Moving Rapidly from Target Discovery to Lead Development: RNAi in the Context of the Emerging Drug Development Paradigm (Journal Club)

That RNAi is now widely employed in the Pharma world for target identification and validation purposes is no longer a secret. That RNAi may immediately lend itself as the therapeutic agent as well has been recognized by Merck, Novartis, and Roche, and it is this widespread familiarity with the technology that is also a major driver for more Big Pharma companies embracing RNAi as a therapeutic platform technology, too.

Today, I’d like to use a paper by Dan Cao on the identification of new host factors for Hepatitis Delta Virus (HDV) replication to illustrate how easy it is now, in theory, to exploit RNAi to dramatically shorten drug development timelines, especially in an environment that demands the rapid development of drugs based on new, innovative targets (Dan et al.: Combined proteomic-RNAi screen for host factors involved in human hepatitis delta virus replication. RNA [epub ahead of print]).

HDV is typically found in the context of a Hepatitis B Virus (HBV) infection as HDV requires HBV to introduce its genetic material into the host cell. Viral replication, however, is independent of HBV, and there are indeed a few patients that have got rid of HBV, but still harbor HDV. While this is uncomfortable already, having both active HBV and HDV is not pleasant at all. Because HDV is a condition thought to be of little pharmaco-economic importance, there is no HDV-specific drug. The identification of viral host factors therefore would immediately yield candidates for the development of RNAi-based drugs specific for HDV, in addition to the basic molecular biology insight they might provide for the study of RNA-directed transcription for which HDV is a great model system.

Targeting host factors is especially attractive for RNA viral infections, because it should be much more difficult for the virus to escape the drug by mutation. There are a number of well-publicized studies identifying host factors for HIV through genome-wide RNAi screens. This, however, demands a dedicated RNAi screening facility and a lot of hard work. Moreover, relying on just one technology, albeit RNAi, should increase false negatives, and especially false positives. The alternative approach would be to search the literature for reported viral host factors and then target such a list of genes by RNAi. The draw-back of this approach is that it will not include new factors and that the quality of reported factors can be quite ‘variable’.

For all these reasons, but particularly because the life-span and budget of a post-doc are limited, Dan decided to take a slightly different tack. The approach relies on employing two functionally independent screens for HDV host factors, but which because they were serially coupled should increase the quality of the identified factors while reducing the overall workload.

In a first screen for HDV-related host factors, the only viral protein (HDAg) was immunoprecipitated and associated proteins identified by mass-spectrometry. Over 100 proteins were such identified, although some were more and some less abundant. Among the most abundant ones were several subunits of the host RNA Polymerase II complex, which carries out viral replication and this therefore was a great validation for the validity of the immunoprecipitation results. The proteome-wide IP-mass spectrometry was relatively painless since the IP eluate could be handed over to the mass-spec core facility almost unprocessed and was then analyzed by a skilled specialist using standard methods (about one week, i.e. negligible turnaround for somebody that has gone through the process once).

Dan then picked about 60 genes from the list and got about 3 siRNAs for each. These were used in a screen for their ability to inhibit HDV replication. Although this still requires hard labor, 60 is a number that is within the reach of a single worker and no complicated screening facility is required. All you basically need is a good screen. Thanks to the existence of genome-wide siRNA libraries (which, of course, is a result of the interest in RNAi), the cost of siRNAs has also come down dramatically compared to 5 to 7 years ago (from $250 an siRNA to about $15-20 an siRNA). In summary, ~1 ½ man years identified about 5-10 genes that both interacted with HDAg and when knocked down strongly inhibited HDV replication.

Although not done in this study, these candidates could now be vetted in the literature for whether they may make for good targets (based on predicted requirement for cell viability etc), and then tested in small animal models of HDV infection for pre-clinical in vivo proof-of-concept using existing siRNA delivery technologies for the liver. After another 2 years of siRNA chemistry and pre-clinical pharm/tox (which will be routine in a company with an established RNAi Therapeutic platform) you could imagine to be in the clinic.

This is just a small example of how RNAi may be ideally suited as a therapeutic modality in an age of personalized medicines where pharmaco-economic principles would demand efficient drug development. An article mentioning GSK’s intention to announce next month an orphan-disease initiative is another sign of the times, and gets me a little bit more excited about Alnylam’s prospects for a platform deal- maybe even this year (see also blog on the neglected disease patent pool donation). Who knows, one day it may not just be the government of Taiwan that is interested in treating HDV anymore.

Abstract:

Combined proteomic-RNAi screen for host factors involved in human hepatitis delta virus replication.

Cao D, Haussecker D, Huang Y, Kay MA.

Department of Pediatrics, Stanford University, Stanford, California 94305-5164, USA.

Human hepatitis delta virus (HDV) is the only animal virus known to replicate its RNA genome using a host polymerase because its only virally encoded proteins, the small and large hepatitis delta antigens (HDAg-S and HDAg-L), lack polymerase activity. Although this makes HDV an ideal model system to study RNA-directed transcription in mammalian cells, little is known about the host factors involved in its replication. To comprehensively identify such host factors, we created a stable cell line carrying a functional FLAG-HDAg-S. Anti-Flag immunopurification and mass spectrometry identified >100 proteins associated with FLAG-HDAg-S, many of which had predicted roles in RNA metabolism. The biological relevance of this screen was strongly supported by the identification of nine out of the 12 subunits of the RNA polymerase II complex thought to mediate HDV replication. To further investigate the significance of these factors for HDV replication, we selected 65 proteins to look for factors that would also affect the accumulation of HDV RNA following siRNA knockdown. Fifteen and three factors were found to regulate HDV RNA accumulation negatively and positively, respectively, upon RNAi knockdown. Our results provide a valuable resource for future research to advance our mechanistic understanding of HDV replication and RNA-directed transcription in mammalian cells.

Capped Small RNAs Expand Universe of Small RNAs

Findings from research by myself and colleagues here in Stanford on the discovery of a new species of distinct small RNAs, capped small RNAs, have just been published online at Nature Structural & Molecular Biology (Haussecker et al.: Capped small RNAs and MOV10 in human hepatitis delta virus replication).

It is becoming clear from large-scale sequencing efforts and hypothesis-driven research such as ours, that siRNAs and microRNAs are just two classes in an ecosystem of other small and non-coding RNAs. This particular class of small RNAs carries mRNA-like 5’ cap structures and was discovered during our studies on human Hepatitis Delta Virus (HDV) replication.

HDV is the smallest virus known to the animal kingdom and is even more so remarkable in that it does not encode for its own polymerase for viral replication, as all other viruses do, and instead relies on host RNA Polymerase II (Pol II) for its replication. HDV accomplishes this with carrying the genetic information for just one non-catalytic protein, the hepatitis delta antigen (HDAg), while its RNA genome calls all the other shots. It is also the only known example of RNA-directed transcription by Pol II in vertebrates, as Pol II is largely thought to use DNA, not RNA, as a transcription template.

Intrigued by this and by the possibility that within the complexity of our transcriptome there may be hidden RNA-directed transcription, vestiges of our RNA World heritage, and HDV may be the key to its understanding, we set out to investigate whether small RNAs would also play a role in HDV replication. Sure enough, a few Northern blots later, it became clear that RNA secondary structures previously associated with the initiation of HDV transcription harbored small RNAs. Long story short, the occurrence of capped small RNAs from hairpin secondary structures suggests that maybe analogous RNAs in our genome could be involved in analogous processes.

As we were studying the capped small RNAs, we also sought to determine the host factors that HDAg interacted with. Knowing this may give us further insights into HDV-related RNA-directed transcription. Intriguingly, the mass-spectrometry screen yielded MOV10 which is a gene that in plants had been implicated in RNA amplification during RNAi. Knocking down MOV10 with siRNAs inhibited HDV RNA accumulation, consistent with a function in RNA-directed transcription in vertebrate cells as well. Given that triphosphorylated small RNAs, which like capped small RNAs, should be derived from short transcription initiation events, had been found during RNAi amplification in worms, we speculate that HDV replication may indeed tap into an evolutionarily conserved process which makes it also more likely that non-viral RNA-directed transcription indeed occurs in our cells.

While RNAi-related factors such as Dicer and Drosha do not appear to play a role in HDV replication, we found in the course of our studies that knockdown of Argonaute 4 (AGO4) significantly affected HDV replication. AGO4 is related to AGO2 which is the Slicer in RNAi that cleaves target mRNA, and yet very little is known about AGO4. Determining the roles and preferences of the various AGO proteins in humans will be an important area of RNAi Therapeutics research as it promises to yield improvements in the design of RNAi triggers both in terms of safety and knockdown potency.

It remains to be seen whether capped small RNAs are specific for RNA-directed transcription, or whether they are a reflection of a more widely used gene regulatory mechanism. In fact, high-throughput sequencing efforts by others indicate a population of small RNAs associated with gene promoter regions. It is possible, although not yet demonstrated, that these are capped, and since the HDV small RNAs are abundant enough to be detected by Northern blot, HDV replication may serve as a model system to understand this type of gene regulation. Finally, it is tempting to speculate whether the modulation of capped small RNAs may be utilized for therapeutic purposes.