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Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5' 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

MRX34, a microRNA (miRNA)-based therapy for cancer, has recently entered clinical trials as the first clinical candidate in its class. It is a liposomal nanoparticle loaded with a synthetic mimic of the tumor suppressor miRNA miR-34a as the active pharmaceutical ingredient. To understand the pharmacokinetic properties of the drug and to rationalize an optimal dosing regimen in the clinic, a method is needed to quantitatively detect the miRNA mimic. Here, we report the development and qualification of a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay in support of pharmacokinetic and toxicokinetic assessments in the nonhuman primate. Detection and quantification were performed on total ribonucleic acid (RNA) isolated from whole blood. The qualified range of the standard curve spans 6 orders of magnitude from 2.5 × 10(-7) to 2.5 × 10(-1) ng per reverse transcription (RT) reaction, corresponding to an estimated blood concentration from 6.2 × 10(-5) to 6.2 × 10(1) ng/mL. Our results demonstrate that endogenous as well as the exogenous miR-34a can be accurately and precisely quantified. The assay was used to establish the pharmacokinetic profile of MRX34, showing a favorable residence time and exposure of the miRNA mimic in whole blood from nonhuman primates.

Porous 75:25 poly(D,L-lactide-co-glycolide) scaffolds reinforced with polyglycolide fibers were prepared with mechanical properties tailored for use in articular cartilage repair. Compression testing was performed to investigate the influence of physiological testing conditions, manufacturing method, anisotropic properties due to predominant fiber orientation, amounts of fiber reinforcement (0 to 20 wt, %), and viscoelasticity via a range of strain rates. Using the same testing modality, the mechanical properties of the scaffolds were compared with pig and goat articular cartilage. Results showed that mechanical properties of the scaffolds under physiological conditions (aqueous, 37 degrees C) were much lower than when tested under ambient conditions. The manufacturing method and anisotropy of the scaffolds significantly influenced the mechanical properties. The compressive modulus and yield strength proportionally increased with increasing fiber reinforcement up to 20%. From 0.01 to 10 mm/mm/min strain rate, the compressive modulus increased in a logarithmic fashion, and the yield strength increased in a semi-log fashion. The compressive modulus of the non-reinforced scaffolds was most similar to the pig and goat articular cartilage when compared using similar testing conditions and modality, but the improvement in yield strength using the stiffer scaffolds with fiber reinforcement could provide needed structural support for in vivo loads.