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Harvard’s Dan Peer Discusses Targeted siRNA Delivery In Vivo

NAME: Dan Peer
POSITION: Junior investigator, Immune Diseases Institute, Harvard Medical School
Research fellow, CBR Institute for Biomedical Research, Harvard Medical School — 2005
PhD, biophysics, Tel-Aviv University — 2004
Instructor, Tel-Aviv University — 2002-2004
MSc, biochemistry, Tel-Aviv University — 1999
Teaching assistant, Tel-Aviv University — 1998-2002
In January, researchers from the lab of Motomu Shimaoka at Harvard’s Immune Disease Institute (formerly known as the CBR Institute for Biomedical Research) published data in Science showing that cyclin D1, a cell cycle-regulatory molecule and established cancer target, also plays a role in inflammation.
To discover this role for cyclin D1, the investigators knocked down its expression in mice using siRNAs delivered via targeted, stabilized nanoparticles.
This week, RNAi News spoke with Dan Peer, the lead author on the Science paper, about the delivery approach.

Let’s start with the lab you work in and its focus.
The [Motomu] Shimaoka lab … is a laboratory of integrin biology and experimental therapeutics. The lab studies how to manipulate leukocyte functions to cure inflammatory diseases, [as well as] different types of cancers.
When I joined the lab, I was coming from the drug-delivery field … and it was a perfect match because … we are trying to deliver [to] and validate the role of cyclin D1 in leukocytes in vivo.
That work included some RNAi delivery aspects. Could you talk a bit about the nanoparticles used in the study?
In order to test our hypothesis that cyclin D1 does play a role in the pathogenesis of inflammatory bowel disease, we had to either knock out the gene or knock it down [using RNAi], which is usually faster. The problem was how to deliver siRNAs into those cells in vivo.
Leukocytes are very hard to transfect; they are resistant to lipid transfections, and if you want to do [your experiments] in an in vivo setting, it is impossible to do electroporation. So we had to use something else.
As a person coming from the drug-delivery field, I thought it would be easier to start with lipid-based particles — basically liposomes. We made multilamellar vesicles … and we extruded them through filters to be about 100 nanometers in diameter. Then, we put [on] two functional layers: one is hyaluronan, which not only acts as a protectant in the process of lyophilization and reconstitution, but also acts as an [in vivo] stabilizing scaffold for antibody binding.
The second layer — and these are the most important factors [of the delivery vehicle] — [comprises] antibodies against beta-7 integrins. We chose those targets for siRNA delivery because [although] integrins are the largest family of adhesion molecules, leukocyte integrins are very specific; the only ones you can find on leukocytes are the beta-7 and beta-2 integrins. They are readily internalized and when they do this, they usually take the carrier with them [into the endosome] in a very fast way.
Are these targeted nanoparticles related to or do they build on the antibody-linked siRNA work from Judy Lieberman’s lab [at Harvard]?
Definitely not. The concept is different. We had been involved in Judy’s integrin-fusion proteins and published a paper with her in 2007 in [Proceedings of the National Academy of Sciences]. But this is a different system because, first, the amount [of siRNAs] you can entrap is about 4,000 molecules in one particle. The amount we can load per fusion protein is about five or six siRNAs.
In this sense, [our delivery approach] is much more robust in terms how much siRNA you can load. And, you get better protection because we have two layers. Just to clarify, we first pre-condense the siRNA with human recombinant protamine, which is a positively charged small protein that nucleates DNA in sperm. Then, this complex is entrapped in a liposome.
The work described in Science was done in mice. Has there been any experimentation with this delivery technique in other animals?
Right now, we’re trying different models. We think of this really as a platform; you can put any antibody on a surface — not only antibodies of integrins — and you can put any siRNAs inside and target them to cells you are interested in.
We’re trying different models because we are interested not only in inflammation but also in cancers, as I mentioned, including hematological cancers. We are also interested in viral infections. So I think the potential [of the delivery approach] is quite huge.
Do you see human therapeutics potential for the technology?
Yes. Definitely.
We are also collaborating with somebody at Dana-Farber [Cancer Institute]. It’s very preliminary, but we are seriously considering a clinical trial [using the technology] within … two years.
Would that be with an siRNA drug?
This remains to be seen [and depends] on in vivo results we’re going to get.
Can you talk about the work going on in collaboration with Dana-Farber?
Not yet. It’s very preliminary.

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