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Silenseed Publishes Data on siRNA Delivery System, Plans for Phase II/III Cancer Study

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Israeli RNAi drug developer Silenseed this week published in vitro and in vivo data demonstrating the potential of its implantable siRNA delivery system to knock down the oncogene KRAS as a treatment for pancreatic cancer.

Meanwhile, as it nears the completion of a phase I trial of its RNAi drug delivery platform — called Local Delivery of RNAi, or LODER — Silenseed is preparing to begin a multinational phase II/III study that could lay the groundwork for US regulatory approval, a company official told Gene Silencing News.

LODER was developed as a means to address the delivery issues facing RNAi therapeutics, Silenseed CEO and Co-founder Amotz Shemi explained.

The system is essentially a miniature biodegradable polymeric matrix containing siRNAs that is implanted into a tumor. It is designed to protect its therapeutic payload from degradation while slowly and steadily releasing it over a period of several months.

Given its oncology focus, Silenseed is initially pursuing pancreatic cancer, a disease with an extremely high fatality rate and one that is most frequently caused by mutations in the tissue-signaling gene KRAS.

In 2010, Silenseed began a phase I trial of LODER in 15 patients with locally advanced pancreatic tumors who received 0.025 mg, 0.75 mg, or 3 mg doses of KRAS-targeting siRNA contained in 1, 2, or 8 LODERs, respectively. Patients also received weekly treatments of the chemotherapeutic gemcitabine.

Two patients were later found to have metastatic disease and were not eligible for analysis. Among the remaining 13 study participants, all showed disease stability after treatment, with a median overall survival of 16 months, according to data presented at this year's American Society of Clinical Oncology meeting.

The phase I trial remains ongoing, Shemi said, since two patients remain alive and are being monitored. However, their outcomes aren't expected to affect the final phase I data significantly.

As such, Silenseed is moving forward with plans to file an investigational new drug application with the US Food and Drug Administration in order to begin a potentially pivotal phase II/III study of its pancreatic cancer treatment next year.

Although the study's protocol has yet to be finalized, the trial is expected to enroll between 100 and 150 patients with unresectable locally advanced pancreatic cancer, who will receive 2.8 mg of anti-KRAS siRNAs contained in eight LODERs, Shemi said. They will also receive FOLFIRINOX, a chemotherapy regimen that combines four chemotherapeutic drugs.

After four months, patients will receive additional LODER treatments until disease progression is halted or they are unable to continue in the study.

The primary endpoint of the study, which is to be run at multiple centers in the US and potentially Europe, is patient survival, he added, but progression-free survival may be evaluated as a secondary endpoint.

Should data from that trial prove positive, and if Silenseed can obtain orphan drug status for its therapy, Shemi said that the study may be sufficient for seeking FDA approval.

Preclinical data

Prior to launching the phase I study, Silenseed and collaborators conducted a number of experiments in culture and in animals to validate the LODER technology, and this week published those data in the Proceedings of the National Academy of Sciences.

They began by examining the system's ability to release siRNAs, incubating LODERs containing 10 micrograms of anti-KRAS siRNAs into phosphate buffer solution, finding that approximately 75 percent of the oligos were released after 60 days.

To assess its ability to prevent siRNA degradation, LODERs bearing a similar amount of the RNAi molecules were placed in phosphate buffer solution or in mouse liver tissue. An analysis showed that the siRNAs remained intact for at least 97 days in the solution and for at least 60 days in the liver tissue.

In terms of LODER's efficacy, the researchers transfected human pancreatic cancer cells with siRNAs against KRAS, or with control siRNAs, they wrote in PNAS. Quantitative PCR showed that siRNA treatment reduced KRAS mRNA levels with an IC50 of 67 picomoles, and 5' RACE analysis validated that the effect was due to RNAi.

Further testing showed that siRNAs delivered with LODER had the same effect on KRAS levels in the cultured pancreatic cancer cells as siRNAs administered directly, and that treatment with the RNAi molecules inhibited the cells' growth in vitro.

Given KRAS' putative role in the epithelial-mesenchymal transition, or EMT, commonly experienced by cancer cells, the investigators conducted a series of experiments to confirm whether KRAS inhibition suppressed EMT and, as a result, cell migration. Indeed, siRNA-treated pancreatic cancer cells "barely migrated" in response to a scratch assay, while untreated cells or those receiving control siRNAs migrated normally, according to the paper.

Turning in vivo, Silenseed and its partners tested whether LODERs containing siRNAs against luciferase could silence their target in both normal and tumor tissues that constitutively express the reporter gene.

To do so, empty and siRNA-loaded LODERs were implanted into the livers of transgenic mice expressing the luciferase gene in their hepatic cells. Imaging analysis confirmed that siRNA treatment led to a significant decrease in luciferase levels compared with empty LODERs. LODERs carrying siRNAs against luciferase were then implanted into synograft tumors, resulting in a reduction of luciferase expression after three days.

To study the potential toxicity of Silenseed's system, LODERs carrying KRAS-directed siRNAs and empty LODERs were implanted into the pancreas of normal mice and rats, with the effects compared with sham operated and untreated animals, and those that received intraperitoneal injections of the siRNAs.

LODER treatment had no apparent effect on animal mortality, behavior, or body and liver weight, the researchers reported in their paper. "Hematological and biochemical tests failed to reveal any statistically significant differences between the tested groups," while "gross and histopathology analyses revealed that all changes were of minimal severity and typical in untreated mice or rats of the same age and strain."

In order to examine LODER's therapeutic efficacy in vivo, the scientists used immunodeficient mice carrying tumors derived from human pancreatic cell lines with KRAS mutations. The animals received intratumoral implantations of either siRNA-containing LODERS or empty LODERS, or were left untreated.

LODER RNAi treatment resulted in significant inhibition of tumor growth, destruction of tumor tissue, and an increase in animal survival. Similar effects were seen in immune proficient animals, while an analysis of the remaining siRNA content of LODERs isolated from tumors after implantation showed that the system protected its RNAi payload from degradation over a 70-day period.

Finally, the Silenseed team established an orthotopic pancreatic cancer mouse model in which luciferase-expressing cancer cells were injected into the tail of the animals' pancreas, according to the PNAS paper. "When tumors were detected, mice were divided into the different treatment groups, keeping a similar average level of luciferase intensity in each group." LODERs were then implanted into the tumor mass.

Luciferase measurements revealed a suppression of tumor growth in siRNA LODER-treated animals throughout the study compared with control mice. More importantly, the overall survival of treated animals was "considerably prolonged" versus control groups.

Further testing showed the LODER treatment had a direct effect on KRAS protein expression in xenograft tumor tissues, and that its effect on tumor cell growth was greater in areas closest to the implantation.

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