By Doug Macron
Harvard University's Immune Disease Institute and the Scripps Research Institute pulled in big bucks from the National Institutes of Health last month, each securing grants worth more than $3 million to advance efforts using RNAi as a tool to solve questions related to immunity and learning.
At the same time, microRNA research continues to garner significant NIH support, with three projects related to the small non-coding RNAs receiving more than $1.3 million in combined funding last month.
RNAi continues to show promise as a therapeutic modality, and its utility as a research tool is well established. Aiming to expand that utility further is Judy Lieberman, a researcher at Harvard's Immune Disease Institute, who received a $3.4 million grant from the National Institute of Allergy and Infectious Diseases to use RNAi to study immunity in humanized mice.
"The immune response to infection or vaccination is the outcome of such a complex network of positive and negative regulatory pathways that it is often difficult to predict from in vitro experiments what will happen in vivo," Lieberman wrote in her grant's abstract.
Further complicating matters is the fact that immune responses in animal models such as rodents frequently differ from those in humans, and that analyses can be affected by the "genetic diversity and unique history of environmental/infectious exposure of each individual," the abstract notes.
At the same time, "experimental manipulations … that have been so powerful for dissecting the molecular basis of mouse immunology are rarely possible in humans," it adds.
However, the combination of RNAi and humanized mice offers the possibility of performing genetic manipulation studies of human immune responses in vivo, the abstract states. To capitalize on this potential, Lieberman and colleagues aim to develop a method to knock down individual genes in CD4+ and CD8+ cells in humanized mice transplanted with human bone marrow, fetal liver, and thymus — so-called BLT mice created by researchers at the University of Texas Southwestern Medical Center.
The BLT mice "reconstitute an immune system that mimics human immune cell distribution in uninfected mice, as well as viral dynamics and B and T cell responses to HIV infection" and will be used in combination with siRNAs to manipulate human immunity in vivo, the grant's abstract states.
With siRNA delivery remaining a key challenge to using RNAi in vivo, especially in lymphocytes, Lieberman's team has developed chimeric small RNAs composed of an aptamer that recognizes human CD4 and has been linked to an siRNA, the abstract states.
These chimeras are able to knock down genes specifically in human CD4+ cells in peripheral blood, human tissue explants, and in BLT mice after intravaginal administration. Using the funding from the NIH, the researchers plan to test and optimize them for systemic knockdown of gene expression in CD4 T cells, monocytes, and macrophages.
They also plan to engineer similar CD8 aptamer-siRNA chimeras for gene knockdown in CD8 T cells and natural killer lymphocytes, the abstract adds.
To test the approach for studying human immunity in vivo, they plan to develop CD4 aptamer-siRNAs designed to knock down the transcription factors FOXP3 and BCL6, which direct CD4 T cell differentiation into suppressive Treg cells and antibody-promoting TFH cells, respectively, the abstract states.
"We will use them to test the hypothesis that enhancing TFH cells and suppressing Treg cells augments the humoral response to two HIV candidate vaccines, a gp120 subunit vaccine and whole killed virus."
Lieberman's grant began on Sept. 30 and runs until the end of September 2013.
Also looking to harness RNAi is Scripps researcher Ronald Davis, who was awarded $3.3 million last month to use the technology to examine neurobiological processes underlying learning, short-term memory, and information retrieval.
"Flies carrying … RNAi transgenes for each individual Drosophila gene will be constructed and then trained and tested for odor learning," according to the grant's abstract. "This will identify the set of genes required for acquisition of information, memory stability over a short period, and/or the retrieval of that stored information."
Data from the project is expected to help uncover the molecular and cellular machinery that controls learning in the flies. "Given that Drosophila 'learning' genes are conserved both structurally and functionally, with some already implicated in human brain disorders, the knowledge gained from this project will also catalyze discoveries about the genetics of human neurological and psychiatric disorders and contribute to the development of drugs to treat impairments in cognition," the abstract notes.
Davis' grant began on Sept. 30 and runs until the end of August 2013.
Research projects related to miRNAs also received significant support from the NIH last month, including one headed by Ohio State University's Karl Obrietan studying expression of the ncRNAs in the mature nervous system.
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"Within the developing mammalian central nervous system, results from Dicer-null mice support a role for microRNAs in neuronal morphogenesis and neuronal survival," the grant's abstract states. "However, relatively little is known about how neuronal activity regulates microRNA expression patterns in the mature nervous system and, importantly, whether [miRNAs] regulate neuronal plasticity and cell viability."
To test the hypothesis that miRNAs play an important role in activity-dependent structural plasticity in the mature nervous system, Obrietan and his colleagues will use deep sequencing to examine activity-dependent expression of non-coding RNA in the hippocampus, as well as the "contribution of transcriptional networks that underlie activity-dependent neuronal plasticity and perform a series of experiments to identify functionally relevant microRNA targets," the abstract adds.
To determine the extent to which miRNAs affect adult neuronal structural plasticity and neuroprotection, the team created an inducible form of Cre-recombinase to disrupt Dicer expression and evaluate the effects of Dicer elimination on neuronal activity.
The investigators also will focus on a particular miRNA, miR-132, in activity-induced structural remodeling in vivo, the abstract notes.
Obrietan's grant, which is worth $396,596 this year, began on Sept. 15 and is set to run until Aug. 31, 2015.
Also at OSU, investigator Kay Huebner was awarded an NIH grant to examine miRNA profiles in triple-negative breast cancer. These tumors are negative for estrogen and progesterone receptors and HER2 and resemble cancers associated with BRCA1 mutations, her grant's abstract states.
"Defining the microRNA expression profiles of TN cancers will allow identification of altered signal pathways and specific target proteins that may lead to development of rationally targeted therapies," the abstract states.
Specifically, Huebner and her collaborators will use the miRNA-expression signatures to "define signal pathways regulated by [miRNAs] that are significantly up- and down-modulated in TN cancers and precursor lesions," she wrote in the abstract.
The grant is worth $548,311 this year. It began on Sept. 30 and will run until the end of July 2015.
At East Carolina University, researcher Alexander Murashov is aiming to use a recently awarded NIH grant to examine whether miRNAs play a role in the toxicity of nanomaterials.
"Recent observations have demonstrated that nanomaterials may be toxic to human tissue and cell cultures, resulting in oxidative stress, inflammatory cytokine production and cell death," but data also suggest that toxicity may extend to the nervous system, according to his grant's abstract. "A significant question remaining to be addressed is how nanoparticles trigger changes in the nerve cells, and what can be done to detect these defects" early.
Murashov and his colleagues plan to focus on carbon nanotubes, which have potential medical applications but may be limited by toxicity, he wrote in the abstract.
Hypothesizing that carbon nanotubes will dysregulate miRNAs in neuronal cells and negatively impact their function, he and his team will determine the effects of direct exposure of carbon nanotubes on neuronal cells in vitro, as well as indirect effects on peripheral neuron regeneration following respiratory carbon nanotube exposure in vivo.
The investigators will then characterize miRNA expression signatures in neuronal cells in response to the nanotube exposures, and see if miRNAs depletion will make neuron regeneration more vulnerable to carbon nanotubes by deleting Dicer in vitro and in vivo.
His grant began on Sept 24 and is slated to run until Aug. 31, 2012. It is worth $404,348 this year.