Fibroblast growth factor 21 (FGF21) gene therapy

Abstract

Described herein is a genetic construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) for use in the treatment and / or prevention of metabolic disorders, wherein therapy comprises expression of the genetic construct in the central nervous system (CNS).

Description

[Background technology]

[0001] The prevalence of diabetes is increasing at an alarming rate and is a major global health problem. Obesity is strongly associated with insulin resistance and type 2 diabetes (T2D) (Moller, DE, and Flier, JS, 1991. N. Engl. J. Med. 325:938-948). Both T2D and obesity increase the risk of death (Peeters, A. et al., 2003. Ann. Intern. Med. 138:24-32) and highly morbid chronic diseases, including cardiovascular disease, hypertension, and certain types of cancer (Haslam, DW et al., 2005, Lancet. 366, 1197-1209; Roberts, DL et al., 2010, Annu. Rev. Med. 61, 301-316). Insulin resistance and obesity-related diseases are subsequently associated with reduced life expectancy and quality of life.

[0002] It is now widely accepted that peripheral tissues, such as adipose tissue, liver, or skeletal muscle, experience chronic low-grade inflammation during obesity, which can contribute to metabolic dysfunction, including the development of insulin resistance (Valdearcos, M. et al., 2015, Annu. Rev. Physiol. 77, 131-160; Hotamisligil, G. et al., 2017, Nature. 542, 177-185). In recent years, a growing body of literature has demonstrated that obesity and insulin resistance are also associated with inflammation in the brain (Guillemot-Legris, O. et al., 2017, Trends Neurosci. 40, 237-253; Beilharz, J. et al., 2016, Behav. 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] Fibroblast growth factor 21 (FGF21), a growth factor secreted primarily by the liver but also by adipose tissue and the pancreas (Muise, ES et al., 2008. Mol. Pharmacol. 74:403-412), has been shown to increase brown adipose tissue (BAT) proliferation and the expression of thermogenic genes in BAT and white adipose tissue (WAT), stimulating energy expenditure (Coskun, T. et al., 2008. Endocrinology 149:6018-6027; Fisher, FM et al., 2012. Genes Dev. 26:271-281; ​​Kharitonenkov, A. et al., 2005. J. Clin. Invest 115:1627-1635; Konishi, M. et al. al.,2000.J.Biol.Chem.275:12119-12122;Tomlinson,E.et al.,2002.Endocrinology 143:1741-1747;Xu,J.et al.,2009.Diabetes 58:250-259).

[0004] Native FGF21 protein exhibits poor pharmacokinetic characteristics. It has a 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, WY and Leung, PS 2016. Med Res Rev. 36(4):672-704; Zhang, J. and Li, Y. 2015. Front Endocrinol (Lausanne). 6:168). Various engineering approaches have been developed to extend the half-life and improve the stability and solubility of FGF21. Currently, two engineered FGF21 mimetics (LY2405319 and PF-05231023) are being tested in humans. Nevertheless, FGF21 mimetics require multiple administrations, which imposes a significant burden on patients. Furthermore, engineered FGF21 mimetics / analogs may present a higher risk of immunogenicity than native FGF21; for example, patients treated with LY2405319 developed injection site reactions, anti-drug antibodies, and severe hypersensitivity reactions (Gaich, G. et al., 2013. Cell Metab. 18(3):333-40). Therefore, the long-term and effective expression provided by a single administration of the vector of the present invention represents a significant advantage over other therapies.

[0005] Given the importance of neuroinflammation, which likely plays a role in the cognitive decline observed in diabetes and obesity as well as in whole-body energy and glucose metabolism, new therapeutic approaches addressing inflammation in the central nervous system (CNS) may be critically important. Recent studies have shown that in the CNS, particularly in the hypothalamus, a key brain region regulating whole-body energy metabolism, FGF21 peripheral metabolic effects may indeed be mediated by FGF21 signaling (D.A. Sarruf et al., Diabetes. 59, 1817-1824 (2010); A.L. Bookout et al., Nat. Med. 19, 1147-1152 (2013); B.M. Owen et al., Cell Metab. 20, 670-677 (2014); N. Douris et al., Endocrinology. 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

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

[0008] In a first aspect, there is provided a genetic construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) for use in therapy, wherein the therapy comprises expression of the genetic construct in the central nervous system (CNS), preferably the brain, and more preferably the hypothalamus. In some embodiments, there is provided a genetic construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) for use in treatment of a metabolic disorder, wherein the therapy comprises expression of the genetic construct in the central nervous system (CNS), preferably the brain, and more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, and most preferably the hypothalamus.

[0009] Preferably, the nucleotide sequence encoding FGF21 is operably linked to a ubiquitous promoter. In a preferred embodiment, the ubiquitous promoter is selected from the group consisting of a CAG promoter and a CMV promoter, and preferably the ubiquitous promoter is a CAG promoter. Preferably, the nucleotide sequence encoding FGF21 is operably linked to a ubiquitous promoter and to at least one target sequence of a microRNA that is expressed in the tissue in which it is desired to prevent the expression of FGF21.

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

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

[0012] Preferably, the target sequence of the microRNA expressed in the heart is selected from SEQ ID NOs: 13 and 21-25, and the target sequence of the microRNA expressed in the liver is selected from SEQ ID NOs: 12 and 14-20.

[0013] More preferably, the gene construct comprises a target sequence for microRNA-122a and a target sequence for microRNA-1.

[0014] Preferably, the ubiquitous promoter is selected from the group consisting of a CAG promoter and a CMV promoter, and preferably the ubiquitous promoter is a CAG promoter.

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

[0016] In a second aspect, there is provided an expression vector comprising the genetic construct of the first aspect for use in therapy, wherein the therapy comprises expression of the genetic construct in the CNS, preferably the brain, more preferably the hypothalamus. In some embodiments, there is provided an expression vector comprising the genetic construct of the first aspect for use in treating a metabolic disorder, wherein the therapy comprises expression of the genetic construct in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus. Preferably, the expression vector is a viral vector.

[0017] Preferably, the expression vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector and a lentiviral vector, preferably the expression vector is an adeno-associated viral vector.

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

[0019] In a third aspect, there is provided a pharmaceutical composition comprising a genetic construct according to the first aspect and / or an expression vector according to the second aspect together with one or more pharmaceutically acceptable ingredients for use in therapy, wherein the therapy comprises expression of the genetic construct in the CNS and / or brain. In some embodiments, there is provided a pharmaceutical composition comprising a genetic construct according to the first aspect and / or an expression vector according to the second aspect together with one or more pharmaceutically acceptable ingredients for use in treatment of a metabolic disorder, wherein the therapy comprises expression of the genetic construct in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

[0020] In a fourth aspect, there is provided a genetic construct for use according to the first aspect and / or an expression vector for use according to the second aspect and / or a pharmaceutical composition for use according to the third aspect, wherein the genetic construct and / or expression vector and / or pharmaceutical composition is administered by intra-CSF administration.

[0021] In a fifth aspect there is provided a genetic construct for use according to the first aspect and / or an expression vector for use according to the second aspect and / or a pharmaceutical composition for use according to the third aspect for use in the treatment and / or prevention of a metabolic disorder, preferably wherein the metabolic disorder is diabetes and / or obesity. In one aspect, the present invention provides: [Item 1] A genetic construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) for use in the treatment and / or prevention of metabolic disorders, wherein said therapy comprises expression of said genetic construct in the central nervous system (CNS), preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus. [Item 2] 2. The genetic construct for use according to item 1, wherein the nucleotide sequence encoding FGF21 is operably linked to a ubiquitous promoter. [Item 3] 3. The gene construct for use according to item 1 or 2, wherein the ubiquitous promoter is selected from the group consisting of a CAG promoter and a CMV promoter, and preferably the ubiquitous promoter is a CAG promoter. [Item 4] 4. A gene construct for use according to any one of items 1 to 3, comprising at least one target sequence of a microRNA expressed in a tissue in which it is desired to prevent the expression of FGF21. [Item 5] 5. The genetic construct for use according to any one of items 1 to 4, wherein the at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the heart and / or liver of a mammal. [Item 6] 6. The genetic construct for use according to any one of items 1 to 5, wherein the nucleotide sequence encoding FGF21 is operably linked to a ubiquitous promoter and to at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart. [Item 7] 7. The gene construct for use according to item 5 or 6, wherein the target sequence of a microRNA expressed in the heart is selected from SEQ ID NOs: 13 and 21 to 25, and the target sequence of a microRNA expressed in the liver is selected from SEQ ID NOs: 12 and 14 to 20. [Item 8] 8. The gene construct for use according to any one of items 5 to 7, comprising a target sequence for microRNA-122a and a target sequence for microRNA-1. [Item 9] 9. The genetic construct for use according to any one of items 1 to 8, wherein the nucleotide sequence encoding FGF21 is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1, 2, or 3; (b) a nucleotide sequence having at least 60% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11; and (c) A nucleotide sequence whose sequence differs from that of the nucleotide sequence in (b) due to the degeneracy of the genetic code. [Item 10] 10. An expression vector comprising the genetic construct according to any one of items 1 to 9 for use in the treatment and / or prevention of metabolic disorders, wherein said therapy comprises expression of said genetic construct in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus. [Item 11] Item 11. The expression vector for use according to item 10, wherein the expression vector is a viral vector. [Item 12] 12. The expression vector for use according to item 10 or 11, wherein the expression vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector and a lentiviral vector, preferably the expression vector is an adeno-associated viral vector. [Item 13] 13. The expression vector for use according to any one of Items 10 to 12, wherein the expression vector is an adeno-associated virus vector of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10, rh8, Cb4, rh74, DJ, 2 / 5, 2 / 1, 1 / 2, or Anc80, preferably an adeno-associated virus vector of serotype 1, 2, or 9. [Item 14] 14. A pharmaceutical composition comprising the gene construct of any one of items 1 to 9 and / or the expression vector of any one of items 10 to 13, together with one or more pharmaceutically acceptable ingredients, for use in the treatment and / or prevention of metabolic disorders, wherein said therapy comprises expression of said gene construct in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus. [Item 15] The gene construct for use according to any one of Items 1 to 9 and / or the expression vector for use according to any one of Items 10 to 13 and / or the pharmaceutical composition for use according to Item 14, wherein the gene construct and / or expression vector and / or pharmaceutical composition is administered by intraCSF administration. [Item 16] The gene construct for use according to any one of Items 1 to 9 and / or the expression vector for use according to any one of Items 10 to 13 and / or the pharmaceutical composition for use according to Item 14, wherein the metabolic disorder is diabetes and / or obesity.

[0022] overview The present inventors have developed an improved gene therapy strategy based on FGF21 gene therapy directed to the central nervous system (CNS) to suppress obesity and / or diabetes. In particular, as detailed in the experimental section, the present inventors have discovered the following unexpected advantages of brain-directed FGF21 gene therapy: The gene constructs and vectors described herein are capable of obtaining strong and widespread overexpression in the brain (Examples 1, 2, 3 and 4).

[0023] The genetic constructs and vectors described herein reduce adipocyte size, decrease fat accumulation in brown adipocytes, increase thermogenesis, decrease circulating triglycerides and free fatty acids, improve pancreatic health (increase number of pancreatic islets and improve beta cell mass), and decrease systemic inflammation (reduction in pro-inflammatory cytokines such as F4 / 80, IL-6, and TNFα) (Example 1.1).

[0024] In a widely used mouse model of obesity and diabetes, expression of FGF21 in the brain significantly reduced body weight gain, adiposity, and liver weight, completely normalized fed blood glucose (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, expression of FGF21 in the brain 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] Thus, aspects and embodiments of the invention described herein solve at least some of the problems and needs described herein.

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

[0029] "Genetic construct," as used herein, has its conventional and ordinary meaning as understood by those of skill in the art in light of the present disclosure. "Genetic construct," also known as an "expression cassette" or "expression construct," refers to a gene or group of genes comprising a gene encoding a protein of interest operably linked to a promoter that controls its expression. The section of this application entitled "General Information" contains further details regarding "genetic constructs." "Operably linked," as used herein, is further explained in the section of this application entitled "General Information."

[0030] In some embodiments, the genetic construct described herein is suitable for expression in mammals.As used herein, "suitable for expression in mammals" can mean that the genetic construct operably linked to the nucleotide sequence to be expressed comprises one or more regulatory sequences selected based on the mammalian host cell used for expression.Preferably, the mammalian host cell used for expression is human, mouse or canine cell.

[0031] In some embodiments, the genetic constructs described herein are for use in therapy. In preferred embodiments, the genetic constructs described herein are for use in the treatment and / or prevention of metabolic disorders. In preferred embodiments, the therapy involves expression of the genetic construct in the CNS, preferably the brain, and more preferably the hypothalamus. In some embodiments, expression of the genetic construct in the brain can refer to expression of the genetic construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, preferably the hypothalamus. Thus, expression of the genetic construct in the brain can refer to expression of the genetic construct in at least one, or at least two, or at least three, or all brain regions selected from the group consisting of the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb. In preferred embodiments, the therapy involves expression of the genetic construct in the hypothalamus. In some embodiments, expression in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb can refer to specific expression in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. In one embodiment, expression does not include expression in the liver, pancreas, adipose tissue, skeletal muscle and / or heart. In some embodiments, expression does not include expression in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, and heart. A description of CNS-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 / 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." Descriptions of "CNS," "brain," and "hypothalamus" are provided in the section entitled "General Information."

[0033] In some embodiments, the genetic constructs described herein are for use in therapy, and the genetic constructs are administered into the cerebrospinal fluid (CSF) (via cisternal, intrathecal, or intraventricular delivery), intraparenchymal, or intranasal administration, with intraCSF administration being preferred.

[0034] The terms "intra-CSF administration," "intranasal administration," "intraparenchymal administration," "intracisternal administration," "intrathecal administration," and "intraventricular administration" as used herein are described in the section of this application entitled "General Information."

[0035] In some embodiments, the genetic constructs described herein comprise a nucleotide sequence encoding FGF21 that is expressed in the CNS, preferably the brain, and more preferably the hypothalamus. In some embodiments, the genetic constructs described herein are suitable for expression in the CNS, preferably the brain, and more preferably the hypothalamus. In some embodiments, expression of a genetic construct in the brain can refer to expression of the genetic construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. Thus, expression of a genetic construct in the brain can refer to expression of the genetic construct in at least one, at least two, at least three, or all brain regions selected from the group consisting of the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb. Expression in the hypothalamus is most preferred. Expression can be assessed as described in the section entitled "General Information."

[0036] In the context of embodiments of the present invention, FGF21 expressed in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb; and genetic constructs suitable for expression within the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb refers to 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% higher, at least 100% higher, at least 150% higher, at least 200% higher, or more) expression of FGF21 within the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb compared to other organs or tissues. The other organs or tissues may be the 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 heart. In one embodiment, expression is not detectable in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. In some embodiments, expression is not detectable in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, and testis. Expression may be assessed as described in the section entitled "General Information."

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

[0038] Thus, in some embodiments, preferred nucleotide sequences encoding FGF21 encode a polypeptide comprising an amino acid sequence having 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%, 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% identity or similarity to SEQ ID NO:1, 2 or 3. SEQ ID NO: 1 represents 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 nucleotide sequence encoding FGF21 present in a genetic construct according to the present invention has 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%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 1 ... 5%, 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 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% identity.

[0039] An explanation of "identity" or "sequence identity" and "similarity" or "sequence similarity" is provided in the section entitled "General Information."

[0040] In some embodiments, the nucleotide sequence encoding human FGF21 present in a genetic construct according to the invention has 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%, 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% identity to SEQ ID NO:4, 5, 6 or 7. SEQ ID NO: 4 is a nucleotide sequence encoding human FGF21. SEQ ID NO: 5 is a codon-optimized nucleotide sequence encoding human FGF21, variant 1. SEQ ID NO: 6 is a codon-optimized nucleotide sequence encoding human FGF21, variant 2. SEQ ID NO: 7 is a codon-optimized nucleotide sequence encoding human FGF21, variant 3. Variant 1, variant 2, and variant 3 encode the same human FGF21 protein and were obtained by different algorithms of codon optimization. An explanation of "codon optimization" is provided in the section entitled "General Information."

[0041] In some embodiments, the nucleotide sequence encoding mouse FGF21 present in a genetic construct according to the invention has 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%, 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% identity to SEQ ID NO:8 or 9. SEQ ID NO: 8 is a nucleotide sequence encoding mouse FGF21. SEQ ID NO: 9 is a codon-optimized nucleotide sequence encoding mouse FGF21.

[0042] In some embodiments, the nucleotide sequence encoding canine FGF21 present in a genetic construct according to the present invention has 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%, 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% identity to SEQ ID NO: 10 or 11. SEQ ID NO: 10 is the nucleotide sequence encoding canine FGF21. SEQ ID NO: 11 is the codon-optimized nucleotide sequence encoding canine FGF21.

[0043] In some embodiments, a genetic construct as described herein is provided, wherein the nucleotide sequence encoding FGF21 is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having 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%, 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 or similarity to the amino acid sequence of SEQ ID NO:1, 2 or 3.

[0044] (b) a nucleotide sequence having 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%, 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 to the nucleotide sequence of SEQ ID NO:4, 5, 6, 7, 8, 9, 10 or 11.

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

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

[0047] The FGF21 encoded by the nucleotide sequence described herein exhibits at least detectable level of activity of FGF21, as known to those skilled in the art.The activity of FGF21 can be to exhibit anti-obesity and / or anti-diabetic effects, as will be described in more detail later herein.The activity of FGF21 can also be to increase insulin sensitivity.This activity can be evaluated by methods known to those skilled in the art, for example, by using insulin tolerance test or glucose tolerance test.

[0048] In some embodiments, the nucleotide sequence encoding FGF21 is operably linked to a ubiquitous promoter.Preferred ubiquitous promoters are selected from CMV promoters and CAG promoters, preferably CAG promoters.In some embodiments, the nucleotide sequence encoding FGF21 is operably linked to a ubiquitous promoter and to at least one target sequence of microRNA that is expressed in the tissue that is desired to prevent the expression of FGF21.

[0049] An explanation of "ubiquitous promoter," "operably linked," and "microRNA" is provided in the section entitled "General Information." As used herein, a "target sequence of a tissue-expressed microRNA" or a "target sequence that binds to a tissue-expressed microRNA" or a "binding site of a tissue-expressed microRNA" refers to a nucleotide sequence that is complementary or partially complementary to at least a portion of a microRNA expressed in said tissue, as described elsewhere herein.

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

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

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

[0053] As described herein, a portion of a microRNA expressed in the liver or a portion of a microRNA expressed in the heart refers to a nucleotide sequence of at least 4, at least 5, at least 6, or at least 7 consecutive nucleotides of the microRNA. The binding site sequence can be fully complementary to at least a portion of the expressed microRNA, meaning that the sequence matches perfectly without any mismatches. Alternatively, the binding site sequence can be partially complementary to at least a portion of the expressed microRNA, meaning that one mismatch can occur within 4, 5, 6, or 7 consecutive nucleotides. A partially complementary binding site preferably contains full or near-perfect complementarity to the seed region of the microRNA, meaning that no mismatches can occur between the seed region of the microRNA and its binding site (full complementarity) or one mismatch can occur within 4, 5, 6, or 7 consecutive nucleotides (near-perfect complementarity). The seed region of a microRNA consists of the 5' region of the microRNA, from about nucleotide 2 to about nucleotide 8 of the microRNA. The portion described herein is preferably the seed region of the microRNA. The degradation of messenger RNA (mRNA) containing the target sequence of the liver-expressed microRNA or the heart-expressed microRNA can be via the RNA interference pathway or direct translational control (inhibition) of mRNA. The present invention is in no way limited to the pathway ultimately used by the miRNA to inhibit the expression of the transgene or the encoded protein.

[0054] In the context of the present invention, a target sequence that binds to a liver-expressed microRNA may be replaced by a nucleotide sequence comprising a nucleotide sequence having 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%, 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 to SEQ ID NOs: 12 or 14-20.

[0055] In a preferred embodiment, the target sequence of a microRNA expressed in the liver may be replaced by a nucleotide sequence comprising a nucleotide sequence having 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%, 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 to SEQ ID NO:12. In a further embodiment, at least one copy of the target sequence of a liver-expressed microRNA set forth in SEQ ID NO: 12 or 14-20 is present in the genetic construct of the present invention. In a further embodiment, 2, 3, 4, 5, 6, 7, or 8 copies of the target sequence of a liver-expressed microRNA set forth in SEQ ID NO: 12 or 14-20 are present in the genetic construct of the present invention. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7, or 8 copies of the sequence miRT-122a (SEQ ID NO: 12) are present in the genetic construct of the present invention. The preferred copy number of the target sequence of a liver-expressed microRNA is 4.

[0056] As used herein, the target sequence of the microRNA expressed in the liver, as known to those skilled in the art, exerts at least detectable level of activity of the target sequence of the microRNA expressed in the liver.The activity of the target sequence of the microRNA expressed in the liver is to bind to its cognate microRNA expressed in the liver, and when operably linked to transgene, to mediate the detargeting of transgene expression in the liver.This activity can be evaluated by measuring the level of transgene expression in the liver at the level of mRNA or protein by standard assays known to those skilled in the art, such as qPCR, Western blot analysis or ELISA.

[0057] In the context of the present invention, the target sequence of a microRNA expressed in the heart may be replaced by a nucleotide sequence comprising a nucleotide sequence having 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%, 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 to SEQ ID NOs: 13 or 21-25.

[0058] In a preferred embodiment, the target sequence of a microRNA expressed in the heart may be replaced by a nucleotide sequence comprising a nucleotide sequence having 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%, 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 to SEQ ID NO:13. In a further embodiment, at least one copy of the target sequence of a microRNA expressed in the heart as set forth in SEQ ID NO: 13 or 21-25 is present in the genetic construct of the present invention. In a further embodiment, 2, 3, 4, 5, 6, 7, or 8 copies of the target sequence of a microRNA expressed in the heart as set forth in SEQ ID NO: 13 or 21-25 are present in the genetic construct of the present invention. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence encoding miRT-1 (SEQ ID NO: 13) are present in the genetic construct of the present invention. The preferred copy number of the target sequence of a microRNA expressed in the heart is 4.

[0059] As used herein, the target sequence of the microRNA expressed in the heart, as known to those skilled in the art, exerts at least detectable level of activity of the target sequence of the microRNA expressed in the heart.The activity of the target sequence of the microRNA expressed in the heart is to bind to its cognate microRNA expressed in the heart, and when operably linked to transgene, to mediate the detargeting of transgene expression in the heart.This activity can be evaluated by measuring the level of transgene expression in the heart at the level of mRNA or protein by standard assay known to those skilled in the art, such as qPCR, Western blot analysis or ELISA.

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

[0061] In some embodiments, the above-mentioned genetic construct is provided, wherein the target sequence of the microRNA expressed in the liver and the target sequence of the microRNA expressed in the heart are selected from the group consisting of SEQ ID NOs: 12 to 25 and / or combinations thereof. In some embodiments, the above-mentioned genetic construct is provided, wherein the target sequence of the microRNA expressed in the heart is selected from SEQ ID NOs: 13 and 21 to 25, and the target sequence of the microRNA expressed in the liver is selected from SEQ ID NOs: 12 and 14 to 20. In some embodiments, the above-mentioned genetic construct is provided, comprising a target sequence of microRNA-122a and a target sequence of microRNA-1.

[0062] In some embodiments, the ubiquitous promoter described herein is selected from the group consisting of a CAG promoter, a CMV promoter, a mini-CMV promoter, a beta-actin promoter, a Rous sarcoma virus (RSV) promoter, an elongation factor 1 alpha (EF1α) promoter, an early growth response factor-1 (Egr-1) promoter, a eukaryotic translation initiation factor 4A (eIF4A) promoter, a ferritin heavy chain-encoding gene (FerH) promoter, a ferritin light chain-encoding gene (FerL) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, a GRP78 promoter, a GRP94 promoter, a heat shock protein 70 (hsp70) promoter, a ubiquitin B promoter, an SV40 promoter, a beta-kinesin promoter, a ROSA26 promoter, and a PGK-1 promoter.

[0063] In a preferred embodiment, the ubiquitous promoter is a CAG promoter. In the examples, it is demonstrated that the CAG promoter is suitable for use in the genetic constructs according to the invention. In some embodiments, the CAG promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:27.

[0064] Another preferred ubiquitous promoter is the cytomegalovirus (CMV) promoter. In some embodiments, the CMV promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:28. Preferably, the CMV promoter is used in conjunction with an intron sequence. In some embodiments, the intron sequence comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:26.

[0065] Another preferred ubiquitous promoter is the mini-CMV promoter. In some embodiments, the mini-CMV promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:36.

[0066] Another preferred ubiquitous promoter is the EF1α promoter. In some embodiments, the EF1α promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:37.

[0067] Another preferred ubiquitous promoter is the RSV promoter. In some embodiments, the RSV promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO: 38.

[0068] In some embodiments, the nucleotide sequence encoding FGF21 is operably linked to a tissue-specific promoter. In preferred embodiments, the tissue-specific promoter is a CNS-specific promoter, more preferably a brain-specific promoter, and most preferably 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 selected from the group consisting of synapsin 1 promoter, neuron-specific enolase (NSE) promoter, calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, tyrosine hydroxylase (TH) promoter, forkhead box A2 (FOXA2) promoter, alpha-internexin (INA) promoter, nestin (NES) promoter, glial fibrillary acidic protein (GFAP) promoter, aldehyde dehydrogenase 1 family member L1 (ALDH1L1) promoter, myelin-associated oligodendrocyte basic protein (MOBP) promoter, homeobox protein 9 (HB9) promoter, and myelin basic protein (MBP) promoter.

[0071] In some embodiments, the brain-specific promoter described herein is selected from the group consisting of synapsin 1 promoter, neuron-specific enolase (NSE) promoter, calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, tyrosine hydroxylase (TH) promoter, forkhead box A2 (FOXA2) promoter, alpha-internexin (INA) promoter, nestin (NES) promoter, glial fibrillary acidic protein (GFAP) promoter, aldehyde dehydrogenase 1 family member L1 (ALDH1L1) promoter, myelin-associated oligodendrocyte basic protein (MOBP) promoter, and myelin basic protein (MBP) promoter.

[0072] In some embodiments, the hypothalamus-specific promoter can be a gonadotropin-releasing hormone (GnRH) promoter.

[0073] In a preferred embodiment, the CNS-specific and / or brain-specific promoter is the synapsin 1 promoter. In some embodiments, the synapsin 1 promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:39.

[0074] Another preferred CNS- and / or brain-specific promoter is the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter. In some embodiments, the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:40.

[0075] Another preferred CNS- and / or brain-specific promoter is the glial fibrillary acidic protein (GFAP) promoter. In some embodiments, the glial fibrillary acidic protein (GFAP) promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:41.

[0076] Another preferred CNS- and / or brain-specific promoter is the nestin promoter. In some embodiments, the nestin promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:42.

[0077] Another preferred CNS-specific promoter is the homeobox protein 9 (HB9) promoter. In some embodiments, the homeobox protein 9 (HB9) promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:43.

[0078] Another preferred CNS- and / or brain-specific promoter is the tyrosine hydroxylase (TH) promoter. In some embodiments, the tyrosine hydroxylase (TH) promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:44.

[0079] Another preferred CNS- and / or brain-specific promoter is the myelin basic protein (MBP) promoter. In some embodiments, the myelin basic protein (MBP) promoter comprises, consists essentially of, or consists of a nucleotide sequence having 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%, 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 to SEQ ID NO:45.

[0080] In some embodiments, the CNS-specific promoters, brain-specific promoters, and / or hypothalamus-specific promoters described herein direct expression of the nucleotide sequence in at least one cell of the CNS and / or brain and / or hypothalamus. Preferably, the promoters direct expression in at least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80%, 90%, or 100% of the cells of the CNS and / or brain and / or hypothalamus. As used herein, CNS-specific promoters and / or brain-specific promoters also encompass promoters that direct expression in specific regions or cell subsets of the CNS and / or brain. Thus, the CNS-specific promoters and / or brain-specific promoters described herein may also direct expression in at least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80%, 90%, or 100% of the cells of the hippocampus, cerebellum, cortex, hypothalamus, and / or olfactory bulb. Expression can be assessed as described in the section entitled "General Information."

[0081] As used herein, promoters (especially when the promoter sequence is described as having a minimum percentage identity with a given SEQ ID NO) should at least exhibit the activity of promoters known to those of skill in the art. Preferably, a promoter described as having a minimum percentage identity with a given SEQ ID NO should control the transcription of the nucleotide sequence to which it is operably linked (i.e., at least the nucleotide sequence encoding FGF21) as assessed by an assay known to those of skill in the art. For example, such an assay may include measuring the expression of a transgene. Expression may be assessed as described in the section entitled "General Information."

[0082] Additional sequences may be present in the genetic constructs of the present invention. Exemplary additional sequences suitable herein include an inverted terminal repeat (ITR), an SV40 polyadenylation signal (SEQ ID NO: 32), a rabbit β-globin polyadenylation signal (SEQ ID NO: 33), and a CMV enhancer sequence (SEQ ID NO: 29). Within the context of the present invention, "ITR" is intended to encompass one 5'ITR and one 3'ITR, respectively, derived from the AAV genome. Preferred ITRs are derived from AAV2 and are represented by SEQ ID NO: 30 (5'ITR) and SEQ ID NO: 31 (3'ITR). Within the context of the present invention, the use of a CMV enhancer sequence (SEQ ID NO: 29) and a CMV promoter sequence (SEQ ID NO: 28) as two separate sequences or as a single sequence (SEQ ID NO: 34) is encompassed. Each of these additional sequences may be present in a genetic construct according to the present invention.

[0083] Additional nucleotide sequences, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, etc., may be operably linked to the nucleotide sequence(s) encoding FGF21.

[0084] In some embodiments, a genetic construct is provided that includes a nucleotide sequence encoding FGF21, which may not include a target sequence for a microRNA expressed in a tissue in which it is desired to prevent expression of FGF21.

[0085] Expression vector The genetic constructs described herein can be placed in an expression vector. Thus, in another aspect, there is provided an expression vector comprising a genetic construct according to 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 for use in therapy. In preferred embodiments, the expression vectors described herein are for use in the treatment and / or prevention of metabolic disorders. In preferred embodiments, therapy involves expression of a gene construct contained in the expression vector in the CNS, preferably the brain, and more preferably the hypothalamus. In some embodiments, expression of a gene construct in the brain can refer to expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, preferably the hypothalamus. Thus, expression of a gene construct in the brain can refer to expression of the gene construct in at least one, at least two, at least three, or all brain regions selected from the group consisting of the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb. In preferred embodiments, therapy involves expression of a gene construct in the hypothalamus. In some embodiments, expression in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb can refer to specific expression in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. In one embodiment, expression does not include expression in the liver, pancreas, adipose tissue, skeletal muscle and / or heart. In some embodiments, expression does not include expression in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, and heart. A description of CNS-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 / or olfactory bulb-specific expression is provided in the section entitled "General Information."

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

[0088] In some embodiments, the expression vectors described herein are for use in therapy, and the expression vectors are administered into the cerebrospinal fluid (CSF) (via cisternal, intrathecal, or intraventricular delivery), intraparenchymal, or intranasal administration, with intraCSF administration being preferred.

[0089] The terms "intra-CSF administration," "intranasal administration," "intraparenchymal administration," "intracisternal administration," "intrathecal administration," and "intraventricular administration" as used herein are described in the section of this application entitled "General Information."

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

[0091] The viral vector may be a viral vector selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors. Adenoviral vectors are also known as adenovirus-derived vectors, adeno-associated viral vectors are also known as adeno-associated virus-derived vectors, retroviral vectors are also known as retrovirus-derived vectors, and lentiviral vectors are also known as lentivirus-derived vectors. A preferred viral vector is an adeno-associated viral vector. A description of "adeno-associated viral vectors" is provided in the section entitled "General Information."

[0092] In some embodiments, the vector is selected from the group consisting of serotype 1 AAV (AAV1), serotype 2 AAV (AAV2), serotype 3 AAV (AAV3), serotype 4 AAV (AAV4), serotype 5 AAV (AAV5), serotype 6 AAV (AAV6), serotype 7 AAV (AAV7), serotype 8 AAV (AAV8), serotype 9 AAV (AAV9), serotype rh10 AAV (AAVrh10), serotype rh8 AAV (AAVrh8), serotype The adeno-associated vector or adeno-associated virus vector or adeno-associated virus-derived vector (AAV) is selected from the group consisting of AAV of Cb4 (AAVCb4), AAV of serotype rh74 (AAVrh74), AAV of serotype DJ (AAVDJ), AAV of serotype 2 / 5 (AAV2 / 5), AAV of serotype 2 / 1 (AAV2 / 1), AAV of serotype 1 / 2 (AAV1 / 2), and AAV of serotype Anc80 (AAVAnc80).

[0093] In preferred embodiments, the vector is an AAV of serotype 1, 2 or 9 (AAV1, AAV2 or AAV9). The Examples demonstrate that these AAV serotypes are suitable for use as expression vectors according to the present invention.

[0094] In a preferred embodiment, the expression vector is AAV1, AAV2, or AAV9, preferably AAV9, and includes a gene construct comprising a nucleotide sequence encoding FGF21. More preferably, such a gene construct includes a CAG promoter comprising, consisting essentially of, or consisting of a nucleotide sequence having at least 60% sequence identical to SEQ ID NO: 27. Even more preferably, such a gene construct includes at least one target sequence of a microRNA expressed in a tissue in which it is desired to prevent expression of FGF21, as described herein.

[0095] In another preferred embodiment, the expression vector is AAV1 and includes a gene construct comprising a nucleotide sequence encoding FGF21, which may not include a target sequence for a microRNA. In one embodiment, the gene construct does not include a target sequence for a miRNA expressed in a tissue in which it is desired to prevent expression of FGF21. More preferably, such a gene construct includes a CAG promoter comprising, consisting essentially of, or consisting of a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: 27.

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

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

[0098] As used herein, "pharmaceutically acceptable ingredients" includes pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents, and / or excipients. Thus, one or more pharmaceutically acceptable ingredients can be selected from the group consisting of pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents, and / or excipients. Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents, and / or excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, 22nd edition. Pharmaceutical Press (2013).

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

[0101] In some embodiments, the compositions described herein are for use in therapy, and the compositions are administered intracerebral spinal fluid (CSF) (via cisternal, intrathecal, or intraventricular delivery), intraparenchymal, or intranasal administration, with intraCSF administration being preferred.

[0102] The terms "intra-CSF administration," "intranasal administration," "intraparenchymal administration," "intracisternal administration," "intrathecal administration," and "intraventricular administration" as used herein are described in the section of this application entitled "General Information."

[0103] Additional compounds may be present in the compositions of the present invention. Such compounds may aid in the delivery of the compositions. In this regard, suitable compounds are those that can deliver the components described herein and form complexes, nanoparticles, micelles, and / or liposomes that are complexed or entrapped in vesicles or liposomes through the cell membrane. Many of these compounds are known in the art. Suitable compounds include polyethyleneimine (PEI) or similar cationic polymers, including polypropyleneimine or polyethyleneimine copolymers (PEC) and derivatives; synthetic amphiphiles (SAINT-18); Lipofectin™, DOTAP. Those skilled in the art will know which type of formulation is most suitable for the compositions described herein.

[0104] Methods and Uses In a further aspect, there is provided a genetic construct as described herein for use in therapy, wherein the therapy comprises expression of the genetic construct in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

[0105] Further provided is an expression vector as described herein for use in therapy, wherein the therapy comprises expression of a gene construct comprised in the expression vector within the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

[0106] Further provided is a pharmaceutical composition as described herein for use in therapy, wherein the therapy comprises expression of a gene construct comprised in the pharmaceutical composition within the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

[0107] In some embodiments the genetic constructs described herein and / or the expression vectors described herein and / or the pharmaceutical compositions described herein are for use in the treatment and / or prevention of metabolic disorders, preferably obesity and / or diabetes, wherein the therapy comprises expression of the genetic construct in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

[0108] In a further aspect, there is provided a method of treatment comprising administering a genetic construct, expression vector or pharmaceutical composition as described herein, said method comprising expression of the genetic construct as described herein in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

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

[0110] In some embodiments, there is provided a method of treatment comprising administering a genetic construct, expression vector or pharmaceutical composition described herein, wherein the method is for treating and / or preventing a metabolic disorder, and the method comprises expression of the genetic construct described herein in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

[0111] In a further aspect, there is provided use of a genetic construct, expression vector or pharmaceutical composition as described herein for the manufacture of a medicament, wherein said medicament comprises expression of a genetic construct as described herein in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

[0112] In some embodiments there is provided a use of a genetic construct, expression vector or pharmaceutical composition described herein for the manufacture of a medicament, wherein said medicament is for the treatment and / or prevention of a metabolic disorder and comprises expression of a genetic construct described herein in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

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

[0114] In some embodiments there is provided a use of a genetic construct, expression vector or pharmaceutical composition as described herein for medical treatment, wherein said medical treatment is for the treatment and / or prevention of a metabolic disorder and comprises expression of a genetic construct as described herein in the CNS, preferably the brain, more preferably the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, most preferably the hypothalamus.

[0115] Within the context of the genetic constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses according to the present invention, "comprising expression of the genetic construct" may be replaced with "causing expression of the genetic construct" or "inducing expression of the genetic construct".

[0116] In the context of the gene constructs, expression vectors, pharmaceutical compositions, methods, and uses according to the present invention, the therapy and / or treatment and / or medicament may include expression of the gene construct in the CNS, preferably the brain, and more preferably the hypothalamus. In some embodiments, expression of the gene construct in the brain may refer to expression of the gene construct in the hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, preferably the hypothalamus. Thus, expression of the gene construct in the brain may refer to expression of the gene construct in at least one, at least two, at least three, or all brain regions selected from the group consisting of the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb. In preferred embodiments, the therapy includes expression of the gene construct in the hypothalamus. In some embodiments, expression in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb may refer to specific expression in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb. In one embodiment, expression does not include expression in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. In some embodiments, expression does not include expression in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, and heart. A description of CNS-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 / or olfactory bulb-specific expression is provided in the section entitled "General Information."

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

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

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

[0120] The terms "intra-CSF administration," "intranasal administration," "intraparenchymal administration," "intracisternal administration," "intrathecal administration," and "intraventricular administration" as used herein are described in the section of this application entitled "General Information."

[0121] Within the context of the genetic constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses according to the present invention, the therapy and / or treatment and / or medicament may be for use in the treatment and / or prevention of metabolic disorders, preferably obesity and / or diabetes. Complications of metabolic disorders may also be encompassed.

[0122] Metabolic disorders can include metabolic syndrome, diabetes, obesity, obesity-related comorbidities, diabetes-related comorbidities, hyperglycemia, insulin resistance, impaired glucose tolerance, fatty liver, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), coronary heart disease (CHD), hyperlipidemia, atherosclerosis, endocrine disorders, osteosarcopenic obesity syndrome (OSO), diabetic nephropathy, chronic kidney disease (CKD), cardiac hypertrophy, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, arthritis, sepsis, ocular neovascularization, neurodegeneration, dementia, and can also include depression, adenoma, and carcinoma.

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

[0124] Obesity can include overweight, central / upper body obesity, peripheral / lower body obesity, morbid obesity, osteosarcopenic obesity syndrome (OSO), childhood obesity, Mendelian (monogenic) syndromic obesity, Mendelian non-syndromic obesity, and polygenic obesity.

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

[0126] In preferred embodiments, the treatment or therapy described herein, or the use or administration of a pharmaceutical agent, does not need to be repeated. In some embodiments, the treatment or therapy described herein, or the use or administration of a pharmaceutical agent, can be repeated every year or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, including intervals between any two of the listed values, years.

[0127] The subject to be treated may be a higher mammal, such as a cat, a rodent (preferably a mouse, rat, gerbil, or guinea pig, more preferably a mouse or rat), a dog, or a human.

[0128] Within the context of the genetic constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses according to the present invention, the genetic constructs and / or expression vectors and / or pharmaceutical compositions described herein preferably exhibit anti-diabetic and / or anti-obesity effects.

[0129] An antidiabetic effect can be achieved if blood glucose disposal is increased, and / or glucose tolerance is improved, and / or insulin sensitivity is increased. This can be assessed using techniques known to those skilled in the art, such as, for example, measuring glycemia, insulinemia, and / or performance of an insulin tolerance test and / or glucose tolerance test, as performed in the experimental section. In this context, "increase" (or "improvement") means at least a detectable increase (or detectable improvement) using an assay known to those skilled in the art. The increase can be at least 5%, 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 at least 100% using an assay such as measuring glycemia, insulinemia, and / or performance of an insulin tolerance test and / or glucose tolerance test.

[0130] An anti-obesity effect can be achieved if body weight, weight gain, and / or body fat percentage are reduced. An anti-obesity effect can also be achieved if body mass index (BMI), waist circumference, waist-to-hip ratio (WHR), and / or waist-to-height ratio (WHtR) are reduced. An anti-obesity effect can also be achieved if the weight of tissues such as the liver is reduced. This can be assessed using techniques known to those skilled in the art, for example, as performed in the experimental section. In this context, "reduction" (or "improvement") means at least a detectable reduction (or detectable improvement) using assays known to those skilled in the art, for example, the assays performed in the experimental section. An anti-obesity effect includes both obesity prevention and obesity reversal.

[0131] Anti-diabetic and / or anti-obesity effects can also be observed if the progression of typical symptoms (e.g., insulitis, beta cell loss, beta cell mass reduction, weight gain) is delayed as assessed by a physician. Reduction of typical symptoms can mean a delay in the progression of symptom onset or complete disappearance of symptoms. Symptoms, and therefore symptom reduction, can also be evaluated using various methods, including clinical examinations and routine laboratory tests, almost the same methods used to diagnose diabetes and / or obesity. Such methods include both macroscopic and microscopic methods, as well as molecular methods, radiological methods such as X-rays, biochemical methods, immunohistochemical methods, etc. Beta cell loss and / or beta cell mass reduction can be evaluated using immunohistochemical methods, preferably as performed in the experimental section.

[0132] Anti-diabetic and / or anti-obesity effects can also be observed when a reduction in systemic inflammation (reduction in inflammatory cytokines such as F4 / 80, IL-6, TNFα, etc.) is assessed. In this context, "reduction" means at least a detectable reduction using an assay known to those skilled in the art. The reduction can be at least 5%, 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 at least 100% using an assay such as the measurement of inflammatory cytokines such as F4 / 80, IL-6, and / or TNFα using techniques known to those skilled in the art, preferably the techniques used in the experimental section (i.e., RT-PCR).

[0133] Within the context of the genetic constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses according to the present invention, the genetic constructs and / or expression vectors and / or pharmaceutical compositions described herein preferably alleviate one or more symptom(s) of a metabolic disorder such as diabetes and / or obesity in an individual, a cell, tissue or organ of said individual, or alleviate one or more characteristic(s) or symptom(s) of a cell, tissue or organ of said individual.

[0134] The genetic constructs and / or expression vectors and / or pharmaceutical compositions described herein are preferably capable of alleviating symptoms or characteristics of a patient or of a cell, tissue or organ of said patient if said symptoms or characteristics are reduced (e.g., no longer detectable or delayed) at least one week, one month, six months, one year or more after treatment with a genetic construct and / or expression vector and / or composition of the invention as described herein.

[0135] The genetic constructs and / or expression vectors and / or pharmaceutical compositions described herein may be suitable for administration to cells, tissues and / or organs in vivo of an individual suffering from or at risk of developing a metabolic disorder such as diabetes and / or obesity, and may be administered in vivo, ex vivo or in vitro. The genetic constructs and / or expression vectors and / or pharmaceutical compositions may be administered directly or indirectly to cells, tissues and / or organs in vivo of an individual suffering from or at risk of developing a metabolic disorder such as diabetes and / or obesity, and may be administered directly or indirectly in vivo, ex vivo or in vitro.

[0136] The mode of administration can be intravenous, intramuscular, intrathecal, intraventricular, intraperitoneal, by inhalation, intranasal, intraocular, and / or intraparenchymal administration. Preferred modes of administration are intranasal, intraparenchymal, and intraCSF (via cisternal, intrathecal, or intraventricular delivery). IntraCSF administration is most preferred.

[0137] The viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions of the present invention can be administered directly or indirectly using suitable means known in the art. In view of the progress made to date, improvements in the means for providing the viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions of the present invention to an individual or the cells, tissues, or organs of the individual are expected. Of course, such future improvements may be incorporated to achieve the above-mentioned effects of the present invention. The viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions can be delivered directly to an individual or the cells, tissues, or organs of the individual. Depending on the disease or condition, the cells, tissues, or organs of the individual may be as described above in this specification. When administering the viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions of the present invention, it is preferable that such viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions be dissolved in a solution compatible with the delivery method.

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

[0139] General information Unless otherwise defined, all technical and scientific terms used herein have the same meaning as customarily and commonly understood by one of ordinary skill in the art to which this invention belongs and are 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 represented by a nucleic acid or nucleotide sequence encoding a protein fragment, polypeptide, peptide, or derived peptide. In the context of the present invention, an FGF21 protein fragment, polypeptide, peptide, or derived peptide, such as fibroblast growth factor 21 (FGF21), is represented by an amino acid sequence.

[0141] It should be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct identified herein by a given sequence identity number (SEQ ID NO:) is not limited to this particular sequence disclosed. Each coding sequence identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct, or is itself a protein fragment or polypeptide or construct or peptide or derived peptide. Throughout this application, whenever a particular nucleotide sequence SEQ ID NO (e.g., take SEQ ID NO:X) that encodes a given protein fragment or polypeptide or peptide or derived peptide is referenced, it may be replaced by the following: i. a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity to SEQ ID NO: X; ii. a nucleotide sequence that differs in sequence from the sequence of the nucleic acid molecule of (i) due to the degeneracy of the genetic code; or iii. A nucleotide sequence encoding an amino acid sequence having at least 60% amino acid identity or similarity to the amino acid sequence encoded by nucleotide sequence SEQ ID NO:X.

[0142] Throughout this application, whenever a particular amino acid sequence SEQ ID NO (for example, take SEQ ID NO: Y), it may be replaced by: a polypeptide comprising an amino acid sequence having at least 60% sequence identity or similarity to amino acid sequence SEQ ID NO: Y.

[0143] Each nucleotide sequence or amino acid sequence described herein based on its percentage identity or similarity (at least 60%) to a given nucleotide sequence or amino acid sequence, respectively, is in further preferred embodiments 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%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, 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 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% identity or similarity.

[0144] Each non-coding nucleotide sequence (i.e., of a promoter or of another regulatory region) can be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity with a specific nucleotide sequence SEQ ID NO: (e.g., SEQ ID NO: A). Preferred nucleotide sequences have 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%, 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% identity to SEQ ID NO:A. In preferred embodiments, such non-coding nucleotide sequences, such as promoters, exhibit or exert at least an activity of such non-coding nucleotide sequences, eg, the activity of a promoter known to those skilled in the art.

[0145] The terms "homology," "sequence identity," "identity," and the like are used interchangeably herein. Sequence identity is described herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, determined by comparing the sequences. The "similarity" or "sequence similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes with the sequence of a second polypeptide. "Identity" and "similarity" can be easily calculated by known methods, including, but not limited to, those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and Transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004.

[0146] Sequence identity or similarity can be calculated based on the full length of two given SEQ ID NOs or a portion thereof. In some embodiments, a portion thereof means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NOs. In preferred embodiments, sequence identity or similarity is determined by comparing the full length of the sequences identified herein. Unless otherwise specified herein, identity or similarity to a given SEQ ID NO refers to identity or similarity based on the full length (i.e., throughout its entire length or as a whole) of the sequence. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as may be determined by the match between strings of such sequences.

[0147] Sequence identity or similarity can be determined by aligning two peptides or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences.Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences across their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman).Then, sequences can be referred to as "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity or similarity (as described below) (for example, when optimally aligned by the program EMBOSS needle or EMBOSS water using default parameters).

[0148] When two sequences have similar lengths, global alignment is preferably used to determine sequence identity or similarity. When sequences have substantially different overall lengths, local alignment, such as that using the Smith-Waterman algorithm, is preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the default parameters of EMBOSS needle and EMBOSS water are used, with a gap opening penalty of 10 (nucleotide sequence) / 10 (protein) and a gap extension penalty of 0.5 (nucleotide sequence) / 0.5 (protein). For nucleotide sequences, the default scoring matrix used is DNAfull, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).

[0149] Alternatively, the percentage of similarity or identity can be determined by searching against public databases using algorithms such as FASTA and BLAST. Thus, the nucleic acid and protein sequences of some embodiments of the present invention can be further used as "query sequences" to search against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present invention. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information, accessible on the World Wide Web at www.ncbi.nlm.nih.gov / .

[0150] When determining the degree of amino acid similarity, one skilled in the art may also take into account so-called conservative amino acid substitutions.

[0151] As used herein, "conservative" amino acid substitutions refer to the interchangeability of residues having 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; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine.Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence is removed and a different residue is inserted in its place.Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: 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; Ile 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 Phe; and Val to Ile or Leu.

[0153] Genes or coding sequences The term "gene" refers to a DNA fragment containing a region (transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell and operably linked to a suitable regulatory region (e.g., a promoter). A gene usually includes several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end), including a polyadenylation termination site and / or a transcription termination site. A chimeric or recombinant gene (such as the FGF21 gene) is a gene that is not normally found in nature, such as a gene in which the promoter is not naturally associated with some or all of the transcribed DNA region. "Gene expression" refers to the process by which a DNA region operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into RNA that is biologically active, i.e., can be translated into a biologically active protein or peptide.

[0154] A "transgene" is herein defined as a gene or coding sequence or nucleic acid molecule (i.e., a molecule encoding FGF21) newly introduced into a cell, i.e., a gene that may be present but not normally expressed in the cell or may be expressed at an insufficient level. In this context, "insufficient" means that the FGF21 is expressed in the cell, but the conditions and / or diseases described herein may still develop. In this case, the present invention enables overexpression of FGF21. A transgene may include a sequence native to the cell, a sequence not naturally occurring in the cell, or a combination of both. A transgene may include a sequence encoding FGF21 and / or an additional protein as previously identified herein, optionally operably linked to appropriate regulatory sequences for expressing the FGF21-encoding sequence in the cell. Preferably, the transgene is not integrated into the genome of the host cell.

[0155] promoter As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, is located upstream in the direction of transcription of the transcription start site of the coding sequence, and is structurally identified by the presence of binding sites for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by the application of a chemical inducer.

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

[0157] An "organ-specific" or "tissue-specific" promoter is a promoter that is active in a specific type of organ or tissue, respectively. Organ-specific and tissue-specific promoters regulate the expression of one or more genes (or coding sequences) primarily in one organ or tissue, but can also allow detectable levels of expression ("leaky") in other organs or tissues. Leaky expression in other organs or tissues means that, as assessed at the mRNA or protein level by standard assays known to those skilled in the art (e.g., qPCR, Western blot analysis, ELISA), expression is at least one-fold, at least two-fold, at least three-fold, at least four-fold, or at least five-fold lower than organ-specific or tissue-specific expression, but still detectable. The maximum number of organs or tissues in which leaky expression can be detected is 5, 6, 7, or 8.

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

[0159] In the context of the present invention, a CNS-specific promoter and / or brain-specific promoter and / or hypothalamus-specific promoter and / or cortex-specific promoter and / or hippocampus-specific promoter and / or cerebellum-specific promoter and / or olfactory bulb-specific promoter can be a promoter that can promote 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% higher, at least 100% higher, at least 150% higher, at least 200% higher, or more) expression of FGF21 in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb compared to other organs or tissues. The other organs or tissues can be the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, etc. Preferably, the other organs are the liver and heart. Expression can be assessed as described elsewhere in the section entitled "General Information."

[0160] Throughout this application, when 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 are mentioned in the context of expression, cell type-specific expression of the cell type(s) that make up the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, respectively, is also envisaged.

[0161] operably linked As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the linked DNA sequences are typically contiguous, and where necessary to link two protein-coding regions, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites or adapters or linkers inserted instead, or by gene synthesis.

[0162] microRNA As used herein, "microRNA" or "miRNA" or "miR" has its customary and ordinary meaning as understood by those skilled in the art in light of the present disclosure. MicroRNA is a small non-coding RNA molecule found in plants, animals, and some viruses that can function in RNA silencing and post-transcriptional regulation of gene expression. The target sequence of microRNA can be referred to as "miRT". For example, the target sequence of microRNA-1 or miRNA-1 or miR-1 can be referred to as miRT-1.

[0163] Proteins and Amino Acids The terms "protein" or "polypeptide" or "amino acid sequence" are used interchangeably and refer to a molecule consisting of a chain of amino acids, without reference to a particular mechanism of action, size, three-dimensional structure, or origin. In the amino acid sequences described herein, amino acids or "residues" are designated by their three-letter symbols. These three letter symbols and their 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, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, 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 (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine. The residue may be any proteinogenic amino acid, but also any non-proteinogenic amino acid, such as D-amino acids and modified amino acids formed by post-translational modifications, and any unnatural amino acid.

[0164] CNS and Brain As used herein, "central nervous system" or "CNS" refers to the part of the nervous system that includes the brain and spinal cord, to which sensory impulses are transmitted and from which motor impulses emanate, thereby coordinating activity throughout the nervous system.

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

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

[0167] Gene constructs The genetic constructs described herein can be prepared using any cloning and / or recombinant DNA techniques known to those skilled in the art, in which the nucleotide sequence encoding FGF21 is expressed in suitable cells, e.g., cultured cells, or cells of a multicellular organism, as described in Ausubel et al., "Current Protocols in Molecular Biology," Greene Publishing and Wiley-Interscience, New York (1987) and Sambrook and Russell (2001, supra), both of which are incorporated herein by reference in their entireties. See also Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site-directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, JA, et al. (1985) Gene 34:315 (describing cassette mutagenesis).

[0168] Expression vector The term "expression vector" or "vector" generally refers to a nucleotide sequence that can express a gene or coding sequence in a host that is compatible with such a sequence. An expression vector carries a genome that can be stabilized in a cell and remain episomal. Within the context of the present invention, a cell can be meant to include a cell that is used to produce a construct or a cell that the construct is administered to. Alternatively, the vector can be integrated into the genome of a cell, for example, by homologous recombination.

[0169] These expression vectors typically contain at least a suitable promoter sequence and may contain a transcription termination signal. Additional elements necessary or useful for expression can also be used as described herein. The nucleic acid or DNA or nucleotide sequence encoding FGF21 is incorporated into a DNA construct that can be introduced into and expressed in in vitro cell culture. Specifically, the DNA construct is suitable for replication in prokaryotic hosts, such as bacteria, for example, E. coli, or can be introduced into cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi, or other eukaryotic cell lines.

[0170] A DNA construct prepared for introduction into a particular host may include a replication system recognized by the host, a DNA segment intended to encode the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term "operably linked" has been previously described herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide. Generally, operably linked DNA sequences are contiguous, and in the case of a signal sequence, contiguous and in reading frame. However, enhancers need not be contiguous with the coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or adapters or linkers inserted in lieu thereof, or by gene synthesis.

[0171] The selection of an appropriate promoter sequence generally depends on the host cell selected for expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters well known in the art (see, e.g., Sambrook and Russell, 2001, supra). The transcriptional regulatory sequence typically includes a heterologous enhancer or promoter recognized by the host. The selection of an appropriate promoter depends on the host, but promoters such as trp, lac, and phage promoters, tRNA promoters, and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell, 2001, supra). Expression vectors contain a replication system and transcriptional and translational regulatory sequences, along with an insertion site for the polypeptide-encoding segment. In most cases, the replication system is only functional in the cells used to produce the vector (bacterial cells such as E. coli). Most plasmids and vectors do not replicate in cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334:31-36. For example, suitable expression vectors can be expressed in yeast, such as S. cerevisiae, insect cells, such as Sf9 cells, mammalian cells, such as CHO cells, and bacterial cells, such as E. coli. Thus, the cell can be a prokaryotic or eukaryotic host cell. The cell can be a cell suitable for culture in liquid or on solid medium.

[0172] Alternatively, the host cell is a cell that is part of a multicellular organism, such as a transgenic plant or transgenic animal.

[0173] The selection of an appropriate promoter sequence generally depends on the host cell selected for expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters well known in the art (see, e.g., Sambrook and Russell, 2001, supra). The transcriptional regulatory sequence typically includes a heterologous enhancer or promoter recognized by the host. The selection of an appropriate promoter depends on the host, but promoters such as trp, lac, and phage promoters, tRNA promoters, and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell, 2001, supra). Expression vectors contain a replication system and transcriptional and translational regulatory sequences, along with an insertion site for the polypeptide-encoding segment. In most cases, the replication system is only functional in the cells used to produce the vector (bacterial cells such as E. coli). Most plasmids and vectors do not replicate in cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334:31-36. For example, suitable expression vectors can be expressed 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. Thus, the cell can be a prokaryotic or eukaryotic host cell. The cell can be a cell suitable for culture in liquid or on solid medium.

[0174] Alternatively, the host cell is a cell that is part of a multicellular organism, such as a transgenic plant or transgenic animal.

[0175] viral vectors Viral Vectors or Viral Expression Vectors Viral gene therapy vectors are vectors that contain the genetic constructs described herein.

[0176] Viral vectors or viral gene therapy vectors are vectors suitable for gene therapy.Vectors suitable for gene therapy are described in Anderson 1998, Nature 392:25-30; Walther and Stein, 2000, Drugs 60:249-71; Kay et al., 2001, Nat. Med. 7:33-40; Russell, 2000, J. Gen. Virol. 81:2573-604; Amado and Chen, 1999, Science 285:674-6; Federico, 1999, Curr. Opin. Biotechnol. 10:448-53; Vigna and Naldini, 2000, J. Gene Med. 2:308-16; Marin et al., 1997, Mol. Med. Today 3:396-403; Peng and Russell, 1999, Curr. Opin. Biotechnol. 10:454-7; Sommerfelt, 1999, J. Gen. Virol. 80:3049-64; Reiser, 2000, Gene Ther. 7:910-3; and references cited therein.

[0177] Particularly suitable gene therapy vectors include adenovirus vectors and adeno-associated virus (AAV) vectors. These vectors infect many dividing and non-dividing cell types, including synovial cells and hepatocytes. The episomal nature of adenovirus vectors and AAV vectors after cell entry makes these vectors suitable for therapeutic use, as described above (Russell, 2000, J. Gen. Virol. 81: 2573-2604; Goncalves, 2005, Virol J. 2(1): 43). AAV vectors are even more preferred because they are known to provide very stable, long-term expression of transgenes (up to 9 years in dogs (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). Preferred adenoviral vectors are modified to reduce the host response, as outlined by Russell (2000, supra). Methods for gene therapy using AAV vectors are described in Wang et al., 2005, J Gene Med. March 9 (Epub ahead of print); Mandel et al., 2004, Curr Opin Mol Ther. 6(5):482-90; and Martin et al., 2004, Eye 18(11):1049-55; Nathwani et al., 2005, J Gene Med. March 9 (Epub ahead of print); et al, N Engl J Med. 2011 Dec 22;365(25):2357-65, Apparailly et al, Hum Gene Ther. 2005 Apr;16(4):426-34.

[0178] Another suitable gene therapy vector includes retroviral vector. In the present invention, the preferred retroviral vector for application is a lentivirus-based expression construct. Lentivirus vectors have the ability to infect and stably integrate into the genome of dividing and non-dividing cells (Amado and Chen, 1999 Science 285:674-6). Methods for constructing and using lentivirus-based expression constructs are described in U.S. Patent No. 6,165,782, U.S. Patent No. 6,207,455, U.S. Patent No. 6,218,181, U.S. Patent No. 6,277,633 and U.S. Patent No. 6,323,031, and Federico (1999, Curr Opin Biotechnol 10:448-53) and Vigna et al. (2000, J Gene Med 2000;2:308-16).

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

[0180] Adeno-associated virus vector (AAV vector) The terms "adeno-associated virus," "AAV virus," "AAV virion," "AAV viral particle," and "AAV particle," used synonymously herein, refer to a viral particle composed of at least one capsid protein of AAV (preferably composed of all capsid proteins of a particular AAV serotype) and an encapsulated polynucleotide of the AAV genome. When particles contain heterologous polynucleotides (i.e., polynucleotides different from the wild-type AAV genome, e.g., transgenes to be delivered to mammalian cells) flanked by AAV inverted terminal repeat sequences, they are typically known as "AAV vector particles" or "AAV viral vectors" or "AAV vectors." AAV refers to viruses belonging to the Dependovirus genus of the Parvoviridae family. The AAV genome is approximately 4.7 Kb in length and consists of single-stranded deoxyribonucleic acid (ssDNA) that can be detected positively or negatively. The present invention also encompasses the use of double-stranded AAV, also known as dsAAV or scAAV. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs), namely, rep and cap. The rep frame consists of four overlapping genes encoding the Rep protein required for the AAV life cycle. The cap frame contains nucleotide sequences that overlap with the capsid proteins VP1, VP2, and VP3, which interact to form the icosahedral capsid (see Carter and Samulski, 2000, and Gao et al., 2004).

[0181] A preferred viral vector or gene therapy vector is an AAV vector. As used herein, the AAV vector preferably includes a recombinant AAV vector (rAAV vector). As used herein, "rAAV vector" refers to a recombinant vector that includes a portion of the AAV genome encapsidated in a protein shell of capsid proteins derived from the AAV serotypes described herein. The portion of the AAV genome can include inverted terminal repeats (ITRs) derived from adeno-associated virus serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, etc. Preferred ITRs are the ITRs of AAV2, represented by the sequence comprising, consisting essentially of, or consisting of SEQ ID NO: 30 (5'ITR) and SEQ ID NO: 31 (3'ITR). The present invention also provides a sequence having as its 5' ITR at least 80% (or 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 98%, at least 99% or 100%) identity to SEQ ID NO: 30. and the use of a sequence having at least 80% (or 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 98%, at least 99% or 100%) identity to SEQ ID NO: 31 as the 3' ITR.

[0182] The protein shell, composed of capsid proteins, can be derived from any AAV serotype. The protein shell can also be referred to as a capsid protein shell. The rAAV vector can be deleted from one or preferably all wild-type AAV genes, but can still contain functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue, and packaging of AAV virions. The ITR sequences can be wild-type sequences, or can have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the wild-type sequence, or can be altered, for example, by nucleotide insertion, mutation, deletion, or substitution, as long as they remain functional. In this context, functionality refers to the ability to directly package the genome into the capsid shell and then allow it to be expressed in infected or target host cells. In the context of the present invention, the capsid protein shell can be of a different serotype from the rAAV vector genome ITRs.

[0183] The nucleic acid molecule represented by the selected nucleic acid sequence is preferably inserted into an expression construct comprising expression control elements operably linked to the above-identified rAAV genome or ITR sequences, e.g., a coding sequence and a 3' termination sequence. The nucleic acid molecule may also be referred to as a transgene.

[0184] "AAV helper functions" generally refer to the corresponding AAV functions required for rAAV replication and packaging that are supplied to the rAAV vector in trans. AAV helper functions complement AAV functions lacking in the rAAV vector, but lack the AAV ITRs (provided by the rAAV vector genome). AAV helper functions include the two major AAV ORFs, i.e., the rep coding region and the cap coding region, or sequences substantially functionally identical thereto. The Rep and Cap regions are well known in the art; see, for example, Chiorini et al. (1999, J. of Virology, Vol. 73(2):1309-1319) or U.S. Patent No. 5,139,941, which are incorporated herein by reference. AAV helper functions can be supplied in an AAV helper construct. Introduction of the helper construct into a host cell can occur, for example, by transformation, transfection, or transduction, before or simultaneously with the introduction of the rAAV genome present in the rAAV vector identified herein. Thus, the AAV helper constructs of the present invention can be selected to provide a desired combination of serotypes, on the one hand, for the capsid protein shell of the rAAV vector, and on the other hand, for the rAAV genome present in the replication and packaging of said rAAV vector.

[0185] An "AAV helper virus" provides additional functions necessary for AAV replication and packaging. Suitable AAV helper viruses include adenovirus, herpes simplex virus (such as HSV types 1 and 2), and vaccinia virus. As described in U.S. Patent No. 6,531,456, which is incorporated herein by reference, the additional functions provided by the helper virus can also be introduced into host cells via a plasmid.

[0186] "Transduction" refers to the delivery of FGF21 to recipient host cells by a viral vector. For example, the transduction of target cells by the rAAV vector of the present invention transfers the rAAV genome contained in the vector into the transduced cells. "Host cells" or "target cells" refer to the cells into which DNA is delivered, such as target muscle cells. AAV vectors can transduce both dividing and non-dividing cells.

[0187] AAV vector production The generation of recombinant AAV (rAAV) for vectoring transgenes has been previously described. See 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. Gene Ther. 2009;20:922-929; and Virag T, et al., Hum. Gene Ther. 2009;20:807-817. These protocols can be used or adapted to generate the AAV of the present invention. In one embodiment, a producer cell line is transiently transfected with a polynucleotide of the present invention (comprising an expression cassette flanked by ITRs) and with construct(s) encoding rep and cap proteins and providing helper functions. In another embodiment, the cell line stably supplies helper functions and is transiently transfected with a polynucleotide of the invention (comprising an expression cassette flanked by ITRs) and with construct(s) encoding the rep and cap proteins. In another embodiment, the cell line stably supplies rep and cap proteins and helper functions and is transiently transfected with a polynucleotide of the invention. In another embodiment, the cell line stably supplies rep and cap proteins and is transiently transfected with a polynucleotide of the invention and a polynucleotide encoding helper functions. In yet another embodiment, the cell line stably supplies a polynucleotide of the invention, the rep and cap proteins and helper functions. Methods of making and using these and other AAV production systems are described in the art.Muzyczka N,et 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. al., U.S. Patent No. 6,268,213; Rabinowitz 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.; al., U.S. Patent No. 7,172,893, Monahan P, et al., U.S. Patent No. 7,201,898, Samulski R, et al. See U.S. Patent No. 7,229,823 and Ferrari F, et al., U.S. Patent No. 7,439,065.

[0188] The rAAV genome present in the rAAV vector comprises at least the nucleotide sequence of the inverted terminal repeat region (ITR) of one of the AAV serotypes (preferably that of serotype AAV2 previously disclosed herein), or a nucleotide sequence substantially identical thereto or having at least 60% identity thereto, and a nucleotide sequence encoding FGF21 (under the control of suitable regulatory elements) inserted between the two ITRs. The vector genome requires the use of flanking 5' and 3' ITR sequences to enable efficient packaging of the vector genome into the rAAV capsid.

[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 can be cloned or produced by chemical synthesis known in the art, for example, using an oligonucleotide synthesizer supplied by Applied Biosystems Inc. (Fosters, CA, USA), or by standard molecular biology techniques. ITRs can be cloned from the AAV viral genome or excised from a vector containing AAV ITRs. Using standard molecular biology techniques, the ITR nucleotide sequences can be ligated at either end to a nucleotide sequence encoding one or more therapeutic proteins, or the AAV sequence between the ITRs can be replaced with a desired nucleotide sequence.

[0190] Preferably, the rAAV genome present in the rAAV vector does not contain nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or cap (capsid) genes. The rAAV genome may further include marker or reporter genes, such as antibiotic resistance genes, genes encoding fluorescent proteins (e.g., gfp), or genes encoding chemically, enzymatically, or otherwise detectable and / or selectable products (e.g., lacZ, aph, etc.) known in the art.

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

[0192] A suitable 3' untranslated sequence may also be operably linked to the nucleotide sequence encoding FGF21. Suitable 3' untranslated regions may be those naturally associated with the nucleotide sequence or may be derived from various genes, such as, for example, the SV40 polyadenylation signal (SEQ ID NO: 32) and the rabbit β-globin polyadenylation signal (SEQ ID NO: 33).

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

[0194] Expression can be evaluated at any time after administration of gene construct, expression vector or composition described herein.In some embodiments herein, expression can be evaluated after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks or more.

[0195] In the context of the present invention, CNS-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 / or olfactory bulb-specific expression refers to 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% higher, at least 100% higher, at least 150% higher, at least 200% higher, or more) expression of FGF21 in the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb compared to other organs or tissues. The other organs or tissues may be the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, etc. Preferably, the other organs are the liver and / or heart. In one embodiment, expression is undetectable in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. In some embodiments, expression is undetectable in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, and testis. Expression can be assessed as described above.

[0196] Throughout this application, when 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 are mentioned in the context of expression, cell type-specific expression of the cell type(s) that make up the CNS and / or brain and / or hypothalamus and / or cortex and / or hippocampus and / or cerebellum and / or olfactory bulb, respectively, is also envisaged.

[0197] Administration As used herein, "intra-CSF administration" refers to direct administration into the CSF, which is located in the subarachnoid space between the arachnoid and pia mater layers of the meninges surrounding the brain. Intra-CSF administration can be achieved by intracisternal, intraventricular, or intrathecal administration. As used herein, "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 either of the bilateral ventricles of the brain. As used herein, "intrathecal administration" includes direct administration into the CSF within the spinal cavity. As used herein, "intraparenchymal administration" refers to direct local administration into any region of the brain parenchyma. As used herein, "intranasal administration" refers to administration through the nasal structures.

[0198] Codon optimization As used herein, "codon optimization" refers to a process used to modify an existing coding sequence or design a coding sequence, for example, to improve translation of a transcribed RNA molecule transcribed from the coding sequence in an expression host cell or organism, or to improve transcription of the coding sequence. Codon optimization includes, but is not limited to, processes that involve selecting codons for a coding sequence to match the codon preferences of the expression host organism, for example, to match the codon preferences of a mammalian, preferably a mouse, dog, or human, expression host. Codon optimization also eliminates elements that may adversely affect RNA stability and / or translation (e.g., termination sequences, TATA boxes, splice sites, ribosome entry sites, repeat and / or GC-rich sequences, and RNA secondary structure or instability motifs). In some embodiments, the codon-optimized sequence exhibits at least a 3%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase in transcription, RNA stability, and / or translation.

[0199] In this specification and the claims, the verb "to comprise" and its conjugations are used in their open-ended sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. Furthermore, the verb "to consist" may be replaced with "to consist essentially of," which means that the peptides or peptidomimetics, media, or compositions described herein may contain additional component(s) other than those specifically identified, where such additional component(s) do not alter the inherent characteristics of the invention. Additionally, the verb "to consist" may be replaced with "to consist essentially of," which means that the methods described herein may include additional step(s) other than those specifically identified, where such additional step(s) do not alter the inherent characteristics of the invention.

[0200] The reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element be present. Thus, the indefinite article "a" or "an" normally 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 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... etc.

[0202] Individual numerical values ​​are described as approximations as if the value were preceded by the word "about" or "approximately." Similarly, numerical values ​​within various ranges specified in this application are described as approximations as if both the minimum and maximum values ​​within the stated range were preceded by the word "about" or "approximately," unless expressly stated otherwise. As used herein, the terms "about" and "approximately," when referring to numerical values, shall have their obvious and ordinary meaning to one of ordinary skill in the art to which the disclosed subject matter most closely pertains or to whom the range or element in question pertains. The amount by which the numerical value varies from an exact numerical boundary depends on many factors. For example, some factors that may be considered include the criticality of the element and / or the effect that variation in a given amount has on the performance of the claimed subject matter, as well as other considerations known to those of ordinary skill in the art. In the absence of considerations to the contrary, the word "about" or "approximately" when used in connection with a numerical value (e.g., about 10) preferably means that the value may be 1% more or less (that is, 10) than the given value.

[0203] As used herein, the term "and / or" indicates that one or more of the stated instances may occur alone or in combination with at least one of the stated instances, up to all of the stated instances.

[0204] Each of the embodiments specified herein can be combined together unless otherwise specified.

[0205] All patent applications, patents, and printed publications cited herein are incorporated herein by reference in their entirety, except for any definitions, disclaimers or disclaimers of subject matter, and except to the extent that the incorporated material contradicts the explicit disclosure herein, in which case the language in the present disclosure will control.

[0206] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, 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. [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 an AAV vector, the effects of FGF21 in the brain when overexpressed in this organ are tested. Three different experiments were performed: Treatment of db / db mice with AAV9-CAG-moFGF21-dmiRT. Dose: 5×10 10 vg / mouse (Example 1).

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

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

[0212] Furthermore, the present inventors also examined the brain transduction efficiency of AAV1-FGF21, AAV2-FGF21, and AAV9-FGF21 vectors after intra-CSF administration in wild-type mice (Example 3).

[0213] dmiRT refers to four copies of miRT-122a and four copies of miRT-1 sequences.

[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 We used non-obese SAMP8 / TaHsd (db / +) and C57Bl / 6J (wild-type) mice. Mice were fed standard chow ad libitum (2018S Teklad Global Diets®, Harlan Labs., Inc., Madison, WI, US) under a 12-h light-dark cycle (lights on at 8:00 AM) and stable temperature (22°C ± 2°C). For tissue sampling, mice were anesthetized with the inhalation anesthetic isoflurane (IsoFlo®, Abbott Laboratories, Abbott Park, IL, US) and decapitated. Tissues of interest were excised and kept at -80°C until analysis. All experimental procedures were approved by the Ethics Committee for Animal and Human Experimentation at the Universitat Autónoma de Barcelona.

[0216] Recombinant AAV vectors Single-stranded AAV vectors of serotypes 1, 2, and 9 were produced by triple transfection of HEK293 cells according to standard methods (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6):423-36). Cells were grown in DMEM 10% FBS in ten roller bottles (850 cm) until 80% confluence. 2The cells were cultured in a flat plate (Corning™, Sigma-Aldrich Co., Saint Louis, MO, US) and co-transfected by calcium phosphate with a plasmid carrying an expression cassette flanked by AAV2 ITRs (SEQ ID NO: 35), a helper plasmid carrying the AAV2 rep gene and AAV for serotype 1, 2, or 9 cap genes, respectively, and a plasmid carrying adenovirus helper functions. The transgene used was a mouse codon-optimized FGF21 coding sequence (SEQ ID NO: 9) driven by an early enhancer / chicken β-actin (CAG) promoter (SEQ ID NO: 27). In Examples 1, 2, and 3, the transgene also contained four tandem repeats of the miRT-122a sequence (5'CAAACACCATTGTCACACTCCA3', SEQ ID NO: 12) and four tandem repeats of the miRT-1 sequence (5'TTACATACTTCTTTACATTCCA3', SEQ ID NO: 13) cloned into the 3' untranslated region of the expression cassette. In Example 4, the cassette did not carry miRT-122a or miRT-1. AAV was purified using an optimized method based on a polyethylene glycol precipitation step and two successive cesium chloride (CsCl) gradients. This second-generation CsCl-based protocol dramatically reduced empty AAV capsids and DNA and protein impurities (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6):423-36). The purified AAV vector was dialyzed against PBS, filtered, and stored at -80°C. Using linearized plasmid DNA as a standard curve, the viral genome titer was determined by quantitative PCR according to the protocol described for AAV2 reference standard (Lock M, et al., Hum. Gene Ther. 2010;21:1273-1285). The vector was constructed according to molecular biology techniques well known in the art.

[0217] In vivo administration of AAV vectors into CSF Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). The skin on the back of the head was shaved from behind the ears to approximately the interscapular space and rinsed with ethanol. The mice were held in a prone position with their heads tilted slightly downward. A 2 mm rostro-caudal incision was made, and a Hamilton syringe was introduced into the cisterna magna at a 45-55° angle, between the back of the head and the C1 vertebra, and 5 μl of the vector dilution was administered. Considering that the CNS is the primary target compartment for vector delivery, the same number of vector genomes / mouse (5 × 10) were administered regardless of body weight. 10 Mice were administered 100 mg / mouse.

[0218] Immunohistochemical and morphometric analysis Tissues were fixed in formalin (Panreac Quimica) for 24 hours, embedded in paraffin, and sectioned. Tissue samples were stained with hematoxylin-eosin, and images were acquired using a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) connected to a video camera with a monitor equipped with image analysis software (analySIS 3.0; Soft Imaging System, Center Valley, PA, EEUU).

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

[0220] RNA analysis Total RNA was obtained from the hypothalamus, cortex, hippocampus, cerebellum, and olfactory bulb using Tripure isolation reagent (Roche Diagnostics Corp., Indianapolis, IN, US), and from white adipose tissue, brown adipose tissue, and liver using Qiazol lysis reagent (Qiagen NV, Venlo, NL) and the RNeasy Mini or RNeasy Micro kits (Qiagen NV, Venlo, NL) for hippocampal samples. To eliminate residual viral genomes, total RNA was treated with DNAse I (Qiagen NV, Venlo, NL). For RT-PCR analysis, 1 μg of RNA sample was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (04379012001, Roche, California, USA). Real-time quantitative PCR was performed on a SmartCycler II® (Cepheid, Sunnyvale, USA) using TB Green Premix Ex Taq II (Takara Bio Europe, France). Data were normalized by the Rplp0 value and analyzed as previously described (Pfaffl, M., Nucleic Acids Res. 2001;29(9):e45).

[0221] The primers used are outlined below: moFgf21-Fw: 5'-CCTAACCAGGACGCCACAAG-3' (SEQ ID NO: 47) moFgf21-Rv: 5'-GTTCCACCATGCTCAGAGGG-3' (SEQ ID NO: 48) Gfap-Fw: 5'-ACAGACTTTCTCCAACCTCCAG-3' (SEQ ID NO: 49) Gfap-Rv: 5'-CCTTCTGACACGGATTTGGT-3' (SEQ ID NO: 50) S100b-Fw: 5'-AACAACGAGCTCTCTCACTTCC-3' (SEQ ID NO: 51) S100b-Rv: 5'-CGTCTCCATCACTTTGTCCA-3' (SEQ ID 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: 59) Il6-Rv: 5'-CATCAGAGGCAAGGAGGAAAAC-3' (SEQ ID 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: 63) Cidea-Rv: 5'-AGGCCAGTTGTGATGACTAAGAC-3' (SEQ ID NO: 64) Tnfa-Fw: 5'-CGGCATGGATCTCAAAGACAAC-3' (SEQ ID NO: 65) Tnfa-Rv: 5'-AGATAGCAAATCGGCTGACG-3' (SEQ ID NO: 66) F4 / 80-Fw: 5'-CTTTGGCTATGGGCTTCCAGTC-3' (SEQ ID NO: 67) F4 / 80-Rv: 5'-GCAAGGAGGACAGAGTTTATC-3' (SEQ ID NO: 68) Rplp0-Fw: 5'-ACTGGTCTAGGACCCGAGAA-3' (SEQ ID NO: 69) Rplp0-Fw: 5'-TCCCACCTTGTCTCCAGTCT-3' (SEQ ID NO: 70) Hormone and metabolite assays Blood glucose levels were measured using a Glucometer Elite™ analyzer (Bayer, Leverkusen, Germany). Brain levels of FGF21 protein were determined using a quantitative sandwich enzyme immunoassay mouse / rat FGF-21 ELISA kit (MF2100, R&D systems, Abingdon, UK) and normalized by the total protein content measured using Bradford reagent (Bio-Rad Protein Assay, Bio-Rad, Germany) in whole brain homogenates. To extract lipids from the liver, approximately 100 mg of frozen samples were weighed and homogenized in chloroform:methanol (2:1) as described by Carr et al. Liver and serum triglycerides were quantified spectrophotometrically using an enzyme assay kit (Horiba-ABX, Montpellier, France). Serum free fatty acids were measured by the acyl-CoA synthase and acyl-CoA oxidase method (Wako Chemicals GmbH, Neuss, Germany). All biochemical parameters were determined using a Pentra 400 analyzer (Horiba-ABX).

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

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

[0225] [Example 1] Intra-CSF administration of AAV9-CAG-moFGF21-dmirT vector reverses obesity and diabetes in db / db mice We evaluated the antidiabetic and antiobesogenic therapeutic potential of AAV-mediated genetic engineering of the brain with FGF21 in 7-week-old male db / db mice, which are deficient in leptin signaling and a widely used genetic model of obesity and diabetes. To this end, we administered 5 × 10 AAVs encoding the mouse codon-optimized FGF21 coding sequence under the control of a CAG ubiquitous promoter, including target sites for the liver-specific miR-122a and cardiac-specific miR-1 (AAV9-CAG-moFGF21-dmiRT). 10 AAV9 vectors in vg / mice were administered locally into the cerebrospinal fluid (CSF) via the cisterna magna. Untreated db / db animals served as controls.

[0226] Intra-CSF administration of the AAV9-CAG-moFGF21-dmiRT vector mediated widespread overexpression of FGF21 in the brain, as evidenced by increased expression levels of the factor within various brain regions, including the hypothalamus, cortex, hippocampus, and cerebellum, 12 weeks after AAV administration (Fig. 1 ).

[0227] While untreated db / db mice continued to gain weight (approximately 50% weight gain) over the 12-week follow-up period, weight gain was clearly reduced in the cohort treated with a vector encoding FGF21 (approximately 20% weight gain) (Figures 2A and 2B). Consistent with this, animals treated with the AAV9-CAG-moFGF21-dmiRT vector showed reduced adiposity and a 60% reduction in liver weight (Figure 2C). Notably, db / db mice with brain-targeted FGF21 gene transfer also showed complete normalization of fed blood glucose, demonstrating the suppression of diabetes in these animals (Figure 3).

[0228] Obesity is associated with brain inflammation (O. Guillemot-Legris, G.G. Muccioli, Trends Neurosci. 40, 237-253 (2017)). Inflammation in this organ was analyzed by the expression of the astrocyte markers Gfap and S100b, the microglial marker Aif1, and inflammatory molecules such as Nfkb, Il1b, and Il6. db / db mice treated intra-CSF with the AAV9-CAG-moFGF21-dmiRT vector showed decreased expression of Gfap, S100b, Aif1, Nfkb, Il1b, and Il6 in the hypothalamus (Figure 4).

[0229] Example 1.1 Histological analysis of white adipose tissue by hematoxylin-eosin staining revealed a decrease in white adipocyte size in eWAT (Figure 10A). In BAT, histological analysis showed relatively low lipid accumulation and relatively numerous multilocular brown adipocytes (Figure 10B). In accordance with these results, the expression levels of Ucp1 and Cidea were highly increased in BAT of FGF21-treated mice (Figure 10C), suggesting increased thermogenesis after AAV-FGF21 CNS administration. AAV9-FGF21-treated db / db mice had decreased hepatic triglyceride content (Figure 11A). In parallel, these mice also had decreased circulating levels of triglycerides and serum free fatty acids (Figures 11B and 11C). Immunohistochemical analysis of the pancreas revealed an increased number of pancreatic islets (Figure 12A) and improved beta cell mass (Figure 12B) in db / db mice after treatment with the AAV9-FGF21 vector.

[0230] Obesity and diabetes are associated with systemic inflammation. In white adipose tissue, immunohistochemical analysis of the inflammatory marker MAC-2 showed reduced macrophage infiltration in AAV9-FGF21-treated mice (Fig. 13A), which was associated with reduced F4 / 80 mRNA expression levels (Fig. 13B). FGF21-treated animals also showed reduced expression levels of the inflammatory cytokines F4 / 80, Il6, and Tnfα in brown adipose tissue and liver (Fig. 13C and Fig. 13D), indicating reduced systemic inflammation after FGF21 gene therapy.

[0231] [Example 2] Intra-CSF administration of AAV9-CAG-moFGF21-dmirT vector reduces body weight gain in SAMP8 mice 5 × 10 male 7-week-old senescence-accelerated mouse prone 8 (SAMP8) mice, a widely used aging mouse model with age-related brain pathology. 10 The AAV9-CAG-moFGF21-dmiRT vector was administered locally into the CSF via the cisterna magna in vg / mice. Untreated SAMP8 animals served as controls.

[0232] Similar to the observations obtained with db / db mice, intra-CSF administration of the AAV9-CAG-moFGF21-dmiRT vector mediated robust overexpression of FGF21 in the hypothalamus, cortex, hippocampus, and cerebellum of SAMP8 mice 14 weeks after AAV administration (Figure 5). FGF21-treated mice exhibited lower body weight gain than untreated mice (Figure 6A and Figure 6B), which paralleled the reduction in liver weight (Figure 6C). Furthermore, the expression of the pro-inflammatory cytokines Il1b and Il6 was reduced in the hypothalamus of SAMP8 mice overexpressing FGF21 in the brain (Figure 7).

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

[0234] To investigate whether several AAV serotypes can efficiently transduce the brain after direct CSF administration via the cisterna magna, we used 5 × 10 AAVs encoding the mouse codon-optimized FGF21 coding sequence under the control of a CAG ubiquitous promoter, including target sites for the liver-specific miR-122a and cardiac-specific miR-1. 10 Wild-type mice were treated with AAV1, AAV2, and AAV9 vectors (AAV1-CAG-moFGF21-dmiRT, AAV2-CAG-moFGF21-dmiRT, and AAV9-CAG-moFGF21-dmiRT, respectively) at 1000 mg / mouse. Untreated wild-type mice served as controls.

[0235] Three weeks after intra-CSF administration of the AAV vector, brain samples were obtained, and RT-PCR analysis showed increased moFGF21 expression in various brain regions, including the hypothalamus, cortex, hippocampus, and cerebellum (Figure 8). Furthermore, overexpression of moFGF21 resulted in increased FGF21 protein content throughout the brain (Figure 9).

[0236] [Example 4] Reversal of obesity and diabetes by intra-CSF administration of AAV1-CAG-moFGF21 vector in db / db mice.

[0237] Encoding the mouse codon-optimized FGF21 coding sequence under the control of the CAG ubiquitous promoter, 5 × 10 10 The anti-diabetic and anti-obesity therapeutic potential of AAV-mediated brain genetic engineering with FGF21 gene therapy was also evaluated in 7-week-old db / db male mice that received a db / db AAV1 vector (AAV1-CAG-moFGF21) administered locally into the cerebrospinal fluid (CSF) via the cisterna magna. Untreated db / db and untreated db / + (non-obese) mice served as controls.

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

[0239] While untreated db / db mice continued to gain weight over the 14-week follow-up period, the cohort treated with a vector encoding AAV1-FGF21 did not (Figure 15). Notably, db / db mice with brain-targeted FGF21 gene transfer also showed complete normalization of fed and fasted blood glucose (Figures 16A and 16B), demonstrating suppression of diabetes in these animals.

[0240] Insulin tolerance tests showed that insulin resistance improved in db / db mice after treatment with the AAV1-FGF21 viral vector (Figure 17), and intraperitoneal glucose tolerance tests in overnight-fasted mice showed that db / db mice treated with AAV1-CAG-moFGF21 had better glucose tolerance than untreated db / db mice (Figure 18). An intraperitoneal pyruvate tolerance test was performed as an index of hepatic gluconeogenesis. After pyruvate challenge, blood glucose levels rose to 600 mg / dl in untreated db / db mice and remained elevated throughout the test, whereas blood glucose levels in FGF21 db / db-treated mice and non-obese treated mice rose to a maximum of 150 mg / dl, thus indicating reduced gluconeogenesis after AAV1-CAG-FGF21 treatment (Figure 19). [Table 4] TIFF0007801046000005.tif63166

[0241] Amino acid sequence of Homo sapiens FGF21 (SEQ ID NO: 1) MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS Nucleotide sequence of Homo sapiens FGF21 (SEQ ID NO: 4) ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGCGCTGCTGACCAGAGCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGATTATTCAAATCTTGGGAGCTCAAGACATCCAGGTTCCTTGCCAGCGGG CCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCGCTTCCTGA. ホモサピエンスFGF21- コドン コドン optimization nucleotide sequence(sequence number 5) ATGGATTCTGATGAGCAGGCTTCGAGCACAGGCGGCCTGTGGGTTTCAGTTCTGGCTGGACTGCTGCTGGAGCCTGTCAGGCACACCCTTACTCAGATAGCAGCCCTCTGCTGCAGTTCGGCGACAAGTGCGGCAGAGATACCTGTACACCGACGACGCCCAGCAGACAGAAGCCCACCTGGAAATCAGAGAGGATGGCACAGTTGGCGAGCCGCCGATCAGTCTCCTGAATCTCTGCTCCAGCGAAGGCCCTGAAGCCTGGTCGATCCTGGGCGTGAAAACCAGCGGTTCCTGTCCAAAGA CCTGACGGGCCCCTGTATGGCAGCCTGCACTTTGATCCTGAGGCCTGCAGCTTCAGAGAGCTGCTGCTTGAGGACGGCTACAACGTGTACCAGTCTGAGGCCCCATGGCCTGCCTCTGCATCTGCCTGGAACAAGAGCCCTCACAGAGATCCGCTCCTAGAGGCCCTGCCAGATTTCTGCCTCTTCCTGGATTGCCTCTGCTCTGCCAGACCTCCTGGAATTCTGGCTCCTCCTGATGTGGGCAGCTCTGATCCTGAGCATGGTCGGACCTAGCCAGGGCAGATCTCTAGCTACTACGCTCTTGA ホモサピエンスFGF21- コドン コドン optimization nucleotide sequence(SEQ ID NO:6) ATGGACAGCGATGAAACCGGGTTCGAGCACAGCGGTCTGTGGGTGTCCGTGCTGGCCGGACTGCTCCTGGGAGCCTGTCAGGCGCACCCCATCCCTGACTCCTCGCCGCTGCTGCAATTCGGCGGACAAGTCCGCCAGAGATACCTGTACACCGACGACGCCCAGCAGACCGAAGCCCACCTGGAAATTCGGGAGGACGGGACTGTGGGAGGCGCTGCAGATCAGTCACCCGAGTCCCTCCTCCAACTGAAGGCCTTGAAGCCCGGCGTGATTCAGATCCTGGGCGTGAAAACTTCCCGCTTCCTTTGCCAACGGCCGGATGGAGCTCTGTACGGATCCCTGCACTTCGACCCCGAAGCCTGCTCATTCCGCGAGCTGCTCCTTGAGGACGGCTATAACGTGTACCAGTCTGAGGCCCATGGACTCCCCCTGCATCTGCCCGGCAACAAGTCCCCTCACCGGGATCCTGCCCCAAGAGGCCCAGCTCGGTTTCTGCCTCTGCCGGGACTGCCTCCAGCGTTGCCCGAACCCCCTGGTATCCTGGCCCCGCAACCACCTGACGTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAGCCAGGGAAGGTCCCCGTCCTACGCATCCTGA Codon-optimized nucleotide sequence of Homo sapiens FGF21-variant 3 (SEQ ID NO: 7) ATGGATTCCGACGAAACTGGATTTGAACATTTCAGGGCTGTGGGTCTCTGTGCTGGCTGGACTGCTGCTGGGGCTTGTCAGGCTCACCCCATCCCTGAGCTCCCCTCTGCTGCAGTTCGGAGGACAGGTGCGGCAGAGATACCTGTATACCGACGATGCCCAGCAGACAGAGGCACACCTGGAGATCAGGGAGGACGGAACCGTGGGAGGAGCAGCCGATCAGTCTCCCGAGAGCCTGCTGCAGCTGAAGGCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAGACATCTCGGTTTCTGTGCCAGCGG CCCGACGGCGCCCTGTACGGCTCCCTGCACTTCGATCCCGAGGCCTGTTCTTTTAGGAGCTGCTGCTGGAGGACGGCTACAACGTGTATCAGAGCGAGGCACACGGCCTGCCACTGCACCTGCCTGGCAATAAGTCCCCTCACCGCGATCCAGCACCCAGGGCCCAGCACGCTTCCTGCCTCTGCCAGGCCTGCCCCTGCCTGCCAGAGCCACCGGCATCCTGGCCCCAGCTCCAGATGTGGCTCCAGCGATCCTCTGTCAATGTGGGGCCAAGTCAGGGGCGGAGTCCTTCATACGCATAA パースコドン optimization FGF21のnucleotide sequence(sequence number 9) ATGGAATGGATGAGAAGCAGAGTGGGCACCCTGGGCCTGTGGGTGCGACTGCTGCTGGCTGTGTTTCTGCTGGGCGTGTACCAGGCCTACCCCATCCCTGACTCTAGCCCCCTGCTGCAGTTTGGCGGACAAGTGCGGCAGAGATACCTGTACACCGACGACGACCAGGACACCGAGGCCCACCTGGAAATCCGCGAGGATGGCACAGTCGTGGGCGCTGCTCACAGAAGCCCTGAGAGCCTGCTGGAACTGAAGGCCCTGAAGCCCGGCGTGATCCAGATCCTGGGCGTGAAGGCCAGCAGATTCCTGTGCCAGCAGCCTGACGGCGCCCTGTACGGCTCTCCTCACTTCGATCCTGAGGCCTGCAGCTTCAGAGAGCTGCTGCTGGAGGACGGCTACAACGTGTACCAGTCTGAGGCCCACGGCCTGCCCCTGAGACTGCCTCAGAAGGACAGCCCTAACCAGGACGCCACAAGCTGGGGACCTGTGCGGTTCCTGCCTATGCCTGGACTGCTGCACGAGCCCCAGGATCAGGCTGGCTTTCTGCCTCCTGAGCCTCCAGACGTGGGCAGCAGCGACCCTCTGAGCATGGTGGAACCTCTGCAGGGCAGAAGCCCCAGCTACGCCTCTTGA Nucleotide sequence of the CAG promoter (SEQ ID NO: 27) Nucleotide sequence of the CMV promoter (SEQ ID NO: 28) GTGATGCGGTTTTGGCAGTAACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT Nucleotide sequence of the CMV enhancer (SEQ ID NO: 29) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACCGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG CMV promoter and CMV enhancer sequence (SEQ ID NO: 34) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT AAV2 5’ ITR (SEQ ID NO: 30) GCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT AGGGGTTCCT AAV2 3’ ITR (SEQ ID NO: 31) AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGC Rabbit β-globin polyadenylation signal (3'UTR and adjacent regions of rabbit β-globin, including the polyA signal) (SEQ ID NO: 33) GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAA AGGTTGGCTATAAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC miRT sequence miRT-122a (SEQ ID NO: 12): 5'CAAACACCATTGTCACACTCCA3', target for microRNA-122a expressed in the liver (accession number to the miRBase database, MI0000442).

[0242] miRT-152 (SEQ ID NO: 14): 5'CCAAGTTCTGTCATGCACTGA3', target for microRNA-152 expressed in the liver (MI0000462).

[0243] miRT-199a-5p (SEQ ID NO: 15): 5'GAACAGGTAGTCTGAACACTGGG3', target for microRNA 199a expressed in the liver (MI0000242).

[0244] miRT-199a-3p (SEQ ID NO: 16): 5'TAACCAATGTGCAGACTACTGT3', a target for microRNA-199a expressed in the liver (MI0000242).

[0245] miRT-215 (SEQ ID NO: 17): 5'GTCTGTCAATTCATAGGTCAT3', target for microRNA-215 expressed in the liver (MI0000291).

[0246] miRT-192 (SEQ ID NO: 18): 5'GGCTGTCAATTCATAGGTCAG3', target for microRNA-192 expressed in the liver (MI0000234).

[0247] miRT-148a (SEQ ID NO: 19): 5'ACAAAGTTCTGTAGTGCACTGA3', target for microRNA-148a expressed in the liver (MI0000253).

[0248] miRT-194 (SEQ ID NO: 20): 5'TCCACATGGAGTTGCTGTTACA3', target for microRNA-194 expressed in the liver (MI0000488).

[0249] miRT-133a (SEQ ID NO: 21): 5'CAGCTGGTTGAAGGGGACCAAA3', target for microRNA-133a expressed in the heart (MI0000450).

[0250] miRT-206 (SEQ ID NO: 22): 5'CCACACACTTCCTTACATTCCA3', target for microRNA-206 expressed in the heart (MI0000490).

[0251] miRT-1 (SEQ ID NO: 13): 5'TTACATACTTCTTTACATTCCA3', target for microRNA-1 expressed in the heart (MI0000651).

[0252] miRT-208a-5p (SEQ ID NO: 23): 5'GTATAACCCGGGCCAAAAGCTC3', a target for microRNA-208a expressed in the heart (MI0000251).

[0253] miRT-208a-3p (SEQ ID NO: 24): 5'ACAAGCTTTTTGCTCGTCTTAT3', a target for microRNA-208a expressed in the heart (MI0000251).

[0254] miRT-499-5p (SEQ ID NO: 25): 5'AAACATCACTGCAAGTCTTAA3', target for microRNA-499 expressed in the heart (MI0003183).

[0255] pAAV-CAG-moFGF21-dmiRT (SEQ ID NO: 35) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAATCGGCC AACGCGCGGG 51 GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT 101 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG 151 GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT 201 GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC 251 TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC 351 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC 401 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT 451 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA 501 GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT 551 CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG 651 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA 701 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA 751 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT 801 TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG 851 ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT 951 CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT 1001 AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA 1051 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA 1101 GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA 1151 TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC 1251 CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG 1301 TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA 1351 CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGTCAAG 1401 GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG 1451 GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG 1551 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA 1601 TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG 1651 CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG 1701 GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC 1751 CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAAACAGG AAGGCAAAAAT GCCGCAAAAA AGGGATAAG 1851 GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT 1901 GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT 1951 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT 2001 GCCACCTGAC GTCTAAGAAA ASSISTANT ASSISTANT ACCTAAAA 2051 ATAGGCGTAT CACGAGGCCC TTTCGTCTCG CGCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA 2151 AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG 2201 GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG 2251 AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA 2301 ATACCGCATC AGGCGATTCC AACATCCAAT AAATCATACA GGCAAGGCAA 2351 CONNECTION AAATAGCCTC CONNECTIONAA GCTAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTAATA CTTTTGCCGGG AGAAGCCTTT 2451 ATTTCAACGC AAGGATAAAA ATTTTTAGAA CCCTCATATA TTTTAAATGC 2501 AATGCCTGAG TAATGTGTAG GTAAAGATTC AAACGGGTGA GAAAGGCCGG 2551 AGACAGTCAA ATCACCATCA ATATGATATT CAACCGTTCT AGCTGATAAA 2601 TTCATGCCGG AGAGGGTAGC TATTTTTGAG AGGTCTCTAC AAAGGCTATC 2651 AGGTCATTGC CTGAGAGTCT GGAGCAAACA AGAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC CCCGGTTGAT AATCAGAAAA 2751 GCCCCAAAA CAGGAAGATT GTATAAGCAA ATATTTAAAT TGTAAGCGTT 2801 AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATC GCTCATTTTT 2851 TAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA 2901 CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA 2951 AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA 3001 TGGCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT 3051 GCCGTAAAGC ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT 3101 TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA 3151 AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCCGGTAA 3201 CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTACTATGGT 3251 TGCTTTGACG AGCACGTATA ACGTGCTTTC CTCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT TAGACAGGAA CGGTACGCCA 3351 GAATCCTGAG AAGTGTTTTT ATAATCAGTG AGGCCACCGA GTAAAAGGT 3401 CTGTCCATCA CGCAAATTAA CCGTTGTCGC AATACTTCTT TGATTAGTAA 3451 TAACATCACT TGCCTGAGTA GAAGAACTCA AACTATCGGC CTTGCTGGTA 3501 ATATCCAGAA CAATATTACC GCCAGCCATT GCAACGGAAT CGCCATTCGC 3551 CATTCAGGCT GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC ACTGAGGCCG CCCGGGCAAA 3651 GCCCGGGCGT CGGGCGACCT TTGGTCGCCC GGCCTCAGTG AGCGAGCGAG 3701 CGCGCAGAGA GGGAGTGGCC AACTCCATCA CTAGGGGTTC CTTGTAGTTA 3751 ATGATTAACC CGCCATGCTA CTTATCTACT CGACATTGAT TATTGACTAG 3801 TATTCATT CGGGGTCATT AGTTCATAGC CCATATATGG 3851 AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC 3951 GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA 4001 CTGCCCACTT GGCAGTACAT CAAGTGTATC ATATGCCAAG TACGCCCCCT 4051 ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG CCCAGTACAT 4101 GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG 4151 CTATTACCAT GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT TTATTTATTT TTTAATTT 4251 TTGTGCAGCG ATGGGGGCGG GGGGGGGGGG GGGCGCGCG CCAGGCGGGG 4301 CGGGGCGGGG CGAGGGGCGG GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA 4351 GCCAATCAGA GCGGCGCGCT CCGAAAGTTT CCTTTTATGG CGAGGCGGCG 4401 GCGGCGGCGG CCCTAAAAA AGCGAAGCGC GCGGCGGGCG GGAGTCGCTG 4451 CGTTGCCTTC GCCCCGTGCC CCGCTCCGCG CCGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG GTGAGCGGGC GGGACGGCCC 4551 TTCTCCTCCG GGCTGTAATT AGCGCTTGGT TTAATGACGG CTTGTTTCTT 4601 TTCTGTGGCT GCGTGAAAGC CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC 4651 GGGGGGAGCG GCTCGGGGGG TGCGTGCGTG TGTGTGTGCG TGGGGAGCGC 4701 CGCGTGCGGC TCCGCGCTGC CCGGCGGCTG TGAGCGCTGC GGGCGCGGCG 4751 CGGGGCTTTG TGCGCTCCGC AGTGTGCGCG AGGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG GAACAAAGGC TGCGTGCGGG 4851 GTGTGTGCGT GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC GGTCGGGCTG 4901 CAACCCCCCC TGCACCCCCC TCCCCGAGTT GCTGAGCACG GCCCGGCTTC 4951 GGGTGCGGGG CTCCGTACGG GGCGTGGCGC GGGGCTCGCC GTGCCGGGCG 5001 GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG GGGCGGGGCC GCCTCGGGCC 5051 GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC CCCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT TTTATGGTAA TCGTGCGAGA 5151 GGGCGCAGGG ACTTCCTTTG TCCCAAATCT GTGCGGAGCC GAAATCTGGG 5201 AGGCGCCGCC GCACCCCCTC TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC 5251 CGGCAGGAAG GAAATGGGCG GGGAGGGCCT TCGTGCGTCG CCGCGCCGCC 5301 GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT GTCCGCGGGG GGACGGCTGC 5351 CTTCGGGGGG GACGGGGCAG GGCGGGGTTC GGCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA TGCCTTCTTC TTTTTCCTAC 5451 AGCTCCTGGG CAACGTGCTG GTTATTGTGC TGTCTCATCA TTTTGGCAAA 5501 GAATTGATTA ATTCGAGCGA ACGCGTCGAG TCGCTCGGTA CGATTTAAAT 5551 TGAATTGGCC TCGAGCGCAA GCTTGAGCTA GCGCCACCAT GGAATGGATG 5601 AGAAGCAGAG TGGGCACCCT GGGCCTGTGG GTGCGACTGC TGCTGGCTGT 5651 GTTTCTGCTG GGCGTGTACC AGGCCTACCC CATCCCTGAC TCTAGCCCCC 5701 TGCTGCAGTT TGGCGGACAA GTGCGGCAGA GATACCTGTA CACCGACGAC 5751 GACCAGGACA CCGAGGCCCA CCTGGAAATC CGCGAGGATG GCACAGTCGT 5801 GGGCGCTGCT CACAGAAGCC CTGAGAGCCT GCTGGAACTG AAGGCCCTGA 5851 AGCCCGGCGT GATCCAGATC CTGGGCGTGA AGGCCAGCAG ATTCCTGTGC 5901 CAGCAGCCTG ACGGCGCCCT GTACGGCTCT CCTCACTTCG ATCCTGAGGC 5951 CTGCAGCTTC AGAGAGCTGC TGCTGGAGGA CGGCTACAAC GTGTACCAGT 6001 CTGAGGCCCA CGGCCTGCCC CTGAGACTGC CTCAGAAGGA CAGCCCTAAC 6051 CAGGACGCCA CAAGCTGGGG ACCTGTGCGG TTCCTGCCTA TGCCTGGACT 6101 GCTGCACGAG CCCCAGGATC AGGCTGGCTT TCTGCCTCCT GAGCCTCCAG 6151 ACGTGGGCAG CAGCGACCCT CTGAGCATGG TGGAACCTCT GCAGGGCAG 6201 AGCCCCAGCT ACGCCTCTTG AGAATGCGGG CCCGGTACCC CCGACGCGGC 6251 CGCTAATTCT AGATCGCGAA CAAACACCAT TGTCACACTC CAGTATACAC 6301 AAACACCATT GTCACACTCC AGATATCACA AACACCATTG TCACACTCCA 6351 ACACCATTGT CACACTCCA GGCTATTCTA GATCGCGAAT 6401 TACATACTTC TTTACATTCC ACATACTTCT TTACATTCCA 6451 GATATCATTA CATACTTCTT GGCGAATTAC ATACTTCTTT 6501 ACATTCCAAG GCTACCTGAG GCCCGGGGGT ACCTCTTAAT TAACTGGCCT 6551 CATGGGCCTT CCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC 6601 CAGTCAGGTG CAGGCTGCCT ATCAGAAGGT GGTGGCTGGT GTGGCCAATG 6651 CCCTGGCTCA CAAATACCAC TGAGATCTTT TTCCCTCTGC CAAAAATTAT 6701 GGGGACATCA TGAAGCCCCT TGAGCATCTG ACTTCTGGCT AATAAAGGAA 6751 ATTTATTTTC ATTGCAATAG TGTGTTGGAA TTTTTTGTGT CTCTCACTCG 6801 GAAGGACATA TGGGAGGGCA AATCATTTAA AACATCAGAA TGAGTATTTG 6851 GTTTAGAGTT TGGCAACATA TGCCCATATG CTGGCTGCCA TGAACAAAGG 6901 TTGGCTATAA AGAGGTCATC AGTATATGAA ACAGCCCCCT GCTGTCCATT 6951 CCTTATTCCA TAGAAAAGCC TTGACTTGAG GTTAGATTTT TTTTATATTT 7001 TGTTTTGTGT TATTTTTTTC TTTAACATCC CTAAAATTTT CCTTACATGT 7051 TTTACTAGCC AGATTTTTCC TCCTCTCCTG ACTACTCCCA GTCATAGCTG 7101 TCCCTCTTCT CTTATGGAGA TCCCTCGACC TGCAGCCCAA GCTGTAGATA 7151 AGTAGCATGG CGGGTTAATC ATTAACTACA AGGAACCCCT AGTGATGGAG 7201 TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC 7251 AAAGGTCGCC CGACGCCCGG GCTTTGCCCGGGCGGCCTCA 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): 6254 to 6514 bp Rabbit β-globin poly(A) signal (3'UTR and 3' flanking region of rabbit β-globin, including the poly(A) signal): 6674-6764 bp AAV2 3'ITR: 7181-7308bp pAAV-CAG-moFGF21 (SEQ ID NO: 46) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT 61 TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG 121 CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG 181 GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG 241 GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT 361 GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC 421 TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG 481 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC 541 TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG 661 TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT 721 CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC 781 ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA 841 TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT 961 TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC 1021 CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT 1081 GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT 1141 GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT 1261 ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT 1321 GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC 1381 TCCGGTTCCC AACGTCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT 1441 AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG 1561 ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT 1621 TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC 1681 ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT 1741 TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG 1861 AAATGTTGAA TACTCATACT CTTCCTTTTT TACTCATT GAGCATTTA TCAGGGTTAT 1921 TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG 1981 CGCACATTTC CCCGAAAGT GCCACCTGAC GTCTAAGAAA CCATTATT ASSISTANT 2041 ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTCTCG CGCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC 2161 GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT 2221 AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC ATATGCGGTG TGAAATACCG 2281 CACAGATTGCG TAAGGAAA ATACCGCATC AGGCGATTCC AACATCAAT AAATCATACA 2341 GGCAAGGCAA AGAATTAGCA AAATTAAGCA ATAAAGCCTC AGAATTAGCA GCTAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTAATA CTTTTGCGGG AGAAGCCTTT ATTTCAACGC 2461 AAGGATAAA ATTTTTAGAA CCCTCATATA TTTTAAATGC AATGCCTGAG TAATGTGTAG 2521 GTAAAGATTC AAACGGGTGA GAAAGGCCGG AGACAGTCAA ATCACCATCA ATATGATATT 2581 CAACCGTTCT AGCTGATAAA TTCATGCCGG AGAGGGTAGC TATTTTTGAG AGGTCTCTAC 2641 AAAGGCTATC AGGTCATTGC CTGAGAGTCT GGAGCAAACA AGAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC CCCGGTTGAT AATCAGAAAA GCCCCAAAAAA 2761 CAGGAAGATT GTATAAGCAA ATATTTAAAT TGTAAGCGTT AATATTTTGT TAAAATTCGC 2821 GTTAAATTTT TGTTAAATC GCTCATTTTT TAACCAATAG GCCGAAATCG GCAAAATCCC 2881 TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG 2941 TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC 3061 ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA AGCCGGCGAA 3121 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT 3181 AGCGGTCACG CTGCCGGTAA CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC 3241 GTACTATGGT TGCTTTGACG AGCACGTATA ACGTGCTTTC CTCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT TAGACAGGA CGGTACGCCA GAATCCTGAG 3361 AAGTGTTTTT ATAATCAGTG AGGCCACCGA GTAAAAGAGT CTGTCCATCA CGCAAATTAA 3421 CCGTTGTCGC AATACTTCTT TGATTAGTA TAACATCACT TGCCTGAGTA GAAGAACTCA 3481 AACTATCGGC CTTGCTGGTA ATTATCGAGA CAATATTACC GCCAGCCATT GCAACGGAAT 3541 CGCCATTCGC CATTCAGGCT GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC ACTGAGGCCG CCCGGGCAAA GCCCGGGCGT 3661 CGGGCGACCT TTGGTCGCCC GGCCTCAGTG AGCGAGCGAG CGCGCAGAGA GGGAGTGGCC 3721 AACTCCATCA CTAGGGGTTC CTTGTAGTTA ATGATTAACC CGCCATGCTA CTTATCTACT 3781 CGACATTGAT TATTGACTAG TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC 3841 CCATATATGG AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG 3961 ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT 4021 CAAGTGTATC ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC 4081 TGGCATTATG CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA 4141 TTAGTCATCG CTATTACCAT GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT TTATTTATTT TTTAATTATT TTGTGCAGCG 4261 ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG CCAGGCGGGG CGGGGCGGGG CGAGGGGCGG 4321 GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA GCCAATCAGA GCGGCGCGCT CCGAAAGTTT 4381 CCTTTTATGG CGAGGCGGCG GCGGCGGCGG CCCTATAAAA AGCGAAGCGC GCGGCGGGCG 4441 GGAGTCGCTG CGTTGCCTTC GCCCCGTGCC CCGCTCCGCG CCGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG GTGAGCGGGC GGGACGGCCC TTCTCCTCCG 4561 GGCTGTAATT AGCGCTTGGT TTAATGACGG CTTGTTTCTT TTCTGTGGCT GCGTGAAAGC 4621 CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC GGGGGGAGCG GCTCGGGGGG TGCGTGCGTG 4681 TGTGTGTGCG TGGGGAGCGC CGCGTGCGGC TCCGCGCTGC CCGGCGGCTG TGAGCGCTGC 4741 GGGCGCGGCG CGGGGCTTTG TGCGCTCCGC AGTGTGCGCG AGGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG GAACAAAGGC TGCGTGCGGG GTGTGTGCGT 4861 GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC GGTCGGGCTG CAACCCCCCC TGCACCCCCC 4921 TCCCCGAGTT GCTGAGCACG GCCCGGCTTC GGGTGCGGGG CTCCGTACGG GGCGTGGCGC 4981 GGGGCTCGCC GTGCCGGGCG GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG GGGCGGGGCC 5041 GCCTCGGGCC GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC CCCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT TTTATGGTAA TCGTGCGAGA GGGCGCAGGG 5161 ACTTCCTTTG TCCCAAATCT GTGCGGAGCC GAAATCTGGG AGGCGCCGCC GCACCCCCTC 5221 TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC CGGCAGGAAG GAAATGGGCG GGGAGGGCCT 5281 TCGTGCGTCG CCGCGCCGCC GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT GTCCGCGGGG 5341 GGACGGCTGC CTTCGGGGGG GACGGGGCAG GGCGGGGTTC GGCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA TGCCTTCTTC TTTTTCCTAC AGCTCCTGGG 5461 CAACGTGCTG GTTATTGTGC TGTCTCATCA TTTTGGCAAA GAATTGATTA ATTCGAGCGA 5521 ACGCGTCGAG TCGCTCGGTA CGATTTAAAT TGAATTGGCC TCGAGCGCAA GCTTGAGCTA 5581 GCGCCACCAT GGAATGGATG AGAAGCAGAG TGGGCACCCT GGGCCTGTGG GTGCGACTGC 5641 TGCTGGCTGT GTTTCTGCTG GGCGTGTACC AGGCCTACCC CATCCCTGAC TCTAGCCCCC 5701 TGCTGCAGTT TGGCGGACAA GTGCGGCAGA GATACCTGTA CACCGACGAC GACCAGGACA 5761 CCGAGGCCCA CCTGGAAATC CGCGAGGATG GCACAGTCGT GGGCGCTGCT CACAGAAGCC 5821 CTGAGAGCCT GCTGGAACTG AAGGCCCTGA AGCCCGGCGT GATCCAGATC CTGGGCGTGA 5881 AGGCCAGCAG ATTCCTGTGC CAGCAGCCTG ACGGCGCCCT GTACGGCTCT CCTCACTTCG 5941 ATCCTGAGGC CTGCAGCTTC AGAGAGCTGC TGCTGGAGGA CGGCTACAAC GTGTACCAGT 6001 CTGAGGCCCA CGGCCTGCCC CTGAGACTGC CTCAGAAGGA CAGCCCTAAC CAGGACGCCA 6061 CAAGCTGGGG ACCTGTGCGG TTCCTGCCTA TGCCTGGACT GCTGCACGAG CCCCAGGATC 6121 AGGCTGGCTT TCTGCCTCCT GAGCCTCCAG ACGTGGGCAG CAGCGACCCT CTGAGCATGG 6181 TGGAACCTCT GCAGGGCAGA AGCCCCAGCT ACGCCTCTTG AGAATGCGGG CCCGGTACCC 6241 CCGACGCGGC CTAACTGGCC TCATGGGCCT TCCGCTCACT GCCCGCTTTC CAGTCGGGAA 6301 ACCTGTCGTG CCAGTCAGGT GCAGGCTGCC TATCAGAAGG TGGTGGCTGG TGTGGCCAAT 6361 GCCCTGGCTC ACAAATACCA CTGAGATCTT TTTCCCTCTG CCAAAAATTA TGGGGACATC 6421 ATGAAGCCCC TTGAGCATCT GACTTCTGGC TAATAAAGGA AATTATTTT CATTGCAATA 6481 GTGTGTTGGA ATTTTTTGTG TCTCTCACTC GGAAGGACAT ATGGGAGGGC AAATCATTTA 6541 AAACATCAGA ATGAGTATTT GGTTTAGAGT TTGGCAACAT ATGCCCATAT GCTGGCTGCC 6601 ATGAACAAAG GTTGGCTATA AAGAGGTCAT CAGTATATGA AACAGCCCCCC TGCTGTCCAT 6661 TCCTTATTCC ATAGAAAAGC CTTGACTTGA GGTTAGATTT TTTTTATATT TTGTTTTGTG 6721 TTATTTTTTT CTTTAACATC CCTAAAATTT TCCTTACATG TTTTACTAGC CAGATTTTTC 6781 CTCCTCTCCT GACTACTCCC AGTCATAGCT GTCCCTCTTC TCTTATGGAG ATCCCTCGAC 6841 CTGCAGCCCA AGCTGTAGAT AAGTAGCATG GCGGGTTAAT CATTAACTAC AAGGAACCCC 6901 TAGTGATGGA GTTGGCCACT CCCTCTCTGC GCGCTCGCTC GCTCACTGAG GCCGGGCGAC 6961 CAAAGGTCGC CCGACGCCCG GGCTTTGCCC GGGCGGCCTC 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 (3'UTR and 3' flanking region of rabbit β-globin, including the poly(A) signal): 6315-6833 bp AAV2 3'ITR: 6892-7024bp Mini-CMV: cmv intermediate-early promoter (SEQ ID NO: 36) TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTAACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTT Nucleotide sequence of EF1α promoter (SEQ ID NO: 37) Nucleotide sequence of RSV promoter (SEQ ID NO: 38) CATGTTTGACAGCTTATCATCGCAGATCCGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGC TTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATAATTCGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATA TAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTG CCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTAAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTGGGTACCAGCT Synapsin 1 promoter (SEQ ID NO: 39) ctgcgctctcaggcacgacacgactcctccgctgcccaccgcagactgaggcagcgctgagtcgccggcgccgcagcgcagatggtcgcgcccgtgcccccctatctcgc ctcgcgtggtgcggtccggctgggccggcggcggcgcggacgcgaccaaggtggccgggaaggggagtttgcgggggaccggcgagtgacgtcagcgcgccttcagtgctga ggcggcggtggcgcgcgccgccaggcgggggcgaaggcactgtccgcggtgctgaagctggcagtgcgcacgcgcctcgccgcatcctgtttcccctccccctctctgatag gggatgcgcaatttggggaatgggggttgggtgcttgtccagtgggtcggggtcggtcgtcaggtaggcacccccaccccgcctcatcctggtcctaaaacccacttgcact Calcium / calmodulin-dependent protein kinase II (CaMKII) promoter (SEQ ID NO: 40) Glial fibrillary acidic protein (GFAP) promoter (SEQ ID NO: 41) cgcgtgatctaacatatcctggtgtggagtaggggacgctgctctgacagaggctcgggggcctgagctggctctgtgagctggggaggaggcagacagccaggccttgtctgcaagcagacctggcagcattgggctggccgccccccagggcctcctcttcatgcccagtgaatgactcaccttggcacagacacaatgttcggggtgggcacagtgcctgcttcccgccgcaccccagcccccctcaaatgccttccgagaagcccattgagcagggggcttgcattgcaccccagcctgacagcctggcatcttgggataaaagcagcacagccccctaggggctgcccttgctgtgtggcgccaccggcggtggagaacaaggctctattcagcctgtgcccaggaaaggggatcaggggatgcccaggcatggacagtgggtggcagggggggagaggagggctgtctgcttcccagaagtccaaggacacaaatgggtgaggggagagctctccccatagctgggctgcggcccaaccccaccccctcaggctatgccagggggtgttgccaggggcacccgggcatcgccagtctagcccactccttcataaagccctcgcatcccaggagcgagcagagccagagcaggttggagaggagacgcatcacctccgctgctcgcggggtctagagtcga Nestin promoter (SEQ ID NO: 42) Homeobox protein 9 promoter (HB9) promoter (SEQ ID NO: 43) tgaataaatttaagcaggctaattaatatataaactagctcaatttgtcaagttgatttgtattttagttaattgtgaaagtaattaccacatggtcaaattaacagct ttctggaaatgaccaagcctgaggttttatttccttcctgggtgaagaaaattcatttttccaagctcttgatgtgatgaataaaagtcataaatctgggtgattggtgc aggcagagtctaaatggcttcatatttcattttaggtttaatagaaatattcatgctctgttttaatgaaattaaattgaagggggatggggctagagtggttagctga tgaattgacaaaaactaatcagctttattgggaaacaggtttaagggcacggacgtgtcaataacgctcagcctgaccccctcttccattagctaggcaggctgattaga Tyrosine hydroxylase (TH) promoter (SEQ ID NO: 44) Myelin basic protein (MBP) promoter (SEQ ID NO: 45)

Claims

1. A composition comprising a recombinant adeno-associated virus (rAAV) vector for use in treating and / or preventing a metabolic disorder selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), diabetes, and / or obesity, wherein the rAAV vector comprises (i) a genetic construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) operably linked to a ubiquitous promoter, and (ii) an AAV capsid protein selected from serotype 1 (AAV1), serotype 2 (AAV2), or serotype 9 (AAV9), wherein the composition is administered by intracerebrospinal fluid (CSF) administration, and wherein FGF21 is expressed in the central nervous system (CNS).

2. The composition for use according to claim 1, wherein the ubiquitous promoter is selected from the group consisting of a CAG promoter and a CMV promoter.

3. 3. The composition for use according to claim 1 or claim 2, wherein the FGF21 is expressed in the brain.

4. The composition for use according to any one of claims 1 to 3, wherein said FGF21 is expressed in the hypothalamus, cortex, hippocampus, cerebellum, and / or olfactory bulb.

5. The composition for use according to any one of claims 1 to 4, wherein said FGF21 is expressed in the hypothalamus.

6. The composition for use according to any one of claims 1 to 5, wherein the gene construct comprises at least one target sequence of a microRNA expressed in a tissue in which it is desired to prevent the expression of FGF21.

7. The composition for use according to claim 6, wherein said at least one target sequence of a microRNA is selected from target sequences that bind to microRNAs expressed in the heart and / or liver of a mammal.

8. 8. The composition for use according to any one of claims 1 to 7, wherein the nucleotide sequence encoding FGF21 is operably linked to at least one target sequence of a microRNA expressed in the liver and to at least one target sequence of a microRNA expressed in the heart.

9. 9. The composition for use according to claim 7 or 8, wherein the target sequence of a microRNA expressed in the heart is selected from SEQ ID NOs: 13 and 21-25, and the target sequence of a microRNA expressed in the liver is selected from SEQ ID NOs: 12 and 14-20.

10. The composition for use according to any one of claims 7 to 9, comprising a target sequence of microRNA-122a and a target sequence of microRNA-1.

11. The composition for use according to any one of claims 1 to 10, wherein the nucleotide sequence encoding FGF21 comprises: (a) any of the nucleotide sequences of SEQ ID NOs: 5, 6, or 7; or (b) a nucleotide sequence that differs from the sequence of the nucleotide sequence of (a) due to the degeneracy of the genetic code.

12. A composition for use according to any one of claims 1 to 11, wherein the AAV capsid protein is of the AAV1 serotype.

13. A composition for use according to any one of claims 1 to 11, wherein the AAV capsid protein is of the AAV2 serotype.

14. A composition for use according to any one of claims 1 to 11, wherein the AAV capsid protein is of the AAV9 serotype.

15. The composition for use according to any one of claims 1 to 14, wherein said metabolic disorder is diabetes and / or obesity.

16. A pharmaceutical composition for use in the treatment and / or prevention of a metabolic disorder selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), diabetes, and / or obesity, comprising: i) an AAV vector comprising: a) a genetic construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) operably linked to a ubiquitous promoter; and b) an AAV capsid protein selected from serotype 1 (AAV1) capsid protein, serotype 2 (AAV2) capsid protein, or serotype 9 (AAV9) capsid protein; and ii) one or more pharmaceutically acceptable ingredients, wherein the AAV vector is administered by intraCSF administration and FGF21 is expressed in the central nervous system (CNS).

17. 17. The pharmaceutical composition for use according to claim 16, wherein the ubiquitous promoter is selected from the group consisting of a CAG promoter and a CMV promoter.

18. A pharmaceutical composition for use according to claim 16 or claim 17, wherein FGF21 is expressed in the brain.

19. A pharmaceutical composition for use according to any one of claims 16 to 18, wherein FGF21 is expressed in the hypothalamus, cortex, hippocampus, cerebellum and / or olfactory bulb.

20. A pharmaceutical composition for use according to any one of claims 16 to 19, wherein FGF21 is expressed in the hypothalamus.

21. The pharmaceutical composition for use according to any one of claims 16 to 20, wherein the AAV capsid protein is of the AAV1 serotype.

22. A pharmaceutical composition described in any one of claims 16 to 20, wherein the AAV capsid protein is of the AAV2 serotype.

23. A pharmaceutical composition described in any one of claims 16 to 20, wherein the AAV capsid protein is of the AAV9 serotype.

24. The pharmaceutical composition for use according to any one of claims 16 to 23, wherein said metabolic disorder is diabetes and / or obesity.

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