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How Can Thermo Fisher Deliver siRNAInto Cells In Vitro Without Transfection?

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NAME: Devin Leake
 
POSITION: Director, biology research and development, Thermo Fisher Scientific
 
BACKGROUND:
 
Senior scientist, Fisher Scientific — 2005-2006
Research scientist, Dharmacon — 2002-2005
Project manager/senior scientist, Atugen USA — 2001-2002
Postdoc, University of Colorado, Denver — 1999-2001
PhD, molecular biology/biochemistry, State University of New York, Stony Brook — 1999
BA, biochemistry, University of Texas at Austin — 1993
 

 
Last week, Thermo Fisher Scientific unveiled its Accell line of siRNAs, which it claims can be absorbed directly into cells in vitro without the need for delivery reagents, viruses, or electroporation.
 
The company also said that the Accell siRNAs, developed by its Dharmacon unit, can penetrate every cell type that has been tested to date.
 
This week, RNAi News spoke with Devin Leake, director of biology research and development at Thermo Fisher Scientific, about the new line of siRNAs.
 
Let’s start with an overview of the Accell siRNAs.
 
The Accell siRNA is a chemically modified siRNA that promotes cellular uptake. So this is not using a formulation, instrumentation, or viral vector to produce transfection.
 
Can you give some details on these particular modifications and how they work?
 
The modification pattern we use we have been working on for several years. It’s obviously proprietary and something our scientists are extremely proud of being able to develop. The results we’ve seen in beta testing have been very positive for delivery into difficult cell lines, as well as many of the adherent cell lines that are typically used within a lab.
 
When looking to develop these kinds of modifications, what are the issues that needed to be addressed?
 
Clearly, you want to consider the stability of the molecule. If you think about the encapsulation that typically happens with an siRNA in lipids, there is a level of protection that is afforded to the molecule. So when you have a molecule that is chemically modified for delivery, you do have to consider the overall nuclease resistance of the molecule so that whatever gets inside the cell is intact and functional.
There are certainly other properties to be aware of. Too heavily modified a molecule may elicit some kind of cellular stress. What we found with the chemical modifications we identified for the Accell siRNA is that they did not elict the pro-inflammatory response.
 
The other concern about siRNAs and delivery is what happens with off-target effects — these non-specific effects or binding to other genes. We found that some of the [features and modifications] we incorporated into other product lines, such as our On-Target Plus [siRNA reagents], the bioinformatics for selecting potent siRNA sequences [with] enhanced specificity — all of these we put into [the Accell siRNAs] and we find … that the off-target signature is dramatically reduced.
 
So these Accell siRNAs are able to be absorbed by all cells, as far as you know.
 
We’ve had everyone in the group [developing the siRNAs] working on their favorite cell type, and we have yet to find a cell type we can’t get this molecule into.
 
What sort of levels of silencing are you seeing with the Accell siRNAs? Does it vary depending on the cell you’re dealing with?
 
There are going to be differences associated with the gene targets, and part of that is related to the detection and part of it is related to sequence. If you look at any siRNA or siRNA pool, you’ll find there is variability in silencing. If you take one specific siRNA or siRNA pool and look at a lot of different cell types, there is variability.
 
We can use the Accell siRNA to get into, [for example], Jurkats, THP1s, or some of the lymphoid-derived cell lines or suspension cell lines, [but] the knockdown [isn’t necessarily as robust as with other cell types.] If you were, say, to use one siRNA in a HeLa cell … you might get 95 percent silencing. In the Jurkat or the THP1, it may be around 80 to 85 percent silencing.
 
You do have some compromises, but at the same time, the ability to get into these difficult-to-transfect cells, without necessarily using instrumentation such as electroporation, is pretty phenomenal.
 
What is the availability of these? Can you get an Accell siRNA for any target you want at this point?
 
Yes.
 
When researchers are thinking about what kinds of siRNAs they want and they are looking at Dharmacon’s portfolio, are there situations where Accell siRNAs would not be the appropriate choice?
 
There are a couple of different reasons for using the different siRNAs that we have. [Dharmacon’s other siRNA] collections are more comprehensive collections at this time. Currently, the library we have for Accell is not extended to the whole genome. That does not necessarily mean that you can’t go to our website and find your gene of interest and order it; you can do that. But as far as buying libraries of Accell, we’re still ramping up in terms of production and inventory.
 
What’s the availability at this point?
 
We are currently working up to what we consider the druggable genome, which is going to be a collection of 8,000 [siRNAs].
 
Are there possible in vivo applications for the Accell technology?
 
That is an active area for collaborations we have. We’ve done some internal studies, and part of the challenge of doing in vivo work is trying to find a model that is working or benchmarking it against something that has been shown to work.
We did some benchmarking studies … [of an Accell siRNA] against the Alnylam [Pharmaceuticals] molecule that was published. The reason why is that we have [observed] and heard from different researchers [about the] the success of that Alnylam molecule.
 
What we saw was that the uptake in specific tissue types with the Accell siRNA is different than the Alnylam molecule, but the efficacy in most of the tissue types we tested was better. So the cellular uptake properties that we see with the Accell siRNA certainly translate to in vivo models.
 
We did see efficacy and that was what we were looking for: some kind of positive control. And these were simple tail vein injections; these were nothing complex — we didn’t look at specific cell types. Those are studies we are conducting either internally or through collaboration. We have several collaborations looking at tumor models or other localized areas within animals, and we’re hoping to get a few of those [studies] published within the next year or two.
 
In those benchmarking studies, you were using the same sequence as Alnylam, but with the Accell modifications, right?
 
Correct.
 
So Dharmacon is actively working on Accell siRNAs appropriate for in vivo use?
 
Yes. Even now, with the [existing] Accell siRNAs … it would be interesting testing them in animal models.
 
When you think about the RNAi research market, obviously delivery remains a challenge. But what are some other hurdles you see as Dharmacon develops as a business and looks to the next stages of research RNAi?
 
I think enabling … more researchers to use RNA interference. We are still a fairly select group of researchers; RNAi is seen as a niche technology still. It’s growing, definitely, but enabling researchers to do and use more RNAi is critical.
That’s really one of the benefits of the Accell siRNA. Granted, initially when we look at Accell, we see the difficult-to-transfect cell lines such as the Jurkats, such as the neuronal lines, and those are quickly addressed in terms of getting the siRNA into the cell.
 

The other opportunity is to have the Accell siRNAs for researchers that have never used RNA interference before. It is a simple process, and I believe that the more people who are using RNA interference, the more applications we’re going to see for RNA interference.