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NIH Grants In May Reveal Wide Variety of Potential Applications for RNA Interference

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The National Institutes of Health has awarded this month a number of grants supporting research exploring a wide variety of uses for RNAi, including developing animal models for diseases, studying protein function in male germ cells, and treating cancer.

One of the grants went to Zuoshang Xu, an associate professor at the University of Massachusetts Medical School who is separately conducting research into whether RNAi can be used to treat amyotrophic lateral sclerosis on behalf of CytRx (see RNAi News, 1/23/2004).

Xu’s project is focused on using RNAi to overcome the difficulties associated with gene knock out technology — specifically, technical complexity, length of time, and high costs.

“RNA interference can mediate sequence-selective suppression of gene expression in a wide variety of eukaryotes by introducing [siRNAs] with sequence homologies to the target gene,” Xu noted in the grant’s abstract. “Recent experiments indicate that small hairpin RNAs transcribed in vivo can trigger degradation of corresponding mRNAs similar to siRNA and achieve effective knockdowns in cultured cells and, in some instances, in vivo.”

However, Xu stated that RNAi has heretofore been limited to gene knockdown, not gene knockout. “I propose to develop … RNAi technology that can mimic knockout conditions in vivo and may serve as an alternative to the conventional gene knockout,” he wrote. “The central idea is to increase the potency of RNAi so that it can achieve virtual knockout.”

Xu’s project has three aims: To develop strategies to increase the potency of RNAi so that it can knock out gene expression in cell cultures; to apply the most effective of these strategies to knock out genes associated with aging diseases in transgenic mice and determine whether animal models for these diseases can be generated; and to develop inducible promoters for shRNA synthesis so that inhibition of gene expression in these models can be controlled spatially and temporally.

Xu’s grant project runs between May 1, 2004 and April 30, 2006, and is worth about $347,000.

A second grant was awarded to Stuart Moss, a spermatogenesis researcher at the University of Pennsylvania, who is also looking for a way to overcome the hurdles of gene knockouts.

“The one problem that people who work in spermatogenesis have is that there’s no cell culture system for spermatogenesis — you can’t take germ cells and put them into tissue culture and watch them develop into mature sperm,” Moss told RNAi News. “So, the only way of really looking at protein function is using knockouts, which is obviously labor intensive and tedious, and you might not even get a phenotype when you’re all done.”

As such, Moss is turning to RNAi as an alternative. In the project, he is setting out to determine whether siRNA and dsRNA can be used to eliminate or reduce a gene product in mouse male germ cells by using AKAP4 — a protein in the sperm fibrous sheath — as a model protein target.

AKAP4, he noted in the grant’s abstract, offers a number of advantages including the availability of reagents against the protein. Additionally, the gene has been disrupted by homologous recombination, so it can be determined if protein reduction by siRNA/dsRNA phenocopies the null, and AKAP4 is so abundant that the generation of a mutant phenotype by siRNA/dsRNA would suggest that the function of other spermatogenic proteins can be studied similarly.

Moss intends to reduce or eliminate AKAP4 in spermatids and sperm firstly by transcribing siRNA corresponding to a region of AKAP4 from the RNA polymerase III promoter U6. DsRNA corresponding to AKAP4 will also be expressed from the spermatid-specific protamine promoter to examine whether AKAP4 can be reduced or eliminated without eliciting an apoptotic response.

Moss said that even if the RNAi approach does not entirely knock out a particular protein, “you could get a hypomorph phenotype. You may see a partial mutant phenotype, which may tell you something about the critical levels [at which] that protein [functions properly and does] what it’s supposed to do.”

Moss’ grant project runs between May 1, 2004 and April 30, 2006, and is worth up to $100,000.

A third grant awarded this month, with more direct clinical applications, went to Puthupparampil Scaria, vice president of synthetic vectors at Intradigm.

According to the grant’s abstract, the company has developed a “prototype trifunctional polymer that provides some of the multiple functions needed for an effective gene therapy vehicle. This polymer conjugate can condense nucleic acid into nanoparticles with a steric protective layer and targeting ligand layer on its surface.”

Scaria stated in the abstract that Intradigm has “obtained tumor growth inhibition and blood vessel growth inhibition using an siRNA against VEGFR2 delivered intravenously using the trifunctional polymer in a Nuero2A tumor model,” and that it intends to develop the technology as a treatment for colon cancer.

Intradigm CSO Martin Woodle recently told RNAi News that his company is strongly leaning toward cancer, specifically colorectal cancer, as the first disease area it expects to pursue in the clinic with its anti-VEGF drug candidate ICS-283 (see RNAi News, 5/07/2004).

The Scaria grant is a phase I SBIR grant worth roughly $100,000, and runs between May 12, 2004 and April 30, 2005.

— DM

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