Engineered tRNAs suppress nonsense mutations in cells and in vivo.

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases. Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation.

Liver lipopolysaccharide binding protein prevents hepatic inflammation in physiological and pathological non-obesogenic conditions.

Lipopolysaccharide binding protein (LBP) knockout mice models are protected against the deleterious effects of major acute inflammation but its possible physiological role has been less well studied. We aimed to evaluate the impact of liver LBP downregulation (using nanoparticles containing siRNA- Lbp) on liver steatosis, inflammation and fibrosis during a standard chow diet (STD), and in pathological non-obesogenic conditions, under a methionine and choline deficient diet (MCD, 5 weeks). Under STD, liver Lbp gene knockdown led to a significant increase in gene expression markers of liver inflammation (Itgax, Tlr4, Ccr2, Ccl2 and Tnf), liver injury (Krt18 and Crp), fibrosis (Col4a1, Col1a2 and Tgfb1), endoplasmic reticulum (ER) stress (Atf6, Hspa5 and Eif2ak3) and protein carbonyl levels.

Efficacy increase of lipid nanoparticles by inclusion of bis(monoacylglycerol)phosphate.

To investigate the effect of incorporating bis(monoacylglycerol)phosphate (BMP) lipid into a lipid nanoparticle and the functional transport of mRNA by the formulated nanoparticles . The nanoparticles were prepared from ionizable lipid, 1,2-distearoyl--glycerol-3-phosphocholine, cholesterol, 1,2-dimyristoyl--glycerol PEG 2000, BMP and formulated mRNA encoding human erythropoietin. We measured the effect of BMP on physicochemical properties and impact on functional efficacy to transport mRNA to its target cells/tissue as measured by protein expression both and .

Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation.

Circulating lipopolysaccharide-binding protein (LBP) is increased in individuals with liver steatosis. We aimed to evaluate the possible impact of liver LBP downregulation using lipid nanoparticle-containing chemically modified LBP small interfering RNA (siRNA) (LNP- UNA-siRNA) on the development of fatty liver. Weekly LNP- UNA-siRNA was administered to mice fed a standard chow diet, a high-fat and high-sucrose diet, and a methionine- and choline-deficient diet (MCD).

Downregulation of peripheral lipopolysaccharide binding protein impacts on perigonadal adipose tissue only in female mice.

Background And Aims: The sexual dimorphism in fat-mass distribution and circulating leptin and insulin levels is well known, influencing the progression of obesity-associated metabolic disease. Here, we aimed to investigate the possible role of lipopolysaccharide-binding protein (LBP) in this sexual dimorphism.

Methods: The relationship between plasma LBP and fat mass was evaluated in 145 subjects.

Lipid nanoparticle delivers phenylalanine ammonia lyase mRNA to the liver leading to catabolism and clearance of phenylalanine in a phenylketonuria mouse model.

Phenylketonuria (PKU) is a genetic disorder affecting around 1 in 12,000 live births (1), caused by a mutation in the phenylalanine hydroxylase (PAH) gene in the liver which facilitates the catabolism of phenylalanine (Phe). Without a functional copy of PAH, levels of Phe in the blood and tissues rise, resulting in potentially life-threatening damage to the central nervous system. (2) Treatment options for PKU are limited, and center around adherence to a strict PKU diet that suffers from poor patient compliance.

Hepatocyte-specific activity of TSC22D4 triggers progressive NAFLD by impairing mitochondrial function.

Objective: Fibrotic organ responses have recently been identified as long-term complications in diabetes. Indeed, insulin resistance and aberrant hepatic lipid accumulation represent driving features of progressive non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis and non-alcoholic steatohepatitis (NASH) to fibrosis. Effective pharmacological regimens to stop progressive liver disease are still lacking to-date.

Development of an mRNA replacement therapy for phenylketonuria.

Phenylketonuria (PKU) is an inborn error caused by deficiencies in phenylalanine (Phe) metabolism. Mutations in the phenylalanine hydroxylase (PAH) gene are the main cause of the disease whose signature hallmarks of toxically elevated levels of Phe accumulation in plasma and organs such as the brain, result in irreversible intellectual disability. Here, we present a unique approach to treating PKU deficiency by using an mRNA replacement therapy.

Synthesis and bioactivity of readily hydrolysable novel cationic lipids for potential lung delivery application of mRNAs.

Lipid nanoparticles (LNPs) mediated mRNA delivery has gained prominence due to the success of mRNA vaccines against Covid-19, without which it would not have been possible. However, there is little clinical validation of this technology for other mRNA-based therapeutic approaches. Systemic administration of LNPs predominantly targets the liver, but delivery to other organs remains a challenge.

A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice.

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression.

Selective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS.

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by expansion of cytosine-adenine-guanine (CAG)-trinucleotide repeats in causative genes. These diseases include spinal and bulbar muscular atrophy (SBMA), Huntington's disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxias. Targeting expanded CAG repeats is a common therapeutic approach to polyQ diseases, but concomitant silencing of genes with normal CAG repeats may lead to toxicity.

CD8 T cells mediate protection against Zika virus induced by an NS3-based vaccine.

Zika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8 T cell response in the HLA-B*0702 transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen.

Property-Driven Design and Development of Lipids for Efficient Delivery of siRNA.

Ionizable cationic lipids are critical components involved in nanoparticle formulations, which are utilized in delivery platforms for RNA therapeutics. While general criteria regarding lipophilicity and measured p in formulation are understood to have impacts on utility , greater granularity with respect to the impacts of the structure on calculated and measured physicochemical parameters and the subsequent performance of those ionizable cationic lipids in studies would be beneficial. Herein, we describe structural alterations made within a lipid class exemplified by , which allow us to tune calculated and measured physicochemical parameters for improved performance, resulting in substantial improvements versus the state of the art at the outset of these studies, resulting in good activity within a range of measured basicity (p = 6.

Anti-HFRS Human IgG Produced in Transchromosomic Bovines Has Potent Hantavirus Neutralizing Activity and Is Protective in Animal Models.

We explored an emerging technology to produce anti-Hantaan virus (HTNV) and anti-Puumala virus (PUUV) neutralizing antibodies for use as pre- or post-exposure prophylactics. The technology involves hyperimmunization of transchomosomic bovines (TcB) engineered to express human polyclonal IgG antibodies with HTNV and PUUV DNA vaccines encoding GG glycoproteins. For the anti-HTNV product, TcB was hyperimmunized with HTNV DNA plus adjuvant or HTNV DNA formulated using lipid nanoparticles (LNP).

Lipid Nanoparticle Formulation Increases Efficiency of DNA-Vectored Vaccines/Immunoprophylaxis in Animals Including Transchromosomic Bovines.

The use of nucleic acid as a drug substance for vaccines and other gene-based medicines continues to evolve. Here, we have used a technology originally developed for mRNA in vivo delivery to enhance the immunogenicity of DNA vaccines. We demonstrate that neutralizing antibodies produced in rabbits and nonhuman primates injected with lipid nanoparticle (LNP)-formulated Andes virus or Zika virus DNA vaccines are elevated over unformulated vaccine.

Anti-miR-17 therapy delays tumorigenesis in MYC-driven hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options. Genomic amplification and/or overexpression of the MYC oncogene is a common molecular event in HCC, thus making it an attractive target for drug therapy. Unfortunately, currently there are no direct drug therapies against MYC.

Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG).

Systemic delivery of factor IX messenger RNA for protein replacement therapy.

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX ()-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care.

Lipid Nanoparticle-Mediated Delivery of Anti-miR-17 Family Oligonucleotide Suppresses Hepatocellular Carcinoma Growth.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies with poor prognosis and urgent unmet medical need. Aberrant expression of multiple members of the miR-17 family are frequently observed in HCC, and their overexpression promotes tumorigenic properties of HCC cells. However, whether pharmacologic inhibition of the miR-17 family inhibits HCC growth remains unknown.

Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic.

MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes, while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells, including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously, the common mechanism conferring the miR-10b-sustained viability of GSC is unknown.

Examples of Tumor Growth Inhibition Properties of Liposomal Formulations of pH-Sensitive Histidinylated Cationic Amphiphiles.

Herein we report on the unexpected cancer cell selective cytotoxicities of the liposomal formulations of aspartic and glutamic acid backbone-based four novel lipids with endosomal pH-sensitive head-groups and aliphatic n-hexadecyl & n-octadecyl hydrophobic tails. Surprisingly, although the formulations killed cancer cells efficiently, they were significantly less cytotoxic in non-cancerous healthy cells. Importantly, intratumoral administration of the liposomal formulations efficiently inhibited growth of melanoma in a syngeneic C57BL/6J mouse tumor model.

MicroRNA-10b inhibition reduces E2F1-mediated transcription and miR-15/16 activity in glioblastoma.

MicroRNA-10b (miR-10b) is commonly elevated in glioblastoma (GBM), while not expressed in normal brain tissues. Targeted inhibition of miR-10b has pleiotropic effects on GBM derived cell lines, it reduces GBM growth in animal models, but does not affect normal neurons and astrocytes. This data raises the possibility of developing miR-10b-targeting GBM therapy.

Direct recognition of superparamagnetic nanocrystals by macrophage scavenger receptor SR-AI.

Scavenger receptors (SRs) are molecular pattern recognition receptors that have been shown to mediate opsonin-independent uptake of therapeutic and imaging nanoparticles, underlying the importance of SRs in nanomedicine. Unlike pathogens, engineered nanomaterials offer great flexibility in control of surface properties, allowing addressing specific questions regarding the molecular mechanisms of nanoparticle recognition. Recently, we showed that SR-type AI/II mediates opsonin-independent internalization of dextran superparamagnetic iron oxide (SPIO) nanoparticles via positively charged extracellular collagen-like domain.