The Intracellular Effects of Anti-Polymerizing RNA Aptamers in Sickled Red Blood Cells

Sickle cell disease (SCD) is the most common genetic disorder, affecting more than 20 million people worldwide. While current FDA-approved therapies mitigate SCD severity and symptoms, their side effects, long-term complications, cost, and biocompatibility remain a concern. Thus, there is a need for more cost-effective and highly targeted SCD therapeutics. Under deoxygenated conditions, hemoglobin monomers “stick” together forming elongated polymers, giving rise to the characteristic elongated, sickled red blood cells (RBCs) morphology. Polymerization of sickle hemoglobin is the root cause of SCD. We previously developed and characterized two hemoglobin-S specific RNA aptamers, DE3A and OX3B, that effectively inhibit the polymerization of sickle hemoglobin under deoxygenated conditions (Purvis et al. 2017). RNA aptamers are short oligonucleotides that bind to their targeted protein with high affinity and specificity by assembling into unique tertiary structures. Both DE3A and OX3B decrease the slope of polymerization and increase the delay time for polymer formation, resulting in decreased polymers. Thus, DE3A and OX3B hold promise as highly targeted anti-polymerizing SCD therapeutics; however, their intracellular effects are still under investigation. Here, we assessed the intracellular effects of the anti-polymerizing aptamers, particularly, DE3A. This is pertinent to developing an efficient and highly targeted SCD therapeutic. Initially, we designed a chimeric RNA aptamer molecule (Tfr-DE3At), containing a previously characterized transferrin receptor aptamer and the truncated form of DE3A, DE3At. DE3At contains DE3A's functional domain that targets and binds to hemoglobin-S. DE3At binds to hemoglobin S with the same affinity and inhibits polymerization of sickle hemoglobin similar to DE3A. The rationale for targeting the transferrin receptor for the delivery of DE3At is because of the increased expression of the transferrin receptors on reticulocytes and mature sickled RBCs. To investigate Tfr-DE3At's ability to bind to human transferrin receptors on mammalian cells, we labeled both the transferrin receptor and Tfr-DE3At with fluorophores and assessed binding via confocal microscopy. Our data show that Tfr-DE3At binds to the transferrin receptor on the surface of both HeLa and RBCs. We also detected intracellular expression of Tfr-DE3At in both cells. Additionally, we detected the Tfr-DE3At aptamer in reticulocytes. These data suggest that the transferrin receptor aptamer can serve as a viable molecule for the internal transport of Tfr-DE3At across cellular membranes. Interestingly, we also showed that DE3At (in the absence of the Tfr aptamer) is internalized in HeLa cells, implying that DE3At can pass through the cellular membrane of HeLa cells. Together our data demonstrate that DE3At and Tfr-DE3At are intracellularly internalized in HeLa cells, reticulocytes, and sickle red cells. Using a novel microfluidic assay, we assessed DE3At and Tfr-DE3At's ability to decrease RBCs' sickling. Both aptamers reduced the sickling of mature sickled RBCs. We also observed a delay in sickling time in mature sickled RBCs in the presence of DE3At and Tfr-DE3At. These data imply that Tfr-DE3At and DE3At reduce RBCs sickling by potentially delaying polymer formation. These data also suggest that DE3At can effectively transport across the red cell membrane. The mechanism by which this occurs remains under investigation. In conclusion, we show that the DE3At aptamer is a novel, highly targeted SCD therapeutic by targeting the root cause of SCD. Also, we demonstrated the targetability of this therapeutic through a transferrin receptor-mediated transport mechanism. Finally, we show that our truncated anti-polymerizing aptamer can pass through cellular membranes. Further studies are ongoing to investigate the mechanism of DE3At transport, the location of internalization, and its specific mechanism of action.