Before there was Twitter, there was Facebook, and before that, Friendster. And who can forget MySpace? There’s a similar trend of successive usurping technologies in the fast-moving quest to develop therapeutics capable of modifying the genome. Since the late nineties, we’ve witnessed the rise of several gene-silencing approaches, from “antisense” oligonucleotides and RNA interference (RNAi) to the latest targeted genome-editing techniques, such as those based on zinc finger nucleases or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology. These rapid developments raise the stakes for companies that have wagered on a particular gene-silencing approach.
Take the case of an approach known as DNA-directed RNAi (ddRNAi). In January, Australia-based Benitec Biopharma received a green light from the US Food and Drug Administration to begin the first human trial of an intravenous viral gene therapy based on ddRNAi. The therapy, dubbed TT-034, is essentially a modified form of adeno-associated virus 8, which naturally infects people but is not pathogenic. In TT-034, the viral DNA has been engineered to encode short hairpin RNAs (shRNAs) that silence three different components of the hepatitis C virus (HCV). The approach is referred to ddRNAi because the shRNA that carries out the gene silencing is continually produced by the cell from a DNA vector.
In the trial slated to begin imminently, patients infected with HCV will receive a single injection of TT-034; if it works, it should eliminate the virus from their livers and provide lasting immunity to the disease. Benitec sees it as a potential alternative to existing HCV antiviral therapies, which can involve injections and daily pills for a period of time and can sometimes have debilitating side effects. But some question the need for an RNAi-based HCV therapy. “The available drugs that have just been approved, or will be approved in the next 6 to 12 months, will essentially eliminate the virus from the majority of patients,” says Ralf Bartenschlager, a virologist at Heidelberg University in Germany who studies HCV.
Others worry about whether the vector is optimal for the approach. “I think this concept is something that is going to work,” says Mark Kay, head of the division of human gene therapy at Stanford University in California, and co-founder of Voyager Therapeutics, which launched in February and aims to develop adeno-associated virus-based gene therapies for neurological disorders. “I just don’t think they’re using the right vector. At the time they started, it was the obvious choice. But as more data has accumulated over time, in my opinion, it’s not the right choice.”
That’s in part because recent research using mice with humanized livers suggests that the version of the virus used in TT-034 doesn’t seem to be as effective at infecting human cells as it is animal cells, says Dirk Haussecker, an independent RNAi consultant based in Germany. But David Suhy, senior vice president of research and development at Tacere Therapeutics, a subsidiary of Benitec that originally developed TT-034, says that the humanized mouse models may not accurately represent the architecture and gene expression patterns of human liver, making it unclear whether the virus will actually be less effective in humans compared to mice. “We firmly believe that the best way to see how the TT-034 compound will work in humans is to test it in humans,” he says.
Betting on bifunctionality
Benitec isn’t the only company pursuing the ddRNAi strategy. Gradalis, which is based in Dallas, Texas, is also testing ddRNAi-based therapies in Phase 1 and 2 clinical trials, but has taken a slightly different approach. The company has developed two different types of therapies, both of which impede cancer cells using a so-called bifunctional shRNA design. In this approach, a DNA plasmid produces an RNA that adopts a structure containing two short hairpins—one that is processed by the cell to a small interfering RNA (siRNA) that binds to complementary mRNAs and causes them to be degraded, and another that is processed to a microRNA that binds to the same mRNA target and blocks protein production. “Therefore, you get increased strength of the knockdown, and it lasts longer,” says Charles Brunicardi, a cancer surgeon at the University of California-Los Angeles, who is involved in the clinical and pre-clinical trials of Gradalis’s gene-silencing therapies.
But based on what is now known about the molecular biology of the cellular RNAi machinery, some question the rationale behind the bifunctional approach. That’s because siRNA-mediated gene silencing is faster and more efficient than microRNA-mediated inhibition, says Shuo Gu, a cancer researcher at the US National Cancer Institute in Frederick, Maryland, who studies the molecular mechanisms of RNAi. Therefore, mRNAs that are tied up in microRNA complexes might be less accessible to RNAi cleavage, which would reduce inhibition, he says. “More needs to be known about the mechanisms before we know what we’re dealing with here.”
A third company, Calimmune, which is headquartered in Tucson, Arizona, has licensed the ddRNAi technology from Benitec to develop an experimental anti-HIV agent known as Cal-1, which is currently in phase 1/2a trials. Calimmune’s construct is derived from a lentivirus, which integrates its genetic material into host cells. The therapy delivers an shRNA that knocks down a receptor known as CCR5, which HIV uses to gain access to T cells of the immune system, and also contains the instructions for making a protein known as C46, which blocks HIV’s ability to bind to T cells.
But actually giving the Cal-1 therapy to patients is a complicated process that involves harvesting stem cells from the bone marrow of HIV-infected patients, treating the cells with the therapy in vitro, and then putting the engineered cells back into the patients, who must undergo a ‘conditioning’ process that obliterates much of their immune system so that the engineered stem cells can take hold and repopulate the blood with HIV-resistant cells. “That’s an additional risk and challenge, and there are obvious potential drawbacks in trying to implement that in a large scale way,” says David Margolis, an HIV expert at the University of North Carolina School of Medicine in Durham.
The road ahead
Several companies have already tried their hand at ddRNAi therapies, but with little success. In 2007, the now defunct Pennsylvania-based biotech company Nucleonics prematurely ended its phase 1 trial of a systemic non-viral ddRNAi therapy for hepatitis B virus** after treating only three patients; the drug failed to knockdown its targets and triggered mild immune responses. Similarly, Amsterdam Molecular Therapeutics once pursued viral ddRNAi therapies, but sold its assets to the newly formed biotech company UniQure in late 2012.*** And Pfizer, which partnered with Tacere to develop TT-034, halted it ddRNAi efforts in 2012 and handed the rights to the therapy back to Tacere.
Even if the latest ddRNAi-based therapies turn out to be safe and effective, there’s tough competition from the latest genome-modifying tools, including zinc finger nucleases (ZFNs) and CRISPR, which use DNA cutting enzymes to specifically alter or inactivate genes of interest. For example, California-based Sangamo BioSciences is currently conducting using a ZFN-based approach to delete both copies of the CCR5 gene in the T cells of patients infected with HIV. The modified cells are then expanded in the lab and transplanted back into patients. And just last month, researchers at the Massachusetts Institute of Technology (MIT) in Cambridge reported using CRISPR to cure adult mice of a genetic disorder caused by a single point mutation.
Daniel Anderson, who led the MIT study but has also studied RNAi, says he is personally very optimistic about the therapeutic potential of the gene-editing approaches, although he admits that they have delivery challenges and raise concerns about the potential for off-target genetic modifications. But he acknowledges that there is room for both RNAi and gene-editing forms of gene therapy. “To me, it’s still pretty early to call winners or losers.”
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**Corrections (14 April): An earlier version of this story incorrectly stated that Nucleonics was working on a therapy for hepatitis C virus, but in fact it was hepatitis B virus. ***This story also stated that Amsterdam Molecular Therapeutics folded in 2012, but it is more accurate to say that the company sold its assets to UniQure, which is developing ddRNAi therapies for Huntington’s disease. Nature Medicine regrets the errors.