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Fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense as well as stop codons (UGA, UAG, UAA), which are usually localized at the 30 of mRNA and drive the release of the polypeptide chain.However, either natural (NTCs) or premature (PTCs) termination codons, the latter arising from nucleotide changes, can undergo a recoding process named ribosome or translational readthrough, which insert specific amino acids (NTCs) or subset(s) depending on the stop codon type (PTCs). This process is particularly relevant for nonsense mutations, a relatively frequent cause of genetic disorders, which impair gene expression at different levels by potentially leading to mRNA degradation and/or synthesis of truncated proteins. As a matter of fact, many efforts have been made to develop efficient and safe readthrough-inducing compounds, which have been challenged in several models of human disease to provide with a therapy. In this view, the dissection of the molecular determinants shaping the outcome of readthrough, namely nucleotide and protein contexts as well as their interplay and impact on protein structure/function, is crucial to identify responsive nonsense mutations resulting in functional full-length proteins. The interpretation of experimental and mechanistic findings is also important to define a possibly clear picture of potential readthrough-favorable features useful to achieve rescue profiles compatible with therapeutic thresholds typical of each targeted disorder, which is of primary importance for the potential translatability of readthrough into a personalized and mutation-specific, and thus patient-oriented, therapeutic strategy.

In hemophilia A, F8 nonsense variants, and particularly those affecting the large factor VIII (FVIII) B domain that is dispensable for coagulant activity, display lower association with replacement therapy-related anti-FVIII inhibitory antibodies as retrieved from multiple international databases. Since null genetic conditions favor inhibitor development, we hypothesized that translational readthrough over premature termination codons (PTC) may contribute to immune tolerance by producing full-length proteins through the insertion of amino acid subset(s). To quantitatively evaluate the readthrough output in vitro, we developed a very sensitive luciferase-based system to detect very low full-length FVIII synthesis from a wide panel (n=45; ~60% patients with PTC) of F8 nonsense variants. PTC not associated with inhibitors displayed higher readthrough-driven expression levels than inhibitor-associated PTC, a novel observation. Particularly, higher levels were detected for B-domain variants (n=20) than for variants in other domains (n=25). Studies on plasma from six hemophilia A patients with PTC, integrated by expression of the corresponding nonsense and readthrough-deriving missense variants, consistently revealed higher FVIII levels for B-domain variants. Only one B-domain PTC (Arg814*) was found among the highly represented PTC not sporadically associated with inhibitors, but with the lowest proportion of inhibitor cases (4 out of 57). These original insights into the molecular genetics of hemophilia A, and particularly into genotype-phenotype relationships related with disease treatment, demonstrate that B-domain features favor PTC readthrough output. This provides a potential molecular mechanism contributing to differential PTC-associated inhibitor occurrence, with translational implications for a novel, experimentally based classification of F8 nonsense variants.

The short half-life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)-mediated recycling of the chimera. However, patients would greatly benefit from further FIX-HSA half-life extension. In the present study, we designed a FIX-HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, which resulted in improved HSA release. Second, insertion of the FIX R338L (Padua) substitution conferred hyperactive features (sevenfold higher specific activity) as for FIX Padua alone. Furthermore, we exploited an engineered HSA (QMP), which conferred enhanced human (h)FcRn binding [dissociation constant (KD) 0·5 nM] over wild-type FIX-HSA (KD 164·4 nM). In hFcRn transgenic mice, Padua-QMP displayed a significantly prolonged half-life (2·7 days, P < 0·0001) versus FIX-HSA (1 day). Overall, we developed a novel FIX-HSA protein with improved activity and extended half-life. These combined properties may result in a prolonged functional profile above the therapeutic threshold, and thus in a potentially widened therapeutic window able to improve HB therapy. This rational engineering of both partners may pave the way for new fusion strategies for the design of engineered biotherapeutics.

Kawasaki disease (KD), although rare (affects about 15 children out of 100,000/year) and self-limiting, it is the second most frequent cause of pediatric acquired heart disease in Western countries. The classic KD is diagnosed by fever and at least 4 clinical signs between conjunctivitis, mucositis, polymorphic rash, erythema and edema of hands and feet or lymphadenopathy; if clinical signs are less than 4 the incomplete KD can be diagnosed together with specific laboratory criteria. Since March 2020, at the time of the greatest spread of the SARS-CoV-2 epidemic in our province, we found over 20 children, admitted to the Pediatrics of our hospital, with clinical pictures very similar to Kawasaki disease. In addition to signs of mucocutaneous inflammation, some children had a significant systemic inflammation with severe heart involvement, up to shock in some cases. In the next months, similar cases were reported in areas of the world with a high spread of the virus, and the scientific community coined the name of Multisystem Inflammatory Syndrome in Children (MIS-C) for this form linked to SARS-CoV-2. KD conventional therapy based on intravenous immunoglobulin, acetylsalicylic acid and systemic steroids resolved the inflammation in all children. This article critically discusses the common characteristics of the two diseases and hypothesizes that they belong to the same pathological condition with a wide spectrum of clinical manifestations; it proposes the diagnostic framework for this new inflammatory condition based on clinical and laboratory characteristics and, finally, outlines the principles of treatment.

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf /ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5′ splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3 ) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf /ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.

Fusion with human serum albumin (HSA), which represents a well-established technique to extend half-life of therapeutic proteins, commonly exploits intervening peptide linkers as key components. Here, we explored the human coagulation factor X (FX) carboxyl-terminal region, previously demonstrated by us to be dispensable for secretion and coagulant activity, as a natural linker for fusion purposes. To test our hypothesis, we compared direct FX-HSA fusion with the designed FX-HSA fusion proteins mimicking the recombinant activated factor VII (rFVIIa)-HSA or factor IX (FIX)-HSA chimeras, both strongly dependent from artificial linkers. Three constructs were produced by direct tandem fusion (FX-HSA) and through flexible (glycine/serine; FX-GS-HSA, mimicking rFVIIa-HSA) or cleavable (incorporating the FX activation site; FX-CL-HSA, mimicking FIX-HSA) linkers. The FX-HSA was efficiently secreted and displayed prolonged plasma persistence in mice. All chimeras possessed remarkable pro-coagulant activity, comparable to FX for FX-HSA (88.7 ± 6.0%) and FX-CL-HSA (98.0 ± 16.4%) or reduced for FX-GS-HSA (55.8 ± 5.4%). Upon incubation with activators, FX-HSA and FX-CL-HSA displayed a correct activation profile while the FX-GS-HSA activation was slightly defective. In fluorogenic-based assays, FX-HSA showed normal activity over time and a specific amidolytic activity (1.0 ± 0.12) comparable to that of FX. Overall, the FX-HSA features indicate that the FX carboxyl-terminal region represents an intrinsic sequence allowing direct tandem fusion. Our results provide the first experimental evidence for i) a coagulation factor fusion protein with biological properties independent from artificial linkers, ii) the suitability of FX carboxyl-terminal region as a natural linker for fusion purposes.

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on hemophilia A, in which the definition of F8 variants’ causative role and association to bleeding phenotypes is crucial for proper classification, genetic counseling, and management of affected individuals. We extensively characterized a large panel of hemophilia A-causing variants (n = 30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed us to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise classification of HA-affected individuals and favored the development of personalized therapeutic approaches.