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Start-Up Calando Shows That Its RNAi Delivery System Works In Vivo

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Calando Pharmaceuticals, an RNAi therapeutics firm founded in February, released last week preclinical data demonstrating that siRNAs delivered with the company's proprietary technology were able to inhibit tumor growth in a mouse model of Ewing's sarcoma without stimulating an interferon response.

The data, which validate Calando's delivery technology rather than forming the basis for a drug-development program, were presented at the American Association for Cancer Research annual meeting in Anaheim, Calif., the company said.

Calando was co-founded by California Institute of Technology researcher Mark Davis and John Rossi of the City of Hope's Beckman Research Institute in order to advance the RNAi technology of Insert Therapeutics, while also developing an undisclosed RNAi technology recently licensed from CalTech (see RNAi News, 2/25/2005). Insert, which Davis also co-founded, has moved away from the RNAi field in order to focus on other drug technologies.

A key portion of the Insert technology is a delivery approach designed to protect siRNAs from degradation when administered in vivo, while enabling the oligos to target specific tissues. Additionally, the technology appears to help the siRNAs fly under the radar of the immune system and avoid triggering an interferon response.

According to Calando, the technology comprises a linear, cyclodextrin-containing polycation that is able to bind to the anionic backbone of an siRNA. When mixed together, the polymer and siRNA self-assemble into nanoparticles, roughly 50 nanometers in diameter, which are protected from nuclease degradation in blood serum.

The cyclodextrin in the polymer, Calando said, allows stabilizing agents to be attached to the surface of the particles. These agents have terminal adamantane groups that form inclusion complexes with cyclodextrin and contain polyethylene glycol, which prevents aggregation while preventing degradation. Additionally, ligands to cell-surface receptors can be covalently attached to the adamantane-PEG modifier, the company notes, which allows the particles to be targeted to tissues of interest.

For the Ewing's sarcoma study, Davis and collaborators at CalTech and the Children's Hospital Los Angeles created a mouse model of the disease using human Ewing's sarcoma cells engineered to express luciferase.

In previous experiments, the researchers found that siRNAs targeting EWS-FLI1, the chimeric gene product of a chromosome translocation found in more than 90 percent of Ewing's family of tumors and necessary for tumor growth, reduced EWS-FLI1 transcripts by as much as 80 percent and inhibited growth of the cultured cell line TC135.

To test these siRNAs in vivo, two groups of 10 mice each were given either a mock therapy or naked siRNA targeting EWS-FLI1, respectively.

Two other groups of 10 mice were given siRNAs targeting EWS-FLI1 using Calando's delivery system, which featured transferrin ligand at the termini of some of the adamantane-PEG conjugates in order to allow the siRNAs to target tumor cells. The first group received siRNAs with a sequence unrelated to EWS-FLI1, while the second group received anti-EWS-FLI1 siRNA.

A final group of 10 mice was given the anti-EWS-FLI1 using the cyclodextrin-containing polymer delivery system minus the tumor targeting ligand. All the mice received 2.5 mg/kg doses via the tail vein at normal venous pressure.

Only the targeted siRNA formulation had anti-tumor effects, Calando said. Furthermore, no abnormalities were observed in IL-12 and interferon-alpha, liver and kidney function, complete blood counts, or pathology of major organs, the company added.

John Petrovich, CEO of Calando, told RNAi News that the researchers have submitted additional data from the Ewing's sarcoma study for publication in an undisclosed peer-reviewed journal. "They're going through the process of responding to review comments right now," he said.

Despite the promising results, Petrovich said it is unlikely that Calando will pursue development of an siRNA-based Ewing's sarcoma treatment in the near term.

"There is some talk about proceeding further to do some further preclinical work in taking this forward, but I'm fairly certain it's not going to be our actual lead project for Calando Pharmaceuticals," he said. "I'm not at liberty to tell you what that lead project is or the area or indication it will be [in], but it will probably be for something that has a bigger market — [Ewing's sarcoma] is a fairly rare form of cancer."

Petrovich added that "we're pretty clear on the indication we want to go after [in our lead program, but] we're giving it a final look-see [and] seeing what outside advisors we might be able to get lined up to support us.

"We're in the final stages of putting all that together and doing some further checking around on it, but we're pretty sure about what we're going to do for the lead program," he noted. While Petrovich declined to offer details about the lead program, Calando's website notes that its delivery technology has also been used to suppress expression of the inflammatory protein Fas in murine livers.

A Lease on Hope

According to Petrovich, a bigger focus for Calando right now is ramping up its operations in order to be up and running by the summer. "We've found a home — we're putting the finishing touches on a lease," he said. "We're going to be adjacent to the City of Hope campus in Duarte, [Calif., in] a research building … formally occupied by Baxter Labs that the City of Hope bought a year or so ago. They're refurbishing the whole building for use by other City of Hope faculty, and there's a reasonably small lab at the front part of the building that we're going to be taking.

"We'll probably be physically in there in about three weeks," Petrovich added.

Meanwhile, Calando is in the final stages of rounding out its staff. Calando's website indicates the company is looking for two research associates, and Petrovich said that the company is also currently interviewing a couple of PhDs.

"We're going to be able to hit the ground running pretty quickly; by around June 1 … I'm hopeful we'll have a full-time scientific staff of four people," he said. In addition to the scientific guidance provided by Davis and Rossi, Petrovich said that Calando also expects to have enlisted the aid of an undisclosed outside scientific consultant by around the beginning of June.

As for Calando's executive team, Petrovich said that "for at least the first year, we'll keep it where I'll be the jack-of-all-trades on the non-scientific side. [In the] long term, we'll probably need to get a chief scientist on board, but … the plan is to keep it where I'll be the non-scientific management team … for the time being.

"You only have a certain amount of money to start out with, and we already want to do more than we raised capital for," he said. "So part of [my] job is for me to get some collaborations lined up to make up that difference, and it's probably premature to get a lot of business development people" in the company.

In terms of collaborations, Petrovich reaffirmed Davis' statement to RNAi News in February that Calando was on track to have secured an industry partnership by the end of the year. Petrovich declined to comment on the status of these talks, but said that work on a collaboration is proceeding.

"In fact, one of the things I'm looking at today is a license agreement related to that collaboration," he said.

— DM

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