Category: Preclinical Development
Purpose: Short interfering RNA (siRNA) are potent nucleic acid-based drugs that interfere with disease driving genes that may otherwise be undruggable. The potential of administering therapeutic siRNA in vivo is limited due to poor pharmacokinetic properties, including rapid renal clearance and nuclease degradation. siRNA-nanocarriers and -conjugates have been explored as means to improve these properties; however, these methods often involve complex synthesis, lack of specificity, and toxicity issues. In this work, we investigate the use of aptamer-siRNA chimeras to improve these limitations. Aptamers are short single-stranded DNA or RNA molecules that can be selected to have high affinity and specificity for biological targets using SELEX (Systematic Evolution of Ligands through Exponential Enrichment). We sought to identify new aptamers that target endogenous albumin, the most abundant serum protein that has an extraordinary circulation half-life of 19 days. Fusion of siRNAs with these aptamers form complexes, termed chimeras, which confer siRNA with the ability to bind to albumin, thereby improving their circulation times and bioavailability for therapeutic delivery.
Methods: Five rounds of SELEX were completed to identify high affinity albumin-binding aptamers; the SELEX procedure utilized 2’-fluoro modified pyrimidine bases to confer superior serum stability over unmodified DNA and RNA counterparts. Chimeras were synthesized by transcribing the 40 nt RNA aptamer with the antisense strand of the siRNA separated by a two uracil base linker. The sense strand is transcribed separately and the two complementary strands are annealed, allowing for cellular Dicer/RISC-complex recognition (Figure 1A). Knockdown potency of firefly luciferase-interfering chimeras was confirmed in vitro following 24 hour incubation with luciferase-expressing cells, as measured with IVIS. Following tail vein injection, half-lives of chimeras were measured in mice using fluorescent intravital microscopy and confirmed in a peptide nucleic acid-based hybridization assay. All animal protocols were approved by the Vanderbilt University IACUC.
Results: Two albumin-binding aptamers were identified, indicated as Clone1 and Clone3 chimeras. The affinity of Clone1 chimera for human serum albumin was measured at 46 nM, exhibiting strong binding. Aptamer-siRNA chimeras were found to retain silencing potency in vitro in MDA-MB-231 luciferase-expressing cells. Both chimeras showed similar luciferase knockdown to siRNA alone (~60%), compared to a scramble chimera (Figure 1B). Albumin-binding chimeras were systemically injected into CD-1 mice and were found to have significantly improved circulation half-lives compared to controls (~3-fold). Clone1 and Clone3 chimeras exhibited half-lives of 26.9 min and 23.2 min, respectively, compared to 9.0 min for the scramble chimera and 8.7 min for siRNA alone (Figure 1C).
Conclusion: Fusing therapeutic siRNAs with albumin-binding aptamers enhances their pharmacokinetic properties and circulation half-life in vivo, without compromising knockdown efficiency. The small size of these chimeras and improved bioavailability suggest potential for enhanced knockdown of in vivo genes. The modular design of these aptamer-siRNA chimeras allows for broad applications of gene targeted therapeutics.