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NYU's Kent Kirshenbaum on Developing a New siRNA Transfection Reagent

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Kent Kirshenbaum
Assistant professor, chemistry
New York University
Name: Kent Kirshenbaum
Position: Assistant professor, chemistry, New York University
Background: Postdoc, California Institute of Technology — 1999-2002
PhD, pharmaceutical chemistry, University of California, San Francisco — 1999
BA, chemistry, Reed College — 1994
 

 
In the June issue of Molecular BioSystems, NYU researcher Kent Kirshenbaum and colleagues reported the development of a new kind of transfection reagent, one that uses a so-called lipitoid to efficiently deliver siRNAs into difficult-to-transfect primary cells.
This week, RNAi News spoke with Kirshenbaum about his research and how he started working in the RNAi field.
 
Let’s begin with a brief overview of your lab.
 
My lab is involved in biomimetic chemistry and bioorganic chemistry, and we’re interested in developing molecules that combine the best attributes of synthetic molecular systems and biomolecular systems. That’s been my research background throughout my PhD and postdoc [work].
 
I did a lot of my PhD work in the area of biomimetic chemistry, designing new sequence-specific oligomers that have the structural and functional properties of polypeptides. It was at that time that I began to do research with a class of molecules that we called peptoids.
 
These are a family of peptide-mimetic molecules that are variations of glycine oligomers in which we’ve taken the side chains and positioned them on the backbone nitrogen atoms. As part of this PhD work, I demonstrated that these molecules were capable of forming very stable helical secondary structures similar to the types of secondary structures that polypeptides can form. Since that time, there have been some laboratories that have gone on to [further] develop these structural attributes in these oligomers, [which] are now termed foldimers.
 
Now, as an assistant professor, I’ve returned to this topic. I’ve also begun to try to develop more elaborate architectures in this system to move beyond simple secondary structures and to try to develop tertiary structures to make more complex protein-mimetic molecules.
 
At the same time that we’ve been tackling these architectural issues, we also realized that by virtue of their biomimetic character, we may be able to obtain some biological activity from these molecules. So we’ve initiated a number of collaborations with researchers at NYU’s medical school to try to develop peptoids for biomedical applications. In particular, we’ve very interested in areas related to molecular imaging, new diagnostic molecules. We have extensive collaborations in the area of new contrast agents for magnetic resonance imaging and we’ve also begun to develop peptoids for transfection, in particular … for the transfection of oligonucleotides of RNA for specific gene knockdown.
 
Was bringing in siRNA an organic thing, or was it something that came about through discussions with colleagues?
 
It turned out that the laboratory immediately adjoining mine was run by a guy named Fabio Piano who is a mover and shaker in the world of functional genomics. Because of my background at UCSF, I’ve always been very open to initiating dialogues with people across disciplinary boundaries, especially with regard to clinical applications.
 
I don’t know if you are familiar with the culture at UC, San Francisco, but traditionally it’s been an extremely compact institution where laboratories are crowded in one next to the other. The reputation is that it has forced people to collaborate because they physically don’t have any other choice but to be in one another’s business. That’s sort of how I approached being here and being next door to Fabio — just finding out what he was doing. I’m always eager to take our molecules and find ways to solve biomedical challenges.
 
What I really love is when I can talk to a biomedical researcher or clinician, and they can come to me with a laundry list of molecular attributes … and [I] can figure out how to design molecules [to meet their needs.] When Fabio began to talk about his work with RNAi, I realized we had an opportunity to create a tool that could facilitate his work. In particular, I knew that certain of our peptidomimetic sequences had previously been shown to be capable of transfection of DNA.
 
Given the fact that these peptoids had been shown effective for DNA transfection, I began to wonder whether we could utilize the same or similar sequences and make them useful for RNA transfection.
 
You just published a paper recently [in Molecular BioSystems]. Could you talk about this transfection reagent and how it works?
 
The transfection reagent that we use is called a lipitoid. This name derives from the two components of this molecule. One is a peptoid oligomer — as I mentioned before, this is an oligomer of N-substituted glycine, which includes cationic side chains. These provide for electrostatic complimentarity with the RNA oligo. The second portion of the molecule is a lipid group. This head group is designed to provide for interaction with the cell membrane and facilitate cell permeability. These lipitoid molecules have previously been shown to be effective for DNA transfection, and its our working hypothesis that they undergo similar electrostatic interactions with RNA oligos and facilitate uptake of those species while protecting them from degradation.
This work was performed in collaboration with Fabio Piano, and also Michele Pagano at NYU’s medical school and Ouathek Ouerfelli at the Memorial Sloan-Kettering Cancer Center.
 
What benefits have you seen with this approach compared with existing transfection reagents?
 
Some of the benefits we saw we anticipated based upon the previous study with DNA. These benefits are the very low cytotoxicity of the peptoid reagent in comparison with other polycationic transfection reagents. In our studies in cell culture, we found extremely low cytotoxicity — in fact, we’d call it negligible cytotoxicity.
 
Some of the benefits were not anticipated, and these include the remarkable efficiency of transfection even in cell types that have previously been described as being difficult or impossible to transfect using chemical transfection reagents. I’m referring here to primary cell types as being traditionally impervious to chemical transfection techniques. In this study, we showed that in a primary cell type, IMR-90 human lung fibroblasts, we were able to get very efficient gene knockdown by transfection of siRNA oligos into these cells.
 
You expect that this reagent might have benefits for therapeutic applications of RNAi?
 
That’s right. Some of the challenges that are facing the therapeutic application of RNAi relate to the stability of the oligos in biological systems and also the ability to provide cell-specific gene knockdown.
 
With regard to stability, we believe that the complexes formed between our lipitoids and the siRNA oligos will protect them from degradation. But we also believe that we might be able to use these peptoid oligomers as a foundation on which to design more cell type-specific transfection reagents that would allow them to be used much more precisely.
 
As far as I know, the use of siRNA oligos in vivo has had to be accompanied by fairly high concentrations because of their low stability, and this can lead to off-target effects. If we were able to use diminished concentrations, in particular if we were able to demonstrate cell-specific targeting, this would allow us to … avoid some of those off-target effects.
 
Have you done any in vivo work with this yet?
 
We’re in the process of initiating those studies. I can’t really comment because we don’t have enough data on hand to talk about that.
 
Is the idea to start talking about possible commercial partnerships, or is that something for NYU’s tech transfer office to deal with?
 
We’re vigorously engaged in that effort, and NYU’s Office of Industrial Liaison is assisting us with that. I can tell you that we have supplied some of our samples to various commercial partners for their evaluation.

I should also mention that one of our collaborators, Ronald Zuckermann … has now begun a laboratory at the Molecular Foundry, which is part of the Lawrence Berkeley laboratories. They’re engaged in providing tools in nanotechnology for use by the scientific community. I know they are also vigorously engaged in the development of these reagents for general use and also would pursue opportunities to engage commercial partners. We work very closely with Ron Zuckermann at the Molecular Foundry and are coordinating our efforts with that group.

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