Scientists from SR Pharma subsidiary Atugen have shown that systemically delivered siRNAs formulated with the company's liposomal technology can trigger an RNA inference effect in the vasculature of mice, although with varied levels of efficacy.
In a separate study, the researchers showed that the company's formulated siRNAs could be used to inhibit tumor growth and metastases in xenograft mouse models, apparently through angiogenesis, but cautioned that their findings were still preliminary.
Still, the researchers are hopeful that the results, which appear in the upcoming issue of Gene Therapy, could eventually lead to the development of RNAi-based pro- and anti-angiogenic therapies an important focus of cancer research.
In the first Gene Therapy study, Atugen formulated its proprietary RNAi molecules, which are 19-mer siRNA duplexes that lack 3' overhangs and have been chemically stabilized with alternating 2'-O-methyl sugar modifications on both strands, with its so-called lipoplex technology that incorporates a mixture of cationic and fusogenic lipids.
This delivery technology "makes use of the nucleic acid-complexing properties of the novel cationic lipid in combination with a neutral lipid, which mediates the endosomal release of the siRNAs after endocytotic uptake of the lipoplex," the researchers wrote in their paper. "A smaller amount of lipid is required to complex the siRNA molecules due to the higher nucleic acid-binding capacity of [the cationic lipid] compared to standard lipids."
"siRNA-lipoplexes enable siRNA-mediated RNAi in vascular endothelial cells, and might pave the way for the development of novel pro- and anti-angiogenic therapies."
According to the researchers, repeated intravenous administration of the siRNA-lipoplexes into the tail vein of mice resulted in no significant toxicity, and no induction of cytokines was observed. A biodistribution analysis indicated a predominantly specific targeting of endothelial cells in the vasculature of several organs, including the lung, heart, liver, and spleen, after a single dosing.
To determine the level of gene-expression knockdown from the siRNA-lipoplexes, the researchers focused on two target genes CD31 and Tie2 that are expressed in endothelial cells only. They found a reduction in the expression of the genes in certain organs such as the lung, liver, and heart, but with "notable differences in terms of efficacy." Other organs, such as the kidney and spleen, "exhibited a strong uptake of fluorescently labeled lipoplexed siRNAs, but did not show any significant inhibition of gene expression."
The researchers concluded that the data demonstrate "that siRNA-lipoplexes enable siRNA-mediated RNAi in vascular endothelial cells, and might pave the way for the development of novel pro- and anti-angiogenic therapies." Additional work still needs to be done on identifying the correct targets for a specific indication, on the optimization of dosing schedules for sustained delivery and RNAi effect, and the assessment of potential risk/benefit parameters, they added.
In the second study, the Atugen researchers again intravenously administered the company's blunt-ended siRNA molecules, which had been formulated with the lipoplex technology, to tumor xenograft mouse models via the tail vein.
The researchers first determined that the siRNAs formulated with the cationic lipid technology were taken by endothelial cells of the blood vessels in the liver and tumor as opposed to unformulated siRNAs, which were completely absent from the tumor vasculature.
"Functionality of [the siRNAs] in endothelial cells was shown by demonstrating down regulation of … CD31 target expression, and also indirectly by a positive readout assay for increased DNA synthesis after PTEN inhibition," according to the Atugen researchers. "The data represent a proof of concept for RNAi in a particular cell type in vivo and reveal the applicability of siRNA-lipoplex technology to selectively trigger RNAi in vascular endothelial cells, including tumor endothelial cells."
The researchers note that CD31 inhibition in endothelial cells with the siRNA-lipoplex molecules "resulted in an inhibition of tumor growth and in reduced metastases formation," likely as a result of "vessel regression upon RNAi-mediated reduction of CD31, which might be influencing … cell survival or proliferation during neovascularization."
"The data represent a proof of concept for RNAi in a particular cell type in vivo and reveal the applicability of siRNA-lipoplex technology to selectively trigger RNAi in vascular endothelial cells, including tumor endothelial cells."
They stressed that this antiangiogenic effect is thus far "speculative and needs to be addressed in the future to fully elucidate the CD31 loss-of-function phenotype on the molecular level during tumor neovascularization."
The researchers also noted that negative siRNA controls also showed some inhibitory effects on tumor growth compared with a sucrose control, which could possibly be the result of "RNAi-independent effects of the formulation" such as unspecific inhibition of endothelial proliferation by the lipids owning to the daily bolus injections into the bloodstream.
The researchers noted that there was no interferon induction observed with the siRNA-lipoplex treatment, and conclude that the knockdown data suggests that RNAi suppression of CD31 was responsible for the observed inhibition of tumor growth. "Therefore, [the] study provides a basis for the development of antiangiogenic cancer therapies based on RNAi," they wrote.
Publication of the data comes on the heels of an SR Pharma announcement that two of Atugen's RNAi molecules are headed for the clinic this year under a collaboration with Quark Biotech, and that Atugen is gearing up to begin its own phase I studies of an RNAi-based therapeutic for cancer in early 2007 (see RNAi News, 4/6/2006).
Doug Macron ([email protected])