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A whole new RNA world

I was surprised to discover the paper I’m about to present to you came out over a week ago. It didn’t cause much of a splash, yet it details a novel treatment for HIV. Ok, that’s overstating it a little bit, but it’s not far off.

In order to explain exactly how this “aptamer-siRNA chimera” (don’t panic, I’ll return to that mouthful soon) works, I’m going to take several large steps back to the central dogma of molecular biology. Every cell possesses a DNA genome. This DNA contains chunks of information called genes. But DNA as DNA doesn’t really do all that much. It can’t metabolize anything or move anything around. What it does do is stably store cellular information, and this information has to be liberated and used. The way the cell does this is, in the majority of cases, is in a two-step mechanism. In the first step the information is read and converted into a messenger RNA (mRNA) in a process termed transcription. As its name suggests, RNA is related to DNA but has certain chemical distinctions. RNA can subsequently be converted into proteins, the cell’s work force, and thus the information encoded in DNA is translated via RNA to protein.

So why not go straight from DNA to protein? The whole process of gene expression that I just described has to be very tightly regulated. Gene expression gone wrong has very serious consequences, cancer being a prime example. As a result, every step is under stringent control. The accessibility of the DNA, the amount of RNA made, the lifetime of the RNA, the amount of protein made and the lifetime of the protein are all crucial in the “life-cycle” of a gene. By having several levels of control, the cell can fine-tune exactly how much active protein it makes.

RNA as a regulator of gene expression

Just to complicate matters a tiny bit, I have to bring up the fact that RNA itself plays an active role in the cell, and more specifically in the regulation of gene expression. In the late 90s Craig Mello and Andrew Fire (2006 Nobel laureates) discovered a process now known as RNA interference or RNAi. They noticed that when they injected RNA complementary to certain genes into the worm Caenorhabditis elegans, the levels of protein encoded by that gene were significantly reduced. It turned out that the injected RNA was recognizing cellular mRNAs and stalling the production of proteins.

Since its discovery, RNAi has been shown to be a highly conserved mechanism for the regulation of gene expression, and exists in a few different flavors. As in the original discovery RNAi-like systems control myriad cellular processes in all kingdoms of life. But more importantly for the paper I want to tell you about (yes, we’re almost done with background) we can hi-jack this molecular pathway for both basic scientific endeavors and medical advances. We can design and synthesize RNAs, transfer them into cells, and use the RNAi pathway to shut down almost any gene we want.

RNA as an antiviral

Almost a hundred years ago Alexander Flemming noticed the antibacterial properties of a Pencillium fungus, and since then many different antibiotics have been successfully used to treat bacterial infections. In the case of viral infections we haven’t been so lucky. Viruses are bizarre entities. There is still considerable debate over whether they are actually “alive”; I even had an immunology professor who swore that they must have come from outer space. They vary in composition, but in general comprise a small genome of DNA or RNA surrounded by a protein shell or capsid, and in some cases a phospholipid envelope stolen from the cell they just infected. But most infuriatingly, they have a huge tolerance for genetic variation. They can mutate at an extraordinary rate and thus thwart our attempts at vaccination or treatment.

In addition to this ability to outwit modern medicine, viruses invade the cells of our body, and in the case of HIV, the genomes of those cells. Once inside a cell they are shielded from our immune system and free to replicate ad nauseam. HIV is a particularly tricky beast, in that it invades particular cells of the immune system, CD4+ T cells, thus not only evading the immune system but also depleting it. This makes treatment difficult, as eradication of the virus damages the host’s immune system. Thus most antiviral treatments for HIV are toxic to the individual taking them.

An attractive hypothesis, therefore, has been to design a non-toxic RNA that could target the viral genome and silence it in an RNAi-like mechanism. The treatment would be highly specific to the virus and leave the infected cells unscathed. While it would not represent a cure, it has the potential to be a much less abrasive treatment.

The idea of RNA as a therapeutic tool has been around for a while, but has been plagued with technical problems. RNA is fairly rapidly degraded in the bloodstream, so chemical modifications are needed to make it stable in an animal host. Further modifications are necessary to ensure cells could ingest the RNA. And of course the RNA needs some form of targeting mechanism to find the right cell.

RNAi-based treatment of HIV

In the case of HIV, an RNA therapeutic is well on the way to becoming a viable treatment. In a paper published last week in Science Translational Medicine, a team lead by Charles Preston Neff at Colorado State University demonstrated that an extensively modified RNA could significantly reduce viral load in a mouse model of chronic HIV infection.

So here’s where the aptamer-siRNA chimera comes in. The “siRNA” part of this molecule is the business end. Its sequence recognizes viral mRNAs within an infected cell and prevents protein production therefore halting viral particle assembly. The “aptamer” part is an RNA sequence that binds tightly to a cell surface protein, gp120, which is displayed on CD4+ T cells that are infected with HIV. Thus the aptamer provides the targeting mechanism. And it is a “chimera” because the whole molecule comprises two different RNA species stuck together, much like the chimera of Greek mythology (part lion, part goat, part snake). Lastly, the whole molecule contains chemical modifications that protect the RNA from degradation.

Chimera Apulia Louvre

The authors had shown previously that this RNA could subdue virus production in a tissue culture scenario, but the next challenge was to show that the chimera could be efficiently delivered to HIV-infected cell in an animal. The animal they chose was a humanized mouse. This mouse has human blood cells circulating and thus can be infected with strains of HIV that can normally infect humans. This is an important feature of the study, as at least one of the many stages of human drug testing, namely that the treatment doesn’t only work in mice, can be bypassed. Having chosen their model system, the authors next injected the HIV+ mice with a chimera preparation every week for five weeks and monitored the amount of virus persisting in the bloodstream. Four of the six mice used in the trial had undetectable levels of the virus by the third week: A striking effect.

Conventional antiviral treatments for HIV have to be taken for the rest of a patient’s life, and often force mutations in the virus that result in drug resistance. The team therefore assessed how long after administration the effect of the chimera lasted. Up to five weeks after the last injection virus levels were three orders of magnitude lower in treated versus untreated control mice. This is equivalent to the difference between a relatively healthy HIV positive individual and someone with full-blown AIDS. In addition, the mutation rate observed in aptamer-siRNA-treated mice was extremely low, suggesting that this therapy is less likely to induce resistant strains of HIV. One potential way to avoid or slow resistance further would be to attack the infection with several chimeras that target different parts of the virus genome.

A whole new treatment

So what’s the plan now? A few more tests need to be performed in animal models before the treatment can progress to human trials. Most importantly, the optimal treatment regimen needs to be determined, i.e. how much chimera should be administered and at what frequency. But from there, the first trials will most likely be conducted in patients with drug-resistant strains of HIV.

And it doesn’t stop at HIV. This treatment could be groundbreaking in the treatment of other viruses, cancer and various genetic conditions. With these key chemical modifications and targeting mechanisms, a major hurdle in the world of RNA therapeutics has been vaulted with style.

Neff, C., Zhou, J., Remling, L., Kuruvilla, J., Zhang, J., Li, H., Smith, D., Swiderski, P., Rossi, J., & Akkina, R. (2011). An Aptamer-siRNA Chimera Suppresses HIV-1 Viral Loads and Protects from Helper CD4+ T Cell Decline in Humanized Mice Science Translational Medicine, 3 (66), 66-66 DOI: 10.1126/scitranslmed.3001581

Katie Ph.D. ABD


CM Doran

Thanks for posting. Helping with resistance is huge! I hope it holds up in later trials…and perhaps other work in infectious disease.

Katie, Ph.D. (ABD)

I can’t wait to see how this plays out! It’s such an elegant designed strategy, it would be phenomenal to see it work out as a viable treatment in this an other situations.

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Charles Preston Neff

Thank you for your nice review of my work. For an update, we have done further trials of the aptamer, evolving the portion that binds to the siRNA to a flexible bridge capable of delivering multiple siRNAs as apposed to the single siRNA per aptamer as done in these studies. With this we have challenged multiple siRNAs targeting host and viral mRNA, with some varying effective levels. However, it appears that the greatest impact of this treatment comes from the Aptamer itself (soaking up the free floating virus by binding directly to gp120) with the siRNAs playing an additionally role of viral suppression, especially after the initial viral loads are depleted, and prolonging efficacy. Clinical trials are being conducted with promising results.

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