA targeted against CCR5, especially if used in conjunction with other therapies.

Still, Roger Pomerantz of the Division of Infectious Diseases, Thomas Jefferson University, Philadelphia, says his own studies have shown that RNAi is not as robust an inhibitor of HIV-1 as previously thought. "It depends on the small interfering RNA that you pick, the phase of the life cycle, [and] how you set up your system." Pomerantz adds that with gene therapy in general, viruses can overcome the treatment through resistance, or even by simply overwhelming the system.

SPECIAL DELIVERY

Even if such problems are overcome, the problem of delivery to cells remains. "In order to use [siRNAs] as a drug, you have to figure out a way either to introduce these small duplex RNAs, to get them through the plasma membrane of cells, or you have to express them, or express precursors of them, in cells," says Lieberman. Some approaches currently under investigation include expression of small hairpin RNAs from a viral vector, which then can be processed into siRNAs in vivo, and direct injection of modified siRNAs into cells.

Rossi's group put the construct they had used in their 2002 hot paper into a lentiviral vector to transduce siRNAs into hematopoietic progenitor cells.⁹ "We've gone a step further," Rossi says. His group took primary progenitor cells from donors, and modified them so that maturing macrophages, T cells, and monocytes would all be highly resistant to HIV infection.

Nevertheless, little is known about possible side effects of RNAi-based therapy. "We don't really understand enough about the complex mechanisms that are involved in RNAi, siRNAs, microRNAs, and what their normal role is in cells," says Chen. "I think the danger is that if we move too quickly, we could start interrupting normal pathways in cells that can cause unwarranted side effects."

While a growing number of scientists question the future of RNAi as an antiviral therapy, some point to new evidence that RNAi may be an ancestral defense against dsRNA-containing viruses. But none downplays the technique's importance as a tool to study viruses such as HIV. Pomerantz cites the CCR5-inhibition studies as an example of how this technique can be used to tease out aspects of the viral life cycle. Says Cullen: "It's having a dramatic effect on the molecular biology of human cells. You can really ask for the first time, what does this protein actually do in the cell, and you could never do that before in a direct way."

Aileen Constans can be contacted at aconstans@the-scientist.com.