Fibroblast growth factor 21 (FGF21) gene therapy

Abstract

To provide a gene construct for use in the treatment of a metabolic disorder in a mammal, particularly a human.SOLUTION: A gene construct provided comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) for use in the treatment and / or prevention of a metabolic disorder, wherein the therapy involves expression of the gene construct in the central nervous system (CNS), preferably in the brain, more preferably in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, and most preferably in the hypothalamus.SELECTED DRAWING: None

Description

[Background technology]

[0001] The prevalence of diabetes is increasing at an alarming rate and is a major health problem worldwide. Obesity is strongly associated with insulin resistance and type 2 diabetes (T2D) (Moller ,DE,and Flier,JS,1991.N.Engl.J.Med.3 25:938-948). Both T2D and obesity increase the risk of death (Pe eters,A.et al.,2003.Ann.Intern.Med.138:2 4-32), highly morbid chronic diseases including cardiovascular disease, hypertension and certain types of cancer. Increased risk (Haslam, DW et al., 2005, Lancet. 36 6,1197-1209;Roberts,DLet al.,2010,Annu .Rev.Med.61,301-316). Insulin resistance and obesity-related diseases are It is subsequently associated with reduced life expectancy and quality of life.

[0002] During obesity, peripheral tissues such as adipose tissue, liver, or skeletal muscle are involved in the development of insulin resistance. It is now widely accepted that there is chronic low-grade inflammation that can cause metabolic dysfunction, including (Valdearcos, M. et al., 2015, Annu. Rev. Ph ysiol.77,131-160;Hotamisligil,GSet al. ,2017,Nature.542,177-185). In recent years, obesity and insulin resistance have become a major concern. A growing body of literature demonstrates that insulin resistance is also associated with inflammation in the brain (Guildwell, 2004). lemot-Legris,O.et al.,2017,Trends Neuros ci.40,237-253;Beilharz,JEet al.,2016,B ehav. Brain Res. 306, 1-7). Furthermore, in animal models and humans Obesity and insulin resistance are associated with cognitive impairment as well as neuroinflammation. (Guillemot-Legris, O. et al., 2017, Trends Neurosci.40,237-253).

[0003] A growth factor secreted primarily by the liver, but also by adipose tissue and the pancreas Fibroblast growth factor 21 (FGF21) (Muise, ES et al., 20 08.Mol.Pharmacol.74:403-412) is a brown adipose tissue (BAT) ) proliferation and expression of thermogenic genes in BAT and white adipose tissue (WAT) It has been shown that it stimulates energy expenditure (Coskun, T. et al. ,2008.Endocrinology 149:6018-6027;Fisher ,FMet al.,2012.Genes Dev.26:271-281;Kh aritonenkov,A.et al.,2005.J.Clin.Invest 115:1627-1635;Konishi,M.et al.,2000.J.Bi ol.Chem.275:12119-12122;Tomlinson,E.et a l.,2002.Endocrinology 143:1741-1747;Xu,J et al., 2009. Diabetes 58:250-259).

[0004] The native FGF21 protein exhibits poor pharmacokinetic characteristics, due to its short half-life. and is susceptible to in vivo proteolysis and in vitro aggregation (Huang, J. et al.,2013.J Pharmacol Exp Ther.346(2): 270-80;So,WYand Leung,PS2016.Med Res Rev.36(4):672-704;Zhang,J.and Li,Y.2015 Front Endocrinol (Lausanne). 6:168). Extending half-life Various engineering methods have been developed to improve the stability and solubility of FGF21. Currently, two engineered FGF21 mimetics (LY2405319 and and PF-05231023) have been tested. Nevertheless, FGF21 mimetics This requires multiple administrations, which places a heavy burden on patients. F21 mimetics / analogs may present a higher immunogenicity risk than native FGF21, e.g. For example, patients treated with LY2405319 experienced injection site reactions, anti-drug antibodies, and severe A severe hypersensitivity reaction occurred (Gaich, G. et al., 2013. Cell Met ab.18(3):333-40). Thus, a single administration of the vector of the present invention The long-lasting and effective effects achieved represent a significant advantage over other therapies.

[0005] Neuroinflammation may contribute to the cognitive decline and overall energy and metabolic decline observed in diabetes and obesity. Given the importance of its likely role in glucose metabolism, New therapeutic approaches to address central nervous system (CNS) inflammation may be of critical importance. The study showed that in the CNS, specifically the hypothalamus, the main part of the brain that regulates whole-body energy metabolism, that the peripheral metabolic effects of FGF21 may indeed be mediated by FGF21 signaling; (DASarruf et al., Diabetes. 59, 181 7-1824(2010);ALBookout et al., Nat.Med. 19,1147-1152(2013);BMOwen et al.,Cell Metab.20,670-677(2014);N.Douris et al.,E ndocrinology.156,2470-2481(2015). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Moller, DE, and Flier, JS, 1991. N. Engl. J. Med. 325:938-948 [Non-patent document 2] Peeters,A.et al.,2003.Ann.Intern.Med.138:24-32 [Non-patent document 3] Haslam,DWet al.,2005,Lancet.366,1197-1209 [Non-patent document 4] Roberts,DLet al.,2010,Annu.Rev.Med.61,301-316 [Non-patent document 5] Valdearcos,M.et al.,2015,Annu.Rev.Physiol.77,131-160 [Non-patent document 6] Hotamisligil,GSet al.,2017,Nature.542,177-185 [Non-Patent Document 7] Guillemot-Legris,O.et al.,2017,Trends Neurosci.40,237-253 [Non-licensed document 8] Beilharz,JEet al.,2016,Behav.Brain Res.306,1-7

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[0007] Aspects of the present disclosure relate to gene therapy methods for use in treating metabolic disorders in mammals, particularly humans. The present invention relates to the medical field, including compositions. [Means for solving the problem]

[0008] In a first aspect, a method for producing a fibroblast growth factor 21 (FGF21)-containing antibody for use in therapy is provided. a gene construct comprising a nucleotide sequence encoding a gene for a central nervous system (CNS) , preferably in the brain, more preferably in the hypothalamus, comprising a gene construct for expression of the gene construct. In some embodiments, a method for treating a metabolic disorder is provided. A genetic construct comprising a nucleotide sequence encoding growth factor 21 (FGF21), The therapy is directed to the central nervous system (CNS), preferably the brain, more preferably the hypothalamus and / or or the cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the visual cortex A genetic construct is provided that includes expression of the genetic construct within the subfloor.

[0009] Preferably, the nucleotide sequence encoding FGF21 is expressed in a ubiquitous promoter. In a preferred embodiment, the ubiquitous promoter is CAG promoter and CMV promoter, preferably ubiquitous The promoter is a CAG promoter. Preferably, the nucleotide sequence encoding FGF21 is The octide sequence is inserted into the ubiquitous promoter and prevents expression of FGF21. operably linked to at least one target sequence of a microRNA expressed in a desired tissue It has been done.

[0010] Preferably, at least one target sequence of the microRNA is located in the mammalian heart and / or Alternatively, the target sequence is selected from target sequences that bind to microRNAs expressed in the liver.

[0011] More preferably, the nucleotide sequence encoding FGF21 is a ubiquitous promoter. and at least one target sequence of a microRNA expressed in the liver, and operably linked to at least one target sequence of a microRNA expressed in the heart. There are.

[0012] Preferably, the target sequences of the microRNAs expressed in the heart are SEQ ID NOs: 13 and 21 25, and the target sequences of the liver-expressed microRNAs are SEQ ID NOs: 12 and 1 Select from 4 to 20.

[0013] More preferably, the gene construct comprises a target sequence of microRNA-122a and a target sequence of microRNA-122b. It contains the target sequence of RNA-1.

[0014] Preferably, the ubiquitous promoter is a CAG promoter or a CMV promoter. Preferably, the ubiquitous promoter is a CAG promoter. be.

[0015] Preferably, the nucleotide sequence encoding FGF21 is selected from the group consisting of: Can be: (a) having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, or 3; a nucleotide sequence encoding a polypeptide comprising an amino acid sequence (b) a nucleotide sequence at least identical to SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11 nucleotide sequences having 60% sequence identity with each other; and (c) a nucleotide sequence whose sequence differs from that of (b) due to the degeneracy of the genetic code Reotide sequence.

[0016] In a second aspect, an expression vector comprising the genetic construct according to the first aspect for use in therapy. The vector, wherein the therapy is a gene targeting vector in the CNS, preferably the brain, more preferably the hypothalamus. In some embodiments, an expression vector is provided that comprises the expression construct of a metabolic disorder. 1. An expression vector comprising the genetic construct according to the first aspect for use in therapy, The therapy is directed to the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or or genes in the hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus An expression vector containing the expression construct is provided. Preferably, the expression vector is a viral vector. He is a ctor.

[0017] Preferably, the expression vector is an adenovirus vector or an adeno-associated virus vector. Preferably, the vector is selected from the group consisting of retroviral vectors and lentiviral vectors. Alternatively, the expression vector is an adeno-associated virus vector.

[0018] Preferably, the expression vector is an avian serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10 , rh8, Cb4, rh74, DJ, 2 / 5, 2 / 1, 1 / 2 or Anc80 adenoviruses More preferably, the expression vector is an associated viral vector of serotype 1, 2 or 9 adeno-associated virus vector.

[0019] In a third aspect, a compound of formula (I) comprising: comprising a genetic construct according to the first aspect and / or an expression vector according to the second aspect A pharmaceutical composition, wherein the therapy comprises expression of a gene construct in the CNS and / or brain. In some embodiments, pharmaceutical compositions are provided comprising: a genetic construct according to the first aspect and / or A pharmaceutical composition comprising an expression vector according to the second aspect, wherein the therapy is directed against CNS, preferably More preferably, the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or is a pharmaceutical agent comprising expression of a gene construct in the cerebellum and / or olfactory bulb, most preferably in the hypothalamus. A composition is provided.

[0020] In a fourth aspect, a genetic construct and / or expression vector and / or pharmaceutical composition The gene construct and the method for use according to the first aspect, wherein the gene construct and the method for use according to the first aspect are administered by intra-CSF administration. and / or an expression vector for use according to the second aspect and / or according to the third aspect. Pharmaceutical compositions for the above uses are provided.

[0021] In a fifth aspect, preferably the metabolic disorder is diabetes and / or obesity. A genetic construct for use according to the first aspect for use in the treatment and / or prevention of Constructs and / or expression vectors for use according to the second aspect and / or third aspect Pharmaceutical compositions for use according to the aspects are provided.

[0022] overview The present inventors have investigated the effects of steroids on the central nervous system (CNS) to suppress obesity and / or diabetes. We have developed an improved gene therapy strategy based on FGF21 gene therapy, specifically directed against the As detailed in the experimental section, the present inventors have demonstrated the efficacy of brain-directed FGF21 gene therapy. We found the following unexpected benefits: The gene constructs and vectors described herein induce potent and widespread hyperactivity in the brain. Expression can be obtained (Examples 1, 2, 3 and 4).

[0023] The genetic constructs and vectors described herein reduce adipocyte size and promote brown adipose tissue production. Reduces fat storage in adipocytes, increases thermogenesis, and reduces circulating triglycerides and free fat Reduces acidity and improves pancreatic health (increases islet number and improves beta cell mass) , reducing systemic inflammation (or reducing inflammatory factors such as F4 / 80, IL-6, and TNFα) cytokines) (Example 1.1).

[0024] Expression of FGF21 in the brain in widely used mouse models of obesity and diabetes However, it significantly reduced body weight gain, body fat accumulation, and liver weight, and completely normalized blood glucose during feeding. Normalized (Example 1), improved insulin resistance, improved glucose tolerance, and reduced gluconeogenesis (Example 4).

[0025] In a widely used aging mouse model with age-related brain pathology, FGF21 in the brain Expression of α-glucan significantly reduced body weight gain and liver weight (Example 2).

[0026] In both mouse models, inflammation in the hypothalamus is reduced (Examples 1 and 2).

[0027] Accordingly, the aspects and embodiments of the present invention described herein are This invention solves at least some of the problems and needs that exist.

[0028] Gene constructs In a first aspect, a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) is provided. A genetic construct containing the sequence is provided.

[0029] A "genetic construct" as described herein is one that will be understood by those skilled in the art in light of the present disclosure. "Genetic construct" has its customary and ordinary meaning. A target gene is also called a "gene construct" and is operably linked to a promoter that controls its expression. "General Information" refers to a gene or group of genes including the gene encoding the target protein. The section of this application entitled "Genetic Constructs" contains further details regarding "genetic constructs." The term "operably linked" as used herein is further explained in the section of this application entitled "General Information." It will be revealed.

[0030] In some embodiments, the genetic constructs described herein are suitable for expression in mammals. As used herein, "suitable for expression in a mammal" means A genetic construct operably linked to the nucleotide sequence to be expressed is used for expression. This may mean including one or more regulatory sequences selected based on the mammalian host cell to be used. Preferably, the mammalian host cells used for expression are human, murine or canine cells. is.

[0031] In some embodiments, the genetic constructs described herein are intended for use in therapy. In a preferred embodiment, the genetic constructs described herein are used for the treatment of metabolic disorders and In a preferred embodiment, the therapy is for use in the treatment and / or prophylaxis of CNS , preferably involving expression of the gene construct in the brain, and more preferably in the hypothalamus. In embodiments, expression of the gene construct in the brain includes expression in the hypothalamus and / or cortex and / or or a genetic construct in the hippocampus and / or cerebellum and / or olfactory bulb, preferably in the hypothalamus Therefore, expression of a gene construct in the brain can refer to expression of the gene construct in the hypothalamus, cortex, and cerebral cortex. At least one or at least two selected from the group consisting of the olfactory bulb, the cerebellum, and the olfactory bulb. may refer to expression of a gene construct in at least three or all brain regions. In embodiments, the therapy comprises expression of a gene construct in the hypothalamus. , CNS and / or brain and / or hypothalamus and / or cortex and / or nasal passages Expression in the equine and / or cerebellum and / or olfactory bulb refers to the CNS and / or brain and and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or In one embodiment, expression may refer to specific expression in the liver, pancreas, adipose tissue, or the olfactory bulb. In some embodiments, expression is in the liver, skeletal muscle, and / or heart. At least one selected from the group consisting of liver, pancreas, adipose tissue, skeletal muscle and heart C does not contain expression in at least two, at least three, at least four, or all organs. NS-specific expression and / or brain-specific expression and / or hypothalamus-specific expression and / or cortex-specific expression and / or hippocampus-specific expression and / or cerebellum-specific expression and A description of olfactory bulb-specific and / or olfactory bulb-specific expression is provided in the section entitled "General Information."

[0032] Expression can be assessed as described in the section entitled "General Information." "CNS," A description of the "brain" and "hypothalamus" is provided in the section entitled "General Information."

[0033] In some embodiments, the genetic constructs described herein are for use in therapy. The gene construct is administered into the CSF (cerebrospinal fluid) (by cisternal, intrathecal or intraventricular delivery). The preferred administration is intra-CSF administration. It is given.

[0034] As used herein, "intra-CSF administration," "intranasal administration," "intraparenchymal administration," "cisternal administration," "intra-CSF ... "Intrathecal administration," "intrathecal administration," and "intraventricular administration" are defined in the section of this application entitled "General Information." is described in.

[0035] In some embodiments, the genetic constructs described herein are administered to the CNS, preferably the brain, More preferably, it contains a nucleotide sequence encoding FGF21 expressed in the hypothalamus. In some embodiments, the genetic constructs described herein are directed to the CNS, preferably the brain, More preferably, it is suitable for expression in the hypothalamus. Expression of the construct is in the hypothalamus and / or cortex and / or hippocampus and / or subthalamus. It can refer to the expression of a gene construct in the brain and / or olfactory bulb. Expression of the construct is in a region selected from the group consisting of the hypothalamus, cortex, hippocampus, cerebellum and olfactory bulb. Genes in at least one, or at least two, or at least three, or all brain regions Expression in the hypothalamus is most preferred. can be evaluated as described in the section entitled "Methods of Quantitative Analysis of Electron Transfer."

[0036] In the context of embodiments of the present invention, the CNS and / or brain and / or hypothalamus and F expressed in the cortex and / or hippocampus and / or cerebellum and / or olfactory bulb GF21; and CNS and / or brain and / or hypothalamus and / or cortex and and / or a gene construct suitable for expression within the hippocampus and / or cerebellum and / or olfactory bulb. is the CNS and / or brain and / or hypothalamus compared to other organs or tissues and / or FG within the cortex and / or hippocampus and / or cerebellum and / or olfactory bulb Preferential or dominant (at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, At least 70% higher, at least 80% higher, at least 90% higher, at least 10 0% higher, at least 150% higher, at least 200% higher, or more) Other organs or tissues include the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, and hematopoietic The other organs may be tissues, lungs, ovaries, spleen, stomach, testes, etc. Preferably, the other organs are liver and In one embodiment, expression is in the liver, pancreas, adipose tissue, skeletal muscle and / or heart. In some embodiments, expression is not detectable in the liver, pancreas, adipose tissue, or the like. , skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach and testis Select at least one, at least two, at least three, at least four or all Expression is assessed as described in the section entitled "General Information." It can be valued.

[0037] The nucleotide sequence encoding FGF21 present in the genetic construct according to the invention is Any FGF21 gene or FGF21 coding sequence, preferably human, mouse or or a FGF21 gene or FGF21 coding sequence derived from a dog; or preferably a human a mutant FGF21 gene or FGF21 coding sequence derived from a mouse or dog; is preferably a codon-optimized FGF21 gene or FGF21 gene derived from human, mouse or dog. It may be derived from the GF21 coding sequence.

[0038] Thus, in some embodiments, preferred nucleotides encoding FGF21 are The sequence may be at least 60%, at least 61%, at least 62%, or at least 64% identical to SEQ ID NO: 1, 2, or 3. 2%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, At least 76%, at least 77%, at least 78%, at least 79%, at least At least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least At least 89%, at least 90%, at least 91%, at least 92%, at least 93% %, at least 94%, at least 95%, at least 96%, at least 97%, at least Amino acids with at least 98%, at least 99% or 100% identity or similarity SEQ ID NO: 1 encodes a polypeptide comprising the amino acid sequence of human FGF21. SEQ ID NO: 2 represents the amino acid sequence of mouse FGF21. SEQ ID NO: 3 represents the amino acid sequence of canine FGF21. In some embodiments, the gene construct according to the present invention includes a sequence of F21. Nucleotide sequences encoding FGF21 that exist are set forth in SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11. At least 61%, at least 62%, at least 63%, at least 64%, at least 65% , at least 66%, at least 67%, at least 68%, at least 69%, less At least 70%, at least 71%, at least 72%, at least 73%, at least 74% %, at least 75%, at least 76%, at least 77%, at least 78%, at least at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical do.

[0039] The descriptions of "identity" or "sequence identity" and "similarity" or "sequence similarity" are General Information.

[0040] In some embodiments, the human FGF21 present in the genetic construct according to the present invention The encoding nucleotide sequence has at least 60% identity with SEQ ID NO: 4, 5, 6 or 7 at least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, At least 79%, at least 80%, at least 81%, at least 82%, at least At least 83%, at least 84%, at least 85%, at least 86%, at least 87% , at least 88%, at least 89%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, at least 99% or 100% identity SEQ ID NO: 4 is the nucleotide sequence encoding human FGF21. 5 is a codon-optimized nucleotide sequence encoding human FGF21, variant 1. Sequence number 6 is the codon-optimized nucleotide sequence encoding human FGF21, variant 2. SEQ ID NO: 7 is a codon-optimized nucleotide sequence encoding human FGF21, variant 3. Mutant 1, Mutant 2, and Mutant 3 encode the same human FGF21 protein. These are sequences obtained by different codon optimization algorithms. A description of the "chemical synthesis" is provided in the section entitled "General Information."

[0041] In some embodiments, the mouse FGF21 present in the genetic construct according to the present invention The nucleotide sequence encoding the 61%, at least 62%, at least 63%, at least 64%, at least 65%, At least 66%, at least 67%, at least 68%, at least 69%, at least At least 70%, at least 71%, at least 72%, at least 73%, at least 74% , at least 75%, at least 76%, at least 77%, at least 78%, less At least 79%, at least 80%, at least 81%, at least 82%, at least 83% %, at least 84%, at least 85%, at least 86%, at least 87%, at least at least 88%, at least 89%, at least 90%, at least 91%, at least 9 2%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical SEQ ID NO: 8 is the nucleotide sequence encoding mouse FGF21. SEQ ID NO: 9 is , is the codon-optimized nucleotide sequence encoding mouse FGF21.

[0042] In some embodiments, the canine FGF21 present in the genetic construct according to the present invention The encoding nucleotide sequence is at least 60% identical to SEQ ID NO: 10 or 11. At least 61%, at least 62%, at least 63%, at least 64%, at least 65% , at least 66%, at least 67%, at least 68%, at least 69%, less At least 70%, at least 71%, at least 72%, at least 73%, at least 74% %, at least 75%, at least 76%, at least 77%, at least 78%, at least at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical SEQ ID NO: 10 is the nucleotide sequence encoding canine FGF21. 1 is the codon-optimized nucleotide sequence encoding canine FGF21.

[0043] In some embodiments, the nucleotide sequence encoding FGF21 is from the group consisting of: Provided is a genetic construct as described herein selected from: (a) an amino acid sequence identical to that of SEQ ID NO: 1, 2, or 3, at least 60%, at least 61% , at least 62%, at least 63%, at least 64%, at least 65%, less At least 66%, at least 67%, at least 68%, at least 69%, at least 70% %, at least 71%, at least 72%, at least 73%, at least 74%, at least at least 75%, at least 76%, at least 77%, at least 78%, at least 7 9%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99% or 100% sequence identity or similarity A nucleotide sequence encoding a polypeptide comprising an amino acid sequence having the properties of:

[0044] (b) a nucleotide sequence at least identical to SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11 At least 60%, at least 61%, at least 62%, at least 63%, at least 64% %, at least 65%, at least 66%, at least 67%, at least 68%, at least at least 69%, at least 70%, at least 71%, at least 72%, at least 7 3%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, At least 87%, at least 88%, at least 89%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, at least 99% or 1 Nucleotide sequences with 00% sequence identity.

[0045] (c) a nucleotide sequence whose sequence differs from that of (b) due to the degeneracy of the genetic code Reotide sequence.

[0046] In a preferred embodiment, the nucleotide sequence encoding FGF21 is a codon-optimized nucleotide sequence. Nucleotide sequences, preferably selected from the sequences of SEQ ID NOs: 5, 6 and 7. Don-optimized human sequences.

[0047] FGF21 encoded by the nucleotide sequences described herein is a polypeptide known to those skilled in the art. As described above, FGF2 exerts at least a detectable level of activity of FGF21. The activity of 1 refers to anti-obesity and / or The activity of FGF21 may also increase insulin sensitivity. This activity can be measured by methods known to those skilled in the art, for example, by measuring insulin activity. This can be assessed by using a glucose tolerance test or an iron tolerance test.

[0048] In some embodiments, the nucleotide sequence encoding FGF21 is a ubiquitous A preferred ubiquitous promoter is the CMV promoter. The promoter is selected from the group consisting of a CAG promoter and a CAG promoter, preferably a CAG promoter. In some embodiments, the nucleotide sequence encoding FGF21 is a ubiquitous promoter. The motor and myelin expressed in tissues where FGF21 expression is desired to be prevented. The cloRNA is operably linked to at least one target sequence of the cloRNA.

[0049] "Ubiquitous promoter," "operably linked to," and "microRNA" A description of "organization" is provided in the section entitled "General Information." "Target sequence of microRNA expressed in tissue" or "binding to microRNA expressed in tissue" The term "target sequence that binds to a target sequence" or "tissue-expressed microRNA binding site" refers to a target sequence that binds to a target sequence or a tissue-expressed microRNA binding site. As described elsewhere, at least some of the microRNAs expressed in the tissues Refers to a complementary or partially complementary nucleotide sequence.

[0050] In some embodiments, at least one target sequence of the microRNA is a target sequence of a mammal. Selected from target sequences that bind to microRNAs expressed in the heart and / or liver .

[0051] In some embodiments, the nucleotide sequence encoding FGF21 is a ubiquitous promoter and at least one target sequence of a microRNA expressed in the liver. and operably linked to at least one target sequence of a microRNA expressed in the heart. It has been done.

[0052] As used herein, "target sequence of a liver-expressed microRNA" or "liver "Target sequence that binds to microRNA expressed in the liver" or "microRNA expressed in the liver" The "binding site of the microRNA" is complementary to or binds to at least a portion of a microRNA expressed in the liver. Similarly, as used herein, "heart-derived" refers to a nucleotide sequence that is partially complementary to the nucleotide sequence of the target gene. "target sequence of microRNA expressed in the heart" or "target sequence of microRNA that binds to microRNA expressed in the heart" The "target sequence" or "binding site of a cardiac-expressed microRNA" refers to a sequence expressed in the heart. a nucleotide sequence complementary or partially complementary to at least a portion of a microRNA Point.

[0053] As described herein, a portion of the microRNAs expressed in the liver or the heart The portion of the microRNAs that is expressed includes at least four of the microRNAs, at least means a nucleotide sequence of 5, at least 6, or at least 7 consecutive nucleotides The binding site sequence is perfectly complementary to at least a portion of the expressed microRNA. This means that the sequence is perfectly matched without any mismatches. Alternatively, the binding site sequence may be a sequence that is specific to at least one of the expressed microRNAs. It may be partially complementary to a portion of the nucleotide sequence, which may be present in 4, 5, 6, or 7 consecutive nucleotides. This means that one mismatch can occur. Partially complementary binding sites are preferably , which contains perfect or near-perfect complementarity to the seed region of the microRNA, There can be no mismatches between the seed region of the microRNA and its binding site (perfect match). complementary), or one mismatch per 4, 5, 6, or 7 consecutive nucleotides This means that the seed region of a microRNA can be easily differentiated (almost perfect complementarity). The 5' region of the microRNA, from about nucleotide 2 to about nucleotide 8 of the microRNA The portion described herein preferably comprises the seed region of the microRNA. Target sequences of liver-expressed microRNAs or heart-expressed microRNAs The degradation of messenger RNA (mRNA), including The present invention may be based on the direct translational control (inhibition) of mRNA. or are ultimately utilized by miRNAs in inhibiting the expression of their encoded proteins. It is in no way limited to the routes that can be taken.

[0054] In the context of the present invention, the target sequence that binds to the liver-expressed microRNA is SEQ ID NO: 12 or 14-20 and at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 6 7%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, At least 81%, at least 82%, at least 83%, at least 84%, at least At least 85%, at least 86%, at least 87%, at least 88%, at least 89% , at least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98% %, at least 99% or 100% sequence identity. The amino acid sequence may be replaced by a nucleotide sequence.

[0055] In a preferred embodiment, the target sequence of the liver-expressed microRNA is SEQ ID NO: 12 and at least 60%, at least 61%, at least 62%, at least 63%, At least 64%, at least 65%, at least 66%, at least 67%, at least 68% %, at least 69%, at least 70%, at least 71%, at least 72%, at least at least 73%, at least 74%, at least 75%, at least 76%, at least 7 7%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least At least 95%, at least 96%, at least 97%, at least 98%, at least 99% or by a nucleotide sequence containing a nucleotide sequence with 100% sequence identity In a further embodiment, the amino acid sequence of SEQ ID NO: 12 or 14-20 may be substituted. At least one copy of the target sequence of the expressed microRNA is present in the gene construct of the present invention. In a further embodiment, the present invention relates to a polypeptide expressed in the liver, as set forth in SEQ ID NO: 12 or 14-20. 2, 3, 4, 5, 6, 7 or 8 copies of the target sequence of the microRNA expressed in the present invention In a preferred embodiment, the sequence miRT-122a (SEQ ID NO: 1) is present in the gene construct. No. 12) are present in the gene construct of the present invention. The preferred copy number of the target sequence of the liver-expressed microRNA is four.

[0056] As used herein, the target sequence of a liver-expressed microRNA is a sequence that is known to those skilled in the art. As is known, the target sequences of microRNAs expressed in the liver are at least detectable. The activity of the target sequence of the microRNA expressed in the liver is Binds to its cognate microRNA expressed in the liver and activates the transgene When operably linked, detargeting of transgene expression in the liver This activity can be measured using standard assays known to those skilled in the art, such as qP CR, mRNA or protein by Western blot analysis or ELISA This can be assessed by measuring the level of transgene expression in the liver.

[0057] In the context of the present invention, the target sequence of a microRNA expressed in the heart is SEQ ID NO: 13 or 21-25 and at least 60%, at least 61%, at least 62%, at least 6 3%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, At least 77%, at least 78%, at least 79%, at least 80%, at least At least 81%, at least 82%, at least 83%, at least 84%, at least 85% , at least 86%, at least 87%, at least 88%, at least 89%, at least At least 90%, at least 91%, at least 92%, at least 93%, at least 94% %, at least 95%, at least 96%, at least 97%, at least 98%, at least Nucleotides containing nucleotide sequences with at least 99% or 100% sequence identity It can be replaced by a sequence.

[0058] In a preferred embodiment, the target sequence of the microRNA expressed in the heart is SEQ ID NO: 13 and at least 60%, at least 61%, at least 62%, at least 63%, At least 64%, at least 65%, at least 66%, at least 67%, at least 68% %, at least 69%, at least 70%, at least 71%, at least 72%, at least at least 73%, at least 74%, at least 75%, at least 76%, at least 7 7%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least At least 95%, at least 96%, at least 97%, at least 98%, at least 99% or by a nucleotide sequence containing a nucleotide sequence with 100% sequence identity In a further embodiment, the cardiac At least one copy of the target sequence of the expressed microRNA is present in the gene construct of the present invention. In a further embodiment, the present invention relates to a cardiac target gene as set forth in SEQ ID NO: 13 or 21-25. 2, 3, 4, 5, 6, 7 or 8 copies of the target sequence of the microRNA expressed in the present invention In a preferred embodiment, miRT-1 (SEQ ID NO: 13) is present in the gene construct. 1, 2, 3, 4, 5, 6, 7 or 8 copies of the encoding nucleotide sequence are Present in the gene construct: a preferred copy of the target sequence of a cardiac-expressed microRNA. -The number is 4.

[0059] As used herein, the target sequence of a microRNA expressed in the heart is a sequence known to those skilled in the art. As is known, at least detectable target sequences of microRNAs expressed in the heart The activity of the target sequences of microRNAs expressed in the heart is Binds to its cognate microRNA expressed in the heart and activates the transgene When operably linked, it mediates detargeting of transgene expression within the heart. The activity of the protein can be determined by standard assays known to those skilled in the art, e.g., qPCR, Western blot analysis. Transgene expression in the heart at the mRNA or protein level by analysis or ELISA This can be assessed by measuring the level of expression.

[0060] In some embodiments, the liver-expressed polypeptide of SEQ ID NO: 12 or 14-20 is At least one copy of the target sequence of microRNA and the sequence set forth in SEQ ID NO: 13 or 21-25 and at least one copy of the target sequence of the microRNA expressed in the heart. In a further embodiment, the gene is present in a genetic construct. 2, 3, 4, 5, 6, 7, or 8 copies of the target sequence of the microRNA expressed in the liver were and the target of a microRNA expressed in the heart as set forth in SEQ ID NO: 13 or 21-25. In some embodiments, 2, 3, 4, 5, 6, 7 or 8 copies of the target sequence are present in the gene construct of the present invention. In a further embodiment, a nucleotide sequence encoding miRT-122a (SEQ ID NO: 12) is 1, 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence of miRT-1 (SEQ ID NO: 13) ) and 1, 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence encoding this In a further embodiment, miRT-122a ( 4 copies of the nucleotide sequence encoding miRT-1 (SEQ ID NO: 12) and 4 copies of the nucleotide sequence encoding miRT-1 (SEQ ID NO: 1 3) and four copies of the nucleotide sequence encoding the will be done.

[0061] In some embodiments, a target sequence of a microRNA expressed in the liver and a target sequence of a microRNA expressed in the heart are The target sequence of the microRNA to be used is a sequence of SEQ ID NO: 12 to 25 and / or In some embodiments, the genetic construct is selected from the group consisting of: In this embodiment, the target sequences of microRNAs expressed in the heart are SEQ ID NOs: 13 and 21 to 25. The target sequence of the microRNA expressed in the liver is selected from SEQ ID NOs: 12 and 14 to In some embodiments, the above genetic construct is selected from the group consisting of: The above gene, including the target sequence of chromosomal RNA-122a and the target sequence of microRNA-1, A child construct is provided.

[0062] In some embodiments, the ubiquitous promoter described herein is a CAG promoter. promoter, CMV promoter, mini-CMV promoter, β-actin promoter -, Rous sarcoma virus (RSV) promoter, elongation factor 1α (EF1α) promoter -, early growth response factor-1 (Egr-1) promoter, eukaryotic translation initiation factor 4A (e IF4A) promoter, ferritin heavy chain coding gene (FerH) promoter, ferritin Ricin light chain coding gene (FerL) promoter, glyceraldehyde-3-phosphate dehydrogenase Hydrogenase (GAPDH) promoter, GRP78 promoter, GRP94 promoter Motor, heat shock protein 70 (hsp70) promoter, ubiquitin B promoter promoter, SV40 promoter, β-kinesin promoter, ROSA26 and PGK- 1 promoter.

[0063] In a preferred embodiment, the ubiquitous promoter is a CAG promoter. Therefore, the CAG promoter is suitable for use in the genetic constructs of the present invention. In some embodiments, the CAG promoter is at least partially substituted with SEQ ID NO: 27. at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, At least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77% , at least 78%, at least 79%, at least 80%, at least 81%, less At least 82%, at least 83%, at least 84%, at least 85%, at least 86% %, at least 87%, at least 88%, at least 89%, at least 90%, at least at least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99% or comprises or consists essentially of a nucleotide sequence with 100% sequence identity, It consists of that.

[0064] Another preferred ubiquitous promoter is the cytomegalovirus (CMV) promoter. In some embodiments, the CMV promoter is SEQ ID NO: 28 and at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, At least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73% , at least 74%, at least 75%, at least 76%, at least 77%, less At least 78%, at least 79%, at least 80%, at least 81%, at least 82% %, at least 83%, at least 84%, at least 85%, at least 86%, at least at least 87%, at least 88%, at least 89%, at least 90%, at least 9 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 % sequence identity to a nucleotide sequence comprising, consisting essentially of, or Preferably, the CMV promoter is used in conjunction with an intron sequence. In some embodiments, the intron sequence is at least 60% identical to SEQ ID NO:26. At least 61%, at least 62%, at least 63%, at least 64%, at least 65% , at least 66%, at least 67%, at least 68%, at least 69%, less At least 70%, at least 71%, at least 72%, at least 73%, at least 74% %, at least 75%, at least 76%, at least 77%, at least 78%, at least at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity It comprises, consists essentially of, or consists of a nucleotide sequence having

[0065] Another preferred ubiquitous promoter is the mini-CMV promoter. In some embodiments, the mini-CMV promoter is a promoter sequence identical to SEQ ID NO: 36 and at least 60 %, at least 61%, at least 62%, at least 63%, at least 64%, at least at least 65%, at least 66%, at least 67%, at least 68%, at least 6 9%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, At least 83%, at least 84%, at least 85%, at least 86%, at least At least 87%, at least 88%, at least 89%, at least 90%, at least 91% , at least 92%, at least 93%, at least 94%, at least 95%, at least At least 96%, at least 97%, at least 98%, at least 99% or 100% comprising, consisting essentially of, or consisting of a nucleotide sequence with sequence identity .

[0066] Another preferred ubiquitous promoter is the EF1α promoter. In an embodiment, the EF1α promoter is at least 60% identical to SEQ ID NO: 37, at least 60% identical to SEQ ID NO: 37. 1%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, At least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83% , at least 84%, at least 85%, at least 86%, at least 87%, at least At least 88%, at least 89%, at least 90%, at least 91%, at least 92% %, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99% or 100% sequence identity The term "nucleotide sequence" refers to a sequence of nucleotides comprising, consisting essentially of, or consisting of a nucleotide sequence comprising:

[0067] Another preferred ubiquitous promoter is the RSV promoter. In some embodiments, the RSV promoter has at least 60%, at least 61%, or both of the amino acid sequence of SEQ ID NO: 38. , at least 62%, at least 63%, at least 64%, at least 65%, less At least 66%, at least 67%, at least 68%, at least 69%, at least 70% %, at least 71%, at least 72%, at least 73%, at least 74%, at least at least 75%, at least 76%, at least 77%, at least 78%, at least 7 9%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least Nucleotides with at least 97%, at least 98%, at least 99% or 100% sequence identity. It comprises, consists essentially of, or consists of a nucleotide sequence.

[0068] In some embodiments, the nucleotide sequence encoding FGF21 is a tissue-specific promoter. In a preferred embodiment, the promoter is operably linked to a tissue-specific promoter. Preferably, the promoter is a CNS-specific promoter, more preferably a brain-specific promoter, most preferably a It is often a hypothalamus-specific promoter.

[0069] A description of "tissue-specific promoters" is provided in the section entitled "General Information."

[0070] In some embodiments, the CNS-specific promoter described herein is 1 promoter, neuron-specific enolase (NSE) promoter, calcium / calcium CaMKII promoter, tyrosine thromboxylase (TH) promoter, forkhead box A2 (FOXA2) promoter Motor, α-internexin (INA) promoter, nestin (NES) promoter Tar, glial fibrillary acidic protein (GFAP) promoter, aldehyde dehydrogenase ALDH1 family member L1 (ALDH1L1) promoter, myelin-associated oligodendrocyte The promoter of drocyte basic protein (MOBP), homeobox protein 9 ( HB9 promoter and myelin basic protein (MBP) promoter is selected from the group.

[0071] In some embodiments, the brain-specific promoters described herein are promoters encoding synapsin 1 promoters. Neuron-specific enolase (NSE) promoter, calcium / calcium CaMKII promoter, tyrosine hydrochloride xylase (TH) promoter, forkhead box A2 (FOXA2) promoter tar, α-internexin (INA) promoter, nestin (NES) promoter , glial fibrillary acidic protein (GFAP) promoter, aldehyde dehydrogenase 1 Family member L1 (ALDH1L1) promoter, myelin-associated oligodendrocyte Myelin basic protein (MOBP) promoter and myelin basic protein (M BP) promoters.

[0072] In some embodiments, the hypothalamus-specific promoter is a gonadotropin-releasing hormone. The promoter may be the GnRH promoter.

[0073] In a preferred embodiment, a CNS-specific promoter and / or a brain-specific promoter In some embodiments, the promoter is a synapsin 1 promoter. The target is at least 60%, at least 61%, at least 62%, or at least at least 63%, at least 64%, at least 65%, at least 66%, at least 6 7%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, At least 81%, at least 82%, at least 83%, at least 84%, at least At least 85%, at least 86%, at least 87%, at least 88%, at least 89% , at least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98% %, at least 99% or 100% sequence identity to the nucleotide sequence of the present invention; essentially becoming or consisting of it.

[0074] Another preferred CNS-specific promoter and / or brain-specific promoter is the CNS promoter. at the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter In some embodiments, a calcium / calmodulin-dependent protein kinase II (CaMKII) promoter is at least 60%, at least 6 1%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, At least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83% , at least 84%, at least 85%, at least 86%, at least 87%, at least At least 88%, at least 89%, at least 90%, at least 91%, at least 92% %, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99% or 100% sequence identity The term "nucleotide sequence" refers to a sequence of nucleotides comprising, consisting essentially of, or consisting of a nucleotide sequence comprising:

[0075] Another preferred CNS-specific promoter and / or brain-specific promoter is Glycine max. In some embodiments, the promoter is a glycosaminoglycan (GFAP) promoter. The fibrillary acidic protein (GFAP) promoter is at least 60% identical to SEQ ID NO: 41; At least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69% , at least 70%, at least 71%, at least 72%, at least 73%, less At least 74%, at least 75%, at least 76%, at least 77%, at least 78% %, at least 79%, at least 80%, at least 81%, at least 82%, at least at least 83%, at least 84%, at least 85%, at least 86%, at least 8 7%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence comprising, consisting essentially of, or consisting of a nucleotide sequence having identity.

[0076] Another preferred CNS-specific promoter and / or brain-specific promoter is the Ness In some embodiments, the nestin promoter is 42 and at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, At least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81% , at least 82%, at least 83%, at least 84%, at least 85%, less At least 86%, at least 87%, at least 88%, at least 89%, at least 90% %, at least 91%, at least 92%, at least 93%, at least 94%, at least at least 95%, at least 96%, at least 97%, at least 98%, at least 9 containing or essentially consisting of a nucleotide sequence with 9% or 100% sequence identity Become or consist of it.

[0077] Another preferred CNS-specific promoter is the homeobox protein 9 (HB9) promoter. In some embodiments, the homeobox protein 9 (HB9) promoter The promoter has at least 60%, at least 61%, or at least 62% identity with SEQ ID NO: 43. , at least 63%, at least 64%, at least 65%, at least 66%, less At least 67%, at least 68%, at least 69%, at least 70%, at least 71% %, at least 72%, at least 73%, at least 74%, at least 75%, at least at least 76%, at least 77%, at least 78%, at least 79%, at least 8 0%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least nucleotide sequences with at least 98%, at least 99%, or 100% sequence identity. Muka, then essentially becomes or consists of it.

[0078] Another preferred CNS-specific promoter and / or brain-specific promoter is tyrosine kinase inhibitor (TCI). In some embodiments, the promoter is a tyrosine hydroxylase (TH) promoter. The hydroxylase (TH) promoter is at least 60% identical to SEQ ID NO:44. At least 61%, at least 62%, at least 63%, at least 64%, at least 65% , at least 66%, at least 67%, at least 68%, at least 69%, less At least 70%, at least 71%, at least 72%, at least 73%, at least 74% %, at least 75%, at least 76%, at least 77%, at least 78%, at least at least 79%, at least 80%, at least 81%, at least 82%, at least 8 3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity It comprises, consists essentially of, or consists of a nucleotide sequence having

[0079] Another preferred CNS-specific promoter and / or brain-specific promoter is myeloid In some embodiments, the promoter is a myelin basic protein (MBP) promoter. The methylbasic protein (MBP) promoter is at least 60% identical to SEQ ID NO: 45, at least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, At least 79%, at least 80%, at least 81%, at least 82%, at least At least 83%, at least 84%, at least 85%, at least 86%, at least 87% , at least 88%, at least 89%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, at least 99% or 100% sequence identity It comprises, consists essentially of, or consists of a nucleotide sequence having the following properties:

[0080] In some embodiments, the CNS-specific promoters described herein, brain-specific promoters, Motor and / or hypothalamus-specific promoters are expressed in the CNS and / or brain and and / or directing expression of said nucleotide sequence in at least one cell of the hypothalamus. Alternatively, the promoter may be a promoter of the CNS and / or brain and / or hypothalamus cells. At least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80%, 90% or 100%. Also, CNS-specific promoters and / or brain-specific A promoter, as used herein, refers to a gene that is expressed in a specific region of the CNS and / or brain. or a subset of cells. The CNS-specific promoters and / or brain-specific promoters described also include promoters for the hippocampus, small intestine, and periphery of the brain. At least 10%, 20%, 30%, 4% of cells in the brain, cortex, hypothalamus, and / or olfactory bulb Expression may be induced to 0%, 40%, 60%, 70%, 80%, 90% or 100%. may be assessed as described in the section entitled "General Information."

[0081] As used herein, a promoter (particularly one in which the promoter sequence is most closely related to a given SEQ ID NO) is a The promoters known to those skilled in the art (when described as having a small percentage of identity) are Preferably, the minimum identity percentage with a given SEQ ID NO. Promoters described as having a percentage were evaluated by assays known to those skilled in the art. If so, the nucleotide sequence to which it is operably linked (i.e., at least FGF The transcription of the nucleotide sequence encoding the gene encoding the target gene should be controlled. For example, The assay may include measuring expression of the transgene. Expression may be measured using the methods described in the section entitled "General Information." can be evaluated as described in section 3.

[0082] Additional sequences may be present in the genetic constructs of the present invention. Additional sequences include the inverted terminal repeats (ITR), the SV40 polyadenylation signal (SEQ ID NO: 1), No. 32), rabbit β-globin polyadenylation signal (SEQ ID NO: 33), CMV enhancer Within the context of the present invention, "ITR" refers to a sequence of a nucleotide sequence that is identical to that of the nucleotide sequence ... It is intended to encompass one 5'ITR and one 3'ITR from the AAV genome. A preferred ITR is derived from AAV2 and is shown in SEQ ID NO: 30 (5'ITR) and SEQ ID NO: It is represented by sequence number 31 (3'ITR). Within the context of the present invention, the CMV enhancer The sequence (SEQ ID NO: 29) and the CMV promoter sequence (SEQ ID NO: 28) were combined into two separate sequences. It is contemplated that these may be used as a string or as a single sequence (SEQ ID NO: 34). Each of the additional sequences may be present in a genetic construct according to the invention.

[0083] Nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, etc. Additional nucleotide sequences, such as a sequence of nucleotides, may be inserted into the nucleotide(s) encoding FGF21. The nucleic acid sequence may be operably linked to a nucleotide sequence.

[0084] In some embodiments, a genetic construct comprising a nucleotide sequence encoding FGF21. A substance that inhibits the expression of FGF21 and is expressed in tissues in which it is desired to inhibit the expression of FGF21. Gene constructs are provided that may not include the target sequence of NA.

[0085] Expression vector The genetic constructs described herein can be placed into an expression vector. In another aspect, an expression vector comprising the genetic construct of any of the preceding embodiments. A description of "Expression Vectors" is provided in the section entitled "General Information."

[0086] In some embodiments, the expression vectors described herein are intended for use in therapy. In a preferred embodiment, the expression vectors described herein are used in the treatment of metabolic disorders and In a preferred embodiment, the therapy is for use in the treatment and / or prophylaxis of CNS , preferably in the brain, more preferably in the hypothalamus, a gene construct contained in an expression vector In some embodiments, expression of the gene construct in the brain includes expression of the gene construct in the hypothalamus and and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, preferably It can refer to the expression of a gene construct in the hypothalamus. Thus, the expression of a gene construct in the brain. The term "hypothalamus," "cortex," "hippocampus," "cerebellum," and "olfactory bulb" refers to at least one selected from the group consisting of: or expression of the gene construct in at least two, or at least three, or all brain regions. In a preferred embodiment, the therapy comprises expression of a gene construct in the hypothalamus. In some embodiments, the CNS and / or brain and / or hypothalamus and / or Expression in the cortex and / or hippocampus and / or cerebellum and / or olfactory bulb is S and / or brain and / or hypothalamus and / or cortex and / or hippocampus and In one embodiment, expression can refer to specific expression in the cerebellum and / or olfactory bulb. does not include expression in the liver, pancreas, adipose tissue, skeletal muscle and / or heart. In embodiments, expression is in a tissue selected from the group consisting of liver, pancreas, adipose tissue, skeletal muscle, and heart. At least one, at least two, at least three, at least four or all of the vessels CNS-specific expression and / or brain-specific expression and / or visual Hypophysis-specific expression and / or cortex-specific expression and / or hippocampus-specific expression and / or a description of cerebellum-specific expression and / or olfactory bulb-specific expression is provided in the section entitled "General Information." is provided to.

[0087] Expression can be assessed as described in the section entitled "General Information." "CNS," A description of the "brain" and "hypothalamus" is provided in the section entitled "General Information."

[0088] In some embodiments, the expression vectors described herein are for use in therapy. The expression vector is administered into the cerebrospinal fluid (CSF) (by cisternal, intrathecal, or intraventricular delivery). The preferred administration is intra-CSF administration. It is given.

[0089] As used herein, "intra-CSF administration," "intranasal administration," "intraparenchymal administration," "cisternal administration," "intra-CSF ... "Intrathecal administration," "intrathecal administration," and "intraventricular administration" are defined in the section of this application entitled "General Information." is described in.

[0090] In some embodiments, the expression vector is a viral expression vector. A description of the current vector is provided in the section entitled "General Information."

[0091] Viral vectors include adenovirus vectors, adeno-associated virus vectors, and retrovirus vectors. A viral vector selected from the group consisting of a viral vector and a lentiviral vector. Adenoviral vectors are also known as adenovirus-derived vectors. Adeno-associated virus vectors are also known as adeno-associated virus-derived vectors. Retroviral vectors are also known as retrovirus-derived vectors. Lentiviral vectors are also known as lentivirus-derived vectors. The viral vector is an adeno-associated viral vector. An explanation of this is provided in the section entitled "General Information."

[0092] In some embodiments, the vector is an AAV of serotype 1 (AAV1), an AAV of serotype 2 (AAV2), or an AAV of serotype 3 (AAV3). AV (AAV2), serotype 3 AAV (AAV3), serotype 4 AAV (AAV4), blood AAV of serotype 5 (AAV5), AAV of serotype 6 (AAV6), AAV of serotype 7 (AA V7), serotype 8 AAV (AAV8), serotype 9 AAV (AAV9), serotype rh1 AAV of serotype 0 (AAVrh10), AAV of serotype rh8 (AAVrh8), and serotype Cb4 AAV of type 1 (AAVCb4), serotype rh74 AAV (AAVrh74), serotype DJ AAV (AAVDJ), serotype 2 / 5 AAV (AAV2 / 5), serotype 2 / 1 AAV (AAV2 / 1), serotype 1 / 2 AAV (AAV1 / 2), and serotype Anc80 AAV (AAVAnc80) The vectors are either viral vectors or adeno-associated virus-derived vectors (AAV).

[0093] In a preferred embodiment, the vector is an AAV of serotype 1, 2 or 9 (AAV1, AA In the examples, these AAV serotypes are used in the expression of the present invention. It has been demonstrated to be suitable for use as a vector.

[0094] In a preferred embodiment, the expression vector is AAV1 or AAV2 or AAV9, preferably A gene construct, preferably AAV9, comprising a nucleotide sequence encoding FGF21. More preferably, such a genetic construct comprises SEQ ID NO: 27 and at least 60 % CAG fragment comprising, consisting essentially of, or consisting of a nucleotide sequence having More preferably, such a genetic construct comprises a promoter as described herein. As shown in Fig. 1, microR is expressed in tissues where FGF21 expression is desired to be prevented. It contains at least one target sequence of NA.

[0095] In another preferred embodiment, the expression vector is AAV1 and encodes FGF21. The gene construct includes a nucleotide sequence that targets a microRNA. In one embodiment, the gene construct is capable of preventing expression of FGF21. It is more preferred that the target sequence of the miRNA expressed in the tissue in which it is desired to express the miRNA. Such a genetic construct may comprise a nucleotide sequence having at least 60% identical sequence identity to SEQ ID NO: 27. The present invention also includes a CAG promoter comprising, consisting essentially of, or consisting of the sequence.

[0096] composition In a further aspect, the genetic construct described above, together with one or more pharmaceutically acceptable ingredients, and / or compositions comprising the above viral vectors.

[0097] Such compositions may be referred to as gene therapy compositions. Preferably, the compositions are pharmaceutical compositions. It is a thing.

[0098] As used herein, a "pharmaceutically acceptable ingredient" includes any ingredient that is pharmaceutically acceptable. Carriers, fillers, preservatives, solubilizers, vehicles, diluents and / or excipients are included. Thus, one or more pharmaceutically acceptable ingredients may be pharmaceutically acceptable carriers, fillers, may be selected from the group consisting of preservatives, solubilizers, vehicles, diluents and / or excipients Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and and / or excipients are described, for example, in Remington: The Science and Practice of Pharmacy,22nd edition.Pharm aceutical Press (2013).

[0099] In some embodiments, the compositions described herein are for use in therapy. In a preferred embodiment, the compositions described herein are used in the treatment of metabolic disorders and / or In a preferred embodiment, the therapy is for use in the prevention of CNS, preferably It involves expression of the gene construct contained in the composition in the brain, more preferably in the hypothalamus. In some embodiments, expression of the gene construct in the brain includes expression of the gene construct in the hypothalamus and / or cortex and and / or the hippocampus and / or the cerebellum and / or the olfactory bulb, preferably in the hypothalamus Thus, expression of a gene construct in the brain can refer to expression of the construct in the hypothalamus, the cortex, at least one or at least two selected from the group consisting of the substantia nigra, hippocampus, cerebellum and olfactory bulb This may refer to expression of a gene construct in one, at least three, or all brain regions. In a preferred embodiment, the therapy comprises expression of a gene construct in the hypothalamus. In this state, the CNS and / or brain and / or hypothalamus and / or cortex and / or Expression in the hippocampus and / or cerebellum and / or olfactory bulb refers to expression in the CNS and / or brain. and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and In one embodiment, expression may refer to specific expression in the liver, pancreas, adipose tissue, or the olfactory bulb. does not include expression in adipose tissue, skeletal muscle, and / or heart. is at least one selected from the group consisting of liver, pancreas, adipose tissue, skeletal muscle and heart. , at least two, at least three, at least four, or all organs. CNS-specific expression and / or brain-specific expression and / or hypothalamus-specific expression and and / or cortex-specific expression and / or hippocampus-specific expression and / or cerebellum-specific expression A description of olfactory bulb-specific and / or olfactory bulb-specific expression is provided in the section entitled "General Information."

[0100] Expression can be assessed as described in the section entitled "General Information." "CNS," A description of the "brain" and "hypothalamus" is provided in the section entitled "General Information."

[0101] In some embodiments, the compositions described herein are for use in therapy. The composition may be administered intracerebral spinal fluid (CSF) (via cisternal, intrathecal, or intraventricular delivery), Administration is by intraperitoneal or intranasal administration, preferably into the CSF.

[0102] As used herein, "intra-CSF administration," "intranasal administration," "intraparenchymal administration," "cisternal administration," "intra-CSF ... "Intrathecal administration," "intrathecal administration," and "intraventricular administration" are defined in the section of this application entitled "General Information." is described in.

[0103] Additional compounds may be present in the compositions of the present invention. Such compounds may aid in the delivery of the composition. In this regard, preferred compounds are those that deliver the components described herein and penetrate cell membranes. Conjugates, nanoparticles, micelles and vesicles complexed or entrapped in liposomes via These compounds are compounds that can form liposomes and / or liposomes. Suitable compounds are polyethyleneimine (PEI), or polypropylene. Propyleneimine or polyethyleneimine copolymers (PEC) and derivatives thereof cationic polymers such as; synthetic amphiphiles (SAINT-18); lipofectin (commercial Those skilled in the art will appreciate that any type of formulation may be used in conjunction with the compositions described herein. You will know what is best for your product.

[0104] Methods and Uses In a further aspect, there is provided a genetic construct as described herein for use in therapy, comprising: The therapy is directed to the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or or genes in the hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus Gene constructs are provided that contain expression constructs.

[0105] 1. An expression vector as described herein for use in therapy, wherein the therapy is directed against the CNS, preferably Preferably the brain, more preferably the hypothalamus and / or the cortex and / or the hippocampus and / or or in the cerebellum and / or olfactory bulb, most preferably in the hypothalamus, contained in an expression vector Further provided is an expression vector comprising the expression gene construct.

[0106] A pharmaceutical composition as described herein for use in therapy, wherein the therapy is directed against CNS, preferably or the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or or the cerebellum and / or olfactory bulb, most preferably the hypothalamus, Pharmaceutical compositions comprising the expression vectors of the constructs are further provided.

[0107] In some embodiments, the genetic constructs described herein and / or the The expression vectors and / or pharmaceutical compositions described herein are useful in treating metabolic disorders, preferably For use in the treatment and / or prevention of obesity and / or diabetes, The CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or is a genetic construct in the hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus Includes the manifestation of things.

[0108] In a further aspect, the genetic construct, expression vector or pharmaceutical composition described herein wherein the method is carried out in the CNS, preferably the brain, more preferably in the brain, by administering Hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or A therapeutic method comprising expression of a gene construct described herein in the olfactory bulb, and most preferably in the hypothalamus. The law is provided.

[0109] In some embodiments, administering the genetic construct, expression vector, or pharmaceutical composition means administering to a subject in need thereof a therapeutically effective amount of a gene construct, expression vector, or pharmaceutical composition It means to administer something.

[0110] In some embodiments, the genetic constructs, expression vectors or pharmaceutical compositions described herein a method of treating a metabolic disorder comprising administering a composition comprising: and / or to prevent or reduce the activity of the CNS, preferably the brain, more preferably the hypothalamus and / or or the cortex and / or the hippocampus and / or the cerebellum and / or the olfactory bulb, most preferably Methods of treatment are provided that involve expression in the hypothalamus of the gene constructs described herein.

[0111] In a further aspect, the genetic constructs, expression vectors, and the like described herein for the manufacture of a pharmaceutical product are The use of a medicament or pharmaceutical composition, wherein said medicament is administered to the CNS, preferably the brain, more preferably Preferably, the hypothalamus and / or the cortex and / or the hippocampus and / or the cerebellum and / or or olfactory bulb, and most preferably the hypothalamus, and Use is provided.

[0112] In some embodiments, the genetic constructs, gene expression vectors, and / or gene products described herein for the manufacture of a medicament are provided. Use of the present vector or pharmaceutical composition, wherein said medicament is for the treatment and / or administration of a metabolic disorder. or prophylactically, and which target the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably Uses are provided which include expression of the genetic constructs described herein in the hypothalamus.

[0113] In a further aspect, the genetic constructs, expression vectors described herein for medical treatment. or the use of a pharmaceutical composition, wherein said medical treatment is directed to the CNS, preferably the brain, and Preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or the expression of a genetic construct as described herein in the olfactory bulb, most preferably in the hypothalamus. Use is provided.

[0114] In some embodiments, the genetic constructs described herein, the expression Use of a vector or pharmaceutical composition, wherein said medical treatment is the treatment of metabolic disorders and / or or for prophylaxis, and which target the CNS, preferably the brain, more preferably the hypothalamus and and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably Uses are provided that include expression of the genetic constructs described herein in the rat or in the hypothalamus.

[0115] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, "including expression of a genetic construct" means "expression of a genetic construct" "causes expression of a gene construct" or "induces expression of a gene construct" may be substituted. .

[0116] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, therapies and / or treatments and / or medicines The method may involve expression of a gene construct in the CNS, preferably in the brain, and more preferably in the hypothalamus. In some embodiments, expression of the gene construct in the brain is in the hypothalamus and / or cortex. Substantia nigra and / or hippocampus and / or cerebellum and / or olfactory bulb, preferably in the hypothalamus Therefore, expression of a gene construct in the brain can refer to expression of a gene construct in the subthalamus. at least one or more selected from the group consisting of the olfactory bulb, the hippocampus, the cerebellum, and the olfactory bulb; may refer to expression of a gene construct in two, at least three, or all brain regions. In a preferred embodiment, the therapy involves expression of a gene construct in the hypothalamus. In embodiments, the CNS and / or brain and / or hypothalamus and / or cortex and Expression in the CNS and / or hippocampus and / or cerebellum and / or olfactory bulb refers to or brain and / or hypothalamus and / or cortex and / or hippocampus and / or subtotal It may refer to specific expression in the brain and / or olfactory bulb. In one embodiment, expression is in the liver, pancreas, In some embodiments, the expression level in the spleen, adipose tissue, skeletal muscle, and / or heart is not included. The expression is in at least one tissue selected from the group consisting of liver, pancreas, adipose tissue, skeletal muscle, and heart. Expression in at least one, at least two, at least three, at least four, or all organs CNS-specific expression and / or brain-specific expression and / or hypothalamus-specific expression Expression and / or cortex-specific expression and / or hippocampus-specific expression and / or cerebellum-specific expression A description of heterologous and / or olfactory bulb-specific expression is provided in the section entitled "General Information." There are.

[0117] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, genetic constructs and / or expression vectors and and / or pharmaceutical compositions are administered by intra-CSF (cerebrospinal fluid) administration (e.g., cisternal, intrathecal, or ventricular). The drug may be administered intravenously (via intravenous delivery).

[0118] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, genetic constructs and / or expression vectors and The and / or pharmaceutical composition may be administered by intraparenchymal administration.

[0119] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, genetic constructs and / or expression vectors and and / or the pharmaceutical composition may be administered by intranasal administration.

[0120] As used herein, "intra-CSF administration," "intranasal administration," "intraparenchymal administration," "cisternal administration," "intra-CSF ... "Intrathecal administration," "intrathecal administration," and "intraventricular administration" are defined in the section of this application entitled "General Information." is described in.

[0121] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, therapies and / or treatments and / or medicines are used for the treatment and / or prevention of metabolic disorders, preferably obesity and / or diabetes. Complications of metabolic disorders may also be included.

[0122] Metabolic disorders include metabolic syndrome, diabetes, obesity, obesity-related comorbidities, and diabetes-related Co-morbidities, hyperglycemia, insulin resistance, impaired glucose tolerance, fatty liver, alcoholic liver disease (A LD), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NA SH), coronary heart disease (CHD), hyperlipidemia, atherosclerosis, endocrine disorders, Steosarcopenic obesity syndrome (OSO), diabetic nephropathy, chronic kidney disease (CKD), heart Hypertrophy, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, arthritis, sepsis, eye bleeding These may include angiogenesis, neurodegeneration, dementia, and may also include depression, adenoma, and carcinoma.

[0123] Diabetes includes prediabetes, hyperglycemia, type 1 diabetes, type 2 diabetes, and maturity-onset diabetes of the young. (MODY), monogenic diabetes, neonatal diabetes, gestational diabetes, unstable diabetes, idiopathic Idiopathic diabetes, drug- or chemical-induced diabetes , stiff man syndrome, lipoatrophic diabetes, and latent autoimmune diabetes in adults (LADA) It may be included.

[0124] Obesity includes overweight, central / upper body obesity, peripheral / lower body obesity, morbid obesity, osteoporosis Copenic Obesity Syndrome (OSO), childhood obesity, Mendelian (monogenic) syndromic obesity (M endelian(monogenic)syndromic obesity), men Mendelian non-syndromic obesity y), polygenic obesity.

[0125] A preferred metabolic disorder is obesity and / or diabetes.

[0126] In a preferred embodiment, the treatment or therapy or use of a pharmaceutical agent described herein is In some embodiments, the treatments described herein may also be administered in a manner that is consistent with the treatment described herein, but the administration does not need to be repeated. or therapy, or the use or administration of a drug, for any two of the listed values, years. Repeated annually or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, including any intervals between It can be repeated.

[0127] The subject to be treated may be a higher mammal, such as a cat, a rodent (preferably a mouse, a rat, etc.). dogs, gerbils and guinea pigs, more preferably mice and rats), dogs or may be a human.

[0128] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, the genetic constructs and / or The expression vector and / or pharmaceutical composition preferably has antidiabetic and / or anti-obesity effects. It shows full effect.

[0129] Increased blood glucose disposal and / or improved glucose tolerance, and / Alternatively, if insulin sensitivity is increased, an antidiabetic effect may be achieved. , as performed in the experimental part, measurements of glycemia, insulinemia, and / or insulin using techniques known to those skilled in the art, such as performance of a glucose tolerance test and / or a glucose tolerance test. In this context, "increase" (or "improvement") can be assessed using a method known to those skilled in the art. means at least a detectable increase (or a detectable improvement) using the assay The increase may be due to glycemia, insulinemia measurements, and / or insulin tolerance tests and / or or using assays such as glucose tolerance test results, At least 10%, at least 20%, at least 30%, at least 40%, at least 50% , at least 60%, at least 70%, at least 80%, at least 90% or less It can be at least a 100% increase.

[0130] An anti-obesity effect may be achieved if body weight, weight gain and / or body fat percentage is reduced. Body Mass Index (BMI), waist circumference, waist-to-hip ratio (WHR) and / or waist-to-hip ratio (WHR) An anti-obesity effect can also be achieved if the weight of the body is reduced relative to its height (WHtR). Even if the amount is reduced, an anti-obesity effect can be achieved. This is shown, for example, in the experimental section. In this regard, the term "decreased (or "improvement") is an assay known to those skilled in the art, e.g., an assay performed in the experimental part. By "anti-obesity" we mean at least a detectable decrease (or a detectable improvement). Actions include both the prevention of obesity and the reversal of obesity.

[0131] If the progression of typical symptoms (e.g., insulitis, beta cell loss, decreased beta cell mass, weight gain) is detected by a physician Anti-diabetic and / or anti-obesity effects were observed even when delayed as assessed by A typical symptom reduction is a slowing of the progression of symptoms or a complete disappearance of symptoms. Symptoms, and therefore symptom reduction, can also be measured using a variety of methods, including clinical testing and and routine laboratory tests similar to those used to diagnose diabetes and / or obesity. Such methods can be evaluated using macroscopic and microscopic methods. methods, as well as molecular methods, radiological methods such as X-rays, biochemical methods, and immunohistochemistry. β-cell loss and / or β-cell mass reduction can be preferably measured by experimental methods. As performed in section, immunohistochemical methods can be used to evaluate.

[0132] The anti-diabetic and / or anti-obesity effects are also due to the reduction of systemic inflammation (F4 / 80, IL-1) This can also be observed when the effects of inflammatory cytokines such as TNFα and IL-6 are assessed. In this context, "reduction" means at least a detectable decrease using assays known to those skilled in the art. The reduction means the reduction of the amount of the sample by techniques known to those skilled in the art, preferably the technique used in the experimental part ( i.e., F4 / 80, IL-6, and / or TNF-α using RT-PCR) At least 5%, at least 1% of the inflammatory cytokines were measured using assays such as 0%, at least 20%, at least 30%, at least 40%, at least 50%, At least 60%, at least 70%, at least 80%, at least 90% or less Both can be a 100% reduction.

[0133] Genetic constructs for use according to the present invention, expression vectors for use, pharmaceutical compositions for use Within the context of pharmaceutical compositions, methods and uses, the genetic constructs and / or The expression vector and / or pharmaceutical composition is preferably administered to an individual, a cell, tissue or one or more metabolic disorders, such as diabetes and / or obesity, in one or more organs alleviating the symptoms of (one or more of) the individual's cells, tissues, or organs; ) to alleviate the characteristics or symptom(s).

[0134] Genetic constructs and / or expression vectors and / or pharmaceutical compositions described herein preferably comprises a genetic construct of the invention and / or The expression vector and / or composition is administered to a subject for at least 1 week, 1 month, 6 months, or 8 months after treatment. After months, a year, or more, the symptoms or characteristics have decreased (e.g., are no longer detectable). or delayed), symptoms or or characteristics can be relaxed.

[0135] Genetic constructs and / or expression vectors and / or pharmaceutical compositions described herein suffers from or is at risk of developing metabolic disorders such as diabetes and / or obesity may be suitable for administration to cells, tissues and / or organs in vivo in an individual. The gene constructs and / or the vectors can be administered in vivo, ex vivo or in vitro. Alternatively, the expression vector and / or pharmaceutical composition may be used to treat metabolic disorders such as diabetes and / or obesity. In vivo cell, tissue and tissue analysis of individuals suffering from or at risk of developing the disorder. and / or may be administered directly or indirectly to an organ, in vivo, ex vivo or in vitro. The compound may be administered directly or indirectly.

[0136] Modes of administration include intravenous, intramuscular, intrathecal, intracerebroventricular, intraperitoneal, by inhalation, intranasal, intraocular and Preferred modes of administration are intranasal, intraparenchymal and CSF. Intra-CSF administration is most preferred. .

[0137] Viral expression constructs and / or viral vectors and / or nucleic acid molecules of the invention and / or the composition is directly or indirectly administered using any suitable means known in the art. In view of the progress made to date, the viral expression constructs and and / or viral vectors and / or nucleic acid molecules and / or compositions to an individual or Improved means for providing cells, tissues, and organs to such individuals are anticipated. Future improvements such as the above may be incorporated to achieve the above-mentioned effects of the present invention. expression construct and / or viral vector and / or nucleic acid molecule and / or The composition may be delivered directly to an individual, to a cell, tissue or organ of said individual. Depending on the disease or condition, the cells, tissues, or organs of the individual may be cultured as described herein above. The viral expression constructs and / or viral vectors of the present invention may When administering nucleic acid molecules and / or compositions, such viral expression constructs and and / or the vector and / or nucleic acid and / or composition in a solution compatible with the delivery method. It is preferable that the solution is dissolved in

[0138] As encompassed herein, the above viral expression constructs, vectors, nucleic acid molecules and and / or a therapeutically effective dose of the composition is preferably administered in a single, unique dose, thus Avoid repeated cycles.

[0139] General information Unless otherwise specified, all technical and scientific terms used herein are have the same meaning as customarily and commonly understood by a person skilled in the art to which the invention pertains. and should be read in light of the present disclosure.

[0140] Sequence identity / similarity In the context of the present invention, a nucleic acid molecule, such as a nucleic acid molecule encoding FGF21, is a protein fragment. Nucleic acids or nucleic acids encoding fragments or polypeptides or peptides or derived peptides In the context of the present invention, fibroblast growth factor 21 (FGF21 FGF21 protein fragment or polypeptide or peptide or derived peptide as The peptides are represented by amino acid sequences.

[0141] Each nucleic acid molecule identified herein by a given sequence identity number (SEQ ID NO:) or The protein fragment or polypeptide or peptide or derived peptide or construct may be It should be understood that there is no limitation to the particular sequences disclosed. Each coding sequence defined herein encodes a given protein fragment or polypeptide or peptide or The polypeptide may encode a derivative peptide or construct, or may itself be a protein fragment or polypeptide. A given peptide or construct or peptide or derived peptide. A specific gene encoding a protein fragment or polypeptide or peptide or a derived peptide Whenever a nucleotide sequence SEQ ID NO: (e.g., SEQ ID NO: X) is referred to, it shall be referred to as You can also replace it with: i. A nucleic acid sequence containing a nucleotide sequence having at least 60% sequence identity to SEQ ID NO:X. leotide sequence; ii. Nucleotides whose sequence differs from the sequence of the nucleic acid molecule in (i) due to the degeneracy of the genetic code an array; or iii. The amino acid sequence encoded by the nucleotide sequence SEQ ID NO: X and at least Nucleotides encoding amino acid sequences with 60% amino acid identity or similarity array.

[0142] Throughout this application, references to specific amino acid sequence SEQ ID NOs (e.g., SEQ ID NO: Y) will be and may be replaced by the following: amino acid sequence SEQ ID NO: Y and at least 60 % sequence identity or similarity.

[0143] Its identity or similarity percentage with a given nucleotide or amino acid sequence, respectively Each nucleotide sequence or In a further preferred embodiment, the amino acid sequence is a given nucleotide or has at least 61%, at least 62%, at least 63%, or at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, At least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77% , at least 78%, at least 79%, at least 80%, at least 81%, less At least 82%, at least 83%, at least 84%, at least 85%, at least 86% %, at least 87%, at least 88%, at least 89%, at least 90%, at least at least 91%, at least 92%, at least 93%, at least 94%, at least 9 5%, at least 96%, at least 97%, at least 98%, at least 99% or has 100% identity or similarity.

[0144] Each non-coding nucleotide sequence (i.e., of a promoter or of another regulatory region) , a specific nucleotide sequence SEQ ID NO (for example, SEQ ID NO: A) with at least 60% Substitution with a nucleotide sequence containing a nucleotide sequence with sequence identity or similarity Preferred nucleotide sequences have at least 60% similarity to SEQ ID NO: A, but at least 60% similarity to SEQ ID NO: B. at least 61%, at least 62%, at least 63%, at least 64%, at least 6 5%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, At least 79%, at least 80%, at least 81%, at least 82%, at least At least 83%, at least 84%, at least 85%, at least 86%, at least 87% , at least 88%, at least 89%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, at least 99% or 100% identity In a preferred embodiment, such non-coding nucleotide sequences, such as promoters, The sequence may be used to determine the activity of such non-coding nucleotide sequences, for example, promoter sequences known to those skilled in the art. exhibit or exert at least the activity of

[0145] The terms "homology," "sequence identity," "identity," and the like are used interchangeably herein. Sequence identity, as used herein, refers to the degree of sequence identity between two or more sequences, as determined by comparing the sequences. A sequence of amino acids (polypeptides or proteins) or two or more nucleic acids (polynucleotides) The "similarity" or "sequence" between two amino acid sequences is described as the relationship between the two sequences. "Similarity" refers to the amino acid sequence of one polypeptide and its conserved amino acid substitutions. "Identity" and "similarity" are determined by comparing the sequences of two polypeptides. "Sexuality" includes, but is not limited to, Bioinformatics and the ell:Modern Computational Approaches in G economics,Proteomics and transcriptomics,X ia X., Springer International Publishing, New York, 2018; and Bioinformatics:Sequence and Genome Analysis,Mount D.,Cold Sprin g Harbor Laboratory Press, New York, 2004 It can be readily calculated by known methods, including those described.

[0146] Sequence identity or similarity may be calculated based on the entire length or a portion of two given SEQ ID NOs. In some embodiments, the portion can be calculated by dividing at least one of both SEQ ID NOs. This means 50%, 60%, 70%, 80%, 90% or 100%. In this context, sequence identity or similarity is determined by comparing the full length of the sequences identified herein. Unless otherwise specified herein, identity or Similarity is defined as the length of the sequence (i.e., over its entire length or as a whole). In the art, "identity" also refers to identity or similarity based on such amino acid or nucleic acid sequences, as can be determined by the match between strings of such sequences It refers to the degree of sequence relatedness between sequences.

[0147] Sequence identity or similarity can be measured as global or local, depending on the length of the two sequences. Use an alignment algorithm to align two peptides or two nucleotide sequences. Sequences of similar length can be determined by alignment. Global alignment algorithms (e.g., For example, alignment is performed using the Needleman-Wunsch method, whereas Sequences of qualitatively different lengths are preferably aligned using local alignment algorithms (e.g. The sequences are then aligned using the Smith-Waterman algorithm. (e.g., the program EMBOSS needl using default parameters) e or EMBOSS water) at least also share a certain minimum percentage of sequence identity or similarity (as described below). (e.g., "substantially identical" or "essentially similar")

[0148] If two sequences have similar lengths, to determine sequence identity or similarity, A global alignment is preferably used. If the sequences have substantially different overall lengths, Local alignments, such as those using the Smith-Waterman algorithm The EMBOSS needle is a Needleman-Wunsch glow A bulk alignment algorithm is used to align two sequences over their entire length (full length). Aligns sequences vertically, maximizing the number of matches and minimizing the number of gaps. water uses the Smith-Waterman local alignment algorithm. Generally, EMBOSS needle and EMBOSS water are used as default. parameters were used, and gap open penalty = 10 (nucleotide sequence) / 10 (protein) and gap extension penalty = 0.5 (nucleotide sequence) / 0. 5 (proteins). For nucleotide sequences, the default scoring used is The matrix is ​​DNAfull, and for proteins, the default scoring matrix is ​​Blo sum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).

[0149] Alternatively, the percentage of similarity or identity can be calculated using algorithms such as FASTA, BLAST, etc. The information may be determined by searching against public databases using algorithms. Thus, the nucleic acid and protein sequences of some embodiments of the present invention are available in public databases. Searches can be performed against the source to, for example, identify other family members or related sequences. Such a search can be performed using the Alts B of chul, et al. (1990) J.Mol.Biol.215:403-10 This can be done using the LASTn and BLASTx programs (version 2.0). BLAST nucleotide searches can be performed to identify sequences homologous to the oxidoreductase nucleic acid molecules of the invention. To obtain the nucleotide sequence, use the NBLAST program, score = 100, word length = BLAST protein searches can be performed to identify proteins homologous to the protein molecules of the invention. To obtain the amino acid sequence, we used the BLASTx program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, use Alt schul et al.,(1997) Nucleic Acids Res.25( 17):3389-3402 using Gapped BLAST. When using the BLAST and Gapped BLAST programs, In this case, use the default parameters of each program (e.g., BLASTx and BL ASTn) can be used. The National Center for Biotechnology Information homepage is accessible on the World Wide Web. Please refer to the page.

[0150] In determining the degree of amino acid similarity, one skilled in the art will recognize so-called conservative amino acid substitutions. may also be taken into consideration.

[0151] As used herein, "conservative" amino acid substitutions are those substitutions that have similar side chains. Examples of classes of amino acid residues for conservative substitutions are shown in the table below. [Table 1] [Table 2] [Table 3]

[0152] For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine and isoleucine, and the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine, and the group of amino acids with amide-containing side chains is asparagine and The group of amino acids with aromatic side chains is glutamine, phenylalanine, tyrosine and and tryptophan, and the group of amino acids with basic side chains is lysine, arginine and and histidine, and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acids substitution groups are valine-leucine-isoleucine, phenylalan ... lysine-arginine, alanine-valine and asparagine- Substitution variants of the amino acid sequences disclosed herein include those of the disclosed sequences at least one residue in has been removed and a different residue inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions are: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Il e to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu, or Tyr ;Ser to Thr;Thr to Ser;Trp to Tyr;Tyr to Trp or P he; and Val to Ile or Leu.

[0153] Genes or coding sequences The term "gene" refers to a gene that is transcribed within a cell into an RNA molecule (e.g., mRNA) and that is encoded by an appropriate regulatory region. A DNA fragment containing a region (transcribed region) operably linked to a region (e.g., a promoter) A gene usually consists of several operably linked fragments, e.g., polynucleotides. a promoter, e.g., 5', including a denylation termination site and / or a transcription termination site; It contains a leader sequence, a coding region, and a 3' untranslated sequence (3' end). A recombinant gene (such as the FGF21 gene) may be, for example, a promoter that is a promoter for the transcribed DNA. A gene that is not normally found in nature, such as a gene that is not naturally associated with part or all of the A region. Gene expression is controlled by appropriate regulatory regions, especially promoters. The DNA region linked to the It refers to the process by which a protein is transcribed into RNA that can be translated into a protein or peptide.

[0154] "Transgene," as used herein, refers to a gene or coding sequence or sequences that are newly introduced into a cell. or nucleic acid molecule (i.e., a molecule encoding FGF21), i.e., a molecule encoding FGF21, may be present, but is not typically It is usually described as a gene that is not expressed or may be expressed at insufficient levels in cells. In this context, "insufficient" means that the FGF21 is expressed in the cell, but not in the cell as described herein. This means that the condition and / or disease described may still develop. The transgene allows for overexpression of FGF21. The transgene may contain sequences that are not naturally occurring, or may contain a combination of both. operably linked to appropriate regulatory sequences for expression of the GF21 coding sequence. FGF21 and / or additional proteins previously identified herein may be used. Preferably, the transgene does not integrate into the genome of the host cell. stomach.

[0155] promoter As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a It functions to regulate the transcription of the coding sequence and directs transcription of the transcription start site of the coding sequence. It is located upstream of the transcription factor binding site, including but not limited to, transcription factor binding sites, repressors, and and activator protein binding sites, as well as the level of transcription from the promoter directly or or any other nucleotide sequence known to those skilled in the art to act to indirectly regulate DNA-dependent RNA polymerase, transcription start site and any other DNA sequence "Constitutive" promoters refer to nucleic acid fragments that are structurally identified by the presence of a binding site for a specific promoter. The promoters are active in most tissues under most physiological and developmental conditions. An "inducible" promoter is one that can be physiologically induced, for example, by application of a chemical inducer. A promoter is a physiologically or developmentally regulated promoter.

[0156] A "ubiquitous promoter" is one that is active in virtually every tissue, organ, and cell of an organism. It is sex.

[0157] "Organ-specific" or "tissue-specific" promoters are promoters that are specific to specific types of organs. Organ-specific promoters and tissue-specific promoters are promoters that are active in the target tissue or organ. A promoter is a promoter that regulates the expression of one or more genes (or coding sequences) primarily within one organ or tissue. Regulate expression but allow expression at detectable levels ("leaky") in other organs or tissues The leaky expression in other organs or tissues can be achieved by standard methods known to those skilled in the art. mRNA by assay (e.g., qPCR, Western blot analysis, ELISA) or relative to organ- or tissue-specific expression when assessed at the protein level. Compared to, at least one-half, at least one-third, at least four-fold This means one-fifth or at least one-fifth but still detectable expression. The maximum number of organs or tissues in which the protein can be detected is 5, 6, 7, or 8.

[0158] "CNS-specific promoter or brain-specific promoter or hypothalamus-specific promoter "motors" can initiate transcription within the CNS and / or brain and / or hypothalamus Yes, but some leakage in other (up to 5, 6, 7 or 8) organs and parts of the body promoters that still allow expression in the CNS and / or brain and / or Transcription within the hypothalamus is associated with the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb, as well as related regions. It can be detected intracellularly, such as in neurons and / or glial cells.

[0159] In the context of the present invention, CNS-specific promoters and / or brain-specific promoters and and / or hypothalamus-specific promoter and / or cortex-specific promoter and and / or a hippocampus-specific promoter and / or a cerebellum-specific promoter and / or Olfactory bulb-specific promoters are expressed in the CNS and / or brain compared to other organs or tissues. and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and and / or a preferential or predominant (at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, At least 60% higher, at least 70% higher, at least 80% higher, at least 90% High, at least 100% high, at least 150% high, at least 200% high, or The promoter may be a promoter capable of promoting expression (or more) in other organs or tissues. The tissues include the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, The other organs may be the stomach, testes, etc. Preferably, the other organs are the liver and heart. The information contained in this document may be evaluated as described elsewhere in the section entitled "General Information."

[0160] Throughout this application, CNS-specific and / or brain-specific and / or hypothalamus-specific and / or cortex-specific and / or hippocampus-specific and / or cerebellum-specific and / or olfactory bulb-specific, when referred to in the context of expression, CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory Cell type-specific expression of each of the cell type(s) comprising the sphere is also envisaged.

[0161] operably linked As used herein, the term "operably linked" refers to a functional relationship. The term "linkage" refers to the linkage of polynucleotide elements in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is "operably linked" if it affects the transcription of the coding sequence. A gene is operably linked to a coding sequence if it has an effect on the coding sequence. The DNA sequences linked are typically contiguous and contain two protein-coding regions. If the sequences need to be concatenated, they must be contiguous and in reading frame. Ligation can be accomplished by using convenient restriction sites or adapters or ligases inserted in place of the restriction sites. This can be achieved by ligation in a car or by gene synthesis.

[0162] microRNA As used herein, "microRNA" or "miRNA" or "miRNA" "R" has its conventional and ordinary meaning as understood by one of ordinary skill in the art in light of this disclosure MicroRNAs function in RNA silencing and post-transcriptional regulation of gene expression. small non-coding RNA molecules found in plants, animals, and some viruses that can The target sequence of a microRNA may be expressed as "miRT." For example, The target sequence of miRNA-1 or miRNA-1 or miR-1 is denoted as miRT-1. obtain.

[0163] Proteins and Amino Acids The terms "protein" or "polypeptide" or "amino acid sequence" are used interchangeably. and derived from a chain of amino acids without reference to a specific mechanism of action, size, three-dimensional structure or origin. In the amino acid sequences described herein, amino acids or "residues" are represented by three letters. These three-letter symbols and the corresponding one-letter symbols are well known to those skilled in the art. and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, and F (Phe) is phenyl. Alanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isopropyl Lysine, K(Lys) is lysine, L(Leu) is leucine, M(Met) is methionine, N(Asn) is asparagine, P(Pro) is proline, Q(Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Va l) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine. , may be any proteinogenic amino acid, but may also be any non-proteinogenic amino acid, e.g. For example, D-amino acids and modified amino acids formed by post-translational modifications, as well as any It can also be an unnatural amino acid.

[0164] CNS and Brain As used herein, "central nervous system" or "CNS" refers to the system that includes the brain and spinal cord. and there the sensory impulses are transmitted, and from there the motor impulses are sent out, and by this the Refers to the part of the nervous system that coordinates the activity of the entire nervous system.

[0165] As used herein, "brain" refers to the central organ of the nervous system, including the cerebrum, brainstem, and The brain consists of the cerebellum, which controls most of the body's activities and processes information received from the sensory organs. , integrate and coordinate and make decisions regarding the commands sent to the rest of the body.

[0166] In particular, as used herein, the "hypothalamus" refers to the nervous system that regulates the activity of the autonomic nervous system and the pituitary gland. regulates body temperature, thirst, hunger and other homeostatic systems, and is involved in sleep and emotional activity It refers to the forebrain region of the hypothalamus, which is involved in the

[0167] Gene constructs All genetic constructs described herein are incorporated by reference in their entirety. Ausubel et al., “Current Protocols in M olecular Biology”, Greene Publishing and Wiley-Interscience, New York (1987) and Samb As described in Brook and Russell (2001, supra), The nucleotide sequence encoding GF21 is then introduced into a suitable cell, e.g., a cultured cell, or a multicellular Any cloned and / or recombinant D known to those skilled in the art that can be expressed in the cells of a bacterial organism. It can be prepared using NA techniques. Kunkel (1985) Proc. Nat. I. Acad. Sci. 82:488 (describing site-directed mutagenesis) and Robert s et al.(1987) Nature 328:731-734 or Wells , JA, et al. (1985) Gene 34:315 (recording cassette mutagenesis) See also the article in the original.

[0168] Expression vector The term "expression vector" or "vector" generally refers to a vector that is compatible with such sequences. refers to a nucleotide sequence capable of directing the expression of a gene or coding sequence in a host. Expression vectors carry genomes that can be stabilized within cells and remain episomal. Within the context of the present invention, a cell is a cell used to generate a construct or a cell that is used to generate a construct. Alternatively, the vector may be used to express, for example, a homologous It can be incorporated into the genome of a cell by recombination or other means.

[0169] These expression vectors typically contain at least a suitable promoter sequence, Termination signals may also be included. Additional factors necessary or helpful in effecting expression are also included herein. Nucleic acids encoding FGF21 or The DNA or nucleotide sequences can be used for introduction into in vitro cell cultures and for the production of in vitro cells. The DNA construct is incorporated into a DNA construct that allows expression in culture. Suitable for replication in a prokaryotic host, e.g., a bacterium, e.g., E. coli or cultured mammalian, plant, insect (e.g., Sf9), yeast, fungi, or other eukaryotic organisms. It can be introduced into a cell line.

[0170] DNA constructs prepared for introduction into a particular host must be replicable and recognized by the host. The system, the intended DNA segment encoding the desired polypeptide, and the polypeptide transcription and translation initiation and termination controls operably linked to the segment encoding the The term "operably linked" is used herein as defined above. For example, a promoter or enhancer encodes a coding sequence if it stimulates the transcription of the sequence. The DNA for the signal sequence is operably linked to the polypeptide. When expressed as a preprotein, the DNA encoding the polypeptide is operably linked to the Generally, operably linked DNA sequences are contiguous and In the case of signal sequences, they are contiguous and in reading frame. The sequences need not be contiguous with the coding sequences they control the transcription of. Linkage may be convenient. Ligation at restriction sites or adapters or linkers inserted in their place This can be achieved by gene synthesis or by gene synthesis.

[0171] The selection of an appropriate promoter sequence is generally selected for the expression of the DNA segment. Examples of suitable promoter sequences include those known in the art for prokaryotic cells. promoters and eukaryotic promoters (e.g., Sambroo, supra). (See, e.g., Kerr and Russell, 2001). Transcriptional regulatory sequences are typically The present invention also includes heterologous enhancers or promoters recognized by the promoter. The selection depends on the host, but trp, lac and phage promoters, tRNA promoters, Promoters such as ATP and glycolytic enzyme promoters are known and available ( See, for example, Sambrook and Russell, 2001, supra. The vector contains a replication system and transcription factor, along with an insertion site for the polypeptide-encoding segment. In most cases, a replication system is used to generate a vector. Most plasmids are functional only in the cells in which they are used (bacterial cells such as E. coli). The vector does not replicate in cells infected with the vector. Examples of workable combinations are given in Sambrook and Russell (2001, supra). ) and Metzger et al. (1988) Nature 334:31-36 For example, suitable expression vectors are suitable for use in yeast, such as S. cerevisiae ( evisiae), for example insect cells, for example Sf9 cells, mammalian cells, for example CHO cells The expression can be in cells and bacterial cells, e.g., E. coli. The host cells may be karyotic or eukaryotic. The cells may be cultured in liquid or on solid media. The cells may be suitable for

[0172] Alternatively, the host cell may be a transgenic plant or animal. A cell is part of a multicellular organism.

[0173] The selection of an appropriate promoter sequence is generally selected for the expression of the DNA segment. Examples of suitable promoter sequences include those known in the art for prokaryotic cells. promoters and eukaryotic promoters (e.g., Sambroo, supra). (See, e.g., Kerr and Russell, 2001). Transcriptional regulatory sequences are typically The present invention also includes heterologous enhancers or promoters recognized by the promoter. The selection depends on the host, but trp, lac and phage promoters, tRNA promoters, Promoters such as ATP and glycolytic enzyme promoters are known and available ( See, for example, Sambrook and Russell, 2001, supra. The vector contains a replication system and transcription factor, along with an insertion site for the polypeptide-encoding segment. In most cases, a replication system is used to generate a vector. Most plasmids are functional only in the cells in which they are used (bacterial cells such as E. coli). The vector does not replicate in cells infected with the vector. Examples of workable combinations are given in Sambrook and Russell (2001, supra). ) and Metzger et al. (1988) Nature 334:31-36 For example, suitable expression vectors can be used in yeast, such as Saccharomyces cerevisiae, insect cells, such as Sf9 cells, mammalian cells, such as CHO cells, and bacterial cells, such as E. coli The cells can be expressed in bacteria, prokaryotic or eukaryotic host cells. The cells may be cells suitable for culture in liquid or on solid media.

[0174] Alternatively, the host cell may be a transgenic plant or animal. A cell is part of a multicellular organism.

[0175] viral vectors Viral vectors or viral expression vectors. Viral gene therapy vectors are defined herein. A vector containing the genetic construct described in the document.

[0176] Viral vectors or viral gene therapy vectors are vectors suitable for gene therapy. Suitable vectors for gene therapy are described in Anderson 1998, Nature 392:25-30;Walther and Stein,2000,Drugs 6 0:249-71;Kay et al.,2001,Nat.Med.7:33-40 ;Russell,2000,J.Gen.Virol.81:2573-604;Am ado and Chen,1999,Science 285:674-6;Fede rico,1999,Curr.Opin.Biotechnol.10:448-53 ;Vigna and Naldini,2000,J.Gene Med.2:308 -16;Marin et al.,1997,Mol.Med.Today 3:39 6-403;Peng and Russell,1999,Curr.Opin.Bi otechnol.10:454-7;Sommerfelt,1999,J.Gen. Virol.80:3049-64;Reiser,2000,Gene Ther.7 :910-3; and the references cited therein.

[0177] Particularly preferred gene therapy vectors include adenoviral vectors and adeno-associated viruses. These vectors are useful in multiple cell types, including synovial cells and hepatocytes. Adenoviral vectors and Due to the episomal nature of AAV vectors, these vectors can be used in combination with other vectors, such as those described above (Rus sell,2000,J.Gen.Virol.81:2573-2604;Gonca lves, 2005, Virol J. 2(1):43), making it suitable for therapeutic use AAV vectors are known to provide highly stable, long-term expression of transgenes. In dogs, the lifespan is up to 9 years (Niemeyer et al., Blood. 2009) Jan 22;113(4):797-806), and approximately 10 years in humans (Buchlis, G.et al.,Blood.2012 Mar 29;119(13):3038- 41), and even more preferred. A preferred adenoviral vector is the 000, supra) are modified to reduce the host response. Methods for gene therapy using AAV vectors are described by Wang et al. 05,J Gene Med.March 9(Epub ahead of prin t), Mandel et al., 2004, Curr Opin Mol Ther .6(5):482-90, and Martin et al., 2004, Eye 1 8(11):1049-55, Nathwani et al, N Engl J Me d.2011 Dec 22;365(25):2357-65,Apparaily et al,Hum Gene Ther.2005 Apr;16(4):426- 34.

[0178] Other suitable gene therapy vectors include retroviral vectors. A preferred retroviral vector for application is a lentiviral-based expression construct. Lentiviral vectors can infect and stably infect the genomes of dividing and non-dividing cells. (Amado and Chen, 1999 Science e 285:674-6) for the construction and use of lentiviral-based expression constructs. This method is described in U.S. Pat. Nos. 6,165,782, 6,207,455, and 6,207,455. No. 6,218,181, U.S. Pat. No. 6,277,633 and U.S. Pat. No. 6,322,493. 3,031 and Federico (1999, Curr Opin Biotech hnol 10:448-53) and Vigna et al. (2000, J Ge ne Med 2000;2:308-16).

[0179] Other suitable gene therapy vectors include adenovirus vectors, herpesvirus vectors, and vectors, polyoma virus vectors, or vaccinia virus vectors.

[0180] Adeno-associated virus vector (AAV vector) The terms "adeno-associated virus," "AAV virus," and "AAV virus" are used synonymously herein. "," "AAV virion," "AAV virus particle," and "AAV particle" refer to a small amount of AAV. At least one capsid protein (preferably all capsids of a particular AAV serotype) The AAV genome is composed of encapsulated polynucleotides. The term refers to a viral particle composed of a heterologous polynucleotide flanked by AAV inverted terminal repeat sequences. nucleotides (i.e., polynucleotides that differ from the wild-type AAV genome, e.g., mammalian When the vector contains a transgene (transgene to be delivered to animal cells), they are typically referred to as "AAV vectors" particle” or “AAV viral vector” or “AAV vector” AAV refers to a virus belonging to the genus Dependovirus in the family Parvoviridae. The V genome is approximately 4.7 Kb in length and is a single-stranded deoxyribonucleic acid (SDA) molecule that can be detected positively or negatively. The present invention also refers to dsAAV or scAAV. This also includes the use of double-stranded AAVs, which contain inverted terminal repeats at both ends of the DNA strand. (ITR) and two open reading frames (ORFs), namely, rep and The rep frame encodes the protein Rep, which is required for the AAV life cycle. It consists of four overlapping genes that encode the capsid protein VP 1, VP2, and VP3 contain overlapping nucleotide sequences, and VP1, VP2, and VP 3 interact to form a capsid with icosahedral symmetry (Carter and S amulski., 2000, and Gao et al., 2004).

[0181] A preferred viral vector or a preferred gene therapy vector is an AAV vector. The AAV vector used herein is preferably a recombinant AAV vector (rAAV). As used herein, "rAAV vector" refers to a vector that is capable of expressing a specific antigen (e.g., an rAAV vector). Encapsidated protein shells of capsid proteins derived from the described AAV serotypes. AAV genome fragments are recombinant vectors containing a portion of the AAV genome that has been modified. Adeno-associated virus serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5 A preferred ITR is SEQ ID NO: 3. 0 (5'ITR) and SEQ ID NO: 31 (3'ITR) The ITR of AAV2 is represented by a sequence consisting of the ITR and a sequence consisting of the ITR. or at least 80% (or at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least At least 86%, at least 87%, at least 88%, at least 89%, at least 90% , at least 91%, at least 92%, at least 93%, at least 94%, at least At least 95%, at least 96%, at least 97%, at least 98%, at least 99% % or 100%) identity to SEQ ID NO: 31 as the 3' ITR. At least 80% (or at least 81%, at least 82%, at least 83%, at least at least 84%, at least 85%, at least 86%, at least 87%, at least 8 8%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) This includes the use of

[0182] The protein shell, composed of capsid proteins, can be derived from any AAV serotype. The protein shell may also be referred to as a capsid protein shell. The vector may be deleted for one or preferably all wild-type AAV genes, The functional ITR sequences may still contain functional ITR nucleic acid sequences. The ITR sequences can be wild-type sequences or can be modified to contain wild-type sequences. At least 80%, at least 85%, at least 90%, at least 95% identical to the wild-type sequence , at least 97%, at least 98%, at least 99% or 100% sequence identity They may have, for example, nucleotide insertions, mutations, In this context, functionality can be achieved by the ability to encapsidate the genome. The virus is directly packaged into the shell and then expressed by the infected or target cell. In the context of the present invention, the capsid protein shell refers to the ability of the rAAV It may be of a different serotype than the vector genome ITRs.

[0183] The nucleic acid molecule represented by the selected nucleic acid sequence is preferably the rA operably linked to the AV genome or ITR sequences, e.g., coding sequences and 3' termination sequences The nucleic acid molecule is inserted between an expression construct containing expression control elements. It may also be called an introgression.

[0184] "AAV helper functions" are generally rAA helper functions that are supplied in trans to the rAAV vector. AAV helper functions refer to the corresponding AAV functions required for replication and packaging of V. rAAV vectors, but complement the AAV functions that are missing from the AAV vectors. The AAV helper functions are provided by the two main AAV genomes. the functional ORFs, i.e., the rep and cap coding regions, or their functional equivalents. The Rep and Cap regions are well known in the art and contain sequences substantially identical to, for example, See, for example, Chiorini et al. (1999, J. .of Virology,Vol 73(2):1309-1319) or U.S. Pat. See US Pat. No. 5,139,941. AAV helper functions can be achieved by using AAV helper constructs. Introduction of the helper construct into the host cell can be carried out, for example, as described herein. The rAAV vectors identified may be shaped prior to or simultaneously with the introduction of the rAAV genome. This can occur by transformation, transfection or transduction. The AAV helper construct, on the other hand, is inserted into the capsid protein shell of the rAAV vector. On the other hand, the rAAV genome exists in the replication and packaging of the rAAV vector. The serotypes can be selected to provide the desired combination of serotypes.

[0185] "AAV helper viruses" contain additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenovirus, herpes simplex virus, and These include viruses (such as HSV types 1 and 2) and vaccinia viruses. As described in U.S. Patent No. 6,531,456, which is incorporated herein by reference, The additional functions provided by the helper virus are introduced into the host cell via the It is also possible.

[0186] "Transduction" refers to the delivery of FGF21 to recipient host cells by a viral vector. For example, transduction of a target cell by an rAAV vector of the invention refers to the transduction of that vector The rAAV genome contained in the "host cell" or "target cell" is introduced into the transduced cell. refers to the cells into which DNA delivery is to occur, such as muscle cells of a subject. AAV vectors are used in dividing cells. Both dividing and non-dividing cells can be transduced.

[0187] AAV vector production The generation of recombinant AAV (rAAV) for vectoring transgenes has been previously described. Ayuso E, et al., Curr. Gene Ther. 2010; 10:423-436, Okada T, et al., Hum. Gene Ther. 2009;20:1013-1021, Zhang H,et al.,Hum.Gen e Ther.2009;20:922-929, and Virag T,et al. See, Hum. Gene Ther. 2009;20:807-817. These protocols can be used or adapted to generate AAVs of the present invention. In one embodiment, the production cell line contains a polynucleotide of the invention (an expression vector flanked by ITRs). The rep and cap proteins are encoded by the ribosomal transcription factor (RITF) and helper functions. The cells are transiently transfected with a construct or constructs that provide the function of the In an embodiment, the cell line stably provides helper functions and expresses the polynucleotides of the invention (IT R) and the rep and cap proteins. The cells are transiently transfected with the loading construct(s). In this state, the cell line stably supplies rep and cap proteins as well as helper functions. , are transiently transfected with a polynucleotide of the present invention. The cell line stably supplies the rep and cap proteins and contains the polynucleotide of the invention. and transiently transfected with a polynucleotide encoding a helper function. In yet another embodiment, the cell line contains the polynucleotides of the invention, rep and c These and other AAV production systems provide stable supply of ap proteins and helper functions. Methods for making and using the same are described in the art. t al., U.S. Patent No. 5,139,941, Zhou X,et al., U.S. Patent No. 5,741,683, Samulski R, et al., U.S. Patent No. 6,057 , 152, Samulski R, et al., U.S. Patent No. 6,204,059, Samulski R, et al., U.S. Patent No. 6,268,213, Rabino witz J, et al., U.S. Patent No. 6,491,907, Zolotukhin S, et al., U.S. Patent No. 6,660,514, Shenk T, et al. , U.S. Patent No. 6,951,753, Snyder R, et al., U.S. Patent No. 7, No. 094,604, Rabinowitz J, et al., U.S. Patent No. 7,172, No. 893, Monahan P, et al., U.S. Patent No. 7,201,898, Sa. Mulski R, et al., U.S. Patent No. 7,229,823, and Ferra See ri F, et al., U.S. Patent No. 7,439,065.

[0188] The rAAV genome present in the rAAV vector is one of the AAV serotypes (preferably Nucleotide sequences of the inverted terminal repeat region (ITR) of AAV2 (previously disclosed in this specification) nucleotide sequence, or a nucleotide sequence substantially identical thereto or at least The nucleotide sequence has 60% identity with the nucleotide sequence of ... a nucleotide sequence encoding FGF21 (under the control of suitable regulatory elements) The vector genome is efficiently packaged into the rAAV capsid. To enable this synthesis, it requires the use of flanking 5' and 3' ITR sequences.

[0189] The complete genomes of several AAV serotypes and the corresponding ITRs have been sequenced ( Chiorini et al.1999, J.of Virology Vol.73 , No. 2, p1309-1319). They are, for example, in Applied Biosystems Oligonucleotides supplied by Stems Inc. (Fosters, CA, USA) By chemical synthesis as known in the art using a nucleotide synthesizer or by standard molecular synthesis. The ITRs can be cloned or generated by molecular biology techniques. cloned from the virus genome or excised from a vector containing AAV ITRs Using standard molecular biology techniques, a gene encoding one or more therapeutic proteins can be generated. ligating an ITR nucleotide sequence at either end to the nucleotide sequence Alternatively, the AAV sequence between the ITRs can be replaced with a desired nucleotide sequence. .

[0190] Preferably, the rAAV genome present in the rAAV vector encodes viral proteins. The nucleotide sequence encoding the AAV rep (replication) or cap (capsid) ) genes. The rAAV genome does not contain a marker or reporter gene, e.g. , antibiotic resistance genes, genes encoding fluorescent proteins (e.g., gfp), or the like. detectable and / or selectable by chemical, enzymatic or other methods known in the art The gene may further comprise a gene encoding a gene product (e.g., lacZ, aph, etc.).

[0191] The rAAV genome present in the above rAAV vector contains a nucleotide sequence encoding FGF21. The gene further comprises a promoter sequence operably linked to the promoter sequence.

[0192] Also, suitable 3' untranslated sequences may be operably linked to the nucleotide sequence encoding FGF21. A suitable 3' untranslated region may be linked to the nucleotide sequence naturally associated with the nucleotide sequence. For example, the SV40 polyadenylation signal (SEQ ID NO: 32) and The polyadenylation signal from various genes, such as the heron β-globin polyadenylation signal (SEQ ID NO: 33), obtain.

[0193] Expression Expression can be assessed by any method known to those of skill in the art. For example, expression can be assessed by any method known to those of skill in the art. Known standard assays, such as qPCR, Western blot analysis or ELISA Measure the level of transgene expression in the liver at the mRNA or protein level by It can be evaluated by

[0194] Expression may occur in any cell after administration of the genetic constructs, expression vectors or compositions described herein. In some embodiments herein, expression can be assessed at 1 week, 2 weeks, or 3 days. , 3 weeks later, 4 weeks later, 5 weeks later, 6 weeks later, 7 weeks later, 8 weeks later, 9 weeks later, 10 weeks later After, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks or more can be evaluated later.

[0195] In the context of the present invention, CNS-specific expression and / or brain-specific expression and / or thalamus Subcellular specific expression and / or cortex specific expression and / or hippocampus specific expression and / or Or cerebellum-specific expression and / or olfactory bulb-specific expression refers to expression that is specific to the olfactory bulb compared to other organs or tissues. , CNS and / or brain and / or hypothalamus and / or cortex and / or nasal passages Preferential or predominant (less than) expression of FGF21 in the equine and / or cerebellum and / or olfactory bulb At least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher , at least 50% higher, at least 60% higher, at least 70% higher, at least 8 0% higher, at least 90% higher, at least 100% higher, at least 150% higher, Other organs or tissues include liver, Pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, etc. Preferably, the other organ is the liver and / or the heart. In one embodiment, , expression is not detectable in liver, pancreas, adipose tissue, skeletal muscle and / or heart. In some embodiments, expression is in the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, or the like. At least one selected from the group consisting of tissue, lung, ovary, spleen, stomach and testis Expression is undetectable in at least two, at least three, at least four, or all organs. It can be evaluated as above.

[0196] Throughout this application, CNS-specific and / or brain-specific and / or hypothalamus-specific and / or cortex-specific and / or hippocampus-specific and / or cerebellum-specific and / or olfactory bulb-specific, when referred to in the context of expression, CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory Cell type-specific expression of each of the cell type(s) comprising the sphere is also envisaged.

[0197] Administration As used herein, "intra-CSF administration" refers to administration of a substance into the arachnoid and soft tissue layers of the meninges that surround the brain. Administration into the CSF refers to the administration of drugs directly into the CSF, which is located in the subarachnoid space between the tunica media and the CSF. This can be done by intracisternal, intracerebroventricular or intrathecal administration. In this case, "intracisternal administration" refers to administration into the cisterna magna, an opening in the subarachnoid space located between the cerebellum and the dorsal surface of the medulla oblongata. As used herein, "intraventricular administration" refers to administration into the bilateral cerebral cortex of the brain. As used herein, "intrathecal administration" refers to administration into either the spinal cord or the ventricles. As used herein, "intraparenchymal administration" includes administration of the medicament directly into the CSF within the spinal cavity. " refers to direct local administration to any region of the brain parenchyma. "Intranasal administration" means administration via the nasal structures.

[0198] Codon optimization As used herein, "codon optimization" refers to the process of modifying an existing coding sequence and to design a coding sequence, e.g., to control the expression of a transcribed RNA molecule transcribed from the coding sequence. To improve translation or transcription of a coding sequence in a host cell or organism Codon optimization refers to the process used to optimize expression of a gene. A process that involves selecting codons for a coding sequence to conform to the codon preferences of the host organism For example, codon preferences of a mammalian, preferably murine, canine or human expression host. Codon optimization may also have adverse effects on RNA stability and / or translation. elements that may affect the transcription (e.g., termination sequences, TATA boxes, splice sites, ribonuclease inflammasome entry sites, repeat and / or GC-rich sequences and RNA secondary structure or instability In some embodiments, the codon-optimized sequence is a sequence that is optimized for transcription, RNA synthesis, and transcriptional regulation. A stability and / or translation of at least 3%, 5%, 10%, 15%, 20%, 25% , 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more indicates an increase in

[0199] In this specification and the claims thereto, the verbs "to comprise" and "to include" are used interchangeably. and its conjugations include the items that follow the word, but exclude items not specifically mentioned. It is used in its non-restrictive sense to mean that it will not be removed. "Consisting of" refers to a peptide or peptidomimetic described herein. , the medium or composition contains additional component(s) other than those specifically identified. "to consist essentia" means to be able to include "(ally of)" may be substituted, and the additional ingredient(s) may be replaced by "(ally of)" ... It does not change the inherent characteristics of Ming. In addition, the verb "to consist" , the method may further comprise one or more additional steps other than those specifically identified herein. "to consist essenti" means that it can include ally of)" and the additional step(s) may be replaced with " Does not change the inherent characteristics of the invention.

[0200] Reference to an element by the indefinite article "a" or "an" indicates that the context requires that only one element is present. Unless expressly required, the present invention does not exclude the possibility of more than one element being present. Thus, the indefinite article "a" or "an" usually means "at least one."

[0201] As used herein, "at least" a particular value means greater than or equal to the particular value. For example, "at least two" should be interpreted as "two or more," i.e., 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, ... etc. .

[0202] Individual numerical values ​​are approximate as if preceded by the word "about" or "approximately." Similarly, the numerical values ​​within the various ranges specified in this application are not necessarily intended to be limiting unless otherwise specified. Unless expressly specified, the word "about" precedes both the minimum and maximum values ​​within a stated range. or are stated as approximations as if preceded by the word "approximately." As used herein, the terms "about" and "approximately" when referring to numerical values ​​are within the meaning of the disclosed to those skilled in the art to which the subject matter to which it is most closely related, or to the scope or elements in question. Quantities that extend beyond their strict numerical bounds have their obvious and ordinary meaning. It depends on factors. For example, some of the factors that may be considered include the criticality of the element and / or The effect of a given amount of variation on the performance of the claimed subject matter, as well as other effects known to those skilled in the art, are discussed. In the absence of considerations to the contrary, the numerical value (e.g., about 10) The terms "about" or "approximately" when used herein preferably mean that the value is within the range of the This means that the value can be 1% or more or less than a given value (say 10).

[0203] As used herein, the term "and / or" refers to one of the listed instances. One or more of the listed cases, alone or in combination with at least one of the listed cases, This indicates that all of the cases listed above can occur.

[0204] Each embodiment specified herein may be combined together unless otherwise specified. This can be done.

[0205] All patent applications, patents and printed publications cited herein are the property of their respective owners. Except for any express or implied disclaimer or disclaimer of subject matter, and unless the incorporated material is The entire disclosure is incorporated herein by reference except to the extent that it conflicts with the disclosure, in which case the language in this disclosure will control. incorporated herein.

[0206] Those skilled in the art will appreciate that the techniques described herein can be used to practice the present invention. Many similar or equivalent methods and materials will be recognized. Indeed, the present invention relates to the The present invention is in no way limited to the methods and materials described.

[0207] The present invention is further illustrated by the following examples which should not be construed as limiting the scope of the invention. So it will be explained further. [Brief explanation of the drawings]

[0208] [Figure 1] Expression of moFGF21 in the brain of db / db mice. Expression levels of the mouse codon-optimized FGF21 (moFgf21) coding sequence were measured in the hypothalamus, cortex, hippocampus, and cerebellum of db / db mice by RTqPCR and normalized by Rplp0 values. Analysis was performed 12 weeks after intraCSF administration of 5 x 1010 vg / mouse of the AAV9-CAG-moFGF21-dmiRT vector. Results are expressed as mean ± SEM (n = 9 mice / group). ND, not detected. [Figure 2]Loss of body and tissue weights in db / db mice after treatment with AAV9-FGF21 vectors. (A) Body weight change. Body weight was measured weekly after AAV administration. (B) Body weight gain. Body weight gain was calculated as a percentage of body weight gained divided by body weight at the time of AAV administration. (C) iWAT, eWAT, mWAT, BAT, and liver weights in untreated and AAV9-FGF21-treated db / db mice. Analysis was performed 12 weeks after intraCSF administration of 5 x 10 vg / mouse of the AAV9-CAG-moFGF21-dmiRT vector. Results are expressed as mean ± SEM (n = 9 mice / group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to untreated mice. iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue; mWAT, mesenteric white adipose tissue; BAT, interscapular brown adipose tissue; L, liver. [Figure 3] Intra-CSF administration of AAV9-FGF21 vector reverses diabetes in db / db mice. Changes in postprandial blood glucose levels in untreated and AAV9-CAG-moFGF21-dmiRT-treated db / db mice after intra-CSF vector administration. Results are expressed as mean ± SEM (n = 9 mice / group). ***p < 0.001 vs. untreated mice. [Figure 4] Treatment of db / db mice with AAV9-FGF21 vectors reduced brain inflammation. Expression levels of astrocyte markers (Gfap and S100b), microglial marker (Aif1), and inflammatory molecules (Nfkb, Il1b, and Il6) in the hypothalamus of db / db mice were measured by RTqPCR and normalized to Rplp0 values. Analysis was performed 12 weeks after intraCSF administration of 5 x 1010 vg / mouse of the AAV9-CAG-moFGF21-dmiRT vector. Results are expressed as mean ± SEM (n = 9 mice / group). *p < 0.05 vs. untreated mice. Gfap, glial fibrillary acidic protein; S100b, calcium-binding protein B; Aif1, allograft inflammatory factor 1; Nfkb, nuclear factor-κB; Il1b, interleukin-1β; Il6, interleukin-6. [Figure 5]Expression of moFGF21 in the brain of SAMP8 mice. Expression levels of the mouse codon-optimized FGF21 (moFGF21) coding sequence were measured in the hypothalamus, cortex, hippocampus, and cerebellum of SAMP8 mice by RTqPCR and normalized by Rplp0 values. Analysis was performed 14 weeks after intraCSF administration of 5 x 1010 vg / mouse of the AAV9-CAG-moFGF21-dmiRT vector. Results are expressed as mean ± SEM (n = 9 mice / group). ND, not detected. [Figure 6] Loss of body and tissue weights in SAMP8 mice after treatment with AAV9-FGF21 vectors. (A) Body weight change. Body weight was measured weekly after AAV administration. (B) Body weight gain. Body weight gain was calculated as a percentage of body weight gained divided by body weight at the time of AAV administration. (C) iWAT, eWAT, mWAT, BAT, and liver weights in untreated and AAV9-FGF21-treated SAMP8 mice. Analysis was performed 14 weeks after intraCSF administration of 5 x 1010 vg / mouse of the AAV9-CAG-moFGF21-dmiRT vector. Results are expressed as mean ± SEM (n = 9 mice / group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to untreated mice. iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue; mWAT, mesenteric white adipose tissue; BAT, interscapular brown adipose tissue; L, liver. [Figure 7] Reduced brain inflammation in SAMP8 mice treated with AAV9-FGF21. Expression levels of astrocyte markers (Gfap and S100b), microglial marker (Aif1), and inflammatory molecules (Nfkb, Il1b, and Il6) in the hypothalamus of SAMP8 mice were measured by RTqPCR and normalized to Rplp0 values. Analysis was performed 14 weeks after intraCSF administration of 5 x 1010 vg / mouse of the AAV9-CAG-moFGF21-dmiRT vector. Results are expressed as mean ± SEM (n = 9 mice / group). **p < 0.01 vs. untreated mice. Gfap, glial fibrillary acidic protein; S100b, calcium-binding protein B; Aif1, allograft inflammatory factor 1; Nfkb, nuclear factor-κB; Il1b, interleukin-1β; Il6, interleukin-6. [Figure 8]Expression of moFGF21 in the brain after intra-CSF administration of AAV1-FGF21, AAV2-FGF21, and AAV9-FGF21 vectors. Three weeks after intra-CSF administration of 5 x 10 vg / mouse of AAV1-CAG-moFGF21-dmiRT, AAV2-CAG-moFGF21-dmiRT, or AAV9-CAG-moFGF21-dmiRT vectors, expression levels of the mouse codon-optimized FGF21 (moFGF21) coding sequence were measured by RTqPCR in the hypothalamus, cortex, hippocampus, and cerebellum of wild-type mice. Results were normalized by the Rplp0 value and are presented as mean ± SEM (n = 5 mice / group). ND, not detected. [Figure 9] FGF21 protein levels in the brain. Three weeks after administration of 5 × 10 vg / mouse of AAV1-CAG-moFGF21-dmiRT, AAV2-CAG-moFGF21-dmiRT, or AAV9-CAG-moFGF21-dmiRT vectors, FGF21 protein content was determined in brain homogenates of wild-type mice by ELISA. Results were normalized by total protein levels and are expressed as mean ± SEM (n = 5 mice / group). ND, not detected. [Figure 10] Treatment with AAV9-FGF21 vectors reduced adiposity and increased thermogenesis. Representative images of hematoxylin and eosin-stained (A) eWAT and (B) BAT sections from AAV9-FGF21-treated and untreated db / db mice. Original magnification: ×200. (C) Expression levels of thermogenic markers (Ucp1 and Cidea) in BAT of db / db mice were measured by RTqPCR and normalized by Rplp0 values. Analysis was performed 12 weeks after intraCSF administration of 5 × 1010 vg / mouse of the AAV9-CAG-moFGF21-dmiRT vector. Results are expressed as mean ± SEM (n = 9 mice / group). ***p < 0.001 vs. untreated mice. Ucp1, uncoupling protein 1; Cidea, cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; eWAT, epididymal white adipose tissue; BAT, brown adipose tissue. [Figure 11]Decreased liver triglyceride content in AAV9-FGF21-treated mice. (A) Liver triglyceride content. (B) Serum triglyceride and (C) serum FFA levels. Analysis was performed 12 weeks after intra-CSF administration of the vector. Results are expressed as mean ± SEM (n = 9 mice / group). *p < 0.05 vs. untreated mice. FFA, free fatty acids. [Figure 12] Improvement of beta cell mass in FGF21-treated db / db mice. After immunohistochemical analysis of pancreatic sections stained with anti-insulin antibody, (A) the number of pancreatic islets and (B) beta cell mass were calculated for untreated and AAV9-FGF21-treated db / db mice. Results are expressed as mean ± SEM (n = 3 mice / group). *p < 0.05 vs. untreated mice. [Figure 13] Reduced inflammation in the adipose tissue and liver of db / db mice after treatment with AAV9-FGF21 vectors. (A) Representative images of MAC-2 immunohistochemistry of eWAT from untreated and AAV9-FGF21-treated db / db mice (n = 6 per group). (B) Expression levels of the inflammatory marker F4 / 80 in eWAT of db / db mice were measured by RTqPCR and normalized to Rplp0 values. Analysis was performed 12 weeks after intra-CSF administration of 5 x 1010 vg / mouse of AAV9-CAG-moFGF21-dmiRT vector. (C-D) Expression levels of the inflammatory markers F4 / 80, Il6, and Tnfa in BAT (C) and liver (D) of db / db mice were measured by RTqPCR and normalized to Rplp0 values. Analysis was performed 12 weeks after intra-CSF administration of 5 x 1010 vg / mouse of AAV9-CAG-moFGF21-dmiRT vector. Results are expressed as mean ± SEM (n = 9 per group). *p<0.05, **p<0.01, and ***p<0.001 versus untreated mice. F4 / 80, adhesion G-protein-coupled receptor E1; Il6, interleukin-6; Tnfa, tumor necrosis factor-α; eWAT, epididymal white adipose tissue; BAT, brown adipose tissue. MAC-2, lectin, galactose-binding, soluble 3; arrow indicates MAC-2 signaling. [Figure 14]Expression of FGF21 in the brain of AAV1-FGF21-treated db / db mice. Expression levels of the mouse codon-optimized FGF21 (moFgf21) coding sequence were measured by RTqPCR in the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb of db / db mice and normalized by Rplp0 values. Analysis was performed 16 weeks after intra-CSF administration of 5 x 1010 vg / mouse of the AAV1-CAG-moFGF21 vector. Results are expressed as mean ± SEM (n = 7 mice / group). ND, not detected. [Figure 15] Weight loss in db / db mice after treatment with the AAV1-CAG-FGF21 vector. After AAV administration, body weights of untreated db / + (non-obese), untreated db / db, and AAV1-CAG-FGF21-treated db / db mice were measured weekly. Results are expressed as mean ± SEM (n = 7 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001 vs. db / + mice. $$$p < 0.001 vs. untreated db / db mice. [Figure 16] Reversal of diabetes in AAV1-FGF21-treated db / db mice. (A) Changes in postprandial blood glucose levels in non-obese (db / +), untreated, and AAV9-CAG-moFGF21-dmiRT-treated db / db mice after intra-CSF vector administration. (B) Fasting blood glucose levels were measured 11 weeks after AAV1-CAG-FGF21 vector administration. Results are expressed as mean ± SEM (n = 7 mice / group). **p < 0.01 and ***p < 0.001 vs. db / + mice. $$$p < 0.001 vs. untreated db / db mice. [Figure 17] Increased insulin sensitivity in AAV1-FGF21-treated db / db mice. Intraperitoneal insulin tolerance test. Lean (db / +), untreated, and AAV9-CAG-moFGF21-dmiRT-treated db / db mice were intraperitoneally injected with 0.75 U insulin / kg body weight, and blood glucose levels were measured at the indicated time points. Tests were performed 14 weeks after AAV administration. Results are expressed as mean ± SEM (n = 7 mice / group). *p<0.05, **p<0.01, and ***p<0.001 vs. db / + mice. $p<0.05 and $$p<0.01 vs. untreated db / db mice. [Figure 18]Treatment with AAV1-CAG-FGF21 improves glucose tolerance. Glucose tolerance was tested 11 weeks after AAV administration in untreated db / + (non-obese), untreated db / db, and AAV1-CAG-FGF21-treated db / db mice after intraperitoneal injection of glucose (1 g / kg body weight). Results are expressed as mean ± SEM (n = 7 mice / group). *p<0.05 and ***p<0.001 vs. db / + mice. $$$p<0.001 vs. untreated db / db mice. [Figure 19] Decreased gluconeogenesis in db / db mice after AAV1-FGF21 administration. Pyruvate tolerance tests were performed on non-obese (db / +), untreated, and AAV9-CAG-moFGF21-dmiRT-treated db / db mice. All groups were intraperitoneally injected with pyruvate (1 g / kg body weight), and blood glucose levels were measured at the indicated time points. Tests were performed 12 weeks after AAV administration. Results are expressed as mean ± SEM (n = 7 mice / group). ***p < 0.001 vs. untreated mice. $$$p < 0.001 vs. untreated db / db mice. DETAILED DESCRIPTION OF THE INVENTION

[0209] [Example] By using AAV vectors, FG expression in this organ when overexpressed in the brain To test the effect of F21, three different experiments were performed: Treatment of db / db mice with AAV9-CAG-moFGF21-dmiRT. Usage amount: 5×10 10 vg / mouse (Example 1).

[0210] Treatment of SAMP8 mice with AAV9-CAG-moFGF21-dmiRT. Usage amount: 5×10 10 vg / mouse (Example 2).

[0211] Treatment of db / db mice with AAV1-CAG-moFGF21. Dose: 5 x10 10 vg / mouse (Example 4).

[0212] Furthermore, the present inventors have demonstrated that AAV1-FGF21, AA Brain transduction efficiency with V2-FGF21 and AAV9-FGF21 vectors was also examined. (Example 3).

[0213] dmiRT is a sequence consisting of four copies of miRT-122a and four copies of miRT-1. Point.

[0214] The CAG-moFGF21-dmiRT gene construct sequence is contained in the sequence of SEQ ID NO: 35. The CAG-moFGF21 gene construct sequence is contained in the sequence of SEQ ID NO:46.

[0215] General Procedure for Examples Target characteristics Male BKS.Cg-+Lepr db / +Lepr db OlaHsd(db / db), BKS.Cg-m+ / +Lepr db / OlaHsd(db / +, non-obese)SAMP8 / TaHsd (SAMP8) and C57Bl / 6J (wild-type) mice were used. The rats were fed a standard diet ad libitum (2018S Teklad Global Diets (registered trademark), Harlan Labs., Inc., Madison, WI, US, 1 They were kept under a 2-hour light / dark cycle (lights on at 8:00 AM) and a stable temperature (22°C ± 2°C). For tissue sampling, mice were administrated with the inhalation anesthetic isoflurane (IsoFlo). (Registered Trademark), Abbott Laboratories, Abbott Park, IL The animals were anesthetized with acetaminophen (US) and decapitated. The tissues of interest were excised and kept at -80°C until analysis. All experimental procedures were supported by the Universitat Autonoma de Barcel ona's Ethics Committee for Animal and Huma Approved by n Experimentation.

[0216] Recombinant AAV vectors Standard method (Ayuso, E. et al., 2010. Curr Gene Th Triple transfection of HEK293 cells according to the method described in (Er. 10(6):423-36). Single-stranded AAV vectors of serotypes 1, 2, and 9 were generated by transfection. 10 roller bottles (850 cm) in DMEM 10% FBS until fluence 2 , flat; Corning(TM), Sigma-Aldrich Co., Saint Louis, MO, US) and transfected with AAV2 ITR by calcium phosphate method. (SEQ ID NO: 35), serotypes 1 and 2, respectively. or a helper plasmid carrying the AAV2 rep gene and AAV cap gene. The plasmid was cotransfected with a plasmid carrying the adenovirus helper function. The transgene used was early enhancer / chicken β-actin (CAG ) promoter (SEQ ID NO: 27) In Examples 1, 2 and 3, the transgene contained an expression cassette. Four tandem repeats of the miRT-122a sequence cloned into the 3' untranslated region of the (5'CAAACACCATTGTCACACTCCA3', SEQ ID NO: 12) and Four tandem repeats of the miRT-1 sequence (5'TTACATACTTCTTTACA In Example 4, the cassette also contained the miRT-TTCCA3' (SEQ ID NO: 13). 122a and miRT-1 were not present. , using an optimized method based on two successive cesium chloride (CsCl) gradients. This second-generation CsCl-based protocol purifies empty AAV capsids. and dramatically reduced DNA and protein impurities (Ayuso, E. et al. l., 2010. Curr Gene Ther. 10(6):423-36). The AAV vector was dialyzed against PBS, filtered, and stored at -80°C. Using linearized plasmid DNA, the AAV2 reference standard was used. Viral genome titers were determined by quantitative PCR according to Coll (Lock M, et al., Hum. Gene Ther. 2010;21:1273-1285). The vectors were constructed according to molecular biology techniques well known in the art.

[0217] In vivo administration of AAV vectors into CSF Intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg) The mice were anesthetized, and the skin on the back of the head was shaved from behind the ears to almost between the shoulder blades. The mouse was placed in a prone position with its head tilted slightly downward. Hamilton injection using a rostro-caudal incision The syringe was introduced into the cisterna magna at a 45-55° angle, between the occipital region and the C1 vertebra, and 5 μl of vector was injected. -dilutions were administered. Considering that the CNS is the main target compartment for vector delivery , the same number of vector genomes / mouse (5 × 10) regardless of body weight 10 vg / mouse) It was administered to the patient.

[0218] Immunohistochemical and morphometric analysis Tissues were fixed in formalin (Panreac Quimica) for 24 hours and then paraffinized. The tissue samples were embedded in PBS and sectioned. The tissue samples were stained with hematoxylin and eosin and analyzed by image analysis. Software (analySIS 3.0; Soft Imaging System, Video camera with monitor (Center Valley, PA, EEUU) A Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) was connected to Images were acquired using a microscope (Japan).

[0219] Immunohistochemistry Tissues were fixed in 10% formalin for 12–24 hours, embedded in paraffin, and sectioned. For immunohistochemical detection, sections were deparaffinized and stained with rat anti-MAC2 (1:50; CL8942AP; Cedarlane) and guinea pig anti-insulin (1:100; I-8510; Sigma-Aldrich) overnight at 4°C. Immunolabeled rabbit anti-rat (1:300; E0467; Dako) and peroxidase-coupled The secondary antibody was purified with rabbit anti-guinea pig (1:300; P0141; Dako). The ABC peroxidase kit (Pierce) was used for immunodetection. Sections were counterstained in Mayer's hematoxylin. Two insulin sections separated by 200 μm were used. The area of ​​all insulin-positive cells in each section was calculated based on the total pancreatic surface area of ​​the section. The percentage of beta cell area within the pancreas was analyzed by dividing by the product. Sea urchin (Casellas et al., 2006), pancreatic weight multiplied by the percentage of β-cell area The β-cell mass was calculated by

[0220] RNA analysis Tripure isolation reagent (Roche Diagnostics Corp., India) (Islandapolis, IN, US) from the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb. et al., and Qiazol lysis reagent (Qiagen NV, Venlo, NL) and hippocampal RNeasy Mini or RNeasy Micro kit (Qia gen NV, Venlo, NL) to study white adipose tissue, brown adipose tissue, and liver. Total RNA was obtained from the 100-kDa strain. To eliminate residual viral genomes, DNAse I (Qiagen) was used. Total RNA was processed using the RT-PCR assay (Berlin NV, Venlo, NL). Transcriptor First Strand cDNA Synthesis Kit (04 379012001, Roche, California, USA) was used. The RNA samples were reverse transcribed using TB Green Premix Ex Taq II (Ta Kara Bio Europe, France) using the SmartCycler Real-time quantification with II® (Cepheid, Sunnyvale, USA) PCR was performed. Data were normalized by Rplp0 values ​​and analyzed as previously described ( Pfaffl, M., Nucleic Acids Res.2001;29(9):e 45)Analyzed.

[0221] The primers used are outlined below: moFgf21-Fw:5'-CCTAACCAGGACGCCACAAG-3' (configuration Column number 47) moFgf21-Rv:5'-GTTCCACCATGCTCAGAGGG-3' (configuration Column number 48) Gfap-Fw:5'-ACAGACTTTCTCCAACCTCCAG-3' (sequence Number 49) Gfap-Rv: 5'-CCTTCTGACACGGATTTGGT-3' (SEQ ID NO: 50) S100b-Fw:5'-AACAACGAGCTCTCTCACTTCC-3' (configuration Column number 51) S100b-Rv: 5'-CGTCTCCATCACTTTGTCCA-3' (SEQ ID NO: No. 52) Aif1-Fw: 5'-TGAGCCAAAGCAGGGATTTG-3' (SEQ ID NO: 53) Aif1-Rv: 5'-TCAAGTTTGGACGGCAGATC-3' (SEQ ID NO: 54) Nfkb-Fw: 5'-GACCACTGCTCAGGTCCACT-3' (SEQ ID NO: 55) Nfkb-Rv: 5'-TGTCACTATCCCGGAGTTCA-3' (SEQ ID NO: 56) Il1b-Fw: 5'-ATGAAGGGCTGCTTCCAAAC-3' (SEQ ID NO: 57) Il1b-Rv: 5'-ATGTGCTGCTGCGAGATTTG-3' (SEQ ID NO: 58) Il6-Fw: 5'-TCGCTCAGGGTCACAAGAAA-3' (SEQ ID NO: 5 9) Il6-Rv: 5'-CATCAGAGGCAAGGAGGAAAAC-3' (SEQ ID NO: No. 60) Ucp1-Fw: 5'-GGCCTCTACGACTCAGTCCA-3' (SEQ ID NO: 61) Ucp1-Rv: 5'-TAAGCCGGCTGAGATCTTGT-3' (SEQ ID NO: 62) Cidea-Fw: 5'-AAACCATGACCGAAGTAGCC-3' (SEQ ID NO: No. 63) Cidea-Rv:5'-AGGCCAGTTTGTGATGACTAAGAC-3'( SEQ ID NO: 64) Tnfa-Fw: 5'-CGGCATGGATCTCAAAGACAAC-3' (sequence Number 65) Tnfa-Rv: 5'-AGATAGCAAATCGGCTGACG-3' (SEQ ID NO: 66) F4 / 80-Fw:5'-CTTTGGCTATGGGCTTCCAGTC-3' (configuration Column number 67) F4 / 80-Rv:5'-GCAAGGAGGACAGAGTTTATC-3' (sequence Number 68) Rplp0-Fw: 5'-ACTGGTCTAGGACCCGAGAA-3' (SEQ ID NO: No. 69) Rplp0-Fw: 5'-TCCCACCTTGTCTCCAGTCT-3' (SEQ ID NO: No. 70) Hormone and metabolite assays Glucometer Elite™ Analyzer (Bayer, Leverkuse Blood glucose levels were measured using a 100-kDa antibody (Bio-Rad, Germany). Brain levels of FGF21 protein were measured using a 100-kDa antibody (Bio-Rad, Germany). Quantitative sandwich enzyme immunoassay mouse / rat FGF-21 ELISA kit ( MF2100, R&Dsystems, Abingdon, UK) and whole brain Bradford reagent (Bio-Rad Protein Assay) in the homogenate Normalized by total protein content measured using a chromatographic method (Bio-Rad, Germany). To extract lipids from the liver, the method described by Carr et al. Approximately 100 mg of frozen sample was weighed and dissolved in chloroform:methanol (2:1) to Homogenized. Enzyme assay kit (Horiba-ABX, Montpellier) Liver triglycerides and serum triglycerides were measured spectrophotometrically using a 1000-kJ / kg / day meter (France). The acyl-CoA synthase and acyl-CoA oxidase methods (Wak Serum free lipids were measured by the National Institute of Chemicals GmbH, Neuss, Germany. All biochemical parameters were measured using a Pentra 400 analyzer (Ho The results were determined using the riba-ABX.

[0222] Insulin tolerance test For the insulin tolerance test, insulin (0.75 IU / kg body weight; Humulin Regular (Eli Lilly, Indianapolis, IN) wake-fed Mice (awake fed mice) were intraperitoneally injected with insulin. Glucose concentrations were determined in blood samples obtained from the tail vein at the time points.

[0223] Glucose tolerance test Conscious mice were fasted overnight (16 hours) and then intraperitoneally injected with glucose (1 g / kg body weight). Blood glucose was measured in tail vein blood samples at the indicated time points.

[0224] Pyruvate tolerance test Conscious mice were fasted overnight (16 hours) and then intraperitoneally injected with pyruvate (1 g / kg body weight). Blood glucose was measured in tail vein blood samples at the indicated time points.

[0225] [Example 1] of AAV9-CAG-moFGF21-dmirT vector in db / db mice Reversal of obesity and diabetes by intra-CSF administration The present inventors have demonstrated that leptin signaling is deficient and that this is a widely used treatment for obesity and diabetes. The study used FGF21 in 7-week-old db / db male mice, a genetic model that has been shown to The antidiabetogenic and antiobesogenic therapeutic potential of AAV-mediated genetic engineering of the brain was evaluated. For this purpose, liver-specific miR-122a and cardiac Included are the target sites of specific miR-1 (AAV9-CAG-moFGF21-dmiRT) The mouse codon-optimized FGF21 coding sequence was placed under the control of a CAG ubiquitous promoter. Code, 5x10 10 The AAV9 vectors in vg / mouse are delivered via the cisterna magna into the cerebrospinal fluid (CSF) The drug was administered locally into the SF. Untreated db / db animals served as controls.

[0226] within various brain regions, including the hypothalamus, cortex, hippocampus, and cerebellum, 12 weeks after AAV administration. AAV9-CAG-moF, as evidenced by increased expression levels of the factor IntraCSF administration of FGF21-dmiRT vector induces widespread overexpression of FGF21 in the brain. was mediated (Figure 1).

[0227] Untreated db / db mice continued to gain weight during the 12-week follow-up period (approximately 50% weight gain in the cohort treated with a vector encoding FGF21 The body weight was significantly reduced (approximately 20% weight gain) (Fig. 2A and Fig. 2B). Animals treated with the AAV9-CAG-moFGF21-dmiRT vector showed increased body fat The mice showed a reduction in fat accumulation and a 60% reduction in liver weight (Figure 2C). Brain-targeted db / db mice with FGF21 gene transfer also showed a complete reduction in blood glucose during feeding. showed normalization, demonstrating suppression of diabetes in these animals (Fig. 3).

[0228] Obesity is associated with brain inflammation (O. Guillemot-Legris, G.G. Mucc) ioli,Trends Neurosci.40,237-253(2017). Inflammation in the organ was assessed by measuring the astrocyte markers Gfap and S100b, as well as microglia. The marker Aif1, as well as inflammatory molecules such as Nfkb, Il1b, and Il The expression of the AAV9-CAG-moFGF21-dmiRT vector was analyzed. Intra-CSF treatment of db / db mice with α-glucan significantly increased the expression of Gfap, S100b, and Ai in the hypothalamus. showed decreased expression of f1, Nfkb, Il1b, and Il6 (Fig. 4).

[0229] Example 1.1 Histological analysis of white adipose tissue by hematoxylin-eosin staining revealed that the Histological analysis of BAT revealed a decrease in white adipocyte size (Fig. 10A). showed relatively low lipid accumulation and relatively many multilocular brown adipocytes (Fig. 10B). These results indicate that the expression levels of Ucp1 and Cidea were significantly increased in FGF21-treated mice. Thermogenesis was highly increased in BAT of mice (Fig. 10C) after AAV-FGF21 CNS administration. In AAV9-FGF21-treated db / db mice, liver triglycerides were increased. In parallel, these mice had decreased triglyceride and Circulating levels of erythropoietin and serum free fatty acids were also reduced (Fig. 11B and Fig. 11C). Histochemical analysis revealed that db / db mice were treated with AAV9-FGF21 vectors It was found that the number of pancreatic islets increased (Fig. 12A) and β-cell mass improved (Fig. 12B). was turned into.

[0230] Obesity and diabetes are associated with systemic inflammation. In white adipose tissue, MAC-2 inflammatory matrix metalloproteinases (MAC-2) are expressed. Immunohistochemical analysis of the marker revealed no significant differences in macrophages in AAV9-FGF21-treated mice. showed a decrease in phage infiltration (Fig. 13A), which was consistent with a decrease in F4 / 80 mRNA expression levels. FGF21-treated animals also showed a significant decrease in brown adipose tissue and liver. The expression levels of inflammatory cytokines F4 / 80, Il6, and Tnfα were reduced (Figure 13C and Figure 13D), demonstrating a reduction in systemic inflammation after FGF21 gene therapy.

[0231] [Example 2] AAV9-CAG-moFGF21-dmirT vector in SAMP8 mice Reduced weight gain with intra-CSF administration Seven-week-old senescence-accelerated mice, a widely used aging mouse model with age-related brain pathology, were used. 5 × 10 10 vg / mouse A The AV9-CAG-moFGF21-dmiRT vector was locally injected into the CSF via the cisterna magna. Untreated SAMP8 animals served as controls.

[0232] Similar to the observations made in db / db mice, AAV9-CAG-moFGF IntraCSF administration of the 21-dmiRT vector resulted in the development of SAMP8 myeloma 14 weeks after AAV administration. mediated strong overexpression of FGF21 in the hypothalamus, cortex, hippocampus, and cerebellum of mice (Figure 1). 5). FGF21-treated mice showed lower weight gain than the untreated cohort (Fig. 6A and and Fig. 6B), which was paralleled by a decrease in liver weight (Fig. 6C). In the hypothalamus of SAMP8 mice overexpressing 21, the inflammatory cytokines Il1b and Il2b were expressed. The expression of Il6 and Il7 was decreased (Fig. 7).

[0233] [Example 3] AAV1-CAG-moFGF21-dmirT, AAV2-CAG-moFGF21 -dmirT and AAV9-CAG-moFGF21-dmirT vectors in CSF Brain transduction after administration.

[0234] Several AAV serotypes efficiently transduce the brain after direct CSF administration via the cisterna magna. To investigate whether liver-specific miR-122a and cardiac-specific miR-122a could be involved in the pathogenesis of heart failure, A mouse codon under the control of a CAG ubiquitous promoter containing a target site for miR-1 5 x 10 encoding the optimized FGF21 coding sequence 10 vg / mouse AAV1, AA V2 and AAV9 vectors (AAV1-CAG-moFGF21-dmiRT, respectively) , AAV2-CAG-moFGF21-dmiRT and AAV9-CAG-moFGF Wild-type mice were treated with 21-dmiRT. As a control, untreated wild-type mice were treated with 21-dmiRT. We used the same material.

[0235] Three weeks after intra-CSF administration of the AAV vector, brain samples were obtained, and RT-PCR analysis revealed that the thalamus Increased expression of moFGF21 was demonstrated in various brain regions, including the subcortex, hippocampus, and cerebellum. Furthermore, overexpression of moFGF21 increased FGF21 protein content throughout the brain (Fig. 8). This resulted in an increase in the amount of erythrocytes (Fig. 9).

[0236] [Example 4] IntraCSF administration of AAV1-CAG-moFGF21 vector in db / db mice Reversing obesity and diabetes with.

[0237] The mouse codon-optimized FGF21 coding sequence was engineered under the control of a CAG ubiquitous promoter. Code, 5x10 10 vg / mouse AAV1 vector (AAV1-CAG-moF GF21) was administered locally into the cerebrospinal fluid (CSF) via the cisterna magna in 7-week-old db / db males. Antidiabetic effects of AAV-mediated genetic engineering of the brain with FGF21 gene therapy in mice The anti-obesity and anti-obesity therapeutic potential of the untreated db / db and untreated db / db mice were also evaluated. db / + (non-obese) mice were used.

[0238] Various brain regions, including the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb, were observed 16 weeks after AAV administration. As evidenced by increased expression levels of the factor in the AAV1-CAG- Intra-CSF administration of moFGF21 vector mediates widespread overexpression of FGF21 in the brain (Figure 14).

[0239] Untreated db / db mice continued to gain weight during the 14-week follow-up period, whereas AAV1 - The cohort treated with the vector encoding FGF21 did not gain weight ( (Figure 15). Notably, db / db mice were brain-targeted with FGF21 gene transfer. also showed complete normalization of fed and fasting blood glucose (Figures 16A and 16B). Suppression of diabetes was demonstrated in these animals.

[0240] Insulin tolerance tests were performed after treatment with AAV1-FGF21 viral vectors. / db showed improved insulin resistance (Figure 17), and overnight fasting mice Intraperitoneal glucose tolerance test was performed on mice treated with AAV1-CAG-moFGF21. The results showed that db / db mice treated with α-glucose agonist β-glucose agonist (α-glucose agonist) had higher glucose tolerance than untreated db / db mice (Figure 1). 18) As an index of hepatic gluconeogenesis, an intraperitoneal pyruvate tolerance test was performed. After the test, blood glucose levels rose to 600 mg / dl in db / db untreated mice and remained elevated throughout the study. The blood levels of FGF21 db / db treated mice and non-obese treated mice remained unchanged. The glucose level increased up to 150 mg / dl, and therefore, AAV1-CAG-FGF2 A decrease in gluconeogenesis after 1 treatment was demonstrated (Figure 19). [Table 4] TIFF2025170785000005.tif63166

[0241] Amino acid sequence of Homo sapiens FGF21 (SEQ ID NO: 1) MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQ FGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPES LLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPE ACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPR GPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGP SQGRSPSYAS Nucleotide sequence of Homo sapiens FGF21 (SEQ ID NO: 4) ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTG TGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCC AGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATT CGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGAT GCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATG GGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCT CCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTTATTCAAATC TTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAG ATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGC CTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAAT GTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGC CAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGG ACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCGCA CTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCG ATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTC CCAGGGCCGAAGCCCCAGCTACGCTTCCTGGA ホモサピエンスFGF21- コドン コドン optimization nucleotide sequence(sequence number 5) ATGGATTCTGATGAGCAGGCTTCGAGCACAGGCGGCCTG TGGGTTTCAGTTCTGGCTGGACTGCTGCTGGGAGCCTGTC AGGCACACCCTTATTCCAGATAGCAGCCCTCTGCTGCAGTT CGGCGGACAAGTGCGGCAGAGATACCTGTACACCGACGAC GCCCAGCAGACAGAAGCCCACCTGGAAATCAGAGAGGATG GCACAGTTGGCGGAGCCGCCGATCAGTCTCCTGAATCTCT GCTCCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATC CTGGGCGTGAAAACCAGCGGTTCCTGTCCAAAAGACCTG ACGGCGCCCTGTATGGCAGCCTGCACTTTGATCCTGAGGC CTGCAGCTTCAGAGAGCTGCTGCTTGAGGACGGCTACAAC GTGTACCAGTCTGAGGCCCATGGCCTGCCTCTGCATCTGC CTGGAAACAAGAGCCCTCACAGAGATCCCGCTCCTAGAGG CCCTGCCAGATTTCTGCCTCTTCCTGGATTGCCTCCTGCT CTGCCAGAGCCTCCTGGAATTCTGGCTCCTCAGCCTCCTG ATGTGGGCAGCTCTGATCCTCTGAGCATGGTCGGACCTAG CCAGGGCAGATCTCTCTAGCTACGCTCTTGA ホモサピエンスFGF21- コドン コドン optimization nucleotide sequence(SEQ ID NO:6) ATGGACAGCGATGAAACCGGGTTCGAGCACAGCGGTCTG TGGGTGTCCGTGCTGGCCGGACTGCTCCTGGGAGCCTGTC AGGCGCACCCCATCCCTGACTCCTCGCCGCTGCTGCAATT CGGCGGACAAGTCCGCCAGAGATACCTGTACACCGACGAC GCCCAGCAGACCGAAGCCCACCTGGAAATTCGGGAGGACG GGACTGTGGGAGCGCTGCAGATCAGTCACCCGAGTCCCT CCTCCAACTGAAGGCCTTGAAGCCCGGCGTGATTCAGATC CTGGGCGTGAAAACTTCCCGCTTCCTTGCCAACGGCCGG ATGGAGCTCTGTACGGATCCCTGCACTTCGACCCCGAAGC CTGCTCATTCCGCGAGCTGCTCCTTGAGGACGGCTATAAC GTGTACCAGTCTGAGGCCCATGGACTCCCCCTGCATCTGC CCGGCAACAAGTCCCCTCACCGGGATCCTGCCCCAAGAGG CCCAGCTCGGTTTCTGCCTCTGCCGGGACTGCCTCCAGCG TTGCCCGAACCCCCTGGTATCCTGGCCCCGCAACCACCTG ACGTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAG CCAGGGAAGGTCCCCGTCCTACGCATCCTGA ホモサピエンスFGF21- コドン コドン optimization nucleotide sequence(SEQ ID NO:7) ATGGATTCCGACGAAACTGGATTTGAACATTTCAGGGCTG TGGGTCTCTGTGCTGGCTGGACTGCTGCTGGGGCTTGTC AGGCTCACCCCATCCCTGACAGCTCCCCTCTGCTGCAGTT CGGAGGACAGGTGCGGCAGAGATACCTGTATACCGACGAT GCCCAGCAGACAGAGGCACACCTGGAGATCAGGGAGGACG GAACCGTGGGAGGAGCAGCCGATCAGTCTCCCGAGAGCCT GCTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATC CTGGGCGTGAAGACATCTCGGTTTCTGTGCCAGCGGCCCG ACGGCGCCCTGTACGGCTCCCTGCACTTCGATCCCGAGGC CTGTTCTTTTAGGGAGCTGCTGCTGGAGGACGGCTACAAC GTGTATCAGAGCGAGGCACACGGCCTGCCACTGCACCTGC CTGGCAATAAGTCCCCTCACCGCGATCCAGCACCCAGGGGG CCCAGCACGCTTCCTGCCTCTGCCAGGCCTGCCCCTGCC CTGCCAGAGCCACCCGGCATCCTGGCCCCCCAGCCTCCAG ATGTGGGCTCCAGCGATCCTCTGTCAATGGTGGGGCCAAG TCAGGGGCGGAGTCCTTCATACGCATCATAA パースコドン optimization FGF21のnucleotide sequence(sequence number 9) ATGGAATGGATGAGAAGCAGAGTGGGCACCCTGGGCCTG TGGGTGCGACTGCTGCTGGCTGTGTTTCTGCTGGGCGGTGT ACCAGGCCTACCCCATCCCTGACTCTAGCCCCCTGCTGCA GTTTGGCGGACAAGTGCGGCAGAGATACCTGTACACCGAC GACGACCAGGACACCGAGGCCCACCTGGAAATCCGCGAGG ATGGCACAGTCGTGGGCGCTGCTCCACAGAAGCCCTGAGAG CCTGCTGGAACTGAAGGCCCTGAAGCCCGGCTGATCCAG ATCCTGGGCGTGAAGGCCAGCAGATTCCTGTGCCAGCAGC CTGACGGCGCCCTGTACGGCTCTCCTCACTTCGATCCTGA GGCCTGCAGCTTCAGAGAGCTGCTGCTGGAGGACGGCTAC AACGTGTACCAGTCTGAGGCCCACGGCCTGCCCCTGAGAC TGCCTCAGAAGGACAGCCCTAACCAGGACGCCACAAGCTG GGGACCTGTGCGGTTCCTGCCTATGCCTGGACTGCTGCAC GAGCCCCAGGATCAGGCTGGCTTTCTGCCTCCTGAGCCTC CAGACGTGGGCAGCAGCGACCCTCTGAGCATGGTGGAACC TCTGCAGGGCAGAAGCCCCAGCTACGCCTCTTGA Nucleotide sequence of CAG promoter (SEQ ID NO: 27) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTC CCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACAT CAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCA ATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC AATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGG CGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGG TGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAA AGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTC GCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCC CGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGC GGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGT TTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGC CTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCG GCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGC CGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGC GGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCG AGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGG GCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGT GGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTG CAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACG GCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGC GGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGG GTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCT CGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTG TCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAA TCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCT GTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTC TAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAG GAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCC GTCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGG GGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTC GGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTA ACCATGTTCATGCCTTCTTCTTTTTCCTACAG Nucleotide sequence of the CMV promoter (SEQ ID NO: 28) GTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAG CGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGA CTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGT TGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTA TATAAGCAGAGCT Nucleotide sequence of the CMV enhancer (SEQ ID NO: 29) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTC CCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACAT CAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCA ATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATG CMV promoter and CMV enhancer sequences (SEQ ID NO: 34) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTC CCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACAT CAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCA ATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT ACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGATT TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACA ACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGG CGTGTACGGTGGGAGGTCTATATAAGCAGAGCT AAV2 5'ITR (SEQ ID NO: 30) GCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAA GC CCGGGCGTCG GGCGACCTTT GGTCGCCCGG CCTCAG TGAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT AGGGGT TCCT AAV2 3'ITR (SEQ ID NO: 31) AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTC TGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGT CGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GT GAGCGAGC GAGCGCGC Rabbit β-globin polyadenylation signal (containing polyA signal, rabbit β-globin bin 3'UTR and adjacent regions) (SEQ ID NO: 33) GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATG AAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAAT TTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCT CTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAA CATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATG CCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAAG AGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCC TTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTT TTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCT AAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTC CTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCT TATGGAGATC miRT sequence miRT-122a (SEQ ID NO: 12): 5'CAAACACCATTGTCACACT CCA3', a target for liver-expressed microRNA-122a (miRBase Database accession number, MI0000442).

[0242] miRT-152 (SEQ ID NO: 14): 5'CCAAGTTCTGTCATGCACTG A3', target for liver-expressed microRNA-152 (MI0000462) .

[0243] miRT-199a-5p (SEQ ID NO: 15): 5'GAACAGGTAGTCTGAA CACTGGG3', a target for liver-expressed microRNA 199a (MI0 000242).

[0244] miRT-199a-3p (SEQ ID NO: 16): 5'TAACCAATGTGCAGAC TACTGT3', target for liver-expressed microRNA-199a (MI00 00242).

[0245] miRT-215 (SEQ ID NO: 17): 5'GTCTGTCAATTCATAGGTCA T3', a target for liver-expressed microRNA-215 (MI0000291) .

[0246] miRT-192 (SEQ ID NO: 18): 5'GGCTGTCAATTCATAGGTCA G3', target for liver-expressed microRNA-192 (MI0000234) .

[0247] miRT-148a (SEQ ID NO: 19): 5'ACAAAGTTCTGTAGTGCAC TGA3', target for liver-expressed microRNA-148a (MI00002 53).

[0248] miRT-194 (SEQ ID NO: 20): 5'TCCACATGGAGTTGCTGTTA CA3', a target for liver-expressed microRNA-194 (MI0000488 ).

[0249] miRT-133a (SEQ ID NO: 21): 5'CAGCTGGTTGAAGGGGACC AAA3', a target for microRNA-133a expressed in the heart (MI00004 50).

[0250] miRT-206 (SEQ ID NO: 22): 5'CCACACACTTCCTTACATTC CA3', a target for cardiac-expressed microRNA-206 (MI0000490 ).

[0251] miRT-1 (SEQ ID NO: 13): 5'TTACATACTTCTTTACATTCCA 3′, target for cardiac-expressed microRNA-1 (MI0000651).

[0252] miRT-208a-5p (SEQ ID NO: 23): 5'GTATAACCCGGGCCAA AAGCTC3', a target for microRNA-208a expressed in the heart (MI00 00251).

[0253] miRT-208a-3p (SEQ ID NO: 24): 5'ACAAGCTTTTTGCTCG TCTTAT3', a target for microRNA-208a expressed in the heart (MI00 00251).

[0254] miRT-499-5p (SEQ ID NO: 25): 5'AAACATCACTGCAAGTC TTAA3', a target for microRNA-499 expressed in the heart (MI00031 83).

[0255] pAAV-CAG-moFGF21-dmiRT (SEQ ID NO: 35) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAA TCGGCC AACGCGCGGG 51 GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGC TTCCTCG CTCACTGACT 101 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CG GTATCAGC TCACTCAAAG 151 GCGGTAATAC GGTTATCCAC AGAATCAGGG GA TAACGCAG GAAAGAACAT 201 GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CC GTAAAAAG GCCGCGTTGC 251 TGGCGTTTTT CCATAGGCTC CGCCCCCCTG AC GAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GG ACTATAAA GATACCAGGC 351 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TC CTGTTCCG ACCCTGCCGC 401 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CG GGAAGCGT GGCGCTTTCT 451 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GT GTAGGTCG TTCGCTCCAA 501 GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GC CCGACCGC TGCGCCTTAT 551 CCGGTAACTA TCGTCTTGAG TCCAACCCGG TA AGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AG AGCGAGGT ATGTAGGCGG 651 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CT ACGGCTAC ACTAGAAGAA 701 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CA GTTACCTT CGGAAAAAGA 751 GTTGGTAGCT CTTGATCCGG CAAACAAACC AC CGCTGGTA GCGGTGGTTT 801 TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AA AAAAAGGA TCTCAAGAAG 851 ATCCTTTGAT CTTTTCTACG GGGTCTGACG CT CAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AA AAGGATCT TCACCTAGAT 951 CCTTTTAAAT TAAAAATGAA GTTTTAAATC AA TCTAAAGT ATATATGAGT 1001 AAACTTGGTC TGACAGTTAC CAATGCTTAA T CAGTGAGGC ACCTATCTCA 1051 GCGATCTGTC TATTTCGTTC ATCCATAGTT G CCTGACTCC CCGTCGTGTA 1101 GATAACTACG ATACGGGAGG GCTTACCATC T GGCCCCAGT GCTGCAATGA 1151 TACCGCGAGA CCCACGCTCA CCGGCTCCAG A TTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT C CTGCAACTT TATCCGCCTC 1251 CATCCAGTCT ATTAATTGTT GCCGGGAAGC T AGAGTAAGT AGTTCGCCAG 1301 TTAATAGTTT GCGCAACGTT GTTGCCATTG C TACAGGCAT CGTGGTGTCA 1351 CGCTCGTCGT TTGGTATGGC TTCATTCAGC T CCGGTTCCC AACGATCAAG 1401 GCGAGTTACA TGATCCCCCA TGTTGTGCAA A AAAGCGGTT AGCTCCTTCG 1451 GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG C CGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT G TCATGCCAT CCGTAAGATG 1551 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA G TCATTCTGA GAATAGTGTA 1601 TGCGGCGACC GAGTTGCTCT TGCCCGGCGT C AATACGGGA TAATACCGCG 1651 CCACATAGCA GAACTTTAAA AGTGCTCATC A TTGGAAAAC GTTCTTCGGG 1701 GCGAAAACTC TCAAGGATCT TACCGCTGTT G AGATCCAGT TCGATGTAAC 1751 CCACTCGTGC ACCCAACTGA TCTTCAGCAT C TTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT G CCGCAAAAA AGGGAATAAG 1851 GGCGACACGG AAATGTTGAA TACTCATACT C TTCCTTTTT NOTE 1901 GAAGCATTTA TCAGGGTTAT TGTCTCATGA G CGGATACAT ATTTGAATGT 1951 ATTTAGAAA ATAAACAAAAT AGGGGTTCCG C GCACATTTC CCCGAAAGT 2001 GCCACCTGAC GTCTAAGAAA ASSISTANCE C ACKNOWLEDGMENTS 2051 ATAGGCGTAT CACGAGGCCC TTTCGTCTCG C GCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG A CGGTCACAG CTTGTCTGTA 2151 AGCGGATGCC GGGAGCAGAC AAGCCCGTCA G GGCGCGTCA GCGGGTGTTG 2201 GCGGGTGTCG GGGCTGGCTT AACTATGCGG C GATTGTACTG 2251 AGAGTGCACC ATATGCGGTG TGAAATACCG C ACAGATGCG GENERATIONAA 2301 ATACCGCATC AGGCGATTCC AACATCCAAT A AATCATACA GGCAAGGCAA 2351 CHANGE AACH CHANGE ATAAAGCCTC A ENDAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTATGA C TTTTGCGGG AGAAGCCTTT 2451 ATTTCAACGC AAGGATAAAA ATTTTAGAA C CCTCATATA TTTAAATGC 2501 AATGCCTGAG TAATGTGTAG GTAAAGATTC A AACGGGTGA GAAAGGCCGG 2551 AGACAGTCAA ATCACCATCA ATATGATATT C AACCGTTCT AGCTGATAAA 2601 TTCATGCCGG AGAGGGTAGC TATTTTTGAG A GGTCTCTAC AAAGGCTATC 2651 AGGTCATTGC CTGAGAGTCT GGAGCAAACA A GAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC C CCGGTTGAT AATCAGAAAA 2751 GCCCCAAAAA CAGGAAGATT GTATAAGCAA A TATTTAAAT TGTAAGCGTT 2801 AATATTTTGT TAAAATTCGC GTTAAATTTT T GTTAAATCA GCTCATTTTT 2851 TAACCAATAG GCCGAAATCG GCAAAATCCC T TATAAATCA AAAGAATAGA 2901 CCGAGATAGG GTTGAGTGTT GTTCCAGTTT G GAACAAGAG TCCACTATTA 2951 AAGAACGTGG ACTCCAACGT CAAAGGGCGA A AAACCGTCT ATCAGGGCGA 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC A AGTTTTTTG GGGTCGAGGT 3051 GCCGTAAAGC ACTAAATCGG AACCCTAAAG G GAGCCCCCG ATTTAGAGCT 3101 TGACGGGGAA AGCCGGCGAA CGTGGCGAGA A AGGAAGGGA AGAAAGCGAA 3151 AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT A GCGGTCACG CTGCGCGTAA 3201 CCACCACACC CGCCGCGCTT AATGCGCCGC T ACAGGGCGC GTACTATGGT 3251 TGCTTTGACG AGCACGTATA ACGTGCTTTC C TCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT T AGACAGGAA CGGTACGCCA 3351 GAATCCTGAG AAGTGTTTTT ATAATCAGTG A GGCCACCGA GTAAAAGAGT 3401 CTGTCCATCA CGCAAATTAA CCGTTGTCGC A ATACTTCTT TGATTAGTAA 3451 TAACATCACT TGCCTGAGTA GAAGAACTCA A ACTATCGGC CTTGCTGGTA 3501 ATATCCAGAA CAATATACC GCCAGCCATT G CAACGGAAT CGCCATTCGC 3551 CATTCAGGCT GCGCAACTGT TGGGAAGGGC G ATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC A CTGAGGCCG CCCGGGCAAA 3651 GCCCGGGCGT CGGGCGACCT TTGGTCGCCC G GCCTCAGTG GCCTCAGTG 3701 CGCGCAGAGA GGGAGTGGCC AACTCCATCA C TAGGGGTTC CTTGTAGTTA 3751 ATGATTACCC CGCCATGCTA CTTATCTACT C GACATTGAT TATTGACTAG 3801 TATTCATT CGGGGTCATT A GTTCATAGC CCATATATGG 3851 AGTTCCGCGT TACATAACTT ACGGTAAATG G CCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG A CGTATGTTC CGTATTAAC 3951 GCCAATAGGG ACTTTCCATT GACGTCAATG G GTGGAGTAT TTACGGTAAA 4001 CTGCCCACTT GGCAGTACAT CAAGTGTATC A TATGCCAAG TACGCCCCCT 4051 ATTGACGTCA ATGACGGTAA ATGGCCCGCC T GGCATTATG CCCAGTACAT 4101 GACCTTATGG GACTTTCCTA CTTGGCAGTA C ATCTACGTA TTAGTCATCG 4151 CTATTACCAT GGTCGAGGTG AGCCCCACGT T CTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT T TATTTATTT TTTAATTATT 4251 TTGTGCAGCG ATGGGGGCGG GGGGGGGGGG G GGGCGCGCG CCAGGCGGGG 4301 CGGGGCGGGG CGAGGGGCGG GGCGGGGCGA G GCGGAGAGG TGCGGCGGCA 4351 GCCAATCAGA GCGGCGCGCT CCGAAAGTTT C CTTTTATGG CGAGGCGGCG 4401 GCGGCGGCGG CCCTATAAAA AGCGAAGCGC G CGGCGGGCG GGAGTCGCTG 4451 CGTTGCCTTC GCCCCGTGCC CCGCTCCGCG C CGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG G TGAGCGGGC GGGACGGCCC 4551 TTCTCCTCCG GGCTGTAATT AGCGCTTGGT T TAATGACGG CTTGTTTCTT 4601 TTCTGTGGCT GCGTGAAAGC CTTGAGGGGC T CCGGGAGGG CCCTTTGTGC 4651 GGGGGGAGCG GCTCGGGGGG TGCGTGCGTG T GTGTGTGCG TGGGGAGCGC 4701 CGCGTGCGGC TCCGCGCTGC CCGGCGGCTG T GAGCGCTGC GGGCGCGGCG 4751 CGGGGCTTTG TGCGCTCCGC AGTGTGCGCG A GGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG G AACAAAGGC TGCGTGCGGG 4851 GTGTGTGCGT GGGGGGGTGA GCAGGGGGTG T GGGCGCGTC GGTCGGGCTG 4901 CAACCCCCCC TGCACCCCCC TCCCCGAGTT G CTGAGCACG GCCCGGCTTC 4951 GGGTGCGGGG CTCCGTACGG GGCGTGGCGC G GGGCTCGCC GTGCCGGGCG 5001 GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG G GGCGGGGCC GCCTCGGGCC 5051 GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC C CCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT T TTATGGTAA TCGTGCGAGA 5151 GGGCGCAGGG ACTTCCTTTG TCCCAAATCT G TGCGGAGCC GAAATCTGGG 5201 AGGCGCCGCC GCACCCCCTC TAGCGGGCGC G GGGCGAAGC GGTGCGGCGC 5251 CGGCAGGAAG GAAATGGGCG GGGAGGGCCT T CGTGCGTCG CCGCGCCGCC 5301 GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT G TCCGCGGGG GGACGGCTGC 5351 CTTCGGGGGG GACGGGGCAG GGCGGGGTTC G GCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA T GCCTTCTTC TTTTTCCTAC 5451 AGCTCCTGGG CAACGTGCTG GTTATTGTGC T GTCTCATCA TTTTGGCAAA 5501 GAATTGATTA ATTCGAGCGA ACGCGTCGAG T CGCTCGGTA CGATTTAAAT 5551 TGAATTGGCC TCGAGCGCAA GCTTGAGCTA G CGCCACCAT GGAATGGATG 5601 AGAAGCAGAG TGGGCACCCT GGGCCTGTGG G TGCGACTGC TGCTGGCTGT 5651 GTTTCTGCTG GGCGTGTACC AGGCCTACCC C ATCCCTGAC TCTAGCCCCC 5701 TGCTGCAGTT TGGCGGACAA GTGCGGCAGA G ATACCTGTA CACCGACGAC 5751 GACCAGGACA CCGAGGCCCA CCTGGAAATC C GCGAGGATG GCACAGTCGT 5801 GGGCGCTGCT CACAGAAGCC CTGAGAGCCT G CTGGAACTG AAGGCCCTGA 5851 AGCCCGGCGT GATCCAGATC CTGGGCGTGA A GGCCAGCAG ATTCCTGTGC 5901 CAGCAGCCTG ACGGCGCCCT GTACGGCTCT C CTCACTTCG ATCCTGAGGC 5951 CTGCAGCTTC AGAGAGCTGC TGCTGGAGGA C GGCTACAAC GTGTACCAGT 6001 CTGAGGCCCA CGGCCTGCCC CTGAGACTGC C TCAGAAGGA CAGCCCTAAC 6051 CAGGACGCCA CAAGCTGGGG ACCTGTGCGG T TCCTGCCTA TGCCTGGACT 6101 GCTGCACGAG CCCCAGGATC AGGCTGGCTT T CTGCCTCCT GAGCCTCCAG 6151 ACGTGGGCAG CAGCGACCCT CTGAGCATGG T GGAACCTCT GCAGGGCAGA 6201 AGCCCCAGCT ACGCCTCTTG AGAATGCGGG C CCGGTACCC CCGACGCGGC 6251 CGCTAATTCT AGATCGCGA CAAACACCAT T GTCACACTC STATUS 6301 AAACACCATT GTCACACTCC AGATTCCA A ACACCATTG TCACACTCCA 6351 AGGCGAAC ACACCATTGT CACACTCCAA G GCTATTCTA GATCGCGAAT 6401 TACATACTTC TTTACATTCC TACTACATT A CATACTTCT TTACATTCCA 6451 GATATCATTA CATACTTCTT TACATTCCAA G GCGAATTAC ATACTTCTTT 6501 ACATTCCAAG GCTACCTGAG GCCCGGGGGT A CCTCTTAAT TAACTGGCCT 6551 CATGGGCCTT CCGCTCACTG CCCGCTTTCC A GTCGGGAAA CCTGTCGTGC 6601 CAGTCAGGTG CAGGCTGCCT ATCAGAAGGT G GTGGCTGGT GTGGCCAATG 6651 CCCTGGCTCA CAAATACCAC TGAGATCTTT T TCCCTCTGC CAAAAATTAT 6701 GGGGACATCA TGAAGCCCCT TGAGCATCTG A CTTCTGGCT GRANDFATHER 6751 ATTTATTTTC ATTGCAATAG TGTGTTGGAA T TTTTTGTGT CTCTCACTCG 6801 GAAGGACATA TGGGAGGGCA AATCATTTAA A ACATCAGAA TGAGTATTTG 6851 GTTTAGAGTT TGGCAACATA TGCCCATATG C TGGCTGCCA TGAACAAAGG 6901 TTGGCTATAA AGAGGTCATC STATING A CAGCCCCCT GCTGTCCATT 6951 CCTTATTCCA TAGAAAAGCC TTGACTTGAG G TTAGATTTT TTTTATATTT 7001 TGTTTTGTGT TTTTTTTTTC TTTAACATCC C TAAAATTTT CCTTACATGT 7051 TTTACTAGCC AGATTTTTCC TCCTCTCCTG A CTACTCCCA GTCATAGCTG 7101 TCCCTCTTCT CTTATGGAGA TCCCTCGACC T GCAGCCCAA GCTGTAGATA 7151 AGTAGCATGG CGGGTTAATC ATTAACTACA A GGAACCCCT AGTGATGGAG 7201 TTGGCCACTC CCTCTCTGCG CGCTCGCTCG C TCACTGAGG CCGGGCGACC 7251 AAAGGTCGCC CGACGCCCGG GCTTTGCCCG G GCGGCCTCA GTGAGCGAGC 7301 GAGCGCGCAG CTGGCGTAA AAV2 5'ITR: 3615-3742bp CAG promoter: 3782-5452bp Mus musculus codon-optimized FGF21 (moFGF21): 5589 to 6221 bp dmiRT (4 copies of miRT-122a and 4 copies of miRT-1): 625 4~6514bp Rabbit β-globin poly(A) signal (containing the 3' poly(A) signal of rabbit β-globin) UTR and 3' flanking region): 6674-6764bp AAV2 3'ITR: 7181-7308bp pAAV-CAG-moFGF21 (SEQ ID NO: 46) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAA TCGGCC AACGCGCGGG GAGAGGCGGT 61 TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTC ACTGACT CGCTGCGCTC GGTCGTTCGG 121 CTGCGGCGAG CGGTATCAGC TCACTCAAAG GC GGTAATAC GGTTATCCAC AGAATCAGGG 181 GATAACGCAG GAAAGAACAT GTGAGCAAAA GG CCAGCAAA AGGCCAGGAA CCGTAAAAG 241 GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CG CCCCCCTG ACGAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GG ACTATAAA GATACCAGGC GTTTCCCCCT 361 GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG AC CCTGCCGC TTACCGGATA CCTGTCCGCC 421 TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CA TAGCTCAC GCTGTAGGTA TCTCAGTTCG 481 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GT GCACGAAC CCCCCGTTCA GCCCGACCGC 541 TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TC CAACCCGG TAAGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AG AGCGAGGT ATGTAGGCGG TGCTACAGAG 661 TTCTTGAAGT GGTGGCCTAA CTACGGCTAC AC TAGAAGAA CAGTATTTGG TATCTGCGCT 721 CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GT TGGTAGCT CTTGATCCGG CAAACAAACC 781 ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AA GCAGGAGA TTACGCGCAG AAAAAAAAGAGA 841 TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GG GTCTGACG CTCAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AA AAGGATCT TCACCTAGAT CCTTTAAAT 961 TAAAAATGAA GTTTTAAATC AATCAAAGT AT ATTACK AAACTTGGTC ATTACK 1021 CAATGCTTAA TCAGTGAGGC ACCTATCTCA G CGATCTGTC TATTTCGTTC ATCCATAGTT 1081 GCCTGACTCC CCGTCGTGTA GATAACTACG A TACGGGAGG GCTTACCATC TGGCCCCAGT 1141 GCTGCAATGA TACCGCGAGA CCCACGCTCA C CGGCTCCAG ATTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT C CTGCAACTT TATCCGCCTC CATCCAGTCT 1261 ATTAATTGTT GCCGGGAAGC END A GTTCGCCAG TTAATAGTTT GCGCAACGTT 1321 GTTGCCATTG CTACAGGCAT CGTGGTGTCA C GCTCGTCGT TTGGTATGGC TTCATTCAGC 1381 TCCGGTTCCC AACGATCAAG GCGAGTTACA T GATCCCCCA TGTTGTGCAA AAAAGCGGTT 1441 AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA A GTAAGTTGG CCGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT G TCATGCCAT CCGTAAGATG CTTTTCTGTG 1561 ACTGGTGAGT ACTCAACCAA GTCATTCTGA G AATAGTGTA TGCGGCGACC GAGTTGCTCT 1621 TGCCCGGCGT CAATACGGGA TAATACCGCG C CACATAGCA GAACTTTAAA AGTGCTCATC 1681 ATTGGAAAAC GTTCTTCGGG GCGAAAACTC T CAAGGATCT TACCGCTGTT GAGATCCAGT 1741 TCGATGTAAC CCACTCGTGC ACCCAACTGA T CTTCAGCAT CTTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT G CCGCAAAAA AGGGAATAAG GGCGACACGG 1861 AAATGTTGAA TACTCATACT CTTCCTTTTT C AATATTATT GAAGCATTTA TCAGGGTTAT 1921 TGTCTCATGA GCGGATACAT ATTTGAATGT A TTTAGAAAA ATAAACAAAT AGGGGTTCCG 1981 CGCACATTTC CCCGAAAAGT GCCACCTGAC G TCTAAGAAA CCATTATTAT CATGACATTA 2041 ACCTATAAAA ATAGGCGTAT CACGAGGCCC T TTCGTCTCG CGCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG A CGGTCACAG CTTGTCTGTA AGCGGATGCC 2161 GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA G CGGGTGTTG GCGGGTGTCG GGGCTGGCTT 2221 AACTATGCGG CATCAGAGCA GATTGTACTG A GAGTGCACC ATATGCGGTG TGAAATACCG 2281 CACAGATGCG TAAGGAGAAA ATACCGCATC A GGCGATTCC AACATCCAAT AAATCATACA 2341 GGCAAGGCAA AGAATTAGCA AAATTAAGCA A TAAAGCCTC AGAGCATAAA GCTAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTAATA C TTTTGCGGG AGAAGCCTTT ATTTCAACGC 2461 AAGGATAAAA ATTTTTAGAA CCCTCATATA T TTTAAATGC AATGCCTGAG TAATGTGTAG 2521 GTAAAGATTC AAACGGGTGA GAAAGGCCGG A GACAGTCAA ATCACCATCA ATATGATATT 2581 CAACCGTTCT AGCTGATAAA TTCATGCCGG A GAGGGTAGC TATTTTTGAG AGGTCTCTAC 2641 AAAGGCTATC AGGTCATTGC CTGAGAGTCT G GAGAAACA AGAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC C CCGGTTGAT AATCAGAAAAA GCCCCAAAA 2761 CAGGAAGATT GTATAAGCAA ATATTTAAAT T GTAAGCGTT AATATTTTGT TAAAATTCGC 2821 GTTAAATTTT TGTTAAATC GCTCATTTTT T AACCAATAG GCCGAAATCG GCAAAATCCC 2881 TTATAAATCA AAAATAGA CCGAGATAGG G TTGAGTGTT GTTCCAGTTT GGAACAAGAG 2941 TCCACTATTA AAGAACGTGG ACTCCAACGT C AAAGGGCGA AAAACCGTCT ATCAGGGCGA 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC A AGTTTTTG GGGTCGAGGT GCCGTAAAGC 3061 ACTAAATCGG AACCCTAAAG GGAGCCCCCG A TTTAGAGCT TGACGGGGAA AGCCGGCGAA 3121 CGTGGCGAGA AAGGAAGGG AGAAAGCGAA A GGAGCGGGC GCTAGGGCGC TGGCAAGTGT 3181 AGCGGTCACG CTGCCGGTAA CCACCACACC C GCCGCGCTT AATGCGCCGC TACAGGGCGC 3241 GTACTATGGT TGCTTTGACG AGCACGTATA A CGTGCTTTC CTCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT T AGACAGGA CGGTACGCCA GAATCCTGAG 3361 AAGTGTTTTT ATAATCAGTG AGGCCACCGA G TAAAAGAGT CTGTCCATCA CGCAATTA 3421 CCGTTGTCGC AATACTTCTT TGATTAGTA T AACATCACT TGCCTGAGTA GAAGAACTCA 3481 AACTATCGGC CTTGCTGGTA ATTACK C FATHER GCCAGCCATT GCAACGGAAT 3541 CGCCATTCGC CATTCAGGCT GCGCAACTGT T GGGAAGGGC GATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC A CTGAGGCCG CCCGGGCAAA GCCCGGGCGT 3661 CGGGCGACCT TTGGTCGCCC GGCCTCAGTG A GCGAGCGAG CGCGCAGAGA GGGAGTGGCC 3721 AACTCCATCA CTAGGGGTTC CTTGTAGTTA A TGATTAACC CGCCATGCTA CTTATCTACT 3781 CGACATTGAT TATTGACTAG TTATTAAT T NOW CGGGGTCATT AGTTCATAGC 3841 CCATATATGG AGTTCCGCGT TACATAACTT A CGGTAAATG GCCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG A CGTATGTTC CCATAGTAAC GCCAATAGGG 3961 ACTTTCCATT GACGTCAATG GGTGGAGTAT T TACGGTAAA CTGCCCACTT GGCAGTACAT 4021 CAAGTGTATC ATATGCCAAG TACGCCCCCT A TTGACGTCA ATGACGGTAA ATGGCCCGCC 4081 TGGCATTATG CCCAGTACAT GACCTTATGG G ACTTTCCTA CTTGGCAGTA CATCTACGTA 4141 TTAGTCATCG CTATTACCAT GGTCGAGGTG A GCCCCACGT TCTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT T TATTTATTT TTTAATTATT TTGTGCAGCG 4261 ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG C CAGGCGGGG CGGGGCGGGG CGAGGGGCGG 4321 GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA G CCAATCAGA GCGGCGCGCT CCGAAAGTTT 4381 CCTTTTATGG CGAGGCGGCG GCGGCGGCGG C CCTATAAAA AGCGAAGCGC GCGGCGGGCG 4441 GGAGTCGCTG CGTTGCCTTC GCCCCGTGCC C CGCTCCGCG CCGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG G TGAGCGGGC GGGACGGCCC TTCTCCTCCG 4561 GGCTGTAATT AGCGCTTGGT TTAATGACGG C TTGTTTCTT TTCTGTGGCT GCGTGAAAGC 4621 CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC G GGGGGAGCG GCTCGGGGGG TGCGTGCGTG 4681 TGTGTGTGCG TGGGGAGCGC CGCGTGCGGC T CCGCGCTGC CCGGCGGCTG TGAGCGCTGC 4741 GGGCGCGGCG CGGGGCTTTG TGCGCTCCGC A GTGTGCGCG AGGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG G AACAAAGGC TGCGTGCGGG GTGTGTGCGT 4861 GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC G GTCGGGCTG CAACCCCCCC TGCACCCCCC 4921 TCCCCGAGTT GCTGAGCACG GCCCGGCTTC G GGTGCGGGG CTCCGTACGG GGCGTGGCGC 4981 GGGGCTCGCC GTGCCGGGCG GGGGGTGGCG G CAGGTGGGG GTGCCGGGCG GGGCGGGGCC 5041 GCCTCGGGCC GGGGAGGGCT CGGGGGAGGG G CGCGGCGGC CCCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT T TTATGGTAA TCGTGCGAGA GGGCGCAGGG 5161 ACTTCCTTTG TCCCAAATCT GTGCGGAGCC G AAATCTGGG AGGCGCCGCC GCACCCCCTC 5221 TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC C GGCAGGAAG GAAATGGGCG GGGAGGGCCT 5281 TCGTGCGTCG CCGCGCCGCC GTCCCCTTCT C CCTCTCCAG CCTCGGGGCT GTCCGCGGGG 5341 GGACGGCTGC CTTCGGGGGG GACGGGGCAG G GCGGGGTTC GGCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA T GCCTTCTTC TTTTTCCTAC AGCTCCTGGG 5461 CAACGTGCTG GTTATTGTGC TGTCTCATCA T TTTGGCAAA GAATTGATTA ATTCGAGCGA 5521 ACGCGTCGAG TCGCTCGGTA CGATTTAAAT T GAATTGGCC TCGAGCGCAA GCTTGAGCTA 5581 GCGCCACCAT GGAATGGATG AGAAGCAGAG T GGGCACCCT GGGCCTGTGG GTGCGACTGC 5641 TGCTGGCTGT GTTTCTGCTG GGCGTGTACC A GGCCTACCC CATCCCTGAC TCTAGCCCCC 5701 TGCTGCAGTT TGGCGGACAA GTGCGGCAGA G ATACCTGTA CACCGACGAC GACCAGGACA 5761 CCGAGGCCCA CCTGGAAATC CGCGAGGATG G CACAGTCGT GGGCGCTGCT CACAGAAGCC 5821 CTGAGAGCCT GCTGGAACTG AAGGCCCTGA A GCCCGGCGT GATCCAGATC CTGGGCGTGA 5881 AGGCCAGCAG ATTCCTGTGC CAGCAGCCTG A CGGCGCCCT GTACGGCTCT CCTCACTTCG 5941 ATCCTGAGGC CTGCAGCTTC AGAGAGCTGC T GCTGGAGGA CGGCTACAAC GTGTACCAGT 6001 CTGAGGCCCA CGGCCTGCCC CTGAGACTGC C TCAGAAGGA CAGCCCTAAC CAGGACGCCA 6061 CAAGCTGGGG ACCTGTGCGG TTCCTGCCTA T GCCTGGACT GCTGCACGAG CCCCAGGATC 6121 AGGCTGGCTT TCTGCCTCCT GAGCCTCCAG A CGTGGGCAG CAGCGACCCT CTGAGCATGG 6181 TGGAACCTCT GCAGGGCAGA AGCCCCAGCT A CGCCTCTTG AGAATGCGGG CCCGGTACCC 6241 CCGACGCGGC CTAACTGGCC TCATGGGCCT T CCGCTCACT GCCCGCTTTC CAGTCGGGAA 6301 ACCTGTCGTG CCAGTCAGGT GCAGGCTGCC T ATCAGAAGG TGGTGGCTGG TGTGGCCAAT 6361 GCCCTGGCTC ACAAATACCA CTGAGATCTT T TTCCCTCTG CCAAAAATTA TGGGGACATC 6421 ATGAAGCCCC TTGAGCATCT GACTTCTGGC T AATAAAGGA AATTATTTT CATTGCAATA 6481 GTGTGTTGGA ATTTTTTGTG TCTCTCACTC G GAAGGACAT ATGGGAGGGC AAATCATTTA 6541 AAACATCAGA ATGAGTATTT GGTTTAGAGT T TGGCAACAT ATGCCCATAT GCTGGCTGCC 6601 ATGAACAAAG GTTGGCTATA AAGAGGTCAT C AGTATATGA AACAGCCCCCC TGCTGTCCAT 6661 TCCTTATTCC ATAGAAAAGC CTTGACTTGA G GTTAGATTT TTTTTATATT TTGTTTTGTG 6721 TTATTTTTTT CTTTAACATC CCTAAAATTT T CCTTACATG TTTTACTAGC CAGATTTTTC 6781 CTCCTCTCCT GACTACTCCC AGTCATAGCT G TCCCTCTTC TCTTATGGAG ATCCCTCGAC 6841 CTGCAGCCCA AGCTGTAGAT AAGTAGCATG G CGGGTTAAT CATTAACTAC AAGGAACCCC 6901 TAGTGATGGA GTTGGCCACT CCCTCTCTGC G CGCTCGCTC GCTCACTGAG GCCGGGCGAC 6961 CAAAGGTCGC CCGACGCCCG GGCTTTGCCC G GGCGGCCTC AGTGAGCGAG CGAGCGCGCA 7021 GCTGGCGTAA AAV2 5'ITR: 3601-3742bp CAG promoter: 3779-5423bp Mus musculus codon-optimized FGF21 (moFGF21): 5588 to 6221 bp Rabbit β-globin poly(A) signal (containing the 3' poly(A) signal of rabbit β-globin) UTR and 3' flanking region): 6315-6833bp AAV2 3'ITR: 6892-7024bp Mini-CMV: cmv intermediate-early promoter (SEQ ID NO: 36) TATGCCAAGTACGCCCCCTATTGACGTCATGACGGTAA ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG CTATTACCATGGTGATGCGGTTTTGGGCAGTACATCAATGG GCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAA ATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC TATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTG CTTAACTGGCTTATCGAAATTAATACGACTCACTATAGGG AGACCCAAGCTT Nucleotide sequence of EF1α promoter (SEQ ID NO: 37) GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCC ACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAA CCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAG TGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGG GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC TTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGC CGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTAT GGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTA CGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGG GAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTC GTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCG CGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCT TTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTG CTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGC GGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCC GCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTT CGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGG ACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCT GGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAA GGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATG GCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGG ACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACAC AAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATG TGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGAT TAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGG GGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTG GGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAA TTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGT TCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCT TCCATTTCAGGTGTCGTGA Nucleotide sequence of the RSV promoter (SEQ ID NO: 38) ​​​​​​​​​​​​​​GGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTT TCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAA TACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCA ACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGA TTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAA GGCAACAGACGGGTCTGACATGGATTGGACGAACCACTAA ATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTC GATACAATAAACGCCATTTGACCATTCACCACATTGGTGT GCACCTCCAAGCTGGGTACCAGCT Synapsin 1 promoter (SEQ ID NO: 39) ! 这里推测原文中的“シナプシン1”是“Synapsin 1”的日语表述,“配列番号”是“SEQ ID NO:”的日语表述 ctgcgctctcaggcacgacacgactcctccgctgcccac cgcagactgaggcagcgctgagtcgccggcgccgcagcgc agatggtcgcgcccgtgcccccctatctcgcgcctcgcgt ggtgcggtccggctgggccggcggcggcgcggacgcgacc aaggtggccgggaaggggagtttgcgggggaccggcgagt gacgtcagcgcgccttcagtgctgaggcggcggtggcgcg cgccgccaggcgggggcgaaggcactgtccgcggtgctga agctggcagtgcgcacgcgcctcgccgcatcctgtttccc ctccccctctctgataggggatgcgcaatttggggaatgg gggttgggtgcttgtccagtgggtcggggtcggtcgtcag gtaggcacccccaccccgcctcatcctggtcctaaaaccc acttgcact Calcium / calmodulin-dependent protein kinase II (CaMKII) promoter Tar (SEQ ID NO: 40) taacattatggccttaggtcacttcatctccatggggtt cttcttctgattttctagaaaatgagatgggggtgcagag agcttcctcagtgacctgcccagggtcacatcagaaatgt cagagctagaacttgaactcagattactaatcttaaattc catgccttgggggcatgcaagtacgatatacagaaggagt gaactcattagggcagatgaccaatgagtttaggaaagaa gagtccagggcagggtacatctacacccccgcccagccc tgggtgagtccagccacgttcacctcattatagttgcctc tctccagtcctaccttgacgggaagcacaagcagaaactg ggacacggagccccaggacccaaatcttcatggtccctct gggaggatgggtggggagagctgtggcagaggcctcagga ggggccctgctgctcagtggtgacagataggggtgagaaa gcagacagagtcattccgtcagcattctgggtctgtttgg tacttcttctcacgctaaggtggcggtgtgatatgcacaa tggctaaaaagcagggagagctggaaagaaacaaggacag agacagaggccaagtcaaccagaccaattcccagaggaag caaagaaaccattacagagactacaagggggaagggaagg agagatgaattagcttcccctgtaaaccttagaacccagc tgttgccagggcaacggggcaatacctgtctcttcagagg agatgaagttgccagggtaactacatcctgtctttctcaa ggaccatcccagaatgtggcacccactagccgttaccata gcaactgcctctttgccccacttaatcccatcccgtctgt taaaagggccctatagttggaggtgggggaggtaggaaga gcgatgatcacttgtggactaagtttgttcgcatcccctt ctccaaccccctcagtacatcaccctgggggaacagggtc cacttgctcctgggcccacacagtcctgcagtattgtgta tataaggccagggcaaagaggagcaggttttaaagtgaaa ggcaggcaggtgttggggaggcagttaccggggcaacggg aacagggcgtttcggaggtggttgccatggggacctggat gctgacgaaggctcgcgaggctgtgagcagccacagtgcc ctgctcagaagccccaagctcgtcagtcaagccggttctc cgtttgcactcaggagcacgggcaggcgagtggcccctag ttctgggggcagcgggg Glial fibrillary acidic protein (GFAP) promoter (SEQ ID NO: 41) cgcgtgatctaacatatcctggtgtggagtaggggacgc tgctctgacagaggctcgggggcctgagctggctctgtga gctggggaggaggcagacagccaggccttgtctgcaagca gacctggcagcattgggctggccgccccccagggcctcct cttcatgcccagtgaatgactcaccttggcacagacacaa tgttcggggtgggcacagtgcctgcttcccgccgcacccc agcccccctcaaatgccttccgagaagcccattgagcagg gggcttgcattgcaccccagcctgacagcctggcatcttg ggataaaagcagcacagccccctaggggctgcccttgctg tgtggcgccaccggcggtggagaacaaggctctattcagc ctgtgcccaggaaaggggatcaggggatgcccaggcatgg acagtgggtggcagggggggagaggagggctgtctgcttc ccagaagtccaaggacacaaatgggtgaggggagagctct ccccatagctgggctgcggcccaaccccaccccctcaggc tatgccagggggtgttgccaggggcacccgggcatcgcca gtctagcccactccttcataaagccctcgcatcccaggag cgagcagagccagagcaggttggagaggagacgcatcacc tccgctgctcgcggggtctagagtcga Nestin promoter (SEQ ID NO: 42) gaaggcagccccccgggaggtcaaaggctgggcacgcggga ggagaggccagagtcagaggctgcgggtatctcagatatg agaaagatgagagaggctcagaagaggtaagaaaaa cacagagaccagagaagggagaagaattagagggagg cagagccgctgtctctacagacatagctggtagagact gggagaagggatgaaccctgagcgcatgaagggaaggaggag gtggctggtggtatatgggaggatgtagctggggccagggaa aagatcctgcactaaaaatctgaagctaaaataacagga cacggggtggagaggcgaaagggggcagattgaggcaga gagactgagaggcctggggatgtgggcattccggtagggc acacagttcacttgtcttctctttttccaggaggccaaag atgctgacctcaagaactcataataccccagtgggggacca ccgcattcatagccctgttacaagaagtgggagatgttcc tttttgtcccagactggaaatccattacatcccgaggctc aggttctgtggtggtcatctctgtgtggcttgttctgtgg gcctacctaaagtcctaagcacagctctcaagcagatccg aggcgactaagatgctagtaggggttgtctggagagaaga gccgaggaggtgggctgtgatggatcagttcagctttcaa ataaaaaggcgtttttatattctgtgtcgagttcgtgaac ccctgtggtgggcttctccatctgtctgggttagtacctg ccactatactggaataaggagacgcctgcttccctcgagt tggctggacaaggttatgagcatccgtgtacttatggggt tgccagcttggtcctggatcgccccgggcccttcccccacc cgttcggttccccaccaccacccgcgctcgtacgtgcgtc tccgcctgcagctcttgactcatcggggcccccgggtcac atgcgctcgctcggctctataggcgccgccccctgcccac cccccgcccgcgctgggagccgcagccgccgccactcctg ctctctctgcgccgccgccgtcaccaccgccaccgccacc ggctgagtctgcagtcctccgaaacgggccctct (HB9) tgaataaatttaagcaggctaattaatatataaactagc tcaatttgtcaagttgatttgtattttagttaattgtgaa agtaattaccacatggtcaaattaacagctttctggaaat gaccaagcctgaggttttattccttcctgggtgaagaaa attcatttttccaagctcttgatgtgatgaataaaagtca taaatctgggtgattggtgcaggcagagtctaaatggctt catatttcattttaggtttaatagaaatattcatgctctg ttttaatgaaattaaattgaaggggatggggctagagtg gttagctgatgaattgacaaaaactaatcagctttattgg gaaacaggtttaagggcacggacgtgtcaataacgctcag cctgaccccctcttccattagctaggcaggctgattaga Tyrosine hydroxylase (TH) promoter (SEQ ID NO: 44) CTGCTAGGGGCTGCTTCCCAGCTACTCCTCTTGGCTCCG TGGCTTGCCTTCCAGCCTGTGTGCTGTCTGGAGAGCCTTT AAAGCCTCACTTCCACCAACTAGAAGTCTCTCCCCAACCC TGCCCTGACCTCAAGTGCACCTCTTCAAAGTCAGGTTTAG CAGCTGCAGCTGGGGGCCCTGAATCCCACCCCTGCTGTCT TCCTTGAAGACAGAAGTGTTGGGAGCTGAGGATCTGGGCT AGAGACTGGCTGTATGATCCAGAGAAGTAGTGTGCTTCTG GGCCTCAGATTTCCCTTCTGTAGAACAGGTTTGTCTGAAA TGGAGAGGTTGGTGCTCCTCTGCAGGGCCTAGTGGGAGTC ACCATGAGTGGTTAAAAGATCCAGCTTGTCTTTTGGTGAG CTTTGAGAGGAGGTAACAGGGCTGAGTTCTGGAAGCCTGA CCAAGGGCAGACTTAAGGGGCCTCTTGGAGTTGTTCTCAT CAAATGGGGATGGGACACAGCTAAAGTGCCCAGGGCTTCT CTGTGCCCACAGATGCTTTAGATCTTGGCACAGTGTGGTC TACCAGCTGTCTCTCTCTGTGTATATATATGTATTTCATA GACAGTGTACAGTGGCCTGGTTTGTGCTATCAGGCTGGAT ATGGACAGAGGCAAGAGTTTGTGGCAGCAGTTATCTCCCA AGAGAGTCCAAAGACATCATGTTTTCAAGTTTAGGCCAGG TGCTACTTGAGAGAGCTCAGACACAGACAAAGGTCTGGAG AGCACATGTCCTCCACCCCCACCTAGCTTCTGTTGCAAGC ACCTCCAGCCGAGACAAGAGAACGAATTAAAAAGCAATAT TTGTGTCAGTGTAAGACATTTGCCGAAAGGTTAAATCCAC ATTCGTGTTGCTGCAGAGCAGCCCCCTATGCAGGATTTGT TAGATACAGCTCCGTCCTACCCTGTGCCAGCTGAGCAAAC GCCAGGCTGGGTGGGGTGGAACCCAGCCTGGGTTTGCCTC ACCCTGCAATCCCCCCAGCACCCTCTAAAAGGAGGACCCTG TGGTGGGCATGCAGACCTAGGGACTGGGCATAGATAACCT TTGGGTTTGGGCAACAGCCCCCACTCCTCAGGATTGAAGG CTAAGGTGCAGCCAGCTCTGCCTTCATGGTGGGAATGTCT CCACGTGACCCTTTCTGGGCTGTGGAGAACACTCAGAGA AGAGTCCTGGGATGCCAGGCAGGCCAGGGATGTGCTGGGC ATGTTGAGACAGGAGTGGGCTAAGCCAGCAGAGTTGCTGA CCCAGGAAGAGTTCAGAAAGGGGCATGGAACATGGGGGAGG GGTCCATAGTGAGAGAGAGCAGGCAGTGCAGAGTAAATAG TCCCTGAGCTGGGGGTTATGGGATTTGCAGGAGCTTGCTC AGAGAAGGCAGAGGAGAGAGATGCTGCGCCAAGCTGGGTATC ACAGAGCCTCAGACTCCTGGAACAGGAACTGTGGGGGTCA GGTCAGCAGGGGAGGTTAGGGAGTGTTCCCTTTGTACTGA CTTAGCATTTATCCTGCTTCTAGGGGGGAAGGGGGCCAG TGGGGGATGCACAGCAAGGCAGTGATGTGGCAGGCAGCCT GCGGGAGCTCCTGGTTCCTGGTGTGAAAAAGCTGGGAAGG AAGAGGGCTGGGTCTGGTAAGTACAGCAGGCAGTTGGCTC CTGAGAGTCCAAGCCCTGTCTAGAGGGTGGAGTGAGATTT CAGAGGGAGAGCTAAACGGGGTGGGGGCTGGGGAGTCCAG GCTTCTGGCTCCTGCTAATACTCAGTGTGCTGGGTCCTCA GAACCTCAGGGTGGCCATTTTCAGGGTGAGAGCTCTGTCC TTTGGCACTTCTGCAGACTCCAGTATCCAGAGGAATAAAG ATGGTACTCTTCCTCAGTTCCCTTAGTGAGAGGACACCTT TCTCTGAAGGGCTTGGGCAGTTGTCCTGAACCATTGCCTG AAGGAAGGACTTGACTCCAGGGACATAGAATGGGCTCAGC ATAAGTCCCCTGTAGTAGAAAGGTCCCCTCTCTGGTCT CCTTAGAGATCCTGTTTCCTTGGCTGAGGAAGCTAGGGTG GATCTTTGTGTAAGTGGGTGTGGATGCTCACTGGAAATCA AAAGGCCCCTTGGTGTTAGACCTTGGGGTGCCATGGGAGA GTTGATCACTGAGTGCGCCCTTACATGGGGGCCAGCTGAG AATGGGGCTGCCTCTAGCTCGAGACCATGATGCAGGGAGT GAGTGGGGGAGTTCAGGATACTCTTAACTAAAGCAGAGGT CTGTCCCCCCAGGGAGGGGAGGTCAGAAGACCCTAGGGAG ATGCCAAAGGCTAGGGTTGGCACCATGTTGCAGGCTGTGT CTTCAAGGAGATGATAATCAGAGGAATCGAACCTGCAAAA GTGGGCCAGTCTTAGATACACTATAGAGGAATAATCTTCT GAAACATTCTGTGTCTCATAGGACCTGCCTGAGGACCCAG CCCCAGTGCCAGCACATACACTGGGGCAGTGAGTAGATAG TATACTTTGTTACATGGGCTGGGGGGACATGGCCTGTGCC CTGGAGGGGACTTGAAGACATCCAAAAAGCTAGTGAGAGG GCTCCTAGATTTATTTGTCTCCAAGGGCTATATATAGCCT TCCTAACATGAACCCTTGGGTAATCCAGCATGGGCGCTCC CATATGCCCTGGTTTGATTAGAGAGCTCTAGATGTCTCCT GTCCCAGAACACCAGCCAGCCCCTGTCTTCATGTCGTGTC TAGGGCGGAGGGTGATTCAGAGGCAGGTGCCTGCGACAGT GGATGCAATTAGATCTAATGGGACGGAGGCCTCTCTCGTC CGTCGCCCTCGCTCTGTGCCCACCCCCGCCTCCCTCAGGC ACAGCAGGCGTGGAGAGGATGCGCAGGAGGTAGGAGGTGG GGGACCCAGAGGGGCTTTGACGTCAGCCTGGCCTTTAAGA GGCCGCCTGCCTGGCAAGGGCCGTGGAGACAGAACTCGGG ACCACCAGCTTGCACT Myelin basic protein (MBP) promoter (SEQ ID NO: 45) caccgtggctttaacacttagagaaaatgcatcccctct aatcaataagtcatcgacagtgggtagatggaggaacggc agtgcgtagtaggatgcgtgcaagcatagtctcgtgcatg ggtgcatagatcgctgggcaggtggacaaggtgggggtgg ataaagaagtgggtagatgattgatgttaggtaaatatca ctgggtggacagatgggtggtaggtggatggatggttaga atagtcagaagagggatggattgataaggtgaacagatga taaatgggtgatagactggaagggttgtcaaaagaggata agggaagtgtgagctagccgtatttctaaggtcagtaata gagttgggagaagaggttaagttacatccatttaaacctc aacacgaagctgagagggaatggacttgctgccgttggtga ggaaagcgttgcatttcccgtgtgcttggttgtgaagtgc tcaggtcccacatgaagcagtcaggttactgcggcttaca gaggagccagatccaaatgccccgagtaagcacgtccccg agccagaggcctccagcggaatccgggagagggattgctc agtgccctgcttccctggactgtaagctgcagaaagatgt gggaagtcctgttctccactgagaacactaaaagcacctt ttgtcaaacgaccgcttcacatctggggcttgtgcactgg tggccttttaaaccagagacaacccacaagatacctaacc tgcggggctctctggtacagtgagcaactcaggaaatgct ttggcttgattgctgtgggctctcaggccatcgccctctg gagtggttcttttaatgagaacctgaagattggcccctga gccatgtataccaagcaagctcaatccaggttagctccct ctggttggggcaagctaacgtgctccttgggccccgcgcg taactgtgcgttttataggagacagctagttcaagacccc aggaagaaagcggctttgtccccctctaggcctcgtacag gcccacattcatatctcattgttgttgcaggggaggcaga tgcgatccagaacaatgggacctcggctgaggacacggcg gtgacagactccaagcacacagcagacccaaagaataact ggcaaggcgcccacccagctgacccagggaaccgccccca cttgatccgcctcttttcccgagatgccccgggaagggag gacaacaccttcaaagacaggccctcagagtccgacgagc ttcagaccatccaagaagatcccacagcagcttccgaaga attctgcagtcgacggtaccgcgggcccgggatc

Claims

1. Fibroblast growth factor 21 (FG) for use in the treatment and / or prevention of metabolic disorders F21), wherein the therapy is a central nervous system nervous system (CNS), preferably the brain, more preferably the hypothalamus and / or cortex and and / or the hippocampus and / or the cerebellum and / or the olfactory bulb, most preferably in the hypothalamus. A genetic construct comprising an expression gene construct.

2. The nucleotide sequence encoding FGF21 is operable with a ubiquitous promoter.

2. The genetic construct for use according to claim 1, wherein the gene is linked to:

3. The ubiquitous promoter comprises a CAG promoter and a CMV promoter. Preferably, the ubiquitous promoter is a CAG promoter.

3. A genetic construct for use according to claim 1 or claim 2.

4. A small number of microRNAs are expressed in tissues where it is desired to prevent the expression of FGF21. A gene for use according to any one of claims 1 to 3, comprising at least one target sequence. Construction.

5. The at least one target sequence of a microRNA is targeted to the heart and / or liver of a mammal. The method according to any one of claims 1 to 4, wherein the target sequence is selected from the group consisting of a target sequence that binds to a microRNA expressed in the liver. A genetic construct for use according to any one of claims 1 to 4.

6. The nucleotide sequence encoding FGF21 is arranged in a ubiquitous promoter. and at least one target sequence of a microRNA expressed primarily in the liver and a microRNA expressed primarily in the heart. operably linked to at least one target sequence of a microRNA to be targeted by said microRNA; A genetic construct for use according to any one of claims 1 to 5.

7. The target sequence of a microRNA expressed in the heart is selected from SEQ ID NOs: 13 and 21-25. The target sequences of the selected microRNAs expressed in the liver are SEQ ID NOs: 12 and 14-20.

7. A genetic construct for use according to claim 5 or 6, selected from:

8. The claimed invention comprises a target sequence of microRNA-122a and a target sequence of microRNA-1. A genetic construct for use according to any one of items 5 to 7.

9. The nucleotide sequence encoding FGF21 is selected from the group consisting of: A genetic construct for use according to any one of claims 1 to 8, comprising: (a) having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, or 3; a nucleotide sequence encoding a polypeptide comprising an amino acid sequence (b) a sequence having at least the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11; a nucleotide sequence having 60% sequence identity with (c) a nucleotide sequence that differs in sequence from the nucleotide sequence of (b) due to the degeneracy of the genetic code; Reotide sequence.

10. A compound according to any one of claims 1 to 9 for use in the treatment and / or prevention of metabolic disorders. an expression vector comprising the genetic construct described above, wherein the therapy is directed to the CNS, preferably the brain, More preferably, the hypothalamus and / or the cortex and / or the hippocampus and / or the cerebellum and / or the olfactory bulb, most preferably the hypothalamus, and Tar.

11. The expression vector for use according to claim 10, wherein the expression vector is a viral vector. Kutar.

12. The expression vector is an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or a selected from the group consisting of viral vectors and lentiviral vectors, preferably The expression vector of claim 10 or 11 is an adeno-associated virus vector. Expression vectors for use.

13. The expression vector is for serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10, and rh8 , Cb4, rh74, DJ, 2 / 5, 2 / 1, 1 / 2 or Anc80 adeno-associated viruses Preferably, the expression vector is an adenovirus vector of serotype 1, 2 or 9. The vector for use according to any one of claims 10 to 12, which is an associated viral vector. Current Vector.

14. one or more pharmaceutically acceptable salts of a compound selected from the group consisting of benzodiazepines, ... The genetic construct according to any one of claims 1 to 9 and / or claim 1 14. A pharmaceutical composition comprising the expression vector according to any one of claims 10 to 13, wherein the therapy is , CNS, preferably brain, more preferably hypothalamus and / or cortex and / or The genetic structure in the hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus A pharmaceutical composition comprising an expression construct.

15. The gene construct and / or expression vector and / or pharmaceutical composition are administered intra-CSF. A genetic construct for use according to any one of claims 1 to 9, administered by oral administration. and / or an expression vector for the use according to any one of claims 10 to 13 and and / or a pharmaceutical composition for use according to claim 14.

16. The method according to any one of claims 1 to 9, wherein the metabolic disorder is diabetes and / or obesity. A genetic construct for use in the method of claim 10 and / or for use in the method of claim 13.

15. An expression vector for use and / or a pharmaceutical composition for use according to claim 14.

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