Composition containing viral vector expressably containing polynucleotide encoding favine protein

Abstract

The present invention relates to a composition comprising a viral vector expressably containing a polynucleotide encoding Favine protein, wherein the viral vector is an adeno-associated viral vector.

Description

A composition comprising a viral vector capable of expressing a polynucleotide encoding the favin protein.

[0001] This specification discloses compositions comprising a viral vector expressibly containing a polynucleotide encoding the favin protein, and non-human test animals administered with the viral vector expressibly containing the polynucleotide encoding the favin protein.

[0002] Fabin, also known as coiled-coil domain containing 3 (Ccdc3), is a secreted protein strongly expressed in the aorta and adipose tissue. Fabin gene expression is known to be elevated in the adipose tissue of obese mice and diabetic mice compared to normal adipose tissue (Non-Patent Literature 1).

[0003] Furthermore, it has been reported that lipid accumulation increases in adipocytes that overexpress the Favine gene (Non-Patent Literature 2). In addition, it has been reported that lipid accumulation is suppressed in adipocytes of mice lacking the Favine gene (hereinafter referred to as "Favine KO mice"), and that age-related lipid accumulation in the liver is also suppressed (Non-Patent Literature 2).

[0004] In humans, it has been reported that the expression level of fabin in the visceral adipose tissue of a subject correlates with the subject's waist circumference and body mass index (BMI) (Non-Patent Document 3).

[0005] Based on these experimental data, favin is considered to be a lipid accumulation factor (Non-Patent Literature 2).

[0006] Patent Document 1 discloses a composition for preventing atherosclerotic diseases and a composition for inhibiting platelet aggregation, which include favin protein or a polynucleotide capable of expressing cDNA encoding favin protein in vivo.

[0007] Japanese Patent Publication No. 2020-176074

[0008] S. Kobayashi et al. ; Biochemical and Biophysical Research Communications Vol. 392, p. 29-35,2010S. Kobayashi et al. ; The Journal of Biological Chemistry Vol. 290, No. 12, p. 7443-7451, 2015S. Ugi et al. ; Obesity Vol. 22, No. 4, p.1070-1077, 2014

[0009] Arteriosclerosis is a cause of ischemic diseases such as myocardial infarction and cerebral infarction. Ischemic diseases often develop due to the formation of blood clots in atherosclerotic plaques within the arteries. Therefore, suppressing the onset or progression of arteriosclerosis contributes to the prevention of ischemic diseases.

[0010] The object of this invention is to provide a composition that can be used to prevent arteriosclerotic diseases.

[0011] The present invention may include the following embodiments: 1. A composition comprising a viral vector expressibly containing a polynucleotide encoding a favin protein, wherein the viral vector is an adeno-associated viral vector. 2. The composition according to 1, wherein the composition is used for at least one of the following purposes: to prevent atherosclerotic disease, to prevent thrombus formation, to prevent platelet aggregation, and to increase the body weight of an animal to be administered, to suppress body weight loss of an animal to be administered, to increase adipose tissue, and / or to suppress the decrease of adipose tissue. 3. The composition according to 2, wherein the atherosclerotic disease is arteriosclerosis, ischemic heart disease, ischemic cerebrovascular disease, or ischemic bowel disease. 4. A viral vector expressibly containing a polynucleotide encoding a favin protein is administered at a dose of 1 × 10⁶ per kg of body weight. ９ vg / times ~ 1×10 １５The composition according to item 1, administered in a dose of vg / dose. Item 5. A non-human test animal administered with a viral vector capable of expressing a polynucleotide encoding favin protein, wherein the test animal has favin protein expressed from the polynucleotide in its blood.

[0012] According to the present invention, a composition that can be used to prevent arteriosclerotic diseases can be provided.

[0013] The alignment of favine proteins from Mus musculus (SEQ ID NO: 1), Rattus norvegicus (SEQ ID NO: 2), Homo sapiens (SEQ ID NO: 3), Danio rerio (SEQ ID NO: 4), and Xenopus tropicalis (SEQ ID NO: 5) is shown. The underlined parts in the figure indicate the endoplasmic reticulum (ER) localization signal sequence. The enclosed areas indicate the coiled-coil domain. The blood Favine concentrations in Favine-AAV infected mice are shown. The effect of Favine-AAV on thrombus formation is shown. a shows the histological image of the carotid artery ligation site in Favine / ApoE DKO mice administered with control AAV. b shows the histological image of the carotid artery ligation site in Favine / ApoE DKO mice administered with Favine-AAV at a dose of 3 × 10^10 vg / mouse. c shows the histological image of the carotid artery ligation site in Favine / ApoE DKO mice administered with Favine-AAV at a dose of 1.5 × 10^10 vg / mouse. This shows the effect of Favine-AAV on body weight and adipose tissue weight. (A) shows the change in body weight after AAV administration. (B) shows the blood concentration of Favine (pg / ml). (C) shows the weight of adipose tissue around the epididymis and subcutaneously at the time of dissection. This shows the inhibitory effect of Favine on platelet aggregation. (A) shows the platelet aggregation rate when mock medium is added. (B) shows the platelet aggregation rate when conditioned medium is added. The vertical axis shows the platelet aggregation rate, and the horizontal axis shows the monitoring time (minutes).

[0014] 1. Explanation of Terms First, we will explain the terms commonly used in this specification.

[0015] The amino acid symbols used in this specification refer to the amino acids shown in Table 1 below.

[0016]

[0017] In this specification, favin is synonymous with coiled-coil domain containing 3 (Ccdc3). The favin protein may be monomeric or dimeric, preferably dimeric. Figure 1 shows the amino acid sequences of monomeric favin proteins from mouse (SEQ ID NO: 1; NCBI Reference Sequence NP_083080.1), rat (SEQ ID NO: 2; NCBI Reference Sequence XP_574081.4), human (SEQ ID NO: 3; NCBI Reference Sequence NP_113643.1), zebrafish (SEQ ID NO: 4; NCBI Reference Sequence NP_001020681.1), and African clawed frog (SEQ ID NO: 5; NCBI Reference Sequence NP_001017103.2). As shown in Figure 1, the favin protein exhibits high homology across animal species, therefore, the animal species is not limited. In this specification, a favin protein is defined as having at least 80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, or 99% identity in its amino acid sequence with any of the above-mentioned favin proteins, and possessing the function of a favin protein. The function of a favin protein is at least the function of inhibiting platelet aggregation.

[0018] Preferably, the favin protein is a protein having the amino acid sequence represented by the following SEQ ID NO: MPXPLLLAAL CLAXSPAPAR ACQLPSEWRP LSEGCRAELA ETIVYAKVLA LHPEXPGLYN YLPWQYQAGE GGLFYSAEVE MLCDQAWGSM LEVPAGSRLN LTGLGYFSCH SHTVVQDYSY FFFXRMDENY NLLPHGVNFQ DAIFPDTQEN RRMFSSLFQF XNCSQGQQLT TFSSDWEVQE DNRLMCSSVQ KALFEEEDHV KKLQQKVATL EKRNRQLRER VKKVKRSLRQ ARKNXRHLEL XNQKLNEKLX XXXAQQHINA XGXXPVRXPY XHG (SEQ ID NO: 6).

[0019] More preferably, the protein has amino acids from the 22nd amino acid to the last amino acid of SEQ ID NO: 6.

[0020] In sequence number 6, the X (Xaa in sequence listing) at positions 3, 14, 55, 124, 161, 235, 241, 250, 251, 252, 253, 261, 263, 264, 268, and 271 represents any amino acid. In sequence number 6, the amino acids at positions 3, 14, 55, 124, 161, 235, 241, 250, 251, 252, 253, 261, 263, 264, 268, and 271 may be deleted. In particular, the amino acids at positions 252, 263, and 264 may be deleted.

[0021] In Sequence ID No. 6, the 3rd and 261st amino acids are preferably nonpolar or basic amino acids. The 14th, 55th, 124th, 241st, 250th, 251st, and 268th amino acids are preferably nonpolar amino acids. The 161st, 235th, 252nd, 253rd, and 271st amino acids are preferably nonpolar or polar amino acids. The 263rd amino acid is preferably a basic amino acid. The 264th amino acid is preferably an acidic amino acid.

[0022] Here, whether an amino acid is nonpolar, polar, acidic, or basic refers to its physical properties at pH 7, that is, whether the amino acid is polar or not in an aqueous solution at pH 7, or whether the amino acid exhibits acidic or basic properties.

[0023] A more preferred favin protein is a protein having the amino acid sequence represented by the following SEQ ID NO: 7.

[0024] MPXPLLLAAL CLAXSPAPAR ACQLPSEWRP LSEGCRAELA ETIVYAKVLA LHPEXPGLYN YLPWQYQAGE GGLFYSAEVE MLCDQAWGSM LEVPAGSRLN LTGLGYFSCH SHTVVQDYSY FFFXRMDENY NLLPHGVNFQ DAIFPDTQEN RRMFSSLFQF XNCSQGQQLT TFSSDWEVQE DNRLMCSSVQ KALFEEEDHV KKLQQKVATL EKRNRQLRER VKKVKRSLRQ ARKNXRHLEL XNQKLNEKLX XXXAQQHINA XGXXPVRXPY XHG (Sequence ID 7), where the 3rd X is leucine or arginine, the 14th X is alanine, valine or glycine, and the 55th X is valine or alanine. The 124th X is valine or leucine, the 161st X is alanine or serine, the 235th X is serine or glycine, the 241st X is valine or alanine, the 250th X is glycine or alanine, the 251st X is glycine or alanine, the 252nd X is serine or proline or means an amino acid deletion, the 253rd X is serine or glycine, the 261st X is leucine or arginine, the 263rd X is arginine or means an amino acid deletion, the 264th X is glutamic acid or means an amino acid deletion, the 268th X is alanine or proline, and the 271st X is leucine or arginine.

[0025] The above favin protein may have a tag peptide attached to its N-terminus and / or C-terminus. Examples of tags include hemagglutinin (HA) tag, c-Myc tag, FLAG tag (trademark), histidine (His) tag, glutathione-S-transferase (GST) tag, V5 tag, Halo tag (trademark), maltose-binding protein (MBP) tag, GFP, IgG-Fc region, etc. In this specification, favin protein includes those to which the above tag peptides have been attached. Preferably, the favin protein is a protein to which the FLAG tag (trademark) has been attached, having an amino acid sequence represented by any of SEQ ID NOs: 1 to 3. More preferably, the FLAG tag (trademark) is attached to the C-terminus of the favin protein.

[0026] The polynucleotide encoding the fabin protein is not limited as long as it includes the polynucleotide encoding the fabin protein described above. The polynucleotide may be DNA or RNA. Preferably, the polynucleotide is cDNA (complementary DNA) encoding the fabin protein. Here, the mouse fabin cDNA sequence is registered as NCBI Reference Sequence: NM_028804.1, the rat fabin cDNA sequence as NCBI Reference Sequence: XM_574081.7, the human fabin cDNA sequence as NCBI Reference Sequence: NM_031455.4, the zebrafish fabin cDNA sequence as NCBI Reference Sequence: NM_001025510.1, and the African clawed frog fabin cDNA as NCBI Reference Sequence: NM_001017103.3, so these cDNAs may be used. The codons of the cDNA may be optimized depending on the species of the cell expressing the cDNA.

[0027] Viral vectors capable of expressing the polynucleotide encoding the favin protein are not limited as long as they include the polynucleotide encoding the favin protein described above and a transcriptional regulatory region for its expression in vivo. The transcriptional regulatory region can be selected depending on the species of organism expressing the polynucleotide encoding the favin protein. When expressing the polynucleotide encoding the favin protein in the human body, it is preferable to use a transcriptional regulatory region that has transcriptional activity in the liver. Examples of transcriptional regulatory regions include the transcriptional regulatory region of antitrypsin (preferably the transcriptional regulatory region of α1-antitrypsin), the transcriptional regulatory region of trans tyretin, the transcriptional regulatory region of cytomegalovirus, the transcriptional regulatory region of chicken β-actin, and the transcriptional regulatory region of synapsin I. The transcriptional regulatory region may be used together with an enhancer region. Examples of enhancer regions include the hepatic control region of the ApoE / C1 gene enhancer and the cytomegalovirus enhancer. Preferably, the combination of an enhancer and a transcriptional regulatory region that expresses a polynucleotide encoding the favin protein is a combination of the hepatic control region of the ApoE / C1 gene enhancer and the transcriptional regulatory region of α1-antitrypsin, or a combination of a cytomegalovirus enhancer and the transcriptional regulatory region of chicken β-actin.

[0028] A viral vector capable of expressing a polynucleotide encoding the favin protein preferably contains a Kozak sequence upstream of the polynucleotide encoding the favin protein. It may also contain a poly-A addition signal downstream of the polynucleotide encoding the favin protein.

[0029] Viral vectors capable of expressing polynucleotides encoding favin protein include adeno-associated virus vectors, adenovirus vectors, retrovirus vectors, lentiviral vectors, and variants of these viral vectors. Preferably, adeno-associated virus vectors are used. Examples of adeno-associated virus vectors include serotype 8 (AAV8), serotype 5 (AAV5), serotype 6 (AAV6), serotype 9 (AAV9), serotype 1 (AAV1), and serotype 2 (AAV2). Preferably, serotype 8, serotype 5, serotype 6, and serotype 9 are used. More preferably, serotype 8 is used. Examples of variants of adeno-associated virus vectors include point mutations, peptide insertion mutations, and directed evolution mutations. More specifically, examples include mutations in AAV8 from amino acid Y to F (e.g., Y733F), AAV8 capsid surface mutation variants, AAV2.5, AAV-DJ, AAV-LK03, AAV-PHP.B, AAV-PHP.eB, AAV2-YF, AAV2-TM, AAV2-N587Q, AAV9.47, AAV-B1, AAV2-RGD, AAV2-NLS, AAV2-brain, AAVrh10, AAVrh74, and the like.

[0030] Preferably, the viral vector containing an expressible polynucleotide encoding the favin protein is an adeno-associated virus vector of serotype 8, serotype 5, serotype 6, and serotype 9, into which an expressible polynucleotide encoding the favin protein having the amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7 is inserted. More preferably, the viral vector containing an expressible polynucleotide encoding the favin protein is an adeno-associated virus vector of serotype 8, serotype 5, serotype 6, and serotype 9, into which an expressible polynucleotide encoding the favin protein having the amino acid sequence represented by SEQ ID NO: 1 to 3 is inserted, and the expression of the favin protein from the polynucleotide is induced by a combination of the hepatic control region of the ApoE / C1 gene enhancer and the transcriptional regulatory region of α1-antitrypsin. As a viral vector capable of expressing a polynucleotide encoding the favin protein, it is more preferable that, for example, a serotype 8 adeno-associated virus vector is used, into which a polynucleotide encoding the favin protein having an amino acid sequence represented by any of SEQ ID NOs: 1 to 3 is inserted in an expressible manner, and the expression of the favin protein from the polynucleotide is induced by a combination of the hepatic control region of the ApoE / C1 gene enhancer and the transcriptional regulatory region of α1-antitrypsin.

[0031] In this specification, “atherosclerotic disease” refers to atherosclerosis itself and diseases caused by atherosclerosis. Atherosclerotic diseases include atherosclerosis; ischemic heart diseases such as angina pectoris and myocardial infarction; ischemic cerebrovascular diseases such as cerebral infarction; and ischemic bowel diseases such as mesenteric ischemia and ischemic colitis. Ischemic heart diseases and ischemic cerebrovascular diseases may include thrombosis and thromboembolism, which result from blood clots formed in blood vessels, particularly arteries.

[0032] The prevention of arteriosclerotic diseases includes inhibiting, reducing, and delaying the onset of arteriosclerotic diseases. The inhibition, reduction, and delay can be determined by evaluating the degree of arteriosclerosis.

[0033] When the artery can be excised, the degree of arteriosclerosis can be evaluated by excising the artery and detecting fat deposition on the vascular wall, calcification of the vascular wall, and the like. The evaluation of fat deposition on the vascular wall can be carried out not only by visual observation but also, for example, by performing Oil Red O staining, Sudan Black B staining, Nile Blue staining, etc., and evaluating from the intensity of the staining. It can be evaluated that the stronger the staining, the more fat is deposited. In addition, the detection of calcification of the vascular wall can be evaluated not only by visual observation but also by performing Kossa staining, Alizarin Red S staining, etc., and evaluating from the intensity of the staining. It can be evaluated that the stronger the staining, the more calcium is deposited.

[0034] When excision of the artery is not possible, the degree of fat deposition on the vascular wall and / or the degree of calcification of the vascular wall can be evaluated by angiography, MRI examination, CT examination, blood pressure pulse wave examination, carotid ultrasonography, and the like.

[0035] Whether or not platelet aggregation is suppressed can be evaluated by measuring platelet aggregation ability by collecting platelets from a subject and monitoring the change in light transmittance associated with platelet aggregation using a platelet aggregation meter in vitro. The change in light transmittance associated with platelet aggregation can be monitored by adding a sample containing platelets to the cuvette of the platelet aggregation meter, adding a platelet aggregation inducer such as epinephrine, protease activated receptor 1-activating peptide, or protease activated receptor 4-activating peptide to the cuvette, and then measuring the light transmittance inside the cuvette for a predetermined time. Platelet aggregation ability can be expressed as the platelet aggregation rate. The platelet aggregation rate can be calculated by setting the light transmittance when platelets are not aggregated to 0%, and the light transmittance when platelets are completely aggregated or when a sample without platelets is set to 100%. If the platelet aggregation rate is lower than the reference value, it can be evaluated that platelet aggregation is suppressed. Conversely, if the platelet aggregation rate is above the reference value, it can be evaluated that platelet aggregation is not suppressed. Reference values ​​can be selected from, for example, the platelet aggregation rate of healthy individuals, the mean, median, third quartile, first quartile, etc., of platelet aggregation rates obtained from multiple healthy individuals.

[0036] Preventing thrombus formation includes inhibiting, reducing, and delaying thrombus formation. Inhibition, reduction, and delay can be determined by evaluating the degree of thrombus formation.

[0037] The degree of thrombus formation can be evaluated by removing an artery, if possible, and measuring the degree of blood clot formation in areas of fatty deposition or calcification on the vessel wall. The degree of blood clot formation can be evaluated by visual observation, as well as by Weigert's fibrin staining and fibrin immunohistochemistry, based on the intensity of the staining. Stronger staining indicates a greater amount of fibrin deposition, and fibrin deposition indicates strong thrombus formation.

[0038] In cases where arterial excision is not possible, evaluation can be performed by confirming the presence and size of a thrombus via angiography, MRI examination, CT examination, ultrasonic blood flow examination, or the like.

[0039] As used herein, "inhibition of body weight reduction" intends that, when the body weight of a subject at a first measurement time point is compared with the body weight at a second measurement time point that is later than the first measurement time point, the reduction in body weight at the second measurement time point is within 0% / month to 2% / month, preferably within 0% / month to 1% / month, relative to the body weight at the first measurement time point. "Body weight gain" intends that, when the body weight of a subject at a first measurement time point is compared with the body weight at a second measurement time point that is later than the first measurement time point, the increase in body weight at the second measurement time point is 2% / month or more, preferably 4% / month or more, more preferably 6% / month or more, relative to the body weight at the first measurement time point. The upper limit of the body weight gain at the second measurement time point is preferably 10% / month. The unit "% / month" intends that the interval between the first measurement time point and the second time point is converted and calculated on a per-month basis.

[0040] As used herein, "inhibition of adipose tissue reduction" intends that, when the weight of adipose tissue at a first measurement time point is compared with the weight of adipose tissue at a second measurement time point that is later than the first measurement time point, the reduction in the weight of adipose tissue at the second measurement time point is within 0% / month to 2% / month, preferably within 0% / month to 1% / month, relative to the body weight at the first measurement time point. "Increase in adipose tissue" intends that, when the body weight at a first measurement time point is compared with the body weight at a second measurement time point that is later than the first measurement time point, the increase in the weight of adipose tissue at the second measurement time point is 2% / month or more, preferably 4% / month or more, more preferably 6% / month or more, relative to the weight of adipose tissue at the first measurement time point. The upper limit of the increase in the weight of adipose tissue at the second measurement time point is preferably 10% / month. The unit "% / month" intends that the interval between the first measurement time point and the second time point is converted and calculated on a per-month basis. Instead of the weight of adipose tissue, the body fat percentage of the subject or the subcutaneous fat thickness of the subject may be used as an evaluation index. When the subject is a non-human animal, a specific organ (for example, testis or the like) may actually be collected to measure the actual weight.

[0041] 2. Compositions One embodiment disclosed herein relates to a composition comprising a viral vector expressibly containing a polynucleotide encoding a favin protein as an active ingredient.

[0042] The composition may be used for pharmaceutical or non-pharmaceutical purposes. Preferably, the composition may be used for at least one of the following purposes: to prevent atherosclerotic diseases, to prevent thrombus formation, to prevent platelet aggregation, and to increase the body weight of the target animal, to suppress body weight loss in the target animal, to increase adipose tissue, and / or to suppress the decrease in adipose tissue.

[0043] The composition can be prepared by combining the above-mentioned active ingredients with a suitable carrier or additive. Examples of carriers and additives used in the preparation of the composition include various types commonly used in conventional pharmaceuticals, depending on the dosage form of the composition, such as excipients, binders, disintegrants, lubricants, colorants, flavoring agents, odorants, and surfactants.

[0044] When preparing the above pharmaceutical composition as an injectable preparation, diluents such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters can be used as carriers; pH adjusters such as sodium citrate, sodium acetate, and sodium phosphate; buffers such as dipotassium phosphate, trisodium phosphate, sodium hydrogen phosphate, and sodium citrate; stabilizers such as sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid; and sugars such as mannitol, inositol, maltose, sucrose, and lactose can be used as molding agents when freeze-dried. In this case, a sufficient amount of glucose or glycerin to prepare an isotonic solution may be included in the pharmaceutical preparation, and ordinary solubilizers, analgesics, local anesthetics, etc., may also be added. Subcutaneous, intramuscular, and intravenous injectable preparations can be manufactured by conventional methods after adding these carriers.

[0045] When the above preparation is an intravenous drip, it can be prepared by dissolving the administered compound in an isotonic electrolyte infusion preparation based on physiological saline, Ringer's solution, or the like.

[0046] When the dosage of the composition is administered systemically, it is 1×10 per 1 kg of body weight in terms of viral amount 9 vg / dose to 1×10 15 vg / dose. Preferably, it is 5×10 per 1 kg of body weight in terms of viral amount 9 vg / dose to 5×10 12 vg / dose, more preferably 5×10 per 1 kg of body weight in terms of viral amount 9 vg / dose to 5×10 11 vg / dose. The number of administrations of the composition can be appropriately set according to the immune response of the individual to whom the composition is administered against the viral antigen derived from the viral vector. The administration of the composition may be performed once, or may be performed twice. If the immune response is within an allowable range, the administration may be performed three or more times.

[0047] When the composition is administered locally, it is 1×10 per 1 cm of target tissue in terms of viral amount 2 9 vg / dose to 1×10 15 vg / dose. Preferably, it is 5×10 9 vg / dose to 5×10 11 vg / dose, more preferably 5×10 per 1 kg of body weight in terms of viral amount 9 vg / dose to 5×10 10 vg / dose. The administration of the composition may be performed once, or may be performed twice. If the immune response is within an allowable range, the administration may be performed three or more times.

[0048] ​The favin protein expressed from the polynucleotide contained in the composition is preferably present in the liver and blood. The favin protein expressed from the polynucleotide is present in the blood for at least two weeks, preferably three weeks, more preferably seven weeks, even more preferably eleven weeks, and most preferably fourteen weeks. For example, even at the 14th week, it is preferable that the favin protein expressed from the polynucleotide is present in the blood at a concentration of, for example, 100 pg / ml or more, preferably 500 pg / ml or more, more preferably 1,000 pg / ml or more, and even more preferably 10,000 pg / ml or more.

[0049] 3. Test Animals One embodiment of the present invention relates to non-human test animals administered a viral vector expressibly containing a polynucleotide encoding favin protein. The test animals have favin protein expressed from the polynucleotide in their blood. The favin protein expressed from the polynucleotide is present in the blood for at least two weeks, preferably three weeks, more preferably seven weeks, even more preferably eleven weeks, and most preferably fourteen weeks. For example, even at the fourteenth week, it is preferable that the amount of favin protein expressed from the polynucleotide present in the blood is, for example, 100 pg / ml or more, preferably 500 pg / ml or more, more preferably 1,000 pg / ml or more, and even more preferably 10,000 pg / ml or more.

[0050] The test animals are not limited as long as they are not human. Examples include mice, rats, rabbits, guinea pigs, dogs, cats, and monkeys. The test animals may be wild-type animals, genetically modified animals, and their offspring. Examples of genetically modified animals include Favine gene-deficient mice, apolipoprotein E gene-deficient mice, and double-deficient mice (Favine gene and apolipoprotein E gene) resulting from crossing these two. Other examples of test animals include low-density lipoprotein (LDL) receptor gene-deficient mice, db / db mice and ob / ob mice (type 2 diabetes model mice), and klotho gene-deficient mice.

[0051] Test animals can be used for drug screening tests, efficacy tests, and as animal models of disease conditions.

[0052] The inventions disclosed herein will be described in more detail below with reference to examples, but the inventions disclosed herein are not to be construed as being limited to the examples.

[0053] Furthermore, the animal experiments disclosed in this embodiment were conducted with the approval of the Osaka University Animal Experiment Ethics Committee.

[0054] 1. Genetically modified mice and rearing: Favine gene knockout mice (hereinafter referred to as "Favine KO mice") were produced by Unitech Co., Ltd., using target disruption of the Favine gene. Details of the production method are reported by S. Kobayashi et al. (Reference: The Journal of Biological Chemistry Vol. 290, No. 12, pp. 7443-7451, 2015).

[0055] The apolipoprotein E gene-deficient mice (hereinafter referred to as "ApoE KO mice"), a model mouse for atherosclerosis, were purchased from Jackson Laboratory, Bar Harbor, ME, based on the mice reported by Y. Okamoto et al. (Reference: Circulation Vol. 106, p. 2767-2770, 2002).

[0056] Mice lacking both the Favine gene and the apolipoprotein E gene (hereinafter referred to as "Favine / ApoE DKO mice") were created by crossing Favine KO mice with ApoE KO mice.

[0057] Each mouse was housed in an animal enclosure with normal temperature control and a 12-hour light-dark cycle.

[0058] 2. Production of Favine-AAV3 Adeno-associated virus serotype 8 containing the mouse Favine-FLAG sequence was produced by the following method. Hereinafter, adeno-associated virus serotype 8 may be referred to as "AAV," and adeno-associated virus serotype 8 containing the mouse Favine-FLAG sequence may be referred to as "Favine-AAV."

[0059] Favine-AAV was obtained by transfecting the cultured cell line Viral Production Cells 2.0 (Thermo Fisher Scientific) with a Favine expression plasmid containing HCRhAAT (enhancer element of the hepatic control region of the ApoE / C1 gene and the human antitrypsin promoter) and the mouse Favine-FLAG sequence, and then obtaining Favine-AAV from the transfected cells according to the manufacturer's protocol. Control AAV was obtained from Viral Production Cells 2.0 that had not been transfected with the Favine expression plasmid. The AAV produced from Viral Production Cells 2.0 was purified using Poros CaptureSelect AAVX, followed by ultracentrifugation to obtain Favine-AAV and control AAV. Titer analysis of Favine-AAV was performed by digital PCR. Particle concentration of control AAV was measured by ultracentrifugation analysis.

[0060] 3. Measurement of Plasma Favine Concentration in Favine-AAV-Infected Mice Wild-type C57 / BL6J mice were administered either Favine-AAV or control AAV via the tail vein at a dose of 10^12 vg / mouse. Plasma Favine concentrations were measured using the Favine ELISA assay system. The results are shown in Figure 2. Plasma Favine concentrations in Favine-AAV-administered mice were confirmed to persist for 80 weeks.

[0061] 4. Evaluation of the effect of Favine on thrombus formation: 3 × 10¹⁶ samples were taken from the tail vein of 10-week-old Favine / ApoE DKO mice. 10 Favine-AAV or control AAV was administered to create a vg / mouse configuration. Three weeks after AAV infection, the left carotid artery was ligated with silk thread. Two weeks after ligation, the carotid artery was excised, fixed with formalin, and then thin sections were prepared. These sections were stained with hematoxylin and eosin (HE) and the tissue was evaluated.

[0062] The results are shown in Figure 3. Figure 3a shows a histological image of the carotid artery ligation site 400 μm away from the ligation site of a Favine / ApoE DKO mouse administered with control AAV. The plasma Favine concentration at the time of dissection of this mouse was 0 pg / ml. Figure 3b shows a histological image of the carotid artery ligation site of a Favine / ApoE DKO mouse administered with Favine-AAV at a dose of 3 × 10^10 vg / mouse. The plasma Favine concentration at the time of dissection of this mouse was 2996 pg / ml. Figure 3c shows a histological image of the carotid artery ligation site of a Favine / ApoE DKO mouse administered with Favine-AAV at a dose of 1.5 × 10^10 vg / mouse. The plasma Favine concentration at the time of dissection of this mouse was 400 pg / ml. Thrombosis due to ligation was observed in Favine / ApoE DKO mice administered with control AAV. On the other hand, in Favine / ApoE DKO mice administered Favine-AAV, almost no thrombus formation was observed at the ligation site regardless of the dosage. These results indicate that Favine-AAV administration suppresses thrombus formation. Furthermore, it was shown that even with low doses of Favine-AAV, blood Favine concentrations were maintained, and a thrombus-suppressing effect was achieved.

[0063] 5. Evaluation of the effect of Favine on weight gain. Favine KO mice were administered Favine-AAV or control AAV via the tail vein at 15 weeks of age. The amount of AAV administered was 10 × 10 for each type of AAV. 10 vg / mouse, or 10x10 11 The mice were administered vg / mice. Body weight was measured over time after AAV administration, and finally, 14 weeks after administration, the mice were dissected and the weight of adipose tissue was measured.

[0064] The results are shown in Figure 4. In Figure 4, “Control” refers to mice administered with control AAV, and “Favine” refers to mice administered with Favine-AAV. Figure 4(A) shows the change in body weight after AAV administration. Mice administered with Favine-AAV showed a greater increase in body weight than mice administered with control AAV three weeks after administration. Furthermore, a higher viral load resulted in a more pronounced increase in body weight, and a sustained increase in body weight over time was observed. Figure 4(B) shows the plasma concentration of Favine in this experimental system. Regardless of the Favine-AAV dosage, plasma Favine concentrations showed a gradual decreasing trend from three weeks after administration, but were maintained even at 14 weeks.

[0065] Figure 4(C) shows the weight of adipose tissue around the epididymis and subcutaneously at the time of dissection. Mice administered with Favine-AAV showed increased adipose tissue weight in both the epididymal and subcutaneous regions compared to mice administered with control AAV. This indicates that administration of Favine leads to an increase in body weight and adipose tissue.

[0066] Generally, the effective viral dose to mice when using AAV is 1 × 10⁻⁶ 12 Although the recommended dosage is vg / mouse, this example demonstrates that Favine administration can be effective even with a lower viral load than previously reported.

[0067] 6. Reference Examples (1) Favine Construct Using Adenovirus Vectors Favine adenovirus was used to infect BL mice, and the levels of Favine protein in the blood and liver were evaluated using Western Blot. The levels of Favine protein in the blood correlated with liver function and were thought to be leaks associated with liver damage, making it unsuitable for a blood Favine expression system. Favine plasmids were rapidly injected intravenously using the hydrodynamics method, and the levels of Favine protein in the blood and liver were evaluated using Western Blot. The levels of Favine protein in the blood correlated with liver function and were thought to be leaks associated with liver damage, making it unsuitable for a blood Favine expression system. On the other hand, as mentioned above, the blood Favine concentration was maintained with adeno-associated virus vectors, indicating that the use of adeno-associated virus vectors is preferable for in vivo expression of Favine.

[0068] (2) Evaluation of the platelet aggregation inhibitory effect of Favine To evaluate the effect of Favine on platelet aggregation, recombinant Favine peptide was used to evaluate its effect on platelet aggregation.

[0069] i. Method Recombinant mouse Favine was prepared by creating a transient expression strain of mouse Favine, collecting the culture supernatant, and preparing it as a conditioned medium. The mouse Favine expression vector was constructed by inserting a polynucleotide (mouse Favine-FLAG) with a FLAG tag attached downstream of the full-length cDNA of the coding region of mouse Favine (CCDC3, NM_028804.1) into a pcDNA3.1(+) vector. Hereafter, this construct will be referred to as pcDNA3.1 mouse Favine-FLAG.

[0070] pcDNA3.1 mouse Favine-FLAG was transfected into HEK293 cells using Lipofectamine 3000 (Thermo Fisher Scientific), and cultured overnight in DMEM medium supplemented with 10% fetal bovine serum. The medium was then replaced with DMEM medium supplemented with 0.5% bovine serum albumin, and the cells were cultured for two days. The culture supernatant was collected, filtered through a 0.45 μm filter, and the filtrate was used as the conditioned medium for mouse Favine. In addition, to prepare a mock medium, an empty pcDNA3.1 vector was transfected into HEK293 cells in the same manner as pcDNA3.1 mouse Favine-FLAG, and the culture supernatant was collected after following the same culture procedure as the cells that had been transfected with pcDNA3.1 mouse Favine-FLAG.

[0071] To prepare platelet-rich plasma (PRP), venous blood was collected from adult males using sodium citrate as an anticoagulant. The collected blood was centrifuged at room temperature for 10 minutes at 250g, and the supernatant was collected to obtain PRP.

[0072] 100 μl of PRP was added to a glass cuvette for platelet aggregation, and 35 μl of conditioned medium or mock medium was added to each cuvette. The cuvette was then pretreated at 37°C for 20 minutes. Subsequently, 15 μl each of PAR1-AP (Protease activated receptor 1-activating peptide) solutions prepared at 200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM were added to the cuvette, and the light transmittance was monitored for 5 minutes using a platelet aggregation meter, with the time of PAR1-AP addition set to 0 minutes. The platelet aggregation rate was calculated with the light transmittance of a state where platelets were not aggregated set to 0%, and platelet-poor plasma (PPP) without platelets set to 100%. PPP was prepared by centrifugation of blood collected using sodium citrate at 3,000 g for 10 minutes at room temperature, and collection of the supernatant.

[0073] ii. Results Figure 5 shows the platelet aggregation rate. Figure 5(A) shows the platelet aggregation rate when mock medium is added. Figure 5(B) shows the platelet aggregation rate when conditioned medium is added. In Figure 6, the symbols a and a' represent the platelet aggregation rate when 200 μM of PAR1-AP is added, the symbols b and b' represent the platelet aggregation rate when 100 μM of PAR1-AP is added, the symbols c and c' represent the platelet aggregation rate when 50 μM of PAR1-AP is added, the symbols d and d' represent the platelet aggregation rate when 25 μM of PAR1-AP is added, and the symbols e and e' represent the platelet aggregation rate when 12.5 μM of PAR1-AP is added.

[0074] When conditioned medium was added, platelet aggregation was suppressed when PAR1-AP was added at 50 μM or 25 μM compared to when mock medium was added (symbols c→c', d→d'). When PAR1-AP was added at 200 μM or 100 μM, no platelet aggregation inhibitory effect was observed, which was thought to be due to the excessive amount of PAR1-AP added.

[0075] These results suggest that Favine has an inhibitory effect on platelet aggregation.

Claims

1. A composition comprising a viral vector capable of expressing a polynucleotide encoding a favin protein, wherein the viral vector is an adeno-associated virus vector.

2. The composition according to claim 1, wherein the composition is used for at least one of the following purposes: to prevent atherosclerotic disease, to prevent thrombus formation, to prevent platelet aggregation, and to increase the body weight of an animal to be administered, to suppress weight loss in an animal to be administered, to increase adipose tissue, and / or to suppress the decrease in adipose tissue.

3. The composition according to claim 2, wherein the arteriosclerotic disease is arteriosclerosis, ischemic heart disease, ischemic cerebrovascular disease, or ischemic bowel disease.

4. A viral vector containing polynucleotides encoding favin protein capable of expression, 1 x 10⁶ per kg of body weight. ９ vg / times ~ 1×10 １５ The composition according to claim 1, administered in a dose of vg / dose.

5. A non-human test animal that has been administered a viral vector capable of expressing a polynucleotide encoding favin protein, wherein the test animal has favin protein expressed from the polynucleotide in its blood.

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