Translational kinetic-pharmacodynamics of mRNA-6231, an investigational mRNA therapeutic encoding mutein interleukin-2.

Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by serving as negative regulators of adaptive immune system effector cell responses. Reduced production or function of Tregs has been implicated in several human autoimmune diseases. The cytokine interleukin 2 plays a central role in promoting Treg differentiation, survival, and function in vivo and may therefore have therapeutic benefits for autoimmune diseases. mRNA-6231 is an investigational, lipid nanoparticle-encapsulated, mRNA-based therapy that encodes a modified human interleukin 2 mutein fused to human serum albumin (HSA-IL2m). Herein, we report the development of a semi-mechanistic kinetic-pharmacodynamic model to quantify the relationship between subcutaneous dose(s) of mRNA-6231, HSA-IL2m protein expression, and Treg expansion in nonhuman primates. The nonclinical kinetic-pharmacodynamic model was extrapolated to humans using allometric scaling principles and the physiological basis of pharmacological mechanisms to predict the clinical response to therapy a priori. Model-based simulations were used to inform the dose selection and design of the first-in-human clinical study (NCT04916431). The modeling approach used to predict human responses was validated when data became available from the phase I clinical study. This validation indicates that the approach is valuable in informing clinical decision-making.

Translational Pharmacokinetic/Pharmacodynamic Model for mRNA-3927, an Investigational Therapeutic for the Treatment of Propionic Acidemia.

Propionic acidemia (PA) is an ultrarare disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC), composed of PCCA and PCCB subunits. An enzyme replacement therapy is being developed using dual messenger RNA (mRNA) therapy composed of lipid nanoparticles (LNPs) encapsulating mRNAs encoding PCCA and PCCB subunits of the PCC enzyme. We herein report on development of a translational semimechanistic pharmacokinetic (PK) and PK/pharmacodynamic (PD) model to quantify the relationship between the mRNA components of mRNA-3927 (an LNP encapsulating PCCA and PCCB mRNAs) and dose levels; PCCA/B mRNA PK and PD responses were assessed as circulating levels of primary disease markers 2-methyl citrate, 3-hydroxypropionate, and propionyl carnitine normalized to acetyl carnitine (C3/C2 ratio) to inform the first-in-human dose range and regimen selection. The translational PK/PD model was developed using preclinical data available in mice with PA, Sprague Dawley rats, and cynomolgus monkeys at dose levels ranging from 0.2 to 9 mg/kg. PCCA/B mRNA PK in mice, rats, and monkeys was adequately described using allometric scaling of volume and clearance parameters. The interspecies preclinical model was scaled allometrically to humans to predict the dose-response relationship in adult and pediatric patients with PA to guide selection of dose range and regimen for the Phase 1 clinical trial (ClinicalTrials.gov Identifier NCT04159103).

Rational Design and In Vivo Characterization of mRNA-Encoded Broadly Neutralizing Antibody Combinations against HIV-1.

Monoclonal antibodies have been used successfully as recombinant protein therapy; however, for HIV, multiple broadly neutralizing antibodies may be necessary. We used the mRNA-LNP platform for in vivo co-expression of 3 broadly neutralizing antibodies, PGDM1400, PGT121, and N6, directed against the HIV-1 envelope protein. mRNA-encoded HIV-1 antibodies were engineered as single-chain Fc (scFv-Fc) to overcome heavy- and light-chain mismatch. In vitro neutralization breadth and potency of the constructs were compared to their parental IgG form. We assessed the ability of these scFv-Fcs to be expressed individually and in combination in vivo, and neutralization and pharmacokinetics were compared to the corresponding full-length IgGs. Single-chain PGDM1400 and PGT121 exhibited neutralization potency comparable to parental IgG, achieving peak systemic concentrations ≥ 30.81 µg/mL in mice; full-length N6 IgG achieved a peak concentration of 974 µg/mL, but did not tolerate single-chain conversion. The mRNA combination encoding full-length N6 IgG and single-chain PGDM1400 and PGT121 was efficiently expressed in mice, achieving high systemic concentration and desired neutralization potency. Analysis of mice sera demonstrated each antibody contributed towards neutralization of multiple HIV-1 pseudoviruses. Together, these data show that the mRNA-LNP platform provides a promising approach for antibody-based HIV treatment and is well-suited for development of combination therapeutics.

Mathematical Modeling and Experimental Validation of Extracellular Vesicle-Mediated Tumor Suppressor MicroRNA Delivery and Propagation in Ovarian Cancer Cells.

Extracellular vesicle (EV)-mediated microRNA transfer and propagation from the donor cell to the recipient cell in the tumor microenvironment have significant implications, including the development of multidrug resistance (MDR). Although miRNA-encapsulated EV have been shown to have functional effects on recipient cells, the quantitative aspects of transfer kinetics and functional effects remain poorly understood. Intracellular events such as degradation of miRNA, loading of miRNA into EVs, cellular release of EVs, and their uptake by recipient cells govern the transfer and functional effect of encapsulated miRNA. Based on these rate-limiting steps, we developed a mathematical model using ordinary differential equations (model 1). We performed coculture experiments using ID8-VEGF ovarian cancer cells to demonstrate EV-mediated propagation of tumor suppressor miRNA Let7b administered with hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles. Using the experimental data and model fitting, we determined the rate constants for the kinetic events involved in the transfer from the donor cells to the recipient cells. In model 2, we performed Let7b transfection experiments in ID8-VEGF cells with HA-PEI nanoparticles to determine the concentration-effect relationship on HMGA2 mRNA levels. Lastly, in model 3, we combined model 1 and model 2 parameters to describe the kinetics and effect relationship of EV-Let7b in recipient cells to predict the minimum number of miRNA copies needed to show functional effects.

A phase 1 trial of lipid-encapsulated mRNA encoding a monoclonal antibody with neutralizing activity against Chikungunya virus.

Chikungunya virus (CHIKV) infection causes acute disease characterized by fever, rash and arthralgia, which progresses to severe and chronic arthritis in up to 50% of patients. Moreover, CHIKV infection can be fatal in infants or immunocompromised individuals and has no approved therapy or prevention. This phase 1, first-in-human, randomized, placebo-controlled, proof-of-concept trial conducted from January 2019 to June 2020 evaluated the safety and pharmacology of mRNA-1944, a lipid nanoparticle-encapsulated messenger RNA encoding the heavy and light chains of a CHIKV-specific monoclonal neutralizing antibody, CHKV-24 ( NCT03829384 ). The primary outcome was to evaluate the safety and tolerability of escalating doses of mRNA-1944 administered via intravenous infusion in healthy participants aged 18-50 years. The secondary objectives included determination of the pharmacokinetics of mRNA encoding for CHKV-24 immunoglobulin heavy and light chains and ionizable amino lipid component and the pharmacodynamics of mRNA-1944 as assessed by serum concentrations of mRNA encoding for CHKV-24 immunoglobulin G (IgG), plasma concentrations of ionizable amino lipid and serum concentrations of CHKV-24 IgG. Here we report the results of a prespecified interim analysis of 38 healthy participants who received intravenous single doses of mRNA-1944 or placebo at 0.1, 0.3 and 0.6 mg kg-1, or two weekly doses at 0.3 mg kg-1. At 12, 24 and 48 h after single infusions, dose-dependent levels of CHKV-24 IgG with neutralizing activity were observed at titers predicted to be therapeutically relevant concentrations (≥1 µg ml-1) across doses that persisted for ≥16 weeks at 0.3 and 0.6 mg kg-1 (mean t1/2 approximately 69 d). A second 0.3 mg kg-1 dose 1 week after the first increased CHKV-24 IgG levels 1.8-fold. Adverse effects were mild to moderate in severity, did not worsen with a second mRNA-1944 dose and none were serious. To our knowledge, mRNA-1944 is the first mRNA-encoded monoclonal antibody showing in vivo expression and detectable ex vivo neutralizing activity in a clinical trial and may offer a treatment option for CHIKV infection. Further evaluation of the potential therapeutic use of mRNA-1944 in clinical trials for the treatment of CHIKV infection is warranted.

Co-Silencing of Tissue Transglutaminase-2 and Interleukin-15 Genes in a Celiac Disease Mimetic Mouse Model Using a Nanoparticle-in-Microsphere Oral System.

Celiac disease is a chronic inflammatory condition characterized by activation of the immune system in response to deamidation of gluten peptides brought about by tissue transglutaminase-2 (TG2). Overexpression of interleukin-15 (IL-15) in the intestinal epithelium and the lamina propria leads to the dysregulation of the immune system, leading to epithelial damage. The goal of this study was to develop an RNA interference therapeutic strategy for celiac disease using a combination of TG2 and IL-15 gene silencing in the inflamed intestine. TG2 and IL-15 silencing siRNA sequences, along with scrambled control, were encapsulated in a nanoparticle-in-microsphere oral system (NiMOS) and administered in a poly(I:C) mouse model of celiac disease. Single TG2 and IL-15 siRNA therapy and the combination showed effective gene silencing in vivo. Additionally, it was found that IL-15 gene silencing alone and combination in the NiMOS significantly reduced other proinflammatory cytokines. The tissue histopathology data also confirmed a reduction in immune cell infiltration and restoration of the mucosal architecture and barrier function in the intestine upon treatment. Overall, the results of this study show evidence that celiac disease can be potentially treated with an oral microsphere formulation using a combination of TG2 and IL-15 RNA interference therapeutic strategies.

Single-Dose Pharmacokinetics and Pharmacodynamics of Transthyretin Targeting N-acetylgalactosamine-Small Interfering Ribonucleic Acid Conjugate, Vutrisiran, in Healthy Subjects.

Vutrisiran (ALN-TTRsc02) is a liver-directed, investigational, small interfering ribonucleic acid drug for the treatment of transthyretin (TTR)-mediated amyloidosis. This phase I, randomized, single-blind, placebo-controlled, single ascending dose study evaluated the pharmacodynamics, pharmacokinetics, and safety profile of subcutaneously administered vutrisiran (5-300 mg) in healthy subjects (n = 80). Vutrisiran treatment achieved potent and sustained TTR reduction in a dose-dependent manner, with mean maximum TTR reduction of 57-97%, maintained for ≥ 90 days post dose. Vutrisiran was rapidly absorbed (peak plasma concentration 3-5 hours post dose), had a short plasma half-life (4.2-7.5 hours), and plasma concentrations increased in a dose-proportional manner. Pharmacodynamic and pharmacokinetic results were similar in Japanese and non-Japanese subjects. Vutrisiran had an acceptable safety profile; the most common treatment-related adverse event was mild, transient injection site reactions in four (6.7%) vutrisiran-treated subjects. The favorable pharmacokinetic, pharmacodynamic, and safety results observed here support vutrisiran's continued clinical development.

Pharmacokinetics and Biodistribution Analysis of Small Interference RNA for Silencing Tissue Transglutaminase-2 in Celiac Disease After Oral Administration in Mice Using Gelatin-Based Multicompartmental Delivery Systems.

Background: RNA interference (RNAi) therapy has tremendous potential in treating diseases that are characterized by overexpression of genes. However, the biggest challenge to utilize the therapy is to engineer delivery systems that can efficiently transport small interfering RNA (siRNA) to appropriate target sites. Our objective in this study was to develop and evaluate multi-compartmental systems for the oral delivery of siRNA that targets the overexpressed TG2 gene (TG2-siRNA) in the small intestine for the treatment of celiac disease (CD). Materials and Methods: Two types of multicompartmental systems were developed and evaluated: (1) a solid-in-solid multicompartmental system featuring "nanoparticle in microsphere oral system (NiMOS)" where type B gelatin nanoparticles containing TG2-siRNA (TG2-NiMOS) were encapsulated within poly(ɛ-caprolactone) (PCL) based microspheres, and (2) a solid-in-liquid multicompartmental system, "Nanoparticle-in-Emulsion (NiE)" consisting of type-B gelatin nanoparticles containing TG2-siRNA encapsulated within safflower oil containing water-in-oil-in-water (W/O/W) multiple emulsion (TG2-NiE). Results: Evaluation of the biodistribution and pharmacokinetics (PK) after a single oral dose of siRNA containing multicompartmental systems to C57BL/6 mice showed that TG2-siRNA was delivered to the small intestine (duodenum, jejunum and ileum), and colon with minimal systemic exposure via both TG2-NiE and TG2-NiMOS systems. TG2-siRNA exposure (AUC0-t) in the duodenum, jejunum, ileum and colon was 56.4-, 34.3-, 85.5- and 35.5-fold greater for the TG2-NiMOS formulation, relative to the TG2-NiE formulation. Conclusion: The results of this study suggest that TG2-NiMOS formulation was more superior than TG2-NiE formulation in facilitating intestinal delivery of siRNA via the oral route of administration and can be potentially used in the treatment of CD.

Patisiran Pharmacokinetics, Pharmacodynamics, and Exposure-Response Analyses in the Phase 3 APOLLO Trial in Patients With Hereditary Transthyretin-Mediated (hATTR) Amyloidosis.

Hereditary transthyretin-mediated (hATTR) amyloidosis is an inherited, rapidly progressive, life-threatening disease caused by deposition of abnormal transthyretin protein. Patisiran is an RNA interference therapeutic comprising a novel, small interfering ribonucleic acid (ALN-18328) formulated in a lipid nanoparticle targeted to inhibit hepatic transthyretin protein synthesis. The lipid nanoparticle also contains 2 novel lipid excipients (DLin-MC3-DMA and PEG2000 -C-DMG). Here we report patisiran pharmacokinetics (PK), pharmacodynamics (PD), and exposure-response analyses from the phase 3 APOLLO trial, in which patients with hATTR amyloidosis with polyneuropathy were randomized 2:1 to receive patisiran 0.3 mg/kg or placebo intravenously every 3 weeks over 18 months. In patisiran-treated patients, mean maximum reduction in serum transthyretin level from baseline was 87.8%. Patisiran PK exposure was stable following chronic dosing. There were no meaningful differences in PK exposure, serum transthyretin reduction, and efficacy (change from baseline in modified Neuropathy Impairment Score+7) across all subgroups analyzed (age, sex, race, body weight, genotype status of valine-to-methionine mutation at position 30 [V30M] and non-V30M, prior use of tetramer stabilizers, mild/moderate renal impairment, and mild hepatic impairment). transthyretin reduction and efficacy were similar across the interpatient PK exposure range for ALN-18328. There was no trend in the incidence of adverse events or serious adverse events across the interpatient PK exposure range for all 3 analytes. Incidence of antidrug antibodies was low (3.4%) and transient, with no impact on PK, PD, efficacy, or safety. The patisiran dosing regimen of 0.3 mg/kg every 3 weeks is appropriate for all patients with hATTR amyloidosis.

Oral nucleic acid therapy using multicompartmental delivery systems.

Nucleic acid-based therapeutics has the potential for treating numerous diseases by correcting abnormal expression of specific genes. Lack of safe and efficacious delivery strategies poses a major obstacle limiting clinical advancement of nucleic acid therapeutics. Oral route of drug administration has greater delivery challenges, because the administered genes or oligonucleotides have to bypass degrading environment of the gastrointestinal (GI) tract in addition to overcoming other cellular barriers preventing nucleic acid delivery. For efficient oral nucleic acid delivery, vector should be such that it can protect encapsulated material during transit through the GI tract, facilitate efficient uptake and intracellular trafficking at desired target sites, along with being safe and well tolerated. In this review, we have discussed multicompartmental systems for overcoming extracellular and intracellular barriers to oral delivery of nucleic acids. A nanoparticles-in-microsphere oral system-based multicompartmental system was developed and tested for in vivo gene and small interfering RNA delivery for treating colitis in mice. This system has shown efficient transgene expression or gene silencing when delivered orally along with favorable downstream anti-inflammatory effects, when tested in a mouse model of intestinal bowel disease. WIREs Nanomed Nanobiotechnol 2018, 10:e1478. doi: 10.1002/wnan.1478 This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

Cosilencing Intestinal Transglutaminase-2 and Interleukin-15 Using Gelatin-Based Nanoparticles in an in Vitro Model of Celiac Disease.

In this study, we have developed a type B gelatin nanoparticle based siRNA delivery system for silencing of intestinal transglutaminase-2 (TG2) and interleukin-15 (IL-15) genes in cultured human intestinal epithelial cells (Caco-2) and murine alveolar macrophage cells (J774A.1). Small interfering RNA (siRNA) targeting the TG2 or IL-15 gene was encapsulated within gelatin nanoparticles using ethanol-water solvent displacement method. Size, charge, and morphology of gelatin nanoparticles were evaluated using a Zetasizer instrument and transmission electron microscopy. siRNA encapsulation efficiency was determined using an siRNA specific stem-loop quantitative polymerase chain reaction (qPCR) assay. Cellular uptake of siRNA-containing gelatin nanoparticles was determined using fluorescent microscopy and stem-loop qPCR assay. siRNA loading in the RISC (RNA-induced silencing complex) was determined by immunoprecipitation of argonaute 2 (AGO2) protein followed by stem-loop qPCR for siRNA quantification. Gene expression analysis of TG2, IL-15, and the proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), was performed using qPCR assays. Efficacy of silencing TG2 and IL-15 knockdown was evaluated in an in vitro model of celiac disease by utilizing immunogenic α-gliadin peptide p31-43 in cultured J774A.1 cells. siRNA-containing gelatin nanoparticles were spherical in shape with mean particle size and charge of 217 ± 8.39 nm and -6.2 ± 0.95 mV, respectively. siRNA loading efficiency within gelatin nanoparticles was found to be 89.3 ± 3.05%. Evaluations of cellular uptake using fluorescent microscopy showed rapid internalization of gelatin nanoparticles within 2 h of dosing, with cytosolic localization of delivered siRNA in Caco-2 cells. Gelatin nanoparticles showed greater intracellular siRNA exposure with a longer half-life, when compared to Lipofectamine-mediated siRNA delivery. Approximately 0.1% of total intracellular siRNA was associated in the RISC complex. A maximum knockdown of 60% was observed at 72 h post siRNA treatment for both TG2 and IL-15 genes, which corresponded to ∼200 copies of RISC associated siRNA. Further, efficacy of gelatin nanoparticle mediated knockdown of TG2 and IL-15 mRNA was tested in an in vitro model of celiac disease. Significant suppression in the levels of proinflammatory cytokines (TNF-α and IFN-γ) was observed in p31-43 stimulated J774A.1 cells upon either IL-15 or IL-15 + TG2 siRNA treatment. The results from this study indicate that gelatin nanoparticle mediated TG2 and IL-15 siRNA gene silencing is a very promising approach for the treatment of celiac disease.

An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia.

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

Multi-compartmental oral delivery systems for nucleic acid therapy in the gastrointestinal tract.

Gene and RNA interference therapies have significant potential for alleviating countless diseases, including many associated with the gastro-intestinal (GI) tract. Unfortunately, oral delivery of genes and small interfering RNA (siRNA) is very challenging due to the extracellular and intracellular barriers. In this review, we discuss the utilization of multi-compartmental delivery systems for oral administration of nucleic acid therapies. Some of the illustrative examples of multi-compartmental systems include solid nanoparticles-in-microsphere, solid nanoparticles-in-emulsion, and liquid nanoparticles-in-emulsion. Using type B gelatin nanoparticles encapsulated in poly(ε-caprolactone) microspheres, we have prepared nanoparticles-in-microsphere oral system (NiMOS) for gene and siRNA delivery for the treatment of inflammatory bowel disease (IBD). The results of these studies show that the multi-compartmental formulations can overcome many of the barriers for effective oral gene and siRNA delivery.

Multi-compartmental nanoparticles-in-emulsion formulation for macrophage-specific anti-inflammatory gene delivery.

PURPOSE: To develop a safe and effective non-viral vector for gene delivery and transfection in macrophages for potential anti-inflammatory therapy. METHODS: Solid nanoparticles-in-emulsion (NiE) multi-compartmental delivery system was designed using plasmid DNA-encapsulated type B gelatin nanoparticles suspended in the inner aqueous phase of safflower oil-containing water-in-oil-in-water (W/O/W) multiple emulsion. Control and NiE formulations were evaluated for DNA delivery and transfection efficiency in J774A.1 adherent murine macrophages. RESULTS: Using green fluorescent protein (GFP) and murine interleukin-10 (mIL-10) expressing plasmid DNA constructs, the NiE formulation was found superior in enhancing intracellular delivery and gene transfection efficiency in cells. Anti-inflammatory effects of transfected mIL-10 were examined by suppression of tumor necrosis factor-alpha (TNFα) and interleukin 1-beta (IL-1ß) production in lipopolysaccharide (LPS)-stimulated cells. CONCLUSIONS: Overall, the results were very encouraging towards development of a macrophage-specific NiE-based multi-compartmental gene delivery strategy that can potentially affect a number of acute and chronic inflammatory diseases.

Role of antibodies in cancer targeting.

The development of chemotherapeutic agents capable of specifically eliminating tumor cells has been a great challenge since these agents cannot differentiate between normal body cells and tumor cells. Enhanced elimination of cancer cells without affecting normal body cells can be achieved by developing strategies which can enable drug targeting. With recent advances in antibody engineering strategies, the development of different antibody-associated tumor-targeted delivery systems for chemotherapy, chemoprevention, and early cancer diagnosis has become possible. In this review, the role of antibodies for cancer diagnosis, chemoprevention, and chemotherapy will be discussed with an emphasis on recent advances in antibody engineering.