By Doug Macron
The National Institutes of Health this month awarded more than $400,000 to fund two research projects investigating the role of microRNAs in vascular development and alpha-synuclein regulation.
The agency also earmarked nearly the same amount to support research into new approaches for siRNA delivery in February.
The first grant was awarded to University of Massachusetts Medical School's Stefania Nicoli, a researcher in the lab of Nathan Lawson, for work examining the miR-221/222 cluster in vascular development.
“The formation of new blood vessels is required for embryonic development, as well as the progression of numerous diseases,” she wrote in the grant's abstract. Model organisms with conserved processes of blood vessel formation and remodeling are a key tool for investigating the molecular mechanisms of vascular development. Among them, the zebrafish is an “ideal genetic system since it is transparent as an embryo, facilitating “direct visualization of endothelial cell behaviors in vivo during blood vessel development.”
Nicoli notes that new research has implicated miRNAs in proper blood vessel development, yet “little is known about the signaling pathways that miRNAs control during this process.” Deep sequencing methods have identified endothelial-expressed miRNAs, and initial functional characterization of these miRNAs suggests that they play “distinct and specific roles in vascular development.”
With funding from the NIH, she and her colleagues will focus on two endothelial miRNAs, miR-221 and miR-222, by generating zebrafish with targeted deletions in miRNA seed sequences, according to the grant's abstract. At the same time, they will create an “endothelial cell-specific transgenic line expressing myc-tagged Argonaute 2,” which will allow for the purification of miR-221 and miR-222 target complexes specifically from endothelial cells through Ago2 immunoprecipitation.
“The detailed phenotypic analysis of miRNA-knockout zebrafish, along with the ability to identify relevant endothelial-cell specific miRNA targets, will [allow the investigators to determine] pathways and cell behaviors miR-221 or miR-222 may be controlling,” she adds. Then, the findings will allow for the dissection of the genetic networks controlled by endothelial miRNAs during vascular development. “
Nicoli's grant runs from Feb. 4 until the end of January 2013, and is worth $87,172 in its first year.
Robert Wood Johnson Medical School's Eunsung Junn was awarded the second grant to continue his research into miRNAs and the regulation of alpha-synuclein, a protein implicated in Parkinson's disease and related disorders.
“Post-mortem investigations have demonstrated fibrillar [alpha-synuclein] aggregates in Lewy bodies and Lewy neurites in affected brain regions in these disorders,” with the protein's concentration in neurons being “critical,” he wrote in the grant's abstract.
Multiplication of the alpha-synuclein gene locus is associated with dominantly inherited Parkinson's disease with symptom onset correlating inversely to gene dosage, he noted, while transgenic animal models expressing wild-type human alpha-synuclein experience “phenotypic changes reminiscent of this disease.” Further, cultured cells become vulnerable to oxidative stresses by alpha-synuclein over-expression.
In 2009, Junn and colleagues reported that miR-7 represses alpha-synuclein expression by targeting the 3'-UTR of the protein's transcript, and that the miRNA could protect cells from oxidative stress.
The grant will support additional investigations into the function of miRNAs including miR-7 in the pathogenesis of Parkinson's disease as it relates to alpha-synuclein regulation using both cellular and mouse models. “We hypothesize that certain miRNAs repress [alpha-synuclein] expression in vivo and that dysfunction of specific miRNA species results in loss of this check mechanism in disease states, leading to increased [protein] expression and ultimately neurodegeneration,” the abstract states.
His grant began on Feb. 15 and runs until Jan. 31, 2012. It is worth $331,450 in 2011.
On the RNAi-delivery side, University of California, Santa Barbara's Joseph Zasadzinski secured NIH funding to advance his development of light-activated nanoparticles for siRNA delivery in cells and in C. elegans.
The approach uses hollow gold nanoshells, which are designed to “strongly absorb physiologically friendly [near-infrared] light and convert this light energy into local heating,” he wrote in the grant's abstract. The nanoshells can also be conjugated into siRNA and DNA by simple thiol chemistry, or linked to liposomes. Also, “water, proteins, [and] lipids ... do not absorb NIR light, so cells in culture are essentially transparent, which eliminates damage during exposure,” Zasadzinski noted in the grant abstract.
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Quick near-infrared light pulses cause the thiol-conjugated siRNA to be released from the nanoshells by breaking the thiol bonds without damaging the oligo payload, he noted, adding that the “well-known bottleneck of endosomal escape can be bypassed by converting the physiologically friendly ... light energy absorbed by the [nanoshells] to heat, creating unstable microbubbles that mechanically rupture endosomes and release siRNA to the cytosol within seconds.”
This enable the use of much lower concentrations of nanoshell-siRNA conjugates, “greatly increases transfection efficiency, and provides spatially and temporally controlled transfection that can be used to pattern cultured cells and address specific structures within C. elegans,” according to the abstract.
With the support of the NIH, Zasadzinski aims to use the hollow gold nanoshell platform to develop “masking and unmasking techniques for activating or inactivating biological processes with remote control to devise simple and scalable methods of lithographic patterning of cultured cells in real time.”
He and his colleagues also plan to develop methods to multiplex the release from a single nanoshell using a combination of thiol and dithiol anchors that “desorb at different energies to release multiple chemical species,” while new silver nanoshells and gold and silver nanorods will be made to “probe other regions of the NIR spectrum and provide simultaneous delivery and imaging opportunities,” the abstract states.
Zasadzinski's grant, which is worth $328,622 in its first year, runs from Feb. 15 until the end of November 2014.
The final grant went to the Massachusetts Institute of Technology's Daniel Siegwart, a postdoc in the lab of Alnylam Pharmaceuticals collaborator Robert Langer. The project aims to explore siRNA delivery with structured polymers made using combinatorial atom transfer radical and reversible addition-fragmentation chain transfer polymerization.
Applying these techniques, Siegwart and his colleagues will try to assemble a library of more than 1,500 structurally distinct core-shell hairy nanoparticles for siRNA delivery, according to the grant's abstact. “The polymers will be synthesized using controlled/living radical polymerization techniques to enable control over architecture, molecular weight, polydispersity, and physical properties,” it notes.
Because delivery hurdles continue to stymie the development of therapeutic siRNAs, the reseachers will also develop a library of core-shell hairy nanoparticles 10 to 80 nanometers in diameter with a cationic core and a tunable and functionalizable shell for siRNA delivery, Siegwart adds in the abstract. “High-throughput synthesis, purification, measurement of [molecualr weight] and particle diameter, complexation with siRNA, and in vitro screening for delivery of siRNA will be performed in series,” the abstract adds. “Through analysis, we anticipate that structural motifs that are important for complexation and delivery will emerge.”
The grant runs from Feb. 1 until the end of Jan. 2013, and is worth $50,474 this year.
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