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).

Ligand conjugate SAR and enhanced delivery in NHP.

N-Acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) are a leading RNA interference (RNAi) platform allowing targeted inhibition of disease-causing genes in hepatocytes. More than a decade of development has recently resulted in the first approvals for this class of drugs. While substantial effort has been made to improve nucleic acid modification patterns for better payload stability and efficacy, relatively little attention has been given to the GalNAc targeting ligand. In addition, the lack of an intrinsic endosomal release mechanism has limited potency. Here, we report a stepwise analysis of the structure activity relationships (SAR) of the components comprising these targeting ligands. We show that there is relatively little difference in biological performance between bi-, tri-, and tetravalent ligand structures while identifying other features that affect their biological activity more significantly. Further, we demonstrate that subcutaneous co-administration of a GalNAc-functionalized, pH responsive endosomal release agent markedly improved the activity and duration of effect for siRNA conjugates, without compromising tolerability, in non-human primates. These findings could address a significant bottleneck for future siRNA ligand conjugate development.

Preclinical Profile of AB-423, an Inhibitor of Hepatitis B Virus Pregenomic RNA Encapsidation.

AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV replication (50% effective concentration [EC50] = 0.08 to 0.27 µM; EC90 = 0.33 to 1.32 µM) with no significant cytotoxicity (50% cytotoxic concentration > 10 µM). Addition of 40% human serum resulted in a 5-fold increase in the EC50s. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants in vitro Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a de novo infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein. In vitro dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the in vitro combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.

Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice.

siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation.

Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, and inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit nonspecific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of influenza infection, the antiviral activity of siRNA is due primarily to immune stimulation elicited by the active siRNA duplexes and is not the result of therapeutic RNA interference (RNAi) as previously reported. We show that the misinterpretation stems from the use of a particular control green fluorescent protein (GFP) siRNA that we identify as having unusually low immunostimulatory activity compared with the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNAs, all of which could elicit significant interferon induction from primary immune cells. This panel included eight active siRNAs against viral, angiogenic, and oncologic targets, the reported therapeutic efficacy of which was based on comparison with the nonimmunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor, and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to delineate more accurately the mechanism of action underlying such studies.

Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference.

BACKGROUND: Ebola virus (EBOV) infection causes a frequently fatal hemorrhagic fever (HF) that is refractory to treatment with currently available antiviral therapeutics. RNA interference represents a powerful, naturally occurring biological strategy for the inhibition of gene expression and has demonstrated utility in the inhibition of viral replication. Here, we describe the development of a potential therapy for EBOV infection that is based on small interfering RNAs (siRNAs). METHODS: Four siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid-lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge. RESULTS: Treatment of guinea pigs with a pool of the L gene-specific siRNAs delivered by PEI polyplexes reduced plasma viremia levels and partially protected the animals from death when administered shortly before the ZEBOV challenge. Evaluation of the same pool of siRNAs delivered using SNALPs proved that this system was more efficacious, as it completely protected guinea pigs against viremia and death when administered shortly after the ZEBOV challenge. Additional experiments showed that 1 of the 4 siRNAs alone could completely protect guinea pigs from a lethal ZEBOV challenge. CONCLUSIONS: Further development of this technology has the potential to yield effective treatments for EBOV HF as well as for diseases caused by other agents that are considered to be biological threats.

Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes.

The systemic application of nucleic acid drugs requires delivery systems that overcome the poor pharmacokinetics, limited biodistribution, and inefficient uptake of nucleic acids. PEGylated liposomes show considerable promise because of their intrinsic ability to accumulate at disease sites and facilitate transfection of target cells. Unlike many viral vectors, PEGylated liposomes are generally considered to be nonimmunogenic. We have developed a PEGylated liposome for the systemic administration of plasmid DNA that achieves high levels of selective gene expression at distal tumor sites. Here we report that the in vivo efficacy and safety of these systems can be severely compromised following repeat administration. This phenomenon is characterized by a loss of disease site targeting, accelerated clearance from the blood, and acute hypersensitivity. These effects are fully attributable to a surprisingly robust, long-lived antibody response generated against polyethylene glycol (PEG) that results from the strong adjuvant effect of the plasmid payload. Importantly, immunogenicity may be substantially reduced by modifying the alkyl chain of the PEG-lipid conjugate, thereby allowing successful repeat dosing of the modified plasmid formulations without adverse side effects. Immunogenicity is a relevant concern for a number of nonviral delivery systems given the potent immunostimulatory properties of many nucleic acid drugs.

Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.

Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications. Many nucleic acids, including double- (dsRNA) and single-stranded RNA (ssRNA), can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.