[go: up one dir, main page]

WO2024261139A1 - Variants de mfn2 et leur utilisation dans le traitement/la prévention de maladies associées à des altérations dans la fonction du réticulum endoplasmique - Google Patents

Variants de mfn2 et leur utilisation dans le traitement/la prévention de maladies associées à des altérations dans la fonction du réticulum endoplasmique Download PDF

Info

Publication number
WO2024261139A1
WO2024261139A1 PCT/EP2024/067267 EP2024067267W WO2024261139A1 WO 2024261139 A1 WO2024261139 A1 WO 2024261139A1 EP 2024067267 W EP2024067267 W EP 2024067267W WO 2024261139 A1 WO2024261139 A1 WO 2024261139A1
Authority
WO
WIPO (PCT)
Prior art keywords
mfn2
seq
nucleic acid
vector
ermit2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/067267
Other languages
English (en)
Inventor
Deborah Paola Naon Elbirt
Antonio Zorzano Olarte
Luca Scorrano
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitat de Barcelona UB
Fundacio Privada Institut de Recerca Biomedica IRB
Fondazione Ricerca Biomedica Avanzata Onlu Vimm
Universita degli Studi di Padova
Original Assignee
Universitat de Barcelona UB
Fundacio Privada Institut de Recerca Biomedica IRB
Fondazione Ricerca Biomedica Avanzata Onlu Vimm
Universita degli Studi di Padova
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitat de Barcelona UB, Fundacio Privada Institut de Recerca Biomedica IRB, Fondazione Ricerca Biomedica Avanzata Onlu Vimm, Universita degli Studi di Padova filed Critical Universitat de Barcelona UB
Publication of WO2024261139A1 publication Critical patent/WO2024261139A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to nucleic acids encoding for mitofusin 2 (Mfn2) variants, nucleic acid vectors comprising said nucleic acids, as well as their use in the treatment of diseases and conditions characterized by endoplasmatic reticulum (ER) stress.
  • Mfn2 mitofusin 2
  • ER endoplasmatic reticulum
  • MCS membrane contact sites
  • Mitochondria-ER MCS are required for Ca2+ signalling (3,4), apoptosis (5,6), mitochondrial fission (7), autophagosome formation (8,9), phosphatidylserine metabolism (10), and even for nutrient sensing in hypothalamic neurons (11).
  • the ER- mitochondria interface is stabilized by 16-30 nm protein bridges between the two organelles
  • the mitochondrial fusion protein Mitofusin (Mfn) 2 tethers the two organelles, regulating communication between them (13-22).
  • Mfn mitochondrial fusion protein
  • previous works have shown that silencing of Mfn2 in mouse embryonic fibroblasts and HeLa cells disrupts ER morphology and loosens ER-mitochondria interactions, thereby reducing the efficiency of mitochondrial Ca2+ uptake (13).
  • Constitutive or acute Mfn2 ablation increases the distance between the ER and mitochondria, the heterotypic interaction between the mitochondria and ER being crucial for transfer of lipids and especially Ca2+ between the two organelles (16).
  • the role of Mfn2 as a tether was furthermore confirmed independently in the heart
  • Mfn2 deletion impacts other facets of ER-mitochondria communication, like phosphatidylcholine (6) and cholesterol (17, 18) transfer to the mitochondria.
  • ER-mitochondria tethers like the ERMES complex in yeast (23) and the ER VAPB-mitochondrial PTPIP51 couple in mammals (24). These tethers are commonly formed by a couple of proteins interacting in trans with its partner on the opposing organelle. However, the molecular partner of MFN2 on the ER that allows tethering without heterotypic organelle fusion is unknown.
  • Alternative splicing expands genome functional repertoire by generating multiple proteins with different intracellular localization and function (25).
  • alternative splicing of the mitochondrial fission executor dynamin related protein 1 gene encodes for a brainspecific isoform regulating dendrite formation independently of mitochondrial fission (26, 27).
  • MFN2 splicing variants play a role in MFN2 function remains unknown.
  • liver disorders such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), steatosis or liver cancer, liver inflammation and liver fibrosis, but also metabolic or genetic diseases and other types of cancer have been linked to ER stress.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • steatosis or liver cancer liver inflammation and liver fibrosis
  • metabolic or genetic diseases and other types of cancer have been linked to ER stress.
  • Mfn2 as an ER-mitochondria tether
  • Mfn2 Manipulation of the expression of full length Mfn2 in gene therapy has been suggested in the prior art.
  • Hernandez-Alvarez et al. (2019) detected reduced Mfn2 expression in liver biopsies from patients with non-alcoholic steatohepatitis (NASH) and showed that liverspecific ablation of Mfn2 in mice provokes inflammation, triglyceride accumulation, fibrosis, and liver cancer (10). They showed that Mfn2 binds phosphatidylserine (PS) and that it can specifically extract PS into membrane domains, favouring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis.
  • PS phosphatidylserine
  • PE mitochondrial phosphatidylethanolamine
  • WO01/25274 A1 discloses Mfn2 and its use in gene therapy to treat degenerative and other disorders involving mitochondria, including myopathies and Alzheimer's disease.
  • WO2022/072793 A1 discloses compositions and methods for modulating (e.g., inhibiting or promoting) expression of certain mitochondrial regulatory proteins, such as Mfn2 with a nucleic acid, polypeptide, or small molecule. Methods for treating diseases related to aberrant autophagy or mitochondrial function, such as familial neurological movement disorders are suggested comprising administering to a subject such a mitofusin modulator.
  • WO 2022/015715 A1 discloses recombinant adeno associated viruses (rAAV) and other vectors and compositions useful for treating a patient having Charcot Marie Tooth Disease comprising a recombinant nucleic acid sequence encoding an engineered human Mfn2 coding sequence operably linked to regulatory sequences which direct expression thereof in a human target cell.
  • rAAV adeno associated viruses
  • WO 2023/081835 A1 discloses a method for improving efficacy in cancer cell therapy by contacting T cells with mitofusin activators such small molecule activator Duvelisib which increases Mfn2 expression.
  • WO 2004/074482 A1 discloses the association between alterations in the Mfn2 gene and the expression thereof and type 2 diabetes and/or obesity and the usefulness of Mfn2 for the early diagnosis and treatment of obesity and type 2 diabetes.
  • US 2021/0017509 A1 provides methods for treating autosomal dominant diseases, such as autosomal dominant optic atrophy or retinopathy in a subject using of a gene editing enzyme with a pair of unique guide RNA sequences that targets both mutant and wildtype forms of autosomal dominant disease-related genes such as MFN2 for destruction in cells, and then supplying the cells with wildtype autosomal dominant disease-related gene cDNA which is codon modified to evade recognition by the guide RNAs.
  • autosomal dominant diseases such as autosomal dominant optic atrophy or retinopathy
  • Mfn2 Whilst manipulation of the expression of Mfn2 has been suggested in the prior art as a gene therapy approach, it has at the same time also been described that overexpression of the full-length protein can lead to unwanted side effects. Mfn2 induces mitochondrial fusion. It has been observed that Mfn2 overexpression contributes to an imbalance in mitochondrial dynamics that are related to different pathologies. Excessive mitochondrial fusion adversely affects cells by modulating the mitochondrial energy balance and altering ROS levels. Overexpression of Mfn2 has furthermore been shown to have a negative effect on mesenchymal cells that are pivotal to tissue homeostasis, repair and regeneration.
  • the present invention therefore relates in one aspect to a nucleic acid encoding a mitofusin 2 (Mfn2) variant comprising or consisting of
  • the mitofusin 2 (Mfn2) variant is not Mfn2 wildtype.
  • the present invention relates to a nucleic acid encoding a mitofusin 2 (Mfn2) variant comprising or consisting of
  • the nucleic acid encoding a mitofusin 2 (Mfn2) variant is a DNA or an RNA.
  • the nucleic acid encoding a mitofusin 2 (Mfn2) variant is an mRNA.
  • the present invention relates to a nucleic acid vector comprising a nucleic acid coding sequence for a mitofusin 2 (Mfn2) variant comprising or consisting of
  • nucleic acid encoding a Mfn2 variant is operably linked to a regulatory sequence that drives the expression of the coding sequence.
  • the present invention relates to a nucleic acid vector comprising a nucleic acid encoding a mitofusin 2 (Mfn2) variant comprising or consisting of
  • the nucleic acid vector is a gene therapy vector.
  • the nucleic acid vector is a non-integrative vector.
  • the nucleic acid vector is a non-viral vector.
  • the nucleic acid vector is selected from an adeno-associated virus (AAV) vector, an adenoviral vector, an integrase deficient lentivirus vector, a pox virus vector, an alphavirus vector, and a herpes virus vector.
  • AAV adeno-associated virus
  • the nucleic acid vector is an AAV vector selected from serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and pseudotyped AAV.
  • the vector is an AAV vector serotype AAV2, AAV8 or AAV9, most preferred AAV8 or AAV9.
  • the nucleic acid vector comprises a regulatory sequence which is a promoter, preferably a constitutive promoter.
  • the regulatory sequence is selected from a liver specific promoter, such as the promotor of human serum albumin or alpha- 1 -antitrypsin, a kidney specific promoter or human CMV (hCMV) promoter.
  • the promoter is a chimeric promoter.
  • the chimeric promoter consists of the Apolipoprotein E/C-l hepatic control region and the human alpha-1 -antitrypsin core promoter, or of two copies of the human alpha 1 microglobulin/bikunin enhancer coupled to the core promoter of human thyroxine-binding globulin (TBG).
  • the nucleic acid vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • the present invention furthermore relates to a drug delivery system comprising the nucleic acid or the nucleic acid vector as described herein, wherein the drug delivery system is selected from liposomes, polymeric nanomicelles, dendrimers, metal-organic frameworks, inorganic nanoparticles, lipid nanoparticles, solid lipid nanoparticles (SLN), nanogels, colloidal carrier systems, microparticles of poly (lactide) (PLA), poly (glycolide) (PGA), or poly (lactide-co-glycolide) (PLGA), preferably in lipid nanoparticles.
  • the drug delivery system is selected from liposomes, polymeric nanomicelles, dendrimers, metal-organic frameworks, inorganic nanoparticles, lipid nanoparticles, solid lipid nanoparticles (SLN), nanogels, colloidal carrier systems, microparticles of poly (lactide) (PLA), poly (glycolide) (PGA), or poly (lactide-co-g
  • the present invention relates to a recombinant adeno-associated virus (rAAV) genome comprising an expression cassette comprising:
  • the nucleotide sequence of the promoter is selected from a liver specific promoter, such as the promotor of human serum albumin or alpha- 1 -antitrypsin, a kidney specific promoter or human CMV (hCMV) promoter.
  • a liver specific promoter such as the promotor of human serum albumin or alpha- 1 -antitrypsin
  • hCMV human CMV
  • the promoter is a chimeric promoter.
  • the chimeric promoter consists of the Apolipoprotein E/C-l hepatic control region and the human alpha-1 -antitrypsin core promoter, or of two copies of the human alpha 1 microglobulin/bikunin enhancer coupled to the core promoter of human thyroxine-binding globulin (TBG).
  • TBG human thyroxine-binding globulin
  • the rAAV genome is selected from serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and pseudotyped AAV.
  • the rAAV genome is of serotype AAV8 or AAV9.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid, nucleic acid vector, the drug delivery system or the rAAV as described herein and a pharmaceutically acceptable carrier or diluent.
  • the present invention relates to a nucleic acid, a nucleic acid vector, the drug delivery system, the rAAV genome, or the pharmaceutical composition as described herein for use in the treatment or prevention of a disease or condition characterized by endoplasmatic reticulum (ER) stress.
  • ER endoplasmatic reticulum
  • the disease or condition is selected from the group consisting of liver disorders, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), steatosis or liver cancer, liver inflammation and liver fibrosis.
  • liver disorders such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), steatosis or liver cancer, liver inflammation and liver fibrosis.
  • the disease or condition is a rare liver disease selected from the group consisting of Wilson disease and Serpinopathies, such as for example alpha-1 antitrypsin deficiency (AATD).
  • AATD alpha-1 antitrypsin deficiency
  • the disease or condition is selected from insulin resistance, diabetes type 2, obesity or atrial fibrillation (AF).
  • AF atrial fibrillation
  • the disease or condition is selected from Cystic Fibrosis (CF), Wolfram syndrome, Interstitial Lung Diseases (ILDs), kidney diseases such as nephrotic syndrome (NS), diabetic nephropathy (DN), acute kidney injury (AKI), maladaptive transition from AKI- chronic kidney disease (CKD) and renal fibrosis, as well as rare kidney diseases, such as Alport syndrome and autosomal dominant tubulointerstitial kidney disease (ADTKD); myopathies, such as RYR1-related myopathies (RYR1-RM) or muscular dystrophy such as Duchenne muscular dystrophy (DMD).
  • CF Cystic Fibrosis
  • ILDs Interstitial Lung Diseases
  • NS nephrotic syndrome
  • DN diabetic nephropathy
  • AKI acute kidney injury
  • CKD maladaptive transition from AKI- chronic kidney disease
  • CKD AKI- chronic kidney disease
  • renal fibrosis as well as rare kidney diseases, such as Alport syndrome and
  • the disease or condition is selected from cancers, such as breast cancer, pancreatic cancer, lung cancer, squamous cell carcinoma and non-squamous cell carcinoma. In one embodiment the disease or condition is Charcot-Marie-Tooth neuropathy.
  • the use in the treatment or prevention of a disease or condition as described herein comprises a step of administering the nucleic acid, the nucleic acid vector, the rAAV genome, or the pharmaceutical composition to a subject in need thereof.
  • the administration is selected from oral, sublingual, buccal, intravenous, intravascular, intramuscular, subcutaneous, intraperitoneal, conjunctival, rectal, transdermal, intrathecal, topical and/or inhalation-mediated administration, preferably the administration intravenous, intramuscular, or intraperitoneal administration.
  • the present invention relates to an mRNA sequence encoding for a mitofusin 2 (Mfn2) variant comprising or consisting of
  • the mitofusin 2 (Mfn2) variant is not Mfn2 wildtype.
  • the present invention relates to an mRNA sequence encoding a mitofusin 2 (Mfn2) variant comprising or consisting of
  • FIG. 1 Schematic representation of the MFN2 splicing variants.
  • A Schematic representation of the MFN2 splicing variants. Different PCR-amplification products obtained from human skeletal cDNA were cloned and sequenced. PCR was performed with primers located on intron 2 and exon 19. Exons are numbered 1 to 19, and the splicing events generate newexons (3a, 3b, 3c, 4a, 4b, 6a, 6b, 13a, 13b, 15a, and 15b). MFN2 corresponds to sequence NM014874.
  • Variant 1 (V1-MFN2, ERMIN2) mRNA is 1330 bp long, is produced by exons 3b and 6b to 15a skipping, and it encodes for ERMIN2, a 41-kDa (predicted M.W.) protein.
  • Variant 2 (V2-MFN2, ERMIT2) mRNA is 1220 bp long, is produced by exon 4b to 13a skipping, and it encodes ERMIT2, a 43-kDa (predicted M.W.) protein. Exons involved in the alternative splicing process and the domains in the produced proteins are indicated.
  • G1 to G5 are GTPase domain motifs.
  • B RPA in HeLa cells.
  • ERMIN2- and ER IT2-specific probes covered the 5'- and 3'- flanking sequences of the skipped region, and 20% of their length was a nonmatching sequence as positive control for RNAse digestion.
  • C Means ⁇ standard errors (SEs) of mRNA of MFN2, ERMIN2, and ERMIT2 levels (normalized to PPIA) in HeLa cells 4 hours after incubation in Earle’s buffered salt solution (EBSS) (starvation), treatment with rotenone (Rot; 5 mM), or thapsigargin (TG; 1 mM).
  • SEs standard errors
  • EBSS buffered salt solution
  • Rot rotenone
  • thapsigargin TG; 1 mM
  • N 8 independent experiments.
  • FIG. 2 MFN2 variants localize at the ER, not the mitochondria.
  • a and B Mfn2 LKO mice were injected with adenoviruses encoding ERMIN2 (A) or ERMIT2 (B), and after 72 hours, liver subcellular fractions were prepared.
  • Equal amounts (40 mg) of protein from total extracts (Total), crude (Mito), pure mitochondria (Pure mito), MAMs, and LMs were separated by SDS-PAGE and immunoblotted using antibodies against MFN2, the MAM markers PS1 (presenilin 1) and FACL4 (long-chain acyl-CoA synthetase 4), the outer mitochondrial membrane marker TOM20 (translocase of the outer mitochondrial membrane 20), and the ER marker SERCA2 (sarcoplasmic ER Ca 2+ ATPase).
  • C Representative confocal images of Mfn2-/- MEFs cotransfected with the indicated plasmids (green), mitochondrially targeted CFP (mito; blue) and ER-dsRED (ER; red). Insets are magnified 7x. Scale bars, 10 mm.
  • Figure 3 ERMIN2 regulates ER morphology.
  • A Representative volume-rendered 3D reconstructions of confocal z-stacks of Mfn2-/- MEFs cotransfected with the indicated HA- tagged constructs and mt-dsRED. Scale bars, 10 mm.
  • C Experiments were performed as in (A), except that Mfn2-/- EFs were cotransfected with ER-YFP. Scale bars, 10 mm.
  • Figure 4 ERMIT2 reconstitutes ER-mitochondria interaction in Mfn2-/ ⁇ MEFs.
  • A Representative volume- rendered 3D reconstructions of confocal z-stacks of Mfn2-/- MEFs cotransfected with ER-YFP, mt-dsRED, and plasmids coding for the indicated HA-tagged proteins. Yellow indicates pseudo-colocalization of the two organelles.
  • (F) Representative electron microscopy images of Mfn2-/- MEFs cotransfected with GFP and the indicated plasmid. GFP-positive cells were sorted, fixed, processed, and analysed by transmission electron microscopy (TEM). Scale bars, 500 nm.
  • FIG. 5 ERMIT2 interacts with mitochondrial mitofusins, tethering ER to mitochondria.
  • Mfn1-/-, Mfn2-/- MEFs were cotransfected with the indicated plasmids and after 24 hours lysed. Egual amounts (400 mg) of protein were immunoprecipitated (IP) using the indicated antibodies, and immunoprecipitates were separated by SDS-PAGE and immunoblotted using the indicated antibodies.
  • FIG. 6 Ermit2-mitofusins interact via their coiled-coil domains.
  • a to C HeLa cells were cotransfected with plasmids encoding for the indicated constructs and after 24 hours lysed. Schemes depict the domains found in the transfected chimeras. Equal amounts (400 mg) of protein were immunoprecipitated (IP) using the indicated antibodies, and immunoprecipitates were separated by SDS-PAGE and immunoblotted using the indicated antibodies.
  • FIG. 7 ERMIT2 licenses ER-mitochondria Ca2+ and lipid transfer in vitro and in vivo.
  • Figure 8 MFN2 Variants expression in human tissues and immunoblot detection in transfected cells.
  • A Real-time PCR amplification products using primers specific for Mfn2, Ermin2 and Ermit2 were plotted against increased concentrations of cDNA template copy number.
  • Figure 9 Primary protein sequence alignment of ERMIN2, ERMIT2 and MFN2. Protein sequence of ERMIN2 and ERMIT2 aligned with full-length MFN2 sequence (accession # NP055689). Boxed area indicates the GTPase domain, G1-G5 the GTPase sequence motifs; TM: transmembrane domains; CC1:coiled coil 1 ; CC2: coiled coil 2.
  • Figure 10 A mouse MFN2 Variant produced by alternative mRNA splicing localizes in MAMs and regulates ER-mitochondria tethering.
  • moV-MFN2 is produced by exon 3 and exon 4b to 17a skipping of mouse Mfn2gene.
  • B Scheme of protein domains in moV-MFN2.
  • TM transmembrane
  • CC2 coiled coil 2.
  • C Mfn2 protein sequence (acc. #: NP_573464.2) aligned with moV-MFN2.
  • D Representative confocal images of Mfn2-/-MEF co-transfected with GFP-moV-MFN2 and mt-RFP or ER-RFP.
  • FIG 11 Endogenous Ermin2 and Ermit2 are not retrieved in pure mitochondria. Equal amounts (40 pg) of protein of the indicated HeLa subcellular fractions were separated by SDS-PAGE and immunoblotted using the indicated antibodies (Mfn2: aa 557-576). Total: whole cell extracts; Mito: mitochondria; MAMs: mitochondria-associated ER membranes, LM: light membranes. ERMIN2 and ERMIT2 are indicated by asterisks.
  • Figure 12 Confocal images of ER and mitochondria in Mfn2-/- MEFs expressing ERMIN2 and ERMIT2. Representative confocal images of Mfn2-/- MEFs co-transfected with the indicated plasmids (green), mitochondrially targeted CFP (mito, blue) and ER-dsRED (ER, red). Scale bars, 10 pm. The images are the individual channels of Fig. 2B.
  • Figure 13 The transmembrane region determines Ermin2 and Ermit2 ER localization.
  • A Scheme of full-length MFN2 indicating the different domains.
  • B Representative confocal images of Mfn2-/- MEFs cotransfected with the indicated eGFP-ERMIT2 deletion mutants and ER-RFP (ER). Scale bars, 10 pm. Numbering is from MFN2 full-length sequence (NP055689).
  • Figure 15 The N- and C-terminal regions of ERMIN2 and ERMIT2 face the cytoplasm.
  • A Representative confocal images of individual GFP, RFP and merged channels of Mfn2-/- MEFs cotransfected with ER-RFP and the indicated constructs of Split super-folder GFP (sfGFP) engineered for efficient self-complementation This construct breaks the sequence of sfGFP between the 10th and the 11th p-strand into two parts: GFP1-10 and GFP11. GFP11 was fused to the N-terminus of ERMIN2 or ERMIT2. Self-complementation is observed as green fluorescence. Scale bars, 10 pm.
  • FIG. 1 (B) Cartoons showing ERMIN2 and ERMIT2 domains and trypsin cleavage sites as predicted by Peptide Cutter indicated by the short vertical lines. The epitopes recognized by the antibodies used are also indicated. Predicted topology and molecular weight of the protected fragments in limited trypsinization assays are indicated.
  • C Trypsin protection assays. MFN2LKO mice were injected with adenoviruses encoding ERMIN2 or ERMIT2. After 2 days livers were harvested, and ER- enriched LM fractions were isolated. Equal amounts (40 pg) of LM fractions were incubated with the indicated amounts of trypsin for 15 min on ice.
  • Figure 16 Efficient specific downregulation ofERMIN2 and ERMIT2in HeLa cells. He La cells were transfected with the indicated siRNA. After 24 h, 40 pg of total cell extract was separated by SDS/PAGE and immunoblotted using the aa 661-751 MFN2 antibody. MFN2, ERMIN2 and ERMIT2 are indicated.
  • Figure 17 Volume rendered3D reconstructionsof confocal z-stacks of ER and mitochondria in Mfn2-/- MEFs.
  • the images are the individual channels of Fig. 4A.
  • Figure 18 ddGFP fluorescent dots increase in Mfn2-/- MEFs expressing Ermit2.
  • A Representative confocal images of Mfn2-/- MEFs cotransfected for 48 h with calnexin- ddGFP-A and Tom20-ddGFP-B and the indicated plasmids. Scale bars, 10 pm.
  • B MeantSE of quantitative analysis of fluorescence intensity measured using Image J (NIH) from experiments as in A (15 cells/condition from 8 independent experiments; open dots). *, p ⁇ 0.05 in a Kruskal-Wallis ANOVA with Dunn’s test for multiple comparison among the indicated conditions.
  • Figure 20 Analysis of ERMIN2 and ERMIT2 interactions.
  • Lysates 400 pg of protein
  • MEFs of the indicated genotype transfected as indicated were immunoprecipitated using anti-GFP antibodies.
  • IP immunoprecipitates
  • B Counts of the CC1-CC2 contacts (two a carbons from different chains at a distance ⁇ 0.8 nm) during the simulations involving mitochondrially anchored MFN1 and MFN2 versus ERMIT2.
  • Figure 21 Ermin2 and Ermit2 restore ER calcium content in Mfn2-/- MEFs.
  • livers were explanted and mean ⁇ SE of mRNA expression levels of ERMIT2 (A), hepatic H2O2 levels (B), levels of hepatic ER stress protein markers in immunoblots for the indicated proteins (C,D) and mRNA expression levels of the inflammation marker TNFa (E) were measured.
  • A mRNA expression levels of ERMIT2
  • B hepatic H2O2 levels
  • C,D hepatic ER stress protein markers in immunoblots for the indicated proteins
  • E mRNA expression levels of the inflammation marker TNFa
  • Figure 23 Ermit2 ameliorates the liver defects of methionine/choline deficient mice.
  • Wt mice were fed with chow or methionine/choline deficient and high fat diet (MCD) for 3 weeks.
  • MCD methionine/choline deficient and high fat diet
  • livers were explanted, and mean ⁇ SE of protein levels of MFN2 (A), ERMIT2 (B), of ER stress protein markers in immunoblots for the indicated proteins (C,D) and of mRNA levels of the inflammation marker TNFa were measured (E).
  • mean ⁇ SE of plasma glucose concentration (F) and body weight (G) at the end of the 3 weeks treatment were measured. *, p ⁇ 0.05 in a one-way ANOVA with Tukey’s mean comparison.
  • Figure 24 Variant reverts ER stress. Hela cells were transfected with indicated plasmid, after 48 hs mRNA or protein expression of ER stress markers were evaluated by Real time PCR or western blot. Graph indicates mRNA of XBPI (X-box-binding protein-1) and its spliced form XBP1 s; protein levels of activating transcription factor 4 (ATF4) normalized to b actin.
  • XBPI X-box-binding protein-1
  • ATF4 activating transcription factor 4
  • Figure 25 Variants corrects a hallmark of ATTD disease by restoring the ER morphology.
  • ER were labelled with ER- YFP and confocal images were acquired.
  • Figure 26 Variants corrects a hallmark of ATTD disease decreasing the protein aggregation.
  • Protein level of a1AT in organelle (ER) fraction were analysed by western blot and refers to control group. The graph cleared shows that a1AT Z mutation increase its retention at organelle fraction and Variants are able to reduce its organelle retention of a1 AT.
  • Figure 27 Anticancer effect of variants associated with G0/G1 arrest and apoptosis in human cell line.
  • HeLa were cells transfected with indicated plasmid and DNA were labelled using DNA staining propidium iodine and measured by flow cytometry. Proportion of cells in each cycle phase were calculated based in DNA content. Bars represent the percentage of cell in different phase of Cell cycle: G0/G1 , S or G2/M. EV: (empty vector).
  • Figure 28 Apoptotic effect of Variants.
  • HeLa cells were transfected with indicated plasmids. After 48 hours apoptotic cells were detected by staining the cells with Annexin V andpropidium Iodide (PI) solution followed by flow cytometry analysis. Propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells. Graph indicates the percentage of early apoptotic positive cells necrotic negative. Annexin V +, PI-.
  • words of approximation such as, without limitation, "about”, “around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by ⁇ 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Accordingly, the term “about” may mean the indicated value ⁇ 5% of its value, preferably the indicated value ⁇ 2% of its value, most preferably the term “about” means exactly the indicated value ( ⁇ 0%).
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • the term “comprises” also encompasses and expressly discloses the terms “consists of and “consists essentially of”.
  • the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.
  • the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and/or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or.”
  • sequence identity refers to a percentage value obtained when two sequences are compared using a pairwise sequence alignment tool.
  • sequence identity is obtained using the global alignment tool “EMBOSS Needle” using the default settings (Rice et al., 2000. Trends Genet. 16(6):276-7; Li et al., 2015. Nucleic Acids Res. 43(W1):W580-4).
  • the global alignment tool is available at: https://www.ebi.ac.uk/Tools/psa/.
  • expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • a "coding region” or “coding sequence” is a portion of a polynucleotide which consists of codons translatable into amino acids.
  • expression cassette is used herein to refer to a polynucleotide sequence that comprises a coding sequence and one or more regulatory sequences, such as promoters and/or enhancers, that are operably linked to the coding sequence and control or affect or drive its expression.
  • Encoding refers to the inherent property of a nucleic acid to serve as a template, whether directly (i.e. a sense strand) or indirectly (i.e. an antisense strand) for synthesis of peptide, polypeptides, proteins, or other nucleic acids (i.e. rRNA, tRNA, microRNA).
  • a nucleic acid can “encode” whether it is the sense strand, antisense strand, or a double-stranded segment thereof.
  • the sense strand directly encodes the rRNA, tRNA, microRNA, or mRNA.
  • the mRNA then serves as the template for translation of a peptide, polypeptide, or protein.
  • the anti-sense strand is generally considered to be the reverse complementary sequence and is sometimes called a “noncoding” strand in the art (although for present purposes “noncoding” is a misnomer because the non-coding strand still “encodes” the genetic information by perpetuating it during semiconservative replication by acting as a template for the polymerization of a new, sense strand). By perpetuating the genetic information, the antisense strand is still encoding the genetic information for, for example, a protein.
  • a nucleic acid encoding X includes sense and antisense sequences or strands whether X is a peptide, a polypeptide, or a protein or X is a sequence that encodes a rRNA, tRNA, microRNA, antisenseRNA, etc.
  • nucleic acid encoding X includes RNA, DNA, and combinations thereof, since nucleic acids are synthesized from transcription, reverse-transcription, and replication, as naturally occurring processes and man-made processes (recombinant biology, molecular biology, etc). Accordingly, a recited nucleic acid sequence contemplates and supports the complementary version thereof, the reverse complementary version thereof, and doublestranded versions thereof.
  • recombinant refers to a piece of DNA, protein, virus, etc. that has been created by combining two or more fragments from different sources.
  • a recombinant AAV genome refers to a genome formed by combining at least two different fragments from different genomes.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects or control the transcription or expression of the coding sequence.
  • promoter as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a coding polynucleotide sequence. In some instances, this sequence may be the core promoter and in other instances, this sequence may also include, or be an enhancer alone and/or other regulatory elements which are required for expression of the gene product.
  • the promoter may comprise enhancer elements, exons, and introns from one or a variety of viruses and animals, and thereby the term “promoter” shall be understood to not be limited to being a non-expressed sequence, nor exclude a non-expressed sequence that is between expressed sequences (introns), nor be limited to exclude an enhancer alone so long as the combination of sequences used to construct the promoter are capable of initiating and/or controlling the specific transcription of a coding polynucleotide sequence.
  • chimeric promoter refers to a promoter that is formed by the combination of two or more regulatory elements, such as an enhancer and a promoter.
  • Enhancer refers to a nucleic acid sequence that when bound by an activator protein activates or increases transcription of a gene or genes. Enhancer sequences can be up stream (i.e., 5') or downstream (i.e., 3 ') relative to the genes they regulate.
  • variant as used throughout the present specification in the context of a nucleic acid sequence will be recognized and understood by the person of ordinary skill in the art, and is e.g. intended to refer to a variant of a nucleic acid sequence derived from another nucleic acid sequence, but excluding the original nucleic acid sequence from which it is derived.
  • a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived.
  • a variant of a nucleic acid sequence may be at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 91%, 93%, 94% or 95% identical to the nucleic acid sequence the variant is derived from.
  • the variant is a functional variant in the sense that the variant has retained at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence where it is derived from.
  • a “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide identity over a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotides of such nucleic acid sequence.
  • a variant of Mfn2 das claimed herein does not include a nucleic acid having the wildtype sequence of Mfn2.
  • variant refers to a protein variant having an amino acid sequence which differs from the original wildtype sequence of the protein through mutation, substitution or deletion.
  • a variant of Mfn2 does not include a protein having the wildtype sequence of Mfn2.
  • a “variant” of a protein may have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to such protein.
  • a variant of a protein comprises a functional variant of the protein, which means, in the context of the invention, that the variant exerts essentially the same, or at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95% of the role as the protein it is derived from.
  • the term "vector” relates to an entity capable of delivering a target gene to the interior of a cell and encompasses all kind of constructs that are suitable for expressing the target gene, such as for example a naked nucleic acid, a virion, or a plasmid.
  • the vector can include not only the expression-region (i.e. a promoter and a nucleic acid encoding a protein or even a nucleic acid), but also some cis-acting genetic component.
  • the cis-acting genetic component can for example provide for packaging within a virion, expression in a cell, replication in a cell, or a combination thereof.
  • a plasmid can comprise an origin of replication (e.g...
  • ori from cytomegalovirus
  • a viral or non-viral genetic code may provide a nucleic acid sequence or protein encoded therein that allows for insertion of the gene of interest into the host genome, thereby providing for the replication of the target gene during the replication of, and within, the host cell’s genome.
  • the “nucleic acid vector” of present invention can be an expression plasmid as commercially available, or also refer to the naked DNA or RNA or mRNA itself.
  • a “virion” is an entire virus particle comprising or consisting of an outer protein shell called a capsid and an inner core of nucleic acid (either ribonucleic or deoxyribonucleic acid).
  • the core confers infectivity, and the capsid provides specificity to the virus.
  • treatment and “therapy”, as used in the present application refer to a set of hygienic, pharmacological, surgical and/or physical means used with the intent to cure and/or alleviate a disease and/or symptoms with the goal of remediating the health problem.
  • treatment and “therapy” include preventive and curative methods, since both are directed to the maintenance and/or reestablishment of the health of an individual or animal.
  • treatment refers to the application or administration of a pharmaceutical composition to a subject who has a disease or condition characterized by ER stress, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward a disease.
  • terapéuticaally effective amount refers to an amount of matter which has a therapeutic effect, and which is able to treat the herein described diseases.
  • the terms “individual”, “patient” or “subject” are used interchangeably in the present application and are not meant to be limiting in any way.
  • the “individual”, “patient” or “subject” can be of any age, sex and physical condition.
  • Mfn2 induces mitochondrial fusion. It has been observed that Mfn2 overexpression contributes to an imbalance in mitochondrial dynamics that are related with different pathologies. Excessive mitochondrial fusion adversely affects cells by modulating the mitochondrial energy balance and altering ROS levels. Overexpression of Mfn2 has furthermore been shown to have a negative effect on mesenchymal cells that are pivotal to tissue homeostasis, repair and regeneration.
  • ERMIN2 for ERMitofusin 2
  • ERMIT2 for ERMitofusin 2 Tether
  • the inventors therefore firstly aimed to understand whether Mfn2 function in ER-mitochondria tethering relied on alternatively spliced MFN2 gene products. They identified two shorter amplicons that were cloned and sequenced.
  • the mRNA of the first variant (“Ermin2” for ERMitofusin 2) was 1 ,330 bp long and resulted from alternative splicing between exon 3a and 3c and between exon 6a and 15b.
  • the mRNA of the second variant (“Ermit2” for ERMitofusin 2 Tether) was 1 ,220 bp long and resulted from alternative splicing between exon 4a and 13b (Fig.1 A).
  • Ribonuclease protection assay confirmed that the mRNAs of ERMIN2 and ERMIT2 were expressed in HeLa cells (Fig.1 B). The inventors could also show that exogenous stress affected the expression of ERMIN2, ERMIT2 and MFN2. Whilst their mRNAs levels were unchanged in starved HeLa cells, they increased in cells exposed to the mitochondrial complex I inhibitor rotenone.
  • the sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor thapsigargin that causes ER stress, induced the expression of ERMIN2 andERMIT2 but not of /WF/V2 (Fig. 1C).
  • the proteins encoded by ERMIN2 and ERMIT2 were predicted to be shorter than Mfn2, partially or completely lacking Mfn2 GTPase and coiled-coil 1 domains but retaining its transmembrane and coiled-coil 2 regions (Fig. 1A and 9).
  • Mfn2 Alternatively spliced, shorter Mfn2 versions were also detected in M. musculus, where Mfn2 also tethers ER to mitochondria.
  • human and mouse Mitofusin 2 genes are alternatively spliced to generate variants shorter than full-length Mfn2, expressed in multiple tissues and induced when ER-mitochondria juxtaposition is pharmacologically increased.
  • Ermin2 and Ermit2 lack several domains found in full-length Mfn2, the inventors addressed whether they were targeted to mitochondria by examining the distribution of Ermin2 or Ermit2 in subcellular fractions purified from Mfn2 knockout livers (Mfn2 LKO ) where they adenovirally expressed the two variants. Ermin2 and Ermit2 were detected in the ER- enriched light membrane fraction (LM) as well as in mitochondria-associated membranes (MAMs), but not in the pure mitochondria fraction (Fig. 2A).
  • LM ER- enriched light membrane fraction
  • MAMs mitochondria-associated membranes
  • the inventors found that re-expressed Mfn2 localized mostly in mitochondria and only partially in the ER, whereas Ermin2 and Ermit2 displayed a pattern like that of the ER marker. Upon closer inspection, Ermin2 was uniformly distributed on the ER and Ermit2 displayed a punctuate staining colocalizing with ER and mitochondria, compatible with the retrieval of the endogenous protein in MAMs (Fig. 20, D; individual channels in Fig.12).
  • the present invention therefore relates in one aspect to a nucleic acid encoding a mitofusin 2 (Mfn2) variant comprising or consisting of
  • the mitofusin 2 (Mfn2) variant is not Mfn2 wildtype.
  • the present invention relates to a nucleic acid encoding a mitofusin 2 (Mfn2) variant comprising or consisting of
  • SEQ ID NO:1 and SEQ ID NO:2 refer to the amino acid sequences of the Mfn2 variants Ermit2 and Ermin2 as shown in table 1 below.
  • SEQ ID NO:1 (Ermit2) and SEQ ID NO:2 (Ermin2) are polypeptides comprising or consisting of an amino acid sequence according to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 as shown in table 1.
  • the nucleic acid encodes for a mitofusin 2 (Mfn2) variant comprising or consisting of an amino acid sequence having a sequence identity of at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to the amino acid sequence depicted in SEQ ID NOs:1 to 8.
  • Table 1 Sequences of Mfn2 variants
  • the nucleic acid encoding a mitofusin 2 (Mfn2) variant is a DNA or an RNA.
  • the nucleic acid is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • said mRNA is packaged in a drug delivery system selected from liposomes, polymeric nanomicelles, dendrimers, metal-organic frameworks, inorganic nanoparticles, lipid nanoparticles, solid lipid nanoparticles (SLN), nanogels, colloidal carrier systems, microparticles of poly (lactide) (PLA), poly (glycolide) (PGA), or poly (lactide-co-glycolide) (PLGA), preferably in lipid nanoparticles.
  • PLA poly (lactide)
  • PGA poly (glycolide)
  • PLA poly (lactide-co-glycolide)
  • nucleic acid sequence encodes for at least the two transmembrane domains TM1 and TM2 of Mfn2, preferably the domains encompassed by amino acids 610- 647 of Mfn2.
  • the present invention relates to an mRNA sequence encoding for a mitofusin 2 (Mfn2) variant comprising or consisting of
  • the mitofusin 2 (Mfn2) variant is not Mfn2 wildtype.
  • the success of gene therapy essentially depends on the availability of suitable vectors.
  • the vector is supposed to transport therapeutic genes efficiently into the nucleus of a target cell and bring it to expression, to stabilize it there and is neither toxic nor provokes an immune response.
  • the vector must be reproducible on industrial scale.
  • the vector according to present invention can be any entity capable of delivering a target gene to the interior of a cell and encompasses all kind of constructs that are suitable for expressing the target gene, such as for example a naked nucleic acid, a virion, or a plasmid.
  • adenoviral vectors occupy a special position among the viral systems. They are characterized by a wide host range, by the capability to infect resting cells and by extremely high titers. The infection efficiency related to the necessary amount of nucleic acid exceeds that of all other viral systems and exceeds that of plain DNA 100 000 times (in i.m. application). Types 4 and 7 adenoviruses have been used on a broad scale as live virus vaccines and have a good safety profile. The extremely low integrational tendency of adenoviruses is favorable as an additional safety aspect, since it minimizes the risk of insertion mutagenesis and oncogenic activation.
  • Adeno associated viral (AAV) vectors are widely adopted for in vivo organ-directed gene therapy, given their remarkable safety and efficacy profile shown in pre-clinical models and clinical trials.
  • AAV-vector based gene transfer to the liver of clotting factors for the treatment of the coagulation disorder haemophilia is among the most successful applications of gene therapy.
  • Lentiviral vectors are attractive vehicles for gene therapy due to their low prevalence of pre-existing immunity against vector components in humans. They can be used as vectors integrative vectors using their intrinsic ability to stably integrate in the genome of target cells, or they can be applied as non-integrative vectors in which case they are integrase deficient LVs. Other vectors, such as plasmids encoding for any of the Mfn2 variants as described herein, can also be used for gene therapy applications. The plasmids can then be encapsulated in or integrated into any form of drug delivery systems.
  • the drug delivery systems can for example be liposomes, polymeric nanomicelles, dendrimers, metal-organic frameworks, inorganic nanoparticles, lipid nanoparticles, solid lipid nanoparticles (SLN), nanogels, colloidal carrier systems, microparticles of poly (lactide) (PLA), poly (glycolide) (PGA), or poly (lactide-co-glycolide) (PLGA).
  • PLA poly (lactide)
  • PGA poly (glycolide)
  • PLGA poly (lactide-co-glycolide)
  • the present invention thus relates to a nucleic acid vector comprising a nucleic acid encoding a mitofusin 2 (Mfn2) variant comprising or consisting of
  • the nucleic acid encodes for a mitofusin 2 (Mfn2) variant comprising or consisting of an amino acid sequence having a sequence identity of at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to the amino acid sequence depicted in SEQ ID NOs:1 to 8.
  • the nucleic acid encoding a mitofusin 2 (Mfn2) variant is a DNA or an RNA.
  • the nucleic acid is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • said mRNA is packaged in a drug delivery system selected from liposomes, polymeric nanomicelles, dendrimers, metal-organic frameworks, inorganic nanoparticles, lipid nanoparticles, solid lipid nanoparticles (SLN), nanogels, colloidal carrier systems, microparticles of poly (lactide) (PLA), poly (glycolide) (PGA), or poly (lactide-co-glycolide) (PLGA), preferably in lipid nanoparticles.
  • PLA poly (lactide)
  • PGA poly (glycolide)
  • PLA poly (lactide-co-glycolide)
  • the nucleic acid sequence encodes for at least the two transmembrane domains TM1 and TM2 of Mfn2, preferably the domains encompassed by amino acids 610- 647 of Mfn2.
  • the nucleic acid sequence encoding a Mfn2 variant is operably linked to a regulatory sequence that drives the expression of the coding sequence.
  • the nucleic acid vector is a gene therapy vector.
  • the nucleic acid vector is a non-integrative vector.
  • the vector is selected from an adeno-associated virus (AAV) vector, an adenoviral vector, an integrase deficient lentivirus vector, a pox virus vector, an alphavirus vector, and a herpes virus vector.
  • AAV adeno-associated virus
  • the nucleic acid vector is an AAV vector selected from serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and pseudotyped AAV.
  • the vector is an AAV vector serotype AAV2, AAV8 or AAV9, most preferred AAV8 or AAV9.
  • the nucleic acid vector comprises a regulatory sequence which is a promoter, preferably a constitutive promoter.
  • a promoter can be selected from the genes that are specifically expressed in liver, such as for the albumin gene.
  • the regulatory sequence is selected from a liver specific promoter, such as the promotor of human serum albumin or alpha- 1 -antitrypsin, a kidney specific promoter or human CMV (hCMV) promoter.
  • a liver specific promoter such as the promotor of human serum albumin or alpha- 1 -antitrypsin
  • hCMV human CMV
  • the promoter is a chimeric promoter.
  • the chimeric promoter consists of the Apolipoprotein E/C-l hepatic control region and the human alpha-1 -antitrypsin core promoter, or of two copies of the human alpha 1 microglobulin/bikunin enhancer coupled to the core promoter of human thyroxine-binding globulin (TBG).
  • TBG human thyroxine-binding globulin
  • the nucleic acid vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).
  • the present invention relates to a recombinant adeno-associated virus (rAAV) genome comprising an expression cassette comprising:
  • the nucleotide sequence of the promoter is the sequence of a liver specific promoter, such as the promoter of human serum albumin or alpha-1 -antitrypsin.
  • the promoter is a chimeric promoter.
  • the chimeric promoter consists of the Apolipoprotein E/C-l hepatic control region and the human alpha-1 -antitrypsin core promoter, or of two copies of the human alpha 1 microglobulin/bikunin enhancer coupled to the core promoter of human thyroxine-binding globulin (TBG).
  • the rAAV genome is selected from serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and pseudotyped AAV.
  • the rAAV genome is of serotype AAV8 or AAV9.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid, the nucleic acid vector, or the rAAV genome as described herein and a pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable diluent” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and, without limiting the scope of the present invention, include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; saltforming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2- phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinis
  • the pharmaceutical composition may be prepared for or be suitable for oral, sublingual, buccal, intravenous, intravascular, intramuscular, subcutaneous, intraperitoneal, conjunctival, rectal, transdermal, intrathecal, topical and/or inhalation-mediated administration.
  • the pharmaceutical composition may be prepared for or be suitable for intravenous and/or intravascular administration.
  • the pharmaceutical composition may be a solution which is suitable for sublingual, buccal and/or inhalation-mediated administration routes.
  • the pharmaceutical composition may be a gel or solution which is suitable for intrathecal, intraestriatal or intracerebroventricular administration.
  • the pharmaceutical composition may be an aerosol which is suitable for inhalation-mediated administration.
  • the pharmaceutical composition may be prepared for administration in the cisterna magna or in the cerebrospinal fluid. Additionally, in other embodiments compatible with the teachings herein, the pharmaceutical composition can be prepared to be delivered directly to the site of the adverse cellular population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.
  • kits may include one or more containers housing the components of the invention and instructions for use. Specifically, such kits may include one or more agents described herein, along with instructions describing the intended application and the proper use of these agents.
  • agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.
  • the pharmaceutical composition is provided in a kit.
  • the kit can further comprise a container housing a pharmaceutically acceptable carrier.
  • a kit can comprise one container housing the nucleic acid vector of the first aspect or the rAAV of the second aspect and a second container housing a buffer suitable for injection of the nucleic acid or the rAAV into a subject.
  • the container can be a syringe.
  • kits disclosed herein can also contain any one or more of the components described herein in one or more containers.
  • the kits can include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject.
  • the kits can include a container housing agents described herein.
  • the agents can be in the form of a liquid, gel or solid (powder).
  • the agents can be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively, it can be housed in a vial or other container for storage.
  • a second container can have other agents prepared sterilely.
  • the kits can include the active agents premixed and shipped in a syringe, vial, tube, or other container.
  • the kits can have one or more or all of the components required to administrate the therapeutic agents to a subject, such as a syringe, topical application devices, or a needle tubing and bag.
  • MFN2 and ERMIT2 increased mitochondrial Ca2+ uptake (Fig. 7A,B).
  • MFN2 and ERMIT2 expression increased the Ca2+ uptake-dependent priming of mitochondrial ATP generation (16, 37) (Fig.7C,D).
  • ERMIT2 expression in Mfn2-/- cells licenses mitochondrial uptake of ER-released Ca2+ and Ca2+-primed mitochondrial ATP production that depends on matrix Ca2+-activated Krebs’ cycle dehydrogenases (38).
  • ERMIT2 lipid transfer between ER and mitochondria was assessed.
  • PS normalized phosphatidylserine
  • PE phosphatidylethanolamine
  • An adenovirus encoding ERMIT2 was intravenously administered to Mfn2LKO mice.
  • ERMIT2 expression reversed the reduced hepatic incorporation of L-serine (L- Ser) into PS and PE detected in control (LacZ) infected Mfn2LKO mice (Fig.7E,F).
  • ERMIT2 reduced the H2O2 accumulation (Fig. 22B), the ER stress (evidenced by accumulation of phosphorylated elF2a and CHOP, Fig. 22C,D) and the increased expression of the inflammation marker TNFa (Fig.22E) observed in the Mfn2LKO cohort.
  • the inventors furthermore assessed the role of the Mfn2 variants in liver diseases.
  • a mouse model with a non-alcoholic steatohepatitis (NASH) like condition was used as an orthogonal model of liver ER stress and inflammation due to MFN2 deficiency (ref.1O and Fig.23A).
  • Intravenous administration of adenoviruses encoding ERMIT2 increased ERMIT2 but not MFN2 levels (Fig.23A,B) and corrected the increased hepatic accumulation of L-Ser into PS and the reduced L-Ser labeling of PE, a proxy of the reduced PS transfer between ER and mitochondria (Fig.7G,H). Accordingly, ERMIT2 reduced the accumulation of markers of ER stress (Fig.23C,D), and inflammation (Fig.
  • ERMIT2 sustains ER to mitochondria lipid transfer and corrects the liver ER stress and inflammation in genetic and environmental mouse models of reduced liver Mfn2 expression, further highlighting the importance of ER-mitochondria juxtaposition in models of NASH.
  • ERMIT2 corrects the ER- mitochondria communication defects caused by reduced MFN2 levels. ERMIT2 is sufficient to license mitochondrial uptake of Ca2+ released from the ER.
  • ERMIN2 and ERMIT2 also normalized ER Ca2+ in Mfn2-deficient cells by modulating the facets of Ca2+ signaling altered by Mfn2 deletion: capacitative Ca2+ entry, SERCA activity, or ER Ca2+ leak (35, 36).
  • ERMIN2 and ERMIT2 also normalized ER Ca2+ in Mfn2-deficient cells by modulating the facets of Ca2+ signaling altered by Mfn2 deletion: capacitative Ca2+ entry, SERCA activity, or ER Ca2+ leak (35, 36).
  • in vivo ERMIT2 delivery to the liver corrected most of the hepatic alterations associated with Mfn2 genetic ablation or depletion in a diet induced model of NASH.
  • ERMIT normalized PS transfer from the ER to mitochondria, a process linked to alterations in lipid metabolism, ER stress, inflammation and fibrosis (10), suggesting that it participates in PS transfer to mitochondria and documenting the importance of MFN2 mediated ER-mitochondria tethering in liver diseases that are characterized by ER stress.
  • the present invention therefore relates to a nucleic acid, a nucleic acid vector, a rAAV genome, or the pharmaceutical composition as described herein for use in the treatment or prevention of a disease or condition characterized by endoplasmatic reticulum (ER) stress.
  • ER endoplasmatic reticulum
  • disease or condition characterized by ER stress refers to diseases in which ER stress can usually be observed as an intermediate symptom with the suitable detection methods as known to the skilled person, and in which the relief of the ER stress leads to an amelioration of the disease.
  • ER stress chronic liver diseases, such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). It has been shown that these conditions are linked to repressed hepatic MFN2 in humans (10). In addition, conditions such as steatosis and liver disease are characterized by ER stress and Unfolded Protein Response (UPR).
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • URR Unfolded Protein Response
  • the disease or condition is therefore selected from the group consisting of liver disorders, such as Non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), steatosis or liver cancer, liver inflammation and liver fibrosis.
  • liver disorders such as Non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), steatosis or liver cancer, liver inflammation and liver fibrosis.
  • ER stress also plays a role in several rare liver diseases.
  • Wilson disease is a rare genetic disorder characterized by the impaired metabolism of copper in the body. It is also known as hepatolenticular degeneration because it primarily affects the liver and central nervous system.
  • Wilson disease there is a mutation in the ATP7B gene, which is responsible for the production of a protein called ATPase copper- transporting beta (ATP7B). This protein is involved in transporting excess copper out of liver cells and into bile for excretion.
  • this transport mechanism is defective, leading to the accumulation of copper in various tissues, especially the liver, brain, and other organs. The excess copper accumulation can cause liver damage, leading to hepatitis, cirrhosis, or liver failure.
  • copper can be released into the bloodstream and deposited in other organs, including the brain, leading to neurological symptoms.
  • neurological symptoms can include tremors, dystonia (abnormal muscle contractions), difficulty with speech and swallowing, psychiatric disturbances, and cognitive impairment.
  • Other organs, such as the kidneys and eyes, may also be affected.
  • Serpinopathies refer to a group of genetic disorders characterized by mutations in genes that encode serine protease inhibitors, known as serpins.
  • Serpins are a family of proteins that regulate the activity of proteases, enzymes involved in various biological processes. When mutations occur in the genes encoding serpins, it can lead to abnormal folding and aggregation of the protein, impairing its function. This can result in a variety of clinical manifestations depending on the specific serpin affected and the tissues or organs involved.
  • AATD alpha-1 antitrypsin deficiency
  • Alpha-1 antitrypsin is a serpin that protects the lungs from damage caused by neutrophil elastase, an enzyme involved in inflammation.
  • AATD abnormal folding and accumulation of alpha-1 antitrypsin protein in the liver lead to liver disease, while reduced levels of functional protein in the lungs increase the risk of lung disease, such as emphysema.
  • Other serpinopathies include angioedema due to C1 inhibitor deficiency (hereditary angioedema), which is caused by mutations in the SERPING1 gene, and antitrypsin Seattle, caused by mutations in the SERPINA1 gene.
  • the symptoms and severity of serpinopathies can vary widely depending on the specific mutation and affected organ systems. Treatment options may include supportive care, management of symptoms, and in some cases, specific therapies such as enzyme replacement therapy or liver transplantation, depending on the specific serpinopathy.
  • the disease or condition is therefore a rare liver disease selected from Wilson disease and Serpinopathies, such as for example alpha-1 antitrypsin deficiency (AATD).
  • AATD alpha-1 antitrypsin deficiency
  • AATD alpha-1 antitrypsin deficiency
  • AATD and Z-AAT diseases characterized by ER stress
  • MUC5B Interstitial Lung Diseases
  • NPHSA nephrotic syndrome
  • SORBS1 diabeticnephropathy
  • ERMIN2 SEQ ID NO:2
  • Fig.25 Mutation in a1AT gene cause protein aggregation due to the incorrect folding of the protein. These aggregates, that accumulate in the ER, interfere with the normal maturation and posterior secretion of the protein.
  • the variants of present invention correct ER morphology and decrease the protein aggregation (Fig 25). Thus, variants correct one of the major hallmarks of AATD disease.
  • the disease or condition is insulin resistance, diabetes type 2, obesity or atrial fibrillation (AF).
  • AF Atrial fibrillation
  • T2D obesity-induced type 2 diabetes
  • AF mitochondrial dysfunction
  • the disease or condition characterized/caused by ER stress is selected from Cystic Fibrosis (CF), Wolfram syndrome, Interstitial Lung Diseases (ILDs), kidney diseases such as nephrotic syndrome (NS), diabetic nephropathy (DN), acute kidney injury (AKI), maladaptive transition from AKI-chronic kidney disease (CKD) and renal fibrosis, as well as rare kidney disease, such as Alport syndrome and autosomal dominant tubulointerstitial kidney disease (ADTKD); myopathies, such as RYR1-related myopathies (RYR1-RM), muscular dystrophy as Duchenne muscular dystrophy (DMD).
  • CF Cystic Fibrosis
  • ILDs Interstitial Lung Diseases
  • NS nephrotic syndrome
  • DN diabetic nephropathy
  • AKI acute kidney injury
  • CKD maladaptive transition from AKI-chronic kidney disease
  • CKD AKI-chronic kidney disease
  • renal fibrosis as well as
  • Cystic Fibrosis is a rare autosomal recessive disease. The incidence of CF is currently 1:3500. It is a monogenic disease affecting the CFTR gene, located on chromosome?. This gene codes for a type of ATP-binding cassette (ABC) transporter, whose function is to transport chloride and sodium ions and other anions such as GSH or bicarbonate. Located mainly in the apical membranes of epithelial cells in many tissues, CFTR can be affected by numerous types of mutations; more than 2000 variants of the CFTR gene are currently described.
  • ABSC ATP-binding cassette
  • CFTR Malfunctioning CFTR often affects ion conductance efficiency through the membrane pores, which changes the characteristics and composition of cellular secretions by changing the composition of the extracellular milieu. This accumulation of events causes organs to become gradually obstructed, eventually leading to fibrosis. CFTR mutations cause ER dysfunction due to an unwanted accumulation of CFTR protein, leading the cell to ER stress (66, 567). Kerbiriou et al. (2008) showed that, as proposed, ATF6 and Grp78 levels were elevated in cells with mutated CFTR (68). This could confirm the existence of ER stress in CF. In another study, Tang et al.
  • Wolfram syndrome is a rare autosomal recessive disorder caused by mutations in the wolframin ER transmembrane glycoprotein (WFS1) gene and characterized by diabetes mellitus, diabetes insipidus, optic atrophy and deafness.
  • WFS1 wolframin ER transmembrane glycoprotein
  • the homozygous Wfs1 mutant mice have a full penetrance and clearly expressed phenotype, whereas heterozygous mutants have a less-pronounced phenotype between the wildtype and homozygous mutant mice.
  • Heterozygous WFS1 mutations have been shown to be significant risk factors for diabetes and metabolic disorders in humans.
  • the disease or condition characterized/caused by ER stress encompasses other rare diseases caused by abnormal calcium sensing and signalling.
  • CaSR calcium-sensing receptor
  • the calcium-sensing receptor (CaSR) provides the major mechanism for the detection of extracellular calcium concentration in several cell types, via the induction of G-protein- coupled signalling. Accordingly, CaSR plays a pivotal role in calcium homeostasis, and the CaSR gene defects are related to diseases characterized by serum calcium level changes. Activating mutations of the CaSR gene cause enhanced sensitivity to extracellular calcium concentration resulting in autosomal dominant hypocalcemia or Bartter-syndrome type V. Inactivating CaSR gene mutations lead to resistance to extracellular calcium. In these cases, familial hypocalciuric hypercalcaemia (FHH1) or neonatal severe hyperparathyroidism (NSHRT) can develop.
  • FHH1 familial hypocalciuric hypercalcaemia
  • NASHRT neonatal severe hyperparathyroidism
  • FHH2 and FHH3 are associated with mutations of genes of partner proteins of calcium signal transduction.
  • the common polymorphisms of the CaSR gene have been reported not to affect the calcium homeostasis itself; however, they may be associated with the increased risk of malignancies (70).
  • the disease or condition is selected from cancers, such as breast cancer, pancreatic cancer, lung cancer, squamous cell carcinoma and non-squamous cell carcinoma.
  • the disease or condition is Charcot-Marie-Tooth neuropathy.
  • the use in the treatment or prevention of a disease or condition as described herein comprises a step of administering the nucleic acid, the nucleic acid vector, the rAAV genome, or the pharmaceutical composition to a subject in need thereof.
  • the administration is selected from oral, sublingual, buccal, intravenous, intravascular, intramuscular, subcutaneous, intraperitoneal, conjunctival, rectal, transdermal, intrathecal, topical and/or inhalation-mediated administration, preferably the administration intravenous, intramuscular, or intraperitoneal administration.
  • the present invention relates to an isolated polypeptide comprising or consisting of the amino acid sequences as described herein above for use in the treatment of the diseases or conditions as described herein above.
  • the present invention relates to an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
  • the isolated peptide has an amino acid sequence of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence depicted in SEQ ID NOs:1 to 8.
  • the invention relates to an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO:1 or SEQ ID NO:2, or an isolated peptide having an amino acid sequence of at least 70% identity to SEQ ID NO:1 or SEQ ID NO:2 for use in the treatment of a disease, comprising administering said polypeptide to a subject in need thereof.
  • the invention relates an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an isolated peptide having an amino acid sequence of at least 70% identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, for use in the treatment of a disease, comprising administering said peptide and a drug delivery system or a molecule that binds to the said peptide to a subject in need thereof.
  • PCRs were performed using human skeletal muscle cDNA and the following primers:
  • Mfn2 intron2 5’-AAGATCTCTCAGCATCCAAAAA-3’
  • Mfn2 exon 19 5’-ATGGCACTTAGGGCTGGCAGCA-3’.
  • Mouse variant amplification was performed using cDNA from mouse embryonic fibroblasts and the primers 5’-AAGCTTGGACAGGTGGAGTCA-3’ and 5’-CAACCAGCCAGCTTT ATTCC-3’.
  • Different motility products were cloned into pCR8 Topo-GW vector (Invitrogen).
  • First-Strand cDNA synthesis was performed using 4 pg of total RNA and the specific Mfn2 primer 5’-TGGCAAGAAGGGAGGCAAGTC-3’ and oligo dT primers incubated at 50°C for 4 h with Superscript IV Reverse Transcriptase (Invitrogen).
  • split super-folder GFP (sfGFP) previously engineered for efficient self-complementation (10) breaks the sequence of sfGFP between the 10 th and the 11 th [3-strand into two parts: GFP1-10 and GFP11, a short, 16 amino acid peptide (57).
  • the GFP1-10 fragment which contains the 3 residues that constitute the GFP chromophore, is nonfluorescent because chromophore maturation requires the conserved E222 residue located on GFPn.
  • the reconstituted GFP becomes fluorescent after the chromophore maturation reaction is completed.
  • GFPn were fused to the (amino) N-terminus of ERMIN2 or ERMIT2 and coexpressed with GFP1.10.
  • pcDNA3.1-GFP(1-10) (Addgene plasmid # 70219) and pEGFP-GFP11-Clathrin light chain (Addgene plasmid # 70217) were from Bo Huang.
  • GFPn was generated by digestion of pEGFP-GFP11-Clathrin light chain using Bglll and Bell.
  • GFPn-Mfn2, GFPn-Ermin2 and GFPn-Ermit2 were generated using Infusion (Takara Technology) and the following primers:
  • siRNA against ERMIN2 and ERMIT2 were synthesized from the following sequences: 5’- GTGATGTGGCCCAACTCTA-3’ and 5’-GACATGATAGATGGCTTGA-3’ respectively; scrambled siRNA was used as control. All siRNAs were obtained from Sigma. Experiments were performed 24 h after transfection.
  • RNA isolation human and mouse tissues were immediately frozen. RNA from liver tissues was extracted using a protocol combining TRIzol reagent (Invitrogen, Carlsbad Ca, USA) and RNAeasy® minikit columns (Qiagen, Alameda, CA, USA), following the manufacturer’s instructions. RNA was reverse transcribed with the Superscript RT IV kit (Invitrogen, Carlsbad Ca, USA). PCR was performed using the ABI Prism 7900 HT realtime PCR machine (Applied Biosystems, USA) and the SYBR® Green PCR Master Mix (Applied Biosystems, USA). Measurements were normalized to PPIA (Cyclophilin A), ARP or p-actin.
  • PPIA Cyclophilin A
  • ARP p-actin
  • Absolute cDNA quantification in real-time PCR was performed using a standard curve method.
  • the amount of plasmid MFN2, ERMIN2 and ERMIT2 was measured using Qubit fluorometric quantification (ThermoFisher scientific) and copy number was calculated using the online application Calculator for determining the number of copies of a template (URI Genomics & Sequencing Center).
  • Real-time PCR was performed using a QuantStudio 6 Real-Time PCR system (Thermo Fisher) using Applied biosystems power Sybr green PCR master mix (AB Applied Bioscience) and the following primers:
  • Probes were designed for ERMIN2 and ERMIT2 so that they covered the flanking sequences at 5’- and -3’ of the skipped region and a non-matching sequence representing -20% of the probe length.
  • PCR was performed using primers that incorporate a SP6 and T7 phage promoter sequence. PCR products were cloned into pGEMT Easy Vector (Promega) and sequenced. Cloned probes were linearized with Sac II restriction enzyme. Retrotranscription and antisense labelled probes were performed using T7 or SP6 phage RNA polymerases (MaxiScript Kit, Ambion) labelled with [a- 32 P] UTP (Perkin Elmer), following manufacturer’s instructions.
  • DNase digestion of template was achieved by phenol/chloroform extraction precipitation with NH4Acetate and linear acrylamide as carrier. Hybridization and digestion were performed using RPA-III Kit (Ambion), Target RNA and positive control RNA obtained from overexpressing cells were hybridized with 4.4 x 10 5 cpm of probe overnight at 56°C. RNase Digestion of unhybridized RNA was performed at 33°C for 60 min with RNase A/T1 mix. Separation and detection of protected fragments were carried out using 8M urea denaturing 5% polyacrylamide gel.
  • the dried gel was exposed at -80°C overnight using an X-ray film (Amersham) or a Molecular Dynamics (Amersham Pharmacia) PhosphoImager plate, which was then scanned with a Typhoon 9200 Bioimaging analyzer (Molecular dynamics, Amersham Pharmacia Biotech, Piscataway, NJ).
  • Mfn 1 ', Mfn1" Mfn2 ⁇ ' ⁇ mouse embryonic fibroblasts were grown in DMEM (Gibco) supplemented with 10% (vol/vol) FBS (Gibco), 1x nonessential amino acids, 2 mM L-glutamine, 100 U/mL penicillin, and 100 pg/mL Streptomycin (Gibco). Cells were maintained at 80-90% confluency. Microscopy experiments were performed at 70% confluency. MEFs were cultured and transfected as described previously using Transfectin (Biorad) or Lipoafectamine 2000 (Invitrogen), following the manufacturer’s instructions (13).
  • siRNA transfection was performed at a final concentration of 250 nM using Oligofectamine (Invitrogen).
  • Oligofectamine Invitrogen
  • 1.6x10 4 cells were seeded onto 24-mm round glass coverslips or 4x10 3 cells onto 13-mm round glass coverslips.
  • images stacks were acquired using a Leica SP5 inverted microscope equipped with confocal or Leica TCS SP2 AOBS, using a 63X1.4 NA Plan Apo objective.
  • Cells were excited using 488nm Ar/ArKr and 561 DPSS (diode pump solid state) lasers.
  • FRET ma x To image the maximum FRET intensity (FRET ma x), cells were treated with 100nM Rapamycin for 15 min and then fixed with 1 % formaldehyde for 10 min. Images were analyzed using PerkinElmer Harmony 3.5 image analysis software. The YFP channel was chosen to mark the region of interest (ROI) and around each ROI, a second boundary was drawn to measure the background (bg) intensity. FRET ba sai and FRET ma x were calculated as:
  • mitochondria aspect ratio was calculated from automatically thresholded images in ImageJ (NIH).
  • images of cells expressing mtRFP orerYFP were processed using the automatic threshold function of ImageJ, followed by deconvolution, 3D reconstruction and surface rendering using the VolumeJ plugin of ImageJ. The interaction between the ER and mitochondria was analyzed using Manders’ colocalization coefficient.
  • Mfn2 ! ' MEFs were co-transfected with the indicated plasmids and with ddGFP-A and ddGFP-B. Forty-eight hours after transfection, ddGFP fluorescence was acquired as previously described (34).
  • Subcellular fractions of HeLa cells (10 9 ) were obtained as described (73). Cells were washed with PBS, suspended in IB (200 mM sucrose, 1 mM EGTA-Tris, and 10 mM Tris- MOPS, pH 7.4), and then disrupted by dounce homogenization. The homogenate was spun at 800 x g for 10 min. The supernatant was recovered and further centrifuged for 10 min at 8000 x g. The resulting pellet (mitochondrial fraction) was collected while the supernatant was further spun for 30 min at 100,000 x g. The resulting pellet (LM fraction) and supernatant (cytosolic fraction) were spun again at 100,000 x g.
  • IB 200 mM sucrose, 1 mM EGTA-Tris, and 10 mM Tris- MOPS, pH 7.4
  • the mitochondrial fraction was further purified by centrifuging twice at 8000 x g for 10 min.
  • the obtained pellet was purified by centrifugation at 95000 x g for 30 minon a 30% Percoll gradient in IB.
  • the mitochondrial layer obtained was washed free ofPercoll and resuspended in IB.
  • Mitochondria-associatedmembranes were identified as an intermediate layer between the light membranesand the mitochondrial fraction on the Percoll gradient, as previously described. 40 pg of protein was separated by SDS-PAGE and immunoblotted as indicated in the figure legends. Liver fractions were purified as previously described (59, 60).
  • Adenoviruses M-ERMIN2 and M-ERMIT2 (1 x10 9 IFU/mouse) were tail-vein injected to reach the liver of Mfn2 LK0 mice.
  • Light membranes (LM) (1 mg/ml) were isolated from livers 3 days after adenovirus delivery and incubated with the indicated trypsin concentrations for 15 min on ice.
  • SBTI (5 mg/ml) was added and incubated for 20 min on ice.
  • Laemmli sample buffer was added, and samples were boiled for 5 min and separated by 12% SDS-PAGE.
  • DynaBeads Forty pl of DynaBeads were crosslinked with 3 pl of anti-GFP (Invitrogen) or 4 pl of anti-HA (Roche) using BS3 (Sulfo-DSS, Thermo Scientific Pierce). Total protein extracts (400 pg) were dissolved in RIPA buffer (1% TritonX-100, 0.1 %SDS, 0.5% deoxycholate, 0.15M NaCI, 5 mM Tris, pH8) and incubated with antibody-crosslinked Dynabeads protein G (Thermo fisher Scientific) for 16 h 4°C in rotation.
  • RIPA buffer 1% TritonX-100, 0.1 %SDS, 0.5% deoxycholate, 0.15M NaCI, 5 mM Tris, pH8
  • Homogenates for western blot analyses were obtained from either cell cultures or tissues.
  • Cells (1.8x10 6 or 80% of confluence) were harvested 24h (for siRNA experiments) or 48h after transfection and lysed in 150 mM NaCI, 1 % Nonidet P-40/0, 0.25% deoxycholate, 1mM EDTA, and 50mM Tris, pH 7.4 in the presence of complete protease inhibitor mixture (Sigma-Aldrich).
  • Tissue samples were homogenized in 10 volumes of lysis buffer using a polytron. Homogenates were rotated for 1 h at 4°C in an orbital shakerand centrifuged at 13,000 rpm for 15 min at 4°C.
  • Peroxidase-conjugated anti-mouse, anti-goat or antirabbit immunoglobulins were used as secondary antibodies.
  • the following antibodies were used in tissue extracts: Mfn2 (1 :1000, Abeam); p-elF2a; elF2a; p-PERK; PERK (1:1000, Cell Signaling); CHOP (1 :1000, Santa Cruz Biotechnologies); [3-actin (1 :10000, Sigma); and a-tubulin (1 :8000, Sigma).
  • the specific proteins were detected by the ECL western blotting detection analysis system (Amersham). Densitometry was performed using ImageJ (NIH).
  • ER Ca 2+ measurement cells grown on 13-mm round glass coverslips at 50% confluence were co-transfected with erAEQ and the plasmids indicated.
  • erAeq the luminal [Ca 2+ ] E R was reduced by incubating cells for 5 min at 37°C with KRB supplemented with 1% FBS, 1 mM EGTA, 10 pM ionomycin and 0.1 mM tBuBHQ.
  • cytosolic and mitochondrial Ca 2+ measurement cells were co-transfected with CytAEQ and mtAEQ and the indicated plasmid. Reconstitution was performed in KRB containing 1 mM Ca 2+ , and measurement and calibration were performed in KRB containing 100 pM EGTA. ATP was used as IP3R agonist.
  • Mfn2 ⁇ ' ⁇ MEFs were first co-transfected with cytosolic mCherry and with ddGFP-A and ddGFP-B. After 24 h cells were transfected with the indicated plasmids. Twenty-four hours after the second transfection, GFP and mCherry fluorescence were evaluated by flow cytometry analysis. Acquisition was stopped when 50,000 FL2+ events were reached. Data are shown as dot plot of FL1 versus side scatter derived from FL2+ gated events.
  • Sorted GFP + cells were cultured in DMEM supplemented with 2 mM glutamine, 1 mM penicillin/streptomycin, 20% (vol/vol) FBS at 37 °C in a fully humidified atmosphere of 95% air and 5% CO2. On the following day, the medium was replaced to remove dead cells. Growing cells were fixed after 8 h and prepared for EM images.
  • MEFs cotransfected with GFP and the indicated plasmids and sorted as described above were fixed with 1.25% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate at pH 7.4 for 1 h at room temperature. Thin sections were imaged on a Tecnai-20 electron microscope (Philips-FEI).
  • Liver biopsies were obtained from NAFLD patients undergoing bariatric surgery; liver biopsies from normal individuals were not collected due to ethical issues. Biopsies of subcutaneous adipose tissue and skeletal muscle were obtained from non-obese subjects. All patients gave written informed consent. The study protocols conformed to the Ethical Guidelines of the 1975 Declaration of Helsinki, revised in 2000, as reflected in a priori approval by the Hospital Sant Joan de Reus (Institutional Review Board, project code: INFLAMED/15-04-30/4prog7), Hospital Joan XXIII, and Human Ethics Committee.
  • mice were anesthetized using isoflurane and sacrificed by cervical dislocation. Tissues used for RNA purification, protein extraction or histology were prepared as described (61, 62). Sample size was not predetermined.
  • MCD protocol male and female mice were randomized to Chow or MCD diet following a blinded draw of their ID. researchers were not blinded to the genotype, the insert of the adenovirus injected, or the diet treatment. All animals were included in the analyses.
  • adenoviruses (1 *10 9 IFU/mouse) were tail vein injected. Livers were isolated 5 days after adenovirus delivery.
  • Ad-LacZ Ad-LacZ
  • M-ERMIN2 and M-ERMIT2 encoding for human ERMIN2 and ERMIT2
  • ERMIN2 and ERMIT2 were cloned by recombination into the pAdeno-CMV-V5 adenoviral vector (Invitrogen) using the Gateway system.
  • Adenoviruses were generated by transfection of the adenoviral expression vectors in a human embryonic kidney cell line (HEK 293). The adenoviruses generated were then amplified at the Unitat de Produccio de Vectors Virals-CBATEG (UniversitatAutonoma de Barcelona).
  • Liver was homogenized use Teflon-glass homogenizer in Isolation Buffer (IB) (225 mM Mannitol, 25 mM Hepes-KOH, 1 mM EGTA, pH7.4 and protease inhibitors) at a ratio of 4 ml of IB for every gram of tissue.
  • IB Isolation Buffer
  • the homogenate was pelleted for 10 min at 1 ,500 x g at 4°C.
  • the supernatant was transferred to a new tube and pelleted again as above, transferred again to a new tube, and pelleted at 13,000 x g for 20 min at 4°C.
  • This new pellet contained the crude mitochondria fraction, comprising mitochondria and MAM, and was used to measure lipid transfer.
  • the supernatant contained the ER fraction, which was pelleted at 100,000 x g for 1 h at 4°C. This pellet was used as a control in the assay.
  • One mg of the fraction was pelleted again and resuspended in 200pL of PS assay buffer [25 mM Hepes-KOH, 10 mM CaCh, 0.4 mM of 3 H-Ser (20-30 pci/pmol) pH 7.4], The mixture was incubated for 45 min at 37°C and the reaction was stopped by adding 3 volumes of chloroform: MeOH (2:1). Lipids were extracted using the Folch Method, dried on an N2 flow and separated by TLC as described (63, 64).
  • Example 1 Human and mouse Mitofusin 2 gene are alternatively spliced
  • Alternative splicing expands the functional repertoire of the human genome by generating multiple proteins with different intracellular localization and function (25). For example, alternative splicing of gene coding for the master mitochondrial fission executor dynamin related protein 1 (Drp1) results in a brain-specific isoform that regulates dendrite formation independently of mitochondrial fission (26, 27).
  • Drp1 master mitochondrial fission executor dynamin related protein 1
  • Ribonuclease protection assay confirmed that the mRNAs of ERMIN2 and ERMIT2 were expressed in HeLa cells (Fig.1 B).
  • Fig.SA Real-time PCR assays to specifically detect mRNAs of ERMIN2, ERMIT2 and MFN2 in human samples.
  • ERMIN2 accounted for 20-25%
  • ERMIT2 for 7-52% of MFN2 mRNA expression in white adipose tissue, skeletal muscle, and liver from human subjects (Fig.8B).
  • SERCA sarcoplasmic/endoplasmic reticulum Ca2+ ATPase
  • ERMIN2 andERMIT2 The proteins encoded by ERMIN2 andERMIT2 were predicted to be shorter than MFN2, partially or completely lacking its GTPase and coiled-coil 1 domains but retaining its transmembrane and coiled-coil 2 regions (Fig.lA and 9).
  • Exogenous HA-tagged ERMIN2 and ERMIT2 expressed in HeLa were detected at around their theoretical molecular masses i.e., 41 and 43 KDa and like full-length MFN2 were recognized by antibodies raised against MFN2 aa557-576 or CC2 domain (Fig.8D).
  • Using the former antibody we retrieved ERMIN2 and ERMIT2 running at their predicted molecular weight in human skeletal muscle, liver, and white adipose tissue extracts.
  • the ratio between ERMIN2 and MFN2 levels was 0.39 in skeletal muscle, 1.8 in liver and 0.17 in WAT, whereas the ERMIT2/MFN2 ratio was 0.3- 0.35 in all
  • ERMIN2 and ERMIT2 lack several domains found in full-length MFN2, we addressed whether they were targeted to mitochondria.
  • Mfn2LKO Mfn2 knockout livers
  • ERMIN2 endogenous ERMIN2 was present exclusively in LM and ERMIT2 in LM and MAMs, but not in the pure mitochondria fraction of HeLa cells, at a difference from full length MFN2 that was retrieved in all three fractions (13) (Fig.11).
  • ERMIN2 and ERMIT2 were coexpressing GFP-tagged MFN2, ERMIN2 and ERMIT2 with a mitochondrially targeted cyan fluorescent protein (mtCFP) and a dsRED fluorescent protein targeted to the ER (ER-RFP) in Mfn2-/- mouse embryonic fibroblasts (MEFs).
  • mtCFP mitochondrially targeted cyan fluorescent protein
  • ER-RFP dsRED fluorescent protein targeted to the ER
  • ERMIN2 and ERMIT2 While re-expressed MFN2 localized mostly in mitochondria and only partially in the ER, ERMIN2 and ERMIT2 displayed a pattern like that of the ER marker. Upon closer inspection, ERMIN2 was uniformly distributed on the ER and ERMIT2 displayed a punctuate staining colocalizing with ER and mitochondria, compatible with the retrieval of the endogenous protein in MAMs (Fig.2C,D; individual channels in Fig.12).
  • GFP-moV-MFN2 at the interface between ER and mitochondria in Mfn2-/- MEFs (Fig.10D,E) and endogenous moV-MFN2 in MAMs and LM fractions purified from MEFs (Fig.lOF).
  • ERMIN2 and ERMIT2 localize only at the ER and ERMIT2 and moV-MFN2 are enriched at the ER-mitochondria interface.
  • GFP fluorescence was detected when we coexpressed GFP1-10 with GFP11-MFN2, GFP11-ERMIN2 and GFP11-ERMIT2, indicating that the N-termini of MFN2, ERMIN2 and ERMIT2 face the cytosol (Fig.15A).
  • Fig.15A To understand whether the C- termini of ERMIN2 and ERMIT2 were exposed to the lumen of the ER, we performed limited trypsin proteolysis assays on LM fractions purified from Mfn2LKO livers infected with adenoviruses expressing ERMIN2 and ERMIT2.
  • ERMIN2 and ERMIT2 C-termini were exposed to the lumen, trypsin would eliminate the epitopes recognized by the antibody against their N-terminus, whereas the CC2 epitope would be protected, and vice versa if the N-terminus was facing the ER lumen (Fig.15B). Both the N-terminal and the CC2 epitopes were lost upon trypsin treatment, indicating that not only the N-terminus, but also the CC2 domain of ERMIN2 and ERMIT2 were accessible to the protease (Fig.15C). Thus, ERMIN2 and ERMIT2 N- and C-termini face the cytosol (Fig.15D).
  • ERMIN2 Fluorescent recovery after photobleaching (FRAP) assays further corroborated that expression of MFN2 and ERMIN2 restored ER connectivity in Mfn2-/- MEFs.
  • ERMIT2 slightly ameliorated ER morphology, it did not improve ER connectivity (Fig.3C,D).
  • siRNAs to specifically downregulate ERMIN2 or ERMIT2 in HeLa cells (Fig.16).
  • Depletion of ERMIN2 or ERMIT2 did not affect mitochondrial morphology (Fig.3E,F).
  • ERMIN2 downregulation altered peripheral ER morphology and diminished ER interconnectivity, as determined by FRAP analysis (Fig.3G).
  • ERMIN2 and ERMIT2 do not modulate mitochondrial morphology, and endogenous ER IN2 is required for ER connectivity even when MFN2 is present.
  • ERMIT2 is retrieved in MAMs and does not regulate ER or mitochondrial morphology
  • Re-expression of MFN2 as well as of ERMIT2 in Mfn2-/- MEFs improved ER-mitochondria pseudocolocalization.
  • ERMIN2 that restored ER morphology did not modify ER-mitochondria juxtaposition (Fig.
  • ERMIN2 did not significantly increase the fraction of Mfn2-/-ddGFP+ cells, expression of MFN2 or ERMIT2 led to a 2- and 1.75-fold increase in ddGFP positivity (Fig.4D,E). Live confocal imaging confirmed that expression of MFN2 and ERMIT2, but not ERMIN2 in Mfn2-/- cells led to a punctuated ddGFP signal, reminiscent of ER-mitochondria contacts (Fig.18).
  • ER-mitochondria contacts in transmission electron microscopy images from where we calculated the ER-mitochondria contact coefficient (ERMICC) that computes the average ER-mitochondria distance and interaction length over the mitochondrial perimeter (16).
  • MFN2 and ERMIT2 but not ERMIN2 increased ER- mitochondria juxtaposition and ERMICC in Mfn2-/- cells (Fig. 4F,G).
  • MFN2 and ERMIT2 indeed decreased the average ER-mitochondria distance measured from >70 interorganellar interactions occurring in the ⁇ 30 nm range in Mfn2-/- cells (Fig.19).
  • moV-MFN2 which resembles ERMIT2
  • ER-mitochondria tethering we analyzed whether moV-MFN2, which resembles ERMIT2, was also involved in ER-mitochondria tethering.
  • Expression of moV-MFN2 in Mfn2-/- cells increased ER- mitochondria juxtaposition, as reflected by increased pseudolocalization of fluorescent proteins targeted to ER and mitochondria (Fig. 10 G, H).
  • ERMIT2 and moV-MFN2 increases ER-mitochondria juxtaposition.
  • a feature of heterotypic tethers is their interaction in trans with homo or heterotypic partners on the surface of the opposite organelle.
  • mitochondrial Mfns were the mitochondrial partners of ERMIT2.
  • ERMIT2 and ERMIN2
  • GFP-tagged ERMIN2 or ERMIT2 immunoprecipitated V5-tagged MFN2ActA and Flag-tagged MFN1 that are restricted to mitochondria (13), although ERMIN2 immunoprecipitated these mitochondrial Mfns somewhat less efficiently (Fig.5A).
  • MFN2 Mfn1, Mfn2-/-
  • MFN2 and ERMIT2 were unable to increase ER-mitochondria tethering unless we coexpressed them with MFN1 that is retrieved only on the surface of mitochondria (13) (Fig.5B,C).
  • ER ERMIT2 interacts with mitochondrial MFN1 for ER-mitochondria tethering.
  • Example 7 Coiled-coil domains mediate Ermin2/Ermit2-mitofusin interaction
  • MFN1, MFN2, ERMIN2 and ERMIT2 We generated multiple deletion mutants of MFN1, MFN2, ERMIN2 and ERMIT2 to address which domains were required for their heterotypic interaction.
  • GTPase domain to the MFN1 and MFN2 mutants containing only the CC1 region, they comparably immunoprecipitated ERMIN2 and ERMIT2 (Fig.6B).
  • MFN1 and MFN2 interact with ERMIN2 or ERMIT2 through their CC1 or CC2 domains.
  • ERMIT2 mutant lacking the pre-TM domain still immunoprecipitated MFN1 , whereas upon deletion of its CC2 domain MFN1 binding was lost (Fig.6C).
  • ERMIT2 and ERMIN2 also interacted in HeLa cells as well as in MEFs lacking Mfn1, Mfn2 or both mitofusins (Fig.20A).
  • Fig.20A we further tested the emerging interaction paradigm between the C- terminal domains of ERMIT2 or ERMIN2 and the CC1/CC2 domains of mitochondrial Mitofusins in an in silico structural modeling analysis.
  • ERMIT2 like MFN2 influenced two facets of ER-mitochondria communication: mitochondrial uptake of ER-released Ca2+(31 , 32) and lipid transfer (2, 10, 33).
  • MFN2, ERMIN2 or ERMIT2 lowered steady state ER Ca2+ levels measured in empty vector (EV) transfected Mfn2-/- MEFs (13, 34) (Fig.21 A, B).
  • IP3 inositol triphosphate
  • a genetically encoded ATP probe targeted to the mitochondrial matrix (3) indicated that MFN2 and ERMIT2 expression increased the Ca2+ uptake-dependent priming of mitochondrial ATP generation (16, 37) (Fig.7C, D).
  • ERMIT2 expression in Mfn2-/- cells licenses mitochondrial uptake of ER-released Ca2+ and Ca2+-primed mitochondrial ATP production that depends on matrix Ca2+-activated Krebs’ cycle dehydrogenases (38).
  • ERMIT2 reduced the H2O2 accumulation (Fig.22B), the ER stress (evidenced by accumulation of phosphorylated elF2a and CHOP, Fig.22 C, D) and the increased expression of the inflammation marker TN Fa (Fig.22E) observed in the Mfn2LKO cohort (10).
  • Example 9 ERMIT2 and its importance of ER-mitochondria juxtaposition in models of NASH.
  • mice fed a methionine-choline deficient high fat diet (MCD) to induce a NASH-like condition represent an orthogonal model of liver ER stress and inflammation due to MFN2 deficiency (ref.10 and Fig.23A).
  • Intravenous administration of adenoviruses encoding ERMIT2 at day 7 of a 3-week MCD feeding protocol increased ERMIT2 but not MFN2 levels (Fig.23A,B) and corrected the increased hepatic accumulation of L-Ser into PS and the reduced L-Ser labeling of PE, a proxy of the reduced PS transfer between ER and mitochondria (Fig.7G,H).
  • ERMIT2 reduced the accumulation of markers of ER stress (Fig.23C,D), and inflammation (Fig.23E), without altering body weight or glycemia (Fig.23F,G).
  • ERMIT2 sustains ER to mitochondria lipid transfer and corrects the liver ER stress and inflammation in genetic and environmental mouse models of reduced liver Mfn2 expression, further highlighting the importance of ER-mitochondria juxtaposition in models of NASH.
  • adeno-associated virus (serotype AAV8 or AAV9) containing Ermit2 (SEQ ID NO:1) or Ermin2 (SEQ ID NO:2) sequences under a liver specific promoter.
  • Mice will be fed with a methionine-choline deficient high fat diet (MCD) to induce a NASH-like condition.
  • MCD methionine-choline deficient high fat diet
  • Adenoviruses encoding Ermin2 will be intravenously administrated at day 1 of a 3-week MCD feeding protocol.
  • ER stress marker inflammation and fibrosis will be evaluated by measuring mRNA expression or protein levels. Steatosis will be assessed by H&E histochemistry techniques.
  • AAV-Ermit2 and AAV-Ermin2 in Wilson disease and Serpinopathies, such as for example alpha-1 antitrypsin deficiency (AATD).
  • AATD alpha-1 antitrypsin deficiency
  • the different mice models will be infected at day 1 , the experiment will be finished after 2 or 3 weeks of initial infection.
  • ER stress marker, inflammation and fibrosis will be evaluated using mRNA and protein expression and by H&E histochemistry techniques.
  • AAVs serotype AAV8 or AAV9 of Ermit2 (SEQ ID NO:1) or Ermin2 (SEQ I D NO:2) under a specific kidney promoter and evaluate the capacity of Ermit2 and Ermin2 to ameliorate the ER stress of different renal diseases.
  • AKI acute kidney injury
  • CKD AKI-chronic kidney disease
  • ADTKD tubulointerstitial kidney disease
  • the different mice models will be infected at day 1 , the experiment will be finished after 2 or 3 weeks of initial infection.
  • ER stress marker, inflammation and fibrosis will be evaluated using mRNA and protein expression and by H&E histochemistry techniques.
  • AAVs serotype AAV8 or AAV9 of Ermit2 (SEQ ID NO:1) or Ermin2 (SEQ ID NO:2) under the hCMV promoter.
  • Ermin2 and Ermit2 on different rare diseases characterized by ER stress and evaluate the capacity of the variants to ameliorate the phenotype of Wolfram syndrome, cystic fibrosis, myopathies and Duchenne muscular dystrophy and Charcot-Marie-Tooth type2A mice models and measure the accumulation of markers of ER stress.
  • the different mice models will be infected at day 1 , the experiment will be finished after 2 or 3 weeks of initial infection.
  • ER stress marker, inflammation and fibrosis will be evaluated using mRNA and protein expression and by H&E histochemistry techniques.
  • Example 11 Expression of MFN2 variants in disease cellular model
  • AATD alpha-1 antitrypsin deficiency
  • ER stress such as for example Wolfram slndrome (WS1 ; WS2), alpha-1 antitrypsin deficiency (AATD and Z-AAT), Interstitial Lung Diseases (MUC5B), nephrotic syndrome (NPHSA, NPHS2), diabeticnephropathy (SORBS1)
  • mRNA and protein level nephrotic syndrome
  • SORBS1 diabeticnephropathy
  • FIG. 25 shows that ERMIN2 (SEQ ID NO:2) recovers the altered ER morphology caused by the overexpression of the mutated protein form. Mutation in a1AT gene cause protein aggregation due to the incorrect folding of the protein. These aggregates, that accumulate in the ER, interfere with the normal maturation and posterior secretion of the protein. Our variants correct ER morphology and decrease the protein aggregation (Fig 25). Thus, variants correct one of the major hallmarks of AATD disease.
  • the variants of present invention decrease the a1AT protein at ER and thus, promote its secretion.
  • the graph clearly shows that a1AT mutation increases its retention at the organelle fraction and that the variants of present invention are able to reduce the retention of a1AT to similar levels than control cells.
  • Mitofusin 2 is an endoplasmic reticulum-mitochondria tether. Proceedings of the National Academy of Sciences of the United States of Americans, 11249-11254 (2016).
  • VAPB mitochondrial protein
  • Mitofusin 2 is an endoplasmic reticulum-mitochondria tether. Proceedings of the National Academy of Sciences of the United States of Americal 13, 11249-11254 (2016).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des acides nucléiques codant pour des variants de mitofusine 2 (Mfn2), des vecteurs d'acides nucléiques comprenant lesdits acides nucléiques, ainsi que leur utilisation dans le traitement de maladies et d'états caractérisés par un stress du réticulum endoplasmique (ER).
PCT/EP2024/067267 2023-06-21 2024-06-20 Variants de mfn2 et leur utilisation dans le traitement/la prévention de maladies associées à des altérations dans la fonction du réticulum endoplasmique Pending WO2024261139A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23382636 2023-06-21
EP23382636.1 2023-06-21

Publications (1)

Publication Number Publication Date
WO2024261139A1 true WO2024261139A1 (fr) 2024-12-26

Family

ID=86942435

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/067267 Pending WO2024261139A1 (fr) 2023-06-21 2024-06-20 Variants de mfn2 et leur utilisation dans le traitement/la prévention de maladies associées à des altérations dans la fonction du réticulum endoplasmique

Country Status (1)

Country Link
WO (1) WO2024261139A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001025274A1 (fr) 1999-10-06 2001-04-12 The Board Of Trustees Of The Leland Stanford Junior University Mitofusines, homologues fzo, et derives fonctionnels de celles-ci
WO2004074482A1 (fr) 2003-02-21 2004-09-02 Universidad De Barcelona Methode de diagnostic du diabete et de l'obesite
WO2019094830A1 (fr) * 2017-11-10 2019-05-16 Washington University Agents de modulation de mitofusine et leurs méthodes d'utilisation
US20210017509A1 (en) 2018-03-23 2021-01-21 The Trustees Of Columbia University In The City Of New York Gene Editing for Autosomal Dominant Diseases
WO2022015715A1 (fr) 2020-07-13 2022-01-20 The Trustees Of The University Of Pennsylvania Compositions utiles pour le traitement de la maladie de charcot-marie-tooth
WO2022072793A1 (fr) 2020-10-02 2022-04-07 University Of Massachusetts Modulateurs de marf/mfn et leurs utilisations
WO2023049846A1 (fr) * 2021-09-24 2023-03-30 The Trustees Of The University Of Pennsylvania Compositions utiles pour le traitement de la maladie de charcot-marie-tooth
WO2023081835A1 (fr) 2021-11-04 2023-05-11 Emory University Multiplication des lymphocytes t à l'aide d'activateurs de la mitofusine et utilisation dans les thérapies anticancéreuses

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001025274A1 (fr) 1999-10-06 2001-04-12 The Board Of Trustees Of The Leland Stanford Junior University Mitofusines, homologues fzo, et derives fonctionnels de celles-ci
WO2004074482A1 (fr) 2003-02-21 2004-09-02 Universidad De Barcelona Methode de diagnostic du diabete et de l'obesite
WO2019094830A1 (fr) * 2017-11-10 2019-05-16 Washington University Agents de modulation de mitofusine et leurs méthodes d'utilisation
US20210017509A1 (en) 2018-03-23 2021-01-21 The Trustees Of Columbia University In The City Of New York Gene Editing for Autosomal Dominant Diseases
WO2022015715A1 (fr) 2020-07-13 2022-01-20 The Trustees Of The University Of Pennsylvania Compositions utiles pour le traitement de la maladie de charcot-marie-tooth
WO2022072793A1 (fr) 2020-10-02 2022-04-07 University Of Massachusetts Modulateurs de marf/mfn et leurs utilisations
WO2023049846A1 (fr) * 2021-09-24 2023-03-30 The Trustees Of The University Of Pennsylvania Compositions utiles pour le traitement de la maladie de charcot-marie-tooth
WO2023081835A1 (fr) 2021-11-04 2023-05-11 Emory University Multiplication des lymphocytes t à l'aide d'activateurs de la mitofusine et utilisation dans les thérapies anticancéreuses

Non-Patent Citations (70)

* Cited by examiner, † Cited by third party
Title
A. DUARTE ET AL.: "Mitochondrial fusion is essential for steroid biosynthesis", PLOS ONE, vol. 7, 2012, pages 45829
A. FRANCO ET AL.: "Burst mitofusin activation reverses neuromuscular dysfunction in murine CMT2A", ELIFE, vol. 9, 2020
A. G. ROCHA ET AL.: "MFN2 agonists reverse mitochondrial defects in preclinical models of Charcot-Marie-Tooth disease type 2A", SCIENCE, vol. 360, 2018, pages 336 - 341, XP055962875, DOI: 10.1126/science.aao1785
A. KASAHARAS. CIPOLATY. CHENG. W. DORNL. SCORRANO: "Mitochondrial fusion directs cardiomyocyte differentiation via calcineurin and Notch signaling", SCIENCE, vol. 342, 2013, pages 734 - 737
A. MOURIER ET AL.: "Mitofusin 2 is required to maintain mitochondrial coenzyme Q levels", J CELL BIOL, vol. 208, 2015, pages 429 - 442
A. OLICHON ET AL.: "OPA1 alternate splicing uncouples an evolutionary conserved function in mitochondrial fusion from a vertebrate restricted function in apoptosis", CELL DEATH. DIFFER., vol. 14, 2007, pages 682 - 692, XP009088983, DOI: 10.1038/sj.cdd.4402048
A. SUGIURA ET AL.: "MITOL regulates endoplasmic reticulum-mitochondria contacts via Mitofusin2", MOL.CELL, vol. 51, 2013, pages 20 - 34, XP028675712, DOI: 10.1016/j.molcel.2013.04.023
A. W. SENIOR ET AL.: "Improved protein structure prediction using potentials from deep learning", NATURE, vol. 577, 2020, pages 706 - 710, XP037038905, DOI: 10.1038/s41586-019-1923-7
B. KORNMANN ET AL.: "An ER-mitochondria tethering complex revealed by a synthetic biology screen.", SCIENCE, vol. 325, 2009, pages 477 - 481
C. GLYTSOU ET AL.: "Mitophagy promotes resistance to BH3 mimetics in acute myeloid leukemia", CANCER DISCOV, 2023
C. GUELLY ET AL.: "Targeted high-throughput sequencing identifies mutations in atlastin-1 as a cause of hereditary sensory neuropathy type I", AM J HUM GENET, vol. 88, 2011, pages 99 - 105
CHENG, S.H. ET AL.: "Defective Intracellular Transport and Processing of CFTR Is the Molecular Basis of Most Cystic Fibrosis", CELL, vol. 63, 1990, pages 827 - 834, XP023884229, DOI: 10.1016/0092-8674(90)90148-8
D. KAMIYAMA ET AL.: "Versatile protein tagging in cells with split fluorescent protein", NATURE COMMUNICATIONS, vol. 7, 2016, pages 11046
D. LIX. LIY. GUANX. GUO: "Mitofusin-2-mediated tethering of mitochondria and endoplasmic reticulum promotes cell cycle arrest of vascular smooth muscle cells in GO/G1 phase", ACTA BIOCHIMICA ET BIOPHYSICASINICA47, 2015, pages 441 - 450
D. NAON ET AL.: "Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum-mitochondria tether", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 113, 2016, pages 11249 - 11254
D. W. HAILEY ET AL.: "Mitochondria supply membranes for autophagosome biogenesis during starvation", CELL, vol. 141, 2010, pages 656 - 667
DE BRITO OLGA MARTINS ET AL: "Mitofusin 2 tethers endoplasmic reticulum to mitochondria", NATURE,, vol. 456, no. 7222, 1 December 2008 (2008-12-01), pages 605 - 610, XP037696551, DOI: 10.1038/NATURE07534 *
E. AREA-GOMEZ ET AL.: "Upregulated function of mitochondria-associated ER membranes in Alzheimer disease", EMBO J., 2012
E. AREA-GOMEZ ET AL.: "Upregulated function of mitochondria-associated ER membranes in Alzheimer disease", THE EMBO JOURNAL, vol. 31, 2012, pages 4106 - 4123
E. AREA-GOMEZ: "Assessing the function of mitochondria-associated ER membranes", METHODS IN ENZYMOLOGY, vol. 547, 2014, pages 181 - 197
F. GIORDANO ET AL.: "PI(4,5)P(2)-dependent and Ca(2+)-regulated ER-PM interactions mediated by the extended synaptotagmins", CELL, vol. 153, 2013, pages 1494 - 1509
F. X. SORIANO ET AL.: "Evidence for a mitochondrial regulatory pathway defined by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, estrogen-related receptor-alpha, and mitofusin 2", DIABETES, vol. 55, 2006, pages 1783 - 1791
G. CSORDAS ET AL.: "Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface", MOL CELL, vol. 39, 2010, pages 121 - 132, XP002779277
G. CSORDAS ET AL.: "Structural and functional features and significance of the physical linkage between ER and mitochondria", J.CELL BIOL., vol. 174, 2006, pages 915 - 921
G. HAJNOCZKYL. D. ROBB-GASPERSM. B. SEITZA. P. THOMAS: "Decoding of cytosolic calcium oscillations in the mitochondria", CELL, vol. 82, 1995, pages 415 - 424
G. ORSO ET AL.: "Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin", NATURE, vol. 460, 2009, pages 978 - 983
GILBERT, A. ET AL.: "ΔF508 CFTR Localizes in the Endoplasmic Reticulum-Golgi Intermediate Compartment in Cystic Fibrosis Cells", EXP. CELL RES., vol. 242, 1998, pages 144 - 152
H. CHEN ET AL.: "Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development", J.CELL BIOL., vol. 160, 2003, pages 189 - 200, XP002329546, DOI: 10.1083/jcb.200211046
J. M. SUSKI ET AL.: "Isolation of plasma membrane-associated membranes from rat liver", NAT PROTOC, vol. 9, 2014, pages 312 - 322, XP037547614, DOI: 10.1038/nprot.2014.016
J. PRUDENT ET AL.: "MAPL SUMOylation of Drp1 Stabilizes an ER/Mitochondrial Platform Required for Cell Death", MOL CELL, vol. 59, 2015, pages 941 - 955, XP029272923, DOI: 10.1016/j.molcel.2015.08.001
J. R. FRIEDMAN ET AL.: "ER Tubules Mark Sites of Mitochondrial Division", SCIENCE, vol. 334, 2011, pages 358 - 362
K. ITOH ET AL.: "A brain-enriched Drp1 isoform associates with lysosomes, late endosomes, and the plasma membrane", J BIOL CHEM, vol. 293, 2018, pages 11809 - 11822
K. ITOH ET AL.: "Brain-specific Drp1 regulates postsynaptic endocytosis and dendrite formation independently of mitochondrial division", ELIFE8, 2019
K. J. DE VOS ET AL.: "VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis", HUM.MOL.GENET., vol. 21, 2012, pages 1299 - 1311
K. SINGARAVELU ET AL.: "Mitofusin 2 Regulates STIM1 Migration from the Ca2+ Store to the Plasma Membrane in Cells with Depolarized Mitochondria", J BIOL CHEM., vol. 286, 2011, pages 12189 - 12201
KERBIRIOU, M.: "Coupling Cystic Fibrosis to Endoplasmic Reticulum Stress: Differential Role of Grp78 and ATF6", BIOCHIM. BIOPHYS. ACTA MOL. BASIS DIS., vol. 1772, 2007, pages 1236 - 1249, XP022384925
L. S. JOUAVILLEP. PINTONC. BASTIANUTTOG. A. RUTTERR. RIZZUTO: "Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming", PROC.NATL.ACAD.SCI.U.S.A., vol. 96, 1999, pages 13807 - 13812
L. SCORRANO ET AL.: "BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis", SCIENCE, vol. 300, 2003, pages 135 - 139
L. SCORRANO ET AL.: "Coming together to define membrane contact sites", NATURE COMMUNICATIONS, vol. 10, 2019, pages 1287
LI JZHANG D: "Disruption of Sarcoplasmic Reticulum-Mitochondrial Contacts Underlies Contractile Dysfunction in Experimental and Human Atrial Fibrillation: A Key Role of Mitofusin 2", J AM HEART ASSOC, vol. 11, 2022, pages 024478
M. EISENBERG-BORDN. SHAIM. SCHULDINERM. BOHNERTA TETHER: "Is a Tether Is a Tether: Tethering at Membrane Contact Sites", DEV CELL, vol. 39, 2016, pages 395 - 409, XP029819211, DOI: 10.1016/j.devcel.2016.10.022
M. GIACOMELLO ET AL.: "Ca2+ hot spots on the mitochondrial surface are generated by Ca2+ mobilization from stores, but not by activation of store-operated Ca2+ channels", MOL CELL, vol. 38, 2010, pages 280 - 290
M. HAMASAKI ET AL.: "Autophagosomes form at ER-mitochondria contact sites", NATURE, vol. 495, 2013, pages 389 - 393
M. I. HERNANDEZ-ALVAREZ ET AL.: "Deficient Endoplasmic Reticulum-Mitochondrial Phosphatidylserine Transfer Causes Liver Disease", CELL, vol. 177, 2019, pages 881 - 895
M. LIESA ET AL.: "Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta", PLOS ONE, vol. 3, 2008, pages 3613
M. R. WIECKOWSKIC. GIORGIM. LEBIEDZINSKAJ. DUSZYNSKIP. PINTON: "Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells", NAT PROTOC, vol. 4, 2009, pages 1582 - 1590
M. SCHNEEBERGER ET AL.: "Mitofusin 2 in POMC Neurons Connects ER Stress with Leptin Resistance and Energy Imbalance", CELL, vol. 155, 2013, pages 172 - 187, XP028729742, DOI: 10.1016/j.cell.2013.09.003
N. BORGESEI. GAZZONIM. BARBERIS. COLOMBOE. PEDRAZZINI: "Targeting of a tail-anchored protein to endoplasmic reticulum and mitochondrial outer membrane by independent but competing pathways", MOL BIOL CELL, vol. 12, 2001, pages 2482 - 2496
N. ISHIHARAY. EURAK. MIHARA: "Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity", J.CELL SCI., vol. 117, 2004, pages 6535 - 6546
O. M. DE BRITOL. SCORRANO: "Mitofusin 2 tethers endoplasmic reticulum to mitochondria", NATURE, vol. 456, 2008, pages 605 - 610, XP037696551, DOI: 10.1038/nature07534
O. M. DE BRITOL. SCORRANO: "Mitofusin 2: a mitochondria-shaping protein with signaling roles beyond fusion", ANTIOXID.REDOX.SIGNAL.10, 2008, pages 621 - 633
P. COSSONA. MARCHETTIM. RAVAZZOLAL. ORCI: "Mitofusin-2 independent juxtaposition of endoplasmic reticulum and mitochondria: an ultrastructural study", PLOS ONE, vol. 7, 2012, pages 46293
P. PINTON ET AL.: "Reduced loading of intracellular Ca2+ stores and downregulation of capacitative Ca2+ influx in Bcl-2-overexpressing cells", J.CELL BIOL., vol. 148, 2000, pages 857 - 862
R. FILADI ET AL.: "Mitofusin 2 ablation increases endoplasmic reticulum-mitochondria coupling", PROC NATL ACAD SCI U S A, vol. 112, 2015, pages 2174 - 2181
R. G. HANSFORD: "Dehydrogenase activation by Ca2+ in cells and tissues", J.BIOENERG.BIOMEMBR, vol. 23, 1991, pages 823 - 854
R. GALMES ET AL.: "ORP5/ORP8 localize to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function", EMBO REP, vol. 17, 2016, pages 800 - 810, XP072235439, DOI: 10.15252/embr.201541108
R. H. BALOHR. E. SCHMIDTA. PESTRONKJ. MILBRANDT: "Altered axonal mitochondrial transport in the pathogenesis of Charcot-Marie-Tooth disease from mitofusin 2 mutations", J.NEUROSCI., vol. 27, 2007, pages 422 - 430, XP055509813, DOI: 10.1523/JNEUROSCI.4798-06.2007
R. RIZZUTOM. BRINIM. MURGIAT. POZZAN: "Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria", SCIENCE, vol. 262, 1993, pages 744 - 747
S. C. ALFORDY. DINGT. SIMMENR. E. CAMPBELL: "Dimerization-dependent green and yellow fluorescent proteins", ACS SYNTH.BIOL1, 2012, pages 569 - 575
S. CIPOLATO. MARTINS DE BRITOB. DAL ZILIOL. SCORRANO: "OPA1 requires mitofusin 1 to promote mitochondrial fusion", PROC.NATL.ACAD.SCI.U.S.A, vol. 101, 2004, pages 15927 - 15932, XP009088997, DOI: 10.1073/pnas.0407043101
S. STAMM ET AL.: "Function of alternative splicing", GENE, vol. 344, 2005, pages 1 - 20, XP004712161, DOI: 10.1016/j.gene.2004.10.022
S. STRACKT. J. WILSONJ. T. CRIBBS: "Cyclin-dependent kinases regulate splice-specific targeting of dynamin-related protein 1 to microtubules", J CELL BIOL, vol. 201, 2013, pages 1037 - 1051
S. ZUCHNER ET AL.: "Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A", NAT.GENET., vol. 36, 2004, pages 449 - 451, XP002329544, DOI: 10.1038/ng1341
T. DANIELE ET AL.: "Mitochondria and melanosomes establish physical contacts modulated by Mfn2 and involved in organelle biogenesis", CURR BIOL, vol. 24, 2014, pages 393 - 403, XP028615643, DOI: 10.1016/j.cub.2014.01.007
T. KOSHIBA ET AL.: "Structural basis of mitochondrial tethering by mitofusin complexes", SCIENCE, vol. 305, 2004, pages 858 - 862
TANG, A.C. ET AL.: "Endoplasmic Reticulum Stress and Chemokine Production in Cystic Fibrosis Airway Cells: Regulation by STAT3 Modulation", J. INFECT. DIS., vol. 215, 2017, pages 293 - 302
TOKE, J.CZIRJÁK, G.ENYEDI, P. ET AL.: "Rare diseases caused by abnormal calcium sensing and signalling.", ENDOCRINE, vol. 71, 2021, pages 611 - 617, XP037411742, DOI: 10.1007/s12020-021-02620-5
V. DEL DOTTOM. FOGAZZAV. CARELLIM. RUGOLOC. ZANNA: "Eight human OPA1 isoforms, long and short: What are they for?", BIOCHIMBIOPHYS ACTA BIOENERG, vol. 1859, 2018, pages 263 - 269, XP055974567, DOI: 10.1016/j.bbabio.2018.01.005
Y. CHEN ET AL.: "Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelleCa(2+) crosstalk", CIRC RES, vol. 111, 2012, pages 863 - 875
Y. L. CAO ET AL.: "MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion", NATURE, vol. 542, 2017, pages 372 - 376

Similar Documents

Publication Publication Date Title
Ma et al. Low-dose metformin targets the lysosomal AMPK pathway through PEN2
AU2007221470B2 (en) Phosphatase inhibitor Protein-1 as a regulator of cardiac function
AU2013263346B2 (en) Methods and compositions for treating amyloid deposits
JP2018521014A (ja) ミスフォールドタンパク質の分解のための組成物及び方法
KR20170116055A (ko) 최적화된 인간 응고 인자 viii 유전자 발현 카세트 및 그의 용도
EP3538546B1 (fr) Compositions, procédés et utilisations thérapeutiques relatifs à la protéine fusogène minion
US20230024933A1 (en) Treatment of aberrant fibroblast proliferation
US20210338838A1 (en) Gene therapy for macular degeneration
AU2016370590A1 (en) Composition for treatment of Crigler-Najjar syndrome
EP2794642A2 (fr) Variants du gène ndi1 de levure et leurs utilisations dans le traitement d'une maladie associée à un dysfonctionnement mitochondrial
US10543252B2 (en) Regulating calcium in excitable cells with a novel protein
Song et al. A novel nonsense mutation in the MIP gene linked to congenital posterior polar cataracts in a Chinese family
US20210079423A1 (en) Compositions and methods to manufacture tumor suppressor fusions
US20220089670A1 (en) Gene Therapy for BEST1 Dominant Mutations
WO2024261139A1 (fr) Variants de mfn2 et leur utilisation dans le traitement/la prévention de maladies associées à des altérations dans la fonction du réticulum endoplasmique
WO2025014986A2 (fr) Thérapie pour une déficience en alpha-1 antitrypsine
JP7109040B2 (ja) 線維化抑制剤
KR20250163297A (ko) 보체 질환 치료제 및 치료방법
KR20240130680A (ko) 염증 치료를 위한 bag3 방법 및 용도
US11325978B2 (en) Compositions and methods for treating beta-globinopathies
CN116925205A (zh) 激活Hippo信号通路的多肽及其用途
US10772938B2 (en) Compositions and methods of increasing longevity or treating cellular stress
Lu et al. Recombinant adenovirus-mediated overexpression of 3β-hydroxysteroid-Δ24 reductase
EP4069313A1 (fr) Méthodes de traitement de l'arthrose
US20250269064A1 (en) Human ependyma-specific promoter and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24735951

Country of ref document: EP

Kind code of ref document: A1