Lipid nanoparticles (LNPs) have been used to successfully deliver small interfering RNAs (siRNAs) to target cells in both preclinical and clinical studies and currently are the leading systems for in vivo delivery. Here, we propose the use of an ordinary differential equation (ODE)-based model as a tool for optimizing LNP-mediated delivery of siRNAs. As a first step, we have used a combination of experimental and computational approaches to develop and validate a mathematical model that captures the critical features for efficient siRNA-LNP delivery in vitro. This model accurately predicts mRNA knockdown resulting from novel combinations of siRNAs and LNPs in vitro. As demonstrated, this model can be effectively used as a screening tool to select the most efficacious LNPs, which can then further be evaluated in vivo. The model serves as a starting point for the future development of next generation models capable of capturing the additional complexity of in vivo delivery.

No abstract

Sickle cell-related vaso-occlusive crises (VOCs) are among the primary clinical manifestations of sickle cell disease (SCD) and are associated with many acute and chronic complications that lead to significant morbidity and mortality. VOCs are caused by the adhesion of leukocytes and sickle erythrocytes to the endothelium, which results in vascular obstruction and tissue ischemia. By blocking the P-selectin- PSGL-1 (P-selectin glycoprotein ligand 1) mediated cell adhesion, crizanlizumab, a recently FDA approved humanized IgG2 anti-P-selectin antibody, reduced the frequency of VOCs in SCD patients and established the proof of principle for this approach (Ataga KI et al., N Engl J Med, 2017). Inclacumab is a novel, fully human IgG4 monoclonal antibody that selectively targets P-selectin and has safely demonstrated sustained anti-cell adhesion effects in over 700 participants including healthy volunteers and patients with cardiovascular disease (Schmitt C et al., J Cardiovasc Pharmacol. 2015; Tardif JC et al., J Am Coll Cardiol, 2013; Morrison M et al., Eur J Clin Pharmacol, 2015; Kling D et al., Thromb Res, 2013). A crystal structure of inclacumab and P-selectin reveals that inclacumab directly binds to an epitope in the PSGL-1 binding region on P-selectin and thus competitively inhibits P-selectin and its ligand interaction. In contrast, crizanlizumab binds to a more distant epitope to the PSGL-1 binding site on P-selectin. To further elucidate differences between the two antibodies, we characterized inclacumab and crizanlizumab in a series of in vitro functional assays including ligand binding affinity, competitive ligand binding by surface plasmon resonance (SPR), P-selectin mediated cell-based adhesion assay and cell-cell interaction with human whole blood samples. In vitro, inclacumab binds to human P-selectin with high affinity and potently suppresses the interaction of P-selectin with its main ligand PSGL-1. Both antibodies exhibited similar binding affinities to P-selectin (KD of 9.9 and 9.1 nM for inclacumab and crizanlizumab, respectively) and comparable potencies at preventing a PSGL-1 mimetic peptide from binding P-selectin (IC50 of 1.9 and 2.2 µg/mL for inclacumab and crizanlizumab, respectively) or blocking the adhesion of PSGL-1 expressing cells to an immobilized P-selectin (IC50 = 430 ng/mL for inclacumab and IC50 = 453 ng/mL for crizanlizumab). However, inclacumab demonstrated greater maximal platelet-leukocyte cell adhesion inhibition in response to thrombin receptor activating peptide (TRAP) in blood samples from both healthy volunteers and subjects with SCD in an in vitro efficacy assay (see figure). Inclacumab is differentiated from crizanlizumab as a fully human monoclonal antibody that directly blocks the PSGL-1 binding region of P-selectin and shows greater maximal inhibition of cell-cell interactions in vitro. At doses up to 20 mg/kg Q4W, which previous clinical trials have shown to be safe and well-tolerated, inclacumab has much greater drug exposure than the approved dose of crizanlizumab (5 mg/kg W0/W2/Q4W) (Ataga KI et al., N Engl J Med, 2017; Schmitt C et al., J Cardiovasc Pharmacol. 2015; Tardif JC et al., J Am Coll Cardiol, 2013). A single dose of inclacumab 20 mg/kg demonstrated full PLA inhibition for ≥84 days in healthy volunteers (Morrison M et al., Eur J Clin Pharmacol, 2015; Kling D et al., Thromb Res, 2013). Inclacumab may allow for a substantially longer and therefore more convenient dosing interval as compared with crizanlizumab. In aggregate, these data suggest that inclacumab has the potential to be a best-in-class P-selectin inhibitor to reduce VOCs in sickle cell disease. Clinical studies of inclacumab in patients with SCD are planned for the 1st half of 2021. Disclosures Geng: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mihaila:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Strutt:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Benz:Roche Pharmaceuticals: Current Employment. Tang:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayer:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Oksenberg:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company.

No abstract

Drug combinations can improve the control of diseases involving redundant and highly regulated pathways. Validating a multi-target therapy early in drug development remains difficult. Small interfering RNAs (siRNAs) are routinely used to selectively silence a target of interest. Owing to the ease of design and synthesis, siRNAs hold promise for combination therapies. Combining siRNAs against multiple targets remains an attractive approach to interrogating highly regulated pathways. Currently, questions remain regarding how broadly such an approach can be applied, since siRNAs have been shown to compete with one another for binding to Argonaute2 (Ago2), the protein responsible for initiating siRNA-mediated mRNA degradation. Mathematical modeling, coupled with in vitro and in vivo experiments, led us to conclude that endosomal escape kinetics had the highest impact on Ago2 depletion by competing lipid-nanoparticle (LNP)-formulated siRNAs. This, in turn, affected the level of competition observed between them. A future application of this model would be to optimize delivery of desired siRNA combinations in vitro to attenuate competition and maximize the combined therapeutic effect.