Silicon Ether Ionizable Lipids Enable Potent mRNA Lipid Nanoparticles with Rapid Tissue Clearance.

The advent of mRNA for nucleic acid (NA) therapeutics has unlocked many diverse areas of research and clinical investigation. However, the shorter intracellular half-life of mRNA compared with other NAs may necessitate more frequent dosing regimens. Because lipid nanoparticles (LNPs) are the principal delivery system used for mRNA, this could lead to tolerability challenges associated with an accumulated lipid burden. This can be addressed by introducing enzymatically cleaved carboxylic esters into the hydrophobic domains of lipid components, notably, the ionizable lipid. However, enzymatic activity can vary significantly with age, disease state, and species, potentially limiting the application in humans. Here we report an alternative approach to ionizable lipid degradability that relies on nonenzymatic hydrolysis, leading to a controlled and highly efficient lipid clearance profile. We identify highly potent examples and demonstrate their exceptional tolerability in multiple preclinical species, including multidosing in nonhuman primates (NHP).

ASL mRNA-LNP Therapeutic for the Treatment of Argininosuccinic Aciduria Enables Survival Benefit in a Mouse Model.

Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5' and 3' untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA.

Optimizing Lipid Nanoparticles for Delivery in Primates.

Lipid nanoparticles (LNPs) are clinically proven to successfully deliver both small interfering RNA (siRNA) therapeutics and larger mRNA payloads for prophylactic vaccine applications. Non-human primates (NHPs) are generally considered to be the most predictive of human responses. However, for ethical and economic reasons, LNP compositions have historically been optimized in rodents. It has been difficult to translate LNP potency data from rodents to NHPs for intravenously (IV) administered products in particular. This presents a major challenge for preclinical drug development. An attempt to investigate LNP parameters, which have historically been optimized in rodents, is carried out, and seemingly innocuous changes are found to result in large potency differences between species. For example, the ideal particle size for NHPs (50-60 nm) is found to be smaller than for rodents (70-80 nm). Surface chemistry requirements are also different, with almost double the amount of poly(ethylene glycol) (PEG)-conjugated lipid needed for maximal potency in NHPs. By optimizing these two parameters, approximately eight-fold increase in protein expression from intravenously administered messenger RNA (mRNA)-LNP in NHP is gained. The optimized formulations are well tolerated when administered repeatedly with no loss of potency. This advancement enables the design of optimal LNP products for clinical development.

Unsaturated, Trialkyl Ionizable Lipids are Versatile Lipid-Nanoparticle Components for Therapeutic and Vaccine Applications.

Lipid nanoparticles (LNPs) have proven a successful platform for the delivery of nucleic acid (NA)-based therapeutics and vaccines, with the ionizable lipid component playing a key role in modulating potency and tolerability. Here, a library of 16 novel ionizable lipids is screened hypothesizing that short, branched trialkyl hydrophobic domains can improve LNP fusogenicity or endosomal escape, and potency. LNPs formulated with the top-performing trialkyl lipid (Lipid 10) encapsulating transthyretin siRNA elicit significantly greater gene silencing and are better tolerated than those with the benchmark Onpattro lipid DLin-MC3-DMA. Lipid 10 also demonstrates superior liver delivery of mRNA when compared to other literature ionizable lipids, is well tolerated, and successfully repeat-doses in nonhuman primates. In a prime-boost hemagglutinin rodent vaccine model, intramuscular administration of Lipid-10 LNP elicits comparable or better antibody titers to the SM-102 and ALC-0315 lipid compositions used in the U.S. Food and Drug Administration approved mRNA COVID vaccines. These data suggest that Lipid 10 is a particularly versatile ionizable lipid, well-suited for both systemic therapeutic and intramuscular vaccine applications and able to successfully deliver diverse NA payloads.

Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine.

Lipid nanoparticle (LNP)-formulated nucleoside-modified mRNA vaccines have proven to be very successful in the fight against the coronavirus disease 2019 (COVID-19) pandemic. They are effective, safe, and can be produced in large quantities. However, the long-term storage of mRNA-LNP vaccines without freezing is still a challenge. Here, we demonstrate that nucleoside-modified mRNA-LNPs can be lyophilized, and the physicochemical properties of the lyophilized material do not significantly change for 12 weeks after storage at room temperature and for at least 24 weeks after storage at 4°C. Importantly, we show in comparative mouse studies that lyophilized firefly luciferase-encoding mRNA-LNPs maintain their high expression, and no decrease in the immunogenicity of a lyophilized influenza virus hemagglutinin-encoding mRNA-LNP vaccine was observed after 12 weeks of storage at room temperature or for at least 24 weeks after storage at 4°C. Our studies offer a potential solution to overcome the long-term storage-related limitations of nucleoside-modified mRNA-LNP vaccines.

Impact of a CXCL12/CXCR4 Antagonist in Bleomycin (BLM) Induced Pulmonary Fibrosis and Carbon Tetrachloride (CCl4) Induced Hepatic Fibrosis in Mice.

Modulation of chemokine CXCL12 and its receptor CXCR4 has been implicated in attenuation of bleomycin (BLM)-induced pulmonary fibrosis and carbon tetrachloride (CCl4)-induced hepatic injury. In pulmonary fibrosis, published reports suggest that collagen production in the injured lung is derived from fibrocytes recruited from the circulation in response to release of pulmonary CXCL12. Conversely, in hepatic fibrosis, resident hepatic stellate cells (HSC), the key cell type in progression of fibrosis, upregulate CXCR4 expression in response to activation. Further, CXCL12 induces HSC proliferation and subsequent production of collagen I. In the current study, we evaluated AMD070, an orally bioavailable inhibitor of CXCL12/CXCR4 in alleviating BLM-induced pulmonary and CCl4-induced hepatic fibrosis in mice. Similar to other CXCR4 antagonists, treatment with AMD070 significantly increased leukocyte mobilization. However, in these two models of fibrosis, AMD070 had a negligible impact on extracellular matrix deposition. Interestingly, our results indicated that CXCL12/CXCR4 signaling has a role in improving mortality associated with BLM induced pulmonary injury, likely through dampening an early inflammatory response and/or vascular leakage. Together, these findings indicate that the CXCL12-CXCR4 signaling axis is not an effective target for reducing fibrosis.