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Cequent Releases Data on RNAi Technology for Multi-Gene Targeting, Addressing Cancer Drug Resistance

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Cequent Pharmaceuticals and collaborators this week unveiled in vitro data from two early-stage programs, one related to a version of the company's transkingdom RNAi technology designed to knock down multiple gene targets and one evaluating whether tkRNAi can be used to overcome multi-drug resistance in cancer cells.

Cequent's core tkRNAi technology involves using attenuated Escherichia coli to transcribe therapeutic shRNAs. The bacteria are designed to express the protein invasion on their surface, which allows them to enter a host cell, as well as listeriolysin, which permits the shRNA payload to escape after bacterial entry.

Although the technology has shown promise in experiments targeting a single gene (see RNAi News, 2/12/2009), like "many other people in the RNAi world, we feel that for some indications it may be beneficial to target more than one gene at a time," Johannes Fruehauf, Cequent’s vice president of research, told RNAi News.

As such, the company has been working for "some time … to adapt our technology to accommodate that need," he said. "This is the first set of data we're reporting on that."

Meanwhile, Cequent has been actively collaborating with a number of academic groups interested in applying tkRNAi to their own areas of interest, Fruehauf noted. Among them is a team from the University Clinic Charite in Berlin that is researching chemotherapy resistance.

The group "approached us to use our technology in their area of expertise … and they are showing the first set of results they have with the [tkRNAi construct] that was made for them," Fruehauf said.

Both sets of data were presented at this year's American Association for Cancer Research annual meeting this week in Denver, Colo.

Multi-Targeting

As part of its effort to developing approaches for inhibiting multiple targets simultaneously, Cequent cloned into a tkRNAi construct a small DNA oligo designed to "serve as a template for expression of hairpin RNA" targeting three oncogenes known to play a role in colon cancer: beta-catenin, K-ras, and cyclin D1, according to the company's AACR poster.

"Another DNA oligo template for hairpin RNA targeting [green fluorescent protein], luciferase, and LacZ served as [a] negative control," the poster noted.

According to the data, the shRNAs were found to be "abundantly expressed" from the expressions plasmids in the bacteria, and were released into the cytosol following bacterial invasion into the cells.

Additionally, "effective" knockdown of beta-catenin, K-ras, and cyclin D1 protein levels was observed, while "inhibition of cell division and cell death were proportional to the degree of oncogene knockdown," the poster states.

However, qPCR analysis revealed significant reductions only in beta-catenin and cyclin D1 mRNA levels, an outcome possibly related to the design of the tkRNAi construct.

Fruehauf noted that whenever a sequence is place in the middle position of the construct, "it doesn’t lead to as efficient a silencing as in the [first and third] positions. We assume it has to do with the way the primary transcript is processed into individual siRNAs, and we are exploring this phenomenon," he added.

These data, while encouraging, indicate that this particular adaptation of tkRNAi "is not quite there yet," Fruehauf conceded.

The data indicate "substantial knockdown of both mRNA and protein levels, but with the single targeting we still get better efficacy at this time," he said. "We are still modifying this construct to allow for more efficient silencing of all three gene targets."

At the same time, Cequent is also engaged in "several parallel efforts to solve multi-targeting," he noted, although he declined to comment on these since they are still preliminary.

Multi-Drug

Although efforts to overcome the resistance of cancer cells to chemotherapeutic agents are varied, Hermann Lage and colleagues from Charite have been exploring the potential of RNAi to address this issue for several years.

In a 2006 review published in Current Drug Targets, Lage noted that the so-called classical multi-drug resistance phenotype results from "decreased cellular drug accumulation mediated by the adenosine triphosphate-binding cassette transporter MDR1/P-glycoprotein encoded by the human MDR1 gene."

In trying to address this problem, he and his colleagues partnered with Cequent to explore the use of tkRNAi to modulate ABCB1-mediated multi-drug resistance in human gastric cancer cells.

The investigators found that the tkRNAi construct was able to enter the cancer cells, reducing ABCB1 mRNA expression by 45 percent. ABCB1 protein levels were also reduced, according to poster data.

Further, resistance to the chemotherapeutic agent daunorubicin was decreased by 60 percent, while drug accumulation was boosted 45 percent over levels in non-resistance cancer cells.

"Overall, tkRNAi does not yet attain the levels of gene silencing seen with conventional short interfering RNAs or non-bacteria-delivered shRNA-encoding vectors," the poster concludes. "But improvements of the tkRNAi-triggered gene-silencing of [multi-drug resistance are] still in progress, and tkRNAi may become a powerful tool for delivery of RNAi effectors for reversal of cancer MDR in [the] future."

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