A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates.

The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option. However, there are concerns whether an acceptable tolerability profile for chronic dosing can be achieved. The efficiency and tolerability of lipid nanoparticles has been attributed to the amino lipid. Therefore, we developed a new series of amino lipids that address this concern. Clear structure-activity relationships were developed that resulted in a new amino lipid that affords efficient mRNA delivery in rodent and primate models with optimal pharmacokinetics. A 1-month toxicology evaluation in rat and non-human primate demonstrated no adverse events with the new lipid nanoparticle system. Mechanistic studies demonstrate that the improved efficiency can be attributed to increased endosomal escape. This effort has resulted in the first example of the ability to safely repeat dose mRNA-containing lipid nanoparticles in non-human primate at therapeutically relevant levels.

Safety Evaluation of Lipid Nanoparticle-Formulated Modified mRNA in the Sprague-Dawley Rat and Cynomolgus Monkey.

The pharmacology, pharmacokinetics, and safety of modified mRNA formulated in lipid nanoparticles (LNPs) were evaluated after repeat intravenous infusion to rats and monkeys. In both species, modified mRNA encoding the protein for human erythropoietin (hEPO) had predictable and consistent pharmacologic and toxicologic effects. Pharmacokinetic analysis conducted following the first dose showed that measured hEPO levels were maximal at 6 hours after the end of intravenous infusion and in excess of 100-fold the anticipated efficacious exposure (17.6 ng/ml) at the highest dose tested.24 hEPO was pharmacologically active in both the rat and the monkey, as indicated by a significant increase in red blood cell mass parameters. The primary safety-related findings were caused by the exaggerated pharmacology of hEPO and included increased hematopoiesis in the liver, spleen, and bone marrow (rats) and minimal hemorrhage in the heart (monkeys). Additional primary safety-related findings in the rat included mildly increased white blood cell counts, changes in the coagulation parameters at all doses, as well as liver injury and release of interferon γ-inducible protein 10 in high-dose groups only. In the monkey, as seen with the parenteral administration of cationic LNPs, splenic necrosis and lymphocyte depletion were observed, accompanied with mild and reversible complement activation. These findings defined a well-tolerated dose level above the anticipated efficacious dose. Overall, these combined studies indicate that LNP-formulated modified mRNA can be administered by intravenous infusion in 2 toxicologically relevant test species and generate supratherapeutic levels of protein (hEPO) in vivo.

SOCS-1/3 participation in FGF-2 signaling to modulate RANK ligand expression in paget's disease of bone.

Paget's disease of bone (PDB) is a chronic focal skeletal disorder characterized by excessive bone resorption followed by disorganized new bone formation. Measles virus nucleocapsid (MVNP) is implicated in pathogenesis of PDB. RANK ligand (RANKL), a critical osteoclastogenic factor expressed on bone marrow stromal/preosteoblast cells is upregulated in PDB. We recently demonstrated that fibroblast growth factor-2 (FGF-2) which induces RANKL expression is elevated in PDB. In this study, we hypothesized that FGF-2 modulates suppressors of cytokine signaling (SOCS) to induce RANKL expression in PDB. We identified increased levels of SOCS-1/3 mRNA expression in bone marrow mononuclear cells derived from patients with PDB compared to normal subjects. Interestingly, conditioned media obtained from MVNP transduced osteoclast progenitor cells significantly increased SOCS-1/3 mRNA expression in stromal/preosteoblast cells. We next examined if SOCS participates in FGF-2 signaling to modulate RANKL gene expression. We showed that FGF-2 stimulation significantly increased SOCS-1/3 expression in human bone marrow stromal/preosteoblast cells. In addition, co-expression of SOCS-1/3 with hRANKL gene promoter-luciferase reporter plasmid in marrow stromal cells demonstrated a significant increase in promoter activity without FGF-2 stimulation. Furthermore, siRNA inhibition of STAT-1 suppresses FGF-2 increased SOCS-1/3 expression in these cells. Thus, our results suggest that SOCS participates in FGF-2 modulation of RANKL expression in PDB.

Accelerated Blood Clearance of Lipid Nanoparticles Entails a Biphasic Humoral Response of B-1 Followed by B-2 Lymphocytes to Distinct Antigenic Moieties.

Accelerated blood clearance (ABC) is a phenomenon in which certain pharmaceutical agents are rapidly cleared from the blood upon second and subsequent administrations. ABC has been observed for many lipid-delivery vehicles, including liposomes and lipid nanoparticles (LNP). Previous studies have demonstrated a role for humoral responses against the polyethylene glycol motifs in clearance, but significant gaps remain in our understanding of the mechanism of ABC, and strategies for limiting the impact of ABC in a clinical setting have been elusive. mRNA therapeutics have great promise, but require chronic administration in encapsulating delivery systems, of which LNP are the most clinically advanced. In this study, we investigate the mechanisms of ABC for mRNA-formulated LNP in vivo and in vitro. We present evidence that ABC of mRNA-formulated LNP is dramatic and proceeds rapidly, based on a previously unrecognized ability of LNP to directly activate B-1 lymphocytes, resulting in the production of antiphosphorylcholine IgM Abs in response to initial injection. Upon repeated injections, B-2 lymphocytes also become activated and generate a classic anti-polyethylene glycol adaptive humoral response. The ABC response to phosphorylcholine/LNP-encapsulated mRNA is therefore a combination of early B-1 lymphocyte and later B-2 lymphocyte responses.

mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials.

BACKGROUND: We evaluated safety and immunogenicity of the first mRNA vaccines against potentially pandemic avian H10N8 and H7N9 influenza viruses. METHODS: Two randomized, placebo-controlled, double-blind, phase 1 clinical trials enrolled participants between December 2015 and August 2017 at single centers in Germany (H10N8) and USA (H7N9). Healthy adults (ages 18-64 years for H10N8 study; 18-49 years for H7N9 study) participated. Participants received vaccine or placebo in a 2-dose vaccination series 3 weeks apart. H10N8 intramuscular (IM) dose levels of 25, 50, 75, 100, and 400 µg and intradermal dose levels of 25 and 50 µg were evaluated. H7N9 IM 10-, 25-, and 50-µg dose levels were evaluated; 2-dose series 6 months apart was also evaluated. Primary endpoints were safety (adverse events) and tolerability. Secondary immunogenicity outcomes included humoral (hemagglutination inhibition [HAI], microneutralization [MN] assays) and cell-mediated responses (ELISPOT assay). RESULTS: H10N8 and H7N9 mRNA IM vaccines demonstrated favorable safety and reactogenicity profiles. No vaccine-related serious adverse event was reported. For H10N8 (N = 201), 100-µg IM dose induced HAI titers ≥ 1:40 in 100% and MN titers ≥ 1:20 in 87.0% of participants. The 25-µg intradermal dose induced HAI titers > 1:40 in 64.7% of participants compared to 34.5% of participants receiving the IM dose. For H7N9 (N = 156), IM doses of 10, 25, and 50 µg achieved HAI titers ≥ 1:40 in 36.0%, 96.3%, and 89.7% of participants, respectively. MN titers ≥ 1:20 were achieved by 100% in the 10- and 25-µg groups and 96.6% in the 50-µg group. Seroconversion rates were 78.3% (HAI) and 87.0% (MN) for H10N8 (100 µg IM) and 96.3% (HAI) and 100% (MN) in H7N9 (50 µg). Significant cell-mediated responses were not detected in either study. CONCLUSIONS: The first mRNA vaccines against H10N8 and H7N9 influenza viruses were well tolerated and elicited robust humoral immune responses. ClinicalTrials.gov NCT03076385 and NCT03345043.

Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines.

mRNA vaccines have the potential to tackle many unmet medical needs that are unable to be addressed with conventional vaccine technologies. A potent and well-tolerated delivery technology is integral to fully realizing the potential of mRNA vaccines. Pre-clinical and clinical studies have demonstrated that mRNA delivered intramuscularly (IM) with first-generation lipid nanoparticles (LNPs) generates robust immune responses. Despite progress made over the past several years, there remains significant opportunity for improvement, as the most advanced LNPs were designed for intravenous (IV) delivery of siRNA to the liver. Here, we screened a panel of proprietary biodegradable ionizable lipids for both expression and immunogenicity in a rodent model when administered IM. A subset of compounds was selected and further evaluated for tolerability, immunogenicity, and expression in rodents and non-human primates (NHPs). A lead formulation was identified that yielded a robust immune response with improved tolerability. More importantly for vaccines, increased innate immune stimulation driven by LNPs does not equate to increased immunogenicity, illustrating that mRNA vaccine tolerability can be improved without affecting potency.

Beta-arrestins 1 and 2 differentially regulate LPS-induced signaling and pro-inflammatory gene expression.

Toll like receptors, the critical receptor family in innate immunity, have been shown to signal via both ERK 1/2 and transcription factor NFkappaB. beta-Arrestins 1 and 2 have recently been implicated in modulation of NFkappaB signaling and ERK 1/2 activation. Using a number of approaches: mouse embryonic fibroblasts (MEF) from wild-type (WT), beta-arrestins knockouts (KO), beta-arrestins 1 and 2 double KO, and MEFs with reconstituted WT beta-arrestins in the double KO cells, RNA interference (siRNA) specific knockdown of beta-arrestins, and overexpression of WT beta-arrestins, it was demonstrated that beta-arrestin 2 positively regulates LPS-induced ERK 1/2 activation and both beta-arrestins 1 and 2 negatively regulate LPS-induced NFkappaB activation. Also beta-arrestin 2 positively regulate LPS-induced IL-6 production and both beta-arrestins 1 and 2 positively regulate LPS-induced IL-8 production. The specific ERK1/2 inhibitor PD98059 significantly decreased LPS-induced IL-6 and IL-8 production suggesting that IL-6 and IL-8 production is, in part, mediated by ERK 1/2 activation. Over expression of wild type beta-arrestins 1 and 2 had no effect on LPS-induced ERK1/2 activation and LPS-induced IL-8 production suggesting that endogenous beta-arrestins 1 and 2 are sufficient to mediate maximum ERK 1/2 activity and IL-8 production. beta-Arrestins thus not only negatively regulate LPS-induced NFkappaB activation but also positively regulate ERK 1/2 activation and specific pro-inflammatory gene expression. Understanding the role of beta-arrestins in regulation of TLR signaling pathways may provide novel insights into control mechanisms for inflammatory gene expression.

Proteasome inhibitors bortezomib and carfilzomib used for the treatment of multiple myeloma do not inhibit the serine protease HtrA2/Omi.

The proteasome inhibitor bortezomib is associated with the development of peripheral neuropathy in patients, but the mechanism by which bortezomib can induce peripheral neuropathy is not fully understood. One study suggested that off-target inhibition of proteases other than the proteasome, particularly HtraA2/Omi, may be the underlying mechanism of the neuropathy. The same study also concluded that carfilzomib, a second proteasome inhibitor that is associated with less peripheral neuropathy in patients than bortezomib, showed no inhibition of HtrA2/Omi. The goal of the work described here was to determine whether either proteasome inhibitors truly affected HtrA2/Omi activity. A variety of methods were used to test the effects of both bortezomib and carfilzomib on HtrA2/Omi activity that included in vitro recombinant enzyme assays, and studies with the human neuroblastoma SH-SY5Y cell line and HtrA2/Omi-knockout mouse embryonic fibroblasts. The compound ucf-101 was used to assess the effects of specific HtrA2/Omi inhibition. In contrast to previously published data, our results clearly demonstrated that neither bortezomib nor carfilzomib inhibited HtrA2/Omi activity in recombinant enzyme assays at concentrations up to 100 µM, while the specific inhibitor ucf-101 did inhibit the enzyme. The proteasome inhibitors did not inhibit HtrA2/Omi activity in either SH-SY5Y cells or mouse embryonic fibroblasts, as determined by expression of the HtrA2/Omi substrates eIF4G1 and UCH-L1. Based on our biochemical and cell-based assays, we conclude that neither bortezomib nor carfilzomib inhibited HtrA2/Omi activity. Therefore, it is unlikely that bortezomib associated peripheral neuropathy is a direct result of off-target inhibition of HtrA2/Omi.

Interleukin-6 induces cellular insulin resistance in hepatocytes.

Interleukin (IL)-6 is one of several proinflammatory cytokines that have been associated with insulin resistance and type 2 diabetes. A two- to threefold elevation of circulating IL-6 has been observed in these conditions. Nonetheless, little evidence supports a direct role for IL-6 in mediating insulin resistance. Here, we present data that IL-6 can inhibit insulin receptor (IR) signal transduction and insulin action in both primary mouse hepatocytes and the human hepatocarcinoma cell line, HepG2. This inhibition depends on duration of IL-6 exposure, with a maximum effect at 1-1.5 h of pretreatment with IL-6 in both HepG2 cells and primary hepatocytes. The IL-6 effect is characterized by a decreased tyrosine phosphorylation of IR substrate (IRS)-1 and decreased association of the p85 subunit of phosphatidylinositol 3-kinase with IRS-1 in response to physiologic insulin levels. In addition, insulin-dependent activation of Akt, important in mediating insulin's downstream metabolic actions, is markedly inhibited by IL-6 treatment. Finally, a 1.5-h preincubation of primary hepatocytes with IL-6 inhibits insulin-induced glycogen synthesis by 75%. These data suggest that IL-6 plays a direct role in insulin resistance at the cellular level in both primary hepatocytes and HepG2 cell lines and may contribute to insulin resistance and type 2 diabetes.

FGF-2 stimulation of RANK ligand expression in Paget's disease of bone.

Receptor activator for nuclear factor-kappaB ligand (RANKL), a critical osteoclastogenic factor expressed in marrow stromal/preosteoblast cells is up-regulated in Paget's disease of bone (PDB). We previously demonstrated that heat-shock factor-2 (HSF-2) is a downstream target of fibroblast growth factor-2 (FGF-2) signaling to induce RANKL expression in bone marrow stromal/preosteoblast cells. In this study, we identified a 2.5-fold increase in serum FGF-2 levels in patients (n = 8) with PDB compared with normal subjects (n = 10). We showed that HSF-2 co-immunoprecipitates with heat-shock protein-27 (HSP-27) and that FGF-2 stimulation significantly increased phospho-HSP-27 levels in marrow stromal cells. Confocal microscopy revealed HSF-2 colocalization with HSP-27 in unstimulated cells and HSF-2 nuclear translocation upon FGF-2 stimulation. We further show that FGF-2 stimulation significantly increased the levels of phosphorylated signal transducers and activators of the transcription (p-STAT-1) in these cells. Western blot analysis confirmed that small interfering RNA suppression of STAT-1 significantly decreased (3.2-fold) RANKL expression and promoter activity in FGF-2-stimulated cells. Chromatin immunoprecipitation assay revealed STAT-1 binding to a putative motif located far upstream (-8 kb) in the hRANKL gene promoter region. These results suggest STAT-1 is a downstream effector of FGF-2 signaling and that elevated levels of FGF-2 stimulates RANKL expression in PDB.

RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation.

Osteoclast differentiation is tightly regulated by receptor activator of NF-kappaB ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity in RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+1 to -1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to +1 bp to -446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from -446 bp to -1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (-1123 bp to -1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in the absence of RANKL. Taken together, our results suggest that RANKL signals through TRAF6 and that NFATc1 is a downstream effector of RANKL signaling to modulate MMP-9 gene expression during osteoclast differentiation.

Toll-like receptor-2 is essential for the development of palmitate-induced insulin resistance in myotubes.

Fatty acids can activate proinflammatory pathways leading to the development of insulin resistance, but the mechanism is undiscovered. Toll like receptor 2 (TLR2) recognizes lipids, activates proinflammatory pathways, and is genetically associated with inflammatory diseases. This study aimed to examine the role of TLR2 in palmitate-induced insulin resistance in C2C12 myotubes. Treatment with palmitate rapidly induced the association of myeloid differentiation factor 88 (MyD88) with the TLR2 receptor, activated the stress-linked kinases p38, JNK, and protein kinase C, induced degradation of IkappaBalpha, and increased NF-kappaB DNA binding. The activation of these pathways by palmitate was sensitive and temporally regulated and occurred within the upper physiologic range of saturated fatty acid concentrations in vivo, suggesting a receptor-mediated event and not simple lipotoxicity. When compared with an equimolar concentration of palmitate, fibroblast-stimulating lipopeptide-1, a known TLR2 ligand, was a slightly more potent activator of signal transduction and interleukin (IL)-6 production. Palmitate inhibited insulin signal transduction in C2C12 cells beginning 1-2 h after exposure and reached a maximum at 12-16 h. An antagonist TLR2 antibody, mAb 2.5, led to a 50-60% decrease in palmitate-induced IL-6 production and partially restored insulin signal transduction, whereas an isotype-matched control antibody had no effect. RNA interference-mediated inhibition of TLR2 and MyD88 expression in C2C12 muscle cells resulted in a near complete inhibition of palmitate-induced insulin resistance and IL-6 production. This study provides strong evidence that TLR2 mediates the initial events of fatty acid-induced insulin resistance in muscle.

Non-CpG-containing antisense 2'-methoxyethyl oligonucleotides activate a proinflammatory response independent of Toll-like receptor 9 or myeloid differentiation factor 88.

Oligonucleotides with a "CpG" motif trigger a proinflammatory response through activation of Toll-like receptor 9 (TLR9) and are being studied to exploit these properties for use as adjuvants and cancer therapies. However, oligonucleotides intended for antisense applications (ASOs) are designed to minimize proinflammatory responses by avoiding CpG motifs and by using chemical modifications [i.e., 2'-methoxyethyl (MOE) sugars and 5-methyl cytosine residues]. Nonetheless, modified ASOs are capable of eliciting a proinflammatory response at high doses, albeit mild compared with CpG oligos. To determine whether this phenomena is TLR-mediated, wild-type, TLR9 knockout, and myeloid differentiation factor 88 (MyD88) knockout mice were treated with a phosphorothioate-modified oligodeoxyribonucleotide CpG optimal oligo (ISIS 12449), and a representative non-CpG 2'-MOE oligonucleotide (ISIS 116847). The non-CpG oligonucleotide had a lower proinflammatory potency relative to ISIS 12449, requiring a >10-fold higher dose in wild-type animals to trigger a proinflammatory response. Furthermore, the inflammatory response to ISIS 12449 at low doses was TLR9 and MyD88-dependent, whereas non-CpG oligonucleotides retained the ability to activate a proinflammatory response in the knockout animals. Animals treated with the non-CpG oligonucleotide exhibited an increased spleen weight, elevated cytokine levels, increased immune cell infiltrates in liver, and an increased level of mRNA for cell surface markers typical of monocyte/macrophage type cells. Bone marrow-derived cells from wild-type and knockout animals treated with non-CpG oligonucleotide responded similarly with the production of MIP-2 and the activation of extracellular signal-regulated kianse1/2. These data implicate a TLR-independent mechanism of activation for non-CpG 2'-MOE oligonucleotides.

Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator of interleukin-6-dependent insulin resistance in hepatocytes.

Interleukin-6 (IL-6) is one of several pro-inflammatory cytokines implicated in insulin resistance during infection, cachexia, and obesity. We recently demonstrated that IL-6 inhibits insulin signaling in hepatocytes (Senn, J. J., Klover, P. J., Nowak, I. A., and Mooney, R. A. (2002) Diabetes 51, 3391-3399). Members of the suppressors of cytokine signaling (SOCS) family associate with the insulin receptor (IR), and their ectopic expression inhibits IR signaling. Since several SOCS proteins are induced by IL-6, a working hypothesis is that IL-6-dependent insulin resistance is mediated, at least in part, by induction of SOCS protein(s) in insulin target cells. To examine the involvement of SOCS protein(s) in IL-6-dependent inhibition of insulin receptor signaling, HepG2 cells were treated with IL-6 (20 ng/ml) for periods from 1 min to 8 h. IL-6 induced SOCS-3 transcript at 30 min with a maximum effect at 1 h. SOCS-3 protein levels were also markedly elevated at 1 h. Transcript and protein levels returned to near basal levels by 2 h. SOCS-3 induction by IL-6 paralleled IL-6-dependent inhibition of IR signal transduction. Ectopically expressed SOCS-3 associated with the IR and suppressed insulin-dependent receptor autophosphorylation, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase, and activation of Akt. SOCS-3 was also a direct inhibitor of insulin receptor autophosphorylation in vitro. In mice exposed to IL-6 for 60-90 min, hepatic SOCS-3 expression was increased. This was associated with inhibition of hepatic insulin-dependent receptor autophosphorylation and IRS-1 tyrosine phosphorylation. These data suggest that induction of SOCS-3 in liver may be an important mechanism of IL-6-mediated insulin resistance.