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National Cancer Center Research Institute s Ochiya on Delivering siRNAs to Tumor Cells

Takahiro Ochiya
Head, Section for Studies on Metastases
Cancer Center Research Institute

At A Glance

Name: Takahiro Ochiya

Position: Head, Section for Studies on Metastases, National Cancer Center Research Institute

Background: PhD, Osaka University -1988

MSc, medical biology, Tsukuba University — 1984

BSc, biology, Tsukuba University — 1982

In the Aug. 23 issue of The Proceedings of the National Academy of Sciences, Takahiro Ochiya and colleagues from the National Cancer Center Research Institute, Sumitomo Pharmaceutical, Koken Bioscience Institute, and Waseda University describe the use of atelocollagen to deliver siRNAs to bone-metastatic prostate tumor cells in mice. The data indicate that the siRNA can be delivered specifically to tumor cells and exist for up to three days.

Recently, Ochiya spoke with RNAi News via e-mail about his findings.

How and when did you begin involving RNAi into your research?

Atelocollagen-mediated gene delivery technology was established in my lab in 1999. [Robert] Langer at [the Massachusetts Institute of Technology] encouraging me to do research in this field [following the] first demonstration that biomaterials such as atelocollagen will be useful for gene delivery in vivo. In our first demonstration, plasmid DNA was used for atelocollagen-mediated gene delivery in vivo, [and] we recently found that our technology is useful for a short nucleotide such as antisense oligos and siRNAs.

Could you describe atelocollagen and how you came to use it for a delivery vehicle for siRNAs?

Atelocollagen is a highly purified type I collagen of calf dermis with pepsin treatment. Collagen is a fibrous protein in the connective tissue that plays an important role in the maintenance of the morphology of tissues and organs. A collagen molecule has an amino acid sequence called a telopeptide on both its N- and C-terminals, which includes most of the antigenicity of collagen. Atelocollagen obtained by pepsin treatment is low in immunogenicity due to free-from telopeptides, and is used clinically for a wide range of purposes including wound healing, vessel prosthesis, bone cartilage substitute, and [as a] hemostatic agent. We have previously demonstrated that atelocollagen complexed with DNA molecules [can be] efficiently transduced into cells via reverse transfection and allows long-term gene expression in mammalian cells. Since atelocollagen allowed increased cellular uptake, nuclease resistance, and prolonged release of genes and oligonucleotides, an atelocollagen complex [is] applicable to efficient delivery of siRNA in vitro. Furthermore, atelocollagen has a characteristic of being low-toxic and non-immunogenic when it is transplanted in vivo. Thus, our gene delivery method through atelocollagen should permit a safe and efficient siRNA-mediated silencing analysis for therapeutics both in local and systemic administration.

How is it delivered and how does it work to specifically target cells of interest?

[An] atelocollagen/siRNA complex is … a nano-sized particle (100 to 200 nm in diameter) and it is easily uptake into cells via endocytosis. At this moment, although we have not modified for targeted delivery, it is possible to incorporate some target molecules such as [antibodies or] ligand peptides for tumor or tissue targeting.

Does atelocollagen have other applications?

Atelocollagen obtained by pepsin treatment is low in immunogenicity due to free-from telopeptides, and is used clinically for a wide range of purposes including wound healing, vessel prosthesis, bone cartilage substitute, and [as a] hemostatic agent.

Could you provide an overview of the data from the PNAS paper?

In this study, we demonstrated that the delivery system remarkably enhanced efficiency of siRNA for inhibition of bone metastasis through systemic administration. The siRNA/atelocollagen complex was formed by mixing siRNA with atelocollagen. Formation of the complex was confirmed by gel electrophoresis, and the diameter of the complex, which can be administered by conventional methods, was presumed to be several micrometers or less by an observation using [a] microscope. To facilitate the detection of metastatic lesions and the effects of the complex on tumor regression, noninvasive optical imaging technologies and bioluminescent human prostate carcinoma cell line PC-3M-luc-C6 were employed. Intracardiac injection of PC-3M-luc-C6 led [to] metastases in the thorax, jaws, and/or legs of mice. Prior to metastasis inhibition study, we tested whether systemic administration of the complex induces [a] gene-silencing effect on the metastatic sites. When the complex containing siRNA against luciferase mRNA was administrated via tail vein, bioluminescence was inhibited by 80 to 90 percent in the whole body including bone metastases. In contrast, treatment of the siRNA alone either unchanged or slightly suppressed photon emission from tumor cells. This result indicates that [the] siRNA/atelocollagen complex [was] delivered siRNA with high integrity and inhibited gene expression in the metastatic sites including the bone metastases. In order to inhibit bone-metastasis, siRNAs against mRNAs relating cell growth were formed [into] complexes with atelocollagen, and the complexes administered via [the] tail vein. When atelocollagen alone, siRNA alone, and the non-specific siRNA/atelocollagen complex were administered, high metastasis was observed. On the other hand, bioluminescence of mice [in which] the complexes of siRNA [that] inhibit cell growth [were administered] was inhibited by 40 to 50 percent in the whole body including the bone metastases, compared with control group. This result indicates that the atelocollagen-mediated systemic delivery of siRNA could be a novel strategy for inhibition of bone-metastatic prostate tumor growth. Furthermore, we confirmed that the efficient growth inhibition of the complex did not induce an interferon response.

Now that this work is complete, what is the next step? Are you going to conduct further animal studies with atelocollagen and RNAi?

[An] ongoing project is … [to target] bone and lymph node metastasis of breast cancer [with an] atelocollagen/siRNA complex. In this ongoing project, we found [the] genes responsible for docetaxicel resistance in patients with breast cancer. SiRNAs [that] inhibit these drug-resistance … genes were studied in animal models with metastasis of human breast tumor.

Where did you get the siRNAs you used in the experiments details in PNAS?

[The] synthetic siRNA in the PNAS work are from Dharmacon Research and RNAi Co. [based in] Japan.

Are you collaborating with any academics on this work? Any industry partners?

We collaborate [on the] development of atelocollagen-mediated delivery technology with Koken, a collagen manufacturing company [in Japan], and Sumitomo Pharmaceuticals [in] Osaka, Japan. They conduct business development of this technology, and recently they have started a feasibility study with Lorus Therapeutics, a Canadian pharmaceutical company, to develop an antisense DNA drug.

In academia … especially in Japan [we collaborate with Yoshifumi] Takei at Nagoya University, [who] is focusing on VEGF-targeting therapy by using our atelocollagen-delivery system.

Where do you see this work heading? Do you envision an RNAi-based prostate cancer treatment? When could something like this be available?

We believe that [the] application of our atelocollagen delivery method for systemic siRNA delivery [is possible] in [a] clinical setting. We are now talking with doctors and trying to find best [targets in] cancer for clinical [research]. As noted previously, a couple of Canadian and US companies [are] interested our technology and [interested in starting a] feasibility study [with] antisense oligos and siRNA. By the way, Sirna Therapeutics contacted me and we will have a meeting next Wednesday. They are interested in our systemic delivery method.

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