Utilizing AAV-mediated LEAPER 2.0 for programmable RNA editing in non-human primates and nonsense mutation correction in humanized Hurler syndrome mice.

BACKGROUND: The endogenous adenosine deaminases acting on RNA (ADAR) have been harnessed to facilitate precise adenosine-to-inosine editing on RNAs. However, the practicability of this approach for therapeutic purposes is still ambiguous due to the variable expression of intrinsic ADAR across various tissues and species, as well as the absence of all-encompassing confirmation for delivery methods. RESULTS: In this study, we demonstrate that AAV-mediated delivery of circular ADAR-recruiting RNAs (arRNAs) achieves effective RNA editing in non-human primates at dosages suitable for therapy. Within a time frame of 4 to 13 weeks following infection, the editing efficiency in AAV-infected cells can reach approximately 80%, with no discernible toxicity, even at elevated dosages. In addition, when AAV-delivered circular arRNAs are systematically administered to a humanized mouse model of Hurler syndrome, it rectifies the premature stop codon precisely and restores the functionality of IDUA enzyme encoded by the Hurler causative gene in multiple organs. CONCLUSIONS: These discoveries considerably bolster the prospects of employing AAV-borne circular arRNAs for therapeutic applications and exploratory translational research.

Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

A Dynamic Mass Redistribution Assay for the Human Sweet Taste Receptor Uncovers G-Protein Dependent Biased Ligands.

The sweet taste receptor is rather unique, recognizing a diverse repertoire of natural or synthetic ligands, with a surprisingly large structural diversity, and with potencies stretching over more than six orders of magnitude. Yet, it is not clear if different cell-based assays can faithfully report the relative potencies and efficacies of these molecules. Indeed, up to now, sweet taste receptor agonists have been almost exclusively characterized using cell-based assays developed with overexpressed and promiscuous G proteins. This non-physiological coupling has allowed the quantification of receptor activity via phospholipase C activation and calcium mobilization measurements in heterologous cells on a FLIPR system, for example. Here, we developed a novel assay for the human sweet taste receptor where endogenous G proteins and signaling pathways are recruited by the activated receptor. The effects of several sweet taste receptor agonists and other types of modulators were recorded by measuring changes in dynamic mass redistribution (DMR) using an Epic® reader. Potency and efficacy values obtained in the DMR assay were compared to those results obtained with the classical FLIPR assay. Results demonstrate that for some ligands, the two assay systems provide similar information. However, a clear bias for the FLIPR assay was observed for one third of the agonists evaluated, suggesting that the use of non-physiological coupling may influence the potency and efficacy of sweet taste receptor ligands. Replacing the promiscuous G protein with a chimeric G protein containing the C-terminal tail 25 residues of the physiologically relevant G protein subunit Gαgustducin reduced or abrogated bias.

Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo.

Current methods for programmed RNA editing using endogenous ADAR enzymes and engineered ADAR-recruiting RNAs (arRNAs) suffer from low efficiency and bystander off-target editing. Here, we describe LEAPER 2.0, an updated version of LEAPER that uses covalently closed circular arRNAs, termed circ-arRNAs. We demonstrate on average ~3.1-fold higher editing efficiency than their linear counterparts when expressed in cells or delivered as in vitro-transcribed circular RNA oligonucleotides. To lower off-target editing we deleted pairings of uridines with off-target adenosines, which almost completely eliminated bystander off-target adenosine editing. Engineered circ-arRNAs enhanced the efficiency and fidelity of editing endogenous CTNNB1 and mutant TP53 transcripts in cell culture. Delivery of circ-arRNAs using adeno-associated virus in a mouse model of Hurler syndrome corrected the pathogenic point mutation and restored α-L-iduronidase catalytic activity, lowering glycosaminoglycan accumulation in the liver. LEAPER 2.0 provides a new design of arRNA that enables more precise, efficient RNA editing with broad applicability for therapy and basic research.

[Research progress of lactoferrin as drug carriers].

Lactoferrin (Lf) is one of the food protein belonged to the innate immune system. Apart from its main biological function of binding and transport of iron ions, lactoferrin also has many other functions and properties such as antibacterial, antiviral, antiparasitic, catalytic, anti-cancer, anti-allergic and radioprotecting. Lf is usually used as additives of food and cosmetics. The research of lactoferrin has been increasingly reported, and the application of lactoferrin as a drug carrier has drawn extensive attention over the recent year. In this paper, researches of lactoferrin as drug carriers are classified and summarized in brain targeting, liver tumor targeting, lung tumor targeting and oral delivery systems according to their different characteristics.

Brain potentials associated with the outcome processing in framing effects.

Framing effect is a cognitive bias referring to the phenomenon that people respond differently to different but objectively equivalent descriptions of the same problem. By measuring event-related potentials, the present study aimed to investigate the neural mechanisms underlying the framing effect, especially how the negative and positive frames influence the outcome processing in our brain. Participants were presented directly with outcomes framed either positively in terms of lives saved or negatively in terms of lives lost in large and small group conditions, and were asked to rate the favorableness of each of them. The behavioral results showed that the framing effect occurred in both group size conditions, with more favorable evaluations associated with positive framing. Compared with outcomes in positive framing condition, a significant feedback-related negativity (FRN) effect was elicited by outcomes in negative framing condition, even though the outcomes in different conditions were objectively equivalent. The results are explained in terms of the associative model of attribute framing effect which states that attribute framing effect occurs as a result of a valence-based associative processing.

Specific alleles of bitter receptor genes influence human sensitivity to the bitterness of aloin and saccharin.

Variation in human taste is a well-known phenomenon. However, little is known about the molecular basis for it. Bitter taste in humans is believed to be mediated by a family of 25 G protein-coupled receptors (hT2Rs, or TAS2Rs). Despite recent progress in the functional expression of hT2Rs in vitro, up until now, hT2R38, a receptor for phenylthiocarbamide (PTC), was the only gene directly linked to variations in human bitter taste. Here we report that polymorphism in two hT2R genes results in different receptor activities and different taste sensitivities to three bitter molecules. The hT2R43 gene allele, which encodes a protein with tryptophan in position 35, makes people very sensitive to the bitterness of the natural plant compounds aloin and aristolochic acid. People who do not possess this allele do not taste these compounds at low concentrations. The same hT2R43 gene allele makes people more sensitive to the bitterness of an artificial sweetener, saccharin. In addition, a closely related gene's (hT2R44's) allele also makes people more sensitive to the bitterness of saccharin. We also demonstrated that some people do not possess certain hT2R genes, contributing to taste variation between individuals. Our findings thus reveal new examples of variations in human taste and provide a molecular basis for them.

Different functional roles of T1R subunits in the heteromeric taste receptors.

The T1R receptors, a family of taste-specific class C G protein-coupled receptors, mediate mammalian sweet and umami tastes. The structure-function relationships of T1R receptors remain largely unknown. In this study, we demonstrate the different functional roles of T1R extracellular and transmembrane domains in ligand recognition and G protein coupling. Similar to other family C G protein-coupled receptors, the N-terminal Venus flytrap domain of T1R2 is required for recognizing sweeteners, such as aspartame and neotame. The G protein coupling requires the transmembrane domain of T1R2. Surprisingly, the C-terminal transmembrane domain of T1R3 is required for recognizing sweetener cyclamate and sweet taste inhibitor lactisole. Because T1R3 is the common subunit in the sweet taste receptor and the umami taste receptor, we tested the interaction of lactisole and cyclamate with the umami taste receptor. Lactisole inhibits the activity of the human T1R1/T1R3 receptor, and, as predicted, blocked the umami taste of l-glutamate in human taste tests. Cyclamate does not activate the T1R1/T1R3 receptor by itself, but potentiates the receptor's response to l-glutamate. Taken together, these findings demonstrate the different functional roles of T1R3 and T1R2 and the presence of multiple ligand binding sites on the sweet taste receptor.

Identification of ligands for two human bitter T2R receptors.

Earlier, a family of G protein-coupled receptors, termed T2Rs, was identified in the rodent and human genomes through data mining. It was suggested that these receptors mediate bitter taste perception. Analysis of the human genome revealed that the hT2R family is composed of 25 members. However, bitter ligands have been identified for only three human receptors so far. Here we report identification of two novel ligand-receptor pairs. hT2R61 is activated by 6-nitrosaccharin, a bitter derivative of saccharin. hT2R44 is activated by denatonium and 6-nitrosaccharin. Activation profiles for these receptors correlate with psychophysical data determined for the bitter compounds in human studies. Functional analysis of hT2R chimeras allowed us to identify residues in extracellular loops critical for receptor activation by ligands. The discovery of two novel bitter ligand-receptor pairs provides additional support for the hypothesis that hT2Rs mediate a bitter taste response in humans.