DNA base editing corrects common hemophilia A mutations and restores factor VIII expression in in vitro and ex vivo models.

Background: Replacement and nonreplacement therapies effectively control bleeding in hemophilia A (HA) but imply lifelong interventions. Authorized gene addition therapy could provide a cure but still poses questions on durability. FVIIIgene correction would definitively restore factor (F)VIII production, as shown in animal models through nuclease-mediated homologous recombination (HR).

3D Printed Materials for Permanent Restorations in Indirect Restorative and Prosthetic Dentistry: A Critical Review of the Literature.

The objective of this study was to review the scientific evidence currently available on 3D printable materials and 3D printing technologies used for the fabrication of permanent restorations, focusing on material properties that are clinically relevant. A literature search was performed on four databases (MEDLINE/PubMed, Scopus, Cochrane Library, Web of Science) for articles published from January 2013 until November 2023, using a combination of free words: (restorative dentistry OR prosthetic dentistry) AND (3D printing OR additive manufacturing OR rapid prototyping) AND materials. Two reviewers screened titles and/or abstracts of 2.

Whole-Exome Sequencing in a Family with an Unexplained Tendency for Venous Thromboembolism: Multicomponent Prediction of Low-Frequency Variant Deleteriousness and of Individual Protein Interaction.

Whole-exome sequencing (WES) in families with an unexplained tendency for venous thromboembolism (VTE) may favor detection of low-frequency variants in genes with known contribution to hemostasis or associated with VTE-related phenotypes. WES analysis in six family members, three of whom affected by documented VTE, filtered for MAF < 0.04 in 192 candidate genes, revealed 22 heterozygous (16 missense and six synonymous) variants in patients.

Translucency of CAD/CAM and 3D Printable Composite Materials for Permanent Dental Restorations.

The aim of the study was to compare the translucency of CAD/CAM and printable composite materials for fixed dental prostheses (FDP). Eight A3 composite materials (7 CAD/CAM and 1 printable) for FPD were used to prepare a total of 150 specimens. CAD/CAM materials, all characterized by two different opacity levels, were: Tetric CAD (TEC) HT/MT; Shofu Block HC (SB) HT/LT; Cerasmart (CS) HT/LT; Brilliant Crios (BC) HT/LT; Grandio Bloc (GB) HT/LT; Lava Ultimate (LU) HT/LT, Katana Avencia (KAT) LT/OP.

Clinically Relevant Properties of 3D Printable Materials for Intraoral Use in Orthodontics: A Critical Review of the Literature.

The review aimed at analyzing the evidence available on 3D printable materials and techniques used for the fabrication of orthodontic appliances, focusing on materials properties that are clinically relevant. MEDLINE/PubMed, Scopus, and Cochrane Library databases were searched. Starting from an initial retrieval of 669 citations, 47 articles were finally included in the qualitative review.

Counteracting the Common Shwachman-Diamond Syndrome-Causing c.258+2T>C Mutation by RNA Therapeutics and Base/Prime Editing.

Shwachman-Diamond syndrome (SDS) represents one of the most common inherited bone marrow failure syndromes and is mainly caused by gene mutations. Only supportive treatments are available, with hematopoietic cell transplantation required when marrow failure occurs. Among all causative mutations, the c.

Not Just Loss-of-Function Variations: Identification of a Hypermorphic Variant in a Patient With a CDKL5 Missense Substitution.

Background And Objectives: CDKL5 deficiency disorder (CDD) is a neurodevelopmental encephalopathy characterized by early-onset epilepsy and impaired psychomotor development. Variations in the X-linked gene coding for a kinase cause CDD. Molecular genetics has proved that almost all pathogenic missense substitutions localize in the N-terminal catalytic domain, therefore underlining the importance for brain development and functioning of the kinase activity.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spf mice, and govern susceptibility to RNA-based therapies.

Background: Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases.

The Asialoglycoprotein Receptor Minor Subunit Gene Contributes to Pharmacokinetics of Factor VIII Concentrates in Hemophilia A.

Background: The asialoglycoprotein receptor (ASGPR) binds with high affinity factor VIII (FVIII) through its -linked oligosaccharides. However, its contribution to the wide inter-individual variation of infused FVIII pharmacokinetics (PK) in hemophilia A (HA) is unknown.

Objective: To investigate the variability in FVIII PK outcomes in relation to genetic variation in the encoding the ASGPR2 subunit.

Dissection of pleiotropic effects of variants in and adjacent to F8 exon 19 and rescue of mRNA splicing and protein function.

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.

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

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 (/) carrying the mutation c.

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.

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.

Splicing Mutations Impairing CDKL5 Expression and Activity Can be Efficiently Rescued by U1snRNA-Based Therapy.

Mutations in the gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve physiological regulation.

Low-Frequency and Rare Variants in Italian Multiple Sclerosis Patients.

In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (-value ≤ 5 × 10). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.

Next-generation sequencing and recombinant expression characterized aberrant splicing mechanisms and provided correction strategies in factor VII deficiency.

Despite the exhaustive screening of gene exons and exon-intron boundaries and promoter region, a significant proportion of mutated alleles remains unidentified in patients with coagulation factor VII deficiency. Here, we applied next-generation sequencing to 13 FVII-deficient patients displaying genotype-phenotype discrepancies upon conventional sequencing, and identified six rare intronic variants. Computational analysis predicted splicing effects for three of them, which would strengthen (c.

Molecular Mechanisms and Determinants of Innovative Correction Approaches in Coagulation Factor Deficiencies.

Molecular strategies tailored to promote/correct the expression and/or processing of defective coagulation factors would represent innovative therapeutic approaches beyond standard substitutive therapy. Here, we focus on the molecular mechanisms and determinants underlying innovative approaches acting at DNA, mRNA and protein levels in inherited coagulation factor deficiencies, and in particular on: (i) gene editing approaches, which have permitted intervention at the DNA level through the specific recognition, cleavage, repair/correction or activation of target sequences, even in mutated gene contexts; (ii) the rescue of altered pre-mRNA processing through the engineering of key spliceosome components able to promote correct exon recognition and, in turn, the synthesis and secretion of functional factors, as well as the effects on the splicing of missense changes affecting exonic splicing elements; this section includes antisense oligonucleotide- or siRNA-mediated approaches to down-regulate target genes; (iii) the rescue of protein synthesis/function through the induction of ribosome readthrough targeting nonsense variants or the correction of folding defects caused by amino acid substitutions. Overall, these approaches have shown the ability to rescue the expression and/or function of potentially therapeutic levels of coagulation factors in different disease models, thus supporting further studies in the future aimed at evaluating the clinical translatability of these new strategies.

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.

Disease-causing variants of the conserved +2T of 5' splice sites can be rescued by engineered U1snRNAs.

The ability of variants of the spliceosomal U1snRNA to rescue splicing has been proven in several human disease models, but not for nucleotide changes at the conserved GT nucleotide of 5' splice sites (5'ss), frequent and associated with severe phenotypes. Here, we focused on variants at the 5'ss of F9 intron 3, leading to factor IX (FIX) deficiency (hemophilia B). Through minigene expression, we demonstrated that all changes induce complete exon 3 skipping, which explains the associated hemophilia B phenotype.

The somatic FAH C.1061C>A change counteracts the frequent FAH c.1062+5G>A mutation and permits U1snRNA-based splicing correction.

In tyrosinaemia type 1(HT1), a mosaic pattern of fumarylacetoacetase (FAH) immunopositive or immunonegative nodules in liver tissue has been reported in many patients. This aspect is generally explained by a spontaneous reversion of the mutation into a normal genotype. In one HT1 patient carrying the frequent FAH c.

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.

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants.

In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5' (c.519A > G) or 3' (c.

Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy.

The c.2101A>G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation.