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Purdue’s Philip Low Discusses Folic Acid-Based Targeted siRNA Delivery

NAME: Philip Low
POSITION: Professor, biochemistry, Purdue University
— Research associate, University of Massachusetts, 1975-1976
— PhD, University of California, San Diego, 1975
— BS, Brigham Young University, 1971
At the fourth annual meeting of the Oligonucleotide Therapeutics Society in Boston last week, Purdue University researcher Philip Low presented data on a novel delivery system that uses folate to direct siRNAs to certain pathogenic cells including cancer cells and activated macrophages.
This week, RNAi News spoke with Low about the technology and plans for developing it into a clinically viable approach for siRNA-based drug delivery.

Let’s start with an overview of the technology. It’s based on the natural targeting properties of folate, correct?
Yes, that’s right. It turns out there is a receptor for the vitamin folic acid that is expressed on roughly 40 percent of all human cancers. It’s not 100 percent on any one cancer type; for example, in ovarian cancer, it’s 90 percent, and in lung cancer it’s 78 percent. On the other hand, in prostate cancer, it’s nearly zero. So it just depends on the cancer type … [but] there is a large fraction of the human cancer types that have high levels of folate receptor. We use folic acid to deliver an attached siRNA specifically to these folate receptor-expressing cancer cells.
Are these receptors expressed on normal cells?
Very few. The other cells that express the folate receptor are activated macrophages, but not resting macrophages or any other hematopoietic cell … and the apical surface of the proximal tubule of the kidney has folate receptors. These receptors are present to capture folate in the filtrate or the developing urine, endocytose it, carry it across the cell by transcytosis, and release it on the basal lateral side of the cell into the bloodstream; it basically is a means of capturing folate in the urine and transporting it back to the bloodstream. If it happens to perform the same function with a folate-linked siRNA molecule, that’s all the better.
Those are the three major accessible cells that have folate receptors. Having said that, there are also some folate receptors on the apical surfaces of some other epithelia, but these are all inaccessible to folate-linked molecules in the blood. … For practical purposes, the only three cells where folic acid receptors are accessible in reasonable numbers are activated macrophages, cancer cells, and the apical surface of the proximal tubule.
Other than the folate, are there other components of the delivery technology? Is there encapsulation of the siRNA?
No. The reason why we’re unique is that in contrast to the strategies pursued by virtually every other group, we directly link our siRNA to this targeting ligand, folate, and don’t incorporate it into a dendrimer or nanoparticle or a liposome or any large particle because it is our view that these larger particles penetrate solid tumors very poorly. I think data bear that out from quite a number of studies.
On the other hand, small molecules can perfuse a solid tumor very easily. We find in our studies that our folate-linked siRNA molecules reach every cell in a solid tumor.
Is there any problem with instability or degradation since these siRNAs aren’t protected in some way?
We haven’t tested that yet. I could give an argument both for and against that. Obviously, there are nucleases on cell surfaces, maybe even some in the blood, and so as this [folate-linked] double-stranded RNA complex circulates, it could easily be degraded.
Having said that, if the molecules are small enough, [this shouldn’t be a problem]. We haven’t really done the kinetic studies with siRNA, but we have [done work] with folate linked to rhodamine, which is a little smaller, and that [agent] saturates a solid tumor following intravenous injection in less than five minutes. So the time that a folate-siRNA complex would have to survive in the bloodstream could be very brief — just a few minutes — because by the end of these few minutes it will either have saturated all receptors on the tumor or it will have been excreted.
In general, we like to build our molecules so that they are either captured by the receptors on the targeted cell or they are eliminated from the body, simply because any extra non-targeted material circulating in the bloodstream can only lead to non-specific toxicity.
At this point, what’s the status of the development of this technology for siRNA delivery?
We have achieved several goals. We have developed good chemistry for linking siRNA to folate and to other targeting ligands, I should mention, [such as] prostate-specific membrane antigen … which is over-expressed on the vast majority of prostate cancers. … And we’ve been able to demonstrate in animal models very good tumor-specific delivery.
We also used the capability of folate to target activated macrophages to deliver siRNA to sites of inflammation including atherosclerotic plaque in the heart, muscle injury … and arthritis. Each of these diseases, and many more, are caused or worsened by activated macrophages … [for example], in atherosclerosis it’s the activated macrophage that is the foam cell that takes in all of the lipids and engorges itself so much that it leads to occlusion of the blood vessel.
An issue discussed at the OTS meeting was endosomal escape. Can you comment on the status of that with regards to the delivery technology?
That’s the obstacle that remains to be surmounted before this can really be considered seriously for clinical development. We have been able to demonstrate that the vast majority of our conjugates are taken up into endosomes. Fortunately, the endosomes are not degrading; that is, they don’t go to lysosomes [rather they] stay in non-lysosomal compartments. But they don’t seem to be able to escape on their own into the cytoplasm.
We have recognized … that an endosomal-escape strategy would be required to achieve the full potency that this delivery [approach] can bring. So we’re working on that right now. I think it’s very solvable and, as a matter of fact, we have some preliminary data suggesting that we know how to do it.
But I won’t claim that openly until we can reproduce it several times.
If this work proves successful, would the technology be commercialized by the company you founded, Endocyte?
Not likely. Endocyte does not want to dilute itself the required amount to develop the technology for building siRNA molecules. Furthermore, we don’t own a lot of the technology involved in developing these stabilized siRNA backbones and other components that make it an effective therapeutic strategy.
Our thought was that we would either license it in a non-exclusive manner to all the companies interested in siRNA delivery or … license it to one company that wanted to own it exclusively.
This would be through Purdue?
Well, Purdue owns the patents — everything that is discovered in my lab is patented by Purdue University. If Endocyte supports the research, they have the right to license it for a fee from the university. I can’t imagine [the company] not wanting to license it; they have certainly supported the work. So I would guess it would go through Endocyte to one of these companies.
And the companies are interested. I have been contacted by, and I won’t mention names, every one of the major players in the field multiple times.

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