Messenger RNA (mRNA) has broad potential for application in biological systems. However, one fundamental limitation to its use is its relatively short half-life in biological systems. Here we develop ...exogenous circular RNA (circRNA) to extend the duration of protein expression from full-length RNA messages. First, we engineer a self-splicing intron to efficiently circularize a wide range of RNAs up to 5 kb in length in vitro by rationally designing ubiquitous accessory sequences that aid in splicing. We maximize translation of functional protein from these circRNAs in eukaryotic cells, and we find that engineered circRNA purified by high performance liquid chromatography displays exceptional protein production qualities in terms of both quantity of protein produced and stability of production. This study pioneers the use of exogenous circRNA for robust and stable protein expression in eukaryotic cells and demonstrates that circRNA is a promising alternative to linear mRNA.
The rapid expansion of the available genomic data continues to greatly impact biomedical science and medicine. Fulfilling the clinical potential of genetic discoveries requires the development of ...therapeutics that can specifically modulate the expression of disease-relevant genes. RNA-based drugs, including short interfering RNAs and antisense oligonucleotides, are particularly promising examples of this newer class of biologics. For over two decades, researchers have been trying to overcome major challenges for utilizing such RNAs in a therapeutic context, including intracellular delivery, stability, and immune response activation. This research is finally beginning to bear fruit as the first RNA drugs gain FDA approval and more advance to the final phases of clinical trials. Furthermore, the recent advent of CRISPR, an RNA-guided gene-editing technology, as well as new strides in the delivery of messenger RNA transcribed in vitro, have triggered a major expansion of the RNA-therapeutics field. In this review, we discuss the challenges for clinical translation of RNA-based therapeutics, with an emphasis on recent advances in delivery technologies, and present an overview of the applications of RNA-based drugs for modulation of gene/protein expression and genome editing that are currently being investigated both in the laboratory as well as in the clinic.
Circular RNAs (circRNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular ...proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.
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•Triphosphorylated and linear RNA contaminants provoke innate immune responses•Purified exogenous circRNA does not trigger RIG-I•circRNA does not trigger TLRs in overexpression cell lines•Nanoformulated circRNA is translated in mouse tissues
Wesselhoeft et al. find that exogenous circular RNAs are able to bypass RNA sensors, thereby avoiding antiviral defense induction upon cellular entry. They report that nanoformulated, synthetic protein-coding circRNA can be translated in mouse tissues, providing evidence for the potential of circRNA as a vector for therapeutic gene expression.
The utility of messenger RNA (mRNA) as a therapy is gaining a broad interest due to its potential for addressing a wide range of diseases, while effective delivery of mRNA molecules to various ...tissues still poses a challenge. This study reports on the design and characterization of new ionizable amino‐polyesters (APEs), synthesized via ring opening polymerization (ROP) of lactones with tertiary amino‐alcohols that enable tissue and cell type selective delivery of mRNA. With a diverse library of APEs formulated into lipid nanoparticles (LNP), structure‐activity parameters crucial for efficient transfection are established and APE‐LNPs are identified that can preferentially home to and elicit effective mRNA expression with low in vivo toxicity in lung endothelium, liver hepatocytes, and splenic antigen presenting cells, including APE‐LNP demonstrating nearly tenfold more potent systemic mRNA delivery to the lungs than vivo‐jetPEI. Adopting tertiary amino‐alcohols to initiate ROP of lactones allows to control polymer molecular weight and obtain amino‐polyesters with narrow molecular weight distribution, exhibiting batch‐to‐batch consistency. All of which highlight the potential for clinical translation of APEs for systemic mRNA delivery and demonstrate the importance of employing controlled polymerization in the design of new polymeric nanomaterials to improve in vivo nucleic acid delivery.
Most polymeric carriers have been developed for short RNAs, while effective delivery of significantly larger mRNA molecules to various tissues poses an additional challenge. This study reports on the design and characterization of new ionizable amino‐polyesters, synthesized via ring opening polymerization of lactones with tertiary amino‐alcohols, that enable tissue and cell type selective delivery of mRNA.
Smart biomaterials have the ability to respond to changes in physiological parameters and exogenous stimuli and continue to impact many aspects of modern medicine. Smart materials can promote ...promising therapies and improve treatment of debilitating diseases. Here, we describe recent advances in the current state-of-the-art design and application of smart biomaterials in tissue engineering, drug delivery systems, medical devices, and immune engineering.
Future application of conjugated (co)polymers, e.g. in electronic devices, requires the availability of up-scalable synthetic procedures. “Conventional” (hetero)aryl-(hetero)aryl coupling schemes ...often produce toxic or environmentally risky by-products. Here, so-called direct arylation schemes without use of organometallic reagents or related anion equivalents came in the focus of attention as simplified alternative to standard coupling procedures. Now, a couple of structurally defined conjugated (co)polymers can be generated in high yield and molecular weight via direct arylation polycondensation. The promising application potential of these materials as active component of organic solar cells has been demonstrated.
The aim of this work is to promote ultrasound as an energy source that is suitable for the enhancement of drying processes, and in particular of biological materials. The study aims at a more ...profound recognition of the interaction between ultrasonic (US) waves and the tissues of fruits and vegetables, which may contribute to an intensification of moisture removal during their drying. Absorption of acoustic energy causes heating and structural changes of the drying material due to a series of rapid material compressions and decompressions. The research hypothesis is based on the expectation that the ultrasound waves may enhance moisture removal from the fruits and vegetables during drying due to a special “heating effect,” “vibration effect,” and “synergistic effect.” Convective ultrasonic-assisted drying tests were conducted experimentally on a new hybrid dryer with ultrasonic equipment, and the effects of ultrasonic enhancement by drying are presented based on a drying model and assessed numerically.
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Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
•PN/AMX was piloted in a two-stage configuration treating real municipal wastewater.•PN was combined with carbon removal to achieve high-rate treatment.•PN was successful in MBBR and controlled with ...DO/TAN ratio and FA concentration.•Nitrogen removal rate averaged in IFAS reactor averaged 0.45 ± 0.10 g N m−2 d−1.•SpAA of the biofilm was equal to 187 ± 46 mg-N g-VSS−1 d−1 at 20 °C.
A novel partial nitritation-anammox (PNA) reactor configuration was piloted for 250 days. Primary effluent from full-scale municipal wastewater treatment plant was treated in a two-stage biofilm system incorporating innovative process control for cold partial nitritation. Partial nitritation was combined with carbon removal in a moving bed biofilm reactor (MBBR) to achieve high-rate treatment and nitritation was obtained with dissolved oxygen to total ammonium nitrogen (DO/TAN) ratio control and free ammonia (FA) for inhibition of nitratation. Effluent from MBBR was directed to an integrated fixed-film activated sludge (IFAS) reactor where nitrogen was removed via anammox. MBBR achieved partial nitritation at 2.0 ± 0.3 g-N m−2 d−1 and nitrogen removal in the IFAS reactor reached 0.45 ± 0.1 g-N m−2 d−1 (55 g-N m−3 d−1). The process performed well at 19 ± 3 °C with an average effluent total inorganic nitrogen (TIN) concentration of 11 ± 4 mg L−1.
The law of conservation of linear momentum is applied to surface gas exchanges, employing scale analysis to diagnose the vertical velocity (w) in the boundary layer. Net upward momentum in the ...surface layer is forced by evaporation (E) and defines non-zero vertical motion, with a magnitude defined by the ratio of E to the air density, as w = E/ρ. This is true even right down at the surface where the boundary condition is w|0 = E/ρ|0 (where w|0 and ρ|0 represent the vertical velocity and density of air at the surface). This Stefan flow velocity implies upward transport of a non-diffusive nature that is a general feature of the troposphere but is of particular importance at the surface, where it assists molecular diffusion with upward gas migration (of H2O, for example) but opposes that of downward-diffusing species like CO2 during daytime. The definition of flux–gradient relationships (eddy diffusivities) requires rectification to exclude non-diffusive transport, which does not depend on scalar gradients. At the microscopic scale, the role of non-diffusive transport in the process of evaporation from inside a narrow tube – with vapour transport into an overlying, horizontal airstream – was described long ago in classical mechanics and is routinely accounted for by chemical engineers, but has been neglected by scientists studying stomatal conductance. Correctly accounting for non-diffusive transport through stomata, which can appreciably reduce net CO2 transport and marginally boost that of water vapour, should improve characterisations of ecosystem and plant functioning.
Implantable medical devices have revolutionized modern medicine. However, immune-mediated foreign body response (FBR) to the materials of these devices can limit their function or even induce ...failure. Here we describe long-term controlled-release formulations for local anti-inflammatory release through the development of compact, solvent-free crystals. The compact lattice structure of these crystals allows for very slow, surface dissolution and high drug density. These formulations suppress FBR in both rodents and non-human primates for at least 1.3 years and 6 months, respectively. Formulations inhibited fibrosis across multiple implant sites-subcutaneous, intraperitoneal and intramuscular. In particular, incorporation of GW2580, a colony stimulating factor 1 receptor inhibitor, into a range of devices, including human islet microencapsulation systems, electrode-based continuous glucose-sensing monitors and muscle-stimulating devices, inhibits fibrosis, thereby allowing for extended function. We believe that local, long-term controlled release with the crystal formulations described here enhances and extends function in a range of medical devices and provides a generalized solution to the local immune response to implanted biomaterials.
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