Associate professor, pathology, University of Maryland School of Medicine
• Assistant professor, University of Maryland School of Medicine — 1998-2006
• Senior staff and endocrinology fellow, National Institute of Diabetes and Digestive and Kidney Diseases — 1989-1994
• Medical staff fellow, National Cancer Institute — 1986-1989
• Resident, pediatrics/internal medicine, Emory University Hospital — 1979-1982
• MD, Emory University — 1979
• BA, math/chemistry, Vanderbilt University — 1974
University of Maryland School of Medicine researcher James Mixson has developed a gene therapy polymer that he is betting will have therapeutic siRNA-delivery applications.
With the support of a four-year grant from the National Institutes of Health, worth $321,589 in its first year, Mixson is now aiming to test whether the technology can deliver siRNAs against Raf-1 into tumors in vivo, and potentially inhibit their growth.
RNAi News spoke with Mixson this week about the technology and the NIH-funded project.
Let's start with an overview of your lab and the research focus there.
We focus primarily on peptide delivery systems of nucleic acids, both [DNA] plasmids and siRNAs. For approximately the last 10 to 12 years we have utilized a peptide composed primarily of histidines and lysines (HK) to deliver nucleic acids.
Initially, we dealt with linear HK peptides, but they were inefficient in delivering the plasmids so we combined them with liposomes. We had a lot of difficulty with the preparation of the linear peptides, but once we mastered that, our ability to make peptides, including branch peptides, exponentially increased. After mastering the system, we spent a significant amount of time with cell culture systems, and then slowly went into tumor xenograft systems.
As I said, we started with linear HK peptides, went to two-branch, three-branch, four-branch, and finally eight-branch polymers. Besides the number of branches, we also alter the histidines and lysines so that we can tailor the peptide to the particular nucleic acid and cell. We have found that these patterns at times hold up even in vivo … [and that] some peptides are particularly effective for siRNA delivery.
We focus primarily on intravenous delivery, systemic delivery to tumor xenografts … [that] are usually subcutaneously implanted. I have used a variety of xenografts, but primarily we have used MDA-MB-435 cells, which were initially thought to be a breast cancer cell line but evidence now shows that it is a melanoma cell line. But it is still an excellent model to look at.
And you've seen siRNA delivery to the tumor with systemic administration?
Yes. We primarily have targeted Raf-1 with our siRNA-delivery system and we get significant inhibition of Raf-1 in the tumor. We get about a 60 percent inhibition with our unmodified polymer.
As far as how the siRNAs are getting to the tumors, does it have to do with the vasculature of the tumor, for instance? Have you explored targeting agents?
So far, we have primarily used an unmodified nanoparticle, so at present, it's not really targeted. The siRNA against Raf-1 targets the mitogenic endothelial cell of the tumor, as well as the oncogene within the tumor.
We have used targeting polymers with plasmids and have seen high expression within the MDA-MB-435 cells with these targeted systems. [Specifically], we use a cyclic RGD attached to [polyethylene glycol], and we expect that we will see similar results with the siRNA. But we also anticipate that the polymer will be different; we may well require more lysines to bind to the smaller siRNA with a greater affinity, but we haven't done those experiments yet.
When you were seeing target gene knockdown, did you do any testing with control siRNAs to confirm that this isn't, say, an immune response but an actual RNAi effect?
When we see our 60 percent reduction, this is a reduction from a control siRNA … [and] we have used a variety of controls.
With Raf-1, we see 60 percent inhibition, [and] targeting the [vascular endothelial growth factor] receptor on mitogenic endothelial cells of a tumor we have seen 50 percent inhibition. We have also looked at … HK-siRNA polyplexes and have found that some of our polymers — some that contain a high content of histidines — result in much lower expression of serum cytokine levels. We feel that this is through the buffering mechanism, similar to how chloroquine inhibits cytokine induction.
So we feel pretty confident that it's not a cytokine effect. We have compared our polymer polyplexes with potent inducers of cytokines such as DOTAP lipoplexes, and [the levels induced by] our nanoparticle are very low, even with very potent siRNA inducers of cytokines.
Now you've gotten a grant from the NIH to advance the technology. Can you talk about what that project is going to involve?
The grant really involves understanding what the optimal unmodified polymer is and reducing [tumors] in xenografts. From there, we can specifically locate the PEG and cyclic RGD ligand on the HK polymer to get the most stable nanoparticle structure.
Unlike many polymer systems, we can insert the PEG ligand at a specific site on the HK polymer. We feel this is important because we think that the ligand and PEG, inappropriately placed, can actually be a disruptive force in the nanoparticle formation.
So we're going to be doing structure-function analyses, looking at various pegylated sites, looking at the size and surface charge, doing atomic force [and nuclear magnetic resonance] microscopy, and optical tweezers methodology to understand the interaction between siRNA and the polymer, as well as the overall structure of the nanoparticle itself. We will correlate these biophysical properties of the different HK siRNA polyplexes with in vivo delivery.
We plan on looking at several different types of tumor xenografts, primarily [using] intravenous delivery, and looking at not only MDA-MB-435 but also several breast cancer xenografts.
We also plan on using the HK siRNA delivery system to look at the treatment of lung metastases in a mouse model.
Is this a strictly in-house effort, or do you have any industry collaborators?
We collaborate with a number of companies. Sirnaomics is one, which is involved with wound healing, as well as several other projects. Aparna [Biosciences is another] and they are going to be modifying our HK polymers with PEG, but they primarily help us with our plasmid delivery systems.
We also collaborate with Intradigm, [which was originally established by the founders of Sirnaomics and Aparna].