CRISPR-Cas9-mediated somatic correction of a one-base deletion in the Ugt1a gene ameliorates hyperbilirubinemia in Crigler-Najjar syndrome mice.

(AAV)-mediated episomal gene replacement therapy for monogenic liver disorders is currently limited in pediatric settings due to the loss of vector DNA, associated with hepatocyte duplication during liver growth. Genome editing is a promising strategy leading to a permanent and specific genome modification that is transmitted to daughter cells upon proliferation. Using genome targeting, we previously rescued neonatal lethality in mice with Crigler-Najjar syndrome. This rare monogenic disease is characterized by severe neonatal unconjugated hyperbilirubinemia, neurological damage, and death. Here, using the CRISPR-Staphylococcus aureus Cas9 (SaCas9) platform, we edited the disease-causing mutation present in the Ugt1a locus of these mice. Newborn mice were treated with two AAV8 vectors: one expressing the SaCas9 and single guide RNA, and the other carrying the Ugt1a homology regions with the corrected sequence, while maintained in a temporary phototherapy setting rescuing mortality. We observed a 50% plasma bilirubin reduction that remained stable for up to 6 months. We then tested different Cas9:donor vector ratios, with a 1:5 ratio showing the greatest efficacy in lowering plasma bilirubin, with partial lethality rescue when more severe, lethal conditions were applied. In conclusion, we reduced plasma bilirubin to safe levels and partially rescued neonatal lethality by correcting the mutant Ugt1a1 gene of a Crigler-Najjar mouse model.

Formation of an invasion-permissive matrix requires TGFß/SNAIL1-regulated alternative splicing of fibronectin.

BACKGROUND: As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFß, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFß target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses. METHODS: We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis. RESULTS: In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFß-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix  that does not sustain TGFß-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis. CONCLUSIONS: Our results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.

Lethality rescue and long-term amelioration of a citrullinemia type I mouse model by neonatal gene-targeting combined to SaCRISPR-Cas9.

Citrullinemia type I is a rare autosomal-recessive disorder caused by deficiency of argininosuccinate synthetase (ASS1). The clinical presentation includes the acute neonatal form, characterized by ammonia and citrulline accumulation in blood, which may lead to encephalopathy, coma, and death, and the milder late-onset form. Current treatments are unsatisfactory, and the only curative treatment is liver transplantation. We permanently modified the hepatocyte genome in lethal citrullinemia mice (Ass1fold/fold) by inserting the ASS1 cDNA into the albumin locus through the delivery of two AAV8 vectors carrying the donor DNA and the CRISPR-Cas9 platform. The neonatal treatment completely rescued mortality ensuring survival up to 5 months of age, with plasma citrulline levels significantly decreased, while plasma ammonia levels remained unchanged. In contrast, neonatal treatment with a liver-directed non-integrative AAV8-AAT-hASS1 vector failed to improve disease parameters. To model late-onset citrullinemia, we dosed postnatal day (P) 30 juvenile animals using the integrative approach, resulting in lifespan improvement and a minor reduction in disease markers. Conversely, treatment with the non-integrative vector completely rescued mortality, reducing plasma ammonia and citrulline to wild-type values. In summary, the integrative approach in neonates is effective, although further improvements are required to fully correct the phenotype. Non-integrative gene therapy application to juvenile mice ensures a stable and very efficient therapeutic effect.

Long-term correction of ornithine transcarbamylase deficiency in Spf-Ash mice with a translationally optimized AAV vector.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTCSpf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic.

Attenuation of neuro-inflammation improves survival and neurodegeneration in a mouse model of severe neonatal hyperbilirubinemia.

All pre-term newborns and a high proportion of term newborns develop neonatal jaundice. Neonatal jaundice is usually a benign condition and self-resolves within few days after birth. However, a combination of unfavorable complications may lead to acute hyperbilirubinemia. Excessive hyperbilirubinemia may be toxic for the developing nervous system leading to severe neurological damage and death by kernicterus. Survivors show irreversible neurological deficits such as motor, sensitive and cognitive abnormalities. Current therapies rely on the use of phototherapy and, in unresponsive cases, exchange transfusion, which is performed only in specialized centers. During bilirubin-induced neurotoxicity different molecular pathways are activated, ranging from oxidative stress to endoplasmic reticulum (ER) stress response and inflammation, but the contribution of each pathway in the development of the disease still requires further investigation. Thus, to increase our understanding of the pathophysiology of bilirubin neurotoxicity, encephalopathy and kernicterus, we pharmacologically modulated neurodegeneration and neuroinflammation in a lethal mouse model of neonatal hyperbilirubinemia. Treatment of mutant mice with minocycline, a second-generation tetracycline with anti-inflammatory and neuroprotective properties, resulted in a dose-dependent rescue of lethality, due to reduction of neurodegeneration and neuroinflammation, without affecting plasma bilirubin levels. In particular, rescued mice showed normal motor-coordination capabilities and behavior, as determined by the accelerating rotarod and open field tests, respectively. From the molecular point of view, rescued mice showed a dose-dependent reduction in apoptosis of cerebellar neurons and improvement of dendritic arborization of Purkinje cells. Moreover, we observed a decrease of bilirubin-induced M1 microglia activation at the sites of damage with a reduction in oxidative and ER stress markers in these cells. Collectively, these data indicate that neurodegeneration and neuro-inflammation are key factors of bilirubin-induced neonatal lethality and neuro-behavioral abnormalities. We propose that the application of pharmacological treatments having anti-inflammatory and neuroprotective effects, to be used in combination with the current treatments, may significantly improve the management of acute neonatal hyperbilirubinemia, protecting from bilirubin-induced neurological damage and death.

Promoterless gene targeting without nucleases rescues lethality of a Crigler-Najjar syndrome mouse model.

Crigler-Najjar syndrome type I (CNSI) is a rare monogenic disease characterized by severe neonatal unconjugated hyperbilirubinemia with a lifelong risk of neurological damage and death. Liver transplantation is the only curative option, which has several limitations and risks. We applied an in vivo gene targeting approach based on the insertion, without the use of nucleases, of a promoterless therapeutic cDNA into the albumin locus of a mouse model reproducing all major features of CNSI Neonatal transduction with the donor vector resulted in the complete rescue from neonatal lethality, with a therapeutic reduction in plasma bilirubin lasting for at least 12 months, the latest time point analyzed. Mutant mice, which expressed about 5-6% of WT Ugt1a1 levels, showed normal liver histology and motor-coordination abilities, suggesting no functional liver or brain abnormalities. These results proved that the promoterless gene therapy is applicable for CNSI, providing therapeutic levels of an intracellular ER membrane-bound enzyme responsible for a lethal liver metabolic disease.

Modulation of bilirubin neurotoxicity by the Abcb1 transporter in the Ugt1-/- lethal mouse model of neonatal hyperbilirubinemia.

Moderate neonatal jaundice is the most common clinical condition during newborn life. However, a combination of factors may result in acute hyperbilirubinemia, placing infants at risk of developing bilirubin encephalopathy and death by kernicterus. While most risk factors are known, the mechanisms acting to reduce susceptibility to bilirubin neurotoxicity remain unclear. The presence of modifier genes modulating the risk of developing bilirubin-induced brain damage is increasingly being recognised. The Abcb1 and Abcc1 members of the ABC family of transporters have been suggested to have an active role in exporting unconjugated bilirubin from the central nervous system into plasma. However, their role in reducing the risk of developing neurological damage and death during neonatal development is still unknown.To this end, we mated Abcb1a/b-/- and Abcc1-/- strains with Ugt1-/- mice, which develop severe neonatal hyperbilirubinemia. While about 60% of Ugt1-/- mice survived after temporary phototherapy, all Abcb1a/b-/-/Ugt1-/- mice died before postnatal day 21, showing higher cerebellar levels of unconjugated bilirubin. Interestingly, Abcc1 role appeared to be less important.In the cerebellum of Ugt1-/- mice, hyperbilirubinemia induced the expression of Car and Pxr nuclear receptors, known regulators of genes involved in the genotoxic response.We demonstrated a critical role of Abcb1 in protecting the cerebellum from bilirubin toxicity during neonatal development, the most clinically relevant phase for human babies, providing further understanding of the mechanisms regulating bilirubin neurotoxicity in vivo. Pharmacological treatments aimed to increase Abcb1 and Abcc1 expression, could represent a therapeutic option to reduce the risk of bilirubin neurotoxicity.

TDP-43 regulates ß-adducin (Add2) transcript stability.

TDP-43 is an RNA-binding protein involved in several steps of mRNA metabolism including transcription, splicing and stability. It is also involved in ALS and FTD, neurodegenerative diseases characterized by TDP-43 nuclear depletion. We previously identified TDP-43 as a binder of the downstream element (DSE) of the ß-Adducin (Add2) brain-specific polyadenylation site (A4 PAS), suggesting its involvement in pre-mRNA 3' end processing. Here, by using chimeric minigenes, we showed that TDP-43 depletion in HeLa and HEK293 cells resulted in down-regulation of both the chimeric and endogenous Add2 transcripts. Despite having confirmed TDP-43-DSE in vitro interaction, we demonstrated that the in vivo effect was not mediated by the TDP-43-DSE interaction. In fact, substitution of the Add2 DSE with viral E-SV40 and L-SV40 DSEs, which are not TDP-43 targets, still resulted in decreased Add2 mRNA levels after TDP-43 downregulation. In addition, we failed to show interaction between TDP-43 and key polyadenylation factors, such as CstF-64 and CPSF160 and excluded TDP-43 involvement in pre-mRNA cleavage and regulation of polyA tail length. These evidences allowed us to exclude the pre-hypothesized role of TDP43 in modulating 3' end processing of Add2 pre-mRNA. Finally, we showed that TDP-43 regulates Add2 gene expression levels by increasing Add2 mRNA stability. Considering that Add2 in brain participates in synapse assembly, synaptic plasticity and their stability, and its genetic inactivation in mice leads to LTP, LTD, learning and motor-coordination deficits, we hypothesize that a possible loss of Add2 function by TDP-43 depletion may contribute to ALS and FTD disease states.

Characterization of the distal polyadenylation site of the ß-adducin (Add2) pre-mRNA.

Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation.

Long-distance regulation of Add2 pre-mRNA3'end processing.

Accurate 3'end processing depends on the correct recognition of polyadenylation regulatory elements by specific protein complexes. In addition to the well-known hexanucleotide motif and downstream sequence element (DSE), less-defined auxiliary elements are usually found to modulate cleavage and polyadenylation. They are generally located in close proximity to the core polyadenylation elements but, in most of the cases, the molecular mechanisms involved are not well defined. We concentrated our studies on the regulation of the mouse ß adducin (Add2) pre-mRNA cleavage and polyadenylation. It contains two proximal erythroid-specific (PAS1 and PAS2-3) and one distal brain-specific (PAS4) polyadenylation sites along the 3'UTR. Using an in vivo approach based in the transfection of minigenes containing the Add2 polyadenylation signals, we previously identified the core regulatory elements responsible for PAS4 activity. Here, we have identified two novel non-canonical cis-acting elements regulating 3'end processing at PAS4, which show long-distance activities. The first of these elements, which spans for 257 nucleotides and is located at more than 5 kb upstream the PAS4, was essential to enable processing at the Add2 PAS4. The second element, located at about 4.5 kb upstream of the PAS4, reduces PAS4 processing. Both elements display long-distance activities and, to our knowledge, long-distance upstream polyadenylation regulatory elements have not been previously described in non-viral eukaryotic transcripts. These results highlight the complexity of the regulatory mechanisms directing Add2 pre-mRNA 3'end processing, and suggests that pre-mRNA 3' end processing of other genes may also be unexpectedly regulated by non-canonical auxiliary elements.

Identification of 3' gene ends using transcriptional and genomic conservation across vertebrates.

BACKGROUND: In higher eukaryotes, gene expression is regulated at different levels. In particular, 3'UTRs play a central role in translation, stability and subcellular localization of transcripts. In recent years, the development of high throughput sequencing techniques has facilitated the acquisition of transcriptional data at a genome wide level. However, annotation of the 3' ends of genes is still incomplete, thus limiting the interpretation of the data generated. For example, we have previously reported two different genes, ADD2 and CPEB3, with conserved 3'UTR alternative isoforms not annotated in the current versions of Ensembl and RefSeq human databases. RESULTS: In order to evaluate the existence of other conserved 3' ends not annotated in these databases we have now used comparative genomics and transcriptomics across several vertebrate species. In general, we have observed that 3'UTR conservation is lost after the end of the mature transcript. Using this change in conservation before and after the 3' end of the mature transcripts we have shown that many conserved ends were still not annotated. In addition, we used orthologous transcripts to predict 3'UTR extensions and validated these predictions using total RNA sequencing data. Finally, we used this method to identify not annotated 3' ends in rats and dogs. As a result, we report several hundred novel 3'UTR extensions in rats and a few thousand in dogs. CONCLUSIONS: The methods presented here can efficiently facilitate the identification of not-yet-annotated conserved 3'UTR extensions. The application of these methods will increase the confidence of orthologous gene models across vertebrates.

CPEB2, CPEB3 and CPEB4 are coordinately regulated by miRNAs recognizing conserved binding sites in paralog positions of their 3'-UTRs.

The cytoplasmic polyadenylation element binding-protein (CPEB) is an RNA-binding protein that participates in translational control. CPEB2, CPEB3 and CPEB4 are paralog proteins very similar among themselves referred as the CPEB2 subfamily. To gain insight into common mechanisms of regulation of the CPEB2 subfamily transcripts, we looked for putative cis-acting elements present in the 3'-UTRs of the three paralogs. We found different families of miRNAs predicted to target all subfamily members. Most predicted target sites for these families are located in paralog positions suggesting that these putative regulatory motifs were already present in the ancestral gene. We validated target sites for miR-92 and miR-26 in the three paralogs using mutagenesis of miRNA-binding sites in reporter constructs combined with over-expression and depletion of miRNAs. Both miR-92 and miR-26 induced a decrease in Luciferase activity associated to a reduction in mRNA levels of the reporter constructs. We also showed that the endogenous miRNAs co-regulate CPEB2, CPEB3 and CPEB4 transcripts, supporting our hypothesis that these genes have a common regulatory mechanism mediated by miRNAs. We also suggest that the ancestral pattern of miRNA-binding motifs was maintained throughout the generation of highly conserved elements in each of the 3'-UTRs.

A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived.

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA(+)FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA(+)FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues.

Unexpected rescue of alpha-synuclein and multimerin1 deletion in C57BL/6JOlaHsd mice by beta-adducin knockout.

Uniform genetic background of inbred mouse strains is essential in experiments with genetically modified mice. In order to assess Add2 (beta-adducin) function, its null mutation was produced in embryonic stem cells derived from 129Sv mouse and the subsequently obtained mouse mutants were backcrossed for 6 generations with C57BL/6JOlaHsd strain. Comparison of brain proteins between mutated and control animals by two-dimensional gels linked to mass spectroscopy analysis showed expression of Snca (alpha-synuclein) in the mutated animals, but unexpectedly not in the control C57BL/6JOlaHsd mice. Comparison between C57BL/6JOlaHsd and C57BL/6NCrl mice confirmed the presence of a deletion encompassing Snca and in addition Mmrn1 (multimerin1) loci in C57BL/6JOlaHsd strain. The segregation of mutated Add2 together with an adjacent part of the chromosome 6 derived from 129Sv mice, rescued the loss of these two genes in knockout mice on C57BL/6JOlaHsd background. The fact that Add2 knockout was compared with the C57BL/6JOlaHsd mouse strain, which is actually a double knockout of Snca and Mmrn1 emphasizes a need for information provided by commercial suppliers and of exact denominations of substrains used in research.

Impaired motor coordination in mice lacking the EDA exon of the fibronectin gene.

The extracellular matrix (ECM) plays an important role in the central nervous system (CNS) by modulating the migration of cells, axons and dendrites of neurons. Fibronectin (FN) is a major component of the ECM in the CNS and plays essential roles in development, cell adhesion and cell migration. Specific FN-isoforms, generated by alternative splicing at three conserved regions, the extra domain B (EDB), extra domain A (EDA) and type III homologies connecting segment (IIICS), have been shown to modulate these processes in vitro and in vivo. The inclusion of the EDA exon in the brain is highly regulated during development and aging, suggesting an important role of this exon in brain function. However, the direct role of FN-isoforms in brain function and behaviour is still obscure. Therefore, to directly assess the role of the FN-EDA exon in vivo, we have generated two mouse strains devoid of EDA exon regulated splicing in the FN gene that constitutively include (EDA(+/+)) or exclude (EDA(-/-)) the EDA exon in all tissues. Here, we show the behavioural consequences of the absence of regulated splicing of the EDA exon in the FN gene. Deletion of the EDA domain in the FN protein results in reduced motor-coordination abilities and vertical exploratory capacity, whereas mice that constitutively include the EDA domain displayed a decrease in locomotory activity in the open field (OF) test. These results strongly suggest that regulated splicing of the EDA exon is necessary for a normal function of the brain.

RNA folding affects the recruitment of SR proteins by mouse and human polypurinic enhancer elements in the fibronectin EDA exon.

In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing.

Alternative splicing of fibronectin: a mouse model demonstrates the identity of in vitro and in vivo systems and the processing autonomy of regulated exons in adult mice.

We have designed a novel approach using genetically engineered mice to make a systematic study of the EDA exon regulation of the fibronectin gene during development and aging. The genome of the mice was modified either by optimization of the EDA natural splice sites or by deleting the EDA region. The previous in vitro observation that the optimization of the splicing sites leads to constitutive inclusion of the EDA exon was confirmed in our animal model. In fact, all the adult tissues of the genetically modified mice showed only EDA(+) FN mRNA, demonstrating the fidelity of in vitro models, despite of the development- and aging-regulated splicing regulation of the EDA exon, and regardless of the presence of exonic elements described within the exon. This result indicates that the splicing regulatory elements of the EDA exon are dispensable in the presence of consensus splicing sites. Moreover, we demonstrate the autonomy of both the EDB and the IIICS alternatively spliced regions in adult mice lacking regulation of the alternative splicing at the EDA exon. We also show here the tight splicing regulation of all three alternative spliced regions of the FN gene at different time-points during development and aging of mice.

Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.