ES2948817B2 - ANIMAL MODEL DEFICIENT IN FACTOR V - Google Patents

Description

DESCRIPTION

ANIMAL MODEL DEFICIENT IN FACTOR V

TECHNIQUE SECTOR

The present invention falls within the sector of diseases related to blood coagulation, specifically with factor V deficiency, also called parahemophilia or Owren's disease. The invention is related to gene editing and the creation of animal models deficient in factor V for the study of treatments for this coagulation deficiency.

BACKGROUND OF THE INVENTION

In humans, the gene that encodes factor V (FV) is called F5 and is located in the 1q24.2 region of the long arm of chromosome 1. It is a large gene, comprising 25 exons and 24 introns, the exon being 13 the largest. The F5 gene is composed of 9132 bp. The mature messenger RNA of the gene, which encodes a protein of 2,224 amino acids, measures about 6.8 kb. In its inactive form, the FV protein has six domains: A1, A2, B, A3, C1 and C2. Domains A1 and A2 constitute the heavy chain; domain B, encoded entirely by exon 13, constitutes the post-translational region; and domains A3, C1 and C2 form the light chain of the FV protein. The weight of the inactive FV protein is about 330 kDa.

Human FV is 80% homologous to murine FV, and the least conserved structure of the protein is the B domain, as is also the case with other animal species and other coagulation factors. The gene that encodes the FV protein in the mouse is 7430 bp long and encodes a protein of 2183 amino acids. FV levels in mice are exceptionally high compared to humans and different reference levels of FV have been proposed.

Factor V deficiency results in bleeding that can occur spontaneously or as a result of trauma or invasive medical procedures. The treatment of choice is currently based on transfusions of fresh frozen plasma (FFP) or coagulation factor concentrates. It is a palliative therapy that is ineffective and lacks specificity for the disease. Depending on the plasma levels of the protein, various degrees of severity are distinguished according to the tendency or risk of bleeding, as well as the severity of the symptoms, leading to a classification of severe (<1%), moderate ( <10%) and mild (>10%).

The interest in the study of factor V deficiency and the search for alternative treatments has led to the development of several strategies. The article by Serrano LJ. (Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations. International Journal of Molecular Sciences.2022; 10: 5802) describes a cell model deficient in factor V, obtained using CRISPR technology. Through additional steps of CRISPR/Cas9 technology for the correction of mutations, it is possible to transform a severe phenotype into a mild or asymptomatic one by obtaining the correction of 41% of the cells mutated in the F5 gene, giving rise to a functional protein that may be sufficient to convert a severe phenotype into a mild or asymptomatic one.

In the same sense, document WO2021229131A1 refers to an in vitro method to recover the expression of the F5 gene using CRISPR/Cas9 technology, using pairs of guides located at a distance of 150 base pairs towards the 5 'end and 150 base pairs towards the 3 end. ' with respect to a mutation located at nucleotide 3279 of the F5 gene. With this invention a tool is provided to cure factor V deficiency.

US2004014057A1, describes oligonucleotides having nuclease-resistant residues for use in the targeted modification of genetic material, including gene mutation, targeted gene repair and gene inactivation. The invention comprises a single-stranded oligonucleotide that has a DNA domain with at least one mismatch with respect to the genetic sequence that is to be altered, in addition to other chemical modifications. Among the examples included, in example 12, table 19, oligonucleotides appear to make targeted modifications in the F5 gene.

To date, some animal models with factor V deficiency have been created. In mice (Cui J, et al. Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V. Nature,1996; 384(6604):66- 8) individuals born at term died a few days later due to hemorrhage due to rupture of blood vessels or because they were subjected to different procedures. In zebrafish (Weyand AC,et al. Analysis of factor V in zebrafish demonstrates minimal levels needed for early hemostasis. Blood Advances,2019; 3(11):1670-80), embryos and larvae tolerated mutations lethal to mammals , but in adulthood they succumbed due to spontaneous hemorrhages.

The animal models deficient in coagulation factor V that have been developed to date do not produce viable individuals, so it would be of great interest to study the disease and its possible treatment, to obtain an animal model deficient in this protein and that would be viable.

EXPLANATION OF THE INVENTION

Animal model deficient in factor V.

An animal model has been obtained in which to test compounds for the treatment of coagulation disorders caused by mutations in the F5 gene that encodes coagulation factor V.

The animal model has been built by introducing a human transgene of the F5 gene with a specific mutation into its genome, and homozygous animals have been obtained. These homozygous animals hardly have clinical signs of alterations in coagulation, but a lengthening of coagulation times such as prothrombin time (PT) and activated partial thromboplastin (aPTT) is observed, as well as a decrease in blood levels. of factor V.

The mutation that has been introduced is located in humans at amino acid 1898 (or 1870 if the signal peptide of the protein is not taken into account). It is the substitution of a threonine for a methionine, due to the C>T mutation located in nucleotide 5895 of the humanF5 gene (5609 without counting the signal peptide), for which the mutation has been named Thr1898Met. The sequence of the human F5 gene is available in GenBank, with accession number: NM_000130.5 (https://www.ncbi.nlm.nih.gov/nuccore/NM_ 000130.5) and that of the human FV protein in UniProt, with the reference P12259 (https://www.uniprot.org/uniprot/P12259).

To insert the mutation that gives rise to Thr1898Met, from a human case, into the animal's gene, CRISPR/Cas9 technology was used and, to obtain the modified animals, microinjection was used.

One aspect of this invention, therefore, relates to an animal model deficient in coagulation factor V that includes a point mutation in a nucleotide of the F5 gene in the animal, which results in the substitution of a threonine for a methionine in the amino acid corresponding to amino acid 1898 of the human protein. Preferably, the animal used is the mouse and the mutation occurs at position 1857 of the FV protein, which we call 1857FTM (Factor Threonine Methionine). The invention also relates to a mouse animal model whose F5 gene includes the C>T 5695 mutation.

The animal with the mutation corresponding to the human Thr1898Met mutation may be heterozygous or homozygous for this mutation. Until now, in the state of the art, animals homozygous for deficiencies in coagulation factor V had been unviable, with litters that did not reach term or animals that died shortly after birth. However, as described herein, with this invention it has been possible to obtain homozygous animals whose litters reach term and present mild clinical signs of coagulation alteration. However, in these homozygous animals, significant changes occur in coagulometry indicators. This implies that the introduced mutation does not cause suffering in the animal, since the clinical signs are mild, but the mutants can be used as an animal model in the study of specific drugs for the treatment of coagulation factor V deficiency.

Another aspect of the invention relates to a method for producing an animal model deficient in coagulation factor V that includes modifying the genome of the animal in the F5 gene through a mutation that results in an amino acid change in the FV protein that reproduces the human mutation Thr1898Met, including the signal peptide of the FV protein in the amino acid numbering. To introduce the mutation, the method includes the gene editing technique and the mutated animals are obtained through assisted reproduction techniques with microinjection of zygotes.

BRIEF DESCRIPTION OF THE DRAWINGS

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 where, with an illustrative and non-limiting nature, the following has been represented. following:

Figure 1. Multiple alignment of FV proteins from different species of vertebrate animals.

Figure 2. Location of the T1857M allele mutations.

Figure 3. Coagulometry results expressed in seconds.

Figure 4. TP results and FV levels expressed in %.

PREFERRED EMBODIMENT OF THE INVENTION

The present invention is illustrated by the following examples, which are not intended to be limiting of its scope.

Example 1. Generation of knock-in mice with a human mutation in F5 A knock-in mouse was constructed by disabling a human gene sequence with which the sequence of the mouse F5 gene was replaced.

Example 1.1. Sequence selection

For the construction of the knock-in mouse, a mutation described by Delev D. and collaborators (Factor 5 mutation profile in Germán patients with homozygous and heterozygous factor Vdeficiency. Haemophilia,2009; 15(5):1143-53), located in the exon 18, domain A3, of the human F5 gene. In the corresponding human FV protein, this mutation is located at amino acid 1898 (also identified as amino acid 1870 when the signal peptide is not taken into account) and results in the change of a threonine to methionine (Thr1898Met, due to the ACG mutation a ATG in the gene) located at nucleotide 5895 of the humanF5 gene (5609 without counting the signal peptide), so it was named Thr1898Met.

In addition, a multiple alignment of protein sequences from different species of vertebrate animals was performed using the alignment tool M-Coffee (Di Tommaso P, et al. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Research,2011; 39(suppl):W13-7), which combines different sequence alignment methods to find out if the described mutations are structurally and functionally important and if they are located in conserved regions. The human FV (reference P12259 in Uniprot; SEQ ID NO: 1), the dog (reference A0A8P0SJS2 in Uniprot; SEQ ID NO: 2), the rabbit (reference G1U6T0 in Uniprot; SEQ ID NO: 3) were included in the alignment. ), mouse (reference O88783 in Uniprot; SEQ ID NO: 4), pig (reference F1RPW2 in Uniprot; SEQ ID NO: 5), zebrafish (reference Q90X47 in Uniprot; SEQ ID NO: 6), guinea pig (reference H0V8V3 in Uniprot; SEQ ID NO: 7), cat (reference M3W922 in Uniprot; SEQ ID NO: 8) and sheep (reference W5PKA9 in Uniprot; SEQ ID NO: 9). It was found that the area corresponding to the selected mutation was highly conserved in all species, including the mouse. In Figure 1, threonine 1898 is indicated with an arrow, conserved in all the species analyzed; The different degrees of conservation are indicated with the following symbols: Asterisk (*), indicates that in said position the residues are 100% identical; colon (:), indicate positions where conserved substitutions have been made; period (.), indicates semiconserved substitutions. In the case of the colon, the sequences may have different amino acids at that position, but their chemical properties are very similar. Non-conserved substitution presents amino acid substitutions with different chemical properties in another protein. In the case of semiconservative changes, it means that different amino acids in the same position have similar chemical properties, but to a lesser extent. Mathematically, in the alignments, these classifications are given by a score in the so-called Gonnet matrix PAM 250 where a score greater than 0.5 indicates that the conservation has strongly similar properties (:) and a score less than 0.5 (. ) indicates that conservation between amino acids presents less similar properties. Negative scores indicate non-conservation of amino acids.

Example 1.2. Elaboration of the 1857FTM mutation in mice

The threonine at position 1898 in the human protein is located at position 1857 in the mouse protein. The 1857FTM mutation was made to reproduce the Thr1898Met mutation in the mouse.

To do this, a 20 bp CRISPR guide (SEQ ID NO: 10 sequence) and a 100 bp template DNA (SEQ ID NO: 11) were designed to emulate the 1898FTM mutation in the mouse. C57CBA F1 hybrid mice were used. The C57CBA F1 is the cross of the first generation -F1- with male CBA/CaOlaHsd and female C57BL/6JOlaHsd-.

The mixture was microinjected into zygotes and subsequently reimplanted into a recipient female (100 ng/^l of mRNA for Cas9, 50 ng/^l of sgRNA and 10 ng/^l of ssDNA). The objective was to obtain a mosaic individual that had the allele of interest, to then segregate it and obtain heterozygous individuals with that allele. Finally, two mutations were observed (Figure 2), one of them the original one that had been planned, which was called 1857FTM (C>T 5695); the other mutation C>T 5675 that did not give rise to any change in the protein since the AAC triplet was transformed into AAT and both encode asparagine (silent mutation) (Sequence SEQ ID NO: 13). In Figure 2, the sequence SEQ ID NO: 13, which contains the two point mutations indicated in mouse, is compared with the fragment that goes from amino acids 137 to 187 of the sequence of the wild animal SEQ ID NO: 12. The methodology used For the generation of the CRISPR guide, the cultivation and collection of zygotes, their microinjection and their implantation in recipient females was carried out according to the protocol described by Lamas-Toranzo and collaborators ('TMEM95 is a sperm membrane protein essential for mammalian fertilization. eLife,2020;9:e53913.) and according to Bermejo Álvarez and collaborators (Utero-tubal embryo transfer and vasectomy in the mouse model. J Vis Exp, 2014:e51214). Pairs of heterozygous animals were obtained that were crossed to obtain a population stable homozygous.

Superovulation was induced in 18 donor mice, 7-8 weeks old, C57BL/6JOlaHsd- hybrids, by intraperitoneal injections of 5 IU of pregnant mare serum gonadotropin (PMSG, Folligon, MSD Animal Health) and an equivalent dose of chorionic gonadotropin. human (hCG, Sigma), with an interval of 48 hours. Induced females were mated with CBA/CaOlaHsd- males and zygotes were recovered from the oviducts.

Microinjections of mouse zygotes were performed with a micromanipulation system (Eppendorf Transferman) equipped with a Leica DMi8 inverted microscope. In the cytoplasm of each zygote, 3-5 μl of a mixture of 100 ng/^l of polyadenylated mRNA coding for Cas9, 50 ng/^l of guide RNA (sgRNA) and 10 ng/^l of template DNA were injected. of single-stranded oligonucleotide, using a filament microinjection needle of approximately 1 ^m in diameter, using positive pressure to move the solution from the needle towards the cytoplasm (Eppendorf Femptojet system). After microinjection, embryos were cultured in EmbryoMax KSOM Mouse Embryo Media (Millipore) at 37°C with 5% CO<2>and 5% O<2>for 4 days, until they reached the blastocyst stage. 14 blastocysts were transferred to a Swiss recipient female using the utero-tubal technique described in Bermejo-Álvarez et al. JoVE 2014.

The studies carried out with animals were reviewed and approved according to Royal Decree 53/2013 and Directive 2010/63/EU. All procedures used were approved by the Faculty of Veterinary Medicine of the Complutense University of Madrid, the Animal Experimentation Ethics Committee of the Complutense University of Madrid, the Ethics Committee of the National Institute of Agricultural and Food Research and Technology (INIA) and the Community of Madrid (PROEX 040/17, PROEX 358.4/21).

Example 1.3. Genotyping of 1857 FTM mutants

To limit traumatic procedures on homozygous individuals, due to the risk of possible bleeding disorders, tissue collection to genotype these mice was carried out taking advantage of the marking of the animals in the ear to obtain the samples, previously administering EMLA anesthetic cream locally. (25 mg/g lidocaine 25 mg/g prilocaine). In this way, the tissue that came off the perforation was used for genotyping without additional suffering for the animals. Said ear marking was carried out at 21 days of age at the time of weaning from the litter.

The individuals of each litter were separated by sex and the individuals were divided per box according to its dimensions. Ear marking was done by drilling a single hole. 6 different positions were established (three in each ear) so that all animals could be distinguished by piercing, the box where they were housed and sex.

When genotyping was carried out, from the samples taken during labeling, the DNA was extracted using a lysis and extraction protocol using Viagen DirectPCR® Lysis Reagent (Viagen Biotech) adding, to 1 ml of lysis reagent, 10 ^l of a proteinase K solution with a concentration of 20mg/ml, and following the manufacturer's instructions.

A PCR was performed with the DNA obtained and the primers F: 5'-AGAGAGCT CCGTCACAACAT-3' (SEQ ID NO: 14) and R: 5'-ACACTTTTACACTTCTGCCCG-3' (SEQ ID NO: 15). The cycles were as follows: 94°C 2 minutes 35 x (94°C 20 seconds, 60°C 30 seconds, 72°C 30 seconds) and a final elongation at 72°C 5 minutes. The PCR product was purified using the GEL/PCR Purification Mini Kit (Favorgen) following the manufacturer's instructions. The amplified fragment was that of the WT sequence detailed in the previous table and identified with SEQ ID NO: 12.

To obtain the sequence of the different individuals and be able to classify them into WT individuals (non-carriers of the mutation), heterozygous individuals (carriers of the 1857FTM mutation in a single allele) and homozygous individuals, the Sanger sequencing procedure was used, which was carried out in the Genomics and Proteomics Unit of the Complutense University of Madrid. The animals necessary for the subsequent coagulometric study were obtained through crosses between heterozygous individuals. These crosses complied with Mendelian laws (50% heterozygous, 25% WT and 25% homozygous).

Example 2. Coagulometry studies

6 WT animals were selected, 6 heterozygotes and 6 homozygotes obtained as described in example 1.3.

To establish standard lines, blood was drawn from the WT animals to obtain plasma pools. The animal was restrained manually without anesthesia, disinfecting the puncture area with chlorhexidine, and a puncture was performed with a 23G needle. in the submandibular sinus and the blood was obtained in 0.5 ml tubes in sodium citrate (3.2%). After extraction, pressure was applied to the puncture site until it was verified that the animal had stopped bleeding, disinfecting again. the area with chlorhexidine, and after the administration of subcutaneous physiological saline, the animal was returned to the box. The animals were allowed to rest for a week until the next extraction, to avoid continued damage to the animal and for volume to recover.

Individual blood extraction from the animals for coagulometry studies was carried out in the same way as the "pools". The animal was restrained without anesthesia, manually, disinfecting the puncture area with chlorhexidine. The sinus puncture submandibular was performed with a 23G needle, obtaining 0.25 ml of blood in 0.25 ml tubes in 3.2% sodium citrate. After extraction, pressure was applied to the incision site until it was verified that the animal had stopped breathing. bleeding, the area was disinfected with chlorhexidine, warm physiological saline was administered three times (0.75 ml) intraperitoneally and the animals were returned to the box.

As already indicated, 6 animals per group were used (WT, heterozygous and homozygous), the plasmas were extracted, the calibration curves were carried out with "pools" of WT individuals and the prothrombin time (PT), the activated partial thromboplastin time (aPTT) and FV levels.

Determinations of FV, TP and aPTT were performed in a STart Max II R viscosity coagulometer (Diagnostica Stago S.A.S. Barcelona, Spain) following the manufacturer's instructions. The programs corresponding to each parameter were strictly followed as indicated in examples 2.1-2.3. To carry out the measurements, a series of cuvettes (Start 4 Cuve, Diagnostica Stago S.A.S. Barcelona, Spain) were placed in the coagulometer to which metal spheres (Diagnostica Stago S.A.S. Barcelona, Spain) were subsequently added. All reagents were reconstituted following the manufacturer's instructions. Once all the reagents and the sample were poured into the cuvettes, the reaction began. The determination was terminated automatically as soon as the clot formed. At the same time that the determinations were carried out, positive and negative controls were carried out with the System control N/P reagents (Diagnostica Stago S.A.S. Barcelona, Spain). A lengthening of coagulation times was observed both in the case of factor V and in the prothrombin and activated partial thromboplastin times, with the difference being statistically significant in homozygous individuals compared to WT individuals.

Example 2.1. Determination of factor V

For the determination of FV, FV-deficient plasma (StaDeficient V Diagnostica Stago S.A.S. Barcelona, Spain) was used, together with neoplastin Cl+5 (Diagnostica Stago S.A.S. Barcelona, Spain), with an international sensitivity index (ISI) of 1.27. . Metal spheres were placed in the calibrator cuvettes that were preheated to 37°C for 3 minutes. Once reconstituted, Cl+5 neoplastin was homogenized and preheated at 37°C for 3 minutes before use. The Owren Koller buffer (Diagnostica Stago S.A.S. Barcelona, Spain) was allowed to rest for 30 minutes to bring it to room temperature and then make the corresponding dilutions. A mixture of 50 μl of FV-deficient plasma and 50 μl of the sample to be analyzed (diluted in Owren Koller buffer) was introduced into the cuvette. After an incubation period of 60 seconds, 100 μl of Neoplastina Cl+5 were added. Calibration curves were calculated using pools of mouse plasma from WT (healthy) individuals following the same procedure as for the calibration curve. The calibration curve performed with the plasma pool was performed using 1 dilutions: 100, 1:200, 1:400 and 1:800. Individual samples were diluted 1:100. The data obtained were expressed in seconds (Figure 3) and percentages (Figure 4).

Example 2.2. Determination of prothrombin time

To calculate PT, neoplastin Cl+5 was used with an ISI of 1.27. Metal spheres were placed in each calibrator cuvette and preheated to 37°C for 3 minutes. Once reconstituted, Neoplastin Cl+5 was homogenized and preheated at 37°C for 3 minutes before use. A total of 50 μl of the sample was introduced into the well and, after 60 seconds of incubation, 100 μl of Neoplastine Cl+5 was added. The Owren Koller buffer was allowed to settle for 30 minutes before making the appropriate dilutions. The calibration curve with the mouse plasma pool was created following the same procedure as with the commercial calibrator. Dilutions 1:3, 1:6, 1:9 and 1:12 were used; individual samples were diluted to 1:3. TP data obtained from individual measurements were expressed in seconds (Figure 3) and percentages (Figure 4); the international normalized ratio (INR) was calculated as the ratio between the TP of the sample and the TP of reference derived from mixing all individual aliquots, raised to the ISI power.

Example 2.3. Determination of activated partial thromboplastin time

The aPTT was measured by placing metal spheres in all the cuvettes of the commercial calibrator and preheating them at 37°C for 3 minutes. A total of 50 μl of a 1:1 dilution of plasma and 50 μl of PPT Automate (Diagnostica Stago S.A.S. Barcelona, Spain) were added to the cuvette; After incubating the mixture for 180 seconds, 50 μl of CaCh 0.025M (Diagnostica Stago S.A.S. Barcelona, Spain) were added. The data obtained was expressed in seconds (Figure 3).

Example 3. Clinical signs

In homozygous individuals with the 1857FTM mutation, bleeding was observed after performing the ear puncture. These hemorrhages were corrected within 10 seconds after applying pressure with sterile gauze, and did not represent any suffering for the animal. Ear hemorrhages after puncture for marking did not occur in WT or heterozygous individuals. In no case were spontaneous internal or external hemorrhages observed.

Claims (9)

1. Non-human animal model deficient in coagulation factor V whose genome comprises a geneF5 with a mutation that results in an amino acid change in the FV protein that reproduces the human mutation Thr1898Met, including in the numbering of the amino acids the signal peptide of the human FV protein. 2. Animal model according to claim 1 in which the animal is a mouse (Mus musculus) and the mutation in the FV protein is located in Thr1857Met. 3. Animal model according to claim 2 in which the mouse includes the C>T 5695 mutation in the F5 gene sequence. 4. Animal model according to any of the preceding claims in which the animal is heterozygous for the mutation. 5. Animal model according to any of claims 1-3 in which the animal is homozygous for the mutation. 6. Animal model according to claim 5 wherein the animal is viable with mild clinical signs of disease and without suffering for the animal. 7. Method for producing an animal model as defined in any of the preceding claims that includes modifying the genome of the animal in the F5 gene by means of a mutation that results in an amino acid change in the FV protein that reproduces the human mutation Thr1898Met, including in the numbering of the amino acids the signal peptide of the FV protein, where the mutation is produced by gene editing and the mutated animals are obtained by assisted reproduction techniques with microinjection of zygotes. 8. Method for producing an animal model according to claim 7 in which the animal used is a mouse and the mutation in the F5 gene sequence occurs in C>T 5695. 9. Use of the animal model defined in any of claims 1-6 to test specific drugs for the treatment of coagulation factor V deficiency.