From cancers to viruses, treatment of diseases is hindered by the emergence of resistance. Resistance is typically thought to arise by mutations in the protein that a drug is targeting. Diseases such as cancer and viruses have very high mutation rates and thus resistance tends to crop up quite quickly. Therefore, scientists have tried to utilize combination drug protocols that target multiple nodes in a particular disease development (e.g. the reverse transcriptase and integrase of a virus). There are two problems with such a method: (a) there must be two distinct drug targets available and (b) the drugs for both targets need to have compatible bioavailability, kinetics, efficacy, toxicity, etc. This scheme has been proving effective, but still does not seem to work, for resistance still occurs. The authors of this work wanted to find a new modality by which viral infections could be targeted and, ideally, cured without resistance.
One target that has not been exploited as of yet is auto-regulatory circuits that are present in viruses, specifically in herpes simplex virus type one (HSV-1) and cytomegalovirus (CMV). In normal viral progression, proteins such as IE86 in CMV and IE175 in HSV-1 are very closely regulated by negatively feeding back on their own promoters. However, if this circuit were to be broken, these proteins would build up in large amounts, kill the host cell, and ultimately decrease viral progression. The authors of this study chose to make a new type of oligonucleotide “drug” that would mimic the domain of the promoter that these IE proteins bind to, titrate away the IE proteins from the promoter they should be acting on and thus deregulate the amount of this cytotoxic protein that is made. They called this new drug modality circuit disruptor oligonucleotide therapy (C-DOTs). With this, infected cells would die and reduce the viral progression (Figure 1).
The authors first had to find an oligonucleotide sequence that could bind the IE proteins and titrate them away from their natural binding partner. For this, they developed a liquid chromatography based assay that could detect IE protein, DNA and the complex of the two together. With this assay they determined that the optimal oligonucleotide size is 28 base pairs. They further validated some of their hits using a GFP attached to the IE86 protein that was driven by the promoter for IE86 that should be shut down once some IE86 is made. When these cells were treated with the 28 base pair oligonucleotide matching the cis-regulatory sequence in the promoter, there was a significant increase in GFP positive cells.
To evaluate C-DOTS for anti-viral activity, the authors took the C-DOT against CMV (C-DOTC) and against HSV-1 (C-DOTH) and treated cells that were infected with the corresponding virus. The authors were able to find something especially interesting – at low multiplicity of infection (MOI) the C-DOT treatment worked as well as the conventional therapy but at higher MOI the C-DOTs worked better. This is not surprising when one considers the fact that the C-DOT therapy takes advantage of increased amounts of the IE proteins that should be regulated closely; in the case of the higher MOI, there are more viral genomes per cell and thus, more opportunities to have increased amount of IE protein to cause cell death and decreased viral load. Amazingly, the authors found that this treatment could be used for a wide range of viral strains, not just the one that they were using to characterize the treatment in the first place.
The biggest concern that the authors were trying to combat was resistance. To test if the C-DOT therapy could do better than conventional therapies (Fomivirsen for CMV and ACV for HSV-1) in terms of resistance strains forming, the authors performed an assay in which the virus was continuously passaged from infected cells to non-infected cells every four days and the virus was tittered during each passaging. They found that the conventional therapies produced resistance within 12 days while the C-DOT therapy amazingly had essentially zero resistance form for up to 40 days in culturing cells, with CMV titers staying below the limit of detection at day 52 (Figure 2). The authors were even able to take this to the next level by implementing their treatment in mice. When C-DOTs were given to mice challenged with the corresponding virus, the authors saw amazing reduction in viral loads (150 fold decrease in the case of HSV-1).
Why I chose this preprint
The simplicity yet brilliance of the work described here is the major reason why I chose this particular preprint- the idea that one can use the viruses’ own regulatory networks to get it to kill itself. This was simply done by titrating away the DNA binding protein using small oligonucleotides. Not only does this work provide a very promising therapy for CMV and HSV-1 described here, but it also provides a new therapeutic modality to test for other diseases such as cancer. A lot of work has been done showing how cancers rewire regulatory networks and so being able to take advantage of that would be an amazing achievement [1-2]. Furthermore, the readability of this particular work made it an especially fun reading experience – the language was concise and the figures were very clear with panels explaining hypothesized results.
What is the difference between Fomivirsen and the newer C-DOTC? How does the resistance arise for Fomivirsen and does this mechanism apply to C-DOTC?
Is there a way that the binding of the C-DOT and IE protein can be compared to the endogenous promoter? If so, is there a way to increase the C-DOT binding to IE protein such that it is more preferential compared to the endogenous location?
The idea that such feedback loops can be targeted for not just viruses but also other diseases such as cancers is very attractive. However, one of my questions is how one can identify targetable feedback loops?
Fish L, Zhang S, Yu JX, et al. Cancer cells exploit an orphan RNA to drive metastatic progression. Nat Med. 2018;24(11):1743-1751.
Bugaj LJ, Sabnis AJ, Mitchell A, et al. Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras-Erk pathway. Science. 2018;361(6405).
Fomivirsen was an antisense oligonucleotide that blocked translation of the IE2 mRNA by binding to the complementary sequence of the IE2 mRNA. Fomivirsen was the first antisense antiviral approved by the FDA. C-DOTs are essentially the exact opposite of antisense oligonucleotides; whereas antisense oligos knockdown expression of a target protein, C-DOTs increase the expression of the target protein to toxic levels.
It is still not perfectly clear how resistance to Fomivirsen arises, but the CMV mutations appear to be in sites that are distinct and removed from the complimentary region in the mRNA—which is consistent with the expression level or half-life of the mRNA being altered by the virus to compensate for the knockdown by fomivirsen. This sort of up regulation of the mRNA is thought to be a common mechanism that cells can use to counter the effects of inhibition. Since the C-DOT increases rather than decreases mRNA levels, you could think that a compensatory escape mechanism would be for the virus to generate mutations that reduced the mRNA lifetime (for example reducing the promoter strength). However, we have shown previously (Teng et al. Cell 2012) that such mutations that reduce IE2 levels confer a severe fitness disadvantage for the virus because the rate of accumulation of IE2 is necessarily much slower. This is why we think that the mutations don’t arise; they don’t have a strong selective advantage.
Regarding your question about increasing C-DOT binding efficiency relative to the native promoter this is a great question and something that we are actively working on. The simplest approaches, such as screening for sequences that have higher binding affinity to IE2, don’t seem to work. We believe this makes some evolutionary sense since the virus likely evolved the optimal binding sequences so finding a better one is unlikely, but we have other ideas. Stay tuned.
Finally, regarding your question of identifying targetable feedback loops, this is another great question. There are some high-throughput approaches to identify feedback loops that are under development. One involves identifying DNA-protein interactions and then focusing on those that are self-regulating. This is actually a major goal of our work and a long-standing goal in the field of systems biology.
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