Skip to main content
Premium Trial:

Request an Annual Quote

New Research Shows siRNAs Suppress Blood, Lymphatic Vessel Growth Regardless of Sequence or Target

Premium

Non-targeted siRNAs suppress blood and lymphatic vessel growth in vivo as effectively as siRNAs directed against a key angiogenic growth factor as a result of immune response activation rather than an RNAi effect, researchers reported this week.

These data, scheduled to appear this week in the online version of the Proceedings of the National Academy of Sciences, suggest that siRNAs inhibit both hemangiogenesis and lymphangiogenesis regardless of their sequence or targets, raising concerns over the possibility of unintended effects with certain RNAi drugs, the investigators added.

"Collectively, our findings reveal a new facet of the unintended effects of siRNA," the team, led by the University of Kentucky's Jayakrishna Ambati, wrote. And while these effects "could be harnessed for therapeutic advantage in angiogenesis-driven diseases … [they] should be carefully monitored in ongoing clinical trials of [siRNA-based] drugs in non-angiogenic diseases."

Still, Ambati stressed that his research should not be interpreted as evidence that siRNA-based therapeutics won't be successful.

"There are workarounds" to overcoming TLR3 activation, the simplest being the use of duplexes shorter than 21 nucleotides, he told RNAi News this week. "It is not like [TLR3 activation] is some sort of impenetrable barrier."

The findings in PNAS build upon research Ambati and colleagues published about a year ago in Nature demonstrating that siRNAs that are 21 nucleotides in length or longer suppress neovascularization, regardless of their sequences or targets, by triggering the double-stranded RNA immune receptor toll-like receptor 3 (see RNAi News, 3/27/2008).

In that work, Ambati and colleagues tested various siRNAs and found that they all suppressed hemangiogenesis in mouse models of choroidal and dermal neovascularization by activating cell-surface TLR3 on blood endothelial cells, rather than because of an RNAi effect.

In that paper, the team examined two unformulated siRNAs — Opko Health's bevasiranib, which targets vascular endothelial growth factor, and Allergan's AGN745, which targets VEGF receptor-1 — that have already been tested in humans as treatments for wet age-related macular degeneration.

For the PNAS study, the investigators sought to "further explore this newly defined intersection between angiogenesis and innate immunity." Specifically, they set out to determine whether "the generic antihemangiogenic effects of siRNAs extended to other well-established and clinically relevant mouse models of neovascularization in response to corneal suture injury or hindlimb ischemia, and whether siRNA-mediated TLR3 activation also suppressed lymphangiogenesis," the wrote in the paper.

To do so, the team examined the effects of two 21-nucleotide-long siRNAs — one directed against VEGF-A and one against firefly luciferase — as well as a 7-nucleotide-long, luciferase-targeted siRNA that has been shown not to activate TLR3.

In the corneal suture-injury model, a single intracorneal injection of the siRNA targeting VEGF-A at the time of suture placement "dramatically suppressed both hemangiogenesis and lymphangiogenesis," Ambati and his team wrote. The 21-nucleotide-long siRNA targeting luciferase suppressed these effects just as effectively, while the 7-nucleotide-long siRNA had no effect.

"These data demonstrate a sequence-independent ability of siRNA to suppress corneal neovascularization that correlates with its ability to activate TLR3," they wrote.

Meanwhile, in the hindlimb ischemia model, the siRNAs were administered intramuscularly at the time of surgery and two days afterwards. Limbs injected with either of the 21-nucleotide-long siRNAs exhibited "suppressed revascularization and diminished perfusion," the team noted. In contrast, the 7-nucleotide-long siRNA had no effect on either hemangiogenesis or lymphangiogenesis.

Having shown in their Nature paper that naked, unmodified siRNAs do not enter mouse or human blood endothelial cells, the investigators studied whether the gene-silencing molecules were internalized by lymphatic endothelial cells. Using time-lapse confocal microscopy, they observed no uptake of naked fluorescein-tagged siRNAs targeting luciferase by mouse lymphatic endothelial cells.

Additional experiments revealed that TLR3 expression was "significantly more abundant" on the surfaces of mouse lymphatic endothelial cells and human dermal lymphatic endothelial cells than in the intracellular compartment, and that siRNAs at least 21 nucleotides long directly activate TLR3.

Based on these findings, the paper's authors determined that 21-nucleotide-long siRNAs are "sequence- and target-independent inhibitors of both blood and lymphatic vessels in multiple systems.

"Coupled with our earlier demonstration that 21-nt siRNAs inhibit hemangiogenesis in mouse models of choroidal and dermal neovascularization, as well the widespread expression of TLR3 on both vascular endothelial cells from various tissue beds, it is likely that these RNA duplexes are generic inhibitors of both components of neovascularization," Ambati and colleagues wrote in PNAS.

While these effects can be "serendipitous" — such as in the case of an siRNA-based drug for an angiogenesis-driver disorder such as AMD — "siRNAs may induce undesirable effects in the context of ischemic vascular disease or physiological cyclic angiogenesis," they cautioned.

"These concerns are particularly salient given ongoing clinical trials of systemically delivered siRNAs," the team added.

Upcoming Research

While Ambati's PNAS paper focused on unmodified siRNAs that are not internalized into a cell, he told RNAi News that he and his colleagues are preparing to publish new data showing that TLR3 activation remains an issue even for siRNAs delivered into the cell using vehicles such as liposomes or nanoparticles.

Based on their unpublished findings, the use of delivery vehicles "doesn't abolish the TLR3-mediated effects," he said. "Most of these kinds of cell-permeating moieties deliver the cargo into the endosomes … which are enriched with TLR3 and other toll-like receptors. So it shouldn't be surprising [that] when you deliver your cargo there, you encounter these immune sensors."

Ambati added that even if siRNAs are able to enter into RISC and trigger a bona fide RNAi effect, "you still retain the potential for intracellular TLR3 activation."

The Scan

Shape of Them All

According to BBC News, researchers have developed a protein structure database that includes much of the human proteome.

For Flu and More

The Wall Street Journal reports that several vaccine developers are working on mRNA-based vaccines for influenza.

To Boost Women

China's Ministry of Science and Technology aims to boost the number of female researchers through a new policy, reports the South China Morning Post.

Science Papers Describe Approach to Predict Chemotherapeutic Response, Role of Transcriptional Noise

In Science this week: neural network to predict chemotherapeutic response in cancer patients, and more.