Albumin-Binding Lipid siRNA Conjugates for Accumulation in the Blood Brain Barrier Vasculature

Introduction:: Small interfering RNA (siRNA) therapies have the potential to treat a wide range of brain diseases due to their ability to selectively silence pathological gene expression. Further, siRNAs can be readily designed to act on targets undruggable by conventional therapeutics with fewer off-target effects. However, clinical translation of siRNA therapies for central nervous system (CNS) disorders is limited by poor pharmacokinetic properties and an inability to permeate the stringent blood-brain barrier (BBB) owing to a negatively charged backbone and short circulation half-life. As such, a common strategy for potentiating siRNA delivery to the brain involves packaging siRNA in lipid-based nanoparticles, which has been explored extensively in the field. However, systemic toxicity and large size diminish the transport efficiency of nanoparticles to the brain. To circumvent these issues and enhance the bioavailability without toxic effects, we developed an albumin-binding lipid-siRNA conjugate (termed EG18). Albumin is a favorable carrier due to its high concentration in blood and potential to bind fatty acid moieties – including our EG18 conjugate. Here, we demonstrate that EG18 accumulates in brain endothelial cells after intravenous administration and potentiates gene silencing in the choroid plexus. Since BBB and choroid plexus dysfunction are hallmarks of many CNS disorders and often promote a positive feedback loop of neurodegeneration, targeting brain vascular genes responsible for leaky vasculature could mitigate this exacerbation of the disease.


Materials and Methods:: Extensive siRNA chemical modifications are required to confer stability in blood - a critical property for systemically administered RNA therapeutics. All RNA was solid-phase synthesized on a Mermade 12 according to the zipper modification scheme presented in Figure 1A. Next, the lipidated sense strands were purified by high-performance liquid chromatography (HPLC), and the anti-sense by ion-exchange chromatography. Before conducting in vivo studies, the molecular weight was confirmed by electrospray LC/MS, and the cellular uptake and gene silencing activity was evaluated carrier-free in a neuroblastoma cell line with flow cytometry and qPCR respectively.

After validating the chemistry and in vitro efficacy of EG18, we sought to test the hypothesis that EG18 accumulates in brain vascular. CY5-tagged EG18 conjugates were administered intravenously at 20 mg/kg into the tail vein of male C57/Bl6J mice. Delivery was measured at 24, 48, and 72 hours with flow cytometry and cryohistology (48 and 72-hour data not shown). Next, to assess PPIB gene silencing with EG18, injections were performed as described above, and after four or eight days the choroid plexus was dissected and CD31+ endothelial cells were isolated with MACS. The mRNA levels were evaluated with RT-qPCR relative to a vehicle control. All animal protocols were approved by the Vanderbilt University IACUC.


Results, Conclusions, and Discussions:: Preliminary validation in vitro

The stability and albumin-binding properties of EG18 have been well established by previous work. Furthermore, the carrier-free knockdown of EG18 was potent with an IC50 at around 100nM. Progress up to this point indicates that EG18 is a viable platform for specific gene silencing and encourages in vivo exploration.

in vivo systemic delivery

When injected intravenously, EG18 exhibits striking accumulation in the brain vasculature, as evidenced by significant accumulation of EG18 into CD31+ endothelial cells, in contrast to free siRNA (no lipid conjugation) (Figure 1B). These findings are further supported by histological examinations, which demonstrate a clear colocalization of EG18 with brain vessels and with the choroid plexus (Figure 1C). In sum, this distribution presents a unique opportunity to target disease-driving genes in the brain endothelium.

After confirming delivery to vessels and the choroid plexus, we subsequently assessed gene silencing using a PPIB targeting siRNA. Excitingly, we observed a 50% reduction in mRNA in the 4th ventricle and lateral ventricle choroid plexus at both 8 and 15 days after a single IV injection (Figure 1D). To our knowledge, this is the first demonstration of gene silencing in the choroid plexus using siRNA conjugates. We expect similar findings in the CD31 isolated cells. These results confirm that EG18 can localize at blood vessels and mediate gene knockdown in the choroid plexus.

Future Directions

Future work will involve imaging with confocal microscopy and laminin staining of basement membranes to investigate the precise location of EG18 accumulation. In addition, single-cell RNA sequencing will provide detailed information on the knockdown profile in specific cell types along the arterio-venous vascular tree. Lastly, we will seek to target the choroid plexus therapeutically, as it experiences undue calcification and inflammation in aged conditions.

Clinical Relevance

The BBB is famously difficult to traverse, particularly for biologics with poor pharmacokinetic properties. Targeting the brain vasculature with EG18 obviates the need to cross the BBB, and is a promising platform for treating undruggable dysfunction in neurodegenerative disease.


Acknowledgements (Optional): :

I sincerely thank the SyBBURE Searle Undergraduate Research program for funding this work and for the mentorship and support I have received as part of the Lippmann lab!


References (Optional): :