By Doug Macron
Just months after US President Barack Obama signed into law the $787 billion American Recovery and Reinvestment Act, which included $10 billion in funding to the National Institutes of Health (see GenomeWeb News, 2/17/2009), the agency has awarded almost $6 million in new grants to support RNAi- and microRNA-related research projects during 2009.
Of that total, more than $2 million has been handed out to finance therapeutic RNAi research, including the development of new delivery approaches and treatments for HIV-1 and amyotrophic lateral sclerosis.
Among these is a two-year grant given to University of Utah researcher Darin Furgeson to develop peptide-based siRNA carriers for targeted delivery across the blood-brain barrier.
“This research will significantly advance siRNA carrier design and specifically target the central nervous system with contemporary, genetically engineered siRNA vehicles,” according to the grant’s abstract. “Compulsory attributes for any siRNA delivery vehicle are: an efficient and protective condensation of the siRNA; explicit tissue- or cell-specific targeting; and a means of disassembly at said site in sufficient concentrations to elicit the desired pharmacological effect.
“Contemporary recombinant techniques will combine the optimal cationic oligo sequence with a distinct selection of CNS-targeting peptides that will transcytose across the BBB for siRNA access to the brain parenchyma,” it adds. “In addition to rigorous physicochemical characterization, we will evaluate siRNA transfection efficiency with brain-derived primary cells, [namely] astrocytes, to mimic the cerebral compartment. Finally, transcytosis capability across a two-layer transwell in vitro model of the BBB to elucidate CNS-targeting potential will be tested.”
This project is set to run from July 27, 2009, to June 30, 2011, and is worth $176,510 in 2009.
Also taking advantage of the new source of NIH funding is Duke University’s Bruce Sullenger, who was awarded a two-year grant to continue his development of an aptamer-based siRNA delivery approach.
With the funding, Sullenger and colleagues will “explore the ability of aptamers to activate cell-surface receptors,” according to the grant’s abstract. In unpublished studies, the researchers have shown that “dimeric versions of aptamers that we have made against two different receptors on T-cells, 4-1BB and OX40, are able to activate these receptors on primary murine T-cells resulting in T-cell proliferation, cytokine release, and enhanced anti-tumor vaccine activity in murine tumor immunotherapy models.”
Sullenger aims to evaluate the activity of these two aptamers, as well as determine whether a recently made third aptamer against stem cell factor receptor, c-Kit, can also “act as an agonist and activate stem cell factor receptor and erythropoiesis,” it adds. “Analysis of these aptamers will include assessing their ability to deliver siRNAs to primary T-cells and T-cell lymphoma and leukemia cell lines, [as well as] erythrocyte precursors and c-Kit positive tumor cells.”
The grant, which is worth $258,960 in 2009, will run between May 24, 2009, and March 30, 2011.
Paul Knight of the State University of New York at Buffalo also nabbed a two-year grant under the American Recovery and Reinvestment Act to support the creation of siRNA-conjugated nanoplexes for pulmonary delivery, primarily as an influenza therapeutic.
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“The primary objective … is to construct an electrostatic complex between a cationic nanoparticle, gold nanorods, and anionic genetic material, [such as] cDNA or siRNA,” according to the grant’s abstract. “These nanoplexes will be engineered such that they can be taken-up and express bioactivity in large airway epithelial cells with little or no untoward cellular or pulmonary responses.”
The siRNA/cDNA constructs are designed to both suppress the translation of the influenza virulence factor NS1 and independently stimulate type I interferon production through activation of the RIG-I pathway as a result of a triphosphate moiety attached to the 5’ end of the siRNA, the abstract notes.
Knight and colleagues also aim to test the prophylactic and therapeutic efficacy of the siRNA/cDNA nanoplex in large airway epithelial cells in vivo, with the goal of filing an investigational new drug application to begin human trials after the completion of the grant work.
The project is worth $385,377 in 2009, and runs from July 22, 2009, until June 30, 2011.
University of Washington investigator Gao Xiaohu received a two-year grant to develop nanocarriers for siRNA delivery and real-time imaging.
“Motivated by our recent exciting preliminary results on nanoparticle-amphipol complexes for siRNA delivery, we will engineer both quantum dots and magnetic nanoparticles, but at two states,” Xiaohu states in the grant’s abstract. “We will first use fluorescent quantum dots as a discovery tool to follow the entire process of siRNA-nanoparticle interaction in cells and small animals to gain fundamental understanding of siRNA transport.
“Toward the end of this project, we will also engineer iron oxide magnetic nanoparticles based on the design principles learned from quantum dots, because iron oxides are of very low toxicity and because iron oxide can be made with nearly identical size and surface properties as quantum dots,” he adds. “If successful, the proposed work will provide novel and fundamental insights into siRNA behavior, and aid in development of siRNA carriers for specific treatment of cancer.”
Xiaohu’s grant runs from Aug. 12, 2009, until July 31, 2011, and is worth $323,700 in 2009.
At the Burnham Institute for Medical Research, University of Massachusetts Medical School researcher Tariq Rana will use the funding from a two-year grant to develop an RNAi-based treatment for ALS.
“The effectiveness of RNAi in slowing down the ALS progression has been demonstrated in vivo using transgene delivered RNAi,” the grant’s abstract states. “However, the transgene-based delivery has problems in clinical application. … We focus on developing siRNA as small molecule drugs.”
Specifically, Rana and colleagues will develop and test “chemically modified, stabilized, cell-permeable, and silencing-competent siRNA in treatment of ALS animal models that express mutant Cu, Zn superoxide dismutase,” the abstract notes. “Findings of proposed research will also offer a new perspective important in developing RNAi-based therapies for other neurodegenerative diseases.”
Rana’s grant runs from Aug. 5, 2009, until the end of July, 2011, and is worth $527,212 in 2009.
To fund his ongoing NIH grant focused on developing an siRNA-based treatment for HIV-1, University of California, Los Angeles, researcher Irvin Chen received $385,000 in additional funding this year under the American Recovery and Reinvestment Act.
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According to his grant’s abstract, he has already developed shRNAs that down-regulate CCR5 in primary T-cells in vitro, protecting them from HIV-1 infection. The project, which began on March 1, 2003, and is set to run until the end of April, 2011, will advance these findings.
Basic Research, Too
In addition to the stimulus money awarded to RNA-related projects with clinical potential, the NIH awarded a number of grants to researchers exploring the basic science of microRNAs, including their potential role in a variety of different diseases, as well as one grant examining the stability of RNAi molecules in human lymphocytes.
Below is an overview of these grants:
• Peter Sarnow at Stanford University received a grant, worth $295,812 in 2009, to advance findings that hepatitis C depends on the liver-specific miR-122 and the host protein RCK for virus production, and determine whether this dependence “presents an Achilles heel that can be used for anti-viral intervention. The grant runs from July 17, 2009, until June 30, 2011.
• Hui Zhang at Thomas Jefferson University received a grant, worth $386,250 in 2009, to study the mechanisms by which miRNAs affect HIV-1 growth since miRNAs have been shown to “directly bind to HIV-1 RNA, inhibit or enhance the production of viral proteins, and … manipulate HIV-1 replication” in many cells targeted by the virus. The project runs from May 22, 2009, until April 30, 2011.
• Thomas Tuschl of Rockefeller University was awarded $11,760 in additional funding this year to support an ongoing grant project studying miRNA gene-regulatory networks and their role in nervous system diseases such as schizophrenia, depression, and autism. The grant began on July 4, 2007, and runs until March 31, 2012.
• Robert Thompson of the University of Michigan at Ann Arbor received a two-year grant to analyze miRNA expression patterns in human post-mortem brain samples taken from normal patients and those with various psychiatric disorders. The grant runs from Aug. 11, 2009, until July 31, 2011, and is worth $270,375 in 2009.
• Creighton University’s Garrett Soukup received a two-year grant to examine the roles of miRNAs in normal mammalian ear development and sensory cell function. The grant is worth $353,846 in 2009, and runs from June 2, 2009, until May 31, 2011.
• Steven Singer at Georgetown University received a one-year grant to study miRNAs in the pathogenesis of giardia, the most common protozoan cause of diarrhea and a category B biodefense agent. The grant runs from July 14, 2009, until Sept. 30, 2010, and is worth $30,700 this year.
• Ohio State University’s Daniel Schoenberg received a two-year grant to study the inactivation of miR-122 using a catalytic, sequence-specific ribozyme as a means of combating hepatitis C infection. The project runs from June 1, 2009, until May 31, 2011, and is worth $225,000 in 2009.
• Mark Hahn from Woods Hole Oceanographic Institution received a two-year grant to establish a zebrafish embryo model for studying the roles of miRNAs in developmental toxicity and teratogenicity. The grant is worth $170,604 this year, and runs from July 16, 2009, until May 31, 2011.
• Jasmine Perez of the Mount Sinai School of Medicine received a two-year grant to study the role of hematopoietic-specific miRNAs in determining influenza virus tropism using miRNA-mediated attenuation technology, with an eye towards developing a flu vaccine. The grant runs from Aug. 13, 2009, until Aug. 12, 2011, and is worth $34,397 in 2009.
• Massachusetts General Hospital’s Anders Naar received a two-year grant to investigate the role of miR-33 in controlling cholesterol/lipid homeostatis in combination with sterol response element binding proteins as part of a broader effort to find novel therapeutic strategies to increase high-density lipoprotein clearance in patients with cardiovascular disease. The project runs from Aug. 5, 2009, until July 30, 2011, and is worth $221,042 in 2009.
• Boston University’s Jining Lu was awarded a two-year grant to study miRNA-mediated gene regulation in lung development. The project began on July 1, 2009, and runs to June 30, 2011. It is worth $246,322 in 2009.
• University of Texas Southwestern Medical Center researcher Qingha Liu received a one-year grant to study the specificity and mechanism of miRNA biogenesis in Drosophila, and to develop a reconstitution assay to explore miRNA-induced silencing complex assembly and its components. The grant runs from Aug. 3, 2009, until July 31, 2010, and is worth $80,000 in 2009.
• George Calin at the University of Texas MD Anderson Cancer Center received a two-year grant to study miRNAs and other non-coding RNAs in colorectal metastasis. The project runs from July 17, 2009, until June 31, 2011, and is worth $335,032 in 2009.
• George Washington University’s Fatah Kashanchi received a two-year grant to study the role of miRNAs derived from the transactivation response element in HIV-1 replication. The grant runs from May 15, 2009, until April 30, 2011, and is worth $234,000 in 2009.
• Carolyn Klinge from the University of Louisville received an additional $16,631 under the stimulus funding to support her ongoing grant project investigating the regulation of miRNA expression in breast cancer cells. The project started on June 1, 2007, and is set to run until Oct. 31, 2009.
• Mingyu Liang of the Medical College of Wisconsin received a two-year grant to discover renal miRNAs that are relevant to the Dahl salt-sensitive rat model of hypertension and renal failure and identify their targets. The project runs from July 1, 2009, until June 30, 2011, and is worth $228,000 in 2009.
• Mingyu Liang also received a separate two-year grant to develop a high-throughput 3’-UTR assay to examine 98 miRNA-target interactions previously identified in kidney regions and a cell model of epithelial mesenchymal transition, as well as more than 1,000 predicted interactions. The project runs from Aug. 13, 2009, until July 31, 2011, and is worth $228,000 in 2009.
• Oklahoma State University, Stillwater, researcher Lin Liu received a two-year grant to conduct miRNA-expression profiling in idiopathic pulmonary fibrosis samples. The project runs from May 11, 2009, until April 30, 2011, and is worth $73,850 in 2009.
• Lin Liu also received a separate two-year grant to study miRNAs in lung samples taken from normal and bronchopulmonary dysplasia patients, with the goal of identifying miRNAs that are dysregulated in the disease. The project runs from June 1, 2009, until May 31, 2011, and is worth $221,500 in 2009.
• University of California, Los Angeles, researcher Dong Sung An received a two-year grant to examine the stability and toxicity of shRNAs in lymphocytes. The grant runs from Aug. 1, 2009, until July 31, 2011, and is worth $235,888 in 2009.