By Doug Macron
Researchers from Santaris Pharma this week published new data that further supports using the company's so-called tiny locked nucleic acid technology to inhibit entire families of microRNAs.
LNAs are nucleic acid analogs in which the ribose ring is locked by a methylene bridge connecting the 2'-O atom with the 4'-O atom. Santaris' lead therapeutic LNA miravirsen, which inhibits miR-122, is 15 nucleotides long. By comparison, tiny LNAs are around 8 nucleotides long and perfectly complementary to an miRNA's seed region.
"Using tiny LNA-based compounds to successfully inhibit entire disease-associated microRNA families provides a new range of opportunities to develop novel microRNA-targeted drugs for both in-house drug discovery programs, as well as with our partners," Santaris CSO Henrik Orum said in a statement.
The latest research, which appears in Nature Genetics, was conducted in collaboration with investigators from Cold Spring Harbor Laboratory. It also comes five months after a Santaris researcher presented data at the sixth annual Oligonucleotide Therapeutics Society meeting showing how the tiny LNAs work both in vitro and in vivo (GSN 10/28/2010).
That presentation “discussed the therapeutic prospect of being able to target entire families of miRNAs with a single oligo and showed early data that suggested that LNA had the attributes to make it work,” Orum told Gene Silencing News this week. “The data presented in the paper in Nature Genetics comprehensively documents that this is indeed the case, thus expanding our pharmacological reach in the miRNA therapeutic space.”
According to the paper, the Santaris and Cold Spring Harbor Lab team first tested the tiny LNAs in culture. They found that transfecting tiny LNAs complementary to nucleotides 2 through 9 in the mature sequence of miR-21 triggered “potent antagonism” of the microRNA in HeLa cells. Tiny LNAs that bind to sequences adjacent to the seed, meanwhile, had no effect on the miRNA.
To see if the oligos could knock down entire miRNA families, the investigators designed a 7-mer complementary to the seed region of miR-221/222 and constructed two reporters with perfect-match target sites for the miRNAs.
“Both reporters were effectively repressed in PC3 cells in accordance with the high expression of miR-221 and miR-222 in this cancer cell line,” they wrote in the paper. Co-transfection of the tiny LNAs into the cells, however, led to “concentration-dependent de-repression of both reporters when assayed either individually or in combination, implying that this tiny LNA is able to inhibit both members of the miR-221/222 family.
“Similarly, transfection of tiny LNAs targeting the seed region of the miR-17, miR-18, and miR-19 families resulted in de-repression of their respective luciferase reporters in HeLa cells,” they added.
Looking in vivo, the researchers intravenously delivered 10 mg/kg doses of miR-21-targeting LNAs over three consecutive days to breast cancer mouse models expressing a luciferase reporter for the miRNA.
Imaging showed a “significant de-repression” of the luciferase reporter in mice treated with the tiny LNAs, indicating that the oligos had been delivered successfully to the tumors. Although there was no effect on tumor development, the results show that the miR-21 inhibitors “can be delivered to solid tumors, suggesting that tiny LNAs targeting cancer-associated miRNAs could be useful for the development of new therapeutic strategies,” according to the paper.
Meanwhile, transcriptional and proteomic profiling indicated that tiny LNAs have “negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny complementary sites,” the researchers wrote.
“Given the emerging roles of miRNA seed families in disease, such as the let-7 family in lung cancer, the miR-19 family in Myc-driven B-cell lymphomas, and the miR-208 family in cardiac remodeling, we envision tiny LNAs as important tools to study the functional overlap within miRNA seed families in animal disease models,” they concluded. “Finally, our data demonstrate in vivo efficacy and the lack of acute toxicities in mice, which imply that tiny LNAs may be well suited to the development of therapeutic strategies aimed at pharmacological inhibition of disease-associated miRNAs.”
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