WO2025257301A1 - Traitement de la neurofibromatose de type 2 par des inhibiteurs de g6pd, acsl3 et/ou oxsm - Google Patents

Traitement de la neurofibromatose de type 2 par des inhibiteurs de g6pd, acsl3 et/ou oxsm

Info

Publication number
WO2025257301A1
WO2025257301A1 PCT/EP2025/066359 EP2025066359W WO2025257301A1 WO 2025257301 A1 WO2025257301 A1 WO 2025257301A1 EP 2025066359 W EP2025066359 W EP 2025066359W WO 2025257301 A1 WO2025257301 A1 WO 2025257301A1
Authority
WO
WIPO (PCT)
Prior art keywords
inhibitor
cells
g6pd
nucleic acid
acsl3
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/EP2025/066359
Other languages
English (en)
Inventor
Aurelio Teleman
Athina KYRKOU
Michael Boutros
Giulia AMBROSI
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.)
Deutsches Krebsforschungszentrum DKFZ
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
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 Deutsches Krebsforschungszentrum DKFZ filed Critical Deutsches Krebsforschungszentrum DKFZ
Publication of WO2025257301A1 publication Critical patent/WO2025257301A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function

Definitions

  • the present invention relates to an inhibitor of an enzyme being involved in promoting cellular reductive capacity, preferably glucose-6-phosphate-dehydrogenase (G6PD) or acyl-CoA synthetase long chain family member 3 (ACSL3) for use in treating neurofibromatosis type 2 or in preventing neurofibromatosis type 2 tumors.
  • G6PD glucose-6-phosphate-dehydrogenase
  • ACSL3 acyl-CoA synthetase long chain family member 3
  • Neurofibromatosis Type 2 is a genetic condition characterized by predisposition to benign tumors of the peripheral nervous system 1. It is caused by homozygous loss-of-function of the homonymous gene NF22, 3 in Schwann cells, the glia that ensheath peripheral nerves. Individuals with NFII typically harbor a germline mutation in one of the two NF2 alleles.
  • NF2 Recurrent, stochastic, somatic loss of the second NF2 allele gives rise to tumors throughout the lifetime of the patients 1. These tumors grow and constrict the nerves. Since cranial nerves are often affected, this leads to balance problems, dizziness, headaches, facial weakness, and loss of hearing or sight. To the best knowledge of the inventors there is currently no approved pharmacological therapy for NFII 4, 5 although bevacizumab is showing great promise 6. Tumors are surgically resected, leading to nerve damage. Since patients recurrently develop new tumors, this results in significant morbidity. NF2 is an upstream regulator of the Hippo/YAP pathway 7, 8.
  • the identified gene products are G6PD (Glucose-6-phosphate dehydrogenase), ACSL3 (Acyl-CoA Synthetase Long Chain Family Member 3), and 3-oxoacyl-ACP (Acyl Carrier Protein) synthase (OXSM). All three gene products are therapeutic novel targets for the treatment of NF2.
  • the present invention relates to an inhibitor of (a) an enzyme being involved in promoting cellular reductive capacity, preferably of glucose-6-phosphate-dehydrogenase (G6PD) or acyl-CoA synthetase long chain family member 3 (ACSL3), or (b) 3-oxoacyl-ACP (Acyl Carrier Protein) synthase (OXSM) for use in treating neurofibromatosis type 2 or in preventing neurofibromatosis type 2 tumors (i.e. tumors caused by neurofibromatosis type 2).
  • G6PD glucose-6-phosphate-dehydrogenase
  • ACSL3 acyl-CoA synthetase long chain family member 3
  • OFSM 3-oxoacyl-ACP (Acyl Carrier Protein) synthase
  • the present invention also relates to the use of an inhibitor of (a) an enzyme being involved in promoting cellular reductive capacity, preferably of glucose-6-phosphate-dehydrogenase (G6PD) or acyl-CoA synthetase long chain family member 3 (ACSL3), or (b) 3-oxoacyl-ACP (Acyl Carrier Protein) synthase (OXSM) for the manufacture of a medicament for the treatment of neurofibromatosis type 2 or for the prevention of neurofibromatosis type 2 tumors.
  • G6PD glucose-6-phosphate-dehydrogenase
  • ACSL3 acyl-CoA synthetase long chain family member 3
  • OXSM 3-oxoacyl-ACP (Acyl Carrier Protein) synthase
  • the present invention likewise relates to a method of treating a subject having neurofibromatosis type 2 or being at risk of developing neurofibromatosis type 2 by administering to the subject a therapeutically effective amount of an inhibitor of (a) an enzyme being involved in promoting cellular reductive capacity, preferably of glucose-6-phosphate-dehydrogenase (G6PD) or acyl-CoA synthetase long chain family member 3 (ACSL3), (b) or 3-oxoacyl-ACP (Acyl Carrier Protein) synthase (OXSM).
  • G6PD glucose-6-phosphate-dehydrogenase
  • ACSL3 acyl-CoA synthetase long chain family member 3
  • OXSM 3-oxoacyl-ACP (Acyl Carrier Protein) synthase
  • G6PD glucose-6-phosphate- dehydrogenase
  • ACSL3 acyl-CoA synthetase long chain family member 3
  • Glucose-6-phosphate-dehydrogenase (EC 1.1.1.49) is a cytosolic enzyme that catalyzes the chemical reaction D-glucose 6-phosphate + NADP+ + H2O ⁇ 6-phospho-D-glucono-1,5-lactone + NADPH + H + .
  • NADPH is required for the reductive biosynthesis of fatty acids, cholesterol, nucleotides, and amino acids and cholesterol and G6PD is required for lipogenesis.
  • G6PD is therefore involved in promoting cellular reductive capacity
  • Glucose-6-phosphate dehydrogenase deficiency (G6PDD), which is the most common enzyme deficiency worldwide, is an inborn error of metabolism that predisposes to red blood cell breakdown. It is an X-linked recessive disorder that results in a defective glucose-6- phosphate dehydrogenase enzyme.
  • Glucose-6-phosphate dehydrogenase is an enzyme which protects red blood cells, which carry oxygen from the lungs to tissues throughout the body. A defect of the enzyme results in the premature breakdown of red blood cells. Most of the affected subjects have no symptoms. G6PD-deficient individuals do not appear to acquire any illnesses more frequently than other people.
  • G6PD A ⁇ and G6PD Mediterranean are the most common in human populations.
  • G6PD A ⁇ has an occurrence of 10% of Africans and African-Americans while G6PD Mediterranean is prevalent in the Middle East.
  • the known distribution of the mutated allele is largely limited to people of Mediterranean origins (Spaniards, Italians, Greeks, Armenians, Sephardi Jews and other Semitic peoples). Both variants are believed to stem from a strongly protective effect against Plasmodium falciparum and Plasmodium vivax malaria.
  • Acyl-CoA synthetase long chain family member 3 (ACSL3) (EC 6.2.1.3) is an enzyme of the long-chain fatty-acid-coenzyme A ligase family.
  • ACSL3 is highly expressed in brain, and preferentially utilizes myristate, arachidonate, and eicosapentaenoate as substrates.
  • 3-oxoacyl-ACP (Acyl Carrier Protein) synthase (OXSM) (EC 2.3.1.41) is a beta-ketoacyl synthetase.
  • the enzyme is required for elongation of fatty acid chains in the mitochondria.
  • OXSM plays a role in the biosynthesis of lipoic acid as well as longer chain fatty acids required for optimal mitochondrial function.
  • OXSM condenses malonyl-ACP with an even-numbered acyl-ACP, extending the growing acyl chain by two carbons and releasing a molecule of carbon dioxide.
  • the condensation reaction catalyzed by OXSM creates a keto-acyl intermediate, and subsequent enzymes in the mtFAS cycle carry out a series of reduction and dehydration reactions to return the newly elongated acyl chain to a fully saturated state, as in cytosolic FAS.
  • OXSM is the key enzyme of the mtFAS II pathway, catalyzing the chain-elongating reaction of the fatty acid synthesis cycle.
  • the inhibitor of G6PD, ACSL3 or OXSM is preferably a specific inhibitor of G6PD, ACSL3 or OXSM, which means that it does not inhibit any other targets when being administered to a subject.
  • the inhibitor of G6PD, ACSL3 or OXSM with increasing preference has the effect that the respective enzyme can catalyze its chemical reaction at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% less efficient as compared to the absence of the inhibitor. Most preferably the inhibitor completely or essentially completely inhibits the catalytic activity of the G6PD, ACSL3 or OXSM.
  • G6PD, ACSL3 or OXSM The nature of the inhibitor of G6PD, ACSL3 or OXSM is not particularly limited.
  • An inhibitor of G6PD, ACSL3 or OXSM can be identified by routine means.
  • the efficiency of several inhibitors e.g. libraries of small molecule inhibitors, antibodies or antibody mimetics
  • High-throughput assays independently of being biochemical, cellular or other assays, generally may be performed in wells of microtiter plates, wherein each plate may contain 96, 384 or 1536 wells.
  • Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of test compounds with the assay mixture is preferably effected by one or more computer-controlled robotic systems including pipetting devices.
  • mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well.
  • said mixture of test compounds may be de-convoluted to identify the one or more test compounds in said mixture giving rise to said activity. Examples of suitable inhibitors and suitable classes of inhibitors will be described herein below.
  • the inhibitors can be administered to the subject at a suitable dose and/or a therapeutically effective amount.
  • the length of treatment needed to observe changes and the interval following treatment for responses to occur vary depending on the desired effect.
  • the particular amounts and/or length of the treatment may be determined by conventional tests which are well known to the person skilled in the art. Suitable tests are, for example, described in Tamhane and Logan (2002), “Multiple Test Procedures for Identifying the Minimum Effective and Maximum Safe Doses of a Drug”, Journal of the American statistical association, 97(457):1-9.
  • Neurofibromatosis type 2 NF2, neurofibromatosis type II, NFII
  • NF2 neurofibromatosis type II, NFII
  • tumours are usually non-cancerous (benign) but may cause a range of symptoms, such as hearing loss that gradually gets worse over time, hearing ringing or buzzing in the ears (tinnitus) and balance problems (particularly when moving in the dark or walking on uneven ground).
  • NF2 neurotrophic factor
  • Current treatment is limited to regular monitoring and treating any problems as they occur.
  • Surgery can be used to remove most tumours, although it carries a risk of causing problems, such as complete deafness or facial weakness. Therefore, the risks and potential benefits need to be carefully considered before treatment.
  • Most people with NF2 eventually develop significant hearing loss and often benefit from using a hearing aid or learning to lip read. Special implants can sometimes be inserted to improve a person's hearing.
  • the inhibition of an enzyme being involved in promoting cellular reductive capacity (e.g. ACSL3 or G6PD) or OXSM can be a cure for NF2 was not known from or suggested in the prior art.
  • the inhibition an enzyme being involved in promoting cellular reductive capacity (e.g. ACSL3 or G6PD) and/or OXSM specifically causes death of cells with NF2 loss-of-function.
  • the inhibitor is an inhibitor of G6PD.
  • the examples show that NF2 mutant Schwann cells are more oxidized than control cells, in part due to reduced expression of genes involved in NADPH generation such as ME1.
  • G6PD G6PD
  • ACSL3 or OXSM the use of an inhibitor of two or all three of G6PD, ACSL3 and OXSM may be advantageous in certain cases. For instance, if any inhibitor of G6PD, ACSL3 or OXSM causes an unwanted side effect this side effect may be reduced or even avoided by lowering the dose of this inhibitor and using in addition a further inhibitor.
  • the inhibitor is an inhibitor of ACSL3 or OXSM.
  • G6PD comprises or consists of the nucleic acid sequence of SEQ ID NO: 1 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and/or comprises or consists of the amino acid sequence of SEQ ID NO: 2 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto;
  • ACSL3 comprises or consists of the nucleic acid sequence of SEQ ID NO: 3 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, and/or comprises or consists of the amino acid sequence of SEQ ID NO: 4 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto; and/or
  • OXSM comprises or consists of the nucleic acid sequence of SEQ ID NO: 5 or a sequence being at least 80%, preferably at least 90% and most preferably at least 9
  • SEQ ID NO: 1 is the nucleotide sequence of human G6PD.
  • SEQ ID NO: 3 is the nucleotide sequence of human ACSL3.
  • SEQ ID NO: 5 is the nucleotide sequence of human OXSM.
  • SEQ ID NO: 2 is the amino acid sequence of human G6PD.
  • SEQ ID NO: 4 is the amino acid sequence of human ACSL3.
  • SEQ ID NO: 6 is the amino acid sequence of human OXSM.
  • the term “percent (%) sequence identity” describes the number of matches (“hits”) of identical nucleotides/amino acids of two or more aligned nucleic acid or amino acid sequences as compared to the number of nucleotides or amino acid residues making up the overall length of the template nucleic acid or amino acid sequences.
  • hits the number of matches of identical nucleotides/amino acids of two or more aligned nucleic acid or amino acid sequences as compared to the number of nucleotides or amino acid residues making up the overall length of the template nucleic acid or amino acid sequences.
  • using an alignment for two or more sequences or subsequences the percentage of amino acid residues or nucleotides that are the same (e.g.
  • nucleotide and amino acid sequence analysis and alignment in connection with the present invention are preferably carried out using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schulffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res.25:3389-3402).
  • BLAST can be used for nucleotide sequences (nucleotide BLAST) and amino acid sequences (protein BLAST).
  • sequence identities of at least 80% identical, preferably at least 90% identical, and most preferred at least 95% are envisaged by the invention.
  • sequence identities of at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100%.
  • the inhibitor is an inhibitor of the nucleic acid molecule encoding the G6PD, ACSL3 or OXSM protein, or is an inhibitor of the G6PD, ACSL3 or OXSM protein.
  • the inhibitor may be an inhibitor of the nucleic acid molecule encoding G6PD, ACSL3 or OXSM, or may be an inhibitor of the protein G6PD, ACSL3 or OXSM. In the former case the inhibitor may interfere with the transcription of the genomic DNA encoding G6PD, ACSL3 or OXSM into mRNA and/or the translation of the mRNA into protein.
  • Interfering with the transcription or translation reduces the amount of the G6PD, ACSL3 or OXSM enzyme in cells thereby achieving that the enzyme can catalyze its chemical reaction less efficient as compared to the absence of the inhibitor.
  • Inhibitors that interfere with the transcription include compounds interfering with the transcriptional machinery and/or its interaction with the promoter of a gene and/or with expression control elements remote from the promoter such as enhancers.
  • Inhibitors that interfere with the translation include compounds interfering with the translational machinery. The compound inhibiting the expression may specifically interfere with the promoter region controlling the expression.
  • Enzyme inhibitors may bind reversibly or irreversibly. Irreversible inhibitors form a chemical bond with the enzyme such that the enzyme is inhibited until the chemical bond is broken. By contrast, reversible inhibitors bind non-covalently and may spontaneously leave the enzyme, allowing the enzyme to resume its function. Reversible inhibitors produce different types of inhibition depending on whether they bind to the enzyme, the enzyme-substrate complex, or both.
  • the term “nucleic acid molecule” in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA.
  • RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrid molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA.
  • the nucleic acid molecule may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phospho
  • Nucleic acid molecules in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
  • proteins e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • intercalators e.g., acridine, psoralen, etc.
  • chelators e.g., metals, radioactive metals, iron, oxidative metals, etc.
  • alkylators e.g., metals, radioactive metals, iron, oxidative metals, etc.
  • nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers.
  • nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2’-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001, 8: 1).
  • LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2’-oxygen and the 4’-carbon.
  • nucleic acids containing modified bases for example thio-uracil, thio-guanine and fluoro-uracil.
  • a nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and/or polypeptides.
  • the nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non- coding regions, and the like.
  • protein as used herein interchangeably with the term “polypeptide” describes linear molecular chains of amino acids, including single chain proteins or their fragments, containing at least 50 amino acids.
  • peptide as used herein describes a group of molecules consisting of up to 49 amino acids, whereas the term “polypeptide” (also referred to as "protein") as used herein describes a group of molecules consisting of at least 50 amino acids.
  • polypeptide as used herein describes a group of molecules consisting with increased preference of at least 15 amino acids, at least 20 amino acids at least 25 amino acids, and at least 40 amino acids.
  • the group of peptides and polypeptides are referred to together by using the term "(poly)peptide”.
  • Polypeptides may further form oligomers consisting of at least two identical or different molecules.
  • the corresponding higher order structures of such multimers are, correspondingly, termed homo- or heterodimers, homo- or heterotrimers etc.
  • peptidomimetics of such proteins/(poly)peptides where amino acid(s) and/or peptide bond(s) have been replaced by functional analogues are also encompassed by the invention.
  • Such functional analogues include all known amino acids other than the 20 gene- encoded amino acids, such as selenocysteine.
  • the terms “(poly)peptide” and “protein” also refer to naturally modified (poly)peptides and proteins where the modification is effected e.g.
  • the inhibitor of the nucleic acid molecule is selected from a small molecule, an aptamer, a siRNA, a shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas (e.g.
  • Cas9 or Cpf1)- based construct a meganuclease, a zinc finger nuclease, and a transcription activator-like (TAL) effector (TALE) nuclease, and/or (ii) the inhibitor of the protein is selected from a small molecule, an antibody or an antigen binding fragment thereof, an antibody mimetic or an aptamer.
  • the "small molecule” as used herein is preferably an organic molecule.
  • Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds.
  • Organic compounds can be natural or synthetic.
  • the organic molecule is preferably an aromatic molecule and more preferably a heteroaromatic molecule.
  • aromaticity is used to describe a cyclic (ring-shaped), planar (flat) molecule with a ring of resonance bonds that exhibits more stability than other geometric or connective arrangements with the same set of atoms.
  • Aromatic molecules are very stable, and do not break apart easily to react with other substances.
  • a heteroaromatic molecule at least one of the atoms in the aromatic ring is an atom other than carbon, e.g.
  • the molecular weight is preferably in the range of 200 Da to 1500 Da and more preferably in the range of 300 Da to 1000 Da.
  • the "small molecule" in accordance with the present invention may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates). Preferably, the small molecule has a molecular weight of less than about 2000 Da, or less than about 1000 Da such as less than about 500 Da, and even more preferably less than about 250 Da.
  • the size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry.
  • the small molecules may be designed, for example, based on the crystal structure of the target molecule, where sites presumably responsible for the biological activity can be identified and verified in in vivo assays such as in vivo high-throughput screening (HTS) assays.
  • Aptamers are nucleic acid molecules or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. These compound molecules have additional research, industrial and clinical applications (Osborne et. al.
  • Nucleic acid aptamers are nucleic acid species that normally consist of (usually short) strands of oligonucleotides. Typically, they have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.
  • Peptide aptamers are usually peptides or proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold.
  • variable peptide loop typically comprises 10 to 20 amino acids, and the scaffold may be any protein having good solubility properties.
  • the bacterial protein Thioredoxin-A is the most commonly used scaffold protein, the variable peptide loop being inserted within the redox-active site, which is a -Cys-Gly-Pro-Cys-loop (SEQ ID NO: 10) in the wild protein, the two cysteins lateral chains being able to form a disulfide bridge.
  • Peptide aptamer selection can be made using different systems, but the most widely used is currently the yeast two- hybrid system.
  • Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies.
  • aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamers' inherently low molecular weight.
  • Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging.
  • Several modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, fusion to albumin or other half-life extending proteins etc. are available to scientists such that the half-life of aptamers can be increased for several days or even weeks.
  • siRNA small interfering RNA
  • siRNA also known as short interfering RNA or silencing RNA
  • siRNA refers to a class of 18 to 30, preferably 19 to 25, most preferred 21 to 23 or even more preferably 21 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology.
  • siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene.
  • RNAi RNA interference
  • siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome.
  • siRNAs naturally found in nature have a well-defined structure: a short double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group.
  • dsRNA short double-strand of RNA
  • -OH 3' hydroxyl
  • This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs.
  • siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest. Essentially any gene for which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA.
  • the double-stranded RNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation.
  • Exogenously introduced siRNAs may be devoid of overhangs at their 3' and 5' ends, however, it is preferred that at least one RNA strand has a 5'- and/or 3'-overhang.
  • one end of the double-strand has a 3'-overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably 2 nucleotides.
  • the other end may be blunt-ended or has up to 6 nucleotides 3'-overhang.
  • any RNA molecule suitable to act as siRNA is envisioned in the present invention.
  • the most efficient silencing was so far obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3'- overhang.
  • the sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et al.2001).2'-deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant.
  • siRNA Delivery of siRNA may be accomplished using any of the methods known in the art, for example by combining the siRNA with saline and administering the combination intravenously or intranasally or by formulating siRNA in glucose (such as for example 5% glucose) or cationic lipids and polymers can be used for siRNA delivery in vivo through systemic routes either intravenously (IV) or intraperitoneally (IP) (Fougerolles et al. (2008), Current Opinion in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular Biology, vol.437: Drug Delivery Systems – Chapter 3: Delivering Small Interfering RNA for Novel Therapeutics).
  • IV intravenously
  • IP intraperitoneally
  • shRNA short hairpin RNA
  • RISC RNA-induced silencing complex
  • si/shRNAs to be used in the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • Suppliers of RNA synthesis reagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).
  • siRNAs or shRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs.
  • RNAs applicable in the present invention are conventionally synthesized and are readily provided in a quality suitable for RNAi.
  • Further molecules effecting RNAi include, for example, microRNAs (miRNA).
  • MRISPR microRNAs
  • Said RNA species are single-stranded RNA molecules.
  • Endogenously presented miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering the degradation of said mRNA transcript through a process similar to RNA interference. Accordingly, exogenous miRNA may be employed as an inhibitor after introduction into the respective cells.
  • a ribozyme from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA is an RNA molecule that catalyses a chemical reaction.
  • ribozymes catalyse either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyse the aminotransferase activity of the ribosome.
  • Non-limiting examples of well-characterised small self- cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes, whereas the group I intron is an example for larger ribozymes.
  • the principle of catalytic self-cleavage has become well established in recent years.
  • the hammerhead ribozymes are characterised best among the RNA molecules with ribozyme activity.
  • hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it appears that catalytic antisense sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site.
  • the basic principle of constructing hammerhead ribozymes is as follows: A region of interest of the RNA, which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each usually with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them. The best results are usually obtained with short ribozymes and target sequences.
  • the conformational change induced in the aptamer upon binding the target molecule can regulate the catalytic function of the ribozyme.
  • An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and/or translation of the target mRNA is reduced or blocked.
  • the antisense nucleic acid molecule may also be an antisense oligonucleotide, such as a LNA-GapmeR, an AntagomiR, or an antimiR.
  • LNA-GapmeRs or simply GapmeRs are potent antisense oligonucleotides used for highly efficient inhibition of mRNA and lncRNA function. GapmeRs function by RNase H dependent degradation of complementary RNA targets. They are an excellent alternative to siRNA for knockdown of mRNA and lncRNA.
  • GapmeRs contain a central stretch of DNA monomers flanked by blocks of LNAs.
  • the GapmeRs are preferably 14-16 nucleotides in length and are optionally fully phosphorothioated.
  • the DNA gap activates the RNAse H- mediated degradation of targeted RNAs and is also suitable to target transcripts directly in the nucleus.
  • LNA-GapmeRs are routinely designed using established algorithms. LNA-GapmeRs to a selected target are commercially available including positive and negative controls, for example, from Exiqon.
  • AntimiRs are oligonucleotide inhibitors that were initially designed to be complementary to a miRNA.
  • AntimiRs against miRNAs have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics.
  • AntimiRs are preferably AntagomiRs.
  • AntagomiRs are synthetic 2-O-methyl RNA oligonucleotides, preferably of 21 to 23 nucleotides which are preferably fully complementary to the selected target RNA. While AntagomiRs were initially designed against miRNAs they may also be designed against mRNAs.
  • AntagomiRs are preferably synthesized with 2 ⁇ -OMe modified bases (2 ⁇ -hydroxyl of the ribose is replaced with a methoxy group), phosphorothioate (phosphodiester linkages are changed to phosphorothioates) on the first two and last four bases, and an addition of cholesterol motif at 3 ⁇ end through a hydroxyprolinol modified linkage.
  • 2 ⁇ -OMe and phosphorothioate modifications improve the bio-stability whereas cholesterol conjugation enhances distribution and cell permeation of the AntagomiRs.
  • Antisense molecules are preferably chemically synthesized using a conventional nucleic acid synthesizer.
  • Suppliers of nucleic acid sequence synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).
  • CRISPR/Cas technologies are applicable in nearly all cells/model organisms and can be used for knock out mutations, chromosomal deletions, editing of DNA sequences and regulation of gene expression.
  • the regulation of the gene expression can be manipulated by the use of a catalytically dead Cas9 enzyme (dCas9) that is conjugated with a transcriptional repressor to repress transcription of a specific gene.
  • dCas9 enzyme catalytically dead Cas9 enzyme
  • CRISPR from Prevotella and Francisella-1 can be fused to synthetic transcriptional repressors or activators to downregulate endogenous promoters, e.g. the promoter which controls gene expression.
  • the DNA-binding domain of zincfinger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) can be designed to specifically recognize the gene or its promoter region or its 5 ⁇ -UTR thereby inhibiting the expression of the gene.
  • Inhibitors provided as inhibiting nucleic acid molecules that target the gene or a regulatory molecule involved in expression are also envisaged herein. Such molecules, which reduce or abolish the gene expression or a regulatory molecule include, without being limiting, meganucleases, zinc finger nucleases and transcription activator-like (TAL) effector (TALE) nucleases.
  • antibody as used in accordance with the present invention comprises, for example, polyclonal or monoclonal antibodies. Furthermore, also derivatives or fragments thereof, which still retain the binding specificity to the target are comprised in the term "antibody”; i.e. antigen binding fragments of (complete) antibodies.
  • Antibody fragments or derivatives comprise, inter alia, Fab or Fab’ fragments, Fd, F(ab')2, Fv or scFv fragments, single domain VH or V-like domains, such as VhH or V- NAR-domains, as well as multimeric formats such as minibodies, diabodies, tribodies or triplebodies, tetrabodies or chemically conjugated Fab’-multimers (see, for example, Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 198; Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc)., vol.
  • the multimeric formats in particular comprise bispecific antibodies that can simultaneously bind to two different types of antigen.
  • the first antigen can be found on the protein of the invention.
  • the second antigen may, for example, be a tumor marker that is specifically expressed on cancer cells or a certain type of cancer cells.
  • bispecific antibodies formats are Biclonics (bispecific, full length human IgG antibodies), DART (Dual-affinity Re-targeting Antibody) and BiTE (consisting of two single-chain variable fragments (scFvs) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847).
  • the term "antibody” also includes embodiments such as chimeric (human constant domain, non- human variable domain), single chain and humanised (human antibody with the exception of non- human CDRs) antibodies.
  • Various techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane (1988) and (1999) and Altshuler et al., 2010, loc. cit.
  • polyclonal antibodies can be obtained from the blood of an animal following immunisation with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples for such techniques are described, e.g. in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999 and include the hybridoma technique originally described by Köhler and Milstein, 1975, the trioma technique, the human B-cell hybridoma technique (see e.g. Kozbor D, 1983, Immunology Today, vol.4, 7; Li J, et al. 2006, PNAS, vol.
  • recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display.
  • a suitable system for the expression of the recombinant (humanised) antibodies may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US patent 6,080,560; Holliger P, Hudson PJ.2005, Nat Biotechnol., vol. 23(9), 11265).
  • antibody mimetics refers to compounds which, like antibodies, can specifically bind antigens, but which are not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.
  • the antibody mimetic is selected from the group consisting of Anticalins, Affibodies, Adnectins, DARPins, Avimers, Nanofitins, Affilins, ⁇ -Wrapins, ADAPT, Monobodies, RasIns, FingRs, Pronectins, Centyrins, Affimers, Adhirons, Affitins, ⁇ Reps, Repebodies, i-bodies, Fynomers and Kunitz domain proteins.
  • Anticalins are an emerging class of clinical-stage biopharmaceuticals with high potential as an alternative to antibodies.
  • Anticalin molecules are generated by combinatorial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold dominated by a central ⁇ -barrel, supporting four structurally variable loops that form a binding site. Reshaping of this loop region results in Anticalin proteins that can recognize and tightly bind a wide range of medically relevant targets, from small molecules to peptides and proteins, as validated by X- ray structural analysis. Their robust format allows for modification in several ways, both as fusion proteins and by chemical conjugation, for example, to tune plasma half-life (Rothe and Skerra (2016) BioDrugs 32, 233–243.).
  • Affibodies in accordance with the present invention, are a family of antibody mimetics derived from the Z-domain of staphylococcal protein A. Affibodies are structurally based on a three-helix bundle domain. An affibody has a molecular mass of around 6 kDa and is stable at high temperatures and under acidic or alkaline conditions. Target specificity is obtained by randomisation of amino acids located in two alpha-helices involved in the binding activity of the parent protein domain (Feldwisch, J & Tolmachev, V. [2012] Methods Mol. Biol.899:103-126).
  • Adnectins and also “Monobodies”, in accordance with the present invention, are based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts an Ig-like sandwich fold with 2 to 3 exposed loops, but lacks the central disulphide bridge (Gebauer, M. & Skerra, A. [2009] Curr. Opin. Chem. Biol. 13:245-255).
  • Adnectins and Monobodies with the desired target specificity can be genetically engineered by introducing modifications into specific loops or other surface areas of the protein.
  • DARPins in accordance with the present invention, are designed ankyrin repeat domains that provide a rigid interface arising from typically three repeats corresponding to an artificial consensus sequence, whereby six positions per repeat are randomised. Consequently, DARPins lack structural flexibility (Gebauer, M. & Skerra, A. [2009] Curr. Opin. Chem. Biol.13:245-255).
  • Binding of target molecules occurs via the A-domain and domains with desired binding specificity can be selected, for example, by phage display techniques.
  • the target specificity of the different A-domains contained in an Avimer may, but do not have to be identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).
  • “Nanofitins” and also an “Affitins” are antibody mimetic proteins that are derived from the DNA binding protein Sac7d of Sulfolobus acidocaldarius.
  • Nanofitins and Affitins usually have a molecular weight of around 7kDa and are designed to specifically bind a target molecule by randomising the amino acids on the binding surface (Mouratou B, Béhar G, Paillard-Laurance L, Colinet S, Pecorari F., (2012) Methods Mol Biol.; 805:315-31 and Koide et al.1998, J. Mol. Biol.284:1141–51).
  • affilins with the desired target specificity is effected, for example, by phage display or ribosome display techniques.
  • affilins have a molecular weight of approximately 10 or 20kDa.
  • the term affilin also refers to di- or multimerised forms of affilins (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).
  • ⁇ -Wrapins designates affibody protein homodimers with a disulfide bond between the pair of Cys28 residues connecting the two identical monomer subunits, referred to as subunits 1 and 2.
  • the scaffold used in engineering ⁇ -wrapins is ⁇ 3, an ⁇ -binding affibody protein that not only prohibits the initial aggregation of ⁇ monomers into toxic forms, but also dissociates pre-formed oligomeric aggregates by sequestering and stabilizing a ⁇ -hairpin conformation of ⁇ monomers (Orr et al. (2016), Computers & Chemical Engineering, 116(4):322-332).
  • ABSD-Derived Affinity Proteins refers to a class of antibody mimetics that has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold (Garousi et al (2015), Cancer Res.; 75(20):4364-71). By diversifying a surface of the domain that is not directly involved in albumin binding, molecules can be selected to bind a novel target and still retain their ability to bind albumin. This strategy has been used to select binders to a number of proteins, for example, the cancer-related epidermal growth factor receptor 3. As used herein “RasIns” are 10FnIII-based antibody mimetics.
  • RasIns are disulfide-free intrabodies. They were shown to be stable inside cells and also when fused with a fluorescent protein label (Cetin et al. (2017), J Mol Biol.; 429(4):562–573).
  • FingRs Fibronectin intrabodies generated with mRNA display
  • the term “FingRs (Fibronectin intrabodies generated with mRNA display)” designates recombinant antibody-like proteins also being based on the 10FnIII scaffold (Gross et al. (2013), Neuron.; 78(6): 971–985.).
  • Pronectins designates recombinant antibody-like proteins being based on the fourteenth type-III scaffold of human fibronectin (14Fn3).
  • the well-characterized fibronectin protein is prevalent throughout the human body.
  • Human fibronectin, an extracellular protein, is naturally abundant in human serum.
  • Intelligent loop-diversity has been designed to closely mimic the natural human repertoire and avoid sequence immunogenicity.
  • the intrinsic properties of a Pronectin align with the pharmacological properties needed to make it a successful drug, including high potency, specificity, stability, favorable small size, and high-yield production in E. coli and yeast (http://www.protelica.com/pronectin_tech.html).
  • the term “Centyrins” designates recombinant antibody-like proteins being based on the consensus tenascin FN3 framework (Tencon) (Diem et al. (2014), Protein Eng., Des. and Sel. 27, 419–429). Centryins against different targets, e.g. human c-MET, rTNF ⁇ and mIL-17A, were generated.
  • Targets e.g. human c-MET, rTNF ⁇ and mIL-17A
  • Affimers refer to small proteins that bind to target molecules with similar specificity and affinity to that of antibodies. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications.
  • these affinity reagents have been optimized to increase their stability, make them tolerant to a range of temperatures and pH, reduce their size, and to increase their expression in E. coli and mammalian cells.
  • cysteine protease inhibitor family of cystatins which function in nature as cysteine protease inhibitors, these 12–14 kDa proteins share the common tertiary structure of an ⁇ -helix lying on top of an anti-parallel ⁇ -sheet (Tiede et al. (2017), eLife.; 6: e24903).
  • the class of recombinant antibody-like proteins designated as “Adhirons” herein is based on a phytocystatin consensus sequence as the scaffold (Tiede et al. (2014) Protein Eng. Des. Sel. 27, 145- 55).
  • the class of recombinant antibody-like proteins designated as “ ⁇ Rep” herein is derived from alpha- helicoidal HEAT-like repeat protein scaffolds.
  • the ⁇ Rep proteins are derived from a natural family of modular proteins comprising alpha-helical repeats, related to HEAT repeats, named after Huntingtin, the elongation factor 3 (EF3), the protein phosphatase 2A (PP2A), and the yeast kinase TOR.
  • Repebodies designates recombinant antibody-like proteins which are composed of leucine-rich repeat (LRR) modules.
  • LRR leucine-rich repeat
  • the binding scaffold of Repebodies is based on variable lymphocyte receptors, which are nonimmunoglobulin antibodies composed of LRR modules in jawless vertebrates.
  • a template scaffold was first constructed by joining consensus repeat modules between the N- and C-capping motifs of variable lymphocyte receptors.
  • the N-terminal domain of the template scaffold was redesigned based on the internalin-B cap by analyzing the modular similarity between the respective repeat units using a computational approach (Lee at al. (2012), Proc Natl Acad Sci; 109(9): 3299-3304).
  • i-bodies refers to recombinant antibody-like proteins built on the scaffold of a human protein and engineered with two loops that mimic the shape of shark antibodies. These loops are responsible for binding or interacting with a particular target (in circulation or on a cell) that is causing disease.
  • the i-body is a human analogue of the antigen binding domain of the shark antibody, which combines the advantages of monoclonal antibodies (high target specificity and affinity) with the beneficial stability features of small molecules (https://www.ibodies.eu/).
  • the term "Fynomer” refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are well-known in the art and have been described e.g. in Grabulovski et al. (2007) JBC, 282, p.
  • a “Kunitz domain peptide” is derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI).
  • BPTI bovine pancreatic trypsin inhibitor
  • APP amyloid precursor protein
  • TFPI tissue factor pathway inhibitor
  • Kunitz domains have a molecular weight of approximately 6kDa and domains with the required target specificity can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).
  • the inhibitor of the nucleic acid molecule comprises or consists of (a) a nucleic acid sequence which comprises or consists of a nucleic acid sequence being complementary to at least 12 continuous nucleotides of a nucleic acid sequence selected from SEQ ID NOs 1, 3 and 5, (b) a nucleic acid sequence which comprises or consists of a nucleic acid sequence which is at least 70% identical to the complementary strand of one or more nucleic acid sequences selected from SEQ ID NOs 1, 3 and 5, (c) a nucleic acid sequence which comprises or consists of a nucleic acid sequence according to (a) or (b), wherein the nucleic acid sequence is DNA or RNA, (d) an expression vector expressing the nucleic acid sequence as defined in any one of (a) to (c), preferably under the control of a glial cells-specific promoter, preferably Schwann cells-specific promoter, or (e) a host comprising the expression vector of (d).
  • nucleic acid sequences as defined in items (a) to (c) of this preferred embodiment comprise or consist of sequences being complementary to nucleotides as defined by one or more of SEQ ID NOs 1, 3 and 5 described herein above.
  • nucleic acid sequences as defined in items (a) to (c) comprise or are antisense nucleic acid sequences.
  • the nucleic acid sequence according to item (a) of this further preferred embodiment of the invention comprises or consists of a sequence which is with increasing preference complementary to at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides of one or more selected from SEQ ID NOs 1, 3 and 5.
  • At least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, or at least 21 nucleotides are preferably a contiguous part of one or more selected from SEQ ID NOs 1, 3 and 5.
  • the format of the nucleic acid sequence according to item (a) is not particularly limited as long as it comprises or consists of at least 12 continuous nucleotides being complementary to a nucleic acid sequence selected from SEQ ID NOs 1, 3 and 5.
  • the nucleic acid sequence according to item (a) comprises or consists of an antisense oligonucleotide.
  • the nucleic acid sequence according to item (a) reflects the above-mentioned basic principle of the antisense technology which is the use of an oligonucleotide for silencing a selected target RNA through the extraordinar specificity of complementary-based pairing. Therefore, it is to be understood that the nucleic acid sequence according to item (a) is preferably in the format of an siRNA, shRNA or an antisense oligonucleotide as defined herein above.
  • the antisense oligonucleotides are preferably LNA-GapmeRs, AntagomiRs, or antimiRs.
  • nucleic acid sequence according to item (b) requiring at least 70% identity to the complementary strand of one or more nucleic acid sequences selected from SEQ ID NOs 1, 3 and 5 is considerably longer than the nucleic acid sequence according to item (a) which comprises an antisense oligonucleotide and comprises at least 12 continuous nucleotides of a nucleic acid sequence selected from SEQ ID NOs 1, 3 and 5.
  • a nucleic acid sequence according to item (b) of the above preferred embodiment of the invention is capable of interacting with, more specifically hybridizing with the target mRNA. By formation of the hybrid the translation of the mRNA is reduced or blocked.
  • sequence identity of the molecule according to item (b) in connection with a sequence selected from SEQ ID NOs 1, 3 and 5 is with increasing preference at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99% and 100%.
  • sequence identity in connection with each of SEQ ID NOs 1, 3 and 5 can be individually selected. Means and methods for determining sequence identity are known in the art. As discussed above, preferably the BLAST (Basic Local Alignment Search Tool) program is used for determining the sequence identity with regard to one or more of SEQ ID NOs 1, 3 and 5.
  • the nucleotide sequences may be RNA or DNA.
  • RNA or DNA encompasses chemically modified RNA nucleotides or DNA nucleotides.
  • RNA comprises the nucleotide U while DNA comprises the nucleotide T.
  • the inhibitor may also be an expression vector or host (e.g. host cell), respectively being capable of producing a nucleic acid sequence as defined in any one of items (a) to (c).
  • An expression vector may be a plasmid that is used to introduce a specific transcript into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes.
  • the plasmid is in general engineered to contain regulatory sequences that act as enhancer and/or promoter regions and lead to efficient transcription of the transcript.
  • the expression vector preferably contains a glial cells-specific promoter, preferably Schwann cells-specific promoter.
  • Preferred promoters Schwann-cell specific promoters shown in SEQ ID NOs 7-9.
  • SEQ ID NO: 7 is the human P0 promoter
  • SEQ ID NO: 8 the human PMP22 promoter
  • SEQ ID NO: 9 the human MBP promoter.
  • Schwann-cell specific promoters that are with increasing preference at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to one of SEQ ID NOs 7-9.
  • Non-limiting examples of expression vectors include prokaryotic plasmid vectors, such as the pUC- series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro
  • Examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Intvitrogen).
  • a suitable vector is selected in accordance with good manufacturing practice.
  • Such vectors are known in the art, for example, from Ausubel et al, Hum Gene Ther.2011 Apr; 22(4):489-97 or Allay et al., Hum Gene Ther. May 2011; 22(5): 595–604.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript.
  • elements such as origin of replication, drug resistance gene, regulators (as part of an inducible promoter) may also be included.
  • the lac promoter is a typical inducible promoter, useful for prokaryotic cells, which can be induced using the lactose analogue isopropylthiol-b-D-galactoside ("IPTG").
  • IPTG lactose analogue isopropylthiol-b-D-galactoside
  • the polynucleotide of interest may be ligated between e.g. the PelB leader signal, which directs the recombinant protein in the periplasm and the gene III in a phagemid called pHEN4 (described in Ghahroudi et al, 1997, FEBS Letters 414:521-526).
  • Additional elements might include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing.
  • Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from retroviruses, e.g., RSV, HTLVI, HIVI, and the early promoter of the cytomegalovirus (CMV).
  • LTRs long terminal repeats
  • CMV cytomegalovirus
  • cellular elements can also be used (e.g., the human actin promoter).
  • vectors can contain one or more origins of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
  • origins of replication include, for example, the Col E1, the SV40 viral and the M 13 origins of replication.
  • the sequences to be inserted into the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods.
  • Transcriptional regulatory elements parts of an expression cassette
  • These elements comprise regulatory sequences ensuring the initiation of the transcription (e.g., translation initiation codon, promoters, enhancers, and/or insulators), internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci.
  • the nucleotide sequence as defined in item (a) of the above preferred embodiment of the invention is operatively linked to such expression control sequences allowing expression in prokaryotic or eukaryotic cells.
  • the host is preferably a host cell, such as a prokaryotic or eukaryotic cell.
  • a suitable eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell or a plant cell.
  • 2457232-14-1) is 4-([5-oxo-6H,7H,8H,9H-cyclohepta[d]pyrimidin-2- yl]amino)thiophene-2-carbonitrile.
  • G6PDi-1 is commercially available.
  • RRX-001 (Cas No. 925206-65-1) is 1-Bromoacetyl-3,3-dinitroazetidine. RRX-001 is commercially available.
  • Dehydroepiandrosterone (DHEA), also known as androstenolone, (Cas No. 53-43-0) is 3 ⁇ - Hydroxyandrost-5-en-17-one. DHEA is commercially available.
  • Polydatin (Cas No.
  • CB63, CB70, CB72, and CB104 are glucose-6-phosphate dehydrogenase inhibitors being described in Preuss et al. (2013), SLAS Discovery, 18(3):286-297.
  • the following compounds 25, 29 and 32 are glucose-6-phosphate dehydrogenase inhibitors being described in Koperniku et al. (2022), J Med Chem.; 65(6): 4403–4423.
  • Triacsin C (Cas No. 76896-80-5) is (2E,4E,7E)-2,4,7-undecatrienal nitrosohydrazone. Triacsin C is commercially available.
  • the inhibitor is formulated as a pharmaceutical composition.
  • pharmaceutically acceptable carrier excipient or diluent
  • a non-toxic solid, semisolid or liquid filler diluent, encapsulating material or formulation auxiliary of any type (see also Handbook of Pharmaceutical Excipients 6ed. 2010, Published by the Pharmaceutical Press). Ways of administering inhibitors to humans are also described, for example, in De Fougerolles et al., Current Opinion in Pharmacology, 2008, 8:280-285.
  • suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc.
  • compositions comprising such carriers can be formulated by well known conventional methods.
  • the subject wherein neurofibromatosis type 2 is to be treated or the neurofibromatosis type 2 tumors are to be prevented is a mammal.
  • mammals are rodents (e.g. mice, rats, hamsters, and genuine pig) and mammalian farm animals (e.g. cow, pig, goat and sheep) and monkeys and apes (e.g. macaque and chimpanzee).
  • the mammal is a human.
  • ERK 42/44 pThr 202/pTyr204.
  • NF2 KO Schwann cells are more packed and less elongated compared to NF2 WT cells. Images taken at full confluence (96hrs timepoint from panel c).
  • E Schematic diagram of the genome-wide synthetic lethality screen to identify genes that are synthetic-lethal with NF2 loss-of- function.
  • F Scatter plot of the abundance of each sgRNA from the library at passage 7 (endpoint) normalized to passage 0 for both NF2-WT (x-axis) and NF2-KO#1 (y-axis) cells. Each dot represents a single sgRNA, and abundance is calculated as the log2 Fold Change (FC) of passage 7 / passage 0.
  • FIG. 1 Top hits showing a differential proliferative/viability effect in NF2 KO cells compared to NF2-WT cells.
  • Figure 2 Validation of NF2/G6PD synthetic lethality (A-C) sgRNA-mediated depletion of G6PD causes reduced viability of NF2-KO cells but not NF2-WT cells.
  • A Relative cell number 4 days after seeding (7 days post infection), assessed by CellTiter-Glo.
  • B Cell toxicity analyzed with CellTox. Values are normalized to total cell number (CellTiter-Glo).
  • n 8-10 mice/group.
  • FIG. 5 NF2-KO Schwann cells are more oxidized than control cells
  • B NF2 KO Schwann cells have reduced AKR1C3 and increased ACSL3 protein levels compare to isogenic NF2-WT controls.
  • D NF2-KO Schwann cells have reduced levels of ME1 protein compared to NF2-WT isogenic controls.
  • H ME1 mRNA levels are significantly reduced in NF2 mutant and cystic vestibular schwannomas from patients compared to control vestibular nerve. Data are re-analyzed from 33.
  • AKR1C1, 2 and 3 mRNA levels are consistently reduced in different types of vestibular schwannomas (VS: sporadic, cystic and NF2) compared to control vestibular nerve. Data are re-analyzed from 33.
  • NF2 and ME1 are synthetic lethal in Schwann cells
  • B G6PD inhibition and ME1 inhibition display synthetic lethality in Schwann cells. Cell number (by CellTiter-Glo) of NF2-WT cells treated +/- G6PDi-1 +/- ME1 inhibitor, normalized to the non-treated DMSO condition.
  • C-D Re-expression of ME1 rescues the sensitivity of NF2-KO Schwann cells to G6PD inhibition.
  • C Immunoblot of ME1 levels in NF2-WT cells, NF2-KO cells, and a monoclonal line of NF2-KO cells transfected to express ME1.
  • D Relative cell numbers of the indicated cell lines 4 days after treatment with 25 ⁇ M G6PDi-1, normalized to untreated cells. ns>0.05, ****p ⁇ 0.0001 by one- way ANOVA and Dunnett’s multiple comparisons test.
  • FIG. 7 Characterization of NF2 KO Schwann cell lines.
  • A Molecular characterization of the two NF2 KO Schwann cell lines (DNA and resulting protein truncations) targeting exons 1 and 2.
  • B NF2-KO cells do not express a truncated form of NF2. Immunoblot of NF2-WT and NF2-KO cells with an antibody that detects the c-terminal region of NF2.
  • C NF2-KO Schwann cells, but not the NF2-WT parental line, can form anchorage-independent clones in soft agar (7 days).
  • G A dose-response curve with G6PDi-1 reveals that 25 ⁇ M G6PDi-1 causes roughly a 50% inhibition in G6PD activity.
  • n 6 biological replicates.
  • H Molecular characterization of the "NF2-/+" heterozygous Schwann cell line (DNA and resulting protein truncations). Allele 1 leads to a frameshift mutation and premature stop codon whereas allele 2 leads to loss of 1 amino acid.
  • I NF2-/+ cells have reduced NF2 protein levels but not reduced YAP phosphorylation, detected via immunoblotting with the indicated antibodies.
  • NF2-/+ cells proliferate like NF2-wildtype cells, indicating that the remaining levels of NF2 in these cells are sufficient to provide full NF2 activity.
  • Figure 9 NF2-KO cells die in response to G6PD inhibition via a caspase-independent cell death mechanism.
  • A NF2-KO cells treated with 25 ⁇ M G6PDi-1, which induces cells death, does not lead to any visible caspase cleavage, detected by immunoblotting.
  • Figure 10 Knockdown of NF2 reduces ME1 and AKR1C3 protein levels also in primary Schwann cells.
  • A Immunoblot analysis of indicated protein levels in primary Schwann cells 96hrs after siRNA- mediated knockdown of NF2 or RLuc (negative control). Numbers indicate protein levels normalized to calnexin.
  • NF2-KO cells rely more strongly on G6PD for NADPH production compared to NF2-WT cells. If G6PD is also inhibited pharmacologically in NF2-KO cells, then the cells die due to oxidative stress.
  • n 3 biological replicates
  • C Pharmacological inhibition of G6PD in NF2-WT Schwann cells leads to lower levels of reduced NADPH normalized to NADP+.
  • n 4 biological replicates. ***p ⁇ 0.001 by two-way ANOVA and Tukey’s multiple comparisons test.
  • FIG. 12 Knockout of NF2 in HEI-193 cells causes them to die upon G6PD inhibition
  • A Knockout of NF2 in HEI-193 cells leads to reduced YAP phosphorylation but no further reduction in ME1 levels, which are already low (see Fig.10B).
  • B Pharmacological inhibition of G6PD with 25 ⁇ M G6PDi-1 kills NF2-KO HEI-193 cells, and the lethality is rescued by NAC, indicating it is due to oxidative stress.
  • Top panels show immuno-blots for the indicated proteins in each of the respective cell lines compared to the NF2- wildtype parental controls.
  • Statistical analysis shown is a one-way ANOVA and Dunnett’s multiple comparisons test. n ⁇ 3 biological replicates.
  • the Examples illustrate the invention: Example 1 - Generation of an isogenic pair of NF2-WT and NF2-KO human Schwann Cells It was aimed to identify genes that cause cell lethality when inhibited in NF2 mutant Schwann cells but do not cause lethality when inhibited in wildtype cells.
  • NF2 was knocked-out by CRISPR/Cas9 using two independent sgRNAs that target its first two coding exons respectively. This yielded two independent lines containing NF2 frameshift mutations, which should lead to premature stop codons, and very short, truncated NF2 peptides (Fig. 7A).
  • NF2-KO lines have a similar proliferation rate as NF2-WT cells at low cell densities (Fig.1B), but then start diverging at higher confluence and fail to arrest proliferation at a cell density that causes contact inhibition in NF2-WT cells (Fig.1C). Instead, at high densities, NF2-KO cells pack very tightly, lose their elongated morphology, and start growing on top of each other (Fig. 1D).
  • NF2-KO cells acquire characteristics of transformed cells such as the ability to form anchorage-independent colonies in a soft-agar assay (Fig. 7C).
  • RNA-seq A comparison of gene expression in NF2-KO vs NF2-WT cells by RNA-seq followed by Gene Ontology enrichment analysis using ShinyGo 18 identified changes in gene sets related to oncogenic signaling (TGF-beta, Hippo, PI3K-Akt) and cell adhesion (Fig.7D).
  • Example 2 Genome-wide sgRNA screen identifies G6PD and ACSL3 as synthetic-lethal partners for NF2 Cas9 was next introduced into the NF2-WT and NF2-KO#1 cells (Fig.
  • Fig. 1E a genome-wide CRISPR/Cas9 screen was performed to identify synthetic-lethal partners for NF2 in Schwann cells.
  • the screen was performed using the Toronto CRISPR Human Knockout Library (TKO v3) 19 with 89916 sgRNAs targeting 17232 human genes.
  • TKO v3 Toronto CRISPR Human Knockout Library
  • the abundance of each sgRNA was measured after 7 cell passages and compared it to the initial sgRNA representation in the library at passage 0, for both NF2- WT and NF2-KO cells (Fig. 1F). This identified many genes required for cell viability in both cell lines (“essential genes”, Fig. 1F).
  • the sgRNAs that caused the largest increase in proliferation were all 6 of the sgRNAs targeting NF2 (green dots, Fig.1F). This indicates that Schwann cell proliferation is extraordinarly sensitive to NF2, possibly explaining why loss of NF2 in people leads predominantly to schwannomas 20. As expected, the sgRNAs targeting NF2 had little-to-no effect on proliferation of NF2-KO cells, since these cells already lack NF2 and overproliferate. Of note, during the sgRNA screen, cells were split soon after reaching confluence, hence sgRNAs that either increase the proliferation rate during exponential growth, or blunt contact inhibition, were enriched by the end of the screen.
  • NF2-KO cells NF2-KO cells
  • Figure 1G shows the top genes sorted by their differential effect on NF2-KO versus NF2- WT cells. any gene that causes increased proliferation of NF2-WT cells was excluded from further consideration, since these are potentially tumor suppressors and could lead to tumors if targeted pharmacologically in patients.
  • G6PD was identified as a top hit, as it causes no proliferative defects in NF2-WT cells but reduces the number of NF2-KO cells, and ACSL3 as a second hit that has a mild negative effect on NF2-WT cells.
  • G6PD in particular caught our attention because 400 million people worldwide are deficient for G6PD, and people with reductions in G6PD activity down to ⁇ 10% of normal have little or no phenotypes as long as oxidative triggers are avoided by lifestyle management 15. This suggests G6PD could potentially be targeted pharmacologically with an acceptable side-effect profile.
  • Example 3 Inhibition of G6PD causes death of NF2-KO Schwann cells and impairs growth of NF2-KO xenograft tumors It was next confirmed that G6PD loss-of-function causes synthetic lethality in combination with NF2 loss-of-function using 4 different targeting modalities – sgRNAs, shRNAs, siRNAs and a small molecule inhibitor.
  • NF2-WT or NF2-KO cells were transduced with sgRNAs targeting either G6PD or a negative control locus (AAVS1) (Fig.2A-C): NF2-WT and NF2- KO cells both showed a similar increase in cell number over the course of 4 days when transduced with negative-control sgRNA (Fig.2A).
  • G6PD loss-of-function Fig.2C
  • Fig.2C showed no significant effect on NF2-WT cell numbers, it reduced the number of NF2-KO cells by roughly 50% at day 4 (Fig.2A).
  • the reduction in cell number was due at least in part to a significant, 6-fold increase in cell death upon G6PD loss-of-function in NF2-KO cells, assessed by CellTox (Fig. 2B).
  • G6PD was knocked-down using an siRNA that targets a different sequence than the sgRNA.
  • siRNA-mediated knockdown of G6PD also reduced the number of NF2-KO but not NF2-WT Schwann cells (Fig. 8A-C).
  • a small-molecule inhibitor for G6PD (G6PDi-1) was recently reported 23. Four days of treatment with this inhibitor reduced the number of NF2-KO cells but not NF2-WT cells (Fig.2D) with a corresponding increase in cell death in NF2-KO cells (Fig. 8D).
  • NF2-WT Schwann cells do not form subcutaneous tumors, despite using Matrigel, precluding us from testing G6PD knockdown in NF2-WT cells in vivo, whereas NF2-KO cells form tumors that grow flat and nodular (Fig.2G).
  • NF2-KO lines were generated stably carrying doxycycline (dox)-inducible shRNA constructs targeting either RLuc as a negative non-targeting control or two different regions of G6PD (Fig. 2H). Again, knockdown of G6PD reduces the proliferation of NF2-KO cells in cell culture (Fig. 2I).
  • NF2-KO Schwann cells reliably generated palpable tumors that grew, both in the presence and in the absence of doxycycline (dox) (Fig.8F, Fig.2J).
  • NF2- KO Schwann cells with a G6PD knockdown (+dox) yielded much fewer palpable tumors (Fig. 8F), and the ones that formed subsequently completely regressed so that no detectable G6PD knockdown tumors were present at the end of the follow-up (Fig.2K).
  • tumors containing G6PD shRNA in the absence of dox reliably formed palpable tumors that grew (Fig.8F), they grew less well than control tumors, probably due to leakiness of the inducible shRNA.
  • G6PD activity can be titrated more precisely pharmacologically than with shRNA-mediated knockdown.
  • a titration of G6PDi-1 was therefore performed and G6PD activity was measured, and it was found that 25 ⁇ M G6PDi-1 leads to roughly 50% inhibition of G6PD (Fig.8G). Hence this is within the range of G6PD inhibition that would be clinically tolerable.
  • Example 5 - G6PD inhibition does not kill NF2+/- heterozygous cells Often NFII patients are heterozygous for NF2 loss-of-function in many of the cells in their bodies, with loss or mutation of the remaining NF2 allele leading to tumor formation in some cells. Hence another important consideration is whether G6PD inhibition kills NF2 heterozygous cells, because this would lead to severe toxicity.
  • CRISPR/Cas9 was used to generate a Schwann cell line that has a frame-shift mutation and a premature stop codon on one NF2 allele (Fig. 8H). The second allele has the loss of a triplet, leaving the rest of the coding sequence in-frame, from which protein is produced (Fig.
  • NF2-/+ This line (“NF2-/+”) therefore has a level of NF2 function that is equal to, or less than, that of heterozygous cells.
  • NF2-/+ cells proliferate like NF2-WT cells (Fig.8J), in agreement with previous studies showing that even a small amount of NF2 protein is sufficient to provide full NF2 activity 24, 25.
  • pharmacological inhibition of G6PD did not lead to lethality of the NF2-/+ cells (Fig.2L).
  • Example 6 Inhibition of ACSL3 causes death of NF2-KO Schwann cells and impairs growth of NF2- KO xenograft tumors
  • ACSL3 knockdown is also synthetic-lethal with NF2 loss-of-function in Schwann cells using different targeting modalities.
  • Knockout or knockdown of ACSL3 using sgRNAs or siRNAs, respectively, impairs viability of NF2-KO cells but not NF2-WT cells (Fig.3A-C, Fig.8A,C).
  • Partial knockdown of ACSL3 via doxycycline-inducible shRNA constructs impairs proliferation of NF2-KO cells in cell culture (Fig.3D-E) and also significantly impairs growth of NF2-KO schwannoma xenografts (Fig. 3F).
  • Example 7 - G6PD and ACSL3 are synthetic lethal with NF2 due to oxidative stress Interestingly, both G6PD and ACSL3 are involved in lipid biogenesis and in fighting oxidative stress.
  • G6PD is the first and rate-limiting enzyme in the pentose phosphate pathway and is the predominant source of NADPH used by cells as a reducing agent to synthesize lipids and to fight oxidative stress 26.
  • ACSL3 is a member of the acyl-CoA synthetase family which conjugates mono-unsaturated fatty acids to coenzyme-A (CoA) for lipid biogenesis, thereby reducing the susceptibility of plasma membrane lipids to oxidation 27. It was therefore asked whether ACSL3 and G6PD are synthetic lethal with NF2 due to oxidative stress and/or lipid biogenesis. Since NF2-KO cells proliferate more than NF2-WT cells, it was hypothesized that they might either have a higher requirement for lipid biogenesis, or a higher requirement for reducing equivalents, which are needed for lipid biogenesis.
  • the synthetic lethality between G6PD and NF2 cannot be rescued by liproxstatin (Fig. 4E) or by supplementing cells with a lipid mix (Fig.4F). Nonetheless, the synthetic lethality with G6PD is rescued by addition of the antioxidant NAC (Fig.4G-H), suggesting again that the underlying cause of the NF2/G6PD synthetic lethality is oxidative stress.
  • the form of cell death induced by combined loss of NF2 and G6PD is not clear. Since it is not rescued by Liproxstatin (Fig.4E) it is not ferroptosis.
  • caspase cleavage was seen when NF2 and G6PD were knock out with sgRNAs (Fig.2C), this is likely due to the viral infection since it is also present in the control cells treated with control sgRNA (lane 1). Indeed, pharmacological inhibition of G6PD in NF2-KO cells does not cause caspase cleavage (Fig.9A). The lethality is also not rescued by Emricasan, a pan-caspase inhibitor (Fig.9B) suggesting they are not dying via caspase-dependent apoptosis, but some other cell death mechanism. As a control, it was verified that the same concentration of Emricasan efficiently blocks caspase cleavage induced by staurosporin (Fig.9C).
  • Example 8 - NF2-KO Schwann cells have a more oxidized redox state It was noticed in the RNA-seq data that NF2-KO cells have lower expression of several enzymes that fight oxidation. For instance, expression of all three members of the aldosterone reductase family 1 (AKR1C1, 2 and 3) which reduce lipid peroxides to lipid alcohols 29 thereby protecting cells from ferroptosis 30, are lower in two independent NF2-KO lines compared to control cells (Fig. 5A). This is also visible at the protein level for the one tested AKR1 member, AKR1C3 (Fig. 5B).
  • GPX4 a glutathione-dependent lipid peroxidase that plays a key role in protecting cells from ferroptosis 30, is also mildly reduced (Fig.5B).
  • the attenuated levels of AKR1C1, AKR1C2, AKR1C3 and GPX4 could explain why NF2-KO cells are sensitized to ferroptosis when ACLS3 is inhibited 30.
  • ME1 Malic Enzyme 1
  • ME1 and G6PD are two of the four enzymes that generate cytosolic NADPH, which is used by cells to fight oxidative stress, with G6PD being the predominant NADPH source and ME1 the second main source 31.
  • NF2-KO cells not only have strongly reduced levels of ME1 (Fig.5D), but also mildly reduced levels of G6PD. Consistent with this, NF2-KO cells have reduced ME1 and G6PD activity (Fig.5E-F) and a lower ratio of reduced NADPH to oxidized NADP+ (Fig.5G).
  • NF2-KO Schwann cells have lower levels of reducing equivalents (NADPH) and lower levels of enzymes that help counteract oxidative stress.
  • NF2 was knocked-down using two independent siRNAs in primary human Schwann cells and found that this leads to a drop in ME1, AKR1C3 and GPX4 also in these cells (Fig. 10A).
  • these primary cells have an independent genetic background from the immortalized ipn02.32 ⁇ Schwann cell line that were used above.
  • the HEI-193 human schwannoma line was analyzed, which has a point mutation that causes a splicing defect in the NF2 transcript, and thereby a partial NF2 loss-of-function 32 .
  • these cells express NF2 'isoform 3' which runs lower than full-length NF2, and they have reduced YAP phosphorylation (Fig. 10B). In addition, they have low levels of ME1 and AKR1C3 (Fig. 10B). Interestingly, HEI-193 cells have elevated levels of ME3, which also synthesizes NADPH, perhaps as a compensatory effect. Next, it was tested whether reduced levels of AKR1, 2, 3 and ME1 can also be observed in primary schwannomas from patients.
  • NF2-KO cells can buffer these redox changes by relying on NADPH produced by G6PD, but if G6PD is also inhibited this leads to cell death.
  • the redox status of cells was first assessed via the ratio of oxidized to reduced glutathione.
  • NF2-KO cells have a lower ratio of reduced NADPH to oxidized NADP+ (Fig.5G)
  • this imbalance does not translate into a change in the basal oxidation state of glutathione in NF2-KO cells compared to NF2-WT cells (Fig. 6A, bars 4 & 7 versus 1) nor in elevated ROS levels (Fig. 11B), suggesting that the remaining NADPH is sufficient for NF2-KO cells to maintain a proper redox balance further downstream.
  • G6PD inhibition of G6PD in wildtype cells reduces the NADPH/NADP+ ratio (Fig.11C) but does not cause significant oxidation of glutathione (bar 2 vs 1, Fig.6A).
  • NF2-KO cells In contrast, G6PD inhibition in NF2-KO cells causes the glutathione pool to become significantly more oxidized (bars 5&8 versus 4&7 respectively, Fig.6A). This is rescued by addition of NAC (Fig.6A), in agreement with the rescue of lethality by NAC (Fig.4G). Thus NF2-KO cells rely more strongly on G6PD for production of reducing equivalents compared to wildtype cells. It was next asked whether inhibition of ME1 in NF2-WT cells recapitulates the phenotype, causing them to become sensitive to G6PD inhibition.
  • NF2-KO HEI-193 cells were generated, and it was found that they have less phospho-YAP compared to the parental HEI-193 line (Fig.12A), indicating that the NF2 in HEI-193 cells is still partially active.
  • Fig.12A parental HEI-193 line
  • knockout of NF2 in HEI-193 cells sensitized them to G6PD inhibition, and this is rescued by NAC, indicating that they die due to oxidative stress (Fig.12B).
  • Example 10 - Synthetic lethality between NF2 and G6PD is specific to Schwann cells
  • the synthetic lethality between NF2 and G6PD could be specific to Schwann cells, or it could be a general phenomenon observed in different cell types.
  • an NF2-KO was introduced using CRISPR/Cas9 into a variety of different cancer and non-cancer cell lines. In some cells such as HeLa cells (cervical cancer line) or human umbilical vein endothelial cells (HUVECs), loss of NF2 does not lead to reduction of YAP phosphorylation, indicating that the NF2 pathway is not active in these cells (Fig.13A-B).
  • NF2 In some cells, such as the osteosarcoma U2OS line, loss of NF2 leads to a strong reduction in YAP phosphorylation (Fig. 13C), however these cells express little ME1 so no change in ME1 expression can be observed. Finally, in some cells such as the colorectal carcinoma HCT116 line or immortalized human fibroblasts, NF2-KO causes a drop in YAP phosphorylation, indicating that the NF2 pathway is functional, but does not cause a drop in ME1 (Fig.13D-E). In all cases, loss of NF2 does not sensitize these cells to pharmacological G6PD inhibition (Fig.13A-E). Thus, the synthetic lethality between NF2 and G6PD seems to be fairly specific for Schwann cells.
  • Example 11 – Methods Chemical compounds G6PDi-1 inhibitor was custom synthesized by Otava Chemicals or purchased from Merck/Sigma (#SML2980).
  • Ferroptosis inhibitor Liproxstatin (#SML1414), chemically defined Lipid Mixture (#L0288), InSolution Staurosporin (#569396), Erastin (#7781) and N-Acetyl L-Cysteine/NAC (#A7250) were purchased from Merck/Sigma.
  • Malic Enzyme 1 inhibitor (#HY-124861) was from Hölzel and Oligomycin (#SAFSO4876) from VWR international.
  • Emricasan (PF 03491390) was purchased from MedChemExpress.
  • HUVEC human umbilical vein endothelial cells
  • huFIB immortalized human fibroblasts
  • HUVEC were cultured on 0,5% gelatin matrix (#INS-SU-10, InScreenex) and expanded in the corresponding complete medium (#INS-ME-1011, InScreenex).
  • HuFIB were grown on collagen coating (#INS-SU-1017) and their respective medium (#INS-ME-1001), both from Inscreenex.
  • NF2 knockouts wild type cells were transfected with px459 plasmids containing sgNF2 sequences.
  • Schwann cell line ipn02.32 ⁇ , HUVEC and HuFib lines were transfected using Metafectene Pro reagent (#T040, Biontex), while HCT116, Hela and U2OS were transfected with Lipofectamine 3000 (#L3000001,ThermoFisher Scientific) following the manufacturer’s instructions.
  • NF2-/+ line transfection of NF2-WT cells was done using PEI reagent (#408727, Sigma) at a ratio 1:3.48hrs post transfection, cells were treated with 1.5 ⁇ g/ml Puromycin (#P9620, Sigma) for 72hrs. After puromycin treatment and expansion of the surviving population, single clone selection was performed via cell dilution.
  • subconfluent cells were infected in the presence of 6 ⁇ g/ml Polybrene with lentiviral particles harboring Lenti-Cas9-2A-Blast (#73310, Addgene). 48hrs post infection, cells were treated with 5 ⁇ g/ml Blasticidin for 72hrs.
  • the resulting PCR products were purified from an agarose gel using the NucleoSpin Gel and PCR Clean-up kit (#740609, Macherey- Nagel) and introduced into the TOPOTM TA CloningTM vector (#450640, Invitrogen). After bacterial transformation of the ligation product, TOPO clones were selected ( ⁇ 10 per cell line) for Sanger sequencing, to detect the indels occurring in NF2. Generation of ME1 re-expressing NF2-KO Schwann cell line The ME1 ORF was amplified from a ME1orf Gateway clone (Clone ID #130654902, GPCF, DKFZ, Heidelberg, Germany) using Phusion enzyme (#M0530L, NEB) according to manufacturer’s description.
  • the oligos for this PCR are provided in Table 2.
  • the ME1 ORF was gel-purified, cloned into PCRII-TOPO (#450640, Thermofischer Scientific) and sequence-verified.
  • the ME1 ORF was then excised from the PCRII-TOPO plasmid using Nhe1 and Not1 restriction enzymes, and ligated into the same sites of a PiggyBAC transposon vector, and verified by restriction mapping.
  • NF2 KO Schwann cell lines were then transfected with this vector together with a PiggyBAC transposase plasmid in a 1:1 ratio using PEI. After puromycin selection, serial dilutions of the surviving population were performed in order to analyze single cell clones.
  • Lentiviral particles were produced by transfecting Lenti-XTM 293T with either 2 nd generation lentiviral packaging system (pMD2.G #12259 and psPAX2 #12260, Addgene) or the 3 rd generation Virapower system (#K497500, Invitrogen), together with the plasmid of interest, using TransIT-LT1 reagent according to the manufacturer’s protocol (#2304, Mirus). Supernatants were collected after 48–72hrs and sterile-filtered (0.45 ⁇ m filters, #SLHV033RS, Merck/Millippore).
  • Target cells were infected with viral supernatants, supplemented with 3.5 ⁇ g/ml Polybrene Transfection reagent (#TR-1003-G, Merck/Millipore). After 48hrs, transduced cells were selected by addition of 3 ⁇ g/ml Puromycin (#P9620, Sigma), for another 48hrs.
  • Generation of Cas9 expressing lines Subconfluent NF2-WT and NF2-KO#1 cells were infected in the presence of 6 ⁇ g/ml Polybrene with lentiviral particles harboring Lenti-Cas9-2A-Blast (#73310, Addgene).
  • NF2-WT/Cas9 and NF2-KO/Cas9 cell lines were infected in the presence of 6 ⁇ g/ml polybrene (#TR-1003-G, Merck Millipore) with the 90k TKO sgRNAs library at a multiplicity of infection (MOI) of 0.3, to achieve 500-fold coverage (individual sgRNA-editing events represented in 500 cells).
  • MOI multiplicity of infection
  • puromycin-containing medium (4 ⁇ g/ml) was added to the infected cells for 48hrs. After selection, a portion of the puromycin-resistant population from each cell line was harvested for freezing and sequencing (T0 start point) and another portion was seeded for further culture expansion.
  • QIAamp DNA Blood Maxi kit #51192, Qiagen.
  • PCR reactions were performed using 1 ⁇ g of genomic or plasmid library DNA, Q5 Hot Start HF polymerase (#M0493L, NEB), and primers harboring the Illumina TruSeq adapter sequences.
  • PCR products were purified using DNA Clean and Concentrator TM-100 (#C1016-50, Zymo Research) and MagSi-NGSprep Plus beads (#SL-MDKT-01500, Steinbrenner).
  • shRNAs sequences targeting G6PD, ACSL3 and control RLuc were cloned into the lentiviral Tet-pLKO-puro vector (#21915, Addgene) according to the standard protocol 46.
  • Schwann cell cancer xenograft experiment All animal experiments were done in accordance with the guidelines of the responsible national authority, and with approval of the local Governmental Authority for Animal Experimentation (Reg michsconcesidium Düsseldorf, Germany, license 35-9185.81/G-30/20). Mice were maintained in a 12hrs light-dark cycle with unrestricted Kliba diet 3307 and water. After adaptation, mice were randomized according to age.
  • doxycycline (#D9891, Sigma; 1mg/ml) was provided via drinking water supplemented with 5% saccharose three days prior to cell transplantation and was continued throughout the experiment. Controls received drinking water with 5% saccharose.
  • Necropsies were taken when one tumor diameter reached 1.3 cm or when any other pre- defined humane endpoint was reached. Animal wellbeing was monitored regularly and animals were euthanized if any humane endpoints were reached in accordance with the approved license.
  • Cell proliferation assays Real time cell growth of semi-confluent cells was monitored using the xCELLigence DP System (OLS, OMNI Life Science).10.000 cells/well were seeded on E plate L8 PET (0.64cm 2 ) and impedance-based real-time proliferation was assessed over 96hrs (96 sweeps with 1hr interval). In the xCELLigence system, impedence correlates with cell number if other cell properties stay equal.
  • NF2-/+ cells vs NF2-WT and NF2-KO cells
  • 100.000 cells/well were seeded in a 12-well plate in biological triplicates.
  • Cells were fixed for 20min at room temperature with ice cold MetOH. Afterwards 2xPBS washes were performed and fixed cells were stained with 0.5% crystal violet (#V5265, Sigma), diluted in 20% methanol/ddH20 for 20min. After staining, extensive washes were performed with ddH2O and plates were allowed to dry. For quantification analysis, 10% acetic acid was added on top of the fixed cells and the Crystal Violet solution was measured at OD590nm, using a spectrophotometer (Biospectrometer, Eppendorf).
  • Protein extraction was performed with 1%SDS in PBS, supplemented with 1x protease and phosphatase inhibitor (#04693159001 and #4906837001 respectively, both from Roche).
  • Cell lysates were sonicated for 10sec to shear DNA (12% amplitude, Branson Digital Sonifier W-250D) and boiled for 3min at 95 o C.
  • Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific) and colorimetric analysis was performed with a Spectrostar Omega plate reader (BMG Labtech, OD562nm). 10-20 ⁇ g of total cell lysates were loaded on SDS-Page gels and transferred to Amersham nitrocellulose membranes (Merck). Description of all antibodies and dilutions used in this study are described in the Table 1.
  • RNA extraction quantitative RT-PCR and RNAseq
  • total RNA was extracted with TRIzol reagent (#15596026, Invitrogen) following manufacturer’s instructions.
  • cDNA was synthesised with the MaximaH minus Reverse Transcriptase (#EP0753, ThermoScientific) using 2 ⁇ g of total RNA as a template. Quantitative PCR was performed using Maxima SYBR Green/Rox (Fermentas), normalised to Rpl13a.
  • RNA interference Human Schwann cells were seeded at a density of 2x10 5 cells per well on six-well plates. The next day, cells were transfected with Lipofectamine RNAiMAX (#13778-500, Invitrogen) with a final concentration of 20nM siRNA per well.
  • siRNAs were used (sequences provided in Table 2): siRLuc : P-002070-01-50 (individual, ThermoFischer) siNF2#1 : siGenome D-003917-18-0002 (individual) siNF2#2 : siGenome D-003917-19-0002 (individual) siG6PD : OnTarget plus LQ-008181-02-0002 (pool of 4) siACSL3: OnTarget plus LQ-010061-00-0002 (pool of 4) Enzymatic activity assays and metabolite levels G6PD activity was assayed using a commercially available kit, according to the manual’s instructions (#MET-5081, Cell Biolabs) and samples were analysed with a Spectrostar Omega plate reader (BMG Labtech, OD450nm).
  • ME1 activity was performed on fresh cytosolic extracts as follows. The cells were collected and resuspended in 100mM Tris-HCl lysis buffer containing 0.02% Digitonin to release cytosolic content but not mitochondrial content. The suspension was incubated on ice for 10 min and then centrifuged at 10000g for 10 min at 4 o C. The supernatant containing the cell cytosolic extract was collected. The extracts were added to a reaction solution containing 100mM Tris-HCl, 1mM MnCl2, 1mM NH4Cl, 100mM KCl and 1mM NADP + final concentrations.
  • ROS reactive oxygen species
  • the general ROS indicator CM-H2DCFDA was used (#C6827, ThermoScientific) and for lipid ROS levels BODIPYTM 581/591 C11 (#D3861, ThermoScientific).
  • Subconfluent cells were cultured overnight in standard conditions and 30min before FACS analysis, cells were incubated with 2.5 ⁇ of the appropriate cell permeant ROS indicator. Oligomycin treatment (5 ⁇ for 30min) was used as a positive control for ROS generation.
  • FACS analysis was performed with Guava easyCyte HT (Millipore), using BlueV (Ex 450/45) and Yellow G (Em 575/25) lasers.
  • NF2 Neurofibromatosis type 2
  • Baser ME Contributors to the International NFMD. The distribution of constitutional and somatic mutations in the neurofibromatosis 2 gene. Hum Mutat 27, 297-306 (2006).
  • Cumpston EC Rhodes SD, Yates CW. Advances in Targeted Therapy for Neurofibromatosis Type 2 (NF2)-Associated Vestibular Schwannomas. Curr Oncol Rep 25, 531-537 (2023). 5.
  • Feltri ML Weaver MR, Belin S, Poitelon Y.
  • the Hippo pathway Horizons for innovative treatments of peripheral nerve diseases. J Peripher Nerv Syst 26, 4-16 (2021). 6.
  • Plotkin SR et al. Multicenter, prospective, phase II study of maintenance bevacizumab for children and adults with NF2-related schwannomatosis and progressive vestibular s chwannoma. Neuro Oncol 25, 1498-1506 (2023).
  • Zheng Y Pan D. The Hippo Signaling Pathway in Development and Disease. Developmental cell 50, 264-282 (2019).
  • Johnson R Halder G. The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment.
  • a small molecule G6PD inhibitor reveals immune dependence on p entose phosphate pathway. Nature chemical biology 16, 731-739 (2020). 24. Shalem O, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84-87 (2014). 25. Yang C, et al. Missense mutations in the NF2 gene result in the quantitative loss of merlin p rotein and minimally affect protein intrinsic function. Proceedings of the National Academy of Sciences of the United States of America 108, 4980-4985 (2011). 26. Stanton RC. Glucose-6-phosphate dehydrogenase, NADPH, and cell survival. IUBMB Life 64, 362-369 (2012). 27.
  • High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD i nhibition in acute myeloid leukemia cells. Leukemia 31, 2326-2335 (2017). 43. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the C RISPR-Cas9 system. Nature protocols 8, 2281-2308 (2013). 44. Imkeller K, Ambrosi G, Boutros M, Huber W. gscreend: modelling asymmetric count ratios in C RISPR screens to decrease experiment size and improve phenotype detection. Genome biology 21, 53 (2020). 45. Hart T, et al.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un inhibiteur d'une enzyme impliquée dans la promotion de la capacité réductrice cellulaire, de préférence la glucose-6-phosphate-déshydrogénase (G6PD) ou l'élément 3 de la famille à longue chaîne d'acyl-CoA synthétase (ACSL3) pour une utilisation dans le traitement de la neurofibromatose de type 2 ou la prévention de tumeurs provoquées par la neurofibromatose de type 2.
PCT/EP2025/066359 2024-06-13 2025-06-12 Traitement de la neurofibromatose de type 2 par des inhibiteurs de g6pd, acsl3 et/ou oxsm Pending WO2025257301A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP24181852 2024-06-13
EP24181852.5 2024-06-13

Publications (1)

Publication Number Publication Date
WO2025257301A1 true WO2025257301A1 (fr) 2025-12-18

Family

ID=91539924

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/066359 Pending WO2025257301A1 (fr) 2024-06-13 2025-06-12 Traitement de la neurofibromatose de type 2 par des inhibiteurs de g6pd, acsl3 et/ou oxsm

Country Status (1)

Country Link
WO (1) WO2025257301A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US6080560A (en) 1994-07-25 2000-06-27 Monsanto Company Method for producing antibodies in plant cells
WO2008022759A2 (fr) 2006-08-21 2008-02-28 Eidgenoessische Technische Hochschule Zürich Protéines de liaison spécifiques et de haute affinité comprenant des domaines sh3 modifiés de kinase fyn
WO2013043232A2 (fr) * 2011-04-08 2013-03-28 Afraxis, Inc. 8-éthyl-6(aryl)pyrido[2,3-d]pyrimidin-7(8h)-ones utilisables en vue du traitement d'affections touchant le système nerveux, ainsi qu'en vue du traitement du cancer
WO2013067423A1 (fr) * 2011-11-04 2013-05-10 Afraxis, Inc. Inhibiteurs de pak pour le traitement de troubles de prolifération cellulaire
US20200147194A1 (en) * 2017-05-19 2020-05-14 Keio University Peptide vaccine and peptide vaccine composition for cranial nerve disease

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US6080560A (en) 1994-07-25 2000-06-27 Monsanto Company Method for producing antibodies in plant cells
WO2008022759A2 (fr) 2006-08-21 2008-02-28 Eidgenoessische Technische Hochschule Zürich Protéines de liaison spécifiques et de haute affinité comprenant des domaines sh3 modifiés de kinase fyn
WO2013043232A2 (fr) * 2011-04-08 2013-03-28 Afraxis, Inc. 8-éthyl-6(aryl)pyrido[2,3-d]pyrimidin-7(8h)-ones utilisables en vue du traitement d'affections touchant le système nerveux, ainsi qu'en vue du traitement du cancer
WO2013067423A1 (fr) * 2011-11-04 2013-05-10 Afraxis, Inc. Inhibiteurs de pak pour le traitement de troubles de prolifération cellulaire
US20200147194A1 (en) * 2017-05-19 2020-05-14 Keio University Peptide vaccine and peptide vaccine composition for cranial nerve disease

Non-Patent Citations (99)

* Cited by examiner, † Cited by third party
Title
"Handbook of Pharmaceutical Excipients", 2010, PHARMACEUTICAL PRESS
ALLAY ET AL., HUM GENE THER, vol. 22, no. 5, May 2011 (2011-05-01), pages 595 - 604
ALTSHULER EPSEREBRYANAYA DVKATRUKHA AG, BIOCHEMISTRY, vol. 75, no. 13, 2010, pages 1584
AUSUBEL ET AL., HUM GENE THER, vol. 22, no. 4, April 2011 (2011-04-01), pages 489 - 97
BARRY ERSIMOV VVALTINGOJER IVENIER O: "Recent Therapeutic Approaches to Modulate the Hippo Pathway in Oncology and Regenerative Medicine", CELLS, vol. 10, 2021
BASER ME: "Contributors to the International NFMD. The distribution of constitutional and somatic mutations in the neurofibromatosis 2 gene", HUM MUTAT, vol. 27, 2006, pages 297 - 306
BEBBINGTON ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 169 - 175
BERTSCHINGER ET AL., PROTEIN ENG DES SEL, vol. 20, no. 2, 2007, pages 57 - 68
BRAASCHCOREY, CHEM BIOL, vol. 8, 2001, pages 1
CETIN ET AL., J MOL BIOL., vol. 429, no. 4, 2017, pages 562 - 573
CHEN L ET AL.: "NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism", NOT METAB, vol. 1, 2019, pages 404 - 415
CHEN X ET AL.: "Modulation of G6PD affects bladder cancer via ROS accumulation and the AKT pathway in vitro", INT J ONCOL, vol. 53, 2018, pages 1703 - 1712
CLEMM VON HOHENBERG K ET AL.: "Cyclin B/CDK1 and Cyclin A/CDK2 phosphorylate DENR to promote mitotic protein translation and faithful cell division", NATURE COMMUNICATIONS, vol. 13, 2022, pages 668
CUMPSTON ECRHODES SDYATES CW: "Advances in Targeted Therapy for Neurofibromatosis Type 2 (NF2)-Associated Vestibular Schwannomas", CURR ONCOL REP, vol. 25, 2023, pages 531 - 537
DE FOUGEROLLES ET AL., CURRENT OPINION IN PHARMACOLOGY, vol. 8, 2008, pages 280 - 285
DEBEB BG ET AL.: "Histone deacetylase inhibitor-induced cancer stem cells exhibit high pentose phosphate pathway metabolism", ONCOTARGET, vol. 7, 2016, pages 28329 - 28339
DENG Y ET AL.: "A reciprocal regulatory loop between TAZ/YAP and G-protein Galphas regulates Schwann cell proliferation and myelination", NATURE COMMUNICATIONS, vol. 8, 2017, pages 15161
DIEM ET AL., PROTEIN ENG., DES. AND SEL, vol. 27, 2014, pages 419 - 429
DORE MPDAVOLI ALONGO NMARRAS GPES GM: "Glucose-6-phosphate dehydrogenase deficiency and risk of colorectal cancer in Northern Sardinia: A retrospective observational study", MEDICINE, vol. 95, 2016, pages e5254
EVANS DG: "Neurofibromatosis type 2 (NF2): a clinical and molecular review", ORPHANET J RARE DIS, vol. 4, 2009, pages 16, XP021060208, DOI: 10.1186/1750-1172-4-16
FELDWISCH, JTOLMACHEV, V, METHODS MOL. BIOL, vol. 899, 2012, pages 103 - 126
FELTRI MLWEAVER MRBELIN SPOITELON Y: "The Hippo pathway: Horizons for innovative treatments of peripheral nerve diseases", J PERIPHER NERV SYST, vol. 26, 2021, pages 4 - 16
FERNER REO'DOHERTY MJ: "Neurofibroma and schwannoma", CURR OPIN NEUROL, vol. 15, 2002, pages 679 - 684, XP009010729, DOI: 10.1097/00019052-200212000-00004
GAROUSI ET AL., CANCER RES., vol. 75, no. 20, 2015, pages 4364 - 71
GE SXJUNG DYAO R: "ShinyGO: a graphical gene-set enrichment tool for animals and plants", BIOINFORMATICS, vol. 36, 2020, pages 2628 - 2629
GEBAUER, MSKERRA, A, CURR. OPIN. CHEM. BIOL., vol. 13, 2009, pages 245 - 255
GEBAUERSKERRA, CURR OPINION IN CHEMICAL BIOLOGY, vol. 13, 2009, pages 245 - 255
GHAHROUDI ET AL., FEBS LETTERS, vol. 414, 1997, pages 521 - 526
GHERGUROVICH JM ET AL.: "A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway", NATURE CHEMICAL BIOLOGY, vol. 16, 2020, pages 731 - 739, XP037175982, DOI: 10.1038/s41589-020-0533-x
GOMEZ-MANZO S ET AL.: "Glucose-6-Phosphate Dehydrogenase: Update and Analysis of New Mutations around the World", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 17, 2016
GRABULOVSKI ET AL., JBC, vol. 282, 2007, pages 3196 - 3204
GROSS ET AL., NEURON., vol. 78, no. 6, 2013, pages 971 - 985
GROVE M ET AL.: "YAP/TAZ initiate and maintain Schwann cell myelination", ELIFE, vol. 6, 2017
GUGEL I ET AL.: "Contribution of mTOR and PTEN to Radioresistance in Sporadic and NF2-Associated Vestibular Schwannomas: A Microarray and Pathway Analysis", CANCERS, 2020, pages 12
HADPECH ET AL., SCIENTIFIC REPORTS, 2017
HARLOW ELANE D: "Cold Spring Harbor Laboratory Press", 1988
HARLOW ELANE D: "Using Antibodies: A Laboratory Manual", 1999, COLD SPRING HARBOR LABORATORY PRESS, pages: 198
HART T ET AL.: "Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens", G3, vol. 7, 2017, pages 2719 - 2727
HART T ET AL.: "High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities", CELL, vol. 163, 2015, pages 1515 - 1526, XP055947114, DOI: 10.1016/j.cell.2015.11.015
HOLLIGER PHUDSON PJ, NAT BIOTECHNOL., vol. 23, no. 9, 2005, pages 11265
IMKELLER KAMBROSI GBOUTROS MHUBER W: "gscreend: modelling asymmetric count ratios in CRISPR screens to decrease experiment size and improve phenotype detection", GENOME BIOLOGY, vol. 21, 2020, pages 53
JEANETTE H ET AL.: "YAP and TAZ regulate Schwann cell proliferation and differentiation during peripheral nerve regeneration", GLIA, vol. 69, 2021, pages 1061 - 1074
JOHNSON RHALDER G: "The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment", NATURE REVIEWS DRUG DISCOVERY, vol. 13, 2014, pages 63 - 79, XP055570286, DOI: 10.1038/nrd4161
JU HQ ET AL.: "Disrupting G6PD-mediated Redox homeostasis enhances chemosensitivity in colorectal cancer", ONCOGENE, vol. 36, 2017, pages 6282 - 6292
KLASSON TD ET AL.: "ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma", CANCER & METABOLISM, vol. 10, 2022, pages 14
KLUG, ANNUAL REVIEW OF BIOCHEMISTRY, vol. 79, 2010, pages 213 - 231
KOIDE ET AL., J. MOL. BIOL., vol. 284, 1998, pages 1141 - 51
KONTERMANNBRINKMANN, DRUG DISCOVERY TODAY, vol. 20, no. 7, 2015, pages 838 - 847
KOPERNIKU ET AL., J MED CHEM, vol. 65, no. 6, 2022, pages 4403 - 4423
KOZBOR D, IMMUNOLOGY TODAY, vol. 4, 1983, pages 7
KUDEYAROV ET AL., MEDICAL AND BIOLOGICAL MEASUREMENTS, vol. 60, 2017, pages 291 - 296
KYRKOU ATHENA ET AL: "G6PD and ACSL3 are synthetic lethal partners of NF2 in Schwann cells", NATURE COMMUNICATIONS, vol. 15, no. 1, 15 June 2024 (2024-06-15), UK, pages 1 - 15, XP093229465, ISSN: 2041-1723, DOI: 10.1038/s41467-024-49298-7 *
LARABA L ET AL.: "Inhibition of YAP/TAZ-driven TEAD activity prevents growth of NF2-null schwannoma and meningioma", BRAIN, vol. 146, 2023, pages 1697 - 1713
LEE, PROC NATL ACAD SCI, vol. 109, no. 9, 2012, pages 3299 - 3304
LEPONT PSTICKNEY JTFOSTER LAMENG JJHENNIGAN RFIP W: "Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity", MUTATION RESEARCH, vol. 637, 2008, pages 142 - 151, XP022392724, DOI: 10.1016/j.mrfmmm.2007.07.015
LI HCHANG UNEUBAUER DRMUIR DFWALLACE MR: "Immortalization of human normal and NF1 neurofibroma Schwann cells", LAB INVEST, vol. 96, 2016, pages 1105 - 1115
LI J ET AL., PNAS, vol. 103, 2006, pages 3557
LIRU ZHANG ET AL: "Identification and validation of transcription factor-driven enhancers of genes related to lipid metabolism in metastatic oral squamous cell carcinomas", BMC ORAL HEALTH, BIOMED CENTRAL LTD, LONDON, UK, vol. 22, no. 1, 15 April 2022 (2022-04-15), pages 1 - 15, XP021301868, DOI: 10.1186/S12903-022-02157-7 *
LU, METHODS IN MOLECULAR BIOLOGY, vol. 437, 2008
MAGTANONG L ET AL.: "Exogenous Monounsaturated Fatty Acids Promote a Ferroptosis-Resistant Cell State", CELL CHEM BIOL, vol. 26, 2019, pages 420 - 432
MELANI ET AL., CANCER RES, vol. 51, 1991, pages 2897 - 2901
MILLER ET AL., NATURE BIOTECHNOLOGY, vol. 29, no. 2, 2011, pages 143 - 148
MINDOS T ET AL.: "Merlin controls the repair capacity of Schwann cells after injury by regulating Hippo/YAP activity", THE JOURNAL OF CELL BIOLOGY, vol. 216, 2017, pages 495 - 510
MORRISON H ET AL.: "The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44", GENES DEV, vol. 15, 2001, pages 968 - 980, XP002190162, DOI: 10.1101/gad.189601
MOURATOU BBÉHAR GPAILLARD-LAURANCE LCOLINET SPECORARI F., METHODS MOL BIOL., vol. 805, 2012, pages 315 - 31
MURPHY ET AL., BIOCHEM J, vol. 227, 1991, pages 277 - 279
ORR ET AL., COMPUTERS & CHEMICAL ENGINEERING, vol. 116, no. 4, 2018, pages 322 - 332
OSBORNE, CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 1, 1997, pages 5 - 9
OWENS, PROC. NATL. ACAD. SCI. USA, vol. 98, 2001, pages 1471 - 1476
PADANAD MS ET AL.: "Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis", CELL REPORTS, vol. 16, 2016, pages 1614 - 1628
PES GMERRIGO ASORO SLONGO NPDORE MP: "Glucose-6-phosphate dehydrogenase deficiency reduces susceptibility to cancer of endodermal origin", ACTA ONCOL, vol. 58, 2019, pages 1205 - 1211
PETRILLI AMFERNANDEZ-VALLE C: "Role of Merlin/NF2 inactivation in tumor biology", ONCOGENE, vol. 35, 2016, pages 537 - 548, XP037749048, DOI: 10.1038/onc.2015.125
PLOTKIN SR ET AL.: "Multicenter, prospective, phase II study of maintenance bevacizumab for children and adults with NF2-related schwannomatosis and progressive vestibular schwannoma", NEURO ONCOL, vol. 25, 2023, pages 1498 - 1506
POULAIN L ET AL.: "High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD inhibition in acute myeloid leukemia cells", LEUKEMIA, vol. 31, 2017, pages 2326 - 2335, XP037653545, DOI: 10.1038/leu.2017.81
PREUSS ET AL., SLAS DISCOVERY, vol. 18, no. 2457232-14-1, 2013, pages 286 - 297
QUAN J: "Acyl-CoA synthetase long-chain 3-mediated fatty acid oxidation is required for TGFbeta1-induced epithelial-mesenchymal transition and metastasis of colorectal carcinoma.", INT J BIOL SCI, vol. 18, 2022, pages 2484 - 2496
RAN FAHSU PDWRIGHT JAGARWALA VSCOTT DAZHANG F: "Genome engineering using the CRISPR-Cas9 system", NATURE PROTOCOLS, vol. 8, 2013, pages 2281 - 2308, XP009174668, DOI: 10.1038/nprot.2013.143
ROSSI SEBASTIANO M ET AL.: "ACSL3-PAI-1 signaling axis mediates tumor-stroma cross-talk promoting pancreatic cancer progression", SCI ADV, vol. 6, 2020
ROTHESKERRA, BIODRUGS, vol. 32, 2018, pages 233 - 243
SCHLATTER ET AL., MABS, vol. 4, no. 4, 2012, pages 1 - 12
SHALEM O ET AL.: "Genome-scale CRISPR-Cas9 knockout screening in human cells", SCIENCE, vol. 343, 2014, pages 84 - 87, XP055115506, DOI: 10.1126/science.1247005
SILVA ET AL., CURR GENE THER, vol. 11, no. 1, 2011, pages 11 - 27
STANTON RC: "Glucose-6-phosphate dehydrogenase, NADPH, and cell survival", IUBMB LIFE, vol. 64, 2012, pages 362 - 369
STEPANOVA DS ET AL.: "An Essential Role for the Tumor-Suppressor Merlin in Regulating Fatty Acid Synthesis", CANCER RESEARCH, vol. 77, 2017, pages 5026 - 5038
STEPHEN F. ALTSCHULTHOMAS L. MADDENALEJANDRO A. SCHAFFERJINGHUI ZHANGZHENG ZHANGWEBB MILLERDAVID J. LIPMAN, NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
STULLSZOKA, PHARMACEUTICAL RESEARCH, vol. 12, no. 4, 1995, pages 465 - 483
TAMHANELOGAN: "Multiple Test Procedures for Identifying the Minimum Effective and Maximum Safe Doses of a Drug", JOURNAL OF THE AMERICAN STATISTICAL ASSOCIATION, vol. 97, no. 457, 2002, pages 1 - 9
TANG DCHEN XKANG RKROEMER G: "Ferroptosis: molecular mechanisms and health implications", CELL RESEARCH, vol. 31, 2021, pages 107 - 125, XP093156462, DOI: 10.1038/s41422-020-00441-1
TIEDE ET AL., ELIFE., vol. 6, 2017, pages e24903
TIEDE ET AL., PROTEIN ENG. DES. SEL, vol. 27, 2014, pages 145 - 55
WEIDLE UH ET AL., CANCER GENOMICS PROTEOMICS, vol. 10, no. 4, 2013, pages 155 - 68
WIEDERSCHAIN D ET AL.: "Single-vector inducible lentiviral RNAi system for oncology target validation", CELL CYCLE, vol. 8, 2009, pages 498 - 504, XP055038165, DOI: 10.4161/cc.8.3.7701
WU J ET AL.: "Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling", NATURE, vol. 572, 2019, pages 402 - 406, XP036861551, DOI: 10.1038/s41586-019-1426-6
WU WEIDONG ET AL: "Glucose-6-phosphate dehydrogenase promotes the proliferation and migration of lung adenocarcinoma cells via the STAT3 signaling pathway", JOURNAL OF MOLECULAR HISTOLOGY, SPRINGER-VERLAG, DORDRECHT, NL, vol. 53, no. 2, 14 January 2022 (2022-01-14), pages 215 - 225, XP037852211, ISSN: 1567-2379, [retrieved on 20220114], DOI: 10.1007/S10735-021-10045-7 *
YANG C ET AL.: "Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 108, 2011, pages 4980 - 4985
YANG LI ET AL: "Construction and validation of a novel ferroptosis-related prognostic signature for lung adenocarcinoma", vol. 12, no. 8, 28 August 2023 (2023-08-28), Hong Kong, pages 1766 - 1781, XP093229450, ISSN: 2218-6751, Retrieved from the Internet <URL:https://pmc.ncbi.nlm.nih.gov/articles/PMC10483076/pdf/tlcr-12-08-1766.pdf> DOI: 10.21037/tlcr-23-351 *
ZHENG JCONRAD M: "The Metabolic Underpinnings of Ferroptosis", CELL METABOLISM, vol. 32, pages 920 - 937
ZHENG YPAN D: "The Hippo Signaling Pathway in Development and Disease", DEVELOPMENTAL CELL, vol. 50, 2019, pages 264 - 282, XP085757425, DOI: 10.1016/j.devcel.2019.06.003
ZHOU L ET AL: "The scaffold protein KSR1, a novel therapeutic target for the treatment of Merlin-deficient tumors", ONCOGENE, vol. 35, no. 26, 9 November 2015 (2015-11-09), pages 3443 - 3453, XP037324511, ISSN: 0950-9232, DOI: 10.1038/ONC.2015.404 *

Similar Documents

Publication Publication Date Title
US10982211B2 (en) tRNA derived small RNAs (tsRNAs) involved in cell viability
JP2018512373A (ja) Ｋｒａｓ突然変異に関連する悪性腫瘍に対する治療のための方法及び組成物
EP2937099A1 (fr) Agent inducteur d&#39;apopotose
CA2494766A1 (fr) Sondes d&#39;arn interferant ciblant des proteines en relation avec le cancer
US20160024597A1 (en) miRNAs AS THERAPEUTIC TARGETS IN CANCER
Herrera-Pérez et al. A comprehensive review on the genetic regulation of cisplatin-induced nephrotoxicity
US20210380988A1 (en) Reducing Prominin2-Mediated Resistance to Ferroptotic Cell Death
US20160108406A1 (en) Method of regulating cftr expression and processing
KR20180057608A (ko) 치료용 올리고뉴클레오타이드
EP3159009B1 (fr) Inhibiteurs de gst-pi et rb1cc1 pour le traitement du cancer
EP3273971A1 (fr) Méthodes de traitement des maladies des motoneureones
WO2025257301A1 (fr) Traitement de la neurofibromatose de type 2 par des inhibiteurs de g6pd, acsl3 et/ou oxsm
WO2024186883A2 (fr) Compositions et méthodes pour le traitement de cancers liés au papillomavirus humain
US11946046B2 (en) Staufen1 regulating agents and associated methods
WO2017106964A1 (fr) Procédés de traitement du cancer par inhibition de protéines de réparation d&#39;adn en utilisant des traitements à base d&#39;antisens
WO2006054555A1 (fr) Composition medicinale pour le traitement du cancer ou du diabete
WO2020014566A2 (fr) Compositions et méthodes de traitement de l&#39;endométriose
WO2025056690A1 (fr) Inhibiteurs de parp1 et de point de contrôle immunitaire pour le traitement de patients atteints d&#39;un cancer surexprimant l&#39;ubqln1 et/ou l&#39;ubqln4
JP2019033741A (ja) 悪性腫瘍に対する治療方法及び治療用組成物
US20230374505A1 (en) Human XIST Antisense Oligonucleotides for X Reactivation Therapy
WO2024182719A1 (fr) Inhibiteurs à base d&#39;arn d&#39;enzymes de modification d&#39;arnt
Johnson The Role of TNRC6 in RNA Interference
WO2025171309A1 (fr) Procédés de réduction ou d&#39;élimination de restriction de flux sanguin et procédés de traitement, d&#39;amélioration et/ou de prévention de maladies ou de troubles associés à la restriction du flux sanguin
Algouneh CRISPR/Cas9 Dropout Screen Identifies MEPCE as a Novel Vulnerability in BRCA1-Mutated Cancers
CA2680058C (fr) Induction d&#39;apoptose et inhibition de proliferation cellulaire par modulation de l&#39;activite carnitine palmitoyltransferase 1c

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: 25730058

Country of ref document: EP

Kind code of ref document: A1