Translational readthrough at F8 nonsense variants in the factor VIII B domain contributes to residual expression and lowers inhibitor association.

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.

An advanced method for the small-scale production of high-quality minicircle DNA.

Minicircle DNA is a promising tool in the field of gene therapy, whose products are increasingly gaining market access. Greater transfection efficiency and longer expression time as well as lower immunogenicity contrast with cost-intensive production, which also stands in the way of a broader use of the advantages of this technology in research. Starting from a commercial minicircle production kit a simple protocol for the cost-effective small-scale production of high-quality minicircle DNA to be used at a research scale has been developed by combining and improving procedures of various publications. An optimized size-exclusion chromatography method led to almost pure minicircle DNA with a superior proportion of the desired supercoiled plasmid conformation. The pharmaceutical potential of the produced minicircle DNA was investigated in vitro by real-time impedance assays in a tumor cell model in case of coded suicide genes as well as by ELISA of the translation product in case of coded human coagulation factor IX.

Fusion of engineered albumin with factor IX Padua extends half-life and improves coagulant activity.

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.

An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency.

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.

An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics.

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.

Noncanonical type 2B von Willebrand disease associated with mutations in the VWF D'D3 and D4 domains.

We observed a 55-year-old Italian man who presented with mucosal and cutaneous bleeding. Results of his blood analysis showed low levels of von Willebrand factor (VWF) antigen and VWF activity (both VWF ristocetin cofactor and VWF collagen binding), mild thrombocytopenia, increased ristocetin-induced platelet aggregation, and a deficiency of high-molecular-weight multimers, all typical phenotypic hallmarks of type 2B von Willebrand disease (VWD). The analysis of the VWF gene sequence revealed heterozygous in cis mutations: (1) c.2771G>A and (2) c.6532G>T substitutions in the exons 21 and 37, respectively. The first mutation causes the substitution of an Arg residue with a Gln at position 924, in the D'D3 domain. The second mutation causes an Ala to Ser substitution at position 2178 in the D4 domain. The patient's daughter did not present the same fatherly mutations but showed only the heterozygous polymorphic c.3379C>T mutation in exon 25 of the VWF gene causing the p.P1127S substitution, inherited from her mother. The in vitro expression of the heterozygous in cis VWF mutant rVWFWT/rVWF924Q-2178S confirmed and recapitulated the ex vivo VWF findings. Molecular modeling showed that these in cis mutations stabilize a partially stretched and open conformation of the VWF monomer. Transmission electron microscopy and atomic force microscopy showed in the heterozygous recombinant form rVWFWT/rVWF924Q-2178S a stretched conformation, forming strings even under static conditions. Thus, the heterozygous in cis mutations 924Q/2178S promote conformational transitions in the VWF molecule, causing a type 2B-like VWD phenotype, despite the absence of typical mutations in the A1 domain of VWF.

A Compensatory U1snRNA Partially Rescues FAH Splicing and Protein Expression in a Splicing-Defective Mouse Model of Tyrosinemia Type I.

The elucidation of aberrant splicing mechanisms, frequently associated with disease has led to the development of RNA therapeutics based on the U1snRNA, which is involved in 5' splice site (5'ss) recognition. Studies in cellular models have demonstrated that engineered U1snRNAs can rescue different splicing mutation types. However, the assessment of their correction potential in vivo is limited by the scarcity of animal models with the targetable splicing defects. Here, we challenged the U1snRNA in the FAH5961SB mouse model of hepatic fumarylacetoacetate hydrolase (FAH) deficiency (Hereditary Tyrosinemia type I, HT1) due to the FAH c.706G>A splicing mutation. Through minigene expression studies we selected a compensatory U1snRNA (U1F) that was able to rescue this mutation. Intriguingly, adeno-associated virus-mediated delivery of U1F (AAV8-U1F), but not of U1wt, partially rescued FAH splicing in mouse hepatocytes. Consistently, FAH protein was detectable only in the liver of AAV8-U1F treated mice, which displayed a slightly prolonged survival. Moreover, RNA sequencing revealed the negligible impact of the U1F on the splicing profile and overall gene expression, thus pointing toward gene specificity. These data provide early in vivo proof-of-principle of the correction potential of compensatory U1snRNAs in HTI and encourage further optimization on a therapeutic perspective, and translation to other splicing-defective forms of metabolic diseases.

Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants.

Nonsense mutations are relatively frequent in the rare X-linked lysosomal α-galactosidase A (α-Gal) deficiency (Fabry disease; FD), but have been poorly investigated. Here, we evaluated the responsiveness of a wide panel (n = 14) of GLA premature termination codons (PTCs) to the RNA-based approach of drug-induced readthrough through expression of recombinant α-Gal (rGal) nonsense and missense variants.We identified four high-responders to the readthrough-inducing aminoglycoside G418 in terms of full-length protein (C56X/W209X, ≥10% of wild-type rGal) and/or activity (Q119X/W209X/Q321X, ~5-7%), resulting in normal (Q119X/Q321X) or reduced (C56X, 0.27 ± 0.11; W209X, 0.35 ± 0.1) specific activity.To provide mechanistic insights we investigated the predicted amino acid substitutions mediated by readthrough (W209C/R, C56W/R), which resulted in correct lysosomal localization and appreciable protein/activity levels for the W209C/R variants. Differently, the C56W/R variants, albeit appreciably produced and localized into lysosomes, were inactive, thus indicating detrimental effects of substitutions at this position.Noticeably, when co-expressed with the functional W209C or W209R variants, the wild-type rGal displayed a reduced specific activity (0.5 ± 0.2 and 0.6 ± 0.2, respectively) that, considering the dimeric features of the α-Gal enzyme, suggested dominant-negative effects of missense variants through their interaction with the wild-type.Overall, we provide a novel mechanism through which amino acids inserted during readthrough might impact on the functional protein output. Our findings may also have implications for the interpretation of pathological phenotypes in heterozygous FD females, and for other human disorders involving dimeric or oligomeric proteins.

An Altered Splicing Registry Explains the Differential ExSpeU1-Mediated Rescue of Splicing Mutations Causing Haemophilia A.

The exon recognition and removal of introns (splicing) from pre-mRNA is a crucial step in the gene expression flow. The process is very complex and therefore susceptible to derangements. Not surprisingly, a significant and still underestimated proportion of disease-causing mutations affects splicing, with those occurring at the 5' splice site (5'ss) being the most severe ones. This led to the development of a correction approach based on variants of the spliceosomal U1snRNA, which has been proven on splicing mutations in several cellular and mouse models of human disease. Since the alternative splicing mechanisms are strictly related to the sequence context of the exon, we challenged the U1snRNA-mediated strategy in the singular model of the exon 5 of coagulation factor (F)VIII gene (F8) in which the authentic 5'ss is surrounded by various cryptic 5'ss. This scenario is further complicated in the presence of nucleotide changes associated with FVIII deficiency (Haemophilia A), which weaken the authentic 5'ss and create/strengthen cryptic 5'ss. We focused on the splicing mutations (c.602-32A > G, c.602-10T > G, c.602G > A, c.655G > A, c.667G > A, c.669A > G, c.669A > T, c.670G > T, c.670+1G > T, c.670+1G > A, c.670+2T > G, c.670+5G > A, and c.670+6T > C) found in patients with severe to mild Haemophilia A. Minigenes expression studies demonstrated that all mutations occurring within the 5'ss, both intronic or exonic, lead to aberrant transcripts arising from the usage of two cryptic intronic 5'ss at positions c.670+64 and c.670+176. For most of them, the observed proportion of correct transcripts is in accordance with the coagulation phenotype of patients. In co-transfection experiments, we identified a U1snRNA variant targeting an intronic region downstream of the defective exon (Exon Specific U1snRNA, U1sh7) capable to re-direct usage of the proper 5'ss (∼80%) for several mutations. However, deep investigation of rescued transcripts from +1 and +2 variants revealed only the usage of adjacent cryptic 5'ss, leading to frameshifted transcript forms. These data demonstrate that a single ExSpeU1 can efficiently rescue different mutations in the F8 exon 5, and provide the first evidence of the applicability of the U1snRNA-based approach to Haemophilia A.

Missense changes in the catalytic domain of coagulation factor X account for minimal function preventing a perinatal lethal condition.

INTRODUCTION: Inherited deficiencies in the coagulation pathway provide diversified models to investigate the molecular bases of perinatal lethality associated with null-like variants. Differently from X-linked haemophilias, homozygous/doubly heterozygous null variants in the rare autosomally inherited deficiency of factor X (FX) might be incompatible with perinatal survival. AIM: To provide experimental evidence about the null/close-to-null FX function. METHODS: The residual secreted (ELISA) and functional (thrombin generation assays) protein levels associated with the novel nonsense (c.1382G>A; p.Trp461Ter) and missense (c.752T>C; p.Leu251Pro) variants, found in the proposita with life-threatening symptoms at birth, were characterized through recombinant (r)FX expression. RESULTS: The rFX-461Ter showed very low secretion and undetectable function. Expression and function of the predicted readthrough-deriving missense variants (rFX-461Tyr, rFX-461Gln) were also severely impaired. These unfavourable features, due to nucleotide and protein sequence constraints, precluded functional readthrough over the 461 stop codon. Differently, the poorly secreted rFX-251Pro variant displayed residual function that was characterized by anti-TFPI aptamer-based amplification or selective inhibition of activated FX function by fondaparinux in plasma and found to be reduced by approximately three orders of magnitude. Similarly to the rFX-251Pro, a group of catalytic domain missense variants cause poorly secreted molecules with modest function in FX-deficient patients with life-threatening symptoms. CONCLUSIONS: Our data, contributing to the knowledge of the very severe FX deficiency forms, support life-saving requirement of trace FX function, clearly exemplified by the dysfunctional but not completely inactive rFX-251Pro variant that, albeit with severely reduced function, is compatible with a residual activity ensuring minimal haemostasis and permitting perinatal survival.

The carboxyl-terminal region of human coagulation factor X as a natural linker for fusion strategies.

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 ±â€¯6.0%) and FX-CL-HSA (98.0 ±â€¯16.4%) or reduced for FX-GS-HSA (55.8 ±â€¯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 ±â€¯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.

Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth.

Although mitochondria play a multifunctional role in cancer progression and Ca2+ signaling is remodeled in a wide variety of tumors, the underlying mechanisms that link mitochondrial Ca2+ homeostasis with malignant tumor formation and growth remain elusive. Here, we show that phosphorylation at the N-terminal region of the mitochondrial calcium uniporter (MCU) regulatory subunit MICU1 leads to a notable increase in the basal mitochondrial Ca2+ levels. A pool of active Akt in the mitochondria is responsible for MICU1 phosphorylation, and mitochondrion-targeted Akt strongly regulates the mitochondrial Ca2+ content. The Akt-mediated phosphorylation impairs MICU1 processing and stability, culminating in reactive oxygen species (ROS) production and tumor progression. Thus, our data reveal the crucial role of the Akt-MICU1 axis in cancer and underscore the strategic importance of the association between aberrant mitochondrial Ca2+ levels and tumor development.

Secretion of wild-type factor IX upon readthrough over F9 pre-peptide nonsense mutations causing hemophilia B.

Pre-peptide regions of secreted proteins display wide sequence variability, even among highly homologous proteins such as coagulation factors, and are intracellularly removed, thus potentially favoring secretion of wild-type proteins upon suppression of nonsense mutations (translational readthrough). As models we selected F9 nonsense mutations with readthrough-favorable features affecting the pre-peptide and pro-peptide regions of coagulation factor IX (FIX), which cause hemophilia B (HB). Only the p.Gly21Ter (c.61G > T) in the variable pre-peptide hydrophobic core significantly responded (secretion, 4.1 ± 0.5% of wild-type; coagulant activity, 4.0 ± 0.3%) to the readthrough-inducer geneticin. Strikingly, for the p.Gly21Ter mutation, the resulting specific coagulant activity (0.96 ± 0.11) was compatible with normal function, thus suggesting secretion of FIX with wild-type features upon readthrough and removal of pre-peptide. Expression of the predicted readthrough-deriving missense variants (Gly21Trp/Cys/Arg) revealed a preserved specific activity (ranging from 0.84 to 0.98), thus supporting our observation. Conversely, rescue of the p.Cys28Ter (c.84T > A) and p.Lys45Ter (c.133A > T) was prevented by constraints of adjacent cleavage sites, a finding consistent with the association of most missense mutations affecting these regions with severe or moderate HB. Overall, our data indicate that suppression of nonsense mutations in the pre-peptide core preserves mature protein features, thus making this class of mutations preferred candidates for therapeutic readthrough.

Specific factor IX mRNA and protein features favor drug-induced readthrough over recurrent nonsense mutations.

Drug-induced readthrough over premature stop codons (PTCs) is a potentially attractive therapy for genetic disorders, but a wide outcome variability has been observed. Through expression studies, we investigated the responsiveness to the readthrough-inducing drug geneticin of 11 rationally selected factor IX (FIX) nonsense mutations, present in 70% (324/469) of hemophilia B (HB) patients with PTCs. Among the predicted readthrough-permissive TGA variants, only 2 (p.W240X and p.R384X) responded with a remarkable rescue of FIX activity. The amounts of rescued full-length FIX protein for the p.W240X (∼9% of recombinant FIX [rFIX]-wild-type [WT]) slightly exceeded activity (5.2 ± 0.6%). FIX antigen for the p.R384X (1.9 ± 0.3%) was remarkably lower than activity (7.5 ± 0.7%). Data indicate novel specific mechanisms producing functional rescue: (1) prevalent reinsertion of the authentic residue (tryptophan), reverting the nonsense effects for the p.W240X, and (2) gain-of-function for the p.R384X, supported by the fourfold increased activity of the most probable readthrough-mediated missense variant (rFIX-R384W). For most PTCs, impaired secretion/function produced by readthrough-mediated amino acid substitutions prevented a significant functional rescue, which requires combinations of favorable FIX messenger RNA (mRNA) sequence and protein features. This rational approach, applicable to other coagulation disorders, helps with interpreting the poor response reported in the few investigated HB patients, and identifies candidate patients eligible for treatment.

Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics.

Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs). However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exploited the hyperactive Sleeping Beauty transposon system (SB100X) to integrate human factor IX (hFIX) minigenes into genomic DNA in vitro and in vivo. We generated stable HEK293 cell lines and C57BL/6 mice harboring splicing-competent hFIX minigenes either wild type (SChFIX-wt) or mutated (SChFIXex5-2C). In both models the SChFIXex5-2C variant, found in patients affected by Hemophilia B, displayed an aberrant splicing pattern characterized by exon 5 skipping. This allowed us to test, for the first time in a genomic DNA context, the efficacy of the snRNA U1-fix9, delivered with an adeno-associated virus (AAV) vector. With this approach, we showed rescue of the correct splicing pattern of hFIX mRNA, leading to hFIX protein expression. These data validate the SB100X as a versatile tool to quickly generate models of human genetic mutations, to study their effect in a stable DNA context and to assess mutation-targeted therapeutic strategies.