Identificación y caracterización de mutaciones de splicing en pacientes con hiperfenilalaninemia; aproximaciones terapéuticas específicas de RNA

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-04-2019En este trabajo nos hemos centrado en el estudio de mutaciones de splicing que resultan en hiperfenilalaninemia (HFA), bien por defectos en la enzima 6-piruvoil-tetrahidropterina sintasa (PTPS) que causa la deficiencia de tetrahidrobiopterina (BH4), cofactor de las hidroxilasas de aminoácidos aromáticos, o bien por defectos en una de dichas hidroxilasas, la fenilalanina hidroxilasa (PAH). Por una parte, hemos estudiado dos nuevas variantes, c.164-672C>T y c.243+3A>G en el gen PTS identificadas en un paciente HFA debido a un defecto en la enzima PTPS. La variante intrónica profunda c.164-672C>T crea un potencial sitio 5’ de splicing que, según los análisis en fibroblastos del paciente y en minigenes, conduce a la inclusión de varios pseudoexones entre los exones 2 y 3 del mRNA del gen PTS. Este efecto pudo ser parcialmente revertido mediante oligonucleótidos antisentido (AONs) de diferente química. La variante c.243+3A>G afecta al sitio natural 5’ de splicing del exón 4 resultando en el skipping del mismo. La sobreexpresión de un U1 snRNA adaptado, perfectamente complementario al sitio 5’ de splicing mutante, permitió recuperar el transcrito normal en el sistema de minigenes, no así en fibroblastos, siendo necesario la optimización futura de esta estrategia. Con el propósito de mejorar la aplicación de la terapia antisentido se ha estudiado un nuevo método de vehiculización de AONs, los micro-minicírculos (miMCs), vectores plasmídicos compuestos solamente por secuencias eucariotas. Para testar la eficacia de los miMCs se utilizó un plásmido parental que expresaba la fusión U7 snRNA-AON dirigido al sitio natural 5’ de splicing del exón 11 del gen PAH con el fin de forzar la exclusión de dicho exón. Los resultados obtenidos tras la producción de los miMCs y la posterior transfección en una línea de hepatoma revelaron una baja eficiencia de los miMCs como vehículo para la terapia con AONs en nuestro modelo experimental, ya que sólo se observó un skipping parcial del exón 11. Por otra parte, el uso de la secuenciación masiva para la captura completa del gen PAH en 7 pacientes con HFA en los que solo se les había encontrado una mutación PAH nos permitió identificar variantes intrónicas profundas. La interpretación de las variantes mediante un sistema de priorización y el uso de herramientas bioinformáticas nos ha permitido seleccionar aquellas variantes intrónicas con un posible efecto sobre el mecanismo de splicing mediante la potencial inclusión de pseudoexones. Sin embargo, el análisis funcional mediante minigenes descartó dicho efecto indicando que las variantes seleccionadas no son causantes de enfermedad. Finalmente, se ha investigado el efecto patogénico de las variantes c.1199+17G>A y c.1199+20G>C, identificadas en el gen PAH en pacientes con HFA. Ambas mutaciones provocan la exclusión del exón 11 en un sistema de minigenes e impiden la unión de U1 snRNP70 a esta región según los ensayos de unión a RNA. El análisis de los minigenes con deleciones y mutaciones puntuales en esta región, junto con la sobreexpresión de U1 snRNAs adaptados nos ha permitido identificar los motivos críticos involucrados en la regulación del correcto splicing del exón 11. Los resultados indicaron que la unión de U1 snRNP corriente abajo del sitio natural 5’ de splicing determina el splicing eficiente del exón 11, proporcionando así una base para el desarrollo de estrategias terapéuticas dirigidas a corregir mutaciones de splicing que afecten al exón 11 del gen PAH y ampliando las funciones de U1 snRNP como potenciador de splicing en ciertos contextos.Este trabajo ha sido posible gracias a un contrato con cargo a un proyecto de la Fundación Ramón Areces (XVII CN) y a la ayuda para estancias breves (short term scientific misssion) de la Acción COST BM1207. UNIVERSIDAD AUTÓNOMA DE MADRID Facultad de Ciencias Departamento de Biología Molecular IDENTIFICACIÓN Y CARACTERIZACIÓN DE MUTACIONES DE SPLICING EN PACIENTES CON HIPERFENILALANINEMIA; APROXIMACIONES TERAPÉUTICAS ESPECÍFICAS DE RN

Endoplasmic reticulum stress and autophagy in homocystinuria patients with remethylation defects

Proper function of endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site as well as perturbation of mitochondria-associated ER membranes (MAMs) have been linked to neurodegenerative and metabolic diseases. Previously, we have observed an increase in ROS and apoptosis levels in patientderived fibroblasts with remethylation disorders causing homocystinuria. Here we show increased mRNA and protein levels of Herp, Grp78, IPR1, pPERK, ATF4, CHOP, asparagine synthase and GADD45 in patient-derived fibroblasts suggesting ER stress and calcium perturbations in homocystinuria. In addition, overexpressed MAM-associated proteins (Grp75, σ-1R and Mfn2) were found in these cells that could result in mitochondrial calcium overload and oxidative stress increase. Our results also show an activation of autophagy process and a substantial degradation of altered mitochondria by mitophagy in patientderived fibroblasts. Moreover, we have observed that autophagy was partially abolished by antioxidants suggesting that ROS participate in this process that may have a protective role. Our findings argue that alterations in Ca homeostasis and autophagy may contribute to the development of this metabolic disorder and suggest a therapeutic potential in homocystinuria for agents that stabilize calcium homeostasis and/or restore the proper function of ER-mitochondria communications.Ministerio de Ciencia e Innovación (SAF2010-15284 to ER), Ministerio de Economía y Competividad: Instituto de Salud Carlos III (PI13/01239 to BP), MITOLAB (S2010/BMD-2402 to BP), Ministerio de Economía y Competividad (SAF2013-43005-R to ER and LRD), and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa”Peer Reviewe

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAMLife-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseasesThis work was supported by grants of Fondazione Telethon Italy (to N.B.‐P.), MIUR (PRIN2017 to N.B.‐P.), of the Swiss National Science Foundation (grant 320030_176088 to J.H.), of the US National Institutes of Health grants (R21NS091654 and R01NS100979 both to G.S.L.), of the Spanish Ministry of Science and Innovation (PID2019-105344RB-I00/AEI/10.13039/501100011033 toL.R.D. and E.R.), and by a Wellcome Trust Investigator Award (110061to D.M.F.v.A.

Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells

The field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients' samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant , which should result in the accurate recapitulation of the splicing defectThis book is based upon work from COST Action DARTER (CA 17103), supported by COST (European Cooperation in Science and Technology)

Functional analysis of novel variants identified in cis in the PCCB gene in a patient with propionic acidemia

Next-generation sequencing has improved the diagnosis of inborn errors of metabolism, allowing rapid confirmation of cases detected by clinical/biochemical studies or newborn screening. The challenge, however, remains for establishing the pathogenicity of the identified variants, especially for novel missense changes or small in-frame deletions. In this work we report a propionic acidemia patient exhibiting a severe neonatal form with coma and hyperammonaemia. Genetic analysis identified the previously described pathogenic PCCB variant p.R512C in the maternal allele and two novel PCCB variants in cis in the paternal allele, p.G246del and p.S322F. Expression analysis in a eukaryotic system confirmed the deleterious effect of the novel missense variant and of the one amino acid deletion, as they both exhibited reduced protein levels and reduced or null PCC activity compared to the wild-type construct. Accordingly, the double mutant resulted in no residual activity. This study increases the knowledge of the genotype-phenotype correlations in the rare disease propionic acidemia and highlights the necessity of functional analysis of novel variants to understand their contribution to disease severity and to accurately classify their pathogenic status. In conclusion, two novel PCCB pathogenic variants have been identified, expanding the current mutational spectrum of propionic acidemiaPID2019-105344RB-I00, PID2022-137238OB-I0

Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene

We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping. The deep-intronic c.164-672C>T variant creates a potential 5' splice site that leads to the inclusion of four overlapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear element AluSq repeat sequence. Two of the identified pseudoexons have been reported previously, activated by different deep-intronic variants, and were also detected at residual levels in control cells. Interestingly, the predominant pseudoexon is nearly identical to a disease causing activated pseudoexon in the F8 gene, with the same 3' and 5' splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sites and/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexon inclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164-672C>T or the previously described c.164-716A>T. With SSO treatment PTPS protein was recovered, illustrating the therapeutic potential of the approach, for patients with different pseudoexon activating variants in the region. In addition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying the antisense strategy in patients with hypomorphic PTS variants with the purpose of upregulating their expression to increase overall protein and activity

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases

RNA solutions to treat inborn errors of metabolism

RNA-based therapies are a new, rapidly growing class of drugs that until a few years ago were being used mainly in research in rare diseases. However, the clinical efficacy of recently approved oligonucleotide drugs and the massive success of COVID-19 RNA vaccines has boosted the interest in this type of molecules of both scientists and industry, as wells as of the lay public. RNA drugs are easy to design and cost effective, with greatly improved pharmacokinetic properties thanks to progress in oligonucleotide chemistry over the years. Depending on the type of strategy employed, RNA therapies offer the versatility to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. Currently, there are more than a dozen RNA-based drugs approved for clinical use, including some for specific inborn errors of metabolism (IEM), and many other in different stages of development. New initiatives in n-of-1 RNA drug development offer new hope for patients with rare diseases and/or ultra-rare mutations. RNA-based therapeutics include antisense oligonucleotides, aptamers, small interfering RNAs, small activating RNAs, microRNAs, lncRNAs and messenger RNAs. Further research and collaborations in the fields of chemistry, biology and medicine will help to overcome major challenges in their delivery to target tissues. Herein, we review the mechanism of action of the different therapeutic approaches using RNA drugs, focusing on those approved or in clinical trials to treat IEM.This work was funded by grant PID2019-105344RB-I00/AEI/10.13039/501100011033 from Spanish Ministry of Science and Innovation and COST (European Cooperation in Science and Technology) Action CA17103. Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces.Peer reviewe

Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site

Phenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3’ splice site, with different exonic mutations affecting exon 11 splicing through disruption of exonic splicing regulatory elements. In this study, we report a novel intron 11 regulatory element, which is involved in exon 11 splicing, as revealed by the investigated pathogenic effect of variants c.1199+17G>A and c.1199+20G>C, identified in PKU patients. Both mutations cause exon 11 skipping in a minigene system. RNA binding assays indicate that binding of U1snRNP70 to this intronic region is disrupted, concomitant with a slightly increased binding of inhibitors hnRNPA1/2. We have investigated the effect of deletions and point mutations, as well as overexpression of adapted U1snRNA to show that this splicing regulatory motif is important for regulation of correct splicing at the natural 5’ splice site. The results indicate that U1snRNP binding downstream of the natural 5’ splice site determines efficient exon 11 splicing, thus providing a basis for development of therapeutic strategies to correct PAH exon 11 splicing mutations. In this work, we expand the functional effects of non-canonical intronic U1 snRNP binding by showing that it may enhance exon definition and that, consequently, intronic mutations may cause exon skipping by a novel mechanism, where they disrupt stimulatory U1 snRNP binding close to the 5’ splice site. Notably, our results provide further understanding of the reported therapeutic effect of exon specific U1 snRNA for splicing mutations in disease.Fundación Ramon Areces , Grant XVII CN to LRD), European Cooperation in Science And Technology, Action BM1207 to LRD), Natur og Univers, Det Frie Forskningsråd; and Novo; Nordisk Fonden (DK)Peer Reviewe