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University of Nebraska's Joseph Vetro on Delivering siRNAs to Tumor Vasculature

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Name: Joseph Vetro
 
Position: Research assistant professor, pharmaceutical sciences, University of Nebraska Medical Center
 
Background: Postdoc, adjunct research associate, University of Kansas — 2001-2004
- PhD, biochemistry/molecular biology, St. Louis University Health Sciences Center — 2001
- BA, chemistry, University of Nebraska at Omaha — 1996
 

 
This month, University of Nebraska Medical Center researcher Joseph Vetro was awarded a two-year grant from the National Institute of Biomedical Imaging and Bioengineering to develop a nanoparticle system, called a nanogel, for the delivery of therapeutic siRNAs to tumors.
 
This week, RNAi News spoke with Vetro about his efforts.
 
Let’s start with a little background on your research focus.
 
I’m a part of the nanomedicine group at the University of Nebraska Medical Center and our major theme is drug delivery. I am currently developing nanocarriers for targeting tumor vasculature.
 
The goal of my work is to block blood vessel recruitment by solid tumors through the targeted delivery of drug — in this case, siRNA — to locally activated microvascular endothelial cells in order to inhibit tumor growth and subsequent metastasis.
 
A few anti-angiogenesis approaches have had some success in the clinic, but the development of alternative modalities is still needed to make the approach clinically effective against a broad range of solid tumors.
 
How long have you been incorporating RNAi into that work?
 
We’ve been developing RNAi for the last three years.
 
A lot of previous work in the nanomedicine group focused on antisense, but antisense has its own well documented set of limitations. Thus, it’s a natural progression from antisense technology to siRNA.
 
Can you give me some background on the nanogel you are developing?
 
The nanogels were previously developed by Serguei Vinogradov. They are a polymer-based nanoparticle that consists of a charged polymer — in this case, polyethylenimine — cross-linked with polyethylene glycol. This produces a highly hydrophilic nanoparticle that swells upon the addition of water — that’s why it’s described as a nanogel.
 
Nanogels form a complex with siRNA through electrostatic interactions between the positively charged PEI chain and the negatively charged siRNA. 
 
As far as how it targets new vasculature …
 
Our current approach is to modify the surface of the nanogel with a 12-mer peptide, K237, that was identified by phage display as an antagonist for VEGF binding to the VEGFR2 receptor.
VEGFR2 is selectively up-regulated on microvascular endothelial cells that are being locally activated and recruited by the tumor.
 
Thus, modifying the nanogel surface with targeting ligands that have a high affinity and specificity for VEGFR2 is expected to increase nanogel affinity for the surface of activated microvascular endothelial cells and increase the localization of siRNA-loaded nanogels to tumor vasculature.
 
Interactions with proteins and untargeted cells of the vascular compartment, however, largely dictate the intrinsic biodistribution of targeted nanoparticles and, consequently, interfere with targeting activated microvascular endothelial cells. Thus, selectively increasing the affinity of nanogels for the surface of activated microvascular endothelial cells, while minimizing nanogel affinity for untargeted cells of the vascular compartment in the presence of blood plasma, is expected to increase the accumulation of nanogel-siRNA complexes within tumor vasculature after systemic administration. 
 
We determine the binding selectivity of targeted nanogels by isolating cells from the vascular compartment of a mouse model and comparing the affinity of targeted nanogels for the surface of the targeted cell, activated microvascular endothelial cells, versus untargeted cells of the vascular compartment in the presence or absence of mouse plasma. The targeted nanogel is then used in the same mouse strain using a model melanoma to assess [and] correlate both biodistribution and efficacy.
 
What’s the logic of going against this specific receptor as opposed to VEGF itself?
 
Blocking VEGF is one approach to indirectly inhibit angiogenesis by interfering with microvascular endothelial cell regulation. Considering that many angiogenic factors in addition to VEGF are present in the later stages of tumor angiogenesis, however, directly inhibiting the function of activated microvascular endothelial cells is likely the most promising therapeutic approach.
 
We are targeting the VEGFR2 receptor only to increase nanogel affinity for activated microvascular endothelial cells and increase its localization to these cells in vivo.
 
Have you published any data on the nanogel technology?
 
A lot of the work is still preliminary at this stage.
 
What’s the status of the research now? Are you in animal models?
 
We will test the siRNA efficacy by targeted nanogel delivery against B16/F10 melanoma in C57Bl/6 mice. B16/F10 is highly metastatic, which is also important because you really want to address the metastases. As you may know, it’s not the primary tumor that is lethal, it’s the metastases from the primary tumor that end up being the thing that’s lethal.
 
And that work has already begun?
 
Yes, that work has already begun. We’ve got a lot of preliminary data, and I’m in the process of isolating the different cell types from the vascular compartment of the mouse model.
 
As far as siRNA design, is that something you do in-house?
 
We’re using pooled siRNA from Dharmacon against the same mRNA target because they have developed unique chemistry to improve siRNA function and have developed very effective algorithms in designing their siRNA pools.
 
What about collaborations?
 
I have two collaborators in-house: Rakesh Singh, who is an expert in tumor microenvironment/ tumor microvasculature … [and] Serguei Vinogradov, who originally developed the nanogel construct.
 
This is a two-year project?
 
Yes, this is a two-year project to show that our approach to developing targeted nanogels — and targeted nanocarriers in general — is effective.
 
We need to first show good activity in vitro against the target cells — activated microvascular endothelial cells — and that we can produce nanogels with highly specific binding affinity for the target cells in the presence of blood plasma or serum protein.
 
We will then assess targeted nanogels into the animal model to see if there is a correlation between the activities in vitro and relative efficacy and biodistribution in the animal model.

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