WO2021229131A1 - In vitro method for recovering the expression of the f5 gene encoding coagulation factor v - Google Patents

In vitro method for recovering the expression of the f5 gene encoding coagulation factor v Download PDF

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WO2021229131A1
WO2021229131A1 PCT/ES2021/070341 ES2021070341W WO2021229131A1 WO 2021229131 A1 WO2021229131 A1 WO 2021229131A1 ES 2021070341 W ES2021070341 W ES 2021070341W WO 2021229131 A1 WO2021229131 A1 WO 2021229131A1
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gene
factor
mutation
guides
expression
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PCT/ES2021/070341
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Spanish (es)
French (fr)
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Antonio Liras Martin
Luis Javier SERRANO RAMOS
Sara Bernal Noguera
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Universidad Complutense De Madrid
Fundació Institut De Recerca De L'hospital De La Santa Creu I Sant Pau
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention falls within the field of diseases related to blood coagulation and, more specifically, to factor V deficiency, also called parahemophilia or Owren's disease. Likewise, it is related to the application of gene editing for the correction of mutations in the gene that codes for coagulation factor V.
  • Factor V is an essential protein that participates in coagulation and plays a key role in the so-called blood coagulation cascade due to its procoagulant and anticoagulant activity. 80% of circulating factor V is produced in the liver and the remaining 20% in the alpha granules of platelets.
  • the gene that expresses this protein (F5) is approximately 80kb in size and is located on chromosome 1q24.2. The coding sequence of the F5 gene is divided into 25 exons and 24 introns and its cDNA is 6914bp in size. So far, approximately 150 point mutations and small insertions and deletions in the gene have been described.
  • Factor V deficiency an ultramaraderie disease occurring in only 1 to 9 per million, is an autosomal recessive bleeding disorder associated with mutations in the F5 gene.
  • This inherited bleeding disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft tissue bleeding to life-threatening bleeding.
  • This alternative is very short-lived (4 hours) and the dosage is very difficult to adjust to the characteristics of the patient, since the concentration of factor V in plasma pools is highly variable depending on the donor (Tabibian S, et al. A Comprehensive OverView of Coagulation Factor V and Congenital Factor V Deficiency. Semin Thromb Hemost, 2019; 45: 523-43).
  • W02010069946A1 a method is described to purify factor V from biological fluids, with the interest of using it in cases of deficiency in this coagulation factor.
  • Patent application W02008059009A2 refers to a method to prevent and treat bleeding in joints, muscles and soft tissues in hemophiliac patients, by administering factor V resistant to APC (activated protein C), which has the ability to inactivate both factor V as coagulation factor VIII, as a hemostatic.
  • APC activate protein C
  • W02010060035A1 describes therapeutic strategies that use variants derived from factor V present in snake venom, and its derivatives, to modulate the coagulation cascade in patients who require it, since factor V in the venom of some snakes presents around a 44% sequence homology with that of mammalian factor V.
  • compositions and methods have also been described to inhibit the expression of certain mutant factor V genes, as in patent application US2011130443A1 in which a double-stranded RNA is described to inhibit the expression of the mutant gene that causes a substitution of G by A at nucleotide 1691 in the factor V gene, which causes an Arg506Gln change.
  • a gene methodology is used, based on gene editing by means of the CRISPR / Cas9 tool, to provide tools to cure (not palliate) this pathology, acting specifically on the mutation that produces this factor V deficiency in coagulation. It is an in vitro method that is not a method of treating the human or animal body through surgery or therapies performed on the body and it is not intended to modify the genetic identity of the human germ line. The objective is to provide the necessary tools that, later, can be used to establish a long-term or permanent and individualized curative treatment for each patient.
  • One aspect of the present invention relates to an in vitro method to recover the expression of the F5 gene encoding coagulation factor V by using the CRISPR / Cas9 methodology.
  • This method includes the use of a pair of guides in which each of the guides is between 18 and 22 nucleotides in length and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to the position of the localized mutation. at nucleotide 3279 of the F5 gene.
  • a pair of guides characterized by SEQ ID NO: 68-69 or a pair of guides characterized by SEQ ID NO: 70-71 is used.
  • a mutation is corrected in nucleotide 3279 of the F5 gene that generates a nonsense mutation in the amino acid sequence of factor V that gives rise to a stop codon (from stop) (p.Trp1093 *).
  • This mutation has been identified by sequencing the F5 gene of a patient with severe factor V deficiency; the sequence obtained is characterized by SEQ ID NO: 65.
  • Another aspect of the present invention relates to an in vitro cell culture in which the expression of the F5 gene encoding coagulation factor V has been recovered.
  • the mutation of the F5 gene identified at nucleotide 3279 that generated a nonsense mutation in the amino acid sequence of factor V that gives rise to a stop codon has been corrected by using the CRISPR / Cas9 methodology including the use of a pair of guides, in which each of the guides is between 18 and 22 nucleotides in length and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to position of the mutation located at nucleotide 3279 of the F5 gene.
  • the pair of guides characterized by SEQ ID NO: 68-69 or the pair of guides characterized by SEQ ID NO: 70-71 is used.
  • the complete sequence of the mutated F5 gene can be that characterized by SEQ ID NO: 65.
  • kits comprising a pair of guides, in which each of the guides has a length between 18 and 22 nucleotides and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to to the position of the mutation located at nucleotide 3279 of the F5 gene.
  • each of the guides has a length between 18 and 22 nucleotides and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to to the position of the mutation located at nucleotide 3279 of the F5 gene.
  • it comprises the pair of guides characterized by SEQ ID NO: 68-69 and / or the pair of guides characterized by SEQ ID NO: 70-71.
  • this kit may comprise a Cas9 protein or a polynucleotide encoding the Cas9 protein.
  • Figure 1 Pathological genetic variants (mutations) found in the patient's F5 gene.
  • Figure 2. Gene editing for correction of mutations using CRISPR / Cas9, according to the state of the art.
  • FIGURE 3 Design of gene editing guides using CRISPR / Cas9, to obtain the mutated cell model and to correct the mutation:
  • Example 1 Hematological and coagulation analysis.
  • Plasma samples were distributed in tubes with 0.105M sodium citrate (1:10). Plasma was obtained by centrifugation at 2000xg for 20 minutes and subsequently stored in aliquots at -80 ° C until use.
  • Hb hemoglobin concentration
  • HCM mean corpuscular hemoglobin
  • MCHC mean corpuscular hemoglobin concentration
  • erythrocyte count hematocrit
  • MCV mean corpuscular volume
  • reticulocyte count white blood cell count
  • platelet count mean platelet volume (MPV)
  • platelet count Everybody
  • Blood samples for analysis of platelet function and clotting times were taken in vacuum blood collection tubes (Vacutainer, Becton Dickinson) with 3.8% sodium citrate and processed on a Function Platelet Analyzer-100 (PFA-100®; Siemens Healthcare Diagnostics AG, Zurich, Switzerland).
  • Collagen / adenosine-5-diphosphate (ADP) cartridges (Dade® PFA, Siemens Healthcare Diagnostics, AG, Zurich, Switzerland) were used to determine bleeding time and platelet aggregation.
  • the cartridges were used at room temperature by adding 1 mL of citrated blood to them.
  • the samples were aspirated under constant vacuum through a capillary and a microscopic opening made in a membrane coated with collagen and ADP.
  • PT prothrombin time
  • AP prothrombin activity
  • aTPP activated partial thromboplastin time
  • F fibrinogen
  • II international normalized index
  • HemosIL® RecombiPlasTin 2G was used to measure prothrombin time (PT) and fibrinogen levels and a highly sensitive thromboplastin reagent, based on recombinant human tissue factor (rhFT), was used.
  • rhFT recombinant human tissue factor
  • the PT reagent included in the RecombiPlasTin 2G kit is converted into a liposomal preparation containing rhFT in a mixture of synthetic phospholipids and combined with calcium chloride, buffer and a preservative.
  • RecombiPlasTin 2G is highly sensitive to extrinsic pathway clotting factors, making it particularly suitable for assays with these factors.
  • RecombiPlasTin 2G's formulation makes it insensitive to therapeutic heparin levels.
  • the addition of the reagent to the patient's plasma in the presence of calcium ions activates the clotting pathway. extrinsic. This eventually results in the conversion of fibrinogen to fibrin, with the formation of a solid gel. Fibrinogen was quantified by relating the absorbance, or scattering of light, during clot formation, with a calibrator. TP results are reported in seconds, percent activity, or INI; fibrinogen levels, in g / L.
  • HemosIL® APTT-SP liquid was used to determine the aTPP value.
  • the aTPP test uses a contact activator that stimulates the production of factor Xlla.
  • the activator is a colloidal silica dispersion with synthetic phospholipids, a buffer and a preservative and provides a contact surface for the interaction of high molecular weight kininogen, kallikrein and factor Xlla.
  • Contact activation occurs at 37 ° C for a specified period of time.
  • the addition of 0.025 M calcium chloride with a preservative triggers a series of reactions that will lead to the formation of clots.
  • Phospholipids are also required to generate the compounds that will act on factor X and prothrombin. The results are expressed in seconds.
  • HemosIL® FV deficient plasma was used for quantitative determination of factor V activity.
  • Factor V activity in plasma was determined using the modified prothrombin time test performed on the patient's citrate-diluted plasma in the presence of plasma. human immunodepleted with factor V. The correction for prolonged clotting time for deficient plasma was proportional to the concentration (percentage of activity) of the specific factor (factor V) in the patient's plasma, which can be obtained by drawing a calibration curve. Reference levels of factor V ranged from 60-130% (0.60-1.30 IU).
  • Hb hemoglobin concentration
  • HCM mean corpuscular hemoglobin
  • MCHC mean corpuscular hemoglobin concentration
  • RBC total erythrocyte count
  • HCT mean corpuscular volume
  • RETIC leukocyte count
  • GB differential GB count
  • PHT platelet count
  • VMP mean platelet volume
  • PCT platelet
  • PT prothrombin time
  • AP prothrombin activity
  • aTPP activated partial thromboplastin time
  • F fibrinogen
  • II international normalized index
  • factor V activity The patient has a severe factor V deficiency with a factor V activity less than 1% of the reference value.
  • alterations in coagulation parameters and factor V activity were consistent with the level of involvement of the allele of the F5 gene and their heterozygous status.
  • Genomic DNA was automatically extracted from previously isolated peripheral leukocytes, by saline precipitation procedures using Autopure instrumentation (Qiagen). Using this kit, ready-to-use genomic DNA is obtained simply and quickly from 200pL whole blood samples. It is based on vacuum centrifugation and the prior separation of leukocytes is not necessary. Neither phenol / chloroform extraction nor alcohol precipitation is required. DNA purified for use in PCR is stored between -25 ° C and -15 ° C.
  • PCR amplification was carried out in a final volume of 25pL containing 1.5mM-2.5mM MgCL (depending on the fragment to be amplified), 200mM of each dNTP, 0.2mM of each primer, 0.5 Taq units DNA polymerase (Ecogen) in manufacturer's recommended buffer and 150-200ng of genomic DNA.
  • Thermal cycler conditions were as follows: 94 ° C for 5 minutes, followed by 30 cycles at 94 ° C for 30 seconds (denaturation step); 58 ° C-60 ° C (depending on the fragment to be amplified) for 30 seconds and 72 ° C for 2 minutes (elongation step), followed by a final extension step of 20 minutes at 72 ° C.
  • the remaining dNTPs and primers remaining in the PCR product mix were removed by using the ExoSAP digestion enzymes (exonuclease I and alkaline phosphatase) (Sigma-Aldrich).
  • ExoSAP digestion enzymes exonuclease I and alkaline phosphatase
  • the bidirectional sequencing reaction of the complete sequence was performed by the Sanger method using the BigDye TM Terminator v1.1 Cycle Sequencing kit.
  • the sequencing reactions of the amplified fragments were purified using the SEQ96 assembly kit on a vacuum manifold, to remove unincorporated labeled terminators and salts before subjecting the sequencing products to capillary electrophoresis on an ABI Prism 3500 Genetic Analyzer. (Applied Biosystems).
  • the chromatograms of the sequences were analyzed using SeqScape v.2.1.1.
  • HGVS Human Genome Variation Society
  • Msn Nonsense mutation
  • D domains (A1, A2, B, A3, C1 and C2)
  • E exons
  • He heavy chain
  • Le light chain
  • ps post-translational cleavage region
  • APCC activated protein C inactivation site
  • TC thrombin activation site
  • the type of mutation was correlated with the levels of factor V activity in the patient and in her parents.
  • the c.3279G> A mutation in the father is associated with an activity of 21% and the 0.2218OT mutation in the mother, with a factor V activity of 62.9%.
  • CRISPR / Cas9 (Regularly Clustered and Interleaved Short Palindromic Repeats) technology represents a significant improvement over other next-generation gene editing tools.
  • the CRISPR / Cas9 system enables site-specific genomic targeting in the genome in virtually any organism.
  • Figure 2 shows the state of the art: the target for CRISPR / cas9 are small DNA sequences, located around the mutation (MUT X -red-), which expresses a non-functional protein (PPT X -red-). It is a strategy of Gene therapy since it is necessary to introduce the complementary guides (in yellow), which confine the mutation and, by means of a small deletion in the genomic DNA inside the nucleus of the cell, correct the reading frame.
  • the functional protein is indicated as PPT V -green-.
  • the CRISPR / Cas type II system is a prokaryotic adaptive immune response system that uses antisense RNA to guide the nucleation of Cas9 to induce DNA cleavage at a specific site. This DNA damage is repaired by cellular DNA repair mechanisms, either through the homologous or non-homologous DNA repair pathway.
  • the CRISPR / Cas9 system has been leveraged to create a simple, programmable RNA method to mediate genome editing in mammalian cells, and can be used to generate knockout (KO) (insertion / deletion) models.
  • KO knockout
  • a single guide RNA sgRNA is generated to direct the Cas9 nuclease to a specific genomic location. Cas9-induced double strand breaks are repaired via the DNA repair pathway.
  • Example 6 Obtaining the cell model and correction of the mutation.
  • HepG2 cell line was used, provided by Dr. Jose Carlos Segovia of the Center for Energy, Environmental and Technological Research.
  • HepG2 cells belong to a line of immortal cells that are derived from well-differentiated hepatocellular carcinoma. These cells are epithelial in morphology, have a modal chromosome number of 55, and are not tumorigenic in mice. They secrete factor V in addition to a wide variety of important proteins, eg, albumin, and the acute phase proteins fibrinogen, alpha 2-macroglobulin, alpha 1 -antitrypsin, transferrin, and plasminogen.
  • important proteins eg, albumin, and the acute phase proteins fibrinogen, alpha 2-macroglobulin, alpha 1 -antitrypsin, transferrin, and plasminogen.
  • PAM protospacer adjacent motif. It is a 3 base pair DNA sequence immediately after the DNA sequence recognized by the Cas9 nuclease.
  • Del 35 bp Deletion (elimination) of a sequence of 35 base pairs, a region that corresponds to the mutated area in the patient with a stop codon (CS, G> A, change from a guanine to an adenine). The alteration of this area by means of the G1 and G2 guides complementary to the mutation, reproduce the mutation in the model.
  • Del 82 bp Deletion (deletion) of an 82 base pair sequence, which is complemented by the G3 and G4 guidelines to give a non-pathological sequence of the gene.
  • Del 49 bp Deletion (deletion) of a sequence of 49 base pairs, which is complemented by the G5 and G6 guides to give a non-pathological sequence of the gene.
  • the methodology was based on introducing the guides and the Cas9 nuclease inside the cell so that they later reach the nucleus where they are internalized.
  • the method of producing a ribonucleoprotein (RNP), formed from the guides and the Cas9 nuclease was used.
  • RNP ribonucleoprotein
  • Obtaining this complex was carried out using a thermocycler protocol based on incubating the 2 elements of the guide (crRNA and tracrRNA) at 95 ° C, 5 min and subsequent addition of Cas 9, incubating 2 minutes at room temperature.
  • this RNP was introduced by means of a 4D-Nucleofector TM System (Lonza, Switzerland) and the nucleofection kit "SF line cells" (Lonza, Switzerland) following the protocol of the commercial company to carry out the nucleofection in said cell line.
  • This procedure has less cellular toxicity, better internalization in the nucleus, greater editing efficiency, less production time, and is cheaper than others.

Abstract

The present invention relates to coagulation factor V deficiency and gene editing (CRISPR) for the correction in vitro of mutations in the F5 gene and the generation of tools to cure this currently untreatable disease. The invention includes an in vitro method for recovering the expression of the F5 gene encoding coagulation factor V, using CRISPR/Cas9 methodology that corrects the new pathological mutation described. The invention also includes cell cultures in vitro wherein the new mutation has been corrected, as well as kits that include the guide pairs used in the CRISPR/Cas9 method to recover the expression of the F5 gene with said mutation.

Description

MÉTODO IN VITRO PARA RECUPERAR LA EXPRESIÓN DEL GEN F5 QUE CODIFICA EL FACTOR V DE LA COAGULACIÓN
Figure imgf000002_0001
IN VITRO METHOD TO RECOVER THE EXPRESSION OF THE F5 GENE CODING COAGULATION FACTOR V
Figure imgf000002_0001
SECTOR DE LA TÉCNICA TECHNICAL SECTOR
La presente invención se encuadra en el sector de las enfermedades relacionadas con la coagulación sanguínea y, más concretamente, con el déficit de factor V, también denominado parahemofilia o enfermedad de Owren. Así mismo, se relaciona con la aplicación de la edición génica para la corrección de mutaciones en el gen que codifica el factor V de la coagulación. The present invention falls within the field of diseases related to blood coagulation and, more specifically, to factor V deficiency, also called parahemophilia or Owren's disease. Likewise, it is related to the application of gene editing for the correction of mutations in the gene that codes for coagulation factor V.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
El factor V es una proteína esencial que participa en la coagulación y que desempeña un papel clave en la llamada cascada de la coagulación de la sangre debido a su actividad procoagulante y anticoagulante. El 80% del factor V circulante se produce en el hígado y el 20% restante en los gránulos alfa de las plaquetas. En humanos, el gen que expresa esta proteína ( F5) tiene un tamaño aproximado de 80kb y se encuentra en el cromosoma 1q24.2. La secuencia codificadora del gen F5 se divide en 25 exones y 24 intrones y su cDNA tiene un tamaño de 6914bp. Hasta ahora, se han descrito, aproximadamente, 150 mutaciones puntuales y pequeñas inserciones y deleciones en el gen. La deficiencia de factor V, una enfermedad ultramara ya que ocurre en solo 1 a 9 por millón, es un trastorno de la coagulación de tipo autosómico recesivo asociado a mutaciones en el gen F5. Este trastorno hereditario de la coagulación se caracteriza clínicamente por un espectro heterogéneo de manifestaciones hemorrágicas que van desde sangrados de mucosas o tejidos blandos, hasta hemorragias potencialmente mortales. En la actualidad no existe un tratamiento ni paliativo ni curativo para esta enfermedad, tan solo la administración de plasma fresco congelado. Esta alternativa es muy poco duradera (4 horas) y la dosificación es muy difícil de ajustar a las características del paciente ya que la concentración de factor V en los acervos de plasma son muy variables según donante (Tabibian S, y col. A Comprehensive OverView of Coagulation Factor V and Congenital Factor V Deficiency. Semin Thromb Hemost, 2019;45:523-43). Factor V is an essential protein that participates in coagulation and plays a key role in the so-called blood coagulation cascade due to its procoagulant and anticoagulant activity. 80% of circulating factor V is produced in the liver and the remaining 20% in the alpha granules of platelets. In humans, the gene that expresses this protein (F5) is approximately 80kb in size and is located on chromosome 1q24.2. The coding sequence of the F5 gene is divided into 25 exons and 24 introns and its cDNA is 6914bp in size. So far, approximately 150 point mutations and small insertions and deletions in the gene have been described. Factor V deficiency, an ultramaraderie disease occurring in only 1 to 9 per million, is an autosomal recessive bleeding disorder associated with mutations in the F5 gene. This inherited bleeding disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft tissue bleeding to life-threatening bleeding. Currently there is no palliative or curative treatment for this disease, only the administration of fresh frozen plasma. This alternative is very short-lived (4 hours) and the dosage is very difficult to adjust to the characteristics of the patient, since the concentration of factor V in plasma pools is highly variable depending on the donor (Tabibian S, et al. A Comprehensive OverView of Coagulation Factor V and Congenital Factor V Deficiency. Semin Thromb Hemost, 2019; 45: 523-43).
En la solicitud de patente US2012270708A1 sobre el tratamiento de coagulopatías con hiperfibrinolisis, se describe la utilización de análogos de trombomodulina (nuevas modificaciones de la proteína) para la elaboración de un medicamento con el que tratar las coagulopatías que cursan con hiperfibrinolisis, como los desórdenes relacionados con la hemofilia entre los que incluye la parahemofilia. In the patent application US2012270708A1 on the treatment of coagulopathies with hyperfibrinolysis, the use of thrombomodulin analogues (new modifications of the protein) is described for the preparation of a drug with which to treat coagulopathies that present with hyperfibrinolysis, such as related disorders with hemophilia including parahemophilia.
En W02010069946A1 se describe un método para purificar el factor V a partir de fluidos biológicos, con el interés de utilizarlo en casos de deficiencia en este factor de la coagulación. In W02010069946A1 a method is described to purify factor V from biological fluids, with the interest of using it in cases of deficiency in this coagulation factor.
La solicitud de patente W02008059009A2 se refiere a un método para prevenir y tratar hemorragias en articulaciones, músculos y tejidos blandos en pacientes hemofílicos, mediante la administración de factor V resistente a APC (proteína C activada), que tiene capacidad para inactivar tanto el factor V como el factor VIII de la coagulación, como hemostático. Patent application W02008059009A2 refers to a method to prevent and treat bleeding in joints, muscles and soft tissues in hemophiliac patients, by administering factor V resistant to APC (activated protein C), which has the ability to inactivate both factor V as coagulation factor VIII, as a hemostatic.
W02010060035A1 describe estrategias terapéuticas que utilizan variantes derivadas del factor V presente en el veneno de serpiente, y sus derivados, para modular la cascada de la coagulación en los pacientes que lo precisan, dado que el factor V del veneno de algunas serpientes presenta alrededor de un 44% de homología en su secuencia con la del factor V de mamíferos. W02010060035A1 describes therapeutic strategies that use variants derived from factor V present in snake venom, and its derivatives, to modulate the coagulation cascade in patients who require it, since factor V in the venom of some snakes presents around a 44% sequence homology with that of mammalian factor V.
También se han descrito composiciones y métodos para inhibir la expresión de determinados genes mutantes del factor V, como en la solicitud de patente US2011130443A1 en la que se describe un ARN de doble cadena para inhibir la expresión del gen muíante que provoca una sustitución de G por A en el nucleótido 1691 en el gen del factor V, que provoca un cambio Arg506Gln. Compositions and methods have also been described to inhibit the expression of certain mutant factor V genes, as in patent application US2011130443A1 in which a double-stranded RNA is described to inhibit the expression of the mutant gene that causes a substitution of G by A at nucleotide 1691 in the factor V gene, which causes an Arg506Gln change.
A pesar de las aportaciones citadas, sigue sin existir un tratamiento frente a esta enfermedad, en gran medida, debido a que algunas de las soluciones que se describen no son rentables económicamente al tratarse de una enfermedad ultramara. En cualquier caso, serían tratamientos paliativos y de baja eficacia en deficiencias severas de factor V. Despite the contributions cited, there is still no treatment for this disease, to a large extent, because some of the solutions described are not economically profitable as they are an ultramaraderie disease. In any case, they would be palliative and low-efficacy treatments in severe deficiencies of factor V.
EXPLICACIÓN DE LA INVENCIÓN EXPLANATION OF THE INVENTION
Método in vitro para recuperar la expresión del gen F5 que codifica el factor V de la coagulación. In vitro method to recover the expression of the F5 gene encoding coagulation factor V.
Debido a que actualmente en el mercado farmacológico no existe un tratamiento curativo ni paliativo específico de esta patología, a excepción del tratamiento genérico para diversas coagulopatías como es el plasma fresco congelado, en esta invención se utiliza una metodología génica, basada en la edición génica mediante la herramienta CRISPR/Cas9, para proporcionar herramientas para curar (no paliar) esta patología, actuando de manera específica sobre la mutación que produce ese déficit de factor V de la coagulación. Se trata de un método in vitro que no es un método de tratamiento del cuerpo humano o animal mediante cirugía o terapias realizadas en el cuerpo y tampoco es para modificar la identidad genética de la línea germinal del ser humano. El objetivo es proporcionar las herramientas necesarias que, posteriormente, podrán ser utilizadas para establecer un tratamiento curativo de larga duración o permanente e individualizado para cada paciente. Due to the fact that currently in the pharmacological market there is no specific curative or palliative treatment for this pathology, with the exception of generic treatment for various coagulopathies such as fresh frozen plasma, in this invention a gene methodology is used, based on gene editing by means of the CRISPR / Cas9 tool, to provide tools to cure (not palliate) this pathology, acting specifically on the mutation that produces this factor V deficiency in coagulation. It is an in vitro method that is not a method of treating the human or animal body through surgery or therapies performed on the body and it is not intended to modify the genetic identity of the human germ line. The objective is to provide the necessary tools that, later, can be used to establish a long-term or permanent and individualized curative treatment for each patient.
Un aspecto de la presente invención se refiere a un método in vitro para recuperar la expresión del gen F5 que codifica el factor V de la coagulación mediante la utilización de la metodología CRISPR/Cas9. Este método incluye la utilización de una pareja de guías en la que cada una de las guías tiene una longitud de entre 18 y 22 nucleótidos y ambas están localizadas entre 150 nucleótidos corriente arriba y 150 nucleótidos corriente abajo con respecto a la posición de la mutación localizada en el nucleótido 3279 del gen F5. Preferentemente, se utiliza una pareja de guías caracterizadas por SEQ ID NO: 68-69 o una pareja de guías caracterizadas por SEQ ID NO: 70-71. Mediante la utilización de la metodología CRISPR/Cas9 con cualquiera de estas parejas de guías, se corrige una mutación en el nucleótido 3279 del gen F5 que genera en la secuencia aminoacídica del factor V una mutación sin sentido que da lugar a un codón stop (de parada) (p.Trp1093*). Esta mutación se ha identificado mediante secuenciación del gen F5 de una paciente que presenta una deficiencia severa de factor V; la secuencia obtenida está caracterizada por SEQ ID NO: 65. Otro aspecto de la presente invención se refiere a un cultivo celular in vitro en el que se ha recuperado la expresión del gen F5 que codifica el factor V de la coagulación. En estas células se ha corregido la mutación del gen F5 identificada en el nucleótido 3279 que generaba en la secuencia aminoacídica del factor V una mutación sin sentido que da lugar a un codón stop (de parada) (p.Trp1093*) mediante la utilización de la metodología CRISPR/Cas9 incluyendo la utilización de una pareja de guías, en la que cada una de las guías tiene una longitud de entre 18 y 22 nucleótidos y ambas están localizadas entre 150 nucleótidos corriente arriba y 150 nucleótidos corriente abajo con respecto a la posición de la mutación localizada en el nucleótido 3279 del gen F5. Preferentemente, se utiliza la pareja de guías caracterizadas porSEQ ID NO: 68-69 o la pareja de guías caracterizadas por SEQ ID NO: 70-71. La secuencia completa del gen F5 mutado puede ser la caracterizada por SEQ ID NO: 65. One aspect of the present invention relates to an in vitro method to recover the expression of the F5 gene encoding coagulation factor V by using the CRISPR / Cas9 methodology. This method includes the use of a pair of guides in which each of the guides is between 18 and 22 nucleotides in length and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to the position of the localized mutation. at nucleotide 3279 of the F5 gene. Preferably, a pair of guides characterized by SEQ ID NO: 68-69 or a pair of guides characterized by SEQ ID NO: 70-71 is used. Using the CRISPR / Cas9 methodology with any of these pairs of guides, a mutation is corrected in nucleotide 3279 of the F5 gene that generates a nonsense mutation in the amino acid sequence of factor V that gives rise to a stop codon (from stop) (p.Trp1093 *). This mutation has been identified by sequencing the F5 gene of a patient with severe factor V deficiency; the sequence obtained is characterized by SEQ ID NO: 65. Another aspect of the present invention relates to an in vitro cell culture in which the expression of the F5 gene encoding coagulation factor V has been recovered. In these cells, the mutation of the F5 gene identified at nucleotide 3279 that generated a nonsense mutation in the amino acid sequence of factor V that gives rise to a stop codon (p.Trp1093 *) has been corrected by using the CRISPR / Cas9 methodology including the use of a pair of guides, in which each of the guides is between 18 and 22 nucleotides in length and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to position of the mutation located at nucleotide 3279 of the F5 gene. Preferably, the pair of guides characterized by SEQ ID NO: 68-69 or the pair of guides characterized by SEQ ID NO: 70-71 is used. The complete sequence of the mutated F5 gene can be that characterized by SEQ ID NO: 65.
Otro aspecto de la invención se refiere a un kit que comprende una pareja de guías, en la que cada una de las guías tiene una longitud de entre 18 y 22 nucleótidos y ambas están localizadas entre 150 nucleótidos corriente arriba y 150 nucleótidos corriente abajo con respecto a la posición de la mutación localizada en el nucleótido 3279 del gen F5. Preferentemente, comprende la pareja de guías caracterizadas por SEQ ID NO: 68-69 y/o la pareja de guías caracterizadas por SEQ ID NO: 70-71. Además, este kit puede comprender una proteína Cas9 o un polinucleótido que codifica la proteína Cas9. Another aspect of the invention refers to a kit comprising a pair of guides, in which each of the guides has a length between 18 and 22 nucleotides and both are located between 150 nucleotides upstream and 150 nucleotides downstream with respect to to the position of the mutation located at nucleotide 3279 of the F5 gene. Preferably, it comprises the pair of guides characterized by SEQ ID NO: 68-69 and / or the pair of guides characterized by SEQ ID NO: 70-71. Furthermore, this kit may comprise a Cas9 protein or a polynucleotide encoding the Cas9 protein.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Para complementar la descripción que se está realizando y con objeto de ayudar a una mejor comprensión de las características de la invención, se acompaña como parte integrante de dicha descripción, un juego de dibujos en donde, con carácter ilustrativo y no limitativo, se ha representado lo siguiente: To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description in which, with an illustrative and non-limiting nature, it has been represented the next:
Figura 1. Variantes genéticas patológicas (mutaciones) encontradas en el gen F5 de la paciente. Figura 2. Edición génica de corrección de mutaciones mediante CRISPR/Cas9, según el estado de la técnica. Figure 1. Pathological genetic variants (mutations) found in the patient's F5 gene. Figure 2. Gene editing for correction of mutations using CRISPR / Cas9, according to the state of the art.
FIGURA 3. Diseño de guías de edición génica mediante CRISPR/Cas9, para la obtención del modelo celular mutado y para la corrección de la mutación: FIGURE 3. Design of gene editing guides using CRISPR / Cas9, to obtain the mutated cell model and to correct the mutation:
A) Obtención del modelo celular mutado. A) Obtaining the mutated cell model.
B) Reversión y corrección de la mutación. B) Reversal and correction of the mutation.
REALIZACIÓN PREFERENTE DE LA INVENCIÓN PREFERRED EMBODIMENT OF THE INVENTION
La presente invención se ilustra adicionalmente mediante los siguientes ejemplos, que no pretenden ser limitativos de su alcance. The present invention is further illustrated by the following examples, which are not intended to be limiting of its scope.
Ejemplo 1. Análisis hematológico y de coagulación. Example 1. Hematological and coagulation analysis.
Se tomaron muestras de sangre de una paciente de 10 años y de sus padres. La niña, procedente del sur de España, presenta una deficiencia severa de factor V (FV:C<1%). Los demás factores de la coagulación presentan niveles normales. Al analizar tanto al padre como a la madre de la niña, resultó que ambos tienen deficiencias del factor V de la coagulación. Blood samples were taken from a 10-year-old patient and her parents. The girl, from the south of Spain, presents a severe deficiency of factor V (FV: C <1%). The other clotting factors have normal levels. When analyzing both the father and the mother of the girl, it turned out that both have deficiencies of factor V coagulation.
Para la toma de muestras, se obtuvo con anterioridad la aprobación del Comité Ético del Hospital 12 de Octubre de Madrid y el procedimiento se siguió de acuerdo con la Declaración de Helsinki. For sampling, the approval of the Ethics Committee of the Hospital 12 de Octubre in Madrid was previously obtained and the procedure was followed in accordance with the Declaration of Helsinki.
Las muestras de sangre se distribuyeron en tubos con 0,105M de citrato sódico (1:10). El plasma se obtuvo mediante centrifugación a 2000xg durante 20 minutos y, posteriormente, se almacenó en alícuotas a -80°C hasta su utilización. The blood samples were distributed in tubes with 0.105M sodium citrate (1:10). Plasma was obtained by centrifugation at 2000xg for 20 minutes and subsequently stored in aliquots at -80 ° C until use.
Se analizaron los siguientes parámetros hematológicos: concentración de hemoglobina (Hb), hemoglobina corpuscular media (HCM), concentración de hemoglobina corpuscular media (CHCM), recuento de eritrocitos, hematocrito, volumen corpuscular medio (VCM), recuento de reticulocitos, recuento de leucocitos, recuento de plaquetas, volumen medio plaquetario (VMP), y plaquetocrito. Todos estos parámetros se obtuvieron en un contador celular automático ADVIA®120 Hematology System; Siemens Healthcare GmbH, Zurich, Suiza). The following hematological parameters were analyzed: hemoglobin concentration (Hb), mean corpuscular hemoglobin (HCM), mean corpuscular hemoglobin concentration (MCHC), erythrocyte count, hematocrit, mean corpuscular volume (MCV), reticulocyte count, white blood cell count , platelet count, mean platelet volume (MPV), and platelet count. Everybody These parameters were obtained in an ADVIA®120 Hematology System automatic cell counter; Siemens Healthcare GmbH, Zurich, Switzerland).
Las muestras de sangre para el análisis de la función plaquetaria y de los tiempos de coagulación se tomaron en tubos para la extracción de sangre por vacío (Vacutainer, Becton Dickinson) con un 3,8% de citrato sódico y se procesaron en un Platelet Function Analyzer-100 (PFA-100®; Siemens Healthcare Diagnostics AG, Zurich, Suiza). Para determinar el tiempo de sangrado y la agregación plaquetaria se utilizaron cartuchos de colágeno/adenosina-5-difosfato (ADP) (Dade® PFA, Siemens Healthcare Diagnostics, AG, Zurich, Suiza). Los cartuchos se utilizaron a temperatura ambiente añadiéndoles 1 mL de sangre tratada con citrato. Las muestras se aspiraron en vacío constante a través de un capilar y una abertura microscópica practicada en una membrana recubierta de colágeno y ADP. Blood samples for analysis of platelet function and clotting times were taken in vacuum blood collection tubes (Vacutainer, Becton Dickinson) with 3.8% sodium citrate and processed on a Function Platelet Analyzer-100 (PFA-100®; Siemens Healthcare Diagnostics AG, Zurich, Switzerland). Collagen / adenosine-5-diphosphate (ADP) cartridges (Dade® PFA, Siemens Healthcare Diagnostics, AG, Zurich, Switzerland) were used to determine bleeding time and platelet aggregation. The cartridges were used at room temperature by adding 1 mL of citrated blood to them. The samples were aspirated under constant vacuum through a capillary and a microscopic opening made in a membrane coated with collagen and ADP.
Todos los parámetros relacionados con la hemostasia, como el tiempo de protrombina (TP), la actividad de protrombina (AP), el tiempo de tromboplastina parcial activada (aTPP) (tiempo de cefalina), el fibrinógeno (F) y el índice normalizado internacional (INI), se determinaron de conformidad con las normas definidas por el Departamento de Hematología del Hospital Universitario de Jaén, España. Se utilizaron los kits HemosIL® (Instrumentation Laboratory; Bedford, Massachusetts, EE.UU.), según las instrucciones del fabricante. All parameters related to hemostasis, such as prothrombin time (PT), prothrombin activity (AP), activated partial thromboplastin time (aTPP) (cephalin time), fibrinogen (F), and the international normalized index (INI), were determined in accordance with the standards defined by the Department of Hematology of the Hospital Universitario de Jaén, Spain. The HemosIL® kits (Instrumentation Laboratory; Bedford, Massachusetts, USA) were used, according to the manufacturer's instructions.
Se utilizó HemosIL® RecombiPlasTin 2G para medir el tiempo de protrombina (TP) y los niveles de fibrinógeno y se utilizó un reactivo de tromboplastina de alta sensibilidad, basado en el factor tisular humano recombinante (rhFT). Tras reconstituir los diluyentes de RecombiPlasTin 2G, el reactivo PT incluido en el kit RecombiPlasTin 2G se convierte en una preparación liposomal que contiene rhFT en una mezcla de fosfolípidos sintéticos y combinado con cloruro de calcio, tampón y un conservante. Como el rhFT no contiene ningún factor de coagulación contaminante, RecombiPlasTin 2G es muy sensible a los factores de coagulación de vías extrínsecas, lo que lo hace especialmente adecuado para los ensayos con esos factores. Por otro lado, la formulación de RecombiPlasTin 2G hace que sea insensible a los niveles de heparina terapéutica. En la prueba de TP, la adición del reactivo al plasma del paciente en presencia de iones de calcio activa la vía de coagulación extrínseca. Esto eventualmente resulta en la conversión de fibrinógeno en fibrina, con la formación de un gel sólido. El fibrinógeno se cuantificó relacionando la absorbancia, o dispersión de la luz, durante la formación del coágulo, con un calibrador. Los resultados del TP se indican en segundos, porcentaje de actividad, o INI; los niveles de fibrinógeno, en g/L. HemosIL® RecombiPlasTin 2G was used to measure prothrombin time (PT) and fibrinogen levels and a highly sensitive thromboplastin reagent, based on recombinant human tissue factor (rhFT), was used. After reconstituting the RecombiPlasTin 2G diluents, the PT reagent included in the RecombiPlasTin 2G kit is converted into a liposomal preparation containing rhFT in a mixture of synthetic phospholipids and combined with calcium chloride, buffer and a preservative. As rhFT does not contain any contaminating clotting factors, RecombiPlasTin 2G is highly sensitive to extrinsic pathway clotting factors, making it particularly suitable for assays with these factors. On the other hand, RecombiPlasTin 2G's formulation makes it insensitive to therapeutic heparin levels. In the PT test, the addition of the reagent to the patient's plasma in the presence of calcium ions activates the clotting pathway. extrinsic. This eventually results in the conversion of fibrinogen to fibrin, with the formation of a solid gel. Fibrinogen was quantified by relating the absorbance, or scattering of light, during clot formation, with a calibrator. TP results are reported in seconds, percent activity, or INI; fibrinogen levels, in g / L.
Para determinar el valor de aTPP se utilizó HemosIL® APTT-SP (líquido). El test de aTPP utiliza un activador de contacto que estimula la producción de factor Xlla. El activador es una dispersión de sílice coloidal con fosfolípidos sintéticos, un tampón y un conservante y proporciona una superficie de contacto para la interacción del kininógeno de alto peso molecular, la calicreína y el factor Xlla. La activación por contacto se produce a 37°C durante un período de tiempo determinado. La adición de cloruro de calcio 0,025 M con un conservante desencadena una serie de reacciones que darán lugar a la formación de coágulos. También son necesarios fosfolípidos para generar los compuestos que actuarán sobre el factor X y la protrombina. Los resultados se expresan en segundos. HemosIL® APTT-SP (liquid) was used to determine the aTPP value. The aTPP test uses a contact activator that stimulates the production of factor Xlla. The activator is a colloidal silica dispersion with synthetic phospholipids, a buffer and a preservative and provides a contact surface for the interaction of high molecular weight kininogen, kallikrein and factor Xlla. Contact activation occurs at 37 ° C for a specified period of time. The addition of 0.025 M calcium chloride with a preservative triggers a series of reactions that will lead to the formation of clots. Phospholipids are also required to generate the compounds that will act on factor X and prothrombin. The results are expressed in seconds.
Se utilizó plasma deficiente de HemosIL® FV para la determinación cuantitativa de la actividad de factor V. La actividad de factor V en el plasma se determinó utilizando la prueba de tiempo de protrombina modificada realizada en el plasma diluido con citrato del paciente en presencia de plasma humano inmunodeplecionado con factor V. La corrección del tiempo de coagulación prolongado para el plasma deficiente fue proporcional a la concentración (porcentaje de actividad) del factor específico (factor V) en el plasma del paciente, que puede obtenerse trazando una curva de calibración. Los niveles de referencia del factor V oscilaban entre el 60-130% (0,60-1 ,30 IU). HemosIL® FV deficient plasma was used for quantitative determination of factor V activity. Factor V activity in plasma was determined using the modified prothrombin time test performed on the patient's citrate-diluted plasma in the presence of plasma. human immunodepleted with factor V. The correction for prolonged clotting time for deficient plasma was proportional to the concentration (percentage of activity) of the specific factor (factor V) in the patient's plasma, which can be obtained by drawing a calibration curve. Reference levels of factor V ranged from 60-130% (0.60-1.30 IU).
Tanto en la niña como en sus progenitores, todos los parámetros hematológicos, incluyendo la concentración de hemoglobina (Hb), la hemoglobina corpuscular media (HCM), la concentración de hemoglobina corpuscular media (CHCM), el recuento total de eritrocitos (RBC), el hematocritos (HCT), el volumen corpuscular medio (VCM), los reticulocitos (RETIC), el recuento de leucocitos (GB), el recuento diferencial de GB, el recuento de plaquetas (PLT), el volumen medio de plaquetas (VMP), y el plaquetocrito (PCT), estaban dentro de los rangos de referencia. En el análisis de coagulación, la niña mostró valores alterados tanto para las vías de coagulación extrínsecas como para las intrínsecas (tiempo de protrombina (TP), la actividad de protrombina (AP), tiempo de tromboplastina parcial activada (aTPP) (tiempo de cefalina), fibrinógeno (F), índice normalizado internacional (INI), y actividad de factor V. La paciente presenta una deficiencia grave de factor V con una actividad de factor V inferior al 1% con respecto al valor de referencia. En sus padres, las alteraciones de los parámetros de coagulación y la actividad del factor V estuvieron en consonancia con el nivel de afectación del alelo del gen F5 y el estado heterocigoto que presentan. In both the girl and her parents, all hematological parameters, including hemoglobin concentration (Hb), mean corpuscular hemoglobin (HCM), mean corpuscular hemoglobin concentration (MCHC), total erythrocyte count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), reticulocytes (RETIC), leukocyte count (GB), differential GB count, platelet count (PLT), mean platelet volume (VMP) , and the platelet (PCT), were within the reference ranges. In the coagulation analysis, the girl showed altered values for both extrinsic and intrinsic coagulation pathways (prothrombin time (PT), prothrombin activity (AP), activated partial thromboplastin time (aTPP) (cephalin time) ), fibrinogen (F), international normalized index (INI), and factor V activity. The patient has a severe factor V deficiency with a factor V activity less than 1% of the reference value. In her parents, alterations in coagulation parameters and factor V activity were consistent with the level of involvement of the allele of the F5 gene and their heterozygous status.
Ejemplo 2. Extracción de ADN. Example 2. DNA extraction.
Tras el consentimiento informado, se recogieron muestras de sangre anticoagulada con EDTA. El ADN genómico se extrajo automáticamente de los leucocitos periféricos aislados previamente, mediante procedimientos de precipitación salina utilizando la instrumentación de Autopure (Qiagen). Mediante este kit se obtiene DNA genómico listo para su uso de forma simple y rápida a partir de muestras de 200pL de sangre entera. Se basa en una centrifugación a vacío y no es necesaria la previa separación de los leucocitos. No se requiere ni la extracción con fenol/cloroformo ni la precipitación con alcohol. El ADN purificado para su uso en PCR se conserva entre -25 °C y -15 °C. Following informed consent, EDTA anticoagulated blood samples were collected. Genomic DNA was automatically extracted from previously isolated peripheral leukocytes, by saline precipitation procedures using Autopure instrumentation (Qiagen). Using this kit, ready-to-use genomic DNA is obtained simply and quickly from 200pL whole blood samples. It is based on vacuum centrifugation and the prior separation of leukocytes is not necessary. Neither phenol / chloroform extraction nor alcohol precipitation is required. DNA purified for use in PCR is stored between -25 ° C and -15 ° C.
Ejemplo 3. Análisis de las mutaciones. Example 3. Analysis of mutations.
Se diseñaron cebadores correspondientes a la secuencia completa que codifica el gen F5 y las regiones adyacentes intrónicas, según la entrada NC_000001.11 (169511951-169586630, complementaria) de la base de datos del NCBI, para obtener productos de PCR (reacción en cadena de la polimerasa) de un tamaño de entre 300 y 600 pb, aproximadamente (Tabla 1). Primers corresponding to the complete sequence encoding the F5 gene and the adjacent intronic regions were designed, according to entry NC_000001.11 (169511951-169586630, complementary) of the NCBI database, to obtain PCR products (chain reaction of polymerase) with a size of between 300 and 600 bp, approximately (Table 1).
Tabla 1. Referencia de los cebadores y tamaño de los fragmentos obtenidos para la secuenciación completa del gen F5 de la paciente en estudio.
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
Table 1. Reference of the primers and size of the fragments obtained for the complete sequencing of the F5 gene of the patient under study.
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
La amplificación por PCR se realizó en un volumen final de 25pL que contenía 1,5mM-2,5mM MgCL (dependiendo del fragmento que se quería amplificar), 200mM de cada dNTP, 0,2mM de cada cebador, 0,5 unidades de Taq ADN polimerasa (Ecogen) en el tampón recomendado por el fabricante y 150-200ng de ADN genómico. Las condiciones del termociclador fueron las siguientes: 94°C durante 5 minutos, seguidos de 30 ciclos a 94°C durante 30 segundos (paso de desnaturalización); 58°C-60°C (dependiendo del fragmento que se deseaba amplificar) durante 30 segundos y 72°C durante 2 minutos (paso de elongación), seguidos de un paso de extensión final de 20 minutos a 72°C. Cuando se completó la amplificación por PCR, se eliminaron los dNTP y los cebadores sobrantes que quedaban en la mezcla de productos de la PCR mediante la utilización de las enzimas de digestión ExoSAP (exonucleasa I y fosfatasa alcalina) (Sigma-Aldrich). Una vez completado el procedimiento de purificación, la reacción de secuenciación bidireccional de la secuencia completa se realizó por el método Sanger utilizando el kit BigDye™ Terminator v1.1 Cycle Sequencing. Las reacciones de secuenciación de los fragmentos amplificados se purificaron mediante el kit de montaje SEQ96 en un colector de vacío, para retirar los terminadores marcados no incorporados y las sales antes de someter a los productos de secuenciación a electroforesis capilar en un analizador genético ABI Prism 3500 (Applied Biosystems). Los cromatogramas de las secuencias se analizaron utilizando SeqScape v.2.1.1. PCR amplification was carried out in a final volume of 25pL containing 1.5mM-2.5mM MgCL (depending on the fragment to be amplified), 200mM of each dNTP, 0.2mM of each primer, 0.5 Taq units DNA polymerase (Ecogen) in manufacturer's recommended buffer and 150-200ng of genomic DNA. Thermal cycler conditions were as follows: 94 ° C for 5 minutes, followed by 30 cycles at 94 ° C for 30 seconds (denaturation step); 58 ° C-60 ° C (depending on the fragment to be amplified) for 30 seconds and 72 ° C for 2 minutes (elongation step), followed by a final extension step of 20 minutes at 72 ° C. When the PCR amplification was complete, the remaining dNTPs and primers remaining in the PCR product mix were removed by using the ExoSAP digestion enzymes (exonuclease I and alkaline phosphatase) (Sigma-Aldrich). One time Once the purification procedure was completed, the bidirectional sequencing reaction of the complete sequence was performed by the Sanger method using the BigDye ™ Terminator v1.1 Cycle Sequencing kit. The sequencing reactions of the amplified fragments were purified using the SEQ96 assembly kit on a vacuum manifold, to remove unincorporated labeled terminators and salts before subjecting the sequencing products to capillary electrophoresis on an ABI Prism 3500 Genetic Analyzer. (Applied Biosystems). The chromatograms of the sequences were analyzed using SeqScape v.2.1.1.
Para describir las mutaciones y las variantes genéticas se utilizó la nomenclatura de la Human Genome Variation Society (HGVS) (den Dunnen JT, y col. HGVS recommendations for the description of sequence variants. 2016 update. Hum Mutation, 2016;37:564-9). Todos los cambios de nucleótidos identificados en la paciente con deficiencia de factor V se analizaron mediante el programa informático Alamut visual v.2.6. software, que integra la información genética y genómica de diferentes fuentes en un entorno coherente y conveniente para describir las variantes utilizando la nomenclatura de la HGVS y ayudar a interpretar su estado patogénico. Además, todos los cambios detectados en la niña se estudiaron en la madre y en el padre para determinar la segregación familiar. To describe mutations and genetic variants, the Human Genome Variation Society (HGVS) nomenclature (den Dunnen JT, et al. HGVS recommendations for the description of sequence variants. 2016 update. Hum Mutation, 2016; 37: 564-) was used. 9). All nucleotide changes identified in the patient with factor V deficiency were analyzed using the Alamut visual software v.2.6. software, which integrates genetic and genomic information from different sources into a consistent and convenient environment to describe variants using the HGVS nomenclature and help interpret their pathogenic status. In addition, all the changes detected in the girl were studied in the mother and in the father to determine family segregation.
A todas las mutaciones deletéreas y a las variantes genéticas se les asignó un número de nucleótido a partir de la primera base traducida del gen F5 según la secuencia de referencia NM_000130.4 de la base de datos NCBI. All deleterious mutations and genetic variants were assigned a nucleotide number from the first translated base of the F5 gene according to the reference sequence NM_000130.4 from the NCBI database.
En el análisis de las mutaciones del gen F5 de la paciente, se identificaron un total de 24 variantes de secuencias diferentes, entre ellas 2 mutaciones sin sentido que dan lugar a un codón stop, 1 mutación por desplazamiento del marco de lectura, 6 mutaciones que dan lugar a cambio de aminoácido, 9 variaciones sinónimas (sin cambio de traducción) y 7 cambios en intrones (Tabla 2 y Figura 1). In the analysis of the F5 gene mutations of the patient, a total of 24 different sequence variants were identified, including 2 nonsense mutations that give rise to a stop codon, 1 reading frame shift mutation, 6 mutations that give rise to amino acid change, 9 synonymous variations (without translation change) and 7 changes in introns (Table 2 and Figure 1).
En la Figura 1, se muestran las dos variantes (mutaciones) patogénicas encontradas en el gen F5 de la paciente con deficiencia severa de factor V. Son dos mutaciones sin sentido que dan lugar a dos codones de parada (stop codon) (CS). Una de ellas, la NM_000130.4: c. 3279G>A, p.Trp1093*, que implica un cambio de una guanina a una adenina (G>A), y la otra, la NM_000130.4: c. 2218C>T, p.Arg740*, ya descrita previamente, que implica un cambio de una citosina a una timina (OT). Se indican las siguientes características: Msn: Mutación sin sentido; dominios (D) (A1, A2, B, A3, C1 y C2); los exones (E), la cadena pesada (He), la cadena ligera (Le) y la región de división postraduccional (ps); sitio de inactivación por proteína C activada (APCC); sitio de activación por trombina (TC). In Figure 1, the two pathogenic variants (mutations) found in the F5 gene of the patient with severe factor V deficiency are shown. They are two nonsense mutations that give rise to two stop codons (CS). One of them, NM_000130.4: c. 3279G> A, p.Trp1093 *, which implies a change from a guanine to one adenine (G> A), and the other, NM_000130.4: c. 2218C> T, p.Arg740 *, already described previously, which involves a change from a cytosine to a thymine (OT). The following characteristics are indicated: Msn: Nonsense mutation; (D) domains (A1, A2, B, A3, C1 and C2); exons (E), heavy chain (He), light chain (Le) and post-translational cleavage region (ps); activated protein C inactivation site (APCC); thrombin activation site (TC).
Tabla 2. Variantes genéticas identificadas en el gen F5 de la paciente.
Figure imgf000014_0001
Table 2. Genetic variants identified in the patient's F5 gene.
Figure imgf000014_0001
*No aplicable. **Datos no disponibles. *Not applicable. ** Data not available.
De los 24 cambios, 17 tienen una frecuencia alélica pequeña (FAM) ³ 5% a la descrita en la base de datos de 1000 genomas (5 sin sentido, 7 sinónimos y 5 variantes intrónicas) (Tabla 2). Los siete cambios restantes no tienen valor FAM porque son raras variantes de secuencias que no están descritas previamente. Of the 24 changes, 17 have a small allelic frequency (FAM) ³ 5% to that described in the database of 1000 genomes (5 nonsense, 7 synonyms and 5 intronic variants) (Table 2). The remaining seven changes have no FAM value because they are rare sequence variants that are not previously described.
Además, también detectamos una correcta segregación familiar de los cambios identificados en la paciente con sus progenitores. En la niña, las dos mutaciones sin sentido (p.Arg740* y p.Trp1093*) están ambas en estado heterocigótico, una mutación la hereda de su madre (NM_000130.4: c. 22180T, p.Arg740*) y la otra la hereda de su padre (NM_000130.4: c. 3279G>A, p.Trp1093*). In addition, we also detected a correct family segregation of the changes identified in the patient with her parents. In the girl, the two mutations without sense (p.Arg740 * and p.Trp1093 *) are both in the heterozygous state, one mutation is inherited from their mother (NM_000130.4: c. 22180T, p.Arg740 *) and the other is inherited from their father (NM_000130. 4: c. 3279G> A, p.Trp1093 *).
El tipo de mutación se correlacionó con los niveles de actividad del factor V en la paciente y en sus progenitores. La mutación c.3279G>A en el padre se relaciona con una actividad del 21% y la mutación 0.2218OT en la madre, con una actividad de factor V del 62,9%. The type of mutation was correlated with the levels of factor V activity in the patient and in her parents. The c.3279G> A mutation in the father is associated with an activity of 21% and the 0.2218OT mutation in the mother, with a factor V activity of 62.9%.
De todas las variantes (mutaciones) encontradas en el gen F5 de la paciente con deficiencia severa de factor V, dos se consideran mutaciones sin sentido patogénicas porque cumplen los criterios de patogenicidad (el propio carácter patógeno de un codón de parada). De ellas, la NM_000130.4: c. 3279G>A, p.Trp1093* es nueva puesto que no está descrita hasta el momento, mientras que la otra, NM_000130.4: c. 2218C>T, p.Arg740*, ya ha sido descrita previamente (Lunghi B, y col. A Novel Factor V Nuil Mutation Detected in a Thrombophilic Patient With Pseudo- Homozygous APC Resistance and in an Asymptomatic Unrelated Subject. Blood, 1998;92:1463-5). Of all the variants (mutations) found in the F5 gene of the patient with severe factor V deficiency, two are considered pathogenic missense mutations because they meet the criteria for pathogenicity (the very pathogenicity of a stop codon). Of these, NM_000130.4: c. 3279G> A, p.Trp1093 * is new since it is not described so far, while the other one, NM_000130.4: c. 2218C> T, p.Arg740 *, has already been described previously (Lunghi B, et al. A Novel Factor V Nuil Mutation Detected in a Thrombophilic Patient With Pseudo-Homozygous APC Resistance and in an Asymptomatic Unrelated Subject. Blood, 1998; 92 : 1463-5).
Ejemplo 4. Edición génica. Example 4. Gene editing.
Una vez conocida la mutación específica de la paciente en estudio, se aplicó la edición génica (corrección de la mutación) mediante la técnica CRISPR/Cas9 (Figura 2) en un modelo celular diseñado en el laboratorio que portaba la misma mutación de la paciente. Once the specific mutation of the patient under study was known, gene editing (correction of the mutation) was applied using the CRISPR / Cas9 technique (Figure 2) in a cell model designed in the laboratory that carried the same mutation of the patient.
La tecnología CRISPR/Cas9 (repeticiones palindrómicas cortas agrupadas e intercaladas regularmente) representa una mejora significativa con respecto a otras herramientas de edición génica de nueva generación. El sistema CRISPR/Cas9 permite la focalización genómica específica de un sitio en el genoma en prácticamente cualquier organismo. CRISPR / Cas9 (Regularly Clustered and Interleaved Short Palindromic Repeats) technology represents a significant improvement over other next-generation gene editing tools. The CRISPR / Cas9 system enables site-specific genomic targeting in the genome in virtually any organism.
La figura 2 muestra el estado de la técnica: la diana para CRISPR/cas9 son pequeñas secuencias de ADN, localizadas alrededor de la mutación (MUT X -roja-), la cual expresa una proteína no funcional (PPT X -roja-). Se trata de una estrategia de terapia génica ya que se precisa introducir las guías (en amarillo) complementarias, que confinan la mutación y mediante una pequeña deleción en el DNA genómico en el interior del núcleo de la célula, corrigen el marco de lectura. La proteína funcional se indica como PPT V -verde-. Figure 2 shows the state of the art: the target for CRISPR / cas9 are small DNA sequences, located around the mutation (MUT X -red-), which expresses a non-functional protein (PPT X -red-). It is a strategy of Gene therapy since it is necessary to introduce the complementary guides (in yellow), which confine the mutation and, by means of a small deletion in the genomic DNA inside the nucleus of the cell, correct the reading frame. The functional protein is indicated as PPT V -green-.
El sistema CRISPR/Cas tipo II es un sistema de respuesta inmune adaptativa procariótica que utiliza ARN no codificante para guiar la nucleación de Cas9 para inducir la división del ADN en un sitio específico. Este daño al ADN se repara mediante mecanismos de reparación celular del ADN, ya sea a través de la vía de reparación del ADN homologa o no homologa. The CRISPR / Cas type II system is a prokaryotic adaptive immune response system that uses antisense RNA to guide the nucleation of Cas9 to induce DNA cleavage at a specific site. This DNA damage is repaired by cellular DNA repair mechanisms, either through the homologous or non-homologous DNA repair pathway.
El sistema CRISPR/Cas9 se ha aprovechado para crear un método simple y programable de ARN para mediar la edición del genoma en las células de mamíferos, y puede ser usado para generar modelos knockout (KO) (inserción/eliminación). Para crear alteraciones genéticas, se genera un único ARN guía (sgARN) para dirigir la nucleasa Cas9 a una ubicación genómica específica. Las roturas de doble cadena inducidas por Cas9 se reparan a través de la vía de reparación del ADN. The CRISPR / Cas9 system has been leveraged to create a simple, programmable RNA method to mediate genome editing in mammalian cells, and can be used to generate knockout (KO) (insertion / deletion) models. To create genetic alterations, a single guide RNA (sgRNA) is generated to direct the Cas9 nuclease to a specific genomic location. Cas9-induced double strand breaks are repaired via the DNA repair pathway.
Ejemplo 5. Criterios para el diseño de guías específicas para CRISPR/Cas9. Example 5. Criteria for the design of specific guidelines for CRISPR / Cas9.
Las guías específicas necesarias para la obtención del modelo celular mutado y para la corrección de la mutación (Figura 3) se diseñaron en función de los mapas genéticos de factor V humano, elaborados gracias a programas informáticos que nos muestran información relevante del gen, así como la secuencia genética completa en forma de ADNc (codificante sin intrones). Para diseñar con la mayor precisión posible las guías de edición génica se utilizó la aplicación web http://crispor.tefor.net/. Los criterios de selección se basaron en la longitud de secuencia que se elimina, el “score”, la especificidad y los posibles “off-target”. Tanto las guías diseñadas como la nucleasa Cas 9 se adquirieron de la casa comercial IDT (USA). The specific guidelines necessary for obtaining the mutated cell model and for correcting the mutation (Figure 3) were designed based on the genetic maps of human factor V, prepared thanks to computer programs that show us relevant information about the gene, as well as the complete genetic sequence in the form of cDNA (coding without introns). To design the gene editing guides as accurately as possible, the web application http://crispor.tefor.net/ was used. The selection criteria were based on the length of the sequence to be eliminated, the "score", the specificity and the possible "off-target". Both the designed guides and the Cas 9 nuclease were purchased from IDT (USA).
Ejemplo 6. Obtención del modelo celular y corrección de la mutación. Example 6. Obtaining the cell model and correction of the mutation.
Tanto para la obtención del modelo celular como para la eliminación de la mutación (Figura 3) se utilizó la línea celular HepG2, cedida por el Dr. José Carlos Segovia del Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas. Las células HepG2 pertenecen a una línea de células inmortales que se derivan de carcinoma hepatocelular bien diferenciado. Estas células son epiteliales en morfología, tienen un número de cromosomas modal de 55 y no son tumorogénicas en ratones. Secretan factor V además de una amplia variedad de proteínas importantes, por ejemplo, albúmina, y las proteínas de fase aguda fibrinógeno, alfa 2-macroglobulina, alfa 1 -antitripsina, transferrina y plasminógeno. Both to obtain the cell model and to eliminate the mutation (Figure 3), the HepG2 cell line was used, provided by Dr. José Carlos Segovia of the Center for Energy, Environmental and Technological Research. HepG2 cells belong to a line of immortal cells that are derived from well-differentiated hepatocellular carcinoma. These cells are epithelial in morphology, have a modal chromosome number of 55, and are not tumorigenic in mice. They secrete factor V in addition to a wide variety of important proteins, eg, albumin, and the acute phase proteins fibrinogen, alpha 2-macroglobulin, alpha 1 -antitrypsin, transferrin, and plasminogen.
Para el desarrollo del modelo celular KO para factor V se usaron las siguientes guías de edición: The following editing guides were used for the development of the KO cell model for factor V:
5 '-CAAT CAG ACATTGCCCT CTA-3 ' (G1) (SEQ ID NO: 66) 5'-GAGGAATTCTGATTATGGTC-3' (G2) (SEQ ID NO: 67) 5 ' -CAAT CAG ACATTGCCCT CTA-3 ' (G1) (SEQ ID NO: 66) 5 ' -GAGGAATTCTGATTATGGTC-3 ' (G2) (SEQ ID NO: 67)
En el caso de las guías usadas para la corrección de la mutación en el modelo KO, se usaron 2 parejas de guías para comparar la efectividad de edición de cada pareja de guías siendo estas: In the case of the guides used for the correction of the mutation in the KO model, 2 pairs of guides were used to compare the editing effectiveness of each pair of guides, these being:
Pareja 1 Couple 1
5 '-AAG AAG ACTT AAGCATTCGT -3 ' (G3) (SEQ ID NO: 68) 5 ' -AAG AAG ACTT AAGCATTCGT -3 ' (G3) (SEQ ID NO: 68)
5 '-TGCCT G ACCAGT GT C ATTT G-3 ' (G4) (SEQ ID NO: 69) 5 ' -TGCCT G ACCAGT GT C ATTT G-3 ' (G4) (SEQ ID NO: 69)
Pareja 2 Couple 2
5 '-TTCCT C AAAT G ACACT GGTC-3 ' (G5) (SEQ ID NO: 70) 5 ' -TTCCT C AAAT G ACACT GGTC-3 ' (G5) (SEQ ID NO: 70)
5 '-CAAT GTCT G ATT G AGGTCT G-3 ' (G6) (SEQ ID NO: 71) 5 ' -CAAT GTCT G ATT G AGGTCT G-3 ' (G6) (SEQ ID NO: 71)
La estrategia para seguir es similar a la de crear el modelo KO, pero en este caso restaurando el marco de lectura correcto del gen, y como consecuencia, la producción de factor V funcional. The strategy to follow is similar to that of creating the KO model, but in this case restoring the correct reading frame of the gene, and as a consequence, the production of functional factor V.
En la Figura 3 se indica: Figure 3 indicates:
• BD: Dominio B. E13: Exón 13. ps: Región de división postraduccional. • BD: Domain B. E13: Exon 13. ps: Post-translational division region.
• PAM: Motivo adyacente protoespaciador (del inglés protospacer adjacent motif). Es una secuencia de ADN de 3 pares de bases inmediatamente después de la secuencia de ADN reconocida por la nucleasa Cas9. • Del 35 bp: Deleción (eliminación) de una secuencia de 35 pares de bases, región que corresponde a la zona mutada en la paciente con un codón stop (CS, G>A, cambio de una guanina a una adenina). La alteración de esta zona mediante las guías G1 y G2 complementarias a la mutación, reproducen la mutación en el modelo. • PAM: protospacer adjacent motif. It is a 3 base pair DNA sequence immediately after the DNA sequence recognized by the Cas9 nuclease. • Del 35 bp: Deletion (elimination) of a sequence of 35 base pairs, a region that corresponds to the mutated area in the patient with a stop codon (CS, G> A, change from a guanine to an adenine). The alteration of this area by means of the G1 and G2 guides complementary to the mutation, reproduce the mutation in the model.
• Del 82 bp: Deleción (eliminación) de una secuencia de 82 pares de bases, que se complementa con las guías G3 y G4 para dar una secuencia no patológica del gen. • Del 82 bp: Deletion (deletion) of an 82 base pair sequence, which is complemented by the G3 and G4 guidelines to give a non-pathological sequence of the gene.
• Del 49 bp: Deleción (eliminación) de una secuencia de 49 pares de bases, que se complementa con las guías G5 y G6 para dar una secuencia no patológica del gen. • Del 49 bp: Deletion (deletion) of a sequence of 49 base pairs, which is complemented by the G5 and G6 guides to give a non-pathological sequence of the gene.
La metodología se basó en introducir las guías y la nucleasa Cas9 en el interior de la célula para que después alcancen el núcleo donde se internalizan. Para ello se utilizó el método de producción de una ribonucleoproteína (RNP), formada a partir de las guías y la nucleasa Cas9. La obtención de este complejo se llevó a cabo mediante un protocolo termociclador basado en incubar los 2 elementos de la guía (crRNA y tracrRNA) a 95°C, 5 min y adición posterior de la Cas 9, incubando 2 minutos a temperatura ambiente. Por último, se introdujo esta RNP mediante un 4D- Nucleofector™ System (Lonza, Switzerland) y el kit de nucleofección “SF line cells” (Lonza, Switzerland) siguiendo el protocolo de la casa comercial para realizar la nucleofección en dicha línea celular. Este procedimiento tiene menos toxicidad celular, mejor internalización en núcleo, mayor eficacia de editado, menos tiempo de producción y es más económico que otros. The methodology was based on introducing the guides and the Cas9 nuclease inside the cell so that they later reach the nucleus where they are internalized. For this, the method of producing a ribonucleoprotein (RNP), formed from the guides and the Cas9 nuclease, was used. Obtaining this complex was carried out using a thermocycler protocol based on incubating the 2 elements of the guide (crRNA and tracrRNA) at 95 ° C, 5 min and subsequent addition of Cas 9, incubating 2 minutes at room temperature. Finally, this RNP was introduced by means of a 4D-Nucleofector ™ System (Lonza, Switzerland) and the nucleofection kit "SF line cells" (Lonza, Switzerland) following the protocol of the commercial company to carry out the nucleofection in said cell line. This procedure has less cellular toxicity, better internalization in the nucleus, greater editing efficiency, less production time, and is cheaper than others.
Ejemplo 7. Confirmación del modelo y de la corrección de la mutación. Example 7. Confirmation of the pattern and correction of the mutation.
Para confirmar el modelo celular mutado tras CRISPR/Cas9, se estudió la proliferación, morfología y cariotipo de las células HepG2. Después se llevó a cabo una citometría de flujo con marcadores específicos de membrana de las células HepG2. Y, por último, se secuenció el ADNc para factor V en las células HepG2. To confirm the cell model mutated after CRISPR / Cas9, the proliferation, morphology and karyotype of HepG2 cells were studied. Flow cytometry was then carried out with HepG2 cell membrane specific markers. And finally, the cDNA for factor V was sequenced in HepG2 cells.
En este caso no se observaron diferencias significativas entre las líneas HepG2 nativas y KO, producidas por edición génica, con respecto a datos de proliferación, morfología y expresión de marcadores de membrana, comprobados por inmunocitometría de flujo, lo que indica que las guías diseñadas y la edición no afectan, al menos, a genes relacionados con el ciclo celular y/o proliferación, estructura celular ni a proteínas de membrana, indicando que la edición génica puede ser una alternativa por sus escasos efectos inespecíficos sobre otros genes, en estudios in vitro. En cuanto a la secuenciación del ADN, la eficacia de editado para producir el modelo KO, con las guías y protocolos de edición génica utilizados, rondaba el 60% de edición precisa, dando lugar a un cultivo celular en el cual el 60% de las células eran KO para el gen de factor V. Posteriormente, y para obtener una línea celular pura se realizó un crecimiento en colonias para lograr un cultivo celular donde el 100% de las células tuviera la mutación derivada de la edición y por tanto fueran KO para el gen de factor V. In this case, no significant differences were observed between the native HepG2 and KO lines, produced by gene editing, with respect to proliferation data, morphology and expression of membrane markers, verified by flow immunocytometry, which indicates that the designed guides and editing do not affect, at least, genes related to the cell cycle and / or proliferation, cell structure or membrane proteins, indicating that gene editing may be an alternative due to its limited non-specific effects on other genes, in in vitro studies. Regarding DNA sequencing, the efficiency of editing to produce the KO model, with the guidelines and gene editing protocols used, was around 60% accurate editing, giving rise to a cell culture in which 60% of the The cells were KO for the factor V gene. Subsequently, and to obtain a pure cell line, a growth in colonies was performed to achieve a cell culture where 100% of the cells had the mutation derived from the edition and therefore were KO for the factor V gene.
Para confirmar la corrección de la mutación tras CRISPR/Cas9, se llevó a cabo de nuevo una citometría de flujo y se secuenció el ADNc para factor V de las líneas celulares para comprobar la eficacia del editado y la corrección (tratamiento) de la mutación. To confirm the correction of the mutation after CRISPR / Cas9, flow cytometry was again carried out and the factor V cDNA of the cell lines was sequenced to check the efficacy of editing and the correction (treatment) of the mutation.
En este caso se obtuvo una precisión de editado diferente entre las distintas parejas de guías de edición génica, a saber, con la pareja 1 la eficacia de editado fue de aproximadamente el 40%, mientras que con la pareja 2, fue del 30%. Aun siendo eficacias más bajas que en el caso anterior del modelo KO, un tratamiento basado en células con una eficacia de corrección del 30-40% supone el paso de un estado o fenotipo grave de la enfermedad a un estado leve o incluso asintomático. La bajada de la eficacia en la corrección de la línea celular KO también puede ser debido a que esa zona del ADN ha sufrido previamente otra edición génica (para desarrollar el modelo KO) lo que puede hacer que el sistema CRISPR pierda eficacia, al editar sobre una zona previamente editada. In this case, a different editing precision was obtained between the different pairs of gene editing guides, namely, with pair 1 the editing efficiency was approximately 40%, while with pair 2, it was 30%. Even though the efficiencies are lower than in the previous case of the KO model, a cell-based treatment with a correction efficiency of 30-40% supposes the transition from a severe state or phenotype of the disease to a mild or even asymptomatic state. The decrease in the efficiency in the correction of the KO cell line may also be due to the fact that that area of the DNA has previously undergone another gene editing (to develop the KO model), which can make the CRISPR system lose efficacy, when editing on a previously edited zone.

Claims

1. Método in vitro para recuperar la expresión del gen F5 que codifica el factor V de la coagulación mediante la utilización de la metodología CRISPR/Cas9 que incluye la utilización de una pareja de guías caracterizadas por SEQ ID NO: 68-69 o una pareja de guías caracterizadas por SEQ ID NO: 70-71 , donde el gen F5 presenta una mutación en el nucleótido 3279. 1. In vitro method to recover the expression of the F5 gene that encodes coagulation factor V by using the CRISPR / Cas9 methodology that includes the use of a pair of guides characterized by SEQ ID NO: 68-69 or a pair of guides characterized by SEQ ID NO: 70-71, where the F5 gene has a mutation at nucleotide 3279.
2. Método in vitro según la reivindicación 1 en el que el gen F5 mutado está caracterizado por SEQ ID NO: 65. 2. In vitro method according to claim 1, wherein the mutated F5 gene is characterized by SEQ ID NO: 65.
3. Cultivo celular in vitro en el que se ha recuperado la expresión del gen F5 que codifica el factor V de la coagulación mediante la utilización del método definido en cualquiera de las reivindicaciones 1-2. 3. In vitro cell culture in which the expression of the F5 gene encoding coagulation factor V has been recovered by using the method defined in any of claims 1-2.
4. Kit que comprende la pareja de guías caracterizadas por SEQ ID NO: 68-69 y/o la pareja de guías caracterizadas por SEQ ID NO: 70-71. 4. Kit comprising the pair of guides characterized by SEQ ID NO: 68-69 and / or the pair of guides characterized by SEQ ID NO: 70-71.
5. Kit según la reivindicación 4 que comprende, además, una proteína Cas9. Kit according to claim 4, further comprising a Cas9 protein.
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EP1958648A1 (en) * 2005-10-28 2008-08-20 Dnavec Corporation Therapeutic method for blood coagulation disorder
US20110130443A1 (en) * 2005-11-09 2011-06-02 Hans-Peter Vornlocher Compositions And Methods For Inhibiting Expression Of Factor V Leiden Mutant Gene
WO2020022803A1 (en) * 2018-07-26 2020-01-30 주식회사 툴젠 Gene editing of anticoagulants

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EP1958648A1 (en) * 2005-10-28 2008-08-20 Dnavec Corporation Therapeutic method for blood coagulation disorder
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WO2020022803A1 (en) * 2018-07-26 2020-01-30 주식회사 툴젠 Gene editing of anticoagulants

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