Background/Purpose Aptamers are RNA or DNA oligonucleotides selected for their capacity to specifically bind and inhibit the function of a target protein.  The effect is similar to neutralizing antibodies that defend the body against pathogenic antigens.  These potentially therapeutic oligonucleotides often have short half-lives, however, because they are rapidly degraded by nucleases in peripheral blood and excreted by the kidneys.  Here we describe an RNA aptamer against IL-17A that resists degradation in serum and decreases the severity of collagen-induced arthritis in DBA/1 mice.

Methods In vitro assay of serum stability.  After substituting a methoxy group for the 2′ hydroxyl group of ribose, we incubated the RNA aptamers in mouse serum for 0.5 to 72 hours at 37°C.  Controls were incubated in phosphate buffered saline.  Next, samples were added to quenching buffer (8 M urea, 10 mM EDTA, 0.05% bromophenol blue).  Then, the RNA fragments were electrophoresed in 20% polyacrylamide gels that contained 8 M urea in TBE buffer.  Bands were visualized by staining the gel with SYBR Green II.  Each fraction of intact aptamer was normalized to its corresponding control.  Pharmacokinetic studies in mice.  Aptamers were PEGylated with 40-kDa polyethylene glycol and conjugated with 3′-inverted deoxythymidine.  Then, these aptamers were infused as a single bolus into the tail vein of a C57BL/6J mouse at 1 mg/kg.  Aptamer concentrations in plasma were measured by enzyme-linked oligosorbent assay.  Collagen-induced arthritis in mice. Mice were immunized with bovine type II collagen in Freund’s complete adjuvant on day 1.  On day 22, they were boosted with bovine type II collagen in Freund’s incomplete adjuvant.  PEGylated aptamers were injected intraperitoneally at 5 mg/kg once a day from day 22 to day 37.  Objective signs of arthritis for each paw were scored using a scale of 0 to 4.

Results In vitro assay of aptamer 17M-340 for serum stability showed very little degradation after 72 hours incubation.  By contrast, prototype aptamer Apt21-2 or 17M-4 were rapidly degraded within 24 hours.  In vivo, the plasma half-life of 17M-340 was 9.3 hours, more than ten-fold that of 17M-4.  Finally, intraperitoneal injection of 17M-340 but not 17M-4 reduced the objective score of collagen-induced arthritis significantly (p ≤ 0.05). 

Conclusion The anti-IL-17A aptamer is 31-base oligoribonucleotide that forms a stem-loop structure.  Substitution of a methoxy group for the 2′ hydroxyl group of ribose at three positions in the stem and one position in the loop markedly increased stability of the aptamer in vivo.  Our findings raise the possibility that RNA aptamers such as 17M-340 may be effective treatments for patients with chronic inflammatory diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/improvement-of-the-stability-of-rna-aptamers-against-interleukin-17a/