NAME: Mark Behlke
POSITION: CSO, vice president of molecular genetics and biophysics, Integrated DNA Technologies
MD, Washington University, St. Louis — 1988
PhD, immunogenetics, Washington University, St. Louis — 1988
BS, biology, Massachusetts Institute of Technology — 1981
A team of researchers from Integrated DNA Technologies, the City of Hope, the University of Oslo, and siRNAsense published a paper this week in Oligonucleotides describing chemical modifications that stabilize 27-mer Dicer-substrate molecules and allow them to evade innate immune responses while maintaining their potency.
This week, RNAi News spoke with one of the study’s co-authors, IDT CSO and vice president of molecular genetics and biophysics Mark Behlke, about the modifications and his company’s plans to market modified Dicer-substrates for in vivo research applications.
Let’s start with a little bit on where you were when you began investigating modifications.
For routine use in tissue culture, we don’t ourselves ever use modified RNAs — I don’t think you need that routinely if you’re doing 21-mer or 27-mer work. But when you go in vivo and all cell types are present, you are going to need some types of modifications, both to confer some kind of nuclease resistance and to block reactivity with the innate immune system.
Depending on what kind of delivery tool you use in vivo, you may or may not need much nuclease resistance. But you’re always going to need protection from triggering the innate immune system. The more that these systems are studied, the more we’re becoming aware that there are a lot of different receptors that can recognize RNA species, and you’ve got to be protected against them.
What sort of immune responses were you seeing when it comes to unmodified 27-mers?
If you even just take, say, peripheral blood mononuclear cells from a human volunteer and transfect with a cationic lipid 21-mers or 27-mers into them, you will find a brisk type 1 interferon response — so you’ll see interferon alpha, TNF alpha, IL6; a whole variety of cytokines are released.
These kinds of things aren’t seen if you are using HeLa cells, HEK293 cells, or any of your standard tissue-culture cell lines, but when you are using natural cells, the chance of having these kinds of reactions increases. If you go in vivo, you are most certainly going to encounter them.
So they are pretty much the same kinds of effects you’d see with standard 21-mers?
Yes. Basically, it’s double-stranded RNA [that causes the immune response], and the same principles apply whether you’re [using] 27- or 21-mers.
When you started looking at different modification patterns, did you start with the ones that had worked for 21-mers?
What we wanted to do was look at a spectrum of different chemical modifications, [with an eye to] those modification patterns that would be good for research applications, pretty much where you need to have what I call a one-size-fits-all [solution] where you take a modification pattern and can put it pretty much any place you want it.
The problem with chemical modification, which I think people are beginning to appreciate, is that as you begin to add more and more chemical modifications to the duplexes, there begins to appear some sequence specificity wherein a chemical modification pattern will work great at one site, but if you put that modification at a different site, it doesn’t work well.
In fact, Sirna [Therapeutics], in the days before they were bought by Merck, had found that their highly modified nucleic acids [exhibited] this behavior so badly that they no longer screened with unmodified oligos. So they did their initial site screening with modified duplexes because they couldn’t make the conversion from unmodified to modified reliably.
What we were looking for was something where people who are working in vitro with our Dicer-substrates could make a conversion to a modification pattern that would be useful to block immune responses if they were seeing them in vitro or if they were [moving into] in vivo. We were looking for something that would be relatively easy to … transition from unmodified to modified.
We’ve looked at a number of modifications. The ones we looked at the most were 2’-fluoro and 2-O-methyl. We’ve also looked at [locked nucleic acids], but in much less detail because … when we got to more extensive LNA modification patterns, there was a significant impact on potency. I don’t know if that is a more significant of an effect for Dicer-substrates than 21-mers … but we didn’t explore the LNA patterns that much more.
One of the other points we had to consider was that the 27-mers needed to be modified in different ways than the 21-mers because we had different requirements: these had to remain Diceable, so by definition we had to leave a domain open that was nuclease sensitive — a consideration you don’t have when modifying 21-mers.
Can you give an overview of the modifications that were the most effective?
The asymmetric Dicer-substrate designs that we described in our Nucleic Acids Research paper in 2005 … have predictable Dicing patterns. So if you have a given molecule, we know where Dicer cleavage is going to occur in the unmodified version. Knowing that, we were able to leave a certain domain of the oligo unmodified, and then place modifications primarily in those regions of the molecule that were going to remain in the 21-mer that was Diced out of the 27-mer.
We tried different modification patterns with 2’-fluoro and 2’-O-methyl, mixed 2’-fluoro and 2’-O-methyl, and found that modification — particularly of one strand or the other strand — with 2’-O-methyl was generally effective, and an alternating pattern was one that seemed to work pretty well at multiple sites.
In general, your rule of thumb would be that with any kind of double-stranded RNA you’d worry about triggering an immune response. But there are some intracellular receptors that can recognize longer RNAs such as the 27-mers but don’t recognize 21-mers.
One of those receptors we believe is [the RNA helicase] RIG-I. The basic biochemistry of that was reported by Bryan William’s group [at Monash University] in a Nature Biotechnology paper in 2006, [which] identified that as the oligos got longer, the RIG-I system would become responsive to them. [It also reported that RIG-I] appeared to be blunt-end sensitive.
There is a glioblastoma cell line, T98G, that will respond to the 27-mers but doesn’t respond to 21-mers. We studied this cell line with the 2’-O-methyl version of the Dicer-substrates because it was a way of isolating responses that were 27-mer-unique and would be different from the standard TLR7 responses that we typically see from a mixed lymphocyte culture.
Importantly, these responses were also blocked by the 2’-O-methyl [modification]. That gives us an added level of control that examines the cell line where there is a 27-mer-specific response that doesn’t respond to 21-mers, and that is equally blocked by the modifications, as were the mixed lymphocyte reactions that we also studied.
These were tested in vitro?
Yes, in the Oligonucleotides paper, these were only tested in vitro. We have had collaborators use these patterns in vivo. However, that work is, as yet, unpublished.
Are you able to give any indication as to whether they are having success with these modifications in vivo?
Yes, they are.
In fact, one case where we were looking at this [in vivo] was where TNF-alpha was the target. When a very low dose [of Dicer-substrates] was [administered] via intraperitoneal injection, we were able to knock out TNF-alpha, but when a higher dose was used, we were actually triggering immune responses and raising TNF-alpha levels. … When we [began using] the 2’-O-methyl modifications, [we] no longer [triggered] an immune response.
Are efforts actively underway at IDT to follow up on these findings?
Absolutely. What we’ve found with the patterns [described] in the Oligonucleotides paper is that we could put modifications in the Dicer-substrate, that these did not interfere with Dicing, that they could be consistent with the continued very high potency we observed with the unmodified compounds, and that even these relatively incomplete modification patterns did improve nuclease stability and were capable of blocking immune responses in the assay systems we looked at.
These patterns are efficacious, but again they were designed to be the one-size-fits-all kind of patterns. If you have a site that is good and you don’t care if your modification pattern doesn’t work at another site, then it’s quite possible to do more extensive modification to it and impart greater nuclease stability.
In fact, there is a group from Japan that has already done that. [Working independently of IDT,] they published a paper in Molecular Therapy using vitamin E as a in vivo delivery tool with Dicer-substrate siRNAs, and using a more extensive chemical modification pattern that was able to protect the molecule more substantially so that what is, in essence, a naked duplex could be delivered by IV injection.
Is IDT marketing these one-size-fits-all modified Dicer-substrates?
We’ve got a new product line coming out that we’re calling the Evader Dicer-substrate siRNAs, which are intended more for people who are going into in vivo applications.
They’ll be orderable off the [IDT] website shortly, but can be obtained right now by contacting our customer service.