This week, RNAi News spoke with Sood about the findings and his plans to further develop the therapy.
Let’s start with a little bit about your lab.
My lab is focused on cancer biology, and we look at several things. Most of the work is focused on neuroendocrine effects on metastasis, on angiogenesis, and also on developing novel [therapeutic] approaches, especially based on RNA interference.
So RNAi has been a part of the lab for a while?
For several years.
Could you talk a bit about [the CCR] paper?
When we first started to get into this work, we were looking to develop an efficient method of delivering siRNA in vivo but couldn’t really find that much that was published [on approaches] that would have high enough efficiency. Over the last several years, with one of my very close collaborators, who is actually a co-author on the paper, Gabe Lopez-Berestein, I started to look at options for in vivo siRNA delivery.
Dr. Lopez had some experience using neutral liposomes as a method of delivery for oligonucleotides, and we hypothesized that this may be an attractive approach for delivering siRNA. About a year ago we had published a proof-of-concept paper targeting [the transmembrane receptor tyrosine kinase] EphA2, and that’s when we did a series of fluorescence-based studies to look at the efficiency of this approach for delivering siRNA [into tumors].
The gist of that was using a neutral nanoparticle, or this neutral liposome, we could get highly efficient delivery in vivo, much more so than using, for instance, cationic liposomes, most of which seem to get hung up in the vasculature — they did not penetrate deeply. Also, naked siRNA similarly did not penetrate that deeply inside the tumor.
With the neutral liposome we could get highly efficient delivery that would penetrate deeply into the tumor, well beyond the vasculature. So we selected a gene for this particular study [published in CCR] called focal adhesion kinase, or FAK, for several reasons. One is that in a series of studies, including array-based studies as well as more correlative protein expression-based studies, FAK is over expressed in a high percentage of ovarian and other cancers. In many tumor types, its over expression is associated with adverse clinical outcomes. In a paper in 2004 [in CCR], we actually showed that FAK over expression is associated with poor overall patient survival.
We did additional studies looking at [FAK’s] functional and mechanistic relevance, and in a paper last year [in the American Journal of Pathology] we showed that FAK plays a direct role in mediating chemotherapy resistance in tumor cells. So there were several reasons why this was a particularly appealing target to go after.
Now FAK is a non-receptor kinase, so it’s completely inside the cell. As such, it’s been a difficult target to develop drugs against. As a result, we felt siRNAs would be an appealing approach as long as we could deliver [them] with high efficiency. So we started to do studies with the FAK-targeted siRNA.
The first thing we always do is a series of kinetic studies. In [the latest CCR paper] we show that using this siRNA we can down regulate FAK expression after a single dose for about six days. At about six to seven days we start to see re-expression of the protein. Again, we did a series of kinetic studies to come up with the optimal dose of the FAK siRNA for subsequent experiments.
Once we had the optimal dose and we knew we could down regulate [FAK] for at least six days, we chose the frequency of delivery as twice a week. Then we did experiments with a series of models in which we first looked at models that would have sensitivity against chemotherapy and found that FAK siRNA decreases by itself tumor growth. But when you combine with taxine chemotherapy, it has much greater effect compared with chemotherapy or FAK-targeted siRNA alone. [This was measured against] a series of controls where we use empty liposomes or a non-targeted siRNA.
We also did experiments with drug-resistance cell lines because the critical reality is most patients with ovarian cancer eventually end up developing relapse cancer that frequently becomes resistant to chemotherapy. We first did [these] experiments using FAK siRNA alone or in combination with chemotherapy and found that even in taxine-resistance cells we could see efficacy when you combine FAK siRNA with chemotherapy.
We also went on to do experiments with platinum-resistant models. Platinum is one of the key drugs in ovarian cancer, and, again, in a relapse setting a lot of patients’ tumors end up developing platinum resistance. Even in platinum-resistance models, we found that FAK siRNA alone or in combination with cisplatinum had substantial efficacy with regards to decreasing tumor growth.
One of the [CCR] reviewers asked us, ‘How tolerable is this therapy?’ So we did some hematological parameters where we looked at hemoglobin levels, white blood count levels, and platelet levels in these animals at baseline then during the course of therapy. We found that there were no substantial decreases in hematologic parameters, suggesting that overall this therapy was well-tolerated.
Then we started to look at mechanisms [saying], ‘Here we see nice efficacy in both chemotherapy-sensitive and -resistant models — how is it this can be working?’ We found that, with repetitive treatment, we could keep FAK levels low even at later time points. We found that microvessel density was substantially decreased. We found that some of the angiogenic factors such as VEGF and MMP9 were also down-regulated once you down regulate FAK. And that makes sense because there are certainly now growing data that these kinds of factors can be affected by focal adhesion kinase.
We more specifically looked at the vasculature by looking at dual co-localization studies where we looked at CD31 and TUNEL, and found that the extent of endothelial cell apoptosis in the tumor goes up substantially when you combine FAK silencing with chemotherapy.
Our thought is that the effects of FAK silencing on tumor cells are both direct, with regard to inhibiting tumor growth, and indirect in that the vasculature of tumors is also being affected.
So you’re starving the tumors as well as directly impacting them.
Correct.
With these encouraging data, what’s the next step?
We have to do additional toxicology studies. We are having discussions with the [US Food and Drug Administration]. Our ultimate goal for these kinds of studies is to be able to develop a therapeutically relevant approach.
We’re going to take a cautious and careful approach, and do formal toxicology studies. Then, we’ll try to develop this as a therapeutic tool for human patients.
Just to jump back, [the siRNAs] were delivered intravenously into the mouse models?
In the past we’ve done intravenous studies, and now we have a paper under review where we did formal intraperitoneal versus intravenous delivery comparisons. We know that with this vehicle, this neutral liposome, even if we inject intraperitoneally we can get enough systemic uptake to deliver [the siRNA drug] inside the tumor. But in [the CCR] paper, we did intraperitoneal injections.
These tox studies, you’re planning to do them again in rodent models?
They’ll be in rodent models, but the FDA will frequently require a second species. We will do whatever they ask us to.
I’d imagine that to get something like this really going forward into the clinic, at some point you’d have to start talking to industry. Has that occurred?
Yes. There have been discussions with industry.
But there hasn’t been anything signed just yet.
No. Talks are ongoing.
