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Researchers Working on Conditional RNAi Gene Silencing in Transgenic Mice

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With the benefits of using RNA interference to knock down gene expression in vivo well established, researchers are looking to take the technology one step further by developing transgenic mice in which the RNAi effect can be controlled.

At Baylor College of Medicine, Olga Cabello and colleagues are using a three-year grant from the National Eye Institute to test whether RNAi can be used to conditionally silence genes in transgenic mice while avoiding the stimulation of an interferon response.

In the four-part project, the researchers are testing whether RNAi can be induced in transgenic mice by using short palindromic RNAs, or by using long interrupted RNAs. The researchers are also exploring whether gene silencing in the mice can be facilitated by the co-expression of Dicer RNAse and an RNAi transgene, as well as whether short temporal RNAs can be used to inhibit translation.

Cabello et al. are evaluating the efficacy of gene activation by trying to block tryrosinase expression in melanocytes and in retinal pigmented epithelium, predicting changes in pigmentation. They are also trying to inhibit Rb in lens fiber cells, resulting in cataracts and microphthalmia.

Then, by setting up the “proper combination of inducer and regulatory system … we would also be able to have developmental regulation of [RNAi],” Paul Overbeek, a collaborator of Cabello’s, told RNAi News.

Overbeek said that the scientists expect to approach the issue in two ways. The first would to use a system in which a Tet-repressor has been linked to “a transactivation transcription factor called VP16,” he said. “You’d link up a [tissue-specific or ubiquitous] promoter for where you’d want your RNAi expressed … to this Tet-transactivator protein.” Then, an RNA transcript would be linked to the Tet-operator, the binding site for the Tet-transactivator.

“The ability of Tet to bind to its binding site is regulated by the presence or absence of a tetracycline homologue called doxycycline,” which could be given to mice in food or water, he added. This will allow an RNAi transcript, and subsequently a particular gene, to be turned on or off in “a fully reversible fashion.”

This so-called Tet-Op doxycycline system is being looked into by Ralph DiLeone and colleagues at the University of Texas Southwestern Medical Center at Dallas (see Proof-of-Principle RNAi story, pg. 1).

The second technique Cabello and colleagues will use is based on the yeast transcription factor GAL-4, said Overbeek.

“A group of investigators here at Baylor took that transcription factor and linked it up to a modified progesterone receptor [that] is particularly good at binding to the progesterone homologue that is used to induce abortions called RU486 or mifepristone,” he said. “In the absence of RU486, that [GAL-4] is sequestered out in the cytoplasm, so it can’t serve as a transcriptional activator.”

But in the presence of mifepristone, GAL-4 transcription factor is brought into the nucleus, where it can bind with a promoter, called UAS (upstream activating sequence), which has been linked to a specific RNAi transcript. So, by controlling the level of mifepristone given to the transgenic mice, the gene knockdown effect can be manipulated.

The researchers have received about $300,000 in NEI funding since Sept. 2002.

It appears, however, that researchers from the University of Virginia have already found success with controlled mammalian RNAi.

According to the University of Virginia Patent Foundation, Heidi Scrable and Tsutomu Sasaki have invented a conditional RNAi system for transgenic mice based on the lac operator-repressor system.

According to the university’s licensing arm, one embodiment of the invention involves the use of an RNAi vector in which the lac operator-repressor system controls the activity of a mouse promoter, which transcribes a shRNA or siRNA molecule. The transcription of the RNA interference can be controlled by altering the amount of the lactose analog IPTG in the drinking water of mice or in the culture medium.

“This system provides a simple means for controlling endogenous gene expression in vivo and in cultured cells, UVPF stated on a portion of its website detailing technologies available for licensing.

Another embodiment of the invention, said UVPF, comprises a construct in which the lac operator-repressor system controls shRNA transcription using operator sequences adjacent to the shRNA, “alleviating the necessity to insert operators into promoters.

“This construct could be inserted downstream of any promoter of interest to generate conditional RNAi in a tissue-specific manner,” UVPF added.

Miette Michie, a licensing associate at UVPF, told RNAi News that an unpublished provisional patent application has been filed on the technology, and that her office has been marketing the invention to potential licensees for “a few months.” She said that UVPF has received interest from a number of companies involved in RNAi, including developers of RNAi-based drugs, but that no deals have been struck.

Michie said that provisional patent applications expire one year after they are filed if they are not converted into formal patent applications. UVPF’s policy, she added, is to offer the rights to an invention back to its inventors if a licensee isn’t found for the technology before its provisional patent application expires.

— DM

 

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