As the year comes to a close, a handful of new RNAi-related grants from the National Institute of Health and the National Science Foundation have been issued. Among them is one supporting continued research into the genetics of systemic RNAi, one funding the elucidation of one of the many cellular roles played by the RNAi machinery, and one seeking to advance the use of RNAi to treat hematologic diseases.
The first grant was awarded to Harvard University’s Craig Hunter by the NSF in support of his research of systemic RNAi-defective (sid) genes. The effort is expected to yield insights into how intracellular RNA transport is accomplished and regulated in Caenorhabditis elegans, which may reveal why the activity is important and how it may be accomplished in mammals.
According to the grant’s abstract, Hunter performed a forward-genetics screen in C. elegans to identify genes required for systemic RNAi. An initial analysis revealed sid mutations in five genes, two of which, sid-1 and sid-2, have been cloned and characterized.
“Sid-1 encodes an apparent channel that allows passive transport of dsRNA into cells,” the abstract states. “Sid-2 encodes an intestine-limited transmembrane protein required for environmental RNAi. Functional analysis of the protein product of sid-2 indicates that it may be the receptor for uptake of dsRNA from the environment,” the abstract notes.
The first stage of the grant project is to complete the analysis of sid-3, sid-4, and sid-5. Then, Hunter plans to repeat the search for systemic RNAi mutants, but to “broaden the selective criteria to identify additional genes that may have essential roles for viability or fertility, or that have tissue-specific roles in RNAi,” the abstract states. “The broader impacts resulting from these activities include … the potential applications that may unfold from a deep understanding of how dsRNAs are trafficked between cells and tissues.”
Hunter’s grant, which is worth $171,505, runs from Jan. 1, 2005 through Dec. 31, 2005.
The second grant, awarded by the National Institute of Child Health and Human Development to William Theurkauf of the University of Massachusetts Medical School, supports a project to examine the molecular mechanisms of mRNA localization and translational silencing essential to embryonic axis specification.
The abstract for the project notes that although mRNA localization and local translation are key elements in a range of processes in plants and animals, the mechanisms driving mRNA transport and silencing in higher eukaryotes are poorly understood.
According to the abstract, axis specification in Drosophila provides a powerful example of coordinated mRNA transport and translational silencing: “Bicoid and oskar mRNA are synthesized in a cluster of nurse cells, assembled into transport particles, and moved through cytoplasmic bridges to the oocyte. Within the oocyte, microtubules are required for bicoid mRNA localization to the anterior pole and oskar mRNA accumulation at the posterior pole,” the abstract states. “Oskar mRNA is silent until mid-oogenesis, when it is localized to the posterior pole of the oocyte, [and] bicoid mRNA remains translationally silent until early embryogenesis.”
In the abstract, Theurkauf notes that he and his colleagues have developed an in vivo assay for bicoid mRNA localization that reveals multiple steps in the anterior mRNA localization pathway, as well as an in vitro mRNA transport-particle assembly system that recapitulates RNA behavior in vivo. Through the grant, the assays will be used to define RNA sequences and trans-acting proteins that drive anterior localization of bicoid mRNA.
Theurkauf and colleagues also have identified a new axis-specification gene, called armitage, that is essential for oskar mRNA translational silencing, and is required for RNAi and efficient assembly of RISC, the abstract states. Mutations in three other RNAi components also disrupt oskar mRNA silencing and axis specification, thus “the RNAi machinery … appears to be essential to embryonic axis specification,” it adds.
The grant project will look to shed light on the function of armitage in RNAi and RISC assembly, the role of RNAi in axis specification, and the possible role for the ATR/Chk2 tumor-suppressor pathway in regulating RNAi.
Theurkauf’s grant project is set to run between Dec. 1, 2004 and Nov. 30, 2009. Details about the value of the grant were not available.
The final grant, worth $362,250 a year for four years, was awarded by the National Heart, Lung, and Blood Institute to Columbia University’s Arthur Bank, who is working to develop novel oncoretroviral vectors that can be used to transfer and express human genes in hematopoietic stem cells, as well as to develop new lentiviral packaging systems.
“Currently, in lentiviral gene transfer, transient systems are used, involving adding several plasmids to human cells in culture for limited times because of the toxicity of the VSV-G envelope,” the grant’s abstract states. “We intend to develop stable lentiviral vector systems comparable to our best oncoretroviral systems in which the required genes are stably integrated into the genome of the cell lines utilized. Supernatants from these stable lines are more easily tested for safety and can generate retroviral supernatants more efficiently and reproducibly in larger amounts than transient supernatants for use in human clinical trials.”
One construct Bank and his colleagues plan to use in both oncoretroviral and lentiviral systems, the abstract notes, is an siRNA directed against human sickle beta-globin gene mRNA, which is expected to decrease beta-s globin protein production.
“In addition, [the researchers] will deliver a normal human beta-globin gene in these systems,” the abstract adds. “The ultimate goal of these experiments is to provide safe and novel systems to deliver and express therapeutic genes in human HSC and to cure or ameliorate hematologic diseases such as sickle cell disease and beta-thalassemia. The gene delivery technology should also be applicable for use in treating other human diseases, as well.”
This project is set to run from Dec. 7, 2004 through Nov. 30, 2008.