Monday, March 1, 2010

Sense-induced Trans-Silencing: Combining the Best of RNAi and Antisense

No, this is not about single-strand RNAi (ssRNAi), nor an anti-antisense rant. This is about my (probably) last publication as a wet-bench scientist [Haussecker et al (2010): Human tRNA-derived small RNAs in the global regulation of RNA silencing], where my co-authors and myself stumbled across a new method of inducing RNAi that combines elements of DNA-directed RNAi (ddRNAi) and single-strand oligo therapeutics. We christened the technology sense-induced trans-silencing (SITS).

It all begins with the discovery of a new type of small RNA

The story starts with our investigations into the zoo of small RNAs that exists in our cells. We had originally reported on capped small RNAs that are generated in the course of hepatitis delta virus replication. In molecular biology, viruses often make for good model systems for studying general gene regulation mechanisms, and so we set out to discover the non-viral cellular counterparts of the HDV small RNAs. Long story short, instead of discovering the cellular counterparts of the HDV small RNAs (these in fact do exist and have since been widely reported by others to be generated during normal transcription initiation of many cellular genes, e.g. this one), we, in the spirit of curiosity-driven basic science, soon homed into a quite peculiar population of small RNAs that were derived from tRNAs.

It turns out that we were not the first to observe them. In fact many others had cloned them in their small RNA sequencing studies. They were, however, never fully investigated because scientists were apprehensive that they were mere degradation products of highly abundant RNAs. Maybe because we were able to visualize them through enzymatic means before Northern Blotting and to us looked like bona fide small silencing RNAs with 5’phosphates (tRNA degradation products are not 5’P), we continued to study their interaction with components of the RNAi pathway. Importantly, the tRNA-derived small RNAs (tsRNAs) were bound by Argonaute proteins, the effectors of RNAi.

A control experiment yields an unexpected result

Since tsRNAs interacted with Argonaute proteins, we went on with the standard assays to show that they had gene silencing capacity: luciferase reporters into which tsRNA target sites had been cloned. As a control, we co-transfected the luciferase reporters with oligonucleotides that were antisense to the tsRNAs (and therefore sense to the target gene), fully expecting that this would block any tsRNA silencing activity. To our surprise, instead of relief from gene silencing, the oligonucleotides actually triggered gene silencing. Based on subsequent mechanistic studies, it seems that while single-stranded 5’-phosphorylated small RNAs preferentially incorporate into Argonautes 3 and 4 which are not good silencers, converting them into double-stranded RNAs through the addition of complementary oligonucleotides now made them the preferred substrate for the most potent gene silencers among the Argonautes: Argonaute 2. Because the oligonucleotide that induces the silencing is sense with respect to the target gene, we termed the method Sense-Induced Gene Silencing, or SITS.

Sense-Induced Trans-Silencing- The Method

To effect SITS, the guide RNA is engineered into a tRNA expression vector and introduced into the target cells. By itself, this has little effect on target gene expression. However, once you introduce an oligo that is complementary to the guide RNA, silencing is triggered. This means that it is possible to tap into the inherently potent RNAi gene silencing pathway without the need for introducing double-stranded RNA which, in the absence of formulation, is harder to do compared to single-stranded oligos, for example as is largely practiced in antisense. The differentiating factor from antisense technology, however, is that the potency of RNAi on a per molecule basis is significantly higher, as will the general pharmacology be quite different for SITS.












You may ask, why not do straight-forward DNA-directed RNAi. I agree that DNA-directed RNAi is extremely promising. One drawback of most ddRNAi methods, however, is that it is a hit-and-run approach with little ability to regulate the degree of silencing after the DNA has been introduced. In the case of SITS, the activity of the guide strand that is derived from the DNA vector is only turned on as long as there is sense oligo present. Regulated ddRNAi is not new per se, but this method distinguishes itself in that it does not require a potentially immunogenic (foreign) protein such as in tetracycline-controlled systems.

Another question (that also a patent examiner might ask) is that in a way this is adding sense and antisense and thereby reconstituting active siRNAs. In a way this is a correct description of the mechanism, but to my knowledge, the temporally separate addition of passenger (‘sense’) and guide oligos (‘antisense’) has not been demonstrated to trigger successful gene silencing. Our method therefore is not only unexpected because the attempt to antagonize a 5’-phosphorylated small RNA did not inhibit gene silencing, but actually induced it, but also in light of such failures. The fact that it does silence could be due to the curious re-direction of the tsRNAs from Ago3/4 to Ago2 that occurs in the SITS setting. Also, unlike is the case of single-strand RNAi where the transfection of a single-stranded guide RNA is sufficient to trigger silencing, tRNA-derived small RNAs curiously do not do the same even when present in high concentrations. Moreover, I am not aware of attempts to provide passenger and guides through a combination of DNA-directed and synthetic means.

Potential uses of SITS

I probably would not be credible if I claimed that SITS was the preferred embodiment for all RNAi Therapeutics approaches. Like ddRNAi versus synthetic siRNA therapeutics, SITS should have its niches. Applications that might benefit from SITS include those that are also considered acceptable for gene therapies. In case you have not noticed, gene therapies really have come a long way in that there are now more and more clinical success stories, albeit often for orphan diseases (orphan diseases are becoming more and more popular in the drug development industry). Neurodegenerative disease, including those of the eyes could be one primary field of use, especially since AAV and lentiviruses have been shown to be well tolerated there and allow for long-term gene expression. Huntington’s Disease comes to mind where shRNA toxicity has been a concern as well as the question whether too much knockdown of the Huntington gene could be detrimental, too. With SITS, you would first introduce an AAV or lentivirus in the limited area affected by the disease and then via one of the pumps administer from time to time the sense oligo for measured gene knockdown. If there are side-effects, you could titrate down the dose. Furthermore, you may not have to get the vector into all the target cells, but where it does, it should be able to promote consistent gene silencing meaning that a critical number of cells could be kept alive for a long time enough to keep the patient functional.

Commercial aspects

Of course, without patents and, tied to it, funding a technology like this will never make it off the ground. If you are interested, you can contact either myself or the Stanford Office of Technology Licensing. One aspect that has kept investors away from gene therapies is a difficult business model that needs to justify $1M price tags for often just one round of treatment. Amsterdam Molecular Therapeutics for example thinks it can do so for their promising lead candidate for a lipoprotein lipase deficiency that may soon be commercialized based on the fact that you get an almost life-long benefit from one round of treatment, as compared to the $150K annually for some of Genzyme’s drugs that need to be taken continually. In the case of SITS, the cost for gene therapy could again be spread as the initial vector administration would be followed by the routine administrations of synthetic oligonucleotides.

And last but not least, back to tsRNA biology

If they are not junk, then what do tsRNAs do? To answer this question and based on the observation that tsRNAs by themselves do not appear to have robust trans-silencing capacity, we tested whether dialing up or down the abundance of tsRNAs would affect the global activity of other classes of small RNAs such as microRNAs or siRNAs because of competition for shared proteins. We in fact observed that when the abundance of tsRNAs was increased, microRNA activity was decreased. Since tRNA and supposedly tsRNA abundance is correlated with cell proliferation, tsRNAs may be part of the answer to the long-standing question why microRNAs, which biology often uses to determine the differentiated, non-cycling state, are globally down-regulated in cancer.

So that’s it from me as a bench scientist. Having been a bench scientist is a unique opportunity to follow and test your ideas. I was very fortunate that in the labs that I worked in, science for science sake, the ultimate expression of freedom, was possible. In an age that tries to quantify and regulate every aspect of our lives, this is sometimes difficult to maintain. It is therefore important to keep in mind, also as policy makers, that the saying about the best inventions being born out of serendipity is not a platitude, it is a reality.


Anonymous said...

Hi Dirk,

Apparently tRNAs have always something up their sleeves! Good luck with future work. What are you going to do?


Dirk Haussecker said...

Yes, maybe tRNAs are coming back into fashion again. It is amazing where tRNAs are all involved in.

For the foreseeable future, I'll continue to follow RNAi Therapeutics and biotech/drug development in general. It is a difficult time though. In my relatively short life, I've never seen the world so scared about investing in (life sciences) innovation. A fallacy in my opinion, because what the economy needs to get out of its malaise is just that.

Anonymous said...

I am sure that there is still a place for innovation and inventors--even today!


David said...

It has been an amazingly informative blog. You have been both courageous and fair minded in your commentary.

All the best for your future as an RNAi industrialist ...

Top regards from Australia and New Zealand!

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