(306c) Albumin-Binding Lipid-siRNA Conjugates Accumulate in Brain Endothelial Cells

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 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. 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, an albumin-binding lipid-siRNA conjugate (termed EG18). Albumin is a favorable carrier due to its relatively high concentration in the blood and its potential to bind versatile moieties – including our EG18 conjugate. Intriguingly, we found that EG18 accumulates in brain endothelial cells after intravenous administration, suggesting that EG18 could be further designed for permeation into the brain parenchyma or optimized for brain vascular accumulation.

siRNA modifications and EG18 structure

Extensive siRNA chemical modifications are required to confer stability in blood - a critical property for systemically administered RNA drugs. Key modifications to the siRNA backbone include a zipper pattern of alternating 2’ O-methyl and 2’ Fluoro base modifications, phosphorothioate bonds capping each end, and a 5’ vinyl phosphate on the antisense strand. Importantly, these modulations do not inhibit the function of the siRNA, however, they allow the siRNA to persist in serum - exposed to nucleases - for significantly longer. The EG18 lipids consist of 18 ethylene glycol (EG) repeated spacer units before the C18 chain (Figure 1A). Subsequent studies sought to better characterize the albumin binding ability of the dual EG18 tails - as it is critical to the mechanistic understanding of biodistribution. The fluorescently labeled EG18-conjugates were ate eluted in the same size-based chromatography fraction as albumin while multiple control conditions did not.

Characterizing cell uptake and gene silencing in vitro

To be a functional siRNA conjugate, EG18 must be able to gain access to the cytosolic space and mediate knockdown; thus, neuroblastoma cells (N2A) were used to evaluate the capability of EG18 in these facets. To elucidate the uptake route, cells were incubated with EG18 at 4 ℃, a temperature where all endocytic events are inhibited. Interestingly, under these conditions, there was no uptake of free siRNA, while EG18 continued to exhibit substantial cell association. The EG18 outperformed other lipid conjugates including the commonly used cholesterol conjugates under this condition. These studies suggest EG18’s mechanism of intercalation of the C18 tails into the phospholipid bilayer. Furthermore, the carrier-free knockdown of EG18-PPIB siRNA was evaluated with a dose curve determining the IC50 at around 100nM. Together, these results indicate that EG18 effectively enters the cytoplasm and mediates knockdown at low doses without the need for transfection reagents.

in vivo systemic delivery

Validation and mechanistic studies are well conducted in vitro; however, to interrogate our hypothesis of beneficial serum albumin binding and brain vascular accumulation, mouse models were used. To test the biodistribution fluorescently tagged EG18 conjugates were administered intravenously at 20 mg/kg to C57/Bl6J mice and delivery was assessed at 24, 48, and 72 hours. Delivery to specific cells of the brain was assessed by flow cytometry using cell-specific antibodies. Strikingly, greater than 95 percent of CD31+ endothelial cells contained EG18 (Figure 1B). These findings are further supported by immunohistology, which demonstrates a clear colocalization of EG18 with brain vessels (Figure 1C). In sum, systemically administered EG18 accumulates in cerebral vasculature and the choroid plexus, presenting a unique opportunity to target disease-driving genes in the brain endothelium.

Future Directions

To follow up our preliminary data on delivery to brain endothelial cells, we will evaluate the knockdown efficacy of EG18-siRNA specifically in these endothelial cells. Elucidating a more well-characterized mechanism of albumin-mediated EG18-siRNA circulation and lipid-based cellular internalization is also of great interest. Future imaging with confocal microscopy and laminin staining of basement membranes is likely to shed insight into this question. In addition, single-cell RNA sequencing will provide detailed information on the knockdown profile in specific cell types along the arterio-venous vascular tree. Following up on observations about the accumulation in the choroid plexus may illuminate a blood-to-CSF transport pathway that could be specifically exploited in future iterations of the EG18-siRNA conjugate.

Clinical Relevance

The BBB is famously difficult to traverse, particularly for biologics with poor pharmacokinetic properties. Here we describe a biotechnology that instead targets the cells comprising the brain vasculature. Since BBB dysfunction occurs in many brain diseases and often promotes a positive feedback loop of deterioration, targeting some of the many brain vascular genes responsible for leaky vasculature could mitigate the exacerbation of the pathology. Additionally, there are several diseases directly caused by genetic malfunction of the brain vasculature that are currently undruggable with small molecules, which could effectively be targeted by EG18-siRNA conjugates.