2022 Annual Meeting

PNA - Peptide Conjugate for Increased siRNA Bioavailability

Small interfering RNA (siRNA) therapies have the potential to treat a wide range of brain diseases due to their selective and efficacious gene silencing ability. However, clinical translation of siRNA therapies is limited by poor pharmacokinetic properties and an inability to permeate the stringent blood-brain barrier (BBB). A common strategy to potentiate siRNA delivery to the brain involves packaging siRNA in nanoparticle structures expressing Angiopep-2 (ANG2) peptides. ANG2 facilitates transcytosis by binding LRP1 expressing brain endothelial cells. However, systemic toxicity and large size diminish transport efficiency. Here, we complement that work by developing a direct siRNA-ANG2 conjugate, where the siRNA contains an overhang, through which a complementary peptide nucleic acid (PNA)-ANG2 construct is annealed. Extensive siRNA chemical modifications confer stability in blood circulation, enabling the ANG2 motifs to promote LRP1-mediated transcytosis at the brain endothelium.

To evaluate the stability of ANG2-PNA conjugated siRNA (si-ANG2), the conjugate was exposed to 50% serum for 72 hours and its degradation was assessed with a non-denaturing polyacrylamide gel. A full phosphorothioate siRNA overhang prevented siRNA degradation and peptide dissociation. Further, the endocytotic capabilities of si-ANG2 were accessed in LRP1-expressing neuroblastoma cells. The cells were incubated with 100nM of si-ANG2 for 2 hours, and cellular uptake of CY5 labeled si-ANG2 was evaluated with flow cytometry. si-ANG2 demonstrated 3-fold greater internalization compared to the unconjugated siRNA. Moreover, to interrogate the hypothesis that si-ANG2 is endocytosed via the proposed LRP1 mediated pathway, the cellular uptake in neuroblastoma cells was measured at 37 ℃ and 4 ℃. At 4 ℃ (when endocytosis is inhibited) there was no significant accumulation of si-ANG2. Thus, si-ANG2 enters the cells via energy-dependent endocytosis, as opposed to passive diffusion.

In sum, we have characterized the serum stability and in vitro properties of a siRNA-peptide conjugate platform for enhancing siRNA bioavailability in the brain. si-ANG2 is stable in serum, opening possibilities for a drug conjugate that can circulate in vivo without degradation. Further, the addition of the ANG2 peptide improves the endocytotic properties of siRNA by facilitating interactions with membrane proteins. However, additional studies to validate the LRP1-mediated endocytosis hypothesis are crucial to understanding the cellular trafficking of the conjugate. Additionally, a mouse model will be used to evaluate brain targeting and pharmacokinetic properties of si-ANG2 delivered systemically. Altogether, a PNA – peptide siRNA conjugate opens the possibility for a modular targeting siRNA platform that could be functionalized for various cell targets.