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Q&A: MIT's Paula Hammond on siRNA Delivery Via All-RNA Microsponges

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hammond2.jpgName:
Paula Hammond

Position:
Professor, chemical engineering, Massachusetts Institute of Technology

Background:
• Associate professor, chemical engineering, Massachusetts Institute of Technology — 2000-2006
• Postdoc, chemistry, Harvard University — 1993-1995
• PhD, chemical engineering, Massachusetts Institute of Technology — 1993
• MS, chemical engineering, Georgia Institute of Technology — 1988

A team of researchers from the Massachusetts Institute of Technology last month provided details on the development of a new siRNA-delivery approach that combines carrier and cargo into a fully RNA microparticle.

Called RNA interference microsponges to reflect their appearance under magnification, they “consist entirely of cleavable RNA strands, and are processed by the cell’s RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm,” according to a report published in Nature Materials.

This week, Gene Silencing News spoke with Paula Hammond, the paper's senior author, about the microsponges.

What prompted this work? Had you been working on siRNA already?

Our lab examines the delivery of biomaterials using self-assembly and electrostatic interactions, and we have projects examining a method called layer-by-layer assembly. But we are also interested in whether we can generate siRNA in a form that could be more easily packaged.

I had a postdoc, first author Jongbum Lee, [who is] now located at University of Seoul, who brought in expertise in DNA assembly techniques, and we talked about how we might be able to use those kinds of approaches to manufacture an siRNA-assembled microparticle.

Can you give an overview of the development of the microsponges and their structure?

Essentially, these are continuous sequences of the same interference RNA with a short sequence after that, which is cleavable by Dicer. Rolling circle transcription allows a DNA plasmid to continuously generate this siRNA strand, and it repeats that cycle again and again. However, rather than getting short pieces, the rolling circle transcription is giving us elongated, macromolecular chains of siRNAs, alternating with [Dicer-susceptible sequences]. We actually have hundreds to thousands of copies of the siRNA in each chain that is generated.

These chains are extremely long, and they form what appear to be polymeric crystals. The way polymers crystallize is that the repeat sequences begin to fold back on themselves and form a sheet-like structure that can radiate outward from a spherical core, which is what we see with the siRNAs.

We believe that we've created an siRNA-polymeric species [of a] high molecular weight, which is generated in high enough concentrations to form superstructures that are crystalline versions of the polymer. They are self-organized and monodisperse, so all these microsponges are the same size.

When you look at these individual sheets, what you're seeing are these crystalline-arranged structures of many, many sequences of siRNA tied together with Dicer-susceptible sequences.

So these things are just RNA?

They are 100 percent RNA. Nothing else.

How did you go about testing out the microsponges?

Once we generated the microsponges, we did several tests to ensure that we had all RNA sequences. We confirmed that the sponges degrade completely in the presence of RNAse, but not with DNAse, and confirmed the secondary structure of the RNA. We essentially did a basic materials analysis of the system, looking at the size distribution and net charge — these are very highly charged because of the RNA sequences.

Then we wrapped up the microsponge with one adsorbed layer of a polycation. This allowed us to compact the microsponge into a nanostructure. Once we did that, we looked at internalization of the microsponge in vitro … and saw that cell uptake was quite high in these systems.

Did you explore where in the cell these siRNAs were aggregating?

They appeared to be in the endosome, but … they manage to get out into the cytosol because we're seeing the translation of the RNAi. They're getting diced and reaching RISC.

And then you looked in vivo?

Once we saw that this was working in vitro and we could see knockdown, which gives a very nice quantitative measure, we moved on to … a mouse model with subcutaneous tumors that [express luciferase] … [Using] an siRNA sequence programmed into the sponge that knocks down the luciferase gene, [we demonstrated that the microsponges could inhibit their target in animals].

How were the microsponges delivered? Directly to the tumor?

Yes, we did an intratumoral injection.

These microparticles are loaded with siRNA, but how do they compare to other delivery particles?

These contain at least three orders of magnitude higher concentrations of siRNAs within a nanoparticle compared to a typical transfection vector. All of the mass we're dealing with is siRNA.

Were you seeing a commensurate level of efficacy given how much siRNA the microsponges contain?

Yes. We were seeing knockdown with much lower quantities of the siRNA microsponge. We did use orders of magnitude lower quantities of these microsponges in these injections, again compared to a traditional transfection media like a liposomal carrier — about 150 times less [was] needed.

Is the goal to use these for therapeutic applications?

That's exactly our interest. We'd like to be able to demonstrate that we can use this kind of approach to more effectively package siRNA.

One of the big issues with traditional siRNAs is that they are short and highly charged … [and] it's not easy to encapsulate it compared with DNA … which is easily condensed. When you pack DNA down, you can package it in Transfectamine or a range of other vehicles fairly effectively. … SiRNA delivery has the additional challenge that one must effectively condense it and package it in a manner that gives you a dense quantity of the siRNA. It frequently doesn't work … in fact the siRNA is often coming out of your vector before you get to the region targeted, or it is too tightly bound to the vehicle to be freed for transfection.

The point here is that we can essentially knit together an siRNA polymer that forms crystalline microstructures. And that crystalline form actually creates something that we can compact down to a nanomaterial size. On top of that, it gives us a very high density of siRNAs. We also think the stability of the siRNAs is preserved, in part, because it is in this crystalline form.

Our group looks at the use of these kinds of approaches to address cancer, and this is where we'd like to go with this methodology. [We plan to] begin to look at siRNA sequences that can be used to address cancer. … The next steps would be to further optimize the material system, but even now we can begin to look at siRNAs with a specific function.

Given the impact that cost of manufacturing has on drug development, do you see the microsponges as having an edge over other delivery approaches?

Yes. The issues with regard to the incorporation of other synthetic materials is greatly lowered because we only need to add a small amount of a polymer that will condense the microsponge to a smaller size compared to a traditional carrier, which is most likely a synthetic polymer. That gives us a huge advantage in terms of safety [and US Food and Drug Administration] concerns.

I think there is also the added advantage that we are talking about lowering dose.

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