Viral expression construct comprising fibroblast growth factor 21 (FGF21) coding sequence

Abstract

To provide novel and safe antifibrotic therapeutics.SOLUTION: The invention relates to viral expression constructs and related viral vectors and nucleic acid molecules and compositions and to their use wherein the constructs and vectors are suitable for expression in mammals and comprise a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) to be expressed in livers, adipose tissue and / or skeletal muscle.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of steroids in the treatment of metabolic disorders in mammals, particularly in humans. for use in the treatment of liver inflammation and / or fibrosis, for the treatment of cancer and / or for use in extending healthy lifespan. The present invention relates to the medical field, including therapeutic compositions. [Background technology]

[0002] The prevalence of diabetes is increasing at an alarming rate and is a major global health problem. is strongly associated with insulin resistance and type 2 diabetes (T2D) (Moller, DE, and Flier, J. S., 1991. N. Engl. J. Med. 325: 938 -948). Moreover, obesity increases the risk of death (Peeters, A.et al.,2003. Ann.Intern.Med. 138 :24-32 ), as well as heart disease, immune dysfunction, high blood pressure, arthritis, neurodegenerative diseases and certain types of It is also a significant risk factor for cancer (Roberst, DL et al., 2010 Annu.Rev.Med. 61 :301-316;Spiegelman,B. M. et al., 1993. J.Biol.Chem. 268 :6823-68 26;Whitmer,RA,2007. Curr.Alzheimer Res . 4 :117-122). Despite the clinical importance of T2D and obesity, Therefore, novel strategies to prevent and combat the T2D-obesity epidemic are needed. There is an urgent need for effective and safe approaches. In recent years, obesity has become an important risk factor for cancer. It is now widely accepted that al., 2010. Annu.Rev.Med. 61 :301-316). Given the prevalence of obesity, novel and safe approaches to managing obesity-related cancer risk are urgently needed. Increased body weight and insulin resistance are important clinical concerns that need to be addressed. Therefore, novel and promising approaches to prevent and reverse obesity and diabetes are being developed. Safe approaches are needed to extend healthy lifespan. Liver fibrosis is caused by extracellular matrix Excessive accumulation of proteins, such as collagen, primarily due to chronic liver inflammation Advanced liver fibrosis leads to cirrhosis, portal hypertension, and liver failure. Novel and safe antifibrotic therapeutic agents are needed.

[0003] Fibroblast growth factor 21 (FGF21) is secreted primarily by the liver but also by adipose tissue and It is a growth factor secreted by the pancreas and the liver (Muise, ES et al., 2008. Mol.Pharmacol. 74 :403-412), brown adipose tissue ( Increased growth of BAT and increased energy production in BAT and white adipose tissue (WAT). It has been shown to increase the expression of thermogenic genes that stimulate energy expenditure (Cosku n,T. et al.,2008. Endocrinology 149 :6018 -6027;Fisher,FM et al.,2012. Genes Dev . 26 :271-281;Kharitonenkov, A. et al.,200 5. J.Clin.Invest 115 :1627-1635;Konishi,M et al., 2000. J.Biol.Chem. 275 :12119-12 122;Tomlinson, E. et al., 2002. Endocrinol ogy 143 :1741-1747;Xu, J. et al., 2009. Dia betes 58 :250-259).

[0004] Overexpression of FGF21 in transgenic mice renders them prone to diet-induced obesity (Kharitonenkov, A. et al., 2005. J. Cli n.Invest 115 :1627-1635), ob / ob mice, db / db mice FGF21 in mice fed a low-fat diet or a high-fat diet (HFD) or in obese ZDF rats Administration of induced a strong reduction in adiposity and significantly lowered blood glucose and triglycerides. This reduces fasting insulin levels and improves insulin sensitivity. (Coskun, T. et al., 2008. Endocrinology 149 :6018-6027;Kharitonenkov, A. et al.,2 005. J.Clin.Invest 115 :1627-1635;Xu,J. e t al.,2009. Diabetes 58 :250-259;Adams,A. C. et al., 2012. PLoS.One. 7 :e38438.;Berg lund, ED et al., 2009. Endocrinology 150 Furthermore, administration of FGF21 to obese diabetic rhesus monkeys Fasting plasma glucose, fructosamine, triglycerides, insulin and glucagon This dramatically reduced the levels of α-glucan and induced a small but significant reduction in body weight (Kharit onenkov, A. et al., 2007. Endocrinology 14 8 :774-781).

[0005] The native FGF21 protein exhibits poor pharmacokinetic properties, due to its short half-life. They are short, prone to proteolysis in vivo, and prone to aggregation in vitro (Huang, J. et al.,2013. J Pharmacol Exp Ther. 34 6(2) :270-80; So,WY . and Leung, P.S. . 2016. Med Res Rev. 36(4) :672-704;Zhang,J. and Li, Y. 2015. Front Endocrinol (Lausanne) . 6 :168) To extend the half-life of FGF21 and improve its stability and solubility, Various engineering approaches have been developed. Currently, two artificial FGF21 mimetics (LY 2405319 and PF-05231023) have been tested in humans. However, these FGF21 mimetics require multiple administrations, which imposes a significant burden on patients. Moreover, artificial FGF21 mimetics / analogs are less immunogenic than native FGF21. For example, patients treated with LY2405319 may be at higher risk of developing , injection site reactions, anti-drug antibodies, and severe hypersensitivity reactions (Gaich, G. et al., 2013. Cell Metab. 18(3) :333-40).

[0006] Therefore, it is possible to develop a method for treating diabetes and / or obesity that does not have all the drawbacks of existing treatments. and / or liver inflammation and / or fibrosis, and / or cancer, and / or There remains a need for new treatments to improve or extend healthy lifespan. [Prior art documents] [Non-patent literature]

[0007] [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] Roberst,DL et al.,2010. Annu.Rev.Med. 61:301-316 [Non-patent document 4] Spiegelman, BM et al., 1993. J.Biol.Chem. 268:6823-6826 [Non-Patent Document 5] Whitmer, RA, 2007. Curr. Alzheimer Res. 4:117-122 [Non-patent document 6] Muise, ES et al., 2008. Mol. Pharmacol. 74:403-412 [Non-Patent Document 7] Coskun, T. et al., 2008. Endocrinology 149:6018-6027 [Non-patent document 8] Fisher, FM et al., 2012. Genes Dev. 26:271-281

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[0008] The present inventors have developed a method for treating liver diseases in order to combat metabolic disorders, preferably diabetes and / or obesity. , adeno-associated virus (AAV) vector-mediated delivery of F to adipose tissue and / or skeletal muscle An improved gene therapy strategy based on GF21 gene transfer was designed. Treatment may also be used to combat liver inflammation and / or fibrosis. The gene therapy of the present invention also provides a method for treating age-related metabolic disorders, preferably diabetes and / or It can also be used to combat obesity and thereby extend healthy lifespan. Such gene therapy may also be used to combat cancer, preferably liver cancer.

[0009] Creation of a single vector gene construct enables in vivo production of native FGF21 This poses a reduced risk of immunogenicity or other toxicities.

[0010] However, those skilled in the art will appreciate that native FGF21 is prone to proteolysis in vivo and Those skilled in the art will recognize that the drug may have a rapid rate of release and / or a rapid rate of clearance in vivo. All vectors tested in the experimental section were able to deliver stable native FGF21 into the bloodstream. It was found that the single gene transfer vector can enable long-term sustained secretion of the Even with single doses, efficacy is maintained.

[0011] Therefore, the generation of such an AAV vector for the in vivo production of native FGF21 is feasible. As demonstrated in the experimental section, this is not conventional for those skilled in the art. DETAILED DESCRIPTION OF THE INVENTION

[0012] Viral expression constructs In a first embodiment, the expression vector is suitable for expression in mammals, and is expressed in liver, adipose tissue and / or The nucleic acid encoding fibroblast growth factor 21 (FGF21) is expressed in the skeletal muscle. Viral expression constructs containing the nucleotide sequences are provided.

[0013] "Viral expression construct," "suitable for mammalian expression," "liver," The definitions of "adipose tissue" and "skeletal muscle" are given in the section entitled "General Definitions." It is provided as follows.

[0014] A preferred nucleic acid encoding FGF21 present in the viral expression construct of the present invention is The nucleotide sequence has at least 60 identical sequences to SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11. %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99 %, 100% identity. Identity is determined in the section entitled "General Definitions." As described in the preceding paragraph, the sequence number may be assessed across the entire sequence number or across a portion thereof. It can be done.

[0015] The human FGF21-encoding gene present in the viral expression construct of the present invention Preferred nucleotide sequences have at least 60%, 65% similarity to SEQ ID NO: 4, 5, 6 or 7. ,70%,75%,80%,85%,90%,95%,97%,98%,99%,100 % identity. Identity is explained in the section entitled "General Definitions." The sequence number may be assessed across the entire sequence or across portions thereof, as desired. SEQ ID NO: 4 is the nucleotide sequence encoding human FGF21. Nucleotide sequence encoding don-optimized human FGF21, variant 1. SEQ ID NO: 6 is the nucleotide sequence encoding codon-optimized human FGF21, SEQ ID NO: 7 is a nucleotide sequence encoding codon-optimized human FGF21. Variant 1, Variant 2 and Variant 3 are They encode the same human FGF21 protein and are optimized by different codon optimization algorithms. The mouse FGF21 present in the viral expression construct of the present invention was expressed as Another preferred nucleotide sequence for coding is at least 60%, 60%, or 60% identical to SEQ ID NO: 8 or 9. 5%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 1 00% identity. Identity is explained in the section entitled "General Definitions." As will be apparent, the sequence may be assessed across the entire sequence or across portions thereof. SEQ ID NO: 8 is the nucleotide sequence encoding mouse FGF21. SEQ ID NO: 9 is a nucleotide sequence encoding codon-optimized mouse FGF21. Another preferred viral expression construct encoding canine FGF21 is The nucleotide sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 ... 5%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity Identity, as explained in the section entitled "General Definitions," The sequence may be assessed across the entire sequence or across portions thereof. is the nucleotide sequence encoding canine FGF21. SEQ ID NO: 11 is the codon-optimized nucleotide sequence encoding modified canine FGF21. The nucleotide sequence may be any FGF21 gene or FGF21 coding sequence, preferably Preferably, it may be of human, murine or canine origin; or preferably human Mutant FGF21 genes or FGF21 coding regions derived from rats, mice, or dogs Codon-optimized FGF21 gene or FGF21 coding sequence It may also be an array.

[0016] As used herein, FGF21 refers to a compound that has less activity than FGF21 known to those skilled in the art. FGF21 activity improves insulin sensitivity and exerts at least detectable levels. This activity is demonstrated by the methods described in the experimental section, preferably in Example 8 or 9. This can be assessed using an insulin tolerance test.

[0017] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is The octide sequence is: (a) an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, or 3; Nucleotide sequence encoding a polypeptide containing the amino acid sequence (b) a nucleic acid sequence having at least the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11; Nucleotide sequences with at least 60% sequence identity (c) A nucleotide sequence that differs in sequence from the sequence in (b) due to the degeneracy of the genetic code. Code layout The above viral expression construct is provided, wherein the viral expression construct is selected from the group consisting of:

[0018] A preferred nucleotide sequence encoding FGF21 suitable for expression in a mammal is: At least 60%, 65%, 70%, 75%, 80%, 85% identical to SEQ ID NO: 1, 2 or 3 , 90%, 95%, 97%, 98%, 99%, and 100% amino acid sequence identity. The identity is defined in the section entitled "General Definitions." As described in the preceding paragraph, the sequence number may be assessed across the entire sequence number or across a portion thereof. SEQ ID NO: 1 is the amino acid sequence of human FGF21. SEQ ID NO: 2 is the amino acid sequence of mouse FGF21. SEQ ID NO: 3 is the amino acid sequence of canine FGF21. is.

[0019] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is The octide sequence and elements a), b), c), d) and e): (a) Liver-specific promoter (b) Adipose tissue-specific promoter (c) Co-localization of ubiquitous promoters and target sequences of microRNAs expressed in the liver. and at least one nucleotide sequence encoding a microRNA expressed in the heart. In combination with at least one nucleotide sequence encoding a target sequence, the combination, which allows specific expression in tissues. (d) skeletal muscle promoter, and (e) Combination of a ubiquitous promoter with an adeno-associated virus (AAV) vector sequence the combination of a nucleotide sequence and a nucleotide sequence that allows specific expression in skeletal muscle. The above viral expression construct is provided, comprising at least one of:

[0020] "Target sequence of microRNA expressed in the liver" or "mRNA expressed in the liver" The term "target sequence of iRNA" or "binding site of microRNA expressed in the liver" refers to Complementary to or partially complementary to at least a portion of a microRNA expressed in the liver Similarly, "a nucleotide sequence of a microRNA expressed in the heart" refers to a nucleotide sequence that is complementary to the "Target sequence" or "Target sequence of miRNA expressed in the heart" or "In the heart The "binding site of the microRNA expressed in the heart" refers to the binding site of the microRNA expressed in the heart. This specification refers to a nucleotide sequence that is at least partially complementary or partially complementary to the nucleotide sequence. A portion of the microRNAs expressed in the liver or heart as defined in the literature The portion of the microRNA that is present in the present invention is at least 5 or at least 6 of the microRNA. The binding site sequence refers to a nucleotide sequence of 10 contiguous nucleotides. It can have perfect complementarity with at least a portion of a microRNA, which means that the sequence is This means that there can be a match and no mismatch. wherein the binding site sequence is partially complementary to at least a portion of the expressed microRNA This means that one mismatch / 5 or 6 consecutive nucleotides can occur. The partially complementary binding site is preferably a seed region of the microRNA. and contains perfect or near perfect complementarity with the seed region of the microRNA and No mismatches between the binding sites (perfect complementarity) or one mismatch per 5 or 6 This means that a sequence of 100 consecutive nucleotides (near perfect complementarity) can occur. The seed region of A is from about nucleotide 2 to about nucleotide 8 of the microRNA. It consists of the 5' region (i.e., 6 nucleotides) of a certain microRNA. The defined portion is preferably the seed region of the microRNA. or a target sequence of a microRNA expressed in the heart. The degradation of messenger RNA (mRNA) is either via the RNA interference pathway or via the m This may be through direct translational control (inhibition) of RNA. is the pathway ultimately utilized by miRNAs in inhibiting the expression of their encoded proteins. It is not limited by road.

[0021] In the context of the present invention, a gene encoding a target sequence of a microRNA expressed in the liver is The nucleotide sequence has at least 60% sequence identity with SEQ ID NO: 12 or 14-23. Alternatively, it may be replaced by a nucleotide sequence containing a similar nucleotide sequence. More preferred nucleotide sequences are those that are at least 60% identical to SEQ ID NO: 12 or 14-23; 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity is defined in the section entitled "General Definitions." As described, the sequence may be assessed across the entire sequence or across portions thereof. In one embodiment, a target sequence of a microRNA expressed in the liver is obtained. The nucleotide sequence to be used has at least 60% sequence identity or similarity with SEQ ID NO: 12. It may be replaced by a nucleotide sequence comprising a nucleotide sequence having the following structure: The nucleotide sequence has at least 60%, 65%, 70%, 75%, 80%, or 90% similarity to SEQ ID NO:12. %, 85%, 90%, 95%, 97%, 98%, 99%, and 100% identity. In one embodiment, the target sequence of a microRNA expressed in the liver is At least one copy of the nucleotide sequence set forth in SEQ ID NO: 12 or 14-23 In a further embodiment, SEQ ID NO: 12 or 14 to 23, which encodes the target sequence of liver-specific microRNA 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence defined in the present invention In a preferred embodiment, miRT122 is present in a viral expression construct. 1, 2, 3, 4, 5, 6, 7 or 8 of the nucleotide sequence encoding a (SEQ ID NO: 12) Eight copies are present in the viral expression construct of the present invention.

[0022] As used herein, the target sequence of a microRNA expressed in the liver is known to those skilled in the art. At least detectable activity of target sequences of microRNAs known to be expressed in the liver The activity of the target sequences of microRNAs expressed in the liver is Binding to cognate liver-expressed microRNAs and transgenes by mediating detargeting of transgene expression in the liver when operably linked to the This activity was confirmed by qPCR in the liver as described in the experimental section. This can be assessed by measuring the expression level of the gene.

[0023] In the context of the present invention, a gene encoding a target sequence of a microRNA expressed in the heart is The nucleotide sequence has at least 60% sequence identity with SEQ ID NO: 13 or 23-30. Alternatively, it may be replaced by a nucleotide sequence containing a similar nucleotide sequence. Preferred nucleotide sequences have at least 60%, 65%, or 70% identity with SEQ ID NO: 13 or 23-30. %, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 10 0% identity. Identity is explained in the section entitled "General Definitions." The sequence number may be assessed across the entire sequence or across a portion thereof, as in In one embodiment, a gene encoding a target sequence of a microRNA expressed in the heart is The nucleotide sequence has at least 60% sequence identity or similarity with SEQ ID NO: 13. The nucleotide sequence may be replaced by a nucleotide sequence containing one or more nucleotide sequences. The peptide sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% similarity to SEQ ID NO: 13. %, 90%, 95%, 97%, 98%, 99%, 100% identity. Further implementation In the form of SEQ ID NO: 1, encoding a target sequence of a microRNA expressed in the heart. At least one copy of the nucleotide sequence defined in paragraphs 13 or 23-30 of this In a further embodiment, a cardiac-specific 13 or 23-30, encoding the target sequence of the target microRNA 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence to be used in the present invention In a preferred embodiment, miRT1 (SEQ ID NO: 1) is present in the expression construct. 3) 1, 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence encoding this present in the viral expression constructs of the invention.

[0024] The activity of target sequences of cardiac-expressed microRNAs is correlated with their cognate cardiac counterparts. The objective of the present invention is to bind to microRNAs expressed by the transgene and operably linked to the transgene. The activity of the IL-1 receptor is to mediate detargeting of transgene expression in the heart when activated. The expression level of the transgene in the heart was measured by qPCR as described in the experimental section. It can be assessed by measuring.

[0025] In one embodiment, a target sequence for a microRNA expressed in the liver is encoded by Nucleotide sequences and target sequences of microRNAs expressed in the heart The nucleotide sequence is selected from the sequences of SEQ ID NOs: 12 to 30 and / or combinations thereof. The above viral expression construct is provided, wherein the viral expression construct is selected from the group consisting of:

[0026] In one embodiment, a target sequence of a microRNA expressed in the liver is At least one copy of the nucleotide sequence defined in SEQ ID NO: 12 or 14 to 23 SEQ ID NO: 13, which encodes the target sequence of a microRNA expressed in the heart or 23-30, wherein at least one copy of the nucleotide sequence is In a further embodiment, the viral expression construct is SEQ ID NO: 12 or 14-23 encoding the target sequence of the expressed microRNA 2, 3, 4, 5, 6, 7 or 8 copies of the defined nucleotide sequence, and SEQ ID NO: 13 or 23 to 30, which encode the target sequence of microRNA expressed in 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence defined in the present invention In a further embodiment, miRT122 is present in a viral expression construct. 1, 2, 3, 4, 5, 6, 7 or 8 of the nucleotide sequence encoding a (SEQ ID NO: 12) 8 copies of the nucleotide sequence encoding miRT1 (SEQ ID NO: 13), and 3, 4, 5, 6, 7 or 8 copies may be combined in the viral expression construct of the present invention. In a further embodiment, a gene encoding miRT122a (SEQ ID NO: 12) is Four copies of the nucleotide sequence, and a nucleotide sequence encoding miRT1 (SEQ ID NO: 13) Four copies of the nucleotide sequence are combined in the viral expression construct of the present invention.

[0027] Definitions of "promoter", "liver-specific promoter", "adipose tissue-specific promoter" ", "ubiquitous promoter," and "skeletal muscle promoter" are all terms under the heading "General Definition." The title is provided in the description section.

[0028] A preferred ubiquitous promoter is the CAG promoter.

[0029] In the context of the present invention, the nucleotide sequence of the CAG promoter is SEQ ID NO: 44. Nucleotides containing nucleotide sequences with at least 60% sequence identity or similarity A preferred nucleotide sequence is SEQ ID NO: 44 and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and 100% identity. The identity is in the section titled "General Definitions." As described in can be assessed.

[0030] Another preferred ubiquitous promoter is the cytomegalovirus (CMV) promoter. - is.

[0031] In the context of the present invention, the nucleotide sequence of the CMV promoter is SEQ ID NO: 45. Nucleotides containing nucleotide sequences with at least 60% sequence identity or similarity A preferred nucleotide sequence is SEQ ID NO: 45 and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% and 100% identity. The identity is in the section titled "General Definitions." As described in can be assessed.

[0032] Preferably, the CMV promoter is used in conjunction with an intron sequence. In the series, the intron sequence has at least 60% sequence identity or similarity with SEQ ID NO:43. It may be replaced by a nucleotide sequence containing a similar nucleotide sequence. The nucleotide sequence has at least 60%, 65%, 70%, 75%, 80%, or 90% similarity to SEQ ID NO:43. %, 85%, 90%, 95%, 97%, 98%, 99%, 100% identical. The nature of the sequence number is determined by the sequence number as explained in the description section entitled "General Definitions." The issue may be assessed in its entirety or in part.

[0033] A preferred liver-specific promoter is the human alpha-1-antitrypsin (hAAT) promoter. It is a meter.

[0034] In the context of the present invention, the nucleotide sequence of the hAAT promoter is SEQ ID NO: 47 A nucleotide sequence having at least 60% sequence identity or similarity with A preferred nucleotide sequence is at least as follows: SEQ ID NO: 47 Also 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% , 99%, and 100% identity. Identity is determined in the section entitled "General Definitions." As explained in the next section, over the entire sequence number or over a portion thereof. can be assessed.

[0035] Preferably, the hAAT promoter is used in conjunction with an intron sequence. The novel intron sequence is a hepatocyte control region (HCR) enhancer from apolipoprotein E. The most preferred intron sequence is the apolipoprotein A1 sequence defined in SEQ ID NO: 53. The HCR enhancer from protein E. In this context, the intron sequence is Contains a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO:53. The preferred nucleotide sequence is SEQ ID NO: 5. 3 and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 9 The identity is determined by the type "General Definition". As explained in the description of the tel, throughout the sequence number or In one embodiment, the hAAT promoter is In a preferred embodiment, the nucleotide sequence is used with 1, 2, 3, 4 or 5 copies of the nucleotide sequence. wherein the hAAT promoter is an apolipoprotein A promoter as defined in SEQ ID NO: 53. It is used with 1, 2, 3, 4 or 5 copies of the HCR enhancer from protein E.

[0036] Other liver-specific promoters include the albumin promoter, major urinary protein promoter, and tar, phosphoenolpyruvate carboxykinase (PEPCK) promoter, liver Liver-enriched protein activator promoter tein activator promoter), transthyretin promoter, Thyroxine-binding globulin promoter, apolipoprotein A1 promoter, liver fat Fatty acid binding protein promoter and phenylalanine hydroxylase promoter is.

[0037] The adipose tissue-specific promoter is adipose protein 2 (aP2, fatty acid binding protein FABP4 promoter, PPARγ promoter, and Ponectin promoter, phosphoenolpyruvate carboxykinase (PEPCK) The promoter is derived from human aromatase cytochrome p450 (p450arom). The mini / aP2 promoter (fat-specific aP2 enhancer and promoter region) aP2 promoter), uncoupling protein 1 (UCP1) promoter -, mini / UCP1 promoter (fat-specific UCP1 enhancer and basal UCP1 promoter), adipsin promoter, leptin promoter Preferred adipose tissue-specific promoters are the α-actin promoter, ... mini / aP2 promoter (SEQ ID NO: 54) and mini / UCP1 promoter (SEQ ID NO: 55). In this context, the adipose tissue-specific promoter sequence is the sequence Nucleotides having at least 60% sequence identity or similarity to SEQ ID NO: 53 or SEQ ID NO: 54 The nucleotide sequence may be replaced by a nucleotide sequence containing the nucleotide sequence. is at least 60%, 65%, 70%, 75%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 0%, 85%, 90%, 95%, 97%, 98%, 99%, and 100% identity. The identity is determined by the sequence, as explained in the section entitled "General Definitions." It may be assessed over the entire number or over parts of it.

[0038] Preferred skeletal muscle promoters are myosin light chain promoter, myosin heavy chain promoter -, desmin promoter, muscle creatine kinase (MCK) promoter, smooth muscle alfa Actin promoter, CK6 promoter, Unc-45 myosin chaperone B promoter, the basal MCK promoter combined with a copy of the MCK enhancer, Enh358MCK promoter (MCK enhancer and 358 bp proximal to the MCK gene) The most preferred skeletal muscle promoter is SEQ ID NO: 5 In this regard, the skeletal muscle promoter is the C5-12 promoter defined in Ref. 6. The target sequence is a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO:56. The nucleotide sequence may be replaced by a nucleotide sequence containing the nucleotide sequence. is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, %, 95%, 97%, 98%, 99%, and 100% identity. As explained in the description entitled "Definition," It may be assessed in whole or in part.

[0039] As used herein, a promoter is a promoter sequence (particularly one whose sequence is represented by a given SEQ ID NO: (defined as having a minimal percentage identity with a sequence of the same or similar sequence) known to those skilled in the art. The promoter activity is at least as high as that of the promoter of the present invention. See the section entitled "General Definitions." Preferably, a given SEQ ID NO: Promoters defined as having minimal percentage identity are known to those skilled in the art. nucleotides operably linked thereto, as assessed in the assays being performed. that regulates the transcription of the sequence (i.e., the nucleotide sequence encoding FGF21) In the context of the present invention, the promoter is an FGF21 nucleotide sequence as defined above. In one embodiment, the promoter is operably linked to a cell-specific and / or tissue-specific, preferably liver, adipose tissue and / or skeletal muscle specific It is unusual.

[0040] Therefore, several viral expression constructs are encompassed by the present invention: comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal; and a viral expression construct comprising element a), comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal; and a viral expression construct comprising element b), comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal; and a viral expression construct comprising element c), comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal; and a viral expression construct comprising element d); comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal; and a viral expression construct comprising element e); a nucleotide sequence encoding FGF21 suitable for expression in a mammal; and a viral expression construct comprising the nucleotide sequences of element b) and element c) in a nucleotide sequence encoding FGF21 suitable for expression in a mammal; and a viral expression construct comprising the nucleotide sequence of element e) and element c).

[0041] In one embodiment, the liver-specific promoter is human alpha 1-antitrypsin (hA AT) promoter, and / or the adipose tissue specific promoter is mini / the ap2 promoter and / or the mini / UCP1 promoter, and and / or the skeletal muscle promoter is a C5-12 promoter, and / or the ubiquitin The TASS promoter is a cytomegalovirus (CMV) promoter and / or a CA The viral expression constructs described herein are provided in which the promoter is a G promoter. do.

[0042] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is The liver-specific promoter comprises the hAAT promoter (sequence Column number 47) viral expression constructs are included.

[0043] In a preferred embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is provided. a) comprising the nucleotide sequence and element a), and is AAV8-hAAT-moFGF21; The constructs are, for example, those shown in Figure 6A as IT Viral expression depicted as R2-hAAT-moFGF21-polyA-ITR2 the sequence of this expression construct is contained in SEQ ID NO:34.

[0044] For this construct, Example 3 surprisingly demonstrated high and Demonstrate stable liver-specific expression. Expression has been shown to be stable for up to one year. (Example 12) A wide range of beneficial therapeutic options for the reversal and treatment of obesity and diabetes. The therapeutic effect was observed in ob / ob mice (Examples 3 and 11), high-fat diet (HFD)-fed mice ( In aged HFD-fed mice (Examples 4, 12-14) and in aged HFD-fed mice (Examples 5, 12-14) Examples 11 and 16 also show significant improvements in fatty liver, hepatitis, and liver fibrosis. Example 15 shows the improvement of obesity-related WAT inflammation. Example 17 shows This indicates the long-term safety of the treatment. Example 18 shows the beneficial effect in preventing liver tumors. Example 19 demonstrates therapeutic potential in a model of type 1 diabetes.

[0045] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is a) adipose tissue-specific promoter containing the mini / aP2 promoter; b) adipose tissue-specific promoter containing the adipose tissue-specific promoter; motor (SEQ ID NO: 54) and / or mini / UCP1 promoter (SEQ ID NO: 5 5) A viral expression construct is also included.

[0046] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is The ubiquitous promoter is a CAG promoter (sequence No. 44), which encodes the target sequence of a microRNA expressed in the liver. at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 12 or 14 to 23; and at least one sequence encoding a target sequence of a microRNA expressed in the heart. A virus whose nucleotide sequence is selected from the group consisting of SEQ ID NO: 13 or 23-30. Expression constructs are included.

[0047] In a preferred embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is provided. AAV9-CAG-moFGF21-dmiRT containing the nucleotide sequence and element c) or AAV8-CAG-moFGF21-dmiRT viral expression construct. The notations dmiRT and doublemiRT are equivalent. The construct is, for example, ITR2-CAG-moFGF21-4x miR in Figure 1A. Viral expression vector depicted as T122a-4x miRT1-polyA-ITR2 the sequence of this expression construct is contained in SEQ ID NO:32.

[0048] Surprisingly, these constructs in Examples 1 and 2 were administered intracellularly in eWAT. We demonstrate high and stable fat-specific expression after genomic DNA damage. A wide range of beneficial therapeutic effects for the treatment of rhesus monkeys were observed in normal mice (Example 1) and ob / o mice. b) mice (Examples 2 and 10). Example 10 also demonstrates significant hepatic steatosis. Reveal improvements.

[0049] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is the skeletal muscle promoter contains the C5-12 promoter (sequence 56) is a viral expression construct.

[0050] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is The ubiquitous promoter is a CMV promoter (sequence No. 45), and the AAV serotype is AAV1. This includes:

[0051] In a preferred embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is provided. a virus comprising a nucleotide sequence and element e) and which is AAV1-CMV-moFGF21; The constructs are, for example, those shown in Figure 11A as IT Viral expression construct depicted as R2-CMV-moFGF21-polyA-ITR2 the sequence of this expression construct is contained in SEQ ID NO:36.

[0052] For this construct, Example 20 demonstrates high and stable skeletal muscle specificity following intramuscular administration. revealing differential expression of steroid hormones, which may have a wide range of applications for the prevention, reversal and treatment of obesity and diabetes. A beneficial therapeutic effect is demonstrated in HFD-fed mice (Examples 6 and 21). Example 20 demonstrates the beneficial effects of preventing obesity and diabetes on extending healthy lifespan. do.

[0053] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is adipose tissue-specific nucleotide sequence and the nucleotide sequences of elements b) and c); The promoter is the mini / aP2 promoter (SEQ ID NO: 54) and / or the mini / UCP1 promoter (SEQ ID NO: 55). can be.

[0054] In one embodiment, a nucleic acid encoding FGF21 suitable for expression in a mammal is nucleotide sequence and the nucleotide sequences of elements e) and c), The motor is a CMV promoter (SEQ ID NO: 45), and the AAV serotype is AAV 1. A viral expression construct comprising:

[0055] All constructs of the present invention are based on those described in the prior art, e.g., Zhang et al. ,EBioMedicine 15(2017) 173-183 element a), which is a particularly liver-specific promoter, preferably hAAT, and / or The ubiquitous promoter and target sequence of a microRNA expressed in the liver are encoded by the At least one nucleotide sequence expressed in the heart, preferably miRT122a At least one nucleotide sequence encoding a target sequence of a microRNA, preferably or a combination with miRT1 that enables specific expression in adipose tissue. element c), which is a combination of, and / or a ubiquitous promoter, preferably CMV In combination with adeno-associated virus (AAV) vector sequences, preferably AAV1 and those containing element e), a combination that allows specific expression in skeletal muscle. Zhang et al. The wild-type mouse FGF21 coding sequence (EF1a-mFGF21) is under the control of ) (Zhang et al., EBioMedicine 15(20 17) 173-183). This construct is described in Examples 23 and 24 of the present invention. In all in vitro and in vivo experiments, All expression cassettes and AAV vectors of the present invention are intended to be used to express the AAV in target tissues or cell types. Higher expression of FGF21 and less FGF21 in off-target tissues This mediates expression of the expression cassettes and AAV vectors of the present invention. This demonstrates the efficiency as well as higher tissue specificity of the CMV construct. -moFGF21 and CAG-moFGF21-double miRT also inhibited EF Higher protein production and expression in HEK293 cells compared to 1a-mFGF21 Furthermore, hAAT-moFGF21 and AAV8-hAAT mediate secretion into the culture medium. AAT-moFGF21 also binds EF1a-mFGF21 and AAV8-EF1a-mFGF21. It mediated the secretion of more FGF21 into the bloodstream than FGF21.

[0056] As explained in more detail in the section entitled "General Definitions," additional Sequences may be present in the viral expression constructs of the present invention. Preferred additional sequences include: contains an inverted terminal repeat (ITR), an SV40 polyadenylation signal (SEQ ID NO: 50), a rabbit β-globin polyadenylation signal (SEQ ID NO: 51), CMV enhancer sequence (SEQ ID NO: 52), sequence number 46) and the HCR enhancer from apolipoprotein E (SEQ ID NO: 53) Within the context of the present invention, "ITR" refers to one 5'ITR and one 3'ITR. The term "ITR" is intended to encompass the ITRs, each derived from the genome of an AAV. from AV2, SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR) Within the context of the present invention, the CMV enhancer sequence (SEQ ID NO: 46) and and the CMV promoter sequence (SEQ ID NO: 45) as two separate sequences or as a single sequence (SEQ ID NO: 52).

[0057] Each of these additional sequences may be present in the viral expression constructs of the invention ( For example, as depicted in Figures 1, 2, 3, 4, 5, 6, 7, 8 and 9, and in Figure 11, 31 and 32).

[0058] In one embodiment, the FGF21 suitable for expression in a mammal as defined above is Nucleotide sequence encoding and elements a) and / or b) and / or c) and and / or a viral expression construct comprising at least one of d) and / or e). The tract is: - ITRs flanking the expression cassette of said construct, - SV40 or rabbit nucleotide sequence located 3' of the nucleotide sequence encoding FGF21 β-globin polyadenylation signal, and / or - a CMV enhancer sequence located 5' to the nucleotide sequence encoding FGF21 or HCR enhancer sequence Further includes:

[0059] In a preferred embodiment, the FGF2 suitable for expression in a mammal as defined above is nucleotide sequence encoding 1 and elements a) and / or b) and / or c a viral expression vector comprising at least one of d) and / or e) The construct further comprises ITRs flanking the expression cassette of the construct, Rabi: - SV40 or rabbit nucleotide sequence located 3' of the nucleotide sequence encoding FGF21 β-globin polyadenylation signal, and / or - a CMV enhancer sequence located 5' to the nucleotide sequence encoding FGF21 or HCR enhancer sequence It may further include:

[0060] These sequences were identified in the experimental section and in some of the constructs identified herein. It was used as such.

[0061] Therefore, in one embodiment, these preferred viral expression controls as defined above are Each construct contains ITRs, SV40 polyadenylation signal, rabbit β-glucan Robin polyadenylation signal, CMV enhancer sequence, and apolipoprotein E There may be an additional sequence selected from the group consisting of HCR enhancer sequences.

[0062] In a preferred embodiment, the viral expression construct is adapted for expression in mammals. A suitable FGF21-encoding nucleotide sequence and elements a) and / or b) and and / or at least one of c) and / or d) and / or e). , where ITR, SV40 polyadenylation signal, rabbit β-globin polyadenylation signal, CMV enhancer sequence, and HCR enhancer sequence. Additional sequences are present. Preferred ITRs are SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR) of AAV2.

[0063] A preferred viral expression construct comprises elements a) and / or b) and / or c) and / or d) and / or e), and the expression cassette comprises 5'ITR and 3'ITR 'It is set to be adjacent to the ITR.

[0064] Other preferred viral expression constructs comprise elements a) and / or b) and / or or c) and / or d) and / or e), and the expression cassette comprises 5'ITR and In addition, the SV40 polyadenylation signal is located adjacent to the 3' ITR. exist.

[0065] Other preferred viral expression constructs comprise elements a) and / or b) and / or or c) and / or d) and / or e), and the expression cassette comprises 5'ITR and and 3'ITR. In addition, the rabbit β-globin polyadenylation A signal is present.

[0066] Other preferred viral expression constructs comprise elements a) and / or b) and / or or c) and / or d) and / or e), and the expression cassette comprises 5'ITR and and 3'ITR. In addition, a CMV enhancer sequence is present. .

[0067] Other preferred viral expression constructs comprise elements a) and / or b) and / or or c) and / or d) and / or e), and the expression cassette comprises 5'ITR and In addition, the HCR from apolipoprotein E is adjacent to the 3'ITR. Enhancer sequences are present.

[0068] The most preferred viral expression constructs designed include: Construct B (represented by a nucleotide sequence comprising SEQ ID NO: 32), Construct D (represented by a nucleotide sequence comprising SEQ ID NO: 34), Construct F (represented by a nucleotide sequence comprising SEQ ID NO: 36), Construct G (represented by a nucleotide sequence comprising SEQ ID NO: 37), Construct H (represented by a nucleotide sequence comprising SEQ ID NO: 38), Construct I (represented by a nucleotide sequence comprising SEQ ID NO: 39), Construct J (represented by a nucleotide sequence comprising SEQ ID NO: 40), Construct K (represented by a nucleotide sequence comprising SEQ ID NO: 41), Construct L (represented by the nucleotide sequence comprising SEQ ID NO: 4) Examples include:

[0069] As one of skill in the art will appreciate, each of these viral expression constructs is a recombinant vector derived from AAV2. These two ITRs (i.e., SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR)) R)) already included.

[0070] Constructs B and G contain the rabbit β-globin polyadenylation signal. Construct F contains the SV40 polyadenylation signal, the CMV enhancer sequence, and the Nucleotide sequence of melanthron (human β-globin and immunoglobulin heavy chain genes) Constructs D and H to L contain the SV40 polynucleotides (introns from Nucleotide sequences of the adenylation signal, HCR enhancer sequence, and chimeric intron sequence (consisting of introns from the human β-globin and immunoglobulin heavy chain genes) ) is included.

[0071] As explained in the general section entitled "General Definitions," Specific nucleotide sequences representing preferred constructs designed herein are: Whenever we mention the SEQ ID NO of a sequence (take SEQ ID NO: A, B or C as an example), teeth: i. Nucleotides with at least 60% sequence identity or similarity to SEQ ID NO: A, B, or C a nucleotide sequence containing the nucleotide sequence; ii. Nucleotide sequences that differ in sequence from the sequence of the nucleic acid molecule in (i) due to the degeneracy of the genetic code. column can be replaced by

[0072] its percentage identity (at least 60%) with each given nucleotide sequence Each of the nucleotide sequences described herein by In each case, the given nucleotide and at least 65%, 70%, 75%, 80%, and 8 5%, 90%, 95%, 97%, 98%, 99% or higher identity. In a preferred embodiment, sequence identity is determined by comparing the entire length of the sequences identified herein. Unless otherwise indicated herein, identity to a given SEQ ID NO: Identification based on the full-length (i.e., over its entire length or as a whole) sequence It means sex or similarity.

[0073] Its minimum identity to the given SEQ ID NO: specified above (i.e., at least 60%) ) is used in conjunction with this construct or viral expression construct. A construct or a viral vector containing this construct or this construct or a composition containing the vector is transfected into cells, preferably hepatocytes, adipose tissue cells or skeletal cells. If the expression of FGF21 can be induced in muscle cells, it is within the scope of the present invention. FGF21 expression can be assessed using techniques known to those of skill in the art. In a preferred embodiment, the expression is assessed as performed in an experimental section.

[0074] In a preferred embodiment, the viral expression construct comprises SEQ ID NOs: 4, 5, 6, 7 , 8, 9, 10 or 11 or a nucleotide sequence containing SEQ ID NO: 4, 5, 6, 7, 8 , 9, 10 or 11 or a sequence having at least 60% identity with SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11 and at least 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 95%, 97%, 98%, 99%, and 100% identity to sequences It is expressed by the following.

[0075] viral vectors In a further embodiment, a viral vector comprising the viral expression construct defined above. The viral vector is an adenoviral vector, an adenovirus vector, or an adenovirus vector. The vector is preferably a viral vector, a retroviral vector, or a lentiviral vector. Preferably, the vector is an adeno-associated virus 1 (AAV1) vector, an adeno-associated virus 8 (AAV8) vector, or ) vector and adeno-associated virus 9 (AAV9) vector It is an adeno-associated virus vector.

[0076] "Viral vectors" and "adeno-associated viral vectors (AAV vectors)" are Further defined in the section entitled "General Definitions."

[0077] In one embodiment, each element as defined hereinabove and an ITR or portion thereof An AAV vector containing a recombinant AAV (rAAV)-based genome containing the fragment is used. Preferred ITRs are those represented by SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR). It is the AAV2 expressed by

[0078] Preferably, the AAV vector is an AAV1 vector, an AAV8 vector, or an AA It is a V9 vector.

[0079] The viral expression constructs and viral vectors of the present invention preferably contain a drug. The medicament is preferably for use in treating metabolic disorders, preferably diabetes and and / or for preventing, delaying, curing, reversing and / or treating obesity. The diabetic disease may be type 1 diabetes, type 2 diabetes, or monogenic diabetes. In this form, the drug prevents, delays, heals, restores and improves liver inflammation and / or fibrosis. In yet another preferred embodiment, the medicament is for the treatment of Preferably, preventing or delaying age-related metabolic disorders, preferably diabetes and / or obesity. , for extending healthy lifespan by curing, restoring and / or treating In yet another preferred embodiment, the medicament is for preventing, delaying cancer, preferably liver cancer. , to cure, restore and / or treat.

[0080] The subject to be treated is a higher mammal, for example a cat, a rodent (preferably a mouse, a rat) , gerbils and guinea pigs, more preferably mice and rats), or dogs, or or a human.

[0081] nucleic acid molecule In a further embodiment, the expression vector is suitable for expression in a mammal, and is expressed in liver, adipose tissue and / or or a mammalian codon-optimized FGF21 encoding FGF21 expressed in skeletal muscle Nucleic acid molecules represented by the nucleotide sequences are provided.

[0082] A definition of "codon optimization" is provided in the section entitled "General Definitions." It is being done.

[0083] In one embodiment, the nucleotide sequence is SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or or 11 of the above nucleic acid molecules have at least 60% sequence identity with the nucleotide sequence of Preferred nucleotide sequences are SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10 or 11 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% , 98%, 99%, and 100% identity.

[0084] composition In a further embodiment, the viral expression construct as defined above and / or a viral vector as defined in claim 1 and / or a nucleic acid molecule as defined above, Compositions containing the compound together with a pharmaceutically acceptable excipient or vehicle are provided.

[0085] This composition is preferably called a gene therapy composition. and / or is a pharmaceutical composition containing an additive.

[0086] Such pharmaceutically acceptable carriers, excipients, preservatives, solubilizers, diluents and / or additives Additives are described, for example, in Remington: The Science and Practice ce of Pharmacy,20th Edition.Baltimore,M D: Lippincott Williams & Wilkins, 2000 It can be given.

[0087] In a preferred embodiment, the composition is preferably a therapeutic agent for treating a metabolic disorder, preferably diabetes. and / or for preventing, delaying, curing, reversing and / or treating obesity. The diabetes may be type 1 diabetes, type 2 diabetes, or monogenic diabetes. In embodiments, the agent prevents, delays, heals, or reverses liver inflammation and / or fibrosis. and / or for treating. In yet another preferred embodiment, the agent is , preferably preventing age-related metabolic disorders, preferably diabetes and / or obesity, To extend healthy lifespan by delaying, curing, reversing and / or treating In yet another preferred embodiment, the medicament is for preventing cancer, preferably liver cancer, The subject to be treated is a higher mammal. Mammals, e.g., cats, rodents (preferably mice, rats, gerbils and guinea pigs) The mammalian animal may be a mammalian animal, such as a mouse, more preferably a mouse or a rat, or a dog, or a human.

[0088] said viral expression constructs, viral vectors and / or nucleic acid molecules and / or Or when the composition can exhibit anti-diabetic and / or anti-obesity effects, viral expression constructs, viral vectors and / or nucleic acid molecules and / or The composition preferably prevents, delays, reduces or eliminates metabolic disorders, preferably diabetes and / or obesity. It is said to be able to cure, restore and / or treat.

[0089] said viral expression constructs, viral vectors and / or nucleic acid molecules and / or or when the composition can exhibit an anti-fibrotic effect, the viral expression construct The viral vector and / or nucleic acid molecule and / or composition is preferably administered to a subject in need thereof, for example, in the liver. for preventing, delaying, healing, reversing and / or treating inflammation and / or fibrosis of It is said that it is possible to

[0090] said viral expression constructs, viral vectors and / or nucleic acid molecules and / or or the composition may exhibit anti-diabetic and / or anti-obesity effects upon aging. When the viral expression construct, viral vector and / or nucleic acid molecule and and / or the composition is preferably a therapeutic agent for age-related metabolic disorders, preferably diabetes and / or or extending healthy lifespan by preventing, delaying, curing, reversing and / or treating obesity It is said that it can be used for stretching.

[0091] said viral expression constructs, viral vectors and / or nucleic acid molecules and / or or when the composition can exhibit an anti-cancer effect, the viral expression construct The viral vector and / or nucleic acid molecule and / or composition is preferably a cancer and preferably for preventing, delaying, curing, ameliorating and / or treating liver cancer. It is said that this can be done.

[0092] Antidiabetic effects occur when blood glucose disposal is improved and / or glucose tolerance is improved. This can be achieved when insulin is released and / or insulin sensitivity is improved. Preferably, using techniques known to those skilled in the art or as performed in the experimental section. It can be assessed as assessed in Examples 8 or 9. In this regard, Improvement (respectively, "improvement") refers to the ability of a particular individual to demonstrate a particular level of activity using assays known to those skilled in the art or experimentally. Assays performed in, for example, blood glucose, blood insulin measurements and / or At least one test should be performed, such as a sputum tolerance test and / or a glucose tolerance test. Improvement means a detectable improvement (respectively a detectable improvement) in an assay, e.g., blood glucose, Blood insulin measurement and / or insulin tolerance test and / or glucose At least 5%, at least 10%, at least 20%, using stress testing, etc. At least 30%, at least 40%, at least 50%, at least 60%, at least It is an improvement of at least 70%, at least 80%, at least 90% or at least 100%. obtain.

[0093] Anti-obesity effects are observed when body weight, weight gain, and / or body fat percentage are reduced. The anti-obesity effect can also be achieved by reducing body mass index (BMI), waist circumference, waist hip size, and This can be achieved when waist-to-height ratio (WHR) and / or waist-to-height ratio (WHtR) are reduced. This can be done using techniques known to those skilled in the art or as performed in the experimental section. In this context, a "reduction" (respectively, an "improvement") can be assessed by those skilled in the art. using assays known to the public or performed in the experimental section. The anti-obesity effect means at least a detectable decrease (respectively, a detectable improvement) in the individual's Obesity prevention and obesity reversal, as assessed by measurements of body weight, BMI, and / or tissue mass It encompasses both.

[0094] Anti-inflammatory effects in the liver include reduced macrophage infiltration and reduced pro-inflammatory cytokines. This can be achieved by using techniques known to those skilled in the art or in the experimental department. In this regard, the terms "reduction" (and "improvement" respectively) "Good" refers to the results obtained using assays known to those skilled in the art or performed in a laboratory setting. By "detectable improvement" is meant at least a detectable decrease (respectively a detectable improvement) using the assay.

[0095] The anti-fibrotic effect in the liver was due to the reduction of extracellular matrix protein accumulation and blood markers. (For example, N-terminal propeptide of type III collagen in plasma, hyaluronic acid, metalloproteinases, Tissue inhibitor of proteinase type 1 (TIMP-1), YKL-40, serum glutamate Oxaloacetic transaminase (SGOT), serum glutamic acid pyruvate transaminase The anti-fibrotic effect can also be achieved by reducing the levels of SGPT (stimulatory receptor agonist) and SGPT. This can be achieved by improving the scoring system, such as Metavir or Ishak. , using techniques known to those skilled in the art or as performed in the experimental section. In this context, "reduction" (respectively "improvement") refers to any of the effects known to those skilled in the art. At least one test was performed using the assays currently used or performed in the experimental section. This means a detectable decrease (respectively a detectable improvement).

[0096] The healthy lifespan extension effect is the anti-diabetic and / or anti-obesity effect as defined herein above. to prevent the onset or progression of age-related metabolic disorders, preferably diabetes and / or obesity. This can be achieved when used to prevent, delay, cure, reverse or treat diseases. The prolonging effect can also be achieved by increasing healthspan, where metabolic disorders, preferably diabetes, are treated. and / or obesity-related symptoms are absent or reduced. The effect of extending healthy lifespan is also Coordination and balance (assessed by the rotarod test), memory (assessed by the object recognition test) (assessed by the tight rope test) and / or neuromuscular co-ordination (assessed by the tight rope test) Improvement, reduction of mitochondrial and metabolic deterioration (involved in metabolism and mitochondrial function) Genes involved in the expression of ATP synthase, such as PGC-1 alpha, ATP synthase, and ERR alpha This can be achieved by measuring the current level of It can be assessed using techniques or as performed in the experimental section. In the present context, "enhancement" (respectively "improvement") refers to the ability of a subject to be at least partially or completely responsive to a particular condition, using assays known to those skilled in the art. or at least a detectable increase using the assays performed in the experimental part (respectively) detectable improvement).

[0097] Anticancer effects can be achieved by reducing the cumulative incidence of cancer over a lifetime. Assessing using techniques known to those skilled in the art or as performed in the experimental section In this context, "reduction" (respectively "improvement") refers to any effect known to those skilled in the art. At least the detection was performed using assays performed in the experimental section. This means a possible decrease (respectively a detectable improvement).

[0098] The anti-diabetic and / or anti-obesity effects were also evaluated by typical symptoms ( This may also be observed when the progression of pancreatic cancer (e.g., insulitis, beta cell loss, weight gain) slows. A reduction in typical symptoms may mean a slowing of the progression of the symptoms or a complete disappearance of the symptoms. The symptoms and therefore the reduction in symptoms can be assessed using a variety of methods. The methods were largely the same as those used in the diagnosis of diabetes and / or obesity. Clinical examination and routine laboratory tests Such methods include macroscopic methods and aboratory tests. Both microscopic methods, as well as molecular methods, X-ray, biochemistry, immunohistochemistry and and others.

[0099] The anti-inflammatory effect on the liver also reduces typical symptoms assessed by a physician (e.g., fatigue, Slowed progression of cold-like symptoms, dark urine, pale stools, abdominal pain, loss of appetite, unexplained weight loss, and jaundice A reduction in typical symptoms may also be seen when the progression of symptoms slows or when symptoms are completely eliminated. Symptoms and therefore symptom reduction can be assessed using a variety of methods. This method is largely the same as that used in the diagnosis of liver fibrosis. Clinical examination and routine clinical testing Such methods include laboratory tests. Both microscopic and molecular-level methods, as well as imaging methods (e.g., elastic imaging) X-ray, MRI, CT, ultrasound, angiography), biochemistry, immunohistochemistry and other Examples include:

[0100] The anti-fibrotic effect in the liver was also evaluated by typical symptoms assessed by a physician (e.g., liver stiffness, jaundice, loss of appetite, difficulty thinking clearly, fluid retention in the limbs or stomach, It may also be observed when the progression of typical symptoms (nausea, unexplained weight loss, weakness) slows. A decrease can mean a slowing of the progression of the onset of symptoms or a complete disappearance of symptoms. Symptom reduction can also be assessed using a variety of methods, most of which involve liver cirrhosis. The same methods as those used in the diagnosis of fibrosis, including clinical testing, examination and routine laboratory tests Such methods include both macroscopic and microscopic methods, as well as Molecular-level methods, imaging methods (elastic imaging, X-ray, MRI, CT, ultrasound, blood ductography), biochemistry, immunohistochemistry and others.

[0101] The effect of extending healthy lifespan also reduces typical symptoms of age-related metabolic disorders as assessed by physicians. It is also observed when the progression of symptoms (e.g., insulin resistance, glucose intolerance, weight gain) slows. A reduction in typical symptoms may mean a slowing of the progression of the onset of symptoms or a complete disappearance of symptoms. Symptoms and therefore symptom reduction can be assessed using a variety of methods, This method is largely the same as that used in diagnosing diabetes and / or obesity. Clinical examination and routine clinical testing Such methods include laboratory tests. both microscopic and molecular methods, as well as X-ray, biochemistry, and immunohistochemistry. science and others.

[0102] Anti-cancer effects are also evaluated by typical symptoms (e.g., tumor size, unknown Weight loss, loss of appetite, feeling very full after eating a small meal, nausea or vomiting, liver problems Enlarged liver, enlarged spleen, pain in the abdomen or near the right shoulder blade, abdominal swelling or fluid retention, itching, A reduction in typical symptoms may also be observed when the progression of the disease (jaundice) slows. This can mean a slowing down or complete disappearance of symptoms. Symptoms and therefore also a reduction in symptoms can be can be assessed using methods that are mostly used in cancer diagnosis. The same method as that used for clinical examination and and routine laboratory tests. , both macroscopic and microscopic methods, as well as molecular methods, imaging imaging methods (X-ray, MRI, CT, ultrasound, angiography), biochemistry, immunohistochemistry and others Examples include:

[0103] Viral expression constructs and / or viral vectors of the invention and / or Use of the nucleic acid molecules and / or compositions for at least 1 week, 1 month, 6 months, 1 year or and after longer treatment, the symptoms or characteristics as defined above were reduced (for example, If the virus is not detectable or slowed down, then the agent ( The expression constructs, viral vectors, nucleic acid molecules, compositions) are preferably administered to patients or can alleviate a symptom or a characteristic of the patient's cells, tissues, or organs. Cut.

[0104] Viral expression constructs and vectors as defined herein for use in accordance with the present invention and / or viral vectors and / or nucleic acid molecules and / or compositions for the treatment of metabolic disorders disorders, such as diabetes and / or obesity, liver inflammation and / or fibrosis, age-related Those who suffer from or are at risk of developing related metabolic disorders and / or cancer suitable for in vivo administration to cells, tissues and / or organs of a subject The viral expression constructs may be obtained and administered in vivo, ex vivo, or in vitro. Tracts and / or viral vectors and / or nucleic acid molecules and / or compositions are associated with metabolic disorders such as diabetes and / or obesity, liver inflammation and / or fibrosis suffer from or develop age-related metabolic disorders, and / or cancer, In vivo, directly or indirectly, to the cells, tissues and / or organs of individuals at risk for the disease. can be administered directly or indirectly in vivo, ex vivo, or in vitro. The modes of administration can be intravenous, subcutaneous, intramuscular, intrathecal, intraarticular, intracerebroventricular, intraperitoneal, intrafatty Intra-tissue, via inhalation, oral, intranasal, intra-liver, intra-visceral, intra-ocular, intra-aural, topical ) and / or retrograde intraductal pancreatic administration This may be due to the pulmonary pancreatic administration Preferred modes of administration are described in the "General Procedures for the Examples" which are part of this application. As described, intramuscular, intravenous or intraadipose tissue.

[0105] Viral expression constructs and / or viral vectors of the invention and / or The nucleic acid molecules and / or compositions can be prepared using any suitable means known in the art. Taking into account the progress already made to date, and injecting the viral expression construct of the present invention into an individual or into a cell, tissue, or organ of said individual. and / or viral vectors and / or nucleic acid molecules and / or compositions Improvements in the means for achieving this are anticipated. Such future improvements will, of course, be expected to eliminate the benefits of the invention as noted above. Viral expression constructs and / or viruses can be incorporated to achieve The vector and / or nucleic acid molecule and / or composition may be administered to an individual, to a cell of said individual, or Depending on the disease or condition, the individual's cells, tissues, or organs may be delivered to the cells. The tissue or organ may be as defined hereinbefore. The present construct and / or viral vector and / or nucleic acid molecule and / or When the composition is administered, such viral expression constructs and / or vectors and / or the nucleic acid and / or composition is dissolved in a solution compatible with the delivery method. is preferred.

[0106] As encompassed herein, a therapeutically effective dose of the above-described viral expression construct The vectors, nucleic acid molecules and / or compositions are preferably administered in a single and unique dose. Therefore, repeated periodic administration is avoided. More preferably, a single dose is administered to skeletal muscle. It is administered into the adipose tissue or intravenously.

[0107] Additional compounds may be present in the compositions of the present invention. The compounds may assist in the delivery of the composition. The following is a list of suitable compounds: complexes or entrapped in vesicles or liposomes for delivery across the cell membrane; Compounds capable of forming nanoparticles, micelles and / or liposomes. Many of these compounds are known in the art. A suitable compound is polyethyleneimine (PEI ), or polypropyleneimine or polyethyleneimine copolymer (PEC) and Similar cationic polymers, including derivatives, synthetic amphiphiles (SAINT-18), Includes Lipofectin™, DOTAP.

[0108] Depending on their identity, the skilled artisan will be able to determine which formulation types are suitable for the compositions defined herein. Understand what works best for you.

[0109] Method / Usage In a further embodiment, a viral expression vector as defined above for use as a medicament is provided. construct and / or viral vector as defined above and / or The present invention provides a nucleic acid molecule and / or a composition as defined above.

[0110] In one embodiment, the viral expression construct and / or viral vector The target and / or nucleic acid molecule and / or composition is / are a therapeutic agent for treating metabolic disorders, preferably diabetes and and / or for use in the treatment and / or prevention of metabolic disorders. Comorbidities may also be included.

[0111] In another embodiment, the viral expression construct and / or viral vector The target and / or nucleic acid molecule and / or composition is / are intended to treat liver inflammation and / or fibrosis. The present invention is provided for use in the treatment and / or prevention of liver inflammation and / or Complications of fibrosis may also be involved.

[0112] In yet another embodiment, the viral expression construct and / or virus The vector and / or nucleic acid molecule and / or composition is preferably an anti-aging agent. to prevent, delay, cure, reverse and / or prevent metabolic disorders, preferably diabetes and / or obesity; The present invention is provided for use in extending healthy lifespan by treating

[0113] In yet another embodiment, the viral expression construct and / or virus The vector and / or nucleic acid molecule and / or composition is a therapeutic agent for the treatment of cancer, preferably liver cancer. Complications of cancer are also included. obtain.

[0114] In a further embodiment, the viral expression construct as defined above and / or a viral vector as defined above and / or a nucleic acid molecule as defined above and / or In the case of a metabolic disorder, preferably diabetes and / or obesity, the method comprises the use of a composition as defined in and methods for preventing, delaying, ameliorating, curing, and / or treating these complications are provided. .

[0115] Such methods preferably comprise the step of: in an individual, in a cell, tissue or organ of said individual; one or more symptoms of metabolic disorders, such as diabetes and / or obesity or for alleviating one or more of the cells, tissues or organs of said individual. and alleviating a plurality of characteristic(s) or symptom(s), the method comprising: The individual is administered a viral expression construct and / or viral vector as defined herein. This includes administering a target and / or nucleic acid molecule and / or composition.

[0116] In a further embodiment, the viral expression construct as defined above and / or a viral vector as defined above and / or a nucleic acid molecule as defined above and / or and the treatment of liver inflammation and / or fibrosis and their complications, comprising the use of a composition as defined in claim 1. Methods for preventing, delaying, ameliorating, curing and / or treating complications are provided.

[0117] Such methods preferably comprise the step of: in an individual, in a cell, tissue or organ of said individual; to alleviate one or more symptoms of liver inflammation and / or fibrosis or one or more characteristics (or combinations) of the cells, tissues or organs of said individual. and the individual is provided with a method for alleviating the symptoms or condition(s) of the individual, the method comprising administering to the individual a therapeutic agent or therapeutic agent as described herein. Viral expression constructs and / or viral vectors and / or or nucleic acid molecules and / or compositions.

[0118] In a further embodiment, the viral expression construct as defined above and / or a viral vector as defined above and / or a nucleic acid molecule as defined above and / or Preferably, the use of a composition as defined in claim 1, wherein the composition is a glycemic control agent, to provide a healthier lifestyle by preventing, delaying, curing, reversing and / or treating diabetes and / or obesity; Methods for extending healthy lifespan are provided.

[0119] Such methods preferably comprise the step of: in an individual, in a cell, tissue or organ of said individual; and one or more age-related metabolic disorders, such as diabetes and / or obesity. to alleviate the symptom(s) or to treat the cells, tissues or organs of said individual alleviating one or more of the characteristics or symptoms of the disease, The method comprises administering to said individual a viral expression construct and / or comprises administering a viral vector and / or a nucleic acid molecule and / or a composition .

[0120] In a further embodiment, the viral expression construct as defined above and / or a viral vector as defined above and / or a nucleic acid molecule as defined above and / or and the use of a composition as defined in claim 1, wherein the composition is a compound selected from the group consisting of acetaminophen, ... Methods for preventing, delaying, ameliorating, curing and / or treating are provided.

[0121] Such methods preferably comprise the step of: in an individual, in a cell, tissue or organ of said individual; To alleviate one or more symptoms of cancer, such as liver cancer or one or more characteristics of the cells, tissues or organs of said individual or alleviating a symptom(s) thereof, the method comprising administering to said individual a therapeutic agent as defined herein. Viral expression constructs and / or viral vectors and / or nucleic acids This includes administering molecules and / or compositions.

[0122] In the context of the present invention, the present invention provides a method for preventing, delaying metabolic disorders, preferably diabetes and / or obesity. and / or for the manufacture of a medicament for the prolongation, recovery, cure and / or treatment of a disease as defined herein. Viral expression constructs and / or viral vectors and / or nucleic acid molecules Uses of the product and / or composition are provided.

[0123] In the context of the present invention, preventing, delaying, healing, or reversing liver inflammation and / or fibrosis. and / or for the manufacture of a medicament for the treatment of a viral outbreak as defined herein. The present construct and / or viral vector and / or nucleic acid molecule and / or Use of the composition is provided.

[0124] In the context of the present invention, it is preferred to treat age-related metabolic disorders, preferably diabetes and and / or prevent, delay, cure, reverse and / or treat obesity to increase healthy lifespan The viral expression constructs defined herein for the manufacture of agents for extending the Use of vectors and / or viral vectors and / or nucleic acid molecules and / or compositions Use is provided.

[0125] In the context of the present invention, cancer, preferably liver cancer, can be prevented, delayed, reversed, cured and and / or the manufacture of a medicament for treating a viral expression vector as defined herein. Constructs and / or viral vectors and / or nucleic acid molecules and / or compositions Use of the object is provided.

[0126] Metabolic disorders include metabolic syndrome, diabetes, obesity, obesity-related comorbidities, and diabetes Disease-related comorbidities, hyperglycemia, insulin resistance, impaired glucose tolerance, fatty liver, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (N ASH), coronary heart disease (CHD), hyperlipidemia, atherosclerosis, endocrine disorders (e andocrinopathy), osteosarcopenic obesity syndrome (OSO), diabetes Diabetic nephropathy, chronic kidney disease (CKD), cardiac hypertrophy, Diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, arthritis, sepsis, ocular neovascularization , neurodegeneration, dementia, and may also include depression, adenoma, carcinoma.

[0127] Diabetes includes prediabetes, hyperglycemia, type 1 diabetes, type 2 diabetes, and early-onset adult-onset diabetes. Diabetes Mellitus (MODY), monogenic diabetes, neonatal diabetes, gestational diabetes, unstable diabetes, Idiopathic diabetes, drug- or chemical-induced diabetes, stiff-man syndrome, lipoatrophy These include adult-onset diabetes and latent autoimmune diabetes in adults (LADA).

[0128] Obesity includes overweight, central / upper body obesity, peripheral / lower body obesity, morbid obesity, osteoporosis, Leucopenic Obesity Syndrome (OSO), childhood obesity, Mendelian (monogenic) syndrome, Mendelian obesity These include non-syndromic obesity and polygenic obesity.

[0129] The metabolic disorders, diabetes, obesity and types of subjects to be treated are as defined herein above. are.

[0130] Liver inflammation and / or fibrosis include autoimmune hepatitis, types A, B, C, and D and viral hepatitis, including hepatitis E, alcoholic hepatitis, and non-alcoholic fatty liver disease. Nonalcoholic Bone Marrow Stem Cell Disease (NASH) and cirrhosis.

[0131] Cancers include astrocytoma, glioma, leukemia, lymphoma, melanoma, myeloma, and neuroblastoma. Cysts, sarcomas (chondrosarcoma, fibrosarcoma, rhabdomyoscaroma) and osteosarcoma), schwannomas, seminomas, and tumors of the bladder, breast, cervix, colon, , endometrium, esophagus, gallbladder, kidneys, liver, lungs, ovaries, prostate, pancreas, rectum, skin, stomach and A preferred cancer is liver cancer, preferably hepatocellular carcinoma. In one embodiment, the method or use is performed in vitro, e.g., using cell culture. Preferably, said methods or uses are in vivo. Each feature of has been previously defined herein.

[0132] In some methods of the invention, viral expression constructs and / or vectors and and / or nucleic acid molecules and / or compositions for treating a metabolic disorder, preferably diabetes, in an individual. and / or in combination with additional compounds known to be used to treat obesity. can be combined.

[0133] In another method of the invention, viral expression constructs and / or vectors and and / or nucleic acid molecules and / or compositions for treating liver inflammation and / or fibrosis. The compounds may be combined with additional compounds known to be used for the treatment of cancer.

[0134] In yet another method of the invention, viral expression constructs and / or vectors and / or the nucleic acid molecules and / or compositions are used to extend healthy lifespan The compound may be combined with additional compounds known to have anti-inflammatory properties.

[0135] In yet another method of the invention, viral expression constructs and / or vectors and / or the nucleic acid molecule and / or composition treats cancer, preferably liver cancer. The compounds may be combined with additional compounds known to be useful for this purpose.

[0136] In a preferred embodiment, the treatment in the use or method according to the present invention is repeated Alternatively, in the uses or methods according to the invention, it is not necessary to use a viral expression construct. The administration of the lactol or the composition may be repeated annually or every 2, 3, 4, 5, or 6 years. It is possible.

[0137] general definition identity / similarity In the context of the present invention, the protein is fibroblast growth factor 21 (FGF21). A fragment or polypeptide or peptide or derived peptide is a peptide represented by an amino acid sequence. can be.

[0138] In the context of the present invention, the nucleic acid molecule encoding FGF21 is a nucleic acid molecule encoding a protein comprising a nucleotide sequence similar to that of the nucleotide sequence ... A nucleic acid encoding a protein fragment or polypeptide or peptide or a derived peptide is represented by a sequence of nucleotides. The nucleic acid molecule may include a regulatory region.

[0139] Given a Sequence Identity Number (Sequence Identity Number ( Each nucleic acid molecule or protein identified herein by SEQ ID NO) The protein fragment or polypeptide or peptide or derived peptide or construct may be It is understood that there is no limitation to the specific sequences disclosed. Each coding sequence identified in the document also encodes a given protein fragment or polypeptide. or a peptide or a derived peptide or construct, Itself a protein fragment or polypeptide or construct or peptide or a derived peptide. Throughout this application, a given protein fragment or polypeptide is The sequence of a peptide or a specific nucleotide sequence encoding a peptide or a derived peptide Every time you mention a column number (take sequence number X as an example), this: i. A nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO:X. nucleotide sequence containing the string; ii. Nucleotide sequences that differ in sequence from the sequence of the nucleic acid molecule in (i) due to the degeneracy of the genetic code. column; or iii. the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: X and at least Nucleotide sequences encoding amino acid sequences with 60% amino acid identity or similarity can be replaced by

[0140] Throughout this application, specific amino acid sequence SEQ ID NOs (taking SEQ ID NO: Y as an example) are used. Every time you mention raising it, this is: Amino acids with at least 60% sequence identity or similarity to the amino acid sequence of SEQ ID NO: Y Polypeptides containing the sequence can be replaced by

[0141] Its identity or similarity percentage with a given nucleotide or amino acid sequence, respectively Each nucleotide sequence described herein is In a further preferred embodiment, the sequence or amino acid sequence is and amino acid sequence with at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identity or similarity. In certain embodiments, sequence identity or similarity is measured over the entire length of the sequences identified herein. Unless otherwise indicated herein, the sequence of a given sequence is determined by comparison. Identity or similarity is not measured in full length (i.e., over its entire length or as a whole). ) refers to identity or similarity based on said sequences.

[0142] Each nucleotide in a non-coding (i.e., promoter or other regulatory region) The code sequence is the sequence number of a specific nucleotide sequence (take sequence number A as an example). Nucleotides containing nucleotide sequences with at least 60% sequence identity or similarity A preferred nucleotide sequence is at least SEQ ID NO: A. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% %, 99%, or 100% identity. Identity is determined by the It may be assessed across the entire SEQ ID NO: or across a portion thereof. In the present invention, such non-coding nucleotide sequences, such as promoters, are at least At least, the activity of such non-coding nucleotide sequences known to those skilled in the art, e.g. To exhibit or exert promoter activity, etc.

[0143] "Sequence identity" refers to the degree of sequence identity between two or more between or between two or more amino acid (polypeptide or protein) sequences of A polynucleotide sequence is defined herein as the relationship between two or more nucleic acid (polynucleotide) sequences. In embodiments, sequence identity is based on the full length of the two given SEQ ID NOs, or The portion is preferably at least 10 of both SEQ ID NOs. %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% In the art, "identity" also sometimes refers to the string of such sequences. "Sequence relatedness" refers to the degree of sequence relatedness between amino acid or nucleic acid sequences as determined by the match between the amino acid and nucleic acid residues.

[0144] "Similarity" between two amino acid sequences refers to the amino acid sequence of one polypeptide and its Conservative amino acid substitutions are determined by comparing the sequence of the second polypeptide. "Identity" and "similarity" can be easily calculated by known methods. Examples include, but are not limited to, Computational Molecular Biology,Lesk,AM,ed.,Oxford University Press, New York, 1988; Biocomputing: Info rmatics and Genome Projects,Smith,DW,e d., Academic Press, New York, 1993; rAnalysis of Sequence Data,Part I,Griff in,AM,and Griffin,HG,eds.,Humana Pre ss,New Jersey,1994; Sequence Analysis in Molecular Biology,von Heine,G.,Academic Press,1987; and Sequence Analysis Prime r, Gribskov, M. and Devereux, J., eds., M Sto. ckton Press, New York, 1991; and Carillo, H. .,and Lipman,D.,SIAM J.Applied Math.,48: 1073 (1988).

[0145] Preferred methods to determine identity are those that give the largest match between the sequences tested. Methods to determine identity and similarity are publicly available. The method for determining identity and similarity between two sequences is codified in computer programs. A preferred computer program method for this purpose is, for example, the GCG program program P (Devereux, J., et al., Nucleic Acids Re search 12(1):387(1984)), BestFit, BLASTP, B LASTN and FASTA (Altschul, S.F. et al., J. Mol. Biol. 215:403-410(1990). BLAST X Pro The BLAST manual is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda ,MD 20894; Altschul,S.,et al.,J.Mol.Biol 215:403-410(1990). The well-known Smith Waterman algorithm Rhythm can also be used to determine identity.

[0146] Preferred parameters for polypeptide sequence comparison include Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443- 453(1970);Hentikoff and Hentikoff,Proc.N Atl. Acad. Sci. USA. 89:10915-10919(1992) Comparison matrix: BLOSSUM62; Gap Penalty :12; and Gap Length Penalty:4. A useful program with a meter is Genetics, Inc., in Madison, WI. It is available from the Computer Group as the "Ogap" program. These parameters are the default parameters for amino acid comparisons (including end gaps). without penalty).

[0147] Preferred parameters for nucleic acid comparison include Algorithm:Needle man and Wunsch, J. Mol. Biol. 48:443-453(19 70);Comparison matrix:matches=+10,mismat ch=0;Gap Penalty:50;Gap Length Penalty:3 Genetics Computer Group in Madison, Wisconsin The Gap program is available from the group. This is the default parameter.

[0148] In determining the degree of amino acid similarity, one skilled in the art will recognize so-called "conservative" amino acid sequences that are apparent to one skilled in the art. Conservative amino acid substitutions are those with residues having similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, , valine, leucine, and isoleucine; amino acids with aliphatic hydroxyl side chains. The acid group is serine and threonine; the amino acid group with amide-containing side chains is The group of amino acids with aromatic side chains is asparagine and glutamine; amino acids with basic side chains; The groups are lysine, arginine, and histidine; and amino acids with sulfur-containing side chains. The preferred conservative amino acid substitutions are cysteine ​​and methionine. The groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-alanine The amino acids disclosed herein are glutamine, alanine-valine, and asparagine-glutamine. Substitution variants of the disclosed amino acid sequences include those in which at least one residue in the disclosed sequence is replaced. Preferably, the amino acid The preferred conservative substitutions for each naturally occurring amino acid are Al a 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 Pr o;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;Tr p to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu be.

[0149] Genes or coding sequences "gene" or "coding sequence" or "nucleic acid" or "nucleotide sequence" "Nucleic" refers to the specific protein, e.g., FGF21, that is "core." The coding sequence is the region of DNA or RNA that encodes the appropriate When placed under the control of a regulatory region, such as a promoter, it is transcribed (DNA) and A gene is a set of several operably linked fragments, e.g., RNA, which are translated into a protein (RNA). The promoter, 5' leader sequence, introns, coding sequence and 3' untranslated sequence It may contain a polyadenylation site or a 3' untranslated region (3'UTR), etc. The chimeric or recombinant gene (e.g., the FGF21 gene) may be Genes not normally found in nature, e.g., genes whose promoters are not naturally part of the transcribed DNA region "Gene expression" refers to the process by which a gene is converted into RNA. The term refers to the process by which a gene is transcribed and / or translated into an active protein.

[0150] promoter As used herein, the term "promoter" refers to a promoter that is a promoter of one or more genes (or a region of the transcription start site of a gene that functions to regulate the transcription of a gene (or coding sequence) A nucleic acid fragment located upstream in the transcription direction, which is the DNA-dependent RNA polymerase enzyme binding sites, transcription initiation sites, and, but not limited to, transcription factor binding sites, Any other DNA sequence containing repressor and activator protein binding sites. act directly or indirectly to regulate the amount of transcription from the promoter, as well as the sequence Structurally specified by the presence of any other nucleotide sequence known to those skilled in the art. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated in response to physiological or developmental conditions. "Organ-specific" or "tissue-specific" promoters are promoters that are specific to the Organ-specific and tissue-active promoters. A tissue-specific promoter primarily expresses one or more genes in one organ or tissue. (or coding sequence) in other organs or tissues as well. It is possible to allow expression of detectable levels ("leaky") into other organs or tissues. Leaky expression in the presence of a gene can be detected by standard assays known to those skilled in the art (e.g., PCR, Waste analysis, etc.). Organ- or tissue-specific expression as assessed by immunoblot analysis, ELISA At least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold or less than Leaky expression means expression that is at least 5-fold lower but still detectable. The maximum number of organs or tissues is 5, 6, 7, or 8. "Adipose tissue-specific promoter "-" is a promoter capable of initiating transcription in adipose tissue, but other (maximum still allow some leaky expression in (5, 6, 7 or 8) organs or body parts. Transcription in adipose tissue is expressed in adipose tissue and adipocytes, e.g., white adipocytes, brown adipocytes. It can be detected in cells, beige adipocytes, preadipocytes, stromal vascular cells, etc. A "liver-specific promoter" is a promoter capable of initiating transcription in the liver. but there is some leakage in other (up to 5, 6, 7 or 8) organs or body parts. Transcription in the liver allows expression in liver tissue and liver cells, e.g., hepatocytes. , Kupffer cells and / or oval cells, etc. A "skeletal muscle promoter" is a promoter capable of initiating transcription in skeletal muscle. but some leakage in other (up to 5, 6, 7 or 8) organs or body parts Transcription in skeletal muscle is expressed in skeletal muscle cells, e.g., myocytes, myoblasts, salivary glands, and the like. It can be detected in stellate cells, etc.

[0151] A "ubiquitous promoter" is a promoter that is expressed in virtually all tissues, organs, and cells of an organism. It is active.

[0152] Organ-specific and / or tissue-specific expression of nucleotide sequences encoding FGF21 Suitable promoters for expression include the human α1-antitrypsin promoter, α1- in combination with the hepatocyte control region (HCR) enhancer from polypoprotein E Antitrypsin promoter, albumin promoter, major urinary protein promoter -, phosphoenolpyruvate carboxykinase (PEPCK) promoter, liver-enriched Protein activator promoter, transthyretin promoter, thyroxine Fatty acid-binding globulin promoter, apolipoprotein A1 promoter, liver fatty acid binding Protein promoter, phenylalanine hydroxylase promoter, adipocyte transcription factor aP2 (also known as fatty acid binding protein 4 (FABP4)) promoter PPARy promoter, adiponectin promoter, phosphoenol Pyrate carboxykinase (PEPCK) promoter, human aromatase cytochrome p The promoter derived from 450 (p450arom) is the mini / aP2 promoter ( (consisting of the fat-specific aP2 enhancer and the basal aP2 promoter), Uncoupling protein 1 (UCP1) promoter, mini / UCP1 promoter (adipose tissue specific (consisting of the heterologous UCP1 enhancer and the basal UCP1 promoter), Leptin promoter, Foxa-2 promoter, myosin light chain promoter, myosin heavy chain promoter, desmin promoter, C5-12 promoter promoter, muscle creatine kinase (MCK) promoter, smooth muscle alpha-actin promoter Motor, CK6 promoter, Unc-45 myosin chaperone B promoter, MC the basal MCK promoter combined with a copy of the K enhancer, Enh358MCK; The promoter (the combination of the MCK enhancer and the 358 bp proximal promoter of the MCK gene) Examples include:

[0153] operably linked "Operably linked" means a control sequence, such as a promoter sequence or a regulatory sequence. The promoter sequence of the desired nucleotide sequence, preferably encoding FGF21, is Preferably, the control or regulatory sequence is FGF2 in the cell and / or subject. directing the transcription and / or production or expression of a nucleotide sequence of interest encoding 1 or a structure appropriately positioned to affect them. For example, a promoter initiates or regulates the transcription or expression of a coding sequence. If the promoter is operably linked to the coding sequence, In this case, the coding sequence is understood to be "under the control of" the promoter. One or more nucleotide sequences and / or elements contained within the construct The component is "configured to be operably linked to an optional nucleotide sequence of interest." As defined herein, once the nucleotide sequence of interest is If present in the nucleic acid sequence, the nucleotide sequence and / or element is The nucleotide sequences and / or elements are all operably linked to the nucleotide sequence of interest. It is understood that the construct is configured so that the

[0154] Viral expression constructs The expression construct is a gene that can remain stable and episomal within the cell. Within the context of the present invention, cells are those cells used to generate constructs. It can also be meant to encompass cells into which the construct is administered. The construct may be integrated into the genome of a cell, e.g., via homologous recombination or otherwise. Particularly preferred expression constructs are those defined herein. The nucleotide sequence encoding FGF21 is constructed in a promoter as defined herein. operably linked, and the promoter is capable of expressing the nucleotide sequence (i.e., Preferably, the promoter is capable of directing the expression of a gene encoding a gene (i.e., a coding sequence). The motor is expressed in at least one cell of a specific organ and / or tissue. Preferably, the promoter directs the expression of a nucleotide sequence in the liver, adipose tissue, or the like. and / or directing expression of said nucleotide sequence in at least one cell of skeletal muscle. Preferably, the promoter is expressed in cells of the liver, adipose tissue and / or skeletal muscle. At least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80% of In the context of the present invention, the expression of the IL-11 receptor is expressed in liver, adipose tissue, or the like. FGF21 expressed in tissues or skeletal muscle is more abundant than in other organs or tissues, such as the liver, Preferential or predominant (at least 10% more) expression of FGF21 in adipose tissue or skeletal muscle , at least 20% more, at least 30% more, at least 40% more, at least 5 0% more, at least 60% more, at least 70% more, at least 80% more, less At least 90% more, at least 100% more, at least 150% more, at least 20 Throughout this application, the term "liver" refers to liver expression in relation to expression. When liver-specific or fat-specific or skeletal muscle-specific is mentioned, liver, fat tissue, and skeletal muscle are respectively mentioned. Cell type-specific expression of the cell type(s) that make up tissue or skeletal muscle is also expected.

[0155] The viral expression constructs of the present invention are nucleic acids in a form suitable for mammalian expression. The nucleic acid sequence is included in the viral expression construct used for expression. operably linked to the nucleotide sequence to be expressed, selected based on the mammalian host cell Preferably, the expression of the gene is carried out in a manner that includes one or more regulatory sequences. The mammalian host cells may be human, murine or canine cells.

[0156] The viral expression constructs of the present invention are capable of expressing the IL-1 receptor in the liver, adipose tissue and / or skeletal muscle. The vector contains a nucleotide sequence that is expressed by the vector.

[0157] As used herein, "adipose tissue" refers to mature adipocytes (i.e., fat cells). and from a combination of small blood vessels, neural tissue, lymph nodes, and stromal vascular fraction (SVF). SVF is a tissue composed of endothelial cells, fibroblasts, and adipocyte precursor cells (i.e. preadipocytes), and immune cells, such as macrophages and T cells In mammals, there are two different types of adipose tissue: white adipose tissue (WAT) and brown adipose tissue (WAT). In mammals, adipose tissue is a multi-storage area. Contained in multi-depot organs. t) includes, but is not limited to, epididymal WAT ( eWAT), inguinal WAT (iWAT), retroperitoneal WAT (rWAT), mesenteric WAT (m These include interscapular BAT (WAT) and interscapular BAT (iBAT).

[0158] As used herein, "skeletal muscle" refers to tissue composed of muscle fibers. Muscle fibers, also known as myofibers, are A single multinucleated or syncytial cell resulting from the fusion of one hundred myoblasts, of which Some persist within mature muscle as undifferentiated cells known as satellite cells. Each muscle fiber is surrounded by connective tissue called the endomysium. The fibers form fascicles or bundles, which themselves The skeletal muscle is surrounded by another layer of connective tissue called the perimysium. Finally, the skeletal muscle is surrounded by a layer of connective tissue called the epimysium. It is formed by groups of fiber bundles surrounded by another layer of connective tissue. Muscles also consist of numerous blood vessels and nerves. The ends of the muscles are bound by the periosteum or other muscle connections. Concentrated towards tendons and aponeuroses, which are dense connective tissue structures that mediate muscle attachment to the fascia. circle.

[0159] As used herein, "liver" refers to a tissue composed of hepatocytes. Alveoli represent approximately 50-70% of the cells in the liver. In addition to hepatocytes, the liver also contains endothelial cells, It is composed of parasinusoidal cells, oval cells, Kupffer cells, and stellate cells (Ito cells). Upon activation by pericytes, astrocytes transform into myofibroblasts. The central vein and portal tract, which contains the anterior terminal branch of the hepatic artery, the hepatic portal vein, the bile canaliculi, and lymphatic vessels A portal track (portal triad) is also found in the liver.

[0160] Such preferred expression constructs are said to comprise an expression cassette. The expression cassette used may contain or contain a nucleotide sequence encoding FGF21. operably connected to a promoter capable of directing expression of said nucleotide sequence; In one embodiment, the expression cassette used herein is Nucleotide sequence encoding F21, promoter, and gene encoding mAb expressed in liver At least one nucleotide sequence encoding a target sequence of a microRNA and At least one nucleotide sequence encoding a target sequence of a microRNA expressed in In one embodiment, the described expression cassette comprises or consists of A microRNA expressed in the liver that is perfectly complementary to the cognate microRNA of and / or a nucleotide sequence encoding a target sequence of a microRNA expressed in the heart. In another embodiment, the described expression cassette contains an imperfectly complementary (1 mismatch / 5 consecutive nucleotides) in the microRNA binding site. In yet another embodiment, the nucleic acid sequence contains one or more nucleotide sequences encoding the nucleic acid sequence(s). In this study, the expression cassettes contained both perfect and imperfect microRNA binding sites. The expression cassette can therefore contain a single complete microRNA. Target site, multiple complete microRNA target sites, single incomplete microRNA target site sites, multiple incomplete microRNA target sites, or complete and incomplete microRNAs The level of regulation can be varied by using nucleotide sequences encoding combinations of target sites. Furthermore, the target sites of different microRNAs can be tailored to produce Nucleotide sequences encoding genes can be used, thus linking genes to multiple microRNAs. The nucleotide sequence encoding the target sequence of the microRNA can be more regulated. The preferred location of the sequence is the 3'UTR. However, it is also possible to use the sequence in the coding sequence or 5'UTR. The nucleotide sequence inserted into any of the R sequences (encoding the target sequence) is also used. It is possible.

[0161] The selection of nucleotide sequences encoding the target sequences of microRNAs is important to achieve the desired expression pattern. The presence of endogenous microRNAs in cells is determined by the expression of the target microRNA. a gene from an expression construct containing a nucleotide sequence encoding a target sequence or Inhibits expression of a coding sequence. is recognized by microRNAs present in that cell type due to expression of the coding sequence. A nucleotide sequence is selected that encodes the target sequence to be cloned.

[0162] Viral expression constructs are intended to be used in gene therapy. A construct that contains a portion of the viral genome as defined later in this specification. Designed to fit The expression constructs disclosed herein contain a nucleotide sequence encoding the FGF21. The recombinant peptide sequence is expressed in a suitable cell, such as a cultured cell or a cell of a multicellular organism. These can be prepared using techniques such as those described in, for example, Ausubel et al., “Current Protocols in Molecular Biology”, Greene Publishing and Wiley-Interscience , New York (1987) and Sambrook and Russell (2 001, supra); both of which are incorporated herein by reference in their entireties. Kunkel (1985) Proc. Natl. Acad. Sci. 82 :488 (which describes site-directed mutagenesis) and Roberts et al. 1987) Nature 328:731-734 or Wells, JA, et al. (1985) Gene 34:315 (which describes cassette mutagenesis) Please refer to.

[0163] Typically, the nucleic acid or nucleotide sequence encoding FGF21 is expressed in an expression construct. The phrase "expression vector" or "vector" is used in Generally, a gene or coding sequence is expressed in a host compatible with the gene or coding sequence. These expression vectors are nucleotide sequences capable of directing the expression of a coding sequence. , typically including at least a suitable promoter sequence and optionally a transcription termination signal. Any additional factors necessary or helpful in causing expression, as described herein, are also included. Nucleic acid, DNA or nucleoside encoding FGF21 can also be used. The peptide sequence is a DNA construct capable of introduction and expression in in vitro cell culture. Specifically, the DNA construct is introduced into a prokaryotic host, such as a bacterium, Suitable for replication in, for example, E. coli, or in cultured mammalian, plant, can be introduced into mammalian, insect (e.g., Sf9), yeast, fungal, or other eukaryotic cell lines. This can be done.

[0164] DNA constructs prepared for introduction into a particular host must be recognized by the host. a replication system for the desired polypeptide, an intended DNA segment encoding the desired polypeptide, and a polypeptide sequence for the desired polypeptide. Transcription and translation initiation and termination sequences operably linked to the peptide-encoding segment. The term "operably linked" is defined herein as having been previously described. For example, a promoter or enhancer may regulate the transcription of a coding sequence. A signal sequence is operably linked to a coding sequence if it stimulates the expression of the gene. A is expressed as a protein precursor involved in the secretion of a polypeptide. Generally, the DNA encoding the polypeptide is operably linked to the DNA encoding the polypeptide. The DNA sequences involved are contiguous, and in the case of signal sequences, contiguous and in reading frame. However, enhancers are located in the coding sequences whose transcription they control. The ligation may be performed by inserting the fragment at or in place of a convenient restriction site. by ligation with adaptors or linkers, or by gene synthesis It is achieved.

[0165] The selection of an appropriate promoter sequence is generally selected for the expression of the DNA segment. The type of promoter sequence will depend on the host cell being used. Examples of suitable promoter sequences are well known in the art. Prokaryotic and eukaryotic promoters that are suitable for use in the genomic DNA include those described in Sambroo et al., supra. (See, e.g., Kerr and Russell, 2001). Transcriptional regulatory sequences are typically , including heterologous enhancers or promoters recognized by the host. The choice of motor sequence depends on the host, but examples include trp, lac, and phage promoters. Promoters such as tRNA promoters and glycolytic enzyme promoters are known. (See, for example, Sambrook and Russell, supra, 2014). (See e.g., 01). Expression vectors contain a replication system and transcriptional and translational regulatory sequences. , together with the insertion site for the polypeptide-encoding segment. The system is the cell (bacterial cell such as E. coli) used to produce the vector. Most plasmids and vectors function only in the cells they infect. Examples of viable cell line and expression vector combinations are available from Sambroo k and Russell (2001, supra) and Metzger et al. ( 1988) Nature 334 :31-36. For example, suitable expression vectors The vectors are yeast, e.g., S. cerevisiae, insect cells, e.g., S f9 cells, mammalian cells, e.g., CHO cells, and bacterial cells, e.g., E. coli The cells can therefore be expressed in prokaryotic or eukaryotic host cells. The cells may be cells suitable for culture in liquid medium or on solid medium.

[0166] Alternatively, the host cell may be part of a multicellular organism, such as a transgenic plant or animal. These are cells that

[0167] viral vectors The viral vector or viral gene therapy vector may comprise a viral expression vector as defined above. A vector containing the construct.

[0168] Viral vectors or viral gene therapy vectors are vectors suitable for gene therapy. Suitable vectors for gene therapy are described in Anderson 1998, Nature 392 :25-30;Walther and Stein,2000,Drugs 6 0 :249-71;Kay et al.,2001, Nat.Med. 7 :33-4 0;Russell,2000,J.Gen.Virol. 81 :2573-604; Amado and Chen, 1999, Science 285 :674-6;Fe derico,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 T her. 7 :910-3; and in the references cited therein. do.

[0169] Particularly preferred gene therapy vectors include adenoviruses and adeno-associated viruses. These vectors are used to encapsulate synovial cells and liver cells. Adenoviruses and AAV vectors infect a wide range of dividing and non-dividing cell types, including The episomal nature of the vectors after cell entry makes these vectors therapeutically effective, as indicated above. Adapt it to your application (Russell, 2000, J. Gen. Virol. 81 :2573-2604;Goncalves,2005,Virol J. 2(1): 43) AAV vectors can provide highly stable, long-term expression of transgenes. is even more preferable because it is known that t al,Blood. 2009 Jan 22;113(4):797-806) and in humans for about 10 years (Buchlis, G. et al., Blood. 2 012 Mar 29;119(13):3038-41). A preferred adenovirus vector The inhibitors reduce the host response, as reviewed by Russell (2000, supra). The gene therapy method using AAV vectors is described by Wang et al. 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, N Engl J Med. 2011 Dec 22;365(25):2357-65,Apparaily et al,Hum Gene Ther. 2005 Apr;16(4):426- 34.

[0170] Another suitable gene therapy vector is a retroviral vector. Preferred retroviral vectors for use in Lentiviral vectors infect dividing and non-dividing cells. These have the ability to stably integrate into the genome (Amado and Chen, 1999 Science 285:674-6). Lentivirus-based expression constructs Methods for the construction and use of constructs are described in U.S. Patent No. 6,165,782, ... Nos. 207,455, 6,218,181, 6,277,633 and 6,32 3,031, and Federico (1999, Curr Opin Biote chnol 10:448-53) and Vigna et al. (2000, JG ene Med 2000;2:308-16).

[0171] Other suitable gene therapy vectors include adenovirus vectors, herpes viruses, vectors, polyoma virus vectors or vaccinia virus vectors .

[0172] The gene therapy vector comprises a nucleotide sequence encoding FGF21 to be expressed, This ensures that the nucleotide sequence is operably linked to the appropriate regulatory sequences. The nodal sequence includes at least a promoter sequence. Suitable promoters for the expression of a nucleotide sequence encoding the CMV promoter include, for example, promoters such as Murine Moloney Leukemia Virus (MMLV), Rous Sarcoma Virus, or or viral long terminal repeat promoters (LTRs) from HTLV-1, etc., SV40 early promoter, CAG promoter, α1-antithrombin Psin promoter, mini / aP2 promoter, mini / UCP1 promoter , C5-12 promoter and herpes simplex virus thymidine kinase promoter Examples include:

[0173] Some inducible promoter systems can be induced by administration of organic or inorganic small molecules. It has been described that such an inducible promoter can be regulated by heavy metals. metallothionine promoters ( Brinster et al. 1982 Nature 296 :39-42;Ma yo et al. 1982 Cell 29 :99-108), RU-486( progesterone antagonists) (Wang et al. 19 94 Proc.Natl.Acad.Sci.USA 91 :8180-8184), Those regulated by steroids (Mader and White, 1993 Pro c.Natl.Acad.Sci.USA 90 :5603-5607), tetracycline Controlled by phosphorus (Gossen and Bujard 1992 Proc .Natl.Acad.Sci.USA 89 :5547-5551; U.S. Pat. No. 64,758;Furth et al. 1994 Proc.Natl.Acad .Sci.USA 91 :9302-9306;Howe et al. 1995 J .Biol.Chem. 270 :14168-14174;Resnitzky et al. 1994 Mol.Cell.Biol. 14 :1669-1679;Sh ockett et al. 1995 Proc.Natl.Acad.Sci.US A 92 :6522-6526), ​​and the tetR polynucleotide as the VP16 activation domain Chimeras composed of peptides and the ligand-binding domain of the estrogen receptor Lance activator (Yee et al., 2002, US 6,432,705) Examples include those controlled by

[0174] The gene therapy vector may further comprise a nucleotide sequence encoding an additional polypeptide. It may include.

[0175] The gene therapy vector is preferably a composition or pharmaceutical composition as defined herein. In this connection, a composition or pharmaceutical composition is a compound as defined herein above. The composition may contain a suitable pharmaceutical carrier as defined above.

[0176] Adeno-associated virus vector (AAV vector) A preferred viral vector or gene therapy vector is an AAV vector. The AAV vector used herein is preferably a recombinant AAV vector (r As used herein, "rAAV vector" refers to a vector that is capable of expressing an AAV gene. Within the protein shell of capsid proteins derived from AAV serotypes, as described in A recombinant vector containing a portion of the AAV genome that is encapsidated. The portion may be an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV The nucleotide sequence may contain inverted terminal repeats (ITRs), such as from AV5. Preferred ITRs are those of SEQ ID NO: A sequence comprising or consisting of SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR) The present invention also provides a method for producing a 5' ITR of AAV2, preferably comprising the steps of: SEQ ID NO: 48 and at least 80% (or at least 81%, 82%, 83%, 84% ,85%,86%,87%,87%,88%,89%,90%,91%,92%,93% , 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity Use of sequences with at least 80% identity to SEQ ID NO: 49 as the 3' ITR. Includes use.

[0177] The protein shell containing the capsid protein is a protein found in AAV serotypes, e.g., AAV1, AA The preferred AAV vectors are those derived from AAV V2, AAV3, AAV4, AAV5, etc. The capsid is an AAV1, AAV3, AAV8, or AAV9 capsid. is from AAV2. The protein shell is also called the capsid protein shell. The rAAV vector may also be designated as a rAAV vector encoding one, or preferably all, of the wild-type AAV genes. A functional ITR nucleic acid sequence may be deleted but still contain functional ITR nucleic acid sequences. The ITR sequences are required for replication, rescue, and packaging of AAV virions. The sequence can be the wild-type sequence or at least 80%, 85%, 90%, It may have 95%, 97%, 98%, 99% or 100% sequence identity or For example, by nucleotide insertion, mutation, deletion or substitution, so long as they remain functional. In this context, functionality means that the genome is directly packaged into the capsid shell. The ability to package and subsequently allow expression in infected host or target cells. In the context of the present invention, the capsid protein shell is a protein derived from an rAAV vector. The ITR may be of a different serotype than the HIV ITR.

[0178] The nucleic acid molecule represented by the selected nucleic acid sequence is preferably an rAAV as specified above. inserted between genomic or ITR sequences, which are operably linked to, for example, a coding sequence. The expression construct comprises an expression regulatory element and a 3'-terminal sequence linked thereto. Such nucleic acid molecules are sometimes referred to as transgenes.

[0179] "AAV helper functions" generally refer to rAAV vectors that are supplied in trans to the rAAV vector. The corresponding AAV functions required for AAV replication and packaging. The looper function replaces the missing AAV function in the rAAV vector, but not in the AAV IT It lacks R (provided by the rAAV vector genome) and AAV helper functions. The two major ORFs of AAV, namely the rep coding region and the cap coding region, Rep and Cap regions or sequences substantially functionally identical thereto. Regions are well known in the art and are described, for example, in Chiorini et al. (1999 ,J. of Virology,Vol 73(2):1309-1319) or U See US Pat. No. 5,139,941, which is incorporated herein by reference. AAV helper functions can be supplied on an AAV helper construct. Introduction of the helper construct into the host cell can be achieved by using the rAAV vectors identified herein. prior to or simultaneously with the introduction of the rAAV genome present in the host, e.g., transformation, transfection, The AAV vectors of the present invention can be generated by transfection or transduction. The looper construct, therefore, on the one hand, encodes the capsid protein sequence of the rAAV vector. The rAAV genome desired for the cell, as well as the rAAV genome present in the rAAV vector, The desired serotype combinations for replication and packaging of the virus were selected. It can be discovered.

[0180] An "AAV helper virus" is a virus that contains the necessary attachment components for AAV replication and packaging. Suitable AAV helper viruses include adenovirus, simple Examples include herpes viruses (such as HSV types 1 and 2) and vaccinia virus. The additional functions provided by this helper virus are also described in US Pat. 456, which can be introduced into the host cell via a plasmid. The documents are incorporated herein by reference.

[0181] A "transgene" is a gene or coding sequence newly introduced into a cell. Nucleic acid molecules (i.e., molecules encoding FGF21), i.e., molecules that may be present in cells, Genes that may not normally be expressed or may be expressed at insufficient levels include: In this context, "insufficient" means that the FGF21 is not present in the cell. Although the condition is manifested in a patient with a pulmonary edema, symptoms and / or diseases as defined herein still occur. In this case, the present invention allows for the overexpression of FGF21. A transgene can contain sequences that are native to the cell, sequences that do not naturally occur in the cell, This may include a combination of both. The transgene encodes FGF21 in the cell. The nucleic acid sequence specified hereinabove can be operably linked to appropriate regulatory sequences for expression of the sequence. The polypeptide may contain sequences encoding FGF21 and / or additional proteins. Preferably, the transgene does not integrate into the genome of the host cell.

[0182] "Transduction" refers to the transfer of FGF21 to recipient host cells by a viral vector. For example, transduction of a target cell by an rAAV vector of the invention refers to delivery of that vector. The term "host cell" refers to a cell that is transduced with the rAAV genome contained in the vector. "Target cell" refers to a cell into which DNA delivery occurs, such as a muscle cell of a subject. AAV vectors can transduce both dividing and non-dividing cells.

[0183] AAV vector production The production of recombinant AAV (rAAV) for transgene vectorization has been previously described. Ayuso E, et al., Curr. Gene Ther. 2010 ;10:423-436, Okada T,et al.,Hum.Gene Ther 2009;20:1013-1021, Zhang H,et al.,Hum.G ene Ther. 2009;20:922-929 and Virag T,et a See I., Hum. Gene Ther. 2009;20:807-817 These protocols may be used or adapted to generate the AAV of the present invention. In one embodiment, the production cell line contains a polynucleotide of the invention (which is adjacent to the ITRs). containing an expression cassette adjacent to the rep and cap proteins, Construct(s) providing helper function are transiently transfected. In another embodiment, the cell line stably provides helper functions and is capable of expressing the polynucleotides of the invention. otide (containing an expression cassette flanked by ITRs), and rep and cap proteins Construct(s) encoding the protein are transiently transfected. In embodiments, the cell line stably expresses the rep and cap proteins and helper functions. The cells are constantly supplied with the polynucleotides of the invention and transiently transfected. In this embodiment, the cell line stably supplies the rep and cap proteins and Polynucleotides encoding oligonucleotides and helper functions are transiently transferred In yet another embodiment, the cell line is transfected with the polynucleotide of the invention, Stable supply of p and cap proteins and helper functions. These and other Methods for making and using AAV production systems have been described in the art. yczka N, et al., US5,139,941, Zhou X, et al. ,US5,741,683, Samulski R,et al.,US6,057,1 52, Samulski R, et al., US6,204,059, Samulsk i R,et al.,US6,268,213, Rabinowitz J,et a l., US 6,491,907, Zolotukhin S, et al., US6, 660,514, Shenk T, et al., US6,951,753, Snyder r R,et al.,US7,094,604,Rabinowitz J,et a l.,US7,172,893, Monahan P,et al.,US7,201, 898, Samulski R, et al., US 7,229,823 and Ferr See Ari F, et al., US Pat. No. 7,439,065.

[0184] The rAAV genome present in the rAAV vector contains the inverted end of one of the AAV serotypes. The nucleotide sequence of the inter-terminal region (ITR) (preferably the serotype as disclosed hereinabove) AAV2), or a nucleotide sequence substantially identical thereto, or Nucleotide sequences with at least 60% identity and inserted between the two ITRs Nucleotide sequence encoding FGF21 (under the control of suitable regulatory elements) The vector genome comprises at least one of the following: It requires the use of flanking 5' and 3' ITR sequences to enable packaging. do.

[0185] The entire genomes and corresponding ITRs of several AAV serotypes have been sequenced ( Chiorini et al. 1999, J. of Virology Vol. 73, No. 2, pp. 1309-1319). These can be cloned and For example, see Applied Biosystems Inc. (Fosters, CA) The oligonucleotides were synthesized using oligonucleotide synthesizers supplied by the company Eppendorf, OH, USA. They can be produced by chemical synthesis using conventional techniques or by standard molecular biology techniques. ITRs can be cloned from the AAV viral genome or AAV IT The ITR nucleotide sequences can be excised from vectors containing R. Using biological techniques, nucleic acids encoding one or more therapeutic proteins are inserted at either end. The desired nucleotide sequence can be ligated to the AAV sequence between the ITRs. It can be replaced with a nucleotide sequence.

[0186] Preferably, the rAAV genome present in the rAAV vector encodes viral proteins. Any nucleotide sequence encoding, for example, the rep (replication) or cap (cap) sequences of AAV The rAAV genome does not contain any marker or reporter genes. genes, e.g., antibiotic resistance genes, genes encoding fluorescent proteins (e.g., gfp) or detectable by chemical, enzymatic or other methods known in the art and / or genes encoding selectable products (e.g., lacZ, aph, etc.) It may further include:

[0187] The rAAV genome present in the rAAV vector contains a nucleotide sequence encoding FGF21. The promoter sequence may further comprise a promoter sequence operably linked to the nucleotide sequence. -sequence in skeletal muscle cells and / or skeletal muscle, in liver cells and / or liver and promoters that confer expression in adipocytes and / or adipose tissue. Examples of such promoters include CMV, CAG, m, as defined herein above. ini / aP2, mini / UCP1, C5-12 and hAAT promoters. can be.

[0188] Suitable 3' untranslated sequences may also be operably linked to the nucleotide sequence encoding FGF21. A suitable 3' untranslated region may be linked to the nucleotide sequence naturally associated with the nucleotide sequence. The vector may be derived from the same gene or from a different gene, e.g., SV4 0 polyadenylation signal (SEQ ID NO: 50) and rabbit β-globin polyadenylation signal Gunal (SEQ ID NO: 51), etc.

[0189] Optionally, additional nucleotide sequences, such as signal sequences, nuclear localization signals, expression Nucleotide sequences encoding enhancers, etc., are inserted into the nucleotide sequences encoding FGF21. It may be operably linked to a nucleotide sequence(s).

[0190] Codon optimization "Codon optimization," as used herein, refers to modifying an existing coding sequence. or to design a coding sequence, e.g., Expression of the transcribed RNA molecule to improve translation in a host cell or organism Codons are a process used to improve transcription of coding sequences. Optimization may include, but is not limited to, adjusting the sequence to match the codon preferences of the expression host organism. to suit the codon preferences of the mammalian, preferably murine, canine or human, expression host. Examples include the process of selecting codons for a coding sequence, such as Codon optimization can also potentially negatively impact RNA stability and / or translation. elements (e.g., termination sequences, TATA boxes, splice sites, ribosome entry sites) sites, repeats and / or GC-rich sequences and RNA secondary structures or instability motifs Remove the (f).

[0191] In this document and in the claims, the verb "to include" is used. "prise" and its inflections mean that the item that follows the word is included. The term "items" is used in an open, non-exclusive sense to refer to, but does not exclude, any items not specifically mentioned. In addition, the verb "to consist" means "to become essentially from" may be replaced by "consist essentially of" which The viral expression constructs, viral vectors, compositions, genes as defined herein The gene therapy composition may contain additional component(s) other than those specifically identified. said additional component(s) does not alter the inherent properties of the present invention.

[0192] In addition, reference to an element by the indefinite article "a" or "an" indicates that the element is unique. Unless the context clearly requires that The indefinite article "a" or "an" therefore usually means "at least one" means.

[0193] The words "approximately" or "about" are used to describe relationships with numerical values. When used in a series (approximately 10, about 10) , preferably means that the value is within 1% of the given value 10.

[0194] All patents and references cited herein are incorporated herein by reference in their entirety. Each embodiment specified herein may be combined unless otherwise indicated. It may be combined.

[0195] The present invention is further illustrated in the following examples, which are not intended to limit the scope of the invention. It is not intended to be limiting, but merely serves to clarify the invention.

[0196] Figure legend: Figure 1. AAV9-CAG-moFGF21-dmiRT vector in C57Bl6 mice (A) AAV-CAG-moFGF21-d prevents obesity by intra-eWAT administration of FGF21. Schematic diagram of the doublemiRT vector. The expression cassette is codon-optimized under the CAG promoter. The mouse FGF21 coding sequence, as well as the 3' untranslated region of the expression cassette The four tandem repeats of the cloned miRT122a sequence and the miRT1 sequence The expression cassette contained four tandem repeats of the ITRs from AAV2. The diagram is not to scale. CAG: chicken β-actin promoter / CMV enzyme enhancer; pA:polyA. (B) Expression levels of FGF21 in metabolic tissues. C57B Codon-optimized mouse F in eWAT, iWAT, iBAT, and liver of l6 mice The expression level of the GF21 coding sequence was measured by RTqPCR, and the Rplp0 value was positive. Normalized (n = 8–11 animals / group). (C) Circulating levels of FGF21 (n = 8–11 animals) (D / E) FGF21R1 (D) and β-Klotho (E) in metabolic tissues. Expression levels of FGF in eWAT, iWAT, iBAT and liver of C57Bl6 mice Expression levels of FGF21 receptor 1 (FGF21R1) and β-Klotho were measured by RT-qPCR. (F) Body weight evolution. Body weight was measured by the RI and normalized to the Rplp0 value (n = 7 animals / group). Measurements were taken weekly (n = 8–11 animals / group). (G) Representative images of animals. (H) Tissue weights. Chow-fed C57Bl6 mice treated with AAV vectors in eWAT and HF mice eWAT, iWAT, rWAT, mWAT, iBAT and Liver weight (n = 8–11 animals / group). Analysis was performed on 10 12 AAV9-CAG-mo of vg eWA of FGF21-doublemiRT or AAV9-CAG-null vector The results were analyzed 14 weeks after intravenous administration. Results are expressed as mean ± SEM. ND, not detected. HFD, high-fat diet. AU, arbitrary units. eWAT, epididymal white adipose tissue. iWAT, Inguinal white adipose tissue. rWAT, retroperitoneal white adipose tissue; mWAT, mesenteric white adipose tissue; iBAT interscapular brown adipose tissue. *p<0.05 vs. AAV9-CAG-nul l chow, ** p<0.01 vs AAV9-CAG-null chow, * ** p<0.001 vs AAV9-CAG-null chow, $ p<0.0 5 vs AAV9-CAG-null HFD, $$ p<0.01 vs AAV9 -CAG-null HFD, $$$ p<0.001 vs AAV9-CAG-nu ll HFD.

[0197] Figure 2. AAV9-CAG-moFGF21-doublemiRT vector administered to eWAT Histological analysis of adipose tissue and liver of C57Bl6 mice intravenously treated with AAV9-C. (A) AAV9-C AG-moFGF21-doublemiRT or AAV9-CAG-null vector Chow-fed C57Bl6 mice and HFD-fed C57Bl mice were treated with α-glucan in the eWAT. Epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and scapular tissue from 6 mice Hematoxylin and eosin staining of interstitial brown adipose tissue (iBAT) and liver sections. Representative images of eWAT white adipocytes. Original magnification ×100. (B) Average area of ​​white adipocytes in eWAT (n (C) Frequency distribution of white adipocyte area in eWAT (n = 4 animals / group). The analysis is 12 vg AAV9-CAG-moFGF21-doublemiRT The study was conducted 14 weeks after intra-eWAT administration of AAV9-CAG-null vector. Expressed as mean ± SEM. HFD, high-fat diet. ** p<0.01 vs. AAV 9-CAG-null chow, *** p<0.001 vs AAV9-CAG- null chow, $$ p<0.01 vs AAV9-CAG-null HFD , $$$ p<0.001 vs AAV9-CAG-null HFD.

[0198] Figure 3. AAV9-CAG-moFGF21-doublemiRT vector administered to eWAT Energy expenditure and insulin sensitivity improved in intravenously treated C57Bl6 mice (AB) Expression levels of UCP1 (A) and Dio2 (B). UCP1 in iWAT. and Dio2 expression levels were measured by RTqPCR and normalized to Rplp0 values ​​( (n = 7 animals / group). (C) Energy metabolism. Energy expenditure (EE) was measured using an indirect open-circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were collected from AAV-injected mice. After 9 weeks of feeding, the rats were harvested during the light cycle (basal state) and the dark cycle (active phase) and measured by body weight. (n = 8–11 animals / group). (D) Liver triglyceride content (n = 8–10 animals) / group). (EF) Serum triglyceride (E) and cholesterol (F) levels (n = (8–11 animals / group). (G) Intraperitoneal insulin tolerance test. Mice were given 0.75 U insulin. / kg body weight intraperitoneal injection and blood glucose levels were measured at the indicated time points. (n = 6-11 animals / group). The test was performed 11 weeks after AAV administration. (H) Fasting Circulating insulin levels at 10 s. Unless otherwise indicated, analyses were performed 12 AAV9-CA in vg G-moFGF21-doublemiRT or AAV9-CAG-null vector The results were expressed as mean ± SEM. , high-fat diet. TG, triglycerides. Chol, cholesterol. *p<0.05 v s AAV9-CAG-null chow, ** p<0.01 vs AAV9-C AG-null chow, *** p<0.001 vs AAV9-CAG-nul l Chow, $ p<0.05 vs AAV9-CAG-null HFD, $$ p<0.01 vs AAV9-CAG-null HFD, $$$ p<0.001 vs AAV9-CAG-null HFD.

[0199] Figure 4. AAV8-CAG-moFGF21-dmiRT vector in ob / ob mice (A) Expression levels of FGF21 in metabolic tissues. Codon-optimized maize in eWAT, iWAT, iBAT and liver of ob / ob mice The expression level of the mouse FGF21 coding sequence was measured by RTqPCR, and Rplp0 (B) Circulating levels of FGF21. (C-D) Body weight (C) and weight gain. (D) Progression. Body weight was measured weekly. (E) Tissue weight. AAV vector was administered into eWAT. eWAT, iWAT, rWAT, mWAT, iBAT and Liver weight. Analysis was performed on 10 10 vg, 5×10 10 vg, 2 × 10 11 vg or 10 12 vg of AAV8-CAG-moFGF21-doublemiRT or 10 12 v The results were analyzed 16 weeks after intra-eWAT administration of 1000 mg of AAV8-CAG-null vector. Expressed as mean ± SEM. n = 7–8 animals / group. ND, not detected. AU, arbitrary. Units: eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue; rWAT, posterior Peritoneal white adipose tissue. mWAT, mesenteric white adipose tissue. iBAT, interscapular brown adipose tissue. * p<0.05 vs AAV8-CAG-null, ** p<0.01 vs A AV8-CAG-null, *** p<0.001 vs AAV8-CAG-nul l.

[0200] Figure 5. AAV8-CAG-moFGF21-doublemiRT vector administered to eWAT Improved insulin sensitivity in ob / ob mice treated intraperitoneally. (A) Intraperitoneal insulin Insulin tolerance test. Ob / ob mice were given an intraperitoneal injection of 0.75 U insulin / kg body weight. Blood glucose levels were measured at the indicated time points. The study was conducted at 9 weeks of AAV administration. (B) Fasting circulating insulin levels 2 months after AAV administration. Results are mean ± SD. Expressed as SEM, n = 7–8 animals / group. * p < 0.05 vs. AAV8 -CAG-null, ** p<0.01 vs AAV8-CAG-null, *** p<0.001 vs. AAV8-CAG-null.

[0201] Figure 6. Immunoglobulin levels of AAV8-hAAT-moFGF21 vector in ob / ob mice Intravenous administration reversed obesity and improved glucose metabolism. (A) AAV-hAAT-mo Schematic diagram of the FGF21 vector. The expression cassette contains human alpha-1 antitrypsin (hAAT). It contained a promoter and a codon-optimized murine FGF21 coding sequence. The expression cassette was flanked by ITRs from AAV2. Diagram not to scale. pA:p (B) Expression levels of FGF21. Codon-optimized FGF21 in the liver of ob / ob mice. The expression level of the mouse FGF21 coding sequence was measured by RTqPCR, and Rp (C) Circulating levels of FGF21 were normalized to lp0 values. (D-E) Body weight (C) and (D) Progression of weight gain. Body weights were measured weekly. (F) Representative images of animals. (G) Histology. Weight. eWAT, iWAT, and rWAT of ob / ob mice intravenously treated with AAV vectors AT, mWAT, iBAT and liver weights. (H) Intraperitoneal insulin tolerance test. ob / ob mice were given an intraperitoneal injection of 0.75 U insulin / kg body weight and at the indicated time points Blood glucose levels were measured in the rats. The study was performed 9 weeks after AAV administration. ) Fasting circulating insulin levels 3 months after AAV administration. Unless otherwise indicated, analyses were performed at 10 11 vg or 5×10 11vg of AAV8-hAAT-moFGF21 or 5 × 1 0 11 The study was performed 20 weeks after intravenous administration of vg AAV8-hAAT-null vector. Results are expressed as mean ± SEM. n = 9–10 animals / group. ND, not detected. U, arbitrary units. eWAT, epididymal white adipose tissue. iWAT, inguinal white adipose tissue. rW AT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue. iBAT, interscapular brown adipose tissue. adipose tissue. * p<0.05 vs AAV8-hAAT-null, ** p<0.01 vs AAV8-hAAT-null, *** p<0.001 vs AAV8-h AAT-null.

[0202] Figure 7. AAV-hAAT-moFGF21 vector in HFD-fed C57bl6 mice Long-term reversal of obesity by intravenous administration of acetaminophen. (A) Circulating levels of FGF21. (B-C) Progression of body weight (C) and weight gain (D). Body weight was measured weekly. Analysis was performed at 10 10 vg too Or 5 x 10 10 vg of AAV8-hAAT-moFGF21 or 5 × 10 10 vg The study was conducted 52 weeks after intravenous administration of the AAV8-hAAT-null vector. Values ​​are expressed as ± SEM, n = 9–12 animals / group. *** p < 0.001 v s AAV8-hAAT-null chow, $$ p<0.01 vs AAV8- hAAT-null HFD, $$$ p<0.001 vs AAV8-hAAT-n ull HFD.

[0203] Figure 8. AAV-hAAT-moFGF21 vector in HFD-fed C57Bl6 mice Long-term improvement in energy expenditure and insulin sensitivity following intravenous administration of ethanol. (A) Energy metabolism. Energy expenditure (EE) was measured by indirect open-circuit calorimetry. Oxygen consumption and Carbon dioxide production was monitored simultaneously. Data were collected 4 weeks after AAV administration, during the light cycle (basal (B) Intraperitoneal injections were taken during the night (night) and dark cycles (active phase) and adjusted for body weight. Insulin tolerance test. C57Bl6 mice were intraperitoneally injected with 0.75 U insulin / kg body weight. The test was conducted after AAV administration and blood glucose levels were measured at the indicated time points. (C) Circulating insulin levels in fasting and fed states. Results are mean values. Expressed as ±SEM, n = 9–12 animals / group. HFD, high-fat diet. * p < 0 .05 vs AAV8-hAAT-null chow, **p<0.01 vs AAV8-hAAT-null chow, ***p<0.001 vs AAV8- hAAT-null chow, $ p<0.05 vs AAV8-hAAT-nul l HFD, $$ p<0.01 vs AAV8-hAAT-null HFD, $$ $ p<0.001 vs AAV8-hAAT-null HFD.

[0204] Figure 9. Immunoglobulin levels of the AAV-hAAT-moFGF21 vector in aged HFD-fed mice. Reversal of obesity by intravenous administration. (A) Circulating levels of FGF21. (B-C) Body weight. (B) and The progression of weight gain (C). Body weight was measured weekly. 10 vg, 2 × 10 10 v g or 5 x 10 10 vg of AAV8-hAAT-moFGF21 or 5 × 10 10 The results were obtained 21 weeks after intravenous administration of the AAV8-hAAT-null vector (vg). Expressed as mean ± SEM, n = 7–8 animals / group. HFD, high-fat diet. *** p<0.05 vs AAV8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8- hAAT-null HFD. $$$ p<0.001 vs AAV8-hAAT-n ull HFD.

[0205] Figure 10. AAV-hAAT-moFGF21 vector expression in aged HFD-fed mice Intravenous administration improves energy expenditure and insulin sensitivity. (A) Energy metabolism Energy expenditure (EE) was measured by indirect open-circuit calorimetry. Oxygen consumption and carbon dioxide production were Data were collected 6 weeks after AAV administration, during the light cycle (basal state) and (B) Intraperitoneal insulin tolerance test. Aged C57Bl6 mice were given an intraperitoneal injection of 0.75 U insulin / kg body weight. Blood glucose levels were measured at the indicated time points. (C) Circulating insulin levels in fasting and fed states. Results are mean ± SEM Expressed as mean ± SD, n = 7–8 animals / group. HFD, high-fat diet. ** p < 0.01 vs AAV8-hAAT-null chow, *** p<0.001 vs AA V8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT- null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD , $$$ p<0.001 vs AAV8-hAAT-null HFD.

[0206] Figure 11. Muscles of AAV-CMV-moFGF21 vector in C57Bl6 mice (A) Schematic diagram of the AAV-CMV-moFGF21 vector. The set includes a cytomegalovirus (CMV) promoter and a codon-optimized mouse The expression cassette contained the FGF21 coding sequence. The ITRs from AAV2 flanked the expression cassette. Diagram not to scale. pA: polyA. (B) Circulating FGF21 levels. ( C-D) Progression of body weight (C) and weight gain (D). Body weight was measured weekly. Results are mean ± Expressed as SEM. n=6-7 animals / group. *p<0.05 vs. AAV1-C MV-null, **p<0.01 vs. AAV1-CMV-null. FG in the figure The F21 label refers to moFGF21 according to the figure legend.

[0207] Figure 12. FGF2 by codon optimization of the nucleotide sequence encoding human FGF21 1 Increased protein production. (A) Wild-type hFGF21 or codon-optimized human FGF Culture media of HEK293 cells transfected with three different versions of the F21 sequence hFGF21 protein levels in the soil. Results are expressed as mean ± SEM. n=3 Well / group. ND, not detected. *p<0.05 vs. non-transfected cells.

[0208] Figure 13. AAV8-CAG-moFGF21-dmiRT vector in ob / ob mice Intra-eWAT administration of acetaminophen.

[0209] Test either A, B null or FGF21-encoding AAV8 vectors (A) eWAT and (B) smears from ob / ob animals injected intra-eWAT at all doses. ) Representative image of hematoxylin-eosin stained liver tissue section. Scale bar: eW 100 μm for AT and 200 μm for liver.

[0210] C Blood glucose in the fed state.

[0211] D Blood insulin in the fed state 3 months after AAV.

[0212] The FGF21 label in the figure refers to moFGF21.

[0213] Data information: All values ​​are expressed as mean ± SEM. (A, B) n = 6 ~9 animals / group. (CH) n=4-8 animals / group. (I) n=6-8 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. Null Shooting group.

[0214] Figure 14. Effects of FGF21 gene transfer into eWAT of ob / ob mice.

[0215] A. At 11 weeks of age, mice were treated with AAV8-CAG-null vector or AAV8-CAG-moF. One of the GF21-dmiRT vectors was administered at four different doses (1 × 10 10 , 5×10 10 , 2 × 10 11 , 1×10 12 vg / mouse) injected into the eWAT of 25-week-old mice Serum adiponectin levels in b / ob animals.

[0216] B (A) qRT-PCR of the macrophage marker F4 / 80 from the same animals. Expression quantification by R C ob / ob mice receiving the AAV8-CAG-moFGF21-dmiRT vector Immunostaining of eWAT sections from the rat for the macrophage-specific marker Mac2 Representative images. n=4–8 / group. Scale bar: 200 μm.

[0217] D Liver weights in all eWAT treatment groups.

[0218] E, F (F) Liver triglycerides and glycerols in the fed state in the same cohort as in (A) and cholesterol content.

[0219] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0220] Data information: All values ​​are expressed as mean ± SEM. (A, B, D) = 4–8 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. Null injection ob / ob group.

[0221] Figure 15. ob / ob mice treated with AAV8-hAAT-moFGF21 vector Reduction of obesity and improvement of insulin sensitivity in mice.

[0222] 1 × 10 of either a null or FGF21-encoding AAV vector 11 Ma or 5 x 10 11 eWAT tissue sections obtained from ob / ob animals injected with vg / mice Representative images of hematoxylin-eosin staining of sections.

[0223] B Serum adiponectin levels in all groups.

[0224] 1 × 10 of either C null or FGF21-encoding AAV vector 11 Ma or 5 x 10 11of liver tissue sections obtained from ob / ob animals injected with vg / mice. Representative images of hematoxylin-eosin staining.

[0225] D. Blood glucose levels at the time of feeding.

[0226] E Fed serum insulin levels at 5 months after AAV.

[0227] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0228] Data information: All data are expressed as mean ± SEM. (A-C, E, G-H) n = 9-10 animals / group. *P<0.05, **P<0.01 and ***P<0. 001 vs. null-injected ob / ob group.

[0229] Figure 16. Effect of FGF21 hepatic gene transfer in ob / ob mice.

[0230] A eW from ob / ob mice that received the AAV8-hAAT-moFGF21 vector Immunohistochemistry for the macrophage-specific marker Mac2 in AT sections. Kale bar: 500 μm.

[0231] B, C Inflammatory markers F4 / 80 (B) and TNF-α ( C) Expression quantification by qRT-PCR.

[0232] D, E Liver weights (D) and (E) obtained from animals belonging to the same experimental groups as in (A). and a representative image (E).

[0233] F, G (A) Liver triglycerides and glycerols in the same cohort under fed conditions and cholesterol content.

[0234] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0235] Data information: All values ​​are expressed as mean ± SEM. (B, DF, HI) n = 9-10 animals / group. *P<0.05, **P<0.01 and ***P<0. 001 vs. null-injected ob / ob group.

[0236] Figure 17. AAV8-hAAT-moFGF21 treatment increases adipose tissue in ob / ob mice It increases the expression of genes involved in glucose uptake and thermogenesis in the rat.

[0237] A, B At 2 months of age, mice were treated with AAV8-hAAT-null vector or AAV8-hAAT- Hepatic PEPC in ob / ob mice injected with either moFGF21 vector Expression quantification by qRT-PCR of K and G6Pase.

[0238] In eWAT, iWAT, and iBAT in the same animals as in CF (A). qRT-PCR of GLUT1 (C), GLUT4 (D), HKI (E), and HKII (F) Expression quantification by PCR.

[0239] G. Relative expression of UCP1 in iBAT in the same cohort as in (A). .

[0240] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0241] Data information: All values ​​are expressed as mean ± SEM. (AG) n=9 ~10 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. Null injection ob / ob group.

[0242] Figure 18. AAV8-mediated hepatic FGF21 gene transfer counteracts HFD-induced obesity.

[0243] A. AAV8-hAAT-moFG as a young adult (upper panel) or adult (lower panel). Epididymis (eWAT), inguinal (iWA) tissue samples from mice treated with F21 vector were collected. T) and retroperitoneal (rWAT) white adipose tissue depot, liver, and quadriceps muscle weights.

[0244] B Circulating levels of FGF21 at different time points after vector administration.

[0245] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0246] Data information: All values ​​are expressed as mean ± SEM. (AD) n=7 ~10 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. Chow-fed null-injected group. # P<0.05, ## P<0.01 and ### P<0. 001 vs. HFD-fed Null-injected group. HFD, high-fat diet.

[0247] Figure 19. Hepatic FGF21 gene transfer counteracts HFD-induced obesity.

[0248] A, B belong to all experimental groups of studies conducted in young adults (A) or adults (B). A representative image of an animal.

[0249] C. Several doses of AAV8-hAAT-mo administered as young adults (left) or adults (right). Epididymal white fat (eWAT) pads obtained at sacrifice from animals treated with FGF21 A representative image of.

[0250] D. Representative images of livers obtained from animals treated as young adults (left) or adults (right). Image.

[0251] E. AAV-derived FGF21 expression in the liver of animals treated as young adults or adults qPCR was performed to detect the coding region of codon-optimized mouse FGF21 (coFGF21). This was carried out using primers that specifically detect the nucleotide sequence.

[0252] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0253] Data information: All values ​​are expressed as mean ± SEM. (E) n = 7–1 0 animals / group. HFD, high-fat diet. ND, not detected.

[0254] Figure 20. AAV8-hAAT-moFGF21-mediated signaling in iBAT and iWAT Increased energy expenditure and decreased fat accumulation.

[0255] A: Mice were fed an HFD from approximately 2 months of age, and after 2 months, mice were transfected with either a null or FGF21-encoding mouse model. Through open field testing in animals (young adults) treated with either vector Assessment of locomotor activity.

[0256] B. Heads of iBAT tissue sections from animals treated as young adults (left) or adults (right). Matoxylin-eosin staining.

[0257] C Western blotting of UCP1 content in iBAT from the same animal cohort as in (A). A representative immunoblot is shown (left). Histograms show the results of two different immunoblots. Densitometric analysis of the immunoblot is depicted (right).

[0258] D. Heads of iWAT tissue sections from animals treated as young adults (left) or adults (right). Matoxylin-eosin staining.

[0259] E. Groups of animals that received the FGF21 vector starting on HFD feeding as young adults or adults. Quantification of Phospho1 expression in iWAT by qRT-PCR.

[0260] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0261] Data information: All values ​​are expressed as mean ± SEM. (A-C) n=7 ~10 animals / group. In (E), n=4 animals / group. In (G), n=7-10 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. chow-fed N ull injection group. # P<0.05 and ### P<0.001 vs. HFD-fed null Shooting group. HFD, high fat diet.

[0262] Figure 21. Energy expenditure 10 months after gene transfer into the liver.

[0263] A Energy expenditure in a cohort of animals that started on a HFD at 2 months of age was measured using AAV8 - 10 months after hAAT-null or AAV8-hAAT-moFGF21 vector delivery Data were acquired during the light and dark cycles.

[0264] B Western blot analysis of UCP1 content in iWAT from the same animal cohort. A comparative immunoblot is shown (left). The graph shows the densitometric content of two different immunoblots. The analysis is shown (right).

[0265] C. Groups of animals that received the FGF21 vector starting on HFD feeding as young adults or adults. Relative expression levels of Serca2b and RyR2 in iWAT in the 2000-2012 study.

[0266] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0267] Data information: All values ​​are expressed as mean ± SEM. (A) n = 7–1 0 animals / group. In (B), n=4 animals / group. In (C), n=7-10 animals / group. * P<0.05, **P<0.01 and ***P<0.001 vs. chow-fed Null l injection group. ### *P<0.001 vs. HFD-fed Null-injected group. HFD, high-fat diet.

[0268] Figure 22. AAV8-hAAT-moFGF21-mediated reversal of islet hyperplasia.

[0269] A. Emptying in animals receiving the FGF21 vector starting on a HFD as young adults. Interventional glucagon levels.

[0270] B. β-cells in the group of animals that received the FGF21 vector starting on HFD feeding as adults amount.

[0271] C 5 × 10 as adults 10 vg / mouse receiving AAV8-hAAT-moFGF21 Representative images of immunostaining for insulin in pancreatic sections from animals. Scale Bar: 400 μm. Inset scale bar: 100 μm.

[0272] D 5 × 10 as young adults (upper panel) or adults (lower panel) 10 vg / mouse Insulin in pancreatic sections from animals that received AAV8-hAAT-moFGF21 Representative images of double immunostaining for IgG (dark grey) and glucagon (light grey). Kale bar: 100 μm.

[0273] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0274] Data information: All values ​​are expressed as mean ± SEM. (A-C) n=7 ~10 animals / group. In (D), n=4-5 animals / group. *P<0.05, **P<0.0 1 and ***P<0.001 vs. chow-fed Null-injected group. # P<0.05, # # P<0.01 and ### P<0.001 vs. HFD-fed Null-injected group. HFD, High fat diet.

[0275] Figure 23. Treatment with AAV8-hAAT-moFGF21 improves glucose tolerance.

[0276] A. Glucose tolerance in mice that started on a HFD as young adults and received the FGF21 vector. In groups, the test was performed after intraperitoneal injection of glucose (2 g / kg body weight).

[0277] B Serum insulin levels during the glucose tolerance test shown in (A).

[0278] Data information: All data are expressed as mean ± SEM. (A-D) n = 7 ~10 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. Chow-fed null-injected group. # P<0.05, ## P<0.01 and ### P<0. 001 vs. HFD-fed Null-injected group. HFD, high-fat diet.

[0279] Figure 24. AAV8-hAAT-moFGF21 treatment reverses WAT hypertrophy and inflammation. .

[0280] A. Young adults (left panel) or adults (right panel) fed chow or HFD. AAV8-hAAT-null or 5 × 10 10 vg / mouse AAV8-hAAT Hematoxylin of eWAT from animals administered either -moFGF21 vector- Representative images of eosin staining. Adipocytes in HFD-fed null-injected mice were larger. However, adipocytes in HFD-fed FGF21-treated animals were reduced in size. Scale bar: 100μm.

[0281] B Morphometry of WAT adipocyte area in animals treated as young adults or adults analysis.

[0282] C, D Circulating levels of adiponectin (C) and leptin (D).

[0283] E 5 x 10 as adults 10 vg / mouse receiving AAV8-hAAT-moFGF21 Immunoprecipitation of eWAT sections from different animals for the macrophage-specific marker Mac2 Immunohistochemistry. Photomicrographs show crown-like structures in the eWAT of HFD-fed null-injected animals. (arrow and inset) in the eWAT of HFD-fed FGF21-treated mice. Scale bars: 200 μm and 50 μm (inset).

[0284] FH Inflammation in animals receiving FGF21 vector starting on HFD as adults qRT-PCR of disease markers F4 / 80 (F), IL1-β (G), and TNF-α (H) Expression quantification by R.

[0285] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0286] Data information: All values ​​are expressed as mean ± SEM. In (B), n = 4 animals n = 7-10 animals / group. *P<0.05, **P<0.01 and and ***P<0.001 vs. chow-fed Null-injected group. # P<0.05, ## P <0.01 and ### P<0.001 vs. HFD-fed Null-injected group. HFD, high fat Fatty food.

[0287] Figure 25. Adipocyte size and proliferation in AAV8-hAAT-moFGF21-treated animals and inflammation.

[0288] A. Chow or HFD feeding was initiated as young adults (top graph) or adults (bottom graph). Starting with AAV8-hAAT-null or 5 × 10 10 vg / mouse AAV8-hA Frequency of adipocyte area in animal groups receiving either the AT-moFGF21 vector cloth.

[0289] B Mac2 immunohistochemistry in eWAT of animals initiated as young adults. Crowns formed by macrophage infiltration in the eWAT of D-fed null-injected mice The cytoplasmic structures are indicated by arrows. Scale bars: 200 μm and 50 μm (inset). .

[0290] Inflammatory markers F4 / 80, C in the same animal cohort as in C-E (B). Relative expression levels of D68 and TNF-α by qRT-PCR.

[0291] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0292] Data information: All values ​​are expressed as mean ± SEM. In (A), n = 4 animals. / group. (CE) n=7-10 animals / group. ***P<0.001 vs. chow Null-injected feeding group. ### P<0.001 vs. HFD-fed Null-injected group. HFD, High fat diet.

[0293] Figure 26. Treatment with a vector encoding FGF21 ameliorates hepatic steatosis and liver inflammation. To make.

[0294] A. Chow or HFD-fed mice were infected with AAV8-hAAT-null or 5 × 10 10 Animals administered either the AAV8-hAAT-moFGF21 vector or the AAV8-hAAT-moFGF21 vector Representative images of hematoxylin-eosin stained liver sections obtained from HFD. AAV significantly induces lipid droplet accumulation in both young and adult mice. Treatment with 8-hAAT-moFGF21 restored this. Scale bar: 100 μm.

[0295] B, C Liver triglyceride and cholesterol contents during feeding in the same animal cohort amount.

[0296] D HFD fed with AAV8-hAAT-null or 5 × 10 10 vg / mouse Macrophages in liver sections from animals receiving the AAV8-hAAT-moFGF21 vector Immunostaining for the size-specific marker Mac-2. Arrows indicate the presence of crown-like structures. Scale bars: 200 μm and 50 μm (inset).

[0297] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0298] Data information: All values ​​are expressed as mean ± SEM. (BC) n=7 ~10 animals / group. **P<0.01 and ***P<0.001 vs. chow-fed Nu ll injection group. ## *P<0.01 vs. HFD-fed Null-injected group. HFD, high-fat diet.

[0299] Figure 27. AAV8-hAAT-moFGF21-mediated reversal of liver fibrosis.

[0300] 5×10 10 vg / mouse AAV8-hAAT-null or AAV8-hAAT Masson's triclosan in HFD-fed animals receiving either the -moFGF21 vector or the -moFGF21 vector Analysis of liver fibrosis through chromatin staining. AAV8-hAAT-moFGF21 treatment (right panel) ) is a readily detectable color in animals treated with the null vector (left panel). The detection of FGF2 fibers (blue) was significantly reduced. Scale bar: 50 μm. The label 1 refers to moFGF21 according to the legend of this figure.

[0301] Figure 28. AAV8-hAAT-moFGF21 treatment ameliorates liver fibrosis.

[0302] A 5×10 10 vg / mouse AAV8-hAAT-null or AAV8-hAA Picrosirius in HFD-fed animals receiving either the T-moFGF21 vector Analysis of liver fibrosis through staining. AAV8-hAAT-moFGF21 treatment (right panel) , readily detectable collagen in animals treated with the null vector (left panel). The detection of fibers (black) was significantly reduced. Scale bar: 50 μm.

[0303] B, C. Mice were fed a HFD as young adults (B) or adults (C) and administered the FGF21 vector. Quantification of collagen 1 expression in the liver by qRT-PCR in animals receiving IFN-γ.

[0304] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0305] Data information: All values ​​are expressed as mean ± SEM. (BC) n=7 ~10 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. Chow-fed null-injected group. # P<0.05 and ### P<0.001 vs. HFD Null-fed group. HFD, high-fat diet.

[0306] Figure 29. No bone abnormalities were observed in animals treated with AAV8-hAAT-moFGF21. The long-term effects of FGF21 gene transfer on bone were investigated in young adults or in adults with the most severe bone loss. High dose (5 × 10 10 AAV8-hAAT-moFGF21 vector (vg / mouse) Comparison of HFD-fed mice treated with null-injected chow or HFD-fed animals Tested by.

[0307] A Total length from tip of nose to base of tail.

[0308] B Tibial length.

[0309] administered either CO null or FGF21-encoding AAV vectors. Tibial epiphysis (CJ) obtained from FD-fed mice at the time of sacrifice, i.e., when the animals were 18 months old. ) and microcomputed tomography (μCT) analysis of the diaphysis (KO).

[0310] Levels of circulating IGFBP1 (P) and IGF1 (Q) measured by P, Q ELISA Bell.

[0311] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0312] Data information: All data are expressed as mean ± SEM. (A, PQ) = 7-10 animals / group. (BO) n = 4 animals / group. **P<0.01 and ** *P<0.001 vs. chow-fed Null-injected group. HFD, high-fat diet; BMD, bone mineral density bone mineral density; BMC, bone mineral content; BV, bone volume; BV / TV, bone volume / tissue volume ratio BS / BV, bone surface / bone volume ratio; Tb.N, trabecular number; Tb.Th, trabecular width; Tb.Sp , trabecular spacing.

[0313] Figure 30. C57Bl6 mice treated with AAV8-hAAT-moFGF21 vector Analysis of glycemic profiles in rats. Blood glucose levels were assessed under fed conditions. AV, 5×10 10 vg or 2×10 11 vg AAV8-hAAT-moFGF21 (n = 13 and 15, respectively) or 2 × 10 11 AAV8-null vector in vg (n=15) IV administration. Treatment with STZ, streptozotocin (5×50 mg / kg) Results are mean values ​​+ SEM. * p<0.05; *** p<0.001 vs. A AV8-hAAT-Null. FGF21 labeling in the figure follows the legend of this figure. Refers to moFGF21.

[0314] Figure 31. Gene transfer of FGF21 into skeletal muscle of healthy animals.

[0315] A 3×10 11 vg / mouse AAV1-CMV-Null or AAV1-CMV- Either of the moFGF21 vectors was injected into the skeletal muscle of healthy animals fed a chow diet. Circulating levels of FGF21 measured after 40 weeks.

[0316] B. AAV1-CMV-Null or AAV1-CMV-moFGF21 vector was administered to the muscle AAV-derived FGF21 expression in muscle and liver of healthy animals injected intramuscularly.

[0317] C. Weight evolution during 40 weeks of follow-up.

[0318] D Tissue wet weights of different muscles, fat pads and liver.

[0319] E, F Liver triglyceride and cholesterol contents in the fed state.

[0320] G Fed serum insulin levels.

[0321] H is assessed and initiated via intraperitoneal injection of insulin (0.75 units / kg body weight). Insulin sensitivity expressed as a percentage of blood glucose.

[0322] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0323] Data information: All values ​​are expressed as mean ± SEM. (AH) n=5 -7 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. N ull injection group.

[0324] Figure 32. AAV1-mediated skeletal muscle FGF21 gene transfer suppresses HFD-induced obesity and insulin resistance. Counteracts thrombolytic resistance.

[0325] A, B Body weight (A) and weight gain of animals treated with AAV1-CMV-moFGF21 (B) Progression of the treatment. C57Bl6 mice were fed a HFD for approximately 12 weeks, and then 3 × 10 11 vg / mouse administered the AAV1-CMV-moFGF21 vector. Obese mice and control chow-fed mice were 3 × 10 11 vg AAV1-C Received MV-null.

[0326] C Circulating levels of FGF21 at different time points after vector administration.

[0327] D, E Fasting blood glucose (D) and α in the same groups of animals as in (A, B). and fed serum insulin (E) levels.

[0328] F. Insulin sensitivity was measured after intraperitoneal injection of insulin (0.75 units / kg body weight). , were determined in all experimental groups. Results are expressed as a percentage of the starting blood glucose level. The calculation was made as follows:

[0329] The FGF21 label in the figure refers to moFGF21 according to the legend of this figure.

[0330] Data information: All values ​​are expressed as mean ± SEM. (AF) HFD Fed mice n=10 animals / group; chow-fed mice n=5 animals / group. ***P<0. 001 vs. HFD-fed null-injection group.

[0331] Figure 33. FGF2 by codon optimization of the nucleotide sequence encoding human FGF21 Increase in circulating levels in vivo. Hydrodynamic transfection of plasmids encoding three different variants of the GF21 sequence Circulating levels of hFGF21 in C57B16 mice treated with IFN-γ. Results are mean ± SEM n = 9–10 mice / group. ND, not detected. Negative control *p<0.05 vs. untreated mice.

[0332] Figure 34. hAAT-moFGF21, CAG-moFGF21-doublemiRT and in vitro FGF21 expression levels induced by the CMV-moFGF21 expression cassette. (A) WT mouse FGF21 coding region under the control of the EF1a promoter. Codon-optimal sequence (EF1a-mFGF21) or under the control of the CMV promoter Modified mouse FGF21 coding sequence (CMV-moFGF21) or miRT Four tandem repeats of the 122a sequence and four tandem repeats of the miRT1 sequence A codon-optimized mouse FGF21 code under the control of a CAG promoter, accompanied by A plasmid encoding the double miRT coding sequence (CAG-moFGF21-doublemiRT) was inserted. FGF21 expression levels in HEK293 cells transfected with α- and β-actin. and C) Intracellular FGF21 protein content in the same cells as in (A). (C) FGF21 protein levels in the culture medium. (D) Regulation of the EF1a promoter. The WT murine FGF21 coding sequence (EF1a-mFGF21) under Codon-optimized murine FGF21 coding sequence under the control of the CMV promoter C2C1 transfected with a plasmid encoding the sequence (CMV-moFGF21) (E) Expression levels of FGF21 in W2 cells under the control of the EF1a promoter. The mouse FGF21 coding sequence (EF1a-mFGF21) or hAAT promoter Codon-optimized mouse FGF21 under the control of motor (hAAT-moFGF2) FGF21 in HepG2 cells transfected with a plasmid encoding 1) Expression levels of wt and codon-optimized FGF21 coding sequences. The results were expressed as mean ± SEM. n=3 wells / group. ND, not detected. *p<0.05 vs. control. ## p<0.001 vs. EF1a-mFGF21.

[0333] Figure 35. Expression of hAAT-moFGF21 and CMV-moFGF21 cassettes Increase in hepatic FGF21 expression and circulating FGF21 levels in vivo. (A) Elongation factor 1 The WT mouse FGF21 coding sequence under the control of the a(EF1a) promoter ( EF1a-mFGF21) or codon-optimized under the control of the CMV promoter Mouse FGF21 coding sequence (CMV-moFGF21) or hAAT promoter A codon-optimized mouse FGF21 coding sequence (hAAT- C57Bl6 mice hydrodynamically injected with a plasmid encoding moFGF21 Expression levels of FGF21 in mouse liver. qPCR was performed on wild-type and codon-optimized FGF21. This was performed using primers that detect both the FGF21 coding sequence and the FGF21 nucleotide sequence. Circulating FGF21 levels in the same cohort as in A). Results are mean ± SEM Expressed as mean ± SD. n=5 mice / group. **p<0.01 vs. EF1a-mFG F21 Figure 36. AAV8-hAAT-moFGF21-mediated hepatic FGF21 expression and FGF Increase in circulating levels of 21 in vivo. (A) 1 × 10 10 vg, 2 × 10 10 vg or 5×10 10 vg, WT mouse under the control of the elongation factor 1a (EF1a) promoter. An AAV8 vector (AAV8-EF1a-m) encoding the FGF21 coding sequence FGF21) or codon-optimized mouse FGF under the control of the hAAT promoter C57Bl6 mice intravenously administered F21 (AAV8-hAAT-moFGF21) FGF21 expression levels in the liver. qPCR was performed to measure the expression levels of wt and codon-optimized FGF21. (B) This was performed using primers that detect both the coding sequence of the Circulating FGF21 levels in the same cohort as in the AAV study. Analysis was performed 2 weeks post-AAV. Results are expressed as mean ± SEM. n = 4–5 mice per group. Control, untreated Mice. **p<0.01 and ***p<0.001 vs. control. # p<0.01 and ### p<0.001 vs. AAV8-EF1a-mFG F21 Figure 37. AAV8-CAG-moFGF21-dmiRT-mediated adipose FGF21 expression In vivo increase (AB) 2 × 10 10 vg, 5×10 10 vg or 1×10 11 WT mouse FGF21 under the control of the elongation factor 1a (EF1a) promoter in vg AAV8 vector encoding the coding sequence (AAV8-EF1a-mFGF21) or four tandem repeats of the miRT122a sequence and four tandem repeats of the miRT1 sequence. Codon-optimized mouse model under the control of the CAG promoter with indem repeats An AAV8 vector encoding the FGF21 coding sequence (AAV8-CAG-moF C57Bl6 mice administered either GF21-doublemiRT or GF21-doublemiRT in the eWAT Expression levels of FGF21 in eWAT (A) or liver (B) of wt Primers were used to detect both the FGF21 coding sequence and the codon-optimized FGF21 coding sequence. Analysis was performed 2 weeks after AAV. Results are expressed as mean ± SEM. n = 4-5 mice / group. Control, untreated mice. eWAT, epididymis (epididymis). *p<0.05, **p<0.01, and Figure 38. Induction of FGF21 expression in skeletal muscle by AAV1-CMV-moFGF21 In vivo growth: (AB) 5 x 10 10 vg, 1×10 11 vg or 3×10 11 v g, WT mouse FGF21 gene under the control of the elongation factor 1a (EF1a) promoter. AAV8 vector (AAV8-EF1a-mFGF21) encoding the FGF2-binding sequence Codon-optimized murine FGF21 coding sequence under the control of the CMV promoter Either of the AAV1 vectors encoding the FGF21 sequence (AAV1-CMV-FGF21) was administered to the muscle. Intramuscular administration of FGF21 in the quadriceps muscle (A) or liver (B) of C57Bl6 mice. Expression levels. qPCR was performed to quantify the expression of the wt and codon-optimized FGF21 coding sequences. The analysis was performed using primers that detect both. The analysis was performed 2 weeks after AAV. The results were Expressed as mean ± SEM. n = 4-5 mice / group. Control, untreated mice * p<0.05, ** p<0.01 and *** p<0.001 vs. control Troll. # p<0.05, ## p<0.01 and ### p<0.001 vs . AAV8-EF1a-mFGF21. [Example]

[0334] General Procedures of the Examples Target characteristics Male C57Bl / 6J mice and B6.V-Lep ob / OlaHsd(ob / ob ) mice were used. The mice were fed a standard diet (2018S Teklad Global Diet ts (registered trademark), Harlan Labs., Inc., Madison, WI, US ) or high-fat diet (TD.88137 Harlan Teklad Madison, The rats were fed free access to 12-hour light-dark cycle (lights on at 8:00 am) and The mice were kept under a constant temperature (22°C ± 2°C). The anesthetic isoflurane (IsoFlo®, Abbott Laboratories) The animals were anesthetized using an anesthesia kit (U.S.A., Abbott Park, IL, US) and decapitated. The tissue was excised and stored at -80°C or in formalin until analysis. , Ethics at the Universitat Autonoma de Barcelona s Committee for Animal and Human Experiments This information has been approved by the entation.

[0335] Recombinant AAV vectors Single-stranded AAV vectors of serotype 1, 8, or 9 were triple transfected in HEK293 cells. The cells were prepared by transfection according to standard methods (Ayuso, E. et al. .,2010. Curr Gene Ther. 10(6) :423-36). cell 10 roller bottles (850cm 2 , Flat; Corning (trademark), Sig DMEM in 100% ethanol (Ma-Aldrich Co., Saint Louis, MO, US) The cells were cultured in 10% FBS until they reached 80% confluence, and the AAV2 ITR-adjacent expression A plasmid carrying a cassette containing the AAV2 rep gene and AAV serotypes 1, 8, or The helper plasmid carrying the cap gene of 9 and the adenovirus helper function The plasmid carrying the gene was co-transfected by the calcium phosphate method. The transgenes were: 1) miRT12 cloned into the 3' untranslated region of the expression cassette; 2a sequence (5'CAAACACCATTGTCACACTCCA3') (SEQ ID NO: 12) and the miRT1 sequence (5'TTACATACTTCTTTA CATTCCA3' (SEQ ID NO: 13) Gallovirus (CMV) early enhancer / chicken beta-actin (CAG) promoter 2) CMV promoter; or 3) human α1-antitrypsin promoter ( Codon-optimized mouse, canine, or human FGF2 driven by human AAT The sequences were those encoding FGF21 or wt FGF21. A null vector was created using a non-coding plasmid carrying a promoter. AAV was purified by a polyethylene glycol precipitation step and two successive cesium chloride precipitations. The purified product was purified using an optimized method based on a CsCl gradient. The CsCl-based protocol for generation of empty AAV capsids as well as DNA and proteins The protein impurities were dramatically reduced (Ayuso, E. et al., 2010. Cu rr Gene Ther. 10(6) :423-36). Purified AAV vectors were The virus was dialyzed against S, filtered, and stored at -80°C. The viral genome titer was determined by comparing the standard curve with the The protocol described for the AAV2 reference standard using linearized plasmid DNA was The results were determined by quantitative PCR according to the method described in Lock M, et al., Hum. G ens Ther. 2010;21:1273-1285). The vector is The construct was constructed according to molecular biology techniques well known in the field.

[0336] In vivo eWAT administration of AAV vectors Use an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). Mice were anesthetized using a syringe. Laparotomy was performed to remove the epididymal white adipose tissue. AA in PBS containing 0.1% Pluronic® F68 (Gibco) The V vector was resuspended and injected directly into the epididymal fat pad. The mice were injected with 50 μL of AAV solution twice (one injection near the testis and the other near the testis). (in the center of the fat pad). Rinse the abdomen with sterile saline solution and use a two-layer approach Closed.

[0337] Systemic administration of AAV vectors Add an appropriate amount of AAV solution to 200 μL of 0.001% Pluronic® The solution was diluted in PBS and injected manually into the lateral tail vein without applying pressure at the moment of injection. Before the experiment, the animals were heated under a 250W infrared heat lamp (Philips NV, Amsterdam). ,NL) for a few minutes to dilate the blood vessels, making the tail vein more visible and easily accessible. A plastic restrainer (Harvard Apparatus, Holli The animals were restrained for injection using a suitable restraining device (Ston, MA, US). Therefore, no anesthesia was used. Animals were injected using a 30-gauge needle.

[0338] Intramuscular administration of AAV vectors Use an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). Mice were anesthetized using a syringe. The hind limbs were shaved, and a total volume of 180 μl of vector was injected into the quadriceps of each hind limb. 6 sites placed in the gastrocnemius and tibialis cranealis muscles The doses were administered intramuscularly at separate injection sites.

[0339] Immunohistochemical and morphometric analysis Tissues were fixed in formalin (Panreac Quimica) for 24 hours and then paraffinized. The tissue samples were embedded in PEG and sectioned. The tissue samples were stained with hematoxylin and eosin. Image analysis Software (analySIS 3.0; Soft Imaging System, Center Valley, PA, EEUU) and 12 plates per animal. , a Nikon Eclipse E800 microscope connected to a video camera with a monitor ( Hematoxylin / Etch (Nikon, Tokyo, Japan) was used to obtain the hematoxylin / etchnem (obtained at 10×). Determine the area of ​​adipocytes in the osin WAT image and calculate the area of ​​each adipocyte in μm 2 was quantified by The mean adipocyte area was calculated for each experimental group, and the distribution of adipocytes according to size classification was compared. Four animals per group were used, with at least 25% of the animals per group. 0 adipocytes were analyzed.

[0340] immunohistochemistry Tissues were fixed in 10% formalin for 12–24 hours, embedded in paraffin, and sectioned. The sections were stained with rat anti-Mac2 antibody (1:50; CL8942AP; Cedarlane). , guinea pig anti-insulin antibody (1:100; I-8510; Sigma-Aldric h) or rabbit anti-glucagon antibody (1:100; 219-01; Signet Lab The sections were incubated overnight at 4°C with biotinylated rabbit anti-rat antibody (1:300 ;E0467; Dako), goat anti-rabbit IgG antibody (Alexa Fluor 568 conjugate) (1:200; A11011; ThermoFisher), goat anti-mouse Lumot IgG antibody (Alexa Fluor 488 conjugate) (1:300; A11073; ThermoFisher) or rabbit anti- peroxidase-conjugated Guinea pig antibody (1:300; P0141; Dako) was used as the secondary antibody. ABC A peroxidase kit (Pierce) was used for immunodetection, and sections were then transferred to a Mayer's slide. Counterstaining was performed in acetoxylin. Hoechst (B2261; Sigma-Aldrich) Picrosirius red staining and Masson's trichrome staining were used for nuclear counterstaining of fluorescent specimens. Fibrosis was assessed using chrome staining. The percentage of beta cell area in the pancreas was calculated as: In two insulin-stained sections separated by 200 μm, insulin+ cells in one section The area of ​​the pancreas was analyzed by dividing the area of ​​the pancreas by the total area of ​​the pancreas in that section. Calculated by multiplying pancreatic weight by the percentage beta cell area as described previously. (Jimenez et al., 2011).

[0341] RNA analysis From fat depots or liver, respectively, QIAzol Lysis Reagent (Q iagen NV, Venlo, NL) or Tripure isolation r eagent(Roche Diagnostics Corp.,Indianapo lis, IN, US), and RNeasy Lipid Tissue Miniki Total RNA was obtained by using a PCR kit (Qiagen NV, Venlo, NL). To remove residual viral genomes, total RNA was purified with DNAse I (Qiagen NV, Ve For RT-PCR, 1 μg of RNA sample was transcribed. iptor First Strand cDNA Synthesis Kit(04 379012001, Roche, California, USA) Real-time quantitative PCR was performed using a SmartCycler II (registered trademark) (Cephe id, Sunnyvale, USA) and EXPRESS SYBRGreen qP CR supermix(Invitrogen(trademark), Life Technolo The data was analyzed using R Normalized to plp0 values ​​and analyzed as previously described (Pfaffl, M., N Ucleic Acids Res. 2001;29(9):e45).

[0342] Hormone and metabolite assays Blood glucose levels were measured using a Glucometer Elite™ analyzer ( FGF21 was measured using a 1000kJ / kg FGF21 antibody (Bayer, Leverkusen, Germany). Circulating levels of FGF- 21 ELISA kit (MF2100, R&D systems, Abingdon, UK) Serum insulin concentrations were determined by Rat Insulin ELISA. Deutsch assay (90010, Crystal Chem INC. Downers The lipids were extracted from the tissues using a 1000-kJ / kg ELISA kit (Grove, IL 60515, USA). To do this, weigh approximately 100 mg of frozen sample and dissolve it in 15 ml of chloroform:methanol (2:1). The mixture was homogenized in 0.05% H2SO4. The aqueous phase was separated and left overnight at 4°C. The lipid phase was removed using a Pasteur pipette and 1 ml of the lower lipid phase was placed in a glass tube. 1 ml of chloroform and 1% Triton X-100 solution were added to the glass tube. The tube is then incubated in a bath at 90°C to evaporate the chloroform. The use of a mixture of chloroform and Triton X-100 ensured that any residual The aqueous particles were removed from the lipid phase by rinsing the walls of the tube with chloroform after evaporation. The sample was concentrated and heated again at 90°C to evaporate the chloroform. Once completely dried and concentrated, add 500 μl of H20 miliQ at 37°C. The triglyceride content was finally determined by the commercial product GPO-PAP (Roch The results were determined using the NIRS Diagnostics (Basel, Switzerland). Serum triglycerides and cholesterol were measured using enzymatic assay kits (Horiba-A All raw materials were quantified spectrophotometrically using a fluororesin (BX, Montpellier, France). Chemical parameters were measured using a Pentra 400 Analyzer (Horiba-ABX ) was determined.

[0343] Blood glucose was determined using a Glucometer Elite™ (Bayer). Glucagon levels were measured using a glucagon radioimmunoassay (#GL-32K, EMD Adiponectin, leptin, and IGFBP1 were measured using a 1000kJ / kg / day (Millipore). and IGF1 were measured using the Mouse Adiponectin ELISA kit ( 80569,Crystal Chem), Mouse Leptin ELISA kit (90030, Crystal Chem), IGFBP1 (Mouse) ELISA Kit (KA3054, Abnova) and m / r IGF-I-ELISA kit ( The determination was performed using E25 (Mediagnost).

[0344] Insulin tolerance test For the insulin tolerance test, insulin (0.75 IU / kg body weight; Humulin Regular; Eli Lilly, Indianapolis, IN) Insulin was injected intraperitoneally into fed mice. The glucose concentration in the blood samples obtained from the blood samples was determined.

[0345] Glucose tolerance test Conscious mice were fasted overnight (16 hours) and given an intraperitoneal glucose infusion (2 g / kg body weight). Blood glucose was measured in tail vein blood samples at the indicated time points. At each time point, venous blood was collected from the tail vein using a tube (Microvette® CB 300 The samples were collected in a centrifuge and immediately centrifuged to separate the serum, which was then used for the analysis. Insulin levels were measured.

[0346] Oxygen measurement Indirect open circuit calorimeter (Oxylet, Panlab, Cornella, Spain) Using this system, oxygen consumption and carbon dioxide production were simultaneously monitored in eight metabolic chambers. The mice were individually housed and allowed to acclimate in metabolic chambers for 24 hours, and then housed in individual cages for a further 24 hours. Data were collected for 3 minutes every 15 minutes. Data were taken from the light and dark cycles. To calculate energy expenditure, the Met data provided by the manufacturer were used. Abolism software was used.

[0347] Transfection of HEK293, C2C12 and HepG2 cells Cells were cultured in 24-well plates and 0.8 μg of DNA / well was added to Lipofectamine-containing media. Transfection was performed using ctamine 2000 according to the manufacturer's instructions (T hermo Fisher Scientific).

[0348] bone analysis Bone volume and structure were evaluated by μCT. Mouse tibiae were treated with neutral buffered formalin (10% ) and scanned with an eXplore Locus CT scanner (General Electric Scanning was performed using a CT (microscope) at a resolution of 27 microns. Four mice per group The trabecular bone in 1 mm3 of the proximal tibial epiphysis and 1.8 mm3 of the cortical tibial diaphysis was analyzed. Parameter MicroView 3D Image Viewer & Analyse The tibia length was calculated using the tibial is tool. Measurements were taken from the medial malleolus (medial malleolus eminence) to the medial malleolus.

[0349] Western blot analysis iWAT and iBAT were lysed in QIAzol Lysis Reagent (Qiagen) ) and the protein fraction was separated from the organic phase according to the manufacturer's instructions. Proteins were separated by 12% SDS-PAGE and purified using rabbit polyclonal anti-UCP1 antibody (ab10983; Abcam) and rabbit polyclonal anti-α-tubulin anti The antibody (ab4074; Abcam) was used for immunoblotting analysis. Performed using ECL Plus detection reagent (Amersham Biosciences) did.

[0350] Open field test Open field testing was performed from 9:00 am to 1:00 pm as previously reported. The study was conducted during the period (Haurigot et al., 2013). Briefly, animals were placed in a horizontal position. and a brightly lit chamber where two bundles of light intersect to detect vertical motion ( The rats were placed in the center of a 41 × 41 × 30 cm (LE 8811; Panlab). The total distance traveled was recorded using a video tracking system. The evaluation was performed using a computer program (SMART Junior; Panlab).

[0351] statistical analysis All values ​​are expressed as mean ± SEM. Differences between groups were evaluated using Student's t test. Comparisons were made by . Differences were considered significant at p<0.05.

[0352] Example Example 1. AAV-CAG-moFGF21-dmiRT in C57B16 mice Prevention of obesity and diabetes by intracellular administration of vectors in the eWAT The present inventors investigated the effect of FGF21 on the adipose tissue of 8-week-old male C57Bl6 mice. The therapeutic potential of AAV-mediated genetic engineering to prevent obesity and diabetes was evaluated. 1 2 CAG encompassing the target sites of miR122 and miR1 in the viral genome (vg) Codon-optimized murine FGF21 coding sequence under the control of a ubiquitous promoter The AAV9 vector encoding the CAG-moFGF21 doublet sequence (AAV9-CAG-moFGF21-doublet) emiRT) (Fig. 1A) into the eWAT (eWAT: epididymal white adipose tissue) Fat-specific overexpression of F21 (Fig. 1B) was similarly associated with high secretion of the protein into the bloodstream (Fig. 1 C) AAV9-CAG-moFGF21-doublemiRT-treated mice We also used AAV9-CAG-null vectors (which retained comparable infectivity but did not carry the transgene). FGF2 receptor 1 (FGF2) expression in eWAT was significantly increased compared to a vector encoding no FGF2 receptor 1 (FGF2) 1R1) (Fig. 1D) and β-Klotho (FGF21 co-expression) in adipose tissue and liver. The CAG-moFGF21-doublemiR showed overexpression (Figure 1E). The T construct is contained in SEQ ID NO: 32 and the CAG-null construct is contained in SEQ ID NO: Included in No. 31.

[0353] Mice fed a chow diet after AAV-mediated FGF21 gene transfer into eWAT showed weight loss (Figures 1F and 1G). When challenged with a high-fat diet (HFD), Animals that overexpressed FGF21 in adipose tissue remained lean throughout the experiment, whereas AAV 9-CAG-null treated mice became progressively obese (Figures 1F and 1G). In response to lower body weight of chow-fed AAV9-CAG-moFGF21-doublet mice, emiRT-treated mice and HFD-fed AAV9-CAG-moFGF21-doublet All emiRT-treated mice showed reduced fat depot and liver weight (Fig. 1H).

[0354] Histological analysis of white adipose tissue by hematoxylin-eosin staining revealed that eWAT and iW Reduction in white adipocyte size in AT (iWAT: inguinal white adipose tissue) and iWAT revealed multiple multilocular fat cells in the depot, indicating that browning of this depot had occurred. This suggests that the AAV9-CAG-moFGF21-doublet We further confirmed the reduction in the average area of ​​white adipocytes in emiRT-treated mice (Figure 2B The frequency distribution of white adipocyte area also differed between groups. -CAG-moFGF21-doublemiRT treated mice and HFD-fed AAV9 -CAG-moFGF21-doublemiRT treated mice showed an increase in the number of small adipocytes The HFD-fed mice exhibited increased numbers of adipocytes and fewer large adipocytes (Figure 2C). White adipose tissue in AV9-CAG-moFGF21-doublemiRT-treated mice The frequency distribution of the cyst area was almost identical to that of the chow-fed AAV9-CAG-null treated animals. The results were almost identical (Fig. 2C). Therefore, in AAV9-CAG-null treated mice, The HFD-induced hypertrophy of adipocytes observed in FGF21-overexpressing mice was Overexpression of UCP1 and Dio2 in iWAT (Figures 3A and 3B) prevented , chow-fed AAV9-CAG-moFGF21-doublemiRT-treated mice and and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice The browning of iWAT in the rats was further confirmed.

[0355] Histological analysis of iBAT (interscapular brown adipose tissue) has demonstrated lipid accumulation in this depot. However, compared with AAV9-CAG-null mice, chow-fed AAV9-CAG-m oFGF21-doublemiRT treated mice and HFD-fed AAV9-CAG-m The results showed that the oFGF21-doublemiRT-treated mice showed less leukemia (Figure 2 A) In response to this result and the browning of iWAT, HF during the light and dark cycles Energy of D-fed AAV9-CAG-moFGF21-doublemiRT-treated mice The consumption of α-glucan (α-glucan) in the HFD-fed AAV9-CAG-null mice (Fig. 3C) was higher than that in the HFD-fed AAV9-CAG-null mice. Overall, these data support the AAV9-CAG-moFGF21-double This suggests that miRT-treated mice had improved thermogenic activity.

[0356] Liver tissue sections showed that AAV9-CAG-nul Decreased lipid accumulation in hepatocytes of mice overexpressing FGF21 compared with l-treated mice Therefore, HFD-fed AAV9-CAG-moFGF21-do In ublemiRT-treated mice, their liver triglyceride (TG) content was normalized. In parallel, TG, total cholesterol, HDL-cholesterol, and L Circulating levels of DL-cholesterol were significantly elevated in HFD-fed mice overexpressing FGF21. The results were normalized (Figures 3E and 3F).

[0357] HFD-fed mice overexpressing FGF21 were compared with HFD-fed AAV9-null-treated mice. chow-fed AAV9-CAG-moFG mice were more insulin sensitive than control mice (Figure 3G). F21-doublemiRT treated mice and HFD fed AAV9-CAG-moFG All F21-doublemiRT-treated mice showed no significant differences in their AAV9-CAG-nu showed a decrease in circulating insulin levels compared to their ll-treated counterparts (Fig. 3H).

[0358] Example 2. AAV-CAG-moFGF21-dmiRT vector in ob / ob mice Intra-eWAT administration of methicone reverses obesity and improves glucose metabolism The present inventors conducted a fat-reducing study using FGF21 in 11-week-old male ob / ob mice. The antidiabetic and antiobesogenic therapeutic potential of AAV-mediated genetic engineering of tissues was evaluated. These mice lack leptin signaling and are a widely used treatment for obesity and diabetes. To achieve this goal, a dose-response study was performed. Mice were given four different doses (10 10 vg, 5×10 10 vg, 2 × 10 11 vg or 10 12 vg) AAV8-CAG-moFGF21-doublemiRT vector ( As a control, ob / ob animals were administered 10 12 vg of AAV8-CAG-null vector was administered into eWAT.

[0359] Intra-eWAT administration of AAV8-CAG-moFGF21-doublemiRT vector demonstrated the specific overexpression of FGF21 in white adipose tissue, as well as the release of the protein into the bloodstream. The 10 mg / kg bw dose-dependently mediated the high secretion of β-lactamase (β-lactamase) (Figures 4A and 4B). 12 For vg A quantity of AAV8-CAG-moFGF21-doublemiRT vector was administered to eWAT and mediated a very strong overexpression of FGF21 in WAT and iWAT (Fig. 4A), resulting in the highest circulating FGF21 levels. In contrast, the lowest dose of 10 10 The AAV8-CAG-moFGF21-doublemiRT vector in vg was used to Treatment with this dose produced only a very moderate overexpression of FGF21 in the control group (Figure 4A). Placed animals had significantly lower serum FGF21 levels compared to AAV8-CAG-null treated animals This is probably because FGF21 acts as a paracrine-autocrine regulator. Therefore, AAV8-CAG-null treated animals The AAV8-CAG-moFGF21-doublemi mice showed progressive weight gain, whereas the AAV8-CAG-moFGF21-doublemi mice showed progressive weight gain. Animals treated with the RT vector showed a reduction in body weight gain proportional to the administered dose of the vector. (Figures 4C and 4D). 12 AAV8-CAG-moFGF in vg Animals treated with the 21-doublemiRT vector showed significantly higher mitochondrial activity during the first 2 weeks after AAV administration. During this period, the weight decreased by approximately 15%, and then increased until it reached the starting weight (Figure 4C and 4D). Therefore, 10 12 vg AAV8-CAG-moFGF21-dou Animals receiving the blemiRT vector in eWAT were treated with AAV8-CAG-null Compared with the control animals, the rats showed a 40% difference in total body weight at the end of the experiment (Figure 4D). 12 vg treated with AAV8-CAG-moFGF21-doublemiRT vector Treated animals showed a significant reduction in steatosis and a 60% reduction in liver weight (Figure 4E). AT weight increased in this cohort of mice, likely due to enhanced thermogenic activity. (Figure 4E).

[0360] 5×10 10 vg, 2 × 10 11 vg or 10 12 AAV8-CAG-moF in vg Animals treated with the GF21-doublemiRT vector were treated with AAV8-CAG-nu showed improved insulin sensitivity compared to 11L-treated mice (Figure 5A). 11 vg or 10 12 vg AAV8-CAG-moFGF21-doublemiRT vector Animals treated with the vector also showed significantly higher phenotypes than animals treated with the AAV8-CAG-null vector. / ob mice showed lower circulating insulin levels than did / ob mice (Fig. 5B).

[0361] Example 3. Immunoglobulin A (AAV-hAAT-moFGF21) vector in ob / ob mice Intravenous administration reverses obesity and improves glucose metabolism We also demonstrated that AAV-mediated genetic alterations in the liver were mediated by HIV-1 in 8-week-old male ob / ob mice. Antidiabetic and antiobesogenic effects mediated by increasing circulating levels of FGF21 using molecular engineering The effects of the drug on ob / ob mice were evaluated. 11 vg or 5×10 11 Liver-specific vg A codon-optimized human α1-antitrypsin (hAAT) promoter under the control of An AAV8 vector (AAV8-hA) encoding the mouse FGF21 coding sequence was used. AT-moFGF21) (Figure 6A) was administered intravenously. 5x10 for b / ob animals 11 vg of AAV8-hAAT-null vector was administered intravenously. The hAAT-moFGF21 construct is contained in SEQ ID NO: 34, and the hAAT-n The ull construct is contained in SEQ ID NO:33.

[0362] Intravenous administration of the AAV8-hAAT-moFGF21 vector significantly reduced FGF2 expression in the liver. Specific overexpression of 1 similarly mediated high secretion of the protein into the bloodstream in a dose-dependent manner ( Figures 6B and 6C). Specifically, 5 × 10 11 vg dose of AAV8-hAAT-mo The FGF21 vector mediated very strong overexpression of FGF21 in the liver (Figure 6B ), achieved the highest circulating FGF21 levels (Figure 6C). Body weight of animals treated with T-moFGF21 vector increased during the first 2 weeks after AAV administration. The AAV8-hAAT-null treatment decreased the expression of α-hAAT by approximately 7% and then slightly increased it. The mice progressively gained weight (Figs. 6D, 6E, and 6F). 11 vg AAV8 Mice administered the -hAAT-moFGF21 vector were treated with AAV8-hAAT-nul The animals gained significantly less weight than the l-treated animals (Figs. 6D, 6E, and 6F). Specifically, AAV8-hAAT-null animals were 11 vg AAV8-hAAT -Compared to 10% weight gain in animals treated with the moFGF21 vector at the end of the experiment In response to their lower body weight, FG was found in the liver. Animals that overexpressed F21 showed a significant increase in leukemia, especially in animals treated with the highest dose of vector. The mice showed a significant reduction in steatosis and a reduction in liver weight of approximately 60% (Figure 6G). iBAT weight increased similarly in both AAV8-hAAT-moFGF21-treated mouse groups (Fig. 6G), which is likely due to the fact that mice administered the AAV8-hAAT-null vector This may be due to the higher thermogenic activity in these animals compared to the control.

[0363] Animals treated with the AAV8-hAAT-moFGF21 vector were Improved insulin sensitivity and circulating insulin levels compared with T-null treated mice showed a decrease in (Figures 6H and 6I).

[0364] Example 4. Immunoglobulin D-associated protein (AAV-hAAT-moFGF21) expression in HFD-fed mice Long-term reversal of obesity and diabetes by intravenous administration We also demonstrated AAV-mediated genetic engineering of the liver in obese C57Bl6 mice. Anti-diabetic and anti-obesogenic effects mediated by increased circulating levels of FGF21 using Nine-week-old male C57Bl6 mice (young adults) were fed an HFD for 9 weeks, and then 10 in 10 vg or 5×1010 vg AAV8-hAAT-moFGF21 vector After AAV administration, AAV8-hAAT-moFGF21-treated mice were treated with IV injection (Fig. 6A). Mice were maintained on HFD for 52 weeks. 10 vg AAV8- hAAT-null and chow-fed C57Bl6 mice were compared with HFD-fed C57Bl6 mice. These latter two cohorts of mice were then administered IV chow or They were maintained on a HFD.

[0365] Intravenous administration of AAV8-hAAT-moFGF21 vector in HFD-fed mice mediated high secretion of FGF21 into the bloodstream in a dose-dependent manner (Fig. 7A).

[0366] HFD-fed AAV8-null treated mice and 10 10 vg AAV8-hAAT-mo No difference in body weight was observed between HFD-fed animals and those receiving the FGF21 vector (Figure 7B and 7C). However, 5 × 10 10 vg AAV8-hAAT-moFGF 21 HFD-fed animals treated with the vector initially lost 20% of their body weight after AAV administration. , followed by progressive weight loss similar to that seen in chow-fed AAV8-hAAT-null treated mice. Remarkably, from 9 weeks after AAV administration, the number of infected cells increased by 5 × 10 10 v HFD-fed animals administered AAV8-hAAT-moFGF21 vector and chow diet There was a statistically significant difference in total body weight and weight gain between fed and AAV8-hAAT-null treated mice. No significant differences were observed (Figures 7B and 7C).

[0367] 5×10 10HFD treated with vg of AAV8-hAAT-moFGF21 vector Energy expenditure during the light and dark cycles of chow-fed mice was significantly higher than that of chow-fed AAV mice. of 8-hAAT-null mice and HFD-fed AAV8-hAAT-null mice The results were higher in chow-fed AAV8-hAAT-null treated animals and and HFD-fed AAV8-hAAT-null treated animals and 10 10 vg AAV8- No difference in energy expenditure was observed between mice administered with the hAAT-moFGF21 vector. Overall, these data suggest that 5 × 10 10 vg AAV8- These results suggest that mice treated with hAAT-moFGF21 had improved thermogenic activity. do.

[0368] 10 10 Animals treated with vg of AAV8-hAAT-moFGF21 vector were Insulin levels were significantly lower in HFD-fed mice than in AV8-hAAT-null mice. showed improved insulin sensitivity, and their insulin sensitivity was improved by the AAV8-hAAT-null vector. The results were similar to those of the chow-fed mice treated with acetaminophen (Fig. 8B). 5×10 10 Animals administered with vg of AAV8-hAAT-moFGF21 vector showed a Insulin levels were significantly lower in chow-fed mice than in AV8-hAAT-null mice. showed improved insulin sensitivity and normalized circulating insulin levels (Figures 8B and 8C). .

[0369] Example 5. AAV-hAAT-moFGF21 vector in aged HFD-fed mice Reversal of obesity and diabetes by intravenous administration of The present inventors also demonstrated the antidiabetic effect of FGF21 in obese aged (adult) C57B16 mice. The anti-obesity and anti-inflammatory effects of the HFD were evaluated. feeding for 10 weeks, then 10 vg, 2 × 10 10 vg or 5×10 10 vg AAV8 The -hAAT-moFGF21 vector was administered intravenously (Figure 6A). 8-hAAT-moFGF21-treated mice were maintained on a HFD for 22 weeks. Then, 5 x 10 10 vg AAV8-hAAT-null chow-fed aged C57Bl 6 mice and HFD-fed aged C57Bl6 mice. Cohorts of mice were then maintained on a chow diet or HFD.

[0370] Intravenous AAV8-hAAT-moFGF21 vector in aged HFD-fed mice Administration mediated high secretion of FGF21 into the bloodstream in a dose-dependent manner (Fig. 9A).

[0371] HFD-fed AAV8-null treated mice and 10 10 vg AAV8-hAAT-mo No difference in body weight was observed between HFD-fed animals and those receiving the FGF21 vector (Figure 9B and 9C). However, 2 × 10 10 vg or 5×10 10 Either vg HFD-fed animals treated with the AAV8-hAAT-moFGF21 vector showed a significant increase in AAV After administration, there was an initial decrease in body weight of 15% and 20%, respectively (Fig. 9B and 9C). After, 2×10 10 Animals treated with AAV8-hAAT-moFGF21 vectors at vg showed progressive weight gain similar to that of chow-fed AAV8-hAAT-null treated mice. However, 5 × 10 10 Animals treated with AAV8-hAAT-moFGF21 vectors (vg) No significant changes in body weight were observed (Figures 9B and 9C). 3 weeks after administration, 5 × 10 10 vg of AAV8-hAAT-moFGF21 vector between HFD-fed animals and chow-fed AAV8-hAAT-null treated mice No statistically significant differences were observed in total body weight and weight gain (Figures 9B and 9C).

[0372] 5×10 10 HFD treated with vg of AAV8-hAAT-moFGF21 vector Energy expenditure during the light and dark cycles of chow-fed mice was significantly higher than that of chow-fed AAV mice. of 8-hAAT-null mice and HFD-fed AAV8-hAAT-null mice It was higher than that (Fig. 10A). 2 × 10 10 vg AAV8-hAAT-moFGF 21 Vector-treated animals showed increased energy expenditure during the light cycle and dark cycle. There was a tendency for energy expenditure to increase during the cycle (Fig. 10A). 10 vg AAV8 -hAAT-moFGF21 vector-treated animals showed increased energy production during the dark cycle Chow-fed AAV8-hAAT-null treated animals showed increased consumption (Figure 10A). and HFD-fed AAV8-hAAT-null treated animals and 10 10 vg AAV8 No difference was observed between mice administered with the -hAAT-moFGF21 vector (Figure 10A). Overall, these data suggest that treatment with AAV8-hAAT-moFGF21 significantly improved the survival of IFN-γ-glucan-1 (FGF21)-dependently induced ... This suggests that the aged mice treated with steroids had improved thermogenic activity.

[0373] 10 10 vg or 2×10 10 vg AAV8-hAAT-moFGF21 vector Animals treated with hAAT-null vector were compared with HFD-fed mice administered with AAV8-hAAT-null vector. showed improved insulin sensitivity compared to AAV8- was similar to that of chow-fed mice treated with the hAAT-null vector (Figure 10B). Remarkably, 5×10 10 vg AAV8-hAAT-moFGF21 vector The animals administered the AAV8-hAAT-null vector were fed a chow diet. showed improved insulin sensitivity compared to 10 mice (Figure 10A). 10 vg, 2× 10 10 vg or 5×10 10 vg AAV8-hAAT-moFGF21 vector Treated animals showed lower hunger than HFD-fed AAV8-hAAT-null treated mice The circulating insulin levels at 2 × 10 1 0 vg or 5×10 10 vg of AAV8-hAAT-moFGF21 vector was administered intravenously. between chow-fed aged animals and chow-fed AAV8-hAAT-null treated mice No differences were observed in circulating insulin levels (Fig. 10C).

[0374] Example 6. Muscle Growth of AAV-CMV-moFGF21 Vector in C57B16 Mice Evaluation of weight loss due to intramuscular administration We also performed AAV-mediated genetic engineering of skeletal muscle in C57Bl6 mice. We evaluated the therapeutic potential of increasing circulating levels of FGF21 by targeting skeletal muscle. Therefore, the CMV promoter and AAV1 serotype were chosen. Although the TASS promoter is a nucleotide sequence, its use with AAV1 capsids has not been previously published. This allows for highly efficient targeting of skeletal muscle without transducing the liver, as in (Mas et al., Diabetes 2006; Callejas et al. al.,Diabetes 2013).

[0375] 3×10 11 vg dose, codon-optimal under the control of the ubiquitous CMV promoter An AAV1 vector (AAV1-) encoding the engineered murine FGF21 coding sequence CMV-moFGF21) (Figure 11A) was administered to 6- to 12-week-old male C57BL6 mice. Muscles in the quadriceps, gastrocnemius and tibialis anterior (tibialis cranealis) of each hind limb Intracutaneous injection (5×10 10 As a control, age-matched rats were administered 100 mg / intramuscularly. 3 x 10 cells in the same muscle of a C57Bl6 animal 11 vg AAV1-CMV-null vector Tar (5 x 10 10 The CMV-moFGF21 construct was administered intramuscularly (vg / muscle). The CMV-null construct is contained in SEQ ID NO: 35. It can be enjoyed.

[0376] Intramuscular administration of the AAV1-CMV-moFGF21 vector significantly increased the amount of FGF21 released into the bloodstream. The AAV1-CMV-moFGF21 vector-treated mice mediated high secretion of IFN-γ-glucan (Fig. 11B). Animals treated with AAV1-CMV-null showed reduced body weight and weight gain compared to AAV1-CMV-null treated mice. (Figures 11C and 11D).

[0377] Example 7. Protein production with codon-optimized human FGF21 nucleotide sequence Increase in To evaluate whether codon optimization can mediate increased FGF21 protein production To transfect HEK293 cells with three different codon-optimized human FGF21 nucleotide sequences, The control was transfected with a plasmid encoding the sequences (SEQ ID NOs: 40 to 42). As a role, non-transfected cells and wild-type hFGF21 coding sequences were used. Three codon-optimized human FGF21 sequences and WT human FGF21 were used. Expression of the FGF21 sequence was under the control of the hAAT promoter (SEQ ID NO: 47). Cells transduced with don-optimized human FGF21 version 1 or 3 exhibited no significant differences compared with wild-type or or codon-optimized FGF21 variant 2, resulting in higher levels of human FGF2 1 can be secreted into the culture medium (Fig. 12), thus confirming the co-expression of variants 1 and 3. Demonstrate increased FGF21 protein production with donor optimization.

[0378] Example 8. Administration of an AAV vector encoding human FGF21 induces obesity in mice Reversal of obesity and diabetes (in vivo experiments demonstrating the activity of FGF21) HFD-fed mice were treated with an AAV vector encoding human FGF21. As a control, the same dose of AAV-null vector was administered to chow-fed and HFD-fed mice. Administered to bait mice.

[0379] Induce browning of WAT and thermogenic activity of BAT, increase energy expenditure, etc. and to evaluate the ability of human FGF21 to improve glucose and energy metabolism. , carry out the following tests: - Weekly measurements of body weight and food and fluid intake - Temperature measurement - Measurement of energy expenditure and respiratory quotient by indirect calorimetry - Blood glucose measurement - Assessment of whole-body glucose disposal by intraperitoneal glucose tolerance test - Intraperitoneal insulin tolerance test to assess insulin sensitivity - Analysis in tissue and serum samples, including: - Examination of human FGF21 overexpression levels in target tissues and the bloodstream - Morphological and histological analysis.

[0380] - Determination of circulating hormone and cytokine levels - Serum metabolic parameters, e.g., free fatty acids, glycerol, triglycerides, cholesterol Determination of sterols and ketone bodies, etc. - Examination of the presence of beige adipocytes in the inguinal fat pad by immunohistochemistry and Browning capacity according to gene expression of classical white, brown, and beige adipocyte markers Rating Example 9: In vitro assay to assess FGF21 activity FGF21 inhibits glucose uptake and GLUT1 expression in 3T3-L1 cells It is expected to increase (Kharitonenkov, A. et al., 200 5. J.Clin.Invest 115 :1627-1635).

[0381] Example 10. AAV8-CAG-moFGF21-dmiR in ob / ob mice Intra-eWAT administration of T vector reverses obesity and improves glucose metabolism: Further research look The present inventors further investigated the effect of FGF21 on adipose tissue A in ob / ob mice. The antidiabetic and antiobesogenic therapeutic potential of AV-mediated genetic engineering was evaluated (Example 2 (See

[0382] received intra-eWAT injection of AAV8-CAG-moFGF21-dmirT vector b / ob mice showed reduced white adipocyte size in the epididymal pad (FIG. 13A). Circulating adiponectin levels also increased with dose (Figure 14A). Inflammation in eWAT was also assessed as a function of vector dose, with macrophage markers -F4 / 80 expression was also reduced (Fig. 14B and C).

[0383] Null vector or the lowest dose of AAV8-CAG-moFGF21-dmir The livers of ob / ob mice injected with T showed accumulation of lipid droplets in hepatocytes (Figure 13 B). 5×10 10 vg / mouse or higher doses of vector encoding FGF21 Administration of turoctocog alfa completely prevented the development of fatty liver (Fig. 13B), which was associated with a decrease in organ weight (Fig. 14D). ) and its correlation with total triglyceride and cholesterol contents (Fig. 14E and F). 5×10 10 Further evidence suggests that the vg / mouse dose represents a threshold for therapeutic efficacy. , obtained from blood glucose and blood insulin analysis. 1 × 10 10 The dose was measured in vg / mouse. It did not alter blood glucose levels in the state and only partially reduced insulin levels. , 5×10 10 Doses of ≥ 1000 mg / mouse completely normalized blood glucose and blood insulin levels. (Figures 13C and D). Overall, this example demonstrates that FGF21 overexpression in adipose tissue To identify the therapeutic potential of

[0384] Example 11. AAV8-hAAT-moFGF21 vector in ob / ob mice Intravenous administration of riboflavin reverses obesity and improves glucose metabolism: Further insights The present inventors further demonstrated that AAV8-hAAT-moFGF2 The antidiabetic and antiobesogenic therapeutic potential of intravenous administration of 1 vector was evaluated (see Example 1). See 3).

[0385] Consistent with their lower body weight, ob / ob animals overexpressed FGF21 in the liver , especially 5×10 11 Animals treated with vg showed a significant decrease in the size of white adipocytes. This was due to the reduction of Mac2 staining in eWAT and the increase of F4 / 80 and Circulating adiponectin, as evidenced by the expression of TNF-α (Fig. 16A-C), This was paralleled by a dose-dependent increase in levels (Fig. 15B) and a decrease in WAT inflammation. Fortunately, 5 x 10 11 vg-treated ob / ob mice showed a “crown-like” It showed a significant reduction in structure (Figure 16A).

[0386] Seven-month-old ob / ob mice showed significant fatty liver, whereas FGF21-treated ob / ob mice showed no significant fatty liver. The liver of the mice did not show lipid accumulation in hepatocytes (Fig. 15C). 60% reduction in organ weight in ob / ob mice receiving thrombus treatment (Fig. 16D and E) Similarly, there was a significant reduction in total liver triglyceride and cholesterol content (Fig. 16F and G ) were consistent with those of ob / o treated with both doses of AAV8-hAAT-moFGF21. b Animals also showed reduced fed blood glucose, and their blood insulin in the fed state was reduced by approximately 70%. (Fig. 15D and E).

[0387] The present inventors have demonstrated that in ob / ob mice after AAV8-hAAT-moFGF21 treatment We investigated whether the decrease in circulating glucose levels observed in the rats was due to the suppression of hepatic gluconeogenesis. Phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphate The expression of G6Pase was assessed by measuring it by qPCR. 1 × 10 showed increased EPCK expression 11 vg AAV8-hAAT-moFGF21 of AAV8-hAAT-moFGF21-treated ob / ob mice, except for the treated animals. No changes in the expression of these enzymes were observed in the liver (Fig. 17A and B). Our results demonstrate AAV-mediated long-term expression of FGF21 in the liver and subsequent circulation. Increased cyclic FGF21 lowers glucose by inhibiting hepatic glucose production suggests that it wasn't.

[0388] The glucose-lowering effect of FGF21 is also due to increased glucose uptake by adipocytes and This is due to increased blood pressure and energy expenditure (Xu J. et al., 2009). AJP Endocrinol.Metab. 297 :E1105-E1114;D ing X. et al.,2012. Cell Metab. 16 :387-3 93;Camacho RC et al.,2013. Eur.J.Pharm acol. 715 :41-45;Emanuelli B. et al.,2014 .Clin.Invest.124 :515-527;Kharitonenkov A. et al., 2005. Endocrinology 148 :774-7 81;Hondares E. et al.,2010. Cell Metab. 11 :206-212;Samms RJ et al.,2015. Cell Rep. 11 Therefore, we investigated the fat tissue of different pads. In the tissues (iWAT, eWAT, and iBAT), important components of the glucose uptake mechanism Components such as glucose transporters Glut1 and Glut4, glucose transporters The enzymes hexokinase I and II (HKI and HKI), and iBAT In the case of AAV8-FGF21-treated ob / In ob mice, Glut1 expression was increased in iWAT and iBAT (Fig. 17C ), Glut4 expression was increased in eWAT, iWAT, and iBAT (Fig. 17D). HKI and HKII were upregulated only in iBAT (Fig. 17E and F). Moreover, UCP1 expression was significantly enhanced by high doses of the AAV8-hAAT-moFGF21 vector. In iBAT of ob / ob mice treated with α-glucan, the expression of α-glucan was increased in iBAT of ob / ob mice treated with α-glucan (Fig. 17G). These results demonstrate that ob / ob mice treated with AAV8-hAAT-moFGF21 vectors The long-term improvement in blood glucose observed in mice is likely due to the growth of white and brown fat cells. This is due to increased glucose uptake and enhanced heat production in iBAT. This suggests that...

[0389] Example 12. AAV8-hAA in HFD-fed mice and HFD-fed aged mice Long-term reversal of obesity and diabetes by intravenous administration of T-moFGF21 vector: Reduced fabric weight and stable expression for up to one year All studies conducted in young adults or adults (see Examples 4 and 5) Representative images of animals from all experimental groups are shown in Figures 19A-B.

[0390] AAV8-hAAT-moFGF21 treatment reversed obesity in young adult or adult mice. In both treated animals, the major white adipose tissue (WAT) depots, e.g. epididymal (eWAT), inguinal (iWAT), and retroperitoneal (rWAT) fat pads This was paralleled by a dose-dependent decrease in liver weight (Fig. 18A and Fig. 19C). The D-induced increase was observed by FGF21 gene transfer with the highest dose of vector used. Complete normalization was observed, whereas quadriceps muscle weight was not altered by diet or AAV delivery (Figure 18A and Fig. 19D).

[0391] AAV8-hAAT-moFGF21-treated mice at both ages showed codon optimization in the liver The results showed that FGF21 was specifically overexpressed in both groups of mice (Fig. 19E). The vector-dependent secretion of FGF21 into the bloodstream remained stable for up to one year after a single injection. This results in a level of

[0392] Example 13. AAV8-hAA in HFD-fed mice and HFD-fed aged mice Long-term reversal of obesity and diabetes by intravenous administration of T-moFGF21 vector: Improving motor activity and investigating the mechanisms of heat production Increased energy expenditure (see Examples 4 and 5) was observed with AAV8-hAAT- This was also seen in animals treated as young adults 10 months after moFGF21 delivery (Fig. 21A).

[0393] This finding supports the AAV8-hAAT-moFGF21-mediated effects on locomotor activity. Consistent with this, the open-label expression of AAV8 in HFD-fed animals receiving AAV8-null vectors was significantly increased. In contrast to the hypoactivity observed during field studies, 10 vg Mice treated with AAV8-hAAT-moFGF21 were treated with chow-fed null injections. As shown in Figure 20A, AAV showed spontaneous locomotor activity comparable to that of the rhesus monkeys. One year after 8-hAAT-moFGF21 delivery, treated animals traveled longer distances and They rested for less time and engaged in more slow movements and exercise than untreated HFD-fed controls. I spent time doing fast exercise.

[0394] Considering that changes in energy expenditure may reflect changes in thermogenesis, the present inventors investigated the effects of brown adipose tissue The degree of activation of BAT was assessed. 10 vg Both mice treated with AAV8-hAAT-moFGF21 showed increased lipid levels in iBAT. The UCP1 protein content in BAT was significantly higher than that in young adults (Fig. 20B). In mice treated with the AAV8-hAAT-moFGF21 vector, (Fig. 20C), and non-shivering FGF21-induced liver injury was observed. This coincided with an increase in heat production.

[0395] Browning of subcutaneous WAT is characterized by the appearance of beige adipocytes and is associated with increased energy expenditure. Browning is also associated with AAV8-hA. To evaluate whether this could explain the increased energy expenditure observed after AT-moFGF21 treatment. Consistent with the weight loss of this pad (Figure 18), a histological evaluation of the iWAT was performed. A) Adipocytes from HFD-fed AAV8-hAAT-moFGF21-treated animals were compared with those from HFD-fed animals. The tumor size was smaller than that of the null-injected animals (Figure 20D). Treatment with the hAAT-moFGF21 vector was performed as young adults or adults. In all animals, at all doses tested, the multilocular beige fat in iWAT The difference in iW between the HFD-fed groups did not result in increased detection of adipocytes (Figure 20D). There was no statistically significant difference in UCP1 protein levels in AT (FIG. 21B).

[0396] Creatinine-driven substrate cycle and sarcoplasmic reticulum Ca2+-ATPase 2b (Se rca2b)-mediated calcium cycling regulates thermogenesis in iWAT independently of UCP1 (Kazak L. et al., 2015. Cell 163 :643-655;Ikeda K. et al.,2017. Nat.Me d. 23 :1454-1465). It is an enzyme involved in the creatinine-driven substrate cycle. Higher levels of expression of a phosphatase orphan 1 (Phospho1) were observed with age. 5 when compared with matched chow-fed and HFD-fed controls. x10 10 of HFD-fed mice treated with vg of AAV8-hAAT-moFGF21 This was observed in iWAT (Fig. 20E), which indicates that the activity of the creatinine-driven cycle is increased. This suggests that the increase was probably a result of FGF21 gene transfer. Regarding the mechanism of thermogenic activity, the chow-fed null treatment animals or HFD-fed null Animals treated with the AAV8-hAAT-moFGF21 vector showed significantly higher IFN-γ levels than animals treated with the AAV8-hAAT-moFGF21 vector compared with those treated with the No difference in the expression level of Serca2b was detected in iWAT of the mice (Figure 21C). On the other hand, iWA of ryanodine receptor 2 (RyR2), another enzyme involved in the same cycle, T expression in null-treated and AAV8-hAAT-moFGF21-treated mice Both were increased by HFD feeding (Figure 21C). Overall, these results suggest that The calcium circulation-dependent thermogenic mechanism is a systemic enlargement observed after AAV-FGF21 treatment. This suggests that it is not involved in improving energy homeostasis.

[0397] Example 14. AAV8-hAA in HFD-fed mice and HFD-fed aged mice Long-term reversal of obesity and diabetes by intravenous administration of T-moFGF21 vector: Lucagon levels, islet hyperplasia and glucose tolerance Moreover, treated with the AAV8-hAAT-moFGF21 vector as young adults HFD-fed animals had reduced circulating levels of glucagon compared to HFD-fed null-treated mice. The results were small (Figure 22A).

[0398] Whereas AAV8-null treated mice developed islet hyperplasia as a result of HFD feeding , AAV8-hAAT-FGF21 vector (2 × 10 10 or 5×10 10 vg / The beta cell mass of treated animals (at the dose of mice) was significantly higher than that of control mice fed a chow diet. The results were similar to those of the HFD-fed AAV8-hAAT-moF mice (Fig. 22B and C). Double immunostaining for insulin and glucagon in pancreatic sections from GF21-treated mice The colors indicate the normal distribution of α- and β-cells in the islets of these animals, with the periphery of the islets showing Glucagon-expressing cells were localized in the center, and insulin-expressing cells were localized in the center (Fig. 22D).

[0399] To assess glucose tolerance in FGF21-treated mice, an intraperitoneal glucose tolerance test (GTT) was performed. TT) (2 g glucose / kg body weight) was performed 10 weeks after AAV administration. 1 x 10 cells of either vector encoding FGF21 10 injected at a dose of vg / mouse HFD-fed animals were glucose intolerant and had significantly elevated circulating insulin levels during the GTT. In contrast, 5 × 10 10 vg / mouse AAV Animals treated with 8-hAAT-moFGF21 were significantly higher than chow-fed control mice. These two experimental groups showed improved glucose clearance when compared (Figure 23A). Insulin levels were indistinguishable between the 5×10 10 In HFD-fed mice treated with AAV8-hAAT-moFGF21 at 1000 mg / mouse The improvement in insulin sensitivity was further confirmed.

[0400] Example 15. HFD by intravenous administration of AAV8-hAAT-moFGF21 vector Recovery of WAT hypertrophy and inflammation associated with HFD feeding induces an increase in WAT adipocyte size (Sattar N. & Gi ll JMR,2014. BMC Med. 12 :123). FGF21 Administration of the loading vector counteracted this increase (Fig. 24A). Morphometric analysis of WAT 1 × 10 as young adults 10 or 5×10 10 of animals treated with the vg vector and 2 x 10 as adults 10 or 5×10 10 White of mice treated with vg vector The area of ​​pigmented adipocytes was found to be similar to that of animals fed a chow diet. (Fig. 24B). In both FGF21-treated animal groups, there was a redistribution of adipocyte size, with more The proportion of small adipocytes was higher (Fig. 25A). Consistent with the recovery, the highest dose of AAV8-hAAT-moFG, regardless of age at treatment initiation, Adiponectin and leptin levels in animals treated with the F21 vector were also positive. normalized (Fig. 24C and D).

[0401] Obesity also induces inflammation in WAT (Hajer GR et al., 20 08. Eur.Heart J. 29 :2959-2571). Therefore, the present invention They analyzed inflammation in this tissue using immunofluorescence assays for the macrophage-specific marker Mac2. The HFD-fed mice were analyzed through staining and expression of pro-inflammatory molecules in eWAT. showed an increased presence of macrophages, which were expressed as "crown-like" structures, whereas young adults 5 x 10 as a body or adult 10 vg Treated with AAV8-hAAT-moFGF21 Animals treated with IFN-γ had no signs of macrophage infiltration (Fig. 24E and Fig. 25B). Macrophage markers F480 and CD68 and the pro-inflammatory cytokine TNF This was in parallel with the normalization of IL-1α and IL-1β expression (Fig. 24F-H and Fig. 25C- E), indicating that FGF21 expression counteracted obesity-associated WAT inflammation.

[0402] Example 16. Fatty liver caused by intravenous administration of AAV8-hAAT-moFGF21 vector , and recovery from liver inflammation and fibrosis Liver histology revealed that all HFD-fed null-treated animals had significant hepatic steatosis at the time of sacrifice. In contrast, 5 × 10 as young adults or adults (Fig. 26A-D). 10 HFD-fed mice receiving vg AAV8-hAAT-moFGF21 exhibited this pathology These histological findings demonstrated a recovery of the 5×10 lipid deposits (Figure 26A). 10 v g Total liver triglycerides and cholesterol in AAV8-hAAT-moFGF21-treated animals This was paralleled by a significant reduction in terol content (Fig. 26B and C). or fed HFD as adults and 5 × 10 10 vg AAV8-hAAT-moFGF2 Animals treated with the .1 vector showed liver leukemia as evidenced by a lack of staining for Mac2. The staining for Mac2 was similar to that of the null-treated HFD-fed mice, which showed no signs of liver inflammation. The results revealed an increased presence of macrophages in the liver of the mice (Fig. 26D). FGF21 gene transfer into the liver reversed liver fibrosis. Collagen fibers were also found in the HFD-fed mice. Picrosirius readings of liver sections from animals injected with a control null vector were readily detectable after chromatin staining or Masson's trichrome staining, whereas AAV It was not detectable in the livers of 8-hAAT-moFGF21-treated mice (Fig. 28A and These mice also showed a significant reduction in hepatic collagen 1 expression (Fig. 28). B and C). Overall, these findings suggest that AAV8-hAAT-moFGF21 treatment protects against the development of HFD-induced nonalcoholic steatohepatitis (NASH). showed.

[0403] Example 17. Long-term safety of liver-directed AAV-FGF21 treatment Pharmacological treatment or transgenic overexpression of FGF21 inhibits bone mineral density through enhanced bone resorption. It is associated with metabolic disturbances, which can lead to bone loss (Wei W. et al. al., 2012. Proc. Natl. Acad. Sci. 109 :3143- 3148;Wang X. et al.,2015. Cell Metab. 22 :811-824;Charoenphandhu N. et al.,2017. J.Bone Miner.Metab. 35 :142-149;Talukdar S. et al., 2016. Cell Metab. 23 :427-440;K im AM et al.,2017. Diabetes,Obes.Metab Therapeutic potential of AAV8-hAAT-moFGF21 for the treatment of obesity and diabetes Considering the nature of the gene expression, we found that the gene expression in bones of animals treated with the highest dose of vector was significantly higher than that in animals treated with the highest dose of vector. The long-term effects of AAV8 transduction were evaluated. - In animals receiving the hAAT-moFGF21 vector, The length of the tibia and the length of the tibia were normal (Fig. 29A and B). Bone structure was examined by micro-computed tomography (μCT). Analysis of the proximal tibia epiphysis was performed. Fed HFD and 5 × 10 10 vg AAV8-hAAT-moFGF21-administered mice The trabecular and cortical bone of mice treated with the null vector was significantly increased compared to age-matched mice treated with the null vector. Specifically, there was no significant difference in bone mineral density (BMD) (Fig. 29C ), bone mineral content (BMC) (Fig. 29D), bone volume (BV) (Fig. 29E), bone volume / mass Bone volume ratio (BV / TV) (Figure 29F), bone surface / bone volume ratio (BS / BV) (Figure 29G), Trabecular number (Tb.N) (Fig. 29H), trabecular width (Tb.Th) (Fig. 29I) or trabecular spacing ( No differences were recorded in the tibia diaphysis (Fig. 29J). Analysis of compact bone revealed significant differences in BMC, BMD, and BMD between HFD-fed, null-injected, or FGF21-treated groups. , BV, BV / TV, or BS / BV showed no differences (Figures 29K-O).

[0404] The pathological effects of FGF21 are at least partly due to the liver's secretion of insulin-like growth factor It has been reported that this is mediated by increased production of interleukin-1 (IGFPB1) Wang X. et al.,2015. Cell Metab. 22 :811- 824). Consistent with the lack of bone changes, high-dose AAV8-hAAT-moFGF21 treatment compared with null-injected HFD-fed mice at 12 months (young adult) or 6 months (adult) ) did not lead to an increase in circulating IGFBP1 protein levels in early treated animals. (Figure 29P). Circulating IGF1 levels were also normal in all experimental groups (Figure 29Q). Overall, these results suggest that AAV-mediated FGF21 gene transfer to the liver, as opposed to bone, is effective. Support the safety of

[0405] Example 18. HFD by intravenous administration of AAV8-hAAT-moFGF21 vector Prevention of induced liver tumors Long-term HFD feeding (>60 weeks) increases the incidence of liver neoplasia in C57BL / 6J mice associated with an increase (Hill-Baskin AE et al., 2009). Hum.Mol.Genet. 18 :2975-2988;Nakagawa H., 2015. World J. Hepatol. 7 :2110). In our tests In this study, the present invention was developed in animals that were started on a HFD as young adults and maintained on this for 60 weeks. They found liver tumors in 66.7% (6 / 9) of animals injected with the null vector. Animals treated with the AAV8-hAAT-moFGF21 vector showed no significant improvement in HFD-induced liver neoplasia. Protected from biological outbreaks: 5×10 10 vg treated with a vector encoding FGF21 0% (0 / 8) of the animals treated showed tumors, and the lowest dose (1 × 10 10 vg) The incidence in the cohort was 40% (4 / 10). Chow-fed mice (0 / 11) None of them developed tumors during the same period (Table 1).

[0406] Table 1. Liver tumor incidence in young adults [Table 1]

[0407] Example 19. AAV8-mediated liver-specific overexpression of FGF21 inhibits STZ-induced hypertension Improved blood sugar material and method animal The present inventors used 9-week-old male C57bl6 mice. The mice were fed a standard diet ad libitum. The mice were then incubated in a 12-hour light-dark cycle (lights on at 08:00). Therefore, mice were given steroids dissolved in 0.1 mol / L citrate buffer (pH 4.5). They received five intraperitoneal injections of treptozotocin (50 mg / kg) on ​​consecutive days. Levels were measured using an analyzer (Glucometer Elite; Bayer, Level Animal care and experimental procedures were carried out by the University of Illinois at the University of Illinois. Ethics C of iversitat Autonoma de Barcelona omittee for Animal and Human Experiment This information has been approved by the competent authority.

[0408] In vivo administration of AAV vectors For systemic administration, AAV vectors were injected into 200 μl of PBS with 0.001% F68 P1 The solution was diluted in Uronic® (Gibco) and injected via the tail vein.

[0409] result To test the protective potential of AAV-derived FGF21 against type 1 diabetes, 5× 10 10 vg or 2×10 11 Codon optima under the control of the hAAT promoter in vg An AAV8 vector encoding fused mouse FGF21 (AAV8-hAAT-moF GF21) was administered intravenously to 9-week-old male C57B16 mice. Control mice were , 2 × 10 11 2 vg of AAV8-hAAT-Null vector. After a week, all animals were treated with streptozotocin (STZ) (5 doses of 50 mg / kg; 1 dose / The diabetic process was induced by treatment with 100mg of acetaminophen (days).

[0410] Analysis of blood glucose levels in mice treated with an AAV8 vector encoding moFGF21 The animals treated were more sensitive than C57Bl6 mice treated with the AAV8-hAAT-Null vector. The results showed that the circulating glucose levels were lower than those of the control group (Figure 30).

[0411] Example 20. Expression of AAV-CMV-moFGF21 vector in C57B16 mice Health benefits from intramuscular administration by preventing age-related weight gain and insulin resistance Extended lifespan Skeletal muscle (Skm) is a readily accessible tissue and a promising candidate for the development of secretable therapeutic proteins. It is used for production (Haurigot V. et al., 2010. J .Clin.Invest. 123 :3254-3271;Callejas D. et al.,2013. Diabetes 62 :1718-1729;Jaen ML et al.,2017. Mol.Ther.Methods Clin. Dev. 6 :1-7). Could Skm be a viable source of circulating FGF21? To investigate this, we developed an optimized mouse FGF21 under the control of the CMV promoter. , a serotype 1 AAV vector that shows high tropism for Skm (Chao L. et al. l., 2000. J.Clin.Invest. 106 :1221-1228;Wu Z. et al., 2006. J. Virol. 80 :9093-9103;L isowski L. et al.,2015. Curr.Opin.Pharma col.24 :59-67) was used (AAV1-CMV-moFGF21). - 5 x 10 10 The dose was 1000 mg / muscle in the quadriceps and calf muscles of 8-week-old C57Bl6 mice. The abdominal and tibialis anterior muscles were injected (total dose, 3 × 10 11 vg / mouse). Control Animals were injected with the same dose of AAV1-CMV-Null vector. The use of normal mice allowed us to evaluate the long-term safety of FGF21 gene therapy. made it even more possible.

[0412] Eleven-month-old animals injected with a vector encoding FGF21 at 8 weeks of age had significantly lower levels of circulating FGF21. showed a significant increase in the number of vector-derived IL-1 serotypes in the three injected muscles (Fig. 31A). This was paralleled by the high expression level of FGF21 in the IL-16 cells (Fig. 31B). Therefore, this combination of vector serotype, promoter, and administration route is transduced in the liver. This did not result in expression of the transgene (Fig. 31B).

[0413] At the end of the approximately 10-month follow-up period, mice receiving intramuscular injections of AAV1-CMV-moFGF21 showed no improvement. The mice maintained their weight at the start of the study and showed a steady increase in weight as the animals aged. The muscle weight was approximately 38% leaner than that of the control (Fig. 31C). The weight of the white and brown reservoirs as well as the liver was significantly affected by the Indeed, the weight of the analyzed WAT pads was reduced by >50% (Fig. Moreover, mice treated with AAV1-CMV-moFGF21 showed increased liver total tumor volume. The liver cholesterol levels were significantly reduced (Fig. 31E). In contrast to the null-injected animals, AAV1-CMV-m Animals treated with oFGF21 exhibited normoglycemia when they were approximately 1 year of age (data not shown). (Fig. 31G). Therefore, FGF21-treated mice , showed significantly improved insulin sensitivity at the end of the study (Figure 31H). This study demonstrates that administration of AAV vectors that deliver therapeutically relevant levels of circulating FGF21 is effective in healthy individuals. It is safe in the long term and reverses age-related increases in weight and insulin resistance This demonstrates that it can be used to

[0414] Example 21. AAV1-CMV-moFGF2 in HFD-fed C57B16 mice Reversal of obesity and diabetes by intramuscular administration of 1 vector.

[0415] Next, we investigated whether im administration of the AAV1-CMV-moFGF21 vector also reduces obesity and To achieve this goal, we evaluated whether cerebrospinal fluid (CEF) could reverse insulin resistance and improve insulin resistance. Two-month-old C57Bl6 mice were fed either chow or HFD for 12 weeks. During these first 3 months of follow-up, the weight of the chow-fed animals increased by 20%, while the HFD-fed animals The fed animals became obese (95% weight gain) (Fig. 32A and B). So, 5 x 10 10 vg / muscle in the quadriceps and gastrocnemius muscles of both limbs of obese C57Bl6 mice. and tibialis anterior muscles (total dose, 3 × 10 11 vg / mouse) as a control. Another cohort of obese mice and a cohort of chow-fed mice were 3 × 10 11 vg AAV1-CMV-null vector was administered. After delivery, mice were maintained on a chow or HFD diet. Animals treated with 21 experienced progressive weight loss (Figures 32A and B). -Reversal of obesity by CMV-FGF21 treatment was paralleled by increased circulating levels of FGF21. (Figure 32C).

[0416] Null-treated mice fed an HFD showed normal fed blood glucose (Fig. 32D). Insulinemia (Fig. 32E), which suggests that these mice develop insulin resistance. In contrast, the AAV1-CMV-moFGF21-treated mice showed a significant increase in the number of infected mice. HFD-fed mice were normoglycemic and normoinsulinic by the end of the study (Figure 3 2D and E). Moreover, animals administered AAV1-CMV-moFGF21 showed showed greater insulin sensitivity than HFD-fed controls (Figure 32F).

[0417] Example 22. FGF21 circulation using codon-optimized human FGF21 nucleotide sequences Increased ring levels.

[0418] To assess whether codon optimization can mediate increased circulating levels of FGF21 Eight-week-old male C57Bl6 mice were transfected with three different vectors under the control of the hAAT promoter. The codon-optimized human FGF21 nucleotide sequence (SEQ ID NOs: 40-42) is encoded by As controls, untreated mice and and encoding the wild-type hFGF21 coding sequence under the control of the hAAT promoter. Mice were hydrodynamically injected with the plasmid.

[0419] material and method In vivo delivery of plasmids into mice by hydrodynamic tail vein injection Plasmid DNA was dissolved in saline at a volume equivalent to approximately 10% of the average animal weight (in grams). (ml) and injected manually into the lateral tail vein in less than 5 seconds. Before injection, the animals were placed under a 250 W infrared heat lamp (Philips) for a few minutes to allow the blood to The tube was dilated to allow for better visualization and access to the tail vein. Animals were immobilized for injection using a restrainer (Harvard Apparatus). No anesthesia was used, as it was not necessary as long as an appropriate restraint device was used. Animals were injected using a 6G 3 / 8 inch gauge hypodermic needle (BD), which The needle gauge is the largest feasible that fits snugly into the vein to be accessed.

[0420] result Mice treated with either codon-optimized human FGF21 version 2 or 3 Higher levels of FGF21 compared to wild-type or codon-optimized FGF21 variant 1 Human FGF21 can be secreted into the circulation (Fig. 33), and therefore variants 2 and 1 and 2 demonstrate increased FGF21 protein production by codon optimization of 3.

[0421] Example 23 hAAT-moFGF21, CAG-moFGF21-double min Expression and protein of FGF21 by RT and CMV-moFGF21 expression cassettes Increased protein production levels in vitro and in vivo material and method In vivo delivery of plasmids into mice by hydrodynamic tail vein injection Plasmid DNA was dissolved in saline at a volume equivalent to approximately 10% of the average animal weight (in grams). (ml) and injected manually into the lateral tail vein in less than 5 seconds. Before injection, the animals were placed under a 250 W infrared heat lamp (Philips) for a few minutes to allow the blood to The tube was dilated to allow for better visualization and access to the tail vein. Animals were immobilized for injection using a restrainer (Harvard Apparatus). No anesthesia was used, as it was not necessary as long as an appropriate restraint device was used. Animals were injected using a 6G 3 / 8 inch gauge hypodermic needle (BD), which The needle gauge is the largest feasible that fits snugly into the vein to be accessed.

[0422] result In vitro HEK293 cells were transfected with WT maize under the control of the elongation factor 1a (EF1a) promoter. Mouse FGF21 coding sequence (EF1a-mFGF21) (Zhang et al .,EBioMedicine 15(2017) 173-183)(SEQ ID NO:57) or codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter Four tandem replicas of the miRT122a sequence (CMV-moFGF21) or miRT122a sequence The CAG promoter is involved in the regulation of the miRT1 gene, with four tandem repeats of the miRT1 sequence. The codon-optimized murine FGF21 coding sequence (CAG-moFGF The control cells were transfected with a plasmid encoding 21-double miRT. Non-transfected cells were used as a control. emiRT-transduced HEK293 cells were transduced with EF1a-mFGF21 Transduced cells expressed higher levels of FGF21 compared to untransduced cells (Figure 34A). Furthermore, HEK29 cells transduced with CAG-moFGF21-double miRT 3 cells also had higher intracellular FGF21 protein content and higher FGF in the culture medium. EF1a-mFGF21 protein levels were shown (Fig. 34B and C). CMV-moFGF21-transduced HEK293 cells expressed similar levels of FGF21 (Fig. 34A), whereas HEK293 cells transduced with CMV-moFGF21 Higher intracellular FGF21 protein content and higher FGF21 protein in the culture medium Protein levels were shown (Figures 34B and C).

[0423] C2C12 cells were transfected with WT mouse FGF21 under the control of the EF1a promoter. ding sequence (EF1a-mFGF21) (Zhang et al., EBioMed icine 15(2017) 173-183) or under the control of the CMV promoter A codon-optimized mouse FGF21 coding sequence (CMV-moFGF21) As a control, non-transfected C2C12 cells transduced with CMV-moFGF21 were used. -Higher levels of FGF compared to mFGF21-transduced or non-transduced cells F21 was expressed (Fig. 34D).

[0424] HepG2 cells were transfected with WT mouse FGF21 under the control of the EF1a promoter. ding sequence (EF1a-mFGF21) (Zhang et al., EBioMed icine 15(2017) 173-183) or under the control of the hAAT promoter A plasmid encoding codon-optimized mouse FGF21 (hAAT-moF GF21). Non-transfected cells were used as a control. HepG2 cells transduced with hAAT-moFGF21 expressed EF1a-mFGF These cells express higher levels of FGF21 compared to cells transduced with FGF21 or non-transduced cells. appeared (Figure 34E).

[0425] In vivo Eight-week-old male C57B16 mice were transfected with 5 μg of the elongation factor 1a (EF1a) promoter. WT mouse FGF21 coding sequence (EF1a-mFGF21) under the control of (Zhang et al.,EBioMedicine 15(2017) 173- 183) or codon-optimized mouse FGF21 under the control of the CMV promoter The coding sequence (CMV-moFGF21) is under the control of the hAAT promoter. Codon-optimized murine FGF21 coding sequence (hAAT-moFGF21) The plasmid encoding the α-glucan was administered to the mice via hydrodynamic injection. 24 hours after plasmid administration Analysis of FGF21 expression levels in the liver of mice treated with hAAT-moFGF21 or CMV- Animals treated with moFGF21 showed significantly higher EF1a-mFGF21 activity than animals receiving EF1a-mFGF21. We found that hAAT- Animals treated with moFGF21 or CMV-moFGF21 showed a significantly higher EF1a-mFGF Animals receiving 21 showed higher circulating levels of FGF21 than animals receiving 21 (Figure 35B).

[0426] Example 24. AAV8-hAAT-mo compared to AAV8-Ef1a-mFGF21 FGF21, AAV8-CAG-moFGF21-doublemiRT and AAV1 -CMC-moFGF21-induced FGF21 expression and circulation in target tissues Increase in levels in vivo Liver expression Male C57Bl6 mice were given 1 × 10 10 vg, 2 × 10 10 vg or 5×10 10 WT mouse FGF21 gene under the control of the elongation factor 1a (EF1a) promoter of vg AAV8 vector (AAV8-EF1a-mFGF21) encoding the FGF2-binding sequence or codon-optimized mouse FGF2 under the control of the liver-specific hAAT promoter 1. An AAV8 vector encoding the coding sequence (AAV8-hAAT-moFGF2 1) was administered intravenously. Two weeks after AAV administration, treatment with AAV8-hAAT-moFGF21 was performed. The animals were treated with AAV8-EF1a-mFGF21 regardless of the vector dose. Higher FGF21 expression levels in the liver and higher circulating levels of FGF21 than in animals treated with acetaminophen. Both bells were shown (Figures 36A and B).

[0427] Expression in fat Male C57Bl6 mice were given 2 × 10 10 vg, 5×10 10 vg or 1×10 11 WT mouse FGF21 under the control of the elongation factor 1a (EF1a) promoter in vg AAV8 vector encoding the coding sequence (AAV8-EF1a-mFGF21) or four tandem repeats of the miRT122a sequence and four tandem repeats of the miRT1 sequence. Codon-optimized mouse model under the control of the CAG promoter with indem repeats An AAV8 vector encoding the FGF21 coding sequence (AAV8-CAG-moF Two weeks after AAV administration, either GF21-doublemiRT or GF21-doublemiRT was administered into the eWAT. After that, animals treated with AAV8-CAG-moFGF21-doublemiRT showed a The expression of FGF21 in WAT was higher in animals treated with AV8-EF1a-mFGF21 than in animals treated with AV8-EF1a-mFGF21. Furthermore, the AAV8-CAG-moFGF21-dou Animals treated with blemiRT were compared with animals administered AAV8-EF1a-mFGF21. The results showed much lower FGF21 expression in the liver than in the control group (Fig. 37B). Intra-eWAT administration of AAV8-CAG-moFGF21-doublemiRT vector We demonstrate efficient elimination of transgene expression in off-target tissues.

[0428] Expression in skeletal muscle Male C57Bl6 mice were given 5 × 10 10 vg, 1×10 11 vg or 3×10 11 WT mouse FGF21 under the control of the elongation factor 1a (EF1a) promoter in vg AAV8 vector encoding the coding sequence (AAV8-EF1a-mFGF21) or codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter either an AAV1 vector encoding the FGF21 coding sequence (AAV1-CMV-FGF21) Two weeks after AAV administration, animals treated with AAV1-CMV-FGF21 were expressed much higher FGF21 expression in skeletal muscle than in animals treated with AAV8-EF1a-mFGF21. The expression level of AAV8-EF1a-mFGF21 was also shown (Figure 38A). Animals treated with AAV1-CMV-F showed high expression of FGF21 in the liver, whereas animals treated with AAV1-CMV-F Intramuscular administration of the GF21 vector efficiently eliminated transgene expression in the liver (Figure 38B ).

[0429] array Sequence number Sequence type 1. Amino acid sequence of Homo sapiens FGF21 2. Amino acid sequence of Mus musculus FGF21 3 Canis lupus familiaris ris) Amino acid sequence of FGF21 4. Nucleotide sequence of Homo sapiens FGF21 column 5. Codon-optimized Homo sapiens FGF2 1 - Nucleotide sequence of variant 1 6 Codon-optimized Homo sapiens FGF2 1- Nucleotide sequence of variant 2 7. Codon-optimized Homo sapiens FGF2 1- Nucleotide sequence of variant 3 8. Nucleotide sequence of Mus musculus FGF21 column 9. Codon-optimized Mus musculus FGF2 Nucleotide sequence of 1 10 Canis lupus familiaris ris) Nucleotide sequence of FGF21 11 Codon-optimized Canis lupus familiaris s familiaris)FGF21 nucleotide sequence 12. Nucleotide sequence encoding miRT122a 13. Nucleotide sequence encoding miRT1 14. Nucleotide sequence encoding miRT152 15. Nucleotide sequence encoding miRT199a-5p 16. Nucleotide sequence encoding miRT199a-3p 17. Nucleotide sequence encoding miRT215 18. Nucleotide sequence encoding miRT192 19. Nucleotide sequence encoding miRT148a 20. Nucleotide sequence encoding miRT194 21. Nucleotide sequence encoding miRT124 22. Nucleotide sequence encoding miRT216 23. Nucleotide sequence encoding miRT125 24. Nucleotide sequence encoding miRT133a 25. Nucleotide sequence encoding miRT206 26. Nucleotide sequence encoding miRT130 27. Nucleotide sequence encoding miRT99 28. Nucleotide sequence encoding miRT208-5p 29. Nucleotide sequence encoding miRT208a-3p 30. Nucleotide sequence encoding miRT499-5p 31 Construct A 32 Construct B 33 Construct C 34 Construct D 35 Construct E 36 Construct F 37 Construct G 38 Construct H 39 Construct I 40 Construct J 41 Construct K 42 Construct L 43 Consists of introns from the human β-globin and immunoglobulin heavy chain genes Nucleotide sequence of the chimeric intron to be Nucleotide sequence of the 44 CAG promoter 45 CMV promoter nucleotide sequence Nucleotide sequence of 46 CMV enhancer Nucleotide sequence of the 47 hAAT promoter 48 Truncated AAV2 5'ITR 49 Truncated AAV2 3'ITR 50 SV40 polyadenylation signal 51 Rabbit β-globin polyadenylation signal 52 CMV promoter and CMV enhancer sequences 53 Hepatocyte control region (HCR) enhancer from apolipoprotein E 54 mini / aP2 promoter 55 mini / UCP1 promoter 56 C5-12 promoter 57 pAAV-EF1a-mmFGF21-pA The amino acid sequence of Homo sapiens FGF21 (SEQ ID NO: 1) ) MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQF GGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESL LQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRG PARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPS QGRSPSYAS The nucleotide sequence of Homo sapiens FGF21 (SEQ ID NO: No. 4) ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGT GGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCA GGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTC GGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATG CCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGG GACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTC CTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCT TGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGA TGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCC TGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATG TTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCC AGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGA CCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCAC TCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGA TGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCC CAGGGCCGAAGCCCCAGCTACGCTTCCTGA Codon-optimized Homo sapiens FGF21-barrier 1 nucleotide sequence (SEQ ID NO: 5) ント1のヌクレオチド配列(配列番号5) ATGGATTCTGATGAGACAGGCTTCGAGCACAGCGGCCTGT GGGTTTCAGTTCTGGCTGGACTGCTGCTGGGAGCCTGTCA GGCACACCCTATTCCAGATAGCAGCCCTCTGCTGCAGTTC GGCGGACAAGTGCGGCAGAGATACCTGTACACCGACGACG CCCAGCAGACAGAAGCCCACCTGGAAATCAGAGAGGATGG CACAGTTGGCGGAGCCGCCGATCAGTCTCCTGAATCTCTG CTCCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCC TGGGCGTGAAAACCAGCCGGTTCCTGTGCCAAAGACCTGA CGGCGCCCTGTATGGCAGCCTGCACTTTGATCCTGAGGCC TGCAGCTTCAGAGAGCTGCTGCTTGAGGACGGCTACAACG TGTACCAGTCTGAGGCCCATGGCCTGCCTCTGCATCTGCC TGGAAACAAGAGCCCTCACAGAGATCCCGCTCCTAGAGGC CCTGCCAGATTTCTGCCCTTCCTGGATTGCCTCCTGCTC TGCCAGAGCCTCCTGGAATTCTGGCTCCTCAGCCTCCTGA TGTGGGCAGCTCTGATCCTCTGAGCATGGTCGGACCTAGC CAGGGCAGATCTCCTAGCTACGCCTCTTGA Codon-optimized Homo sapiens FGF21-Barrier Nucleotide sequence of nucleotide 2 (SEQ ID NO: 6) ATGGACAGCGATGAAACCGGGTTCGAGCACAGCGGTCTGT GGGTGTCCGTGCTGGCCGGACTGCTCCTGGGAGCCTGTCA GGCGCACCCCATCCCTGACTCCTCGCCGCTGCTGCAATTC GGCGGACAAGTCCGCCAGAGATACCTGTACACCGACGACG CCCAGCAGACCGAAGCCCACCTGGAAATTCGGGAGGACGG GACTGTGGGAGGCGCTGCAGATCAGTCACCCGAGTCCCTC CTCCAACTGAAGGCCTTGAAGCCCGGCGTGATTCAGATCC TGGGCGTGAAAACTTCCCGCTTCCTTTGCCAACGGCCGGA TGGAGCTCTGTACGGATCCCTGCACTTCGACCCCGAAGCC TGCTCATTCCGCGAGCTGCTCCTTGAGGACGGCTATAACG TGTACCAGTCTGAGGCCCATGGACTCCCCCTGCATCTGCC CGGCAACAAGTCCCCTCACCGGGATCCTGCCCCAAGAGGC CCAGCTCGGTTTCTGCCTCTGCCGGGACTGCCTCCAGCGT TGCCCGAACCCCCTGGTATCCTGGCCCCGCAACCACCTGA CGTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAGC CAGGGAAGGTCCCCGTCCTACGCATCCTGA Codon-optimized Homo sapiens FGF21-barrier 3 nucleotide sequence (SEQ ID NO: 7) ント3のヌクレオチド配列(配列番号7) ATGGATTCCGACGAAACTGGATTTGAACATTCAGGGCTGT GGGTCTCTGTGCTGGCTGGACTGCTGCTGGGGGCTTGTCA GGCTCACCCCATCCCTGACAGCTCCCCTCTGCTGCAGTTC GGAGGACAGGTGCGGCAGAGATACCCTGTATACCGACGATG CCCAGCAGACAGAGGCACACCTGGAGATCAGGGAGGACGG AACCGTGGGAGGAGCAGCCGATCAGTCTCCCGAGAGCCTG CTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCC TGGGCGTGAAGACATCTCGGTTTCTGTGCCAGCGGCCCGA CGGCGCCCTGTACGGCTCCCTGCACTTCGATCCCGAGGCC TGTTCTTTTAGGGAGCTGCTGCTGGAGGACGGCTACAACG TGTATCAGAGCGAGGCACACGGCCTGCCACTGCACCTGCC TGGCAATAAGTCCCTCACCGCGATCCAGCACCCAGGGGC CCAGCACGCTTCCTGCCTCTGCCAGGCCTGCCCCCTGCCC TGCCAGAGCCACCCGGCATCCTGGCCCCCAGCCTCCAGA TGTGGGCTCCAGCGATCCTCTGTCAATGGTGGGGCCAAGT CAGGGGCGGAGTCCTTCATACGCATCATAA Nucleotide sequence encoding miRT122a (target sequence of microRNA 122a) ) (SEQ ID NO: 12) 5' CAAACACCATTGTCACACTCCA 3' Nucleotide sequence encoding miRT1 (target sequence of microRNA 1) (SEQ ID NO: 13) 5' TTACATACTTCTTTACATTCCA 3' Nucleotide sequence encoding miRT152 (target sequence of microRNA 152) ( SEQ ID NO: 14) 5' CCAAGTTCTGTCATGCACTGA 3' Nucleotide sequence encoding miRT199a-5p (target of microRNA 199a) Target sequence) (SEQ ID NO: 15) 5' GAACAGGTAGTCTGAACACTGGG 3' Nucleotide sequence encoding miRT199a-3p (target of microRNA 199a) Target sequence) (SEQ ID NO: 16) 5' TAACCAATGTGCAGACTACTGT 3' Nucleotide sequence encoding miRT215 (target sequence of microRNA 215) ( SEQ ID NO: 17) 5' GTCTGTCAATTCATAGGTCAT 3' Nucleotide sequence encoding miRT192 (target sequence of microRNA 192) ( SEQ ID NO: 18) 5' GGCTGTCAATTCATAGGTCAG 3' Nucleotide sequence encoding miRT148a (target sequence of microRNA 148a) ) (SEQ ID NO: 19) 5' ACAAAGTTCTGTAGTGCACTGA 3' Nucleotide sequence encoding miRT194 (target sequence of microRNA 194) ( SEQ ID NO: 20) 5' TCCACATGGAGTTGCTGTTACA 3' Nucleotide sequence encoding miRT124 (target sequence of microRNA 124) ( SEQ ID NO: 21) 5' GGCATTCACCGCGTGCCTTA 3' Nucleotide sequence encoding miRT216 (target sequence of microRNA 216) ( SEQ ID NO: 22) 5' TCACAGTTGCCAGCTGAGATTA 3' Nucleotide sequence encoding miRT125 (target sequence of microRNA 125) ( SEQ ID NO: 23) 5' TCACAGGTTAAAGGGTCTCAGGGA 3' Nucleotide sequence encoding miRT133a (target sequence of microRNA 133a) ) (SEQ ID NO: 24) 5' CAGCTGGTTGAAGGGGACCAAA 3' Nucleotide sequence encoding miRT206 (target sequence of microRNA 206) ( SEQ ID NO: 25) 5' CCACACACTTCCTTACATTCCA 3' Nucleotide sequence encoding miRT130 (target sequence of microRNA 130) ( SEQ ID NO: 26) 5' ATGCCCTTTTAACATTGCACTG 3' Nucleotide sequence encoding miRT99 (target sequence of microRNA 99) (sequence Number 27) 5' CACAAGATCGGATCTACGGGTT 3' Nucleotide sequence encoding miRT208-5p (target of microRNA 208a) Sequence) (SEQ ID NO: 28) 5' GTATAACCCGGGCCAAAAGCTC 3' Nucleotide sequence encoding miRT208a-3p (target of microRNA 208a) Target sequence) (SEQ ID NO: 29) 5' ACAAGCTTTTTGCTCGTCTTAT 3' Nucleotide sequence encoding miRT499-5p (cardiac-specific microRNA 4 99 target sequence) (SEQ ID NO: 30) 5'AAACATCACTGCAAGTCTTAA 3' Nucleotide sequence of the CAG promoter (SEQ ID NO: 44) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATGGGACTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTT CTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCA ATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGA TGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGC GGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGT GCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTC CTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAA GCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCG CCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCC GGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCG GGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTT TAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCC TTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGG CTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCC GCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCG GGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGA GGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGG CTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTG GGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGC AACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGG CCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGG TGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTC GGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGT CGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCT AGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGG AAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCG TCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGG GACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA CCATGTTCATGCCTTCTTCTTTTTCCTACAG Nucleotide sequence of the CMV promoter (SEQ ID NO: 45) GTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGC GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGAC TTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTT GACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTAT ATAAGCAGAGCT Nucleotide sequence of CMV enhancer (SEQ ID NO: 46) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATG Nucleotide sequence of the hAAT promoter (SEQ ID NO: 47) GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAG TGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGAC TGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGAC GCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTA AGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCG<000454​​​​​​​​​​​​​GCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAG C CCGGGCGTCG GGCGACCTTT GGTCGCCCGG CCTCAGT GAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT AGGGGTT CCT Truncated AAV2 3'ITR (SEQ ID NO: 49) AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTCT GCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTC GCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTG AGCGAGC GAGCGCGC SV40 polyadenylation signal (SEQ ID NO: 50) TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAAT GCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCT ATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAG TT Rabbit β-globin polyadenylation signal (SEQ ID NO: 51) GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGA AGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATT TATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTC TCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAAC ATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGC CCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGA GGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCT TATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTT TATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTA AAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCC TCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTT ATGGAGATC CMV promoter and CMV enhancer sequences (SEQ ID NO: ********) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA CACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTT Note: In the translation of "CMVプロモーターおよびCMVエンハンサー配列(配列番号52)", the "配列番号52" is replaced with "SEQ ID NO: ********" as the specific sequence number is not provided in the original text for accurate translation. You may need to adjust it according to the actual situation.CCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGC GTGTACGGTGGGAGGTCTATATAAGCAGAGCT Hepatocyte control region (HCR) enhancer from apolipoprotein E (SEQ ID NO: 53) CAGAGAGGTCTCTGACCTCTGCCCCAGCTCCAAGGTCAGC AGGCAGGGAGGGCTGTGTGTTTGCTGTTTGCTGCTTGCAA TGTTTGCCCATTTTAGGGACATGAGTAGGCTGAAGTTTGT TCAGTGTGGACTTCAGAGGCAGCACACAAACAGC miniaP2 promoter (SEQ ID NO: 54) GATTA ACCCGCCATG CTACTTATCT ACTCGACATT GATTATT GAC TAGGGGAATT CCAGCAGGAA TCAGGTAGCT GGAGAATCGC ACAGAGC CAT GCGATTCTTG GCAAGCCATG CGACAAAGGC AGAAATGCAC ATTTCAC CCA GAGAGAAGGG ATTGATGTCA GCAGGAAGTC ACCACCCAGA GAGCAAA TGG AGTTCCCAGA TGCCTGACAT TTGCCTTCTT ACTGGATCAG AGTTCAC TAG TGGAAGTGTC ACAGCCCAAA CACTCCCCCA AAGCTCAGCC CTTCCTT GCC TTGTAACAAT CAAGCCGCTC CTGGATGAAC TGCTCCGCCC TCTGTCT CTT TGGCAGGGTT GGAGCCCACT GTGGCCTGAG CGACTTCTAT GGCTCCC TTT TCTGTGATTT TCATGGTTTC TGAGCTCTTT TCCCCCGCTT TATGATT TTC TCTTTTTGTC TCTCTCTTGC TAAACCTCCT TCGTATAT GCCCTCT CAG GTTTCATTTC TGAATCATCT ACTGTGAACT ATTCCCATTG TTTGCCA GAA GCCCCCTGGT TCTTCCTTCT AGACACCAGG CAAGGGGCAG GAGGTAA GAG GCAGGAGTCC ATAAAACAGC CCTGAGAGCC TGCTGGGTCA GTGCCTG CTG TCAGAA miniUCP1 Container (Optional Number 55) GACGTCACAG TGGGTCAGTC ACCCTTGATC ACACTGC ACC AGTCTTCACC TTTCCACGCT TCCTGCCAGA GCATGAATCA GGCTCTC TGG GGATACCGGC CTCACCCCTA CTGAGGCAA CTTTCTCCCA CTTCTCA GAG GCTCTGAGGG CAGCAAGGTC AGCCCTTTCT TTGGAATCTA GAACCAC TCC CTGTCTTGAG CTGACATCAC AGGGCAGGCA GATGCAGCAG GGAAGGG CCT GGGACTGGGA CGTTCATCCT ACAAGAAAGC TGTGGAACTT TTCAGCA ACA TCTCAGAAAT CAGATCGCAC TTATTCAAAG GAGCCAGGCC CTGCTCT GCG CCCTGGTGGA GGCTCCTCAT GTGAAGAGTG ACAAAAGGCA CCATGTT GTG GATACGGGGC GAAGCCCCTC CGGTGTGTCC TCCAGGCATC ATCAGGA ACT AGTGCCAAAG CAGAGGTGCT GGCCAGGGCT TTGGGAGTGA CGCGCGT CTG GGAGGCTTGT GCGCCCAGGG CACGCCCCTG CCGATTCCCA CTAGCAG GTC TTGGGGGACC TGGGCCGGCT CTGCCCCTCC TCCAGCAATC GGGCTAT AAA GCTCTTCCAA GTCAGGGCGC AGAAGTGCCG GGCGATCCGG GCTTAAA GAG CGAGAGGAAG GGACGCTCAC CTTTGAGCTC CTCCACAAAT AGCCCTG GTG GCTGCCACAG AAGTTCGAAG TTGAGAGTTC GG C5-12 promoter (SEQ ID NO: 56) CGGCCGTCCG CCTTCGGCAC CATCCTCACG ACACCCA AAT ATGGCGACGG GTGAGGAATG GTGGGGAGTT ATTTTTAGAG CGGTGAGGAA GGTGGGC AGG CAGCAGGTGT TGGCGCTCTA AAAATAACTC CCGGGAGTTA TTTTTAGAGC GGAGGAA TGG TGGACACCCA AATATGGCGA CGGTTCCTCA CCCGTCGCCA TATTTGGGTG TCCGCCC TCG GCCGGGGCCG CATTCCTGGG GGCCGGGCGG TGCTCCCGCC CGCCTCGATA AAAGGCT CCG GGGCCGGCGG CGGCCCACGA GCTACCCGGA GGAGCGGGAG GCGCCA pAAV-EF1a-mmFGF21-pA (SEQ ID NO: 57) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGC CCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCG GCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCA ACTCCATCACTAGGGGTTCCTGCGGCCGCGGCTCCGGTGC CCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAG AAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGA GAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTAT ATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACG [[ID=3८]]GGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTC CCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTG CCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGA TCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGG CCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTG AGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTG GTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCT CTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTT TTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCT GCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGAC GGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGG GCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTC TCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCG CCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGT CGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGG CCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCG GGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGG CCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGA GTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGC TTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTT ATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGA AGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAA TTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGC CTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGT GTCGTGAGGAATTTCGACTGCTAGCACGCGTGATATCAAT GGAATGGATGAGATCTAGAGTTGGGACCCTGGGACTGTGG GTCCGACTGCTGCTGGCTGTCTTCCTGCTGGGGGTCTACC AAGCATACCCCATCCCTGACTCCAGCCCCCTCCTCCAGTT TGGGGGTCAAGTCCGGCAGAGGTACCTCTACACAGATGAC GACCAAGACACTGAAGCCCACCTGGAGATCAGGGAGGATG GAACAGTGGTAGGCGCAGCACACCGCAGTCCAGAAAGTCT CCTGGAGCTCAAAGCCTTGAAGCCAGGGGTCATTCAAATC CTGGGTGTCAAAGCCTCTAGGTTTCTTTGCCAACAGCCAG ATGGAGCTCTCTATGGATCGCCTCACTTTGATCCTGAGGC CTGCAGCTTCAGAGAACTGCTGCTGGAGGACGGTTACAAT GTGTACCAGTCTGAAGCCCATGGCCTGCCCCTGCGTCTGC CTCAGAAGGACTCCCCAAACCAGGATGCAACATCCTGGGG ACCTGTGCGCTTCCTGCCCATGCCAGGCCTGCTCCACGAG CCCCAAGACCAAGCAGGATTCCTGCCCCCAGAGCCCCCAG ATGTGGGCTCCTCTGACCCCCTGAGCATGGTAGAGCCTTT ACAGGGCCGAAGCCCCAGCTATGCGTCCTGAGATATCAAA GAATTCTAAGCTTGTCGACGAATGCAATTGTTGTTAATTA ATTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAAT AAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTT TTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAAT GTATCTTAGTCGAGTTAATTAACGGCGGCCGCAGGAACCC CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCT CGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTA CGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAA CCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGG GTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAG CGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTT CTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATC GGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGT AGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGA CGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCA AACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTT GATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAA AAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAA CAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGT ACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGAC ACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCT GCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCC GGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA AACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTT ATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCA GGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT GTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAA AGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTA TTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAG TTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACA GCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTT TCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCG GTATTATCCCGGTATTGACGCCGGGCAAGAGCAACTCGGTC GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC ACCAGTCACAGAAAAAGCATCTTACGGATGGCATGACAGATA AGAGAATTATGCAGTGCTGCCATACCATGAGTGATAACA CTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTA ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCA TACCAAACGACGAGCGTGACACACGATGCCTGTAGCAAT GGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGG CGGATAAAGTTGCAGGACACTTCTGCGCTCGGCCCTTCC GGCTGGCTGGTTTTATTGCTGATAAATCTGGAGCCGGTGAG CGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAG TCAGGCAACTATGGATGAACGAATAGACAGATCGCTGAG ATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACC AAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCA TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGAT AATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC ACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGG ATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGAC CTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATC CGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGAT GCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAA CGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTT GCTCACATGT Elongation factor 1 alpha promoter: 150 to 1327 (1178 bp) Mus musculus FGF21:1359 (633bp ) SEQ ID NO: 57 also contains the truncated AAV2 5' and 3' ITRs and SV40 polyA. (SEQ ID NOs: 48, 49 and 50, already included in the sequence listing). [Brief explanation of the drawings]

[0430] [Figure 1]Figure 1. Prevention of obesity by intra-eWAT administration of the AAV9-CAG-moFGF21-dmiRT vector in C57B16 mice. (A) Schematic diagram of the AAV-CAG-moFGF21-doublemiRT vector. The expression cassette contained a CAG promoter, a codon-optimized mouse FGF21 coding sequence, and four tandem repeats of the miRT122a sequence and four tandem repeats of the miRT1 sequence cloned into the 3' untranslated region of the expression cassette. ITRs from AAV2 flanked the expression cassette. Diagram not to scale. CAG: chicken β-actin promoter / CMV enhancer; pA: polyA. (B) Expression levels of FGF21 in metabolic tissues. Expression levels of the codon-optimized mouse FGF21 coding sequence in eWAT, iWAT, iBAT, and liver of C57Bl6 mice were measured by RTqPCR and normalized to Rplp0 values ​​(n = 8–11 animals / group). (C) Circulating levels of FGF21 (n = 8–11 animals / group). (D-E) Expression levels of FGF21R1 (D) and β-Klotho (E) in metabolic tissues. Expression levels of FGF21 receptor 1 (FGF21R1) and β-Klotho (E) in eWAT, iWAT, iBAT, and liver of C57Bl6 mice were measured by RTqPCR and normalized to Rplp0 values ​​(n = 7 animals / group). (F) Body weight evolution. Body weight was measured weekly (n = 8–11 animals / group). (G) Representative images of animals. (H) Tissue weights. eWAT, iWAT, rWAT, mWAT, iBAT, and liver weights (n = 8–11 animals / group) from chow-fed and HFD-fed C57Bl6 mice treated with AAV vectors in eWAT. Analysis was performed 14 weeks after intra-eWAT administration of 1012 vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vectors. Results are expressed as mean ± SEM. ND, not detected. HFD, high-fat diet. AU, arbitrary units. eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue; rWAT, retroperitoneal white adipose tissue; mWAT, mesenteric white adipose tissue; iBAT, interscapular brown adipose tissue.* p<0.05 vs AAV9-CAG-null chow, ** p<0.01 vs AAV9-CAG-null chow, *** p<0.001 vs AAV9-CAG-null chow, $ p<0.05 vs AAV9-CAG-null HFD, $$ p<0.01 vs AAV9-CAG-null HFD, $$$ p<0.001 vs AAV9-CAG-null HFD. [Figure 2] Figure 2. Histological analysis of adipose tissue and liver from C57Bl6 mice treated with the AAV9-CAG-moFGF21-doublemiRT vector in eWAT. (A) Representative images stained with hematoxylin and eosin of sections of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), interscapular brown adipose tissue (iBAT), and liver from chow-fed and HFD-fed C57Bl6 mice treated with the AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vector in eWAT. Original magnification ×100. (B) Average area of ​​eWAT white adipocytes (n = 4 animals / group). (C) Frequency distribution of eWAT white adipocyte area (n = 4 animals / group). Analysis was performed 14 weeks after intraeWAT administration of 1012vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vector. Results are expressed as mean ± SEM. HFD, high-fat diet. ** p<0.01 vs. AAV9-CAG-null chow, *** p<0.001 vs. AAV9-CAG-null chow, $$ p<0.01 vs. AAV9-CAG-null HFD, $$$ p<0.001 vs. AAV9-CAG-null HFD. [Figure 3]Figure 3. Improved energy expenditure and insulin sensitivity in C57Bl6 mice treated with AAV9-CAG-moFGF21-double miRT vector in eWAT. (A-B) UCP1 (A) and Dio2 (B) expression levels. UCP1 and Dio2 expression levels in iWAT were measured by RTqPCR and normalized to Rplp0 values ​​(n = 7 animals / group). (C) Energy metabolism. Energy expenditure (EE) was measured using indirect open-circuit calorimetry. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were acquired 9 weeks after AAV administration during the light cycle (basal state) and dark cycle (active phase) and adjusted for body weight (n = 8–11 animals / group). (D) Liver triglyceride content (n = 8–10 animals / group). (EF) Serum triglyceride (E) and cholesterol (F) levels (n = 8–11 animals / group). (G) Intraperitoneal insulin tolerance test. Mice were given an intraperitoneal injection of 0.75 U insulin / kg body weight, and blood glucose levels were measured at the indicated time points (n = 6–11 animals / group). Tests were performed 11 weeks after AAV administration. (H) Fasting circulating insulin levels. Unless otherwise indicated, analyses were performed 14 weeks after intra-eWAT administration of 1012 vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vectors. Results are expressed as mean ± SEM. HFD, high-fat diet. TG, triglycerides. Chol, cholesterol. * p<0.05 vs AAV9-CAG-null chow, ** p<0.01 vs AAV9-CAG-null chow, *** p<0.001 vs AAV9-CAG-null chow, $ p<0.05 vs AAV9-CAG-null HFD, $$ p<0.01 vs AAV9-CAG-null HFD, $$$ p<0.001 vs AAV9-CAG-null HFD. [Figure 4]Figure 4. Reversal of obesity by intra-eWAT administration of AAV8-CAG-moFGF21-dmiRT vector in ob / ob mice. (A) Expression levels of FGF21 in metabolic tissues. Expression levels of the codon-optimized mouse FGF21 coding sequence in eWAT, iWAT, iBAT, and liver of ob / ob mice were measured by RTqPCR and normalized to Rplp0 values. (B) Circulating levels of FGF21. (C-D) Progression of body weight (C) and weight gain (D). Body weight was measured weekly. (E) Tissue weights. Weights of eWAT, iWAT, rWAT, mWAT, iBAT, and liver of ob / ob mice treated with AAV vectors in eWAT. Analysis was performed 16 weeks after intraeWAT administration of 10 vg, 5 x 10 vg, 2 x 10 vg, or 10 vg of AAV8-CAG-moFGF21-doublemiRT or 10 vg of AAV8-CAG-null vector. Results are expressed as mean ± SEM. n = 7–8 animals per group. ND, not detected. AU, arbitrary units. eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue; rWAT, retroperitoneal white adipose tissue; mWAT, mesenteric white adipose tissue; iBAT, interscapular brown adipose tissue. * p < 0.05 vs. AAV8-CAG-null; ** p < 0.01 vs. AAV8-CAG-null; *** p < 0.001 vs. AAV8-CAG-null. [Figure 5] Figure 5. Improved insulin sensitivity in ob / ob mice treated with the AAV8-CAG-moFGF21-doublemiRT vector in eWAT. (A) Intraperitoneal insulin tolerance test. ob / ob mice were given an intraperitoneal injection of 0.75 U insulin / kg body weight, and blood glucose levels were measured at the indicated time points. The test was performed 9 weeks after AAV administration. (B) Fasting circulating insulin levels 2 months after AAV administration. Results are expressed as mean ± SEM, n = 7–8 animals / group. * p < 0.05 vs. AAV8-CAG-null, ** p < 0.01 vs. AAV8-CAG-null, *** p < 0.001 vs. AAV8-CAG-null. [Figure 6]Figure 6. Intravenous administration of AAV8-hAAT-moFGF21 vector reverses obesity and improves glucose metabolism in ob / ob mice. (A) Schematic diagram of the AAV-hAAT-moFGF21 vector. The expression cassette contained the human α1-antitrypsin (hAAT) promoter and a codon-optimized murine FGF21 coding sequence. ITRs from AAV2 flanked the expression cassette. The diagram is not to scale. pA:polyA. (B) Expression levels of FGF21. Expression levels of the codon-optimized murine FGF21 coding sequence in the liver of ob / ob mice were measured by RTqPCR and normalized to the Rplp0 value. (C) Circulating levels of FGF21. (D-E) Progression of body weight (C) and weight gain (D). Body weight was measured weekly. (F) Representative images of animals. (G) Tissue weights. (H) Weights of eWAT, iWAT, rWAT, mWAT, iBAT, and liver from ob / ob mice intravenously treated with AAV vectors. (H) Intraperitoneal insulin tolerance test. Ob / ob mice were given an intraperitoneal injection of 0.75 U insulin / kg body weight, and blood glucose levels were measured at the indicated time points. The test was performed 9 weeks after AAV administration. (I) Fasting circulating insulin levels 3 months after AAV administration. Unless otherwise indicated, analyses were performed 20 weeks after intravenous administration of 10 vg or 5 × 10 vg of AAV8-hAAT-moFGF21 or 5 × 10 vg of AAV8-hAAT-null vector. Results are expressed as mean ± SEM. n = 9–10 animals per group. ND, not detected. AU, arbitrary unit. eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue; rWAT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue; iBAT Interscapular brown adipose tissue. * p<0.05 vs AAV8-hAAT-null, ** p<0.01 vs AAV8-hAAT-null, *** p<0.001 vs AAV8-hAAT-null. [Figure 7]Figure 7. Long-term reversal of obesity by intravenous administration of AAV-hAAT-moFGF21 vector in HFD-fed C57bl6 mice. (A) Circulating levels of FGF21. (B-C) Progression of body weight (C) and weight gain (D). Body weight was measured weekly. Analysis was performed 52 weeks after intravenous administration of 1010 vg or 5x1010 vg of AAV8-hAAT-moFGF21 or 5x1010 vg of AAV8-hAAT-null vector. Results are expressed as mean ± SEM, n = 9–12 animals per group. *** p<0.001 vs. AAV8-hAAT-null chow; $$ p<0.01 vs. AAV8-hAAT-null HFD; $$$ p<0.001 vs. AAV8-hAAT-null HFD. [Figure 8] Figure 8. Long-term improvement in energy expenditure and insulin sensitivity following intravenous administration of the AAV-hAAT-moFGF21 vector in HFD-fed C57Bl6 mice. (A) Energy metabolism. Energy expenditure (EE) was measured using an indirect open-circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were obtained 4 weeks after AAV administration during the light cycle (basal state) and dark cycle (active phase) and adjusted for body weight. (B) Intraperitoneal insulin tolerance test. C57Bl6 mice were given an intraperitoneal injection of 0.75 U insulin / kg body weight, and blood glucose levels were measured at the indicated time points. The test was performed 7 weeks after AAV administration. (C) Circulating insulin levels in the fasted and fed states. Results are expressed as mean ± SEM, n = 9–12 animals per group. HFD, high-fat diet. * p<0.05 vs AAV8-hAAT-null chow, ** p<0.01 vs AAV8-hAAT-null chow, *** p<0.001 vs AAV8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD, $$$ p<0.001 vs AAV8-hAAT-null HFD. [Figure 9]Figure 9. Reversal of obesity by intravenous administration of AAV-hAAT-moFGF21 vector in aged HFD-fed mice. (A) Circulating levels of FGF21. (B-C) Progression of body weight (B) and weight gain (C). Body weight was measured weekly. Analysis was performed 21 weeks after intravenous administration of 1010 vg, 2 x 1010 vg, or 5 x 1010 vg of AAV8-hAAT-moFGF21 or 5 x 1010 vg of AAV8-hAAT-null vector. Results are expressed as mean ± SEM, n = 7–8 animals per group. HFD, high-fat diet. *** p<0.05 vs. AAV8-hAAT-null chow; $ p<0.05 vs. AAV8-hAAT-null HFD; $$ p<0.01 vs. AAV8-hAAT-null HFD. $$$ p<0.001 vs AAV8-hAAT-null HFD. [Figure 10] Figure 10. Improved energy expenditure and insulin sensitivity following intravenous administration of the AAV-hAAT-moFGF21 vector in aged HFD-fed mice. (A) Energy metabolism. Energy expenditure (EE) was measured using an indirect open-circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were obtained 6 weeks after AAV administration during the light cycle (basal state) and dark cycle (active phase) and adjusted for body weight. (B) Intraperitoneal insulin tolerance test. Aged C57B16 mice were given an intraperitoneal injection of 0.75 U insulin / kg body weight, and blood glucose levels were measured at the indicated time points. The test was performed 9 weeks after AAV administration. (C) Circulating insulin levels in the fasted and fed states. Results are expressed as mean ± SEM, n = 7–8 animals per group. HFD, high-fat diet. ** p<0.01 vs AAV8-hAAT-null chow, *** p<0.001 vs AAV8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD, $$$ p<0.001 vs AAV8-hAAT-null HFD. [Figure 11]Figure 11. Weight loss following intramuscular administration of the AAV-CMV-moFGF21 vector in C57Bl6 mice. (A) Schematic diagram of the AAV-CMV-moFGF21 vector. The expression cassette contained a cytomegalovirus (CMV) promoter and a codon-optimized murine FGF21 coding sequence. ITRs from AAV2 flanked the expression cassette. Diagram not to scale. pA:polyA. (B) Circulating FGF21 levels. (C-D) Progression of body weight (C) and weight gain (D). Body weight was measured weekly. Results are expressed as mean ± SEM. n = 6–7 animals / group. * p<0.05 vs. AAV1-CMV-null, ** p<0.01 vs. AAV1-CMV-null. The FGF21 label in the figure refers to moFGF21 according to the figure legend. [Figure 12] Figure 12. Increased FGF21 protein production by codon optimization of the nucleotide sequence encoding human FGF21. (A) hFGF21 protein levels in the culture medium of HEK293 cells transfected with wild-type hFGF21 or three different versions of the codon-optimized human FGF21 sequence. Results are expressed as mean ± SEM. n = 3 wells / group. ND, not detected. * p < 0.05 vs. non-transfected cells. [Figure 13] Figure 13. Intra-eWAT administration of AAV8-CAG-moFGF21-dmiRT vector in ob / ob mice. A, B Representative images of hematoxylin-eosin staining of (A) eWAT and (B) liver tissue sections obtained from ob / ob animals intra-eWAT injected with either null or AAV8 vectors encoding FGF21 at all doses tested. Scale bar: 100 μm for eWAT, 200 μm for liver. C Blood glucose in the fed state. D Blood insulin in the fed state 3 months after AAV administration. The FGF21 label in the figure refers to moFGF21. Data description: All values ​​are expressed as mean ± SEM. n = 6–9 animals / group for (A, B). n = 4–8 animals / group for (CH). n = 6–8 animals / group for (I). *P<0.05, **P<0.01, and ***P<0.001 vs. the null-injected group. [Figure 14] Figure 14. Effect of FGF21 gene transfer into the eWAT of ob / ob mice. A. Serum adiponectin levels in 25-week-old ob / ob animals injected intraeWAT at 11 weeks of age with either the AAV8-CAG-null vector or the AAV8-CAG-moFGF21-dmiRT vector at four different doses (1 × 10, 5 × 10, 2 × 10, and 1 × 10 vg / mouse). B. Quantification of the expression of the macrophage marker F4 / 80 by qRT-PCR in the same animals as in (A). C. Representative images of immunostaining for the macrophage-specific marker Mac2 in eWAT sections from ob / ob mice that received the AAV8-CAG-moFGF21-dmiRT vector. n = 4–8 / group. Scale bar: 200 μm. D. Liver weights in all intraeWAT treatment groups. E, F Liver triglyceride and cholesterol content in the fed state in the same cohort as in (A). The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data information: All values ​​are expressed as mean ± SEM. n = 4–8 animals / group in (A, B, D). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the null-injected ob / ob group. [Figure 15]Figure 15. Reduction of obesity and improvement of insulin sensitivity in ob / ob mice treated with AAV8-hAAT-moFGF21 vector. A. Representative images of hematoxylin-eosin staining of eWAT tissue sections obtained from ob / ob animals injected with either null- or FGF21-encoding AAV vectors at 1 x 10 or 5 x 10 vg / mouse. B. Serum adiponectin levels in all groups. C. Representative images of hematoxylin-eosin staining of liver tissue sections obtained from ob / ob animals injected with either null- or FGF21-encoding AAV vectors at 1 x 10 or 5 x 10 vg / mouse. D. Fed blood glucose levels. E. Fed serum insulin levels 5 months after AAV. FGF21 labeling in the figure refers to moFGF21 according to the figure legend. Data information: All data are expressed as mean ± SEM. In (AC, E, GH), n=9-10 animals / group. *P<0.05, **P<0.01 and ***P<0.001 vs. the null-injected ob / ob group. [Figure 16] Figure 16. Effect of FGF21 hepatic gene transfer in ob / ob mice. A. Immunohistochemistry for the macrophage-specific marker Mac2 in eWAT sections from ob / ob mice receiving the AAV8-hAAT-moFGF21 vector. Scale bar: 500 μm. B, C. Quantification of the expression of inflammatory markers F4 / 80 (B) and TNF-α (C) by qRT-PCR in the same mouse cohort. D, E. Liver weights (D) and representative images (E) obtained from animals belonging to the same experimental group as in (A). F, G. Hepatic triglyceride and cholesterol content in the fed state in the same cohort as in (A). The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data presentation: All values ​​are expressed as mean ± SEM. n = 9–10 animals / group in (B, D, H). *P<0.05, **P<0.01 and ***P<0.001 versus the null-injected ob / ob group. [Figure 17]Figure 17. AAV8-hAAT-moFGF21 treatment increases the expression of genes involved in glucose uptake and thermogenesis in adipose tissue of ob / ob mice. A, B qRT-PCR quantification of hepatic PEPCK and G6Pase in ob / ob mice injected with either the AAV8-hAAT-null vector or the AAV8-hAAT-moFGF21 vector at 2 months of age. C qRT-PCR quantification of GLUT1 (C), GLUT4 (D), HKI (E), and HKII (F) in eWAT, iWAT, and iBAT in the same animals as in (A). G Relative expression of UCP1 in iBAT in the same cohort as in (A). The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data information: All values ​​are expressed as mean ± SEM. n = 9–10 animals / group in (A-G). *P<0.05, **P<0.01 and ***P<0.001 versus the null-injected ob / ob group. [Figure 18] Figure 18. AAV8-mediated hepatic FGF21 gene transfer counteracts HFD-induced obesity. A. Weights of epididymal (eWAT), inguinal (iWAT), and retroperitoneal (rWAT) white adipose tissue depots, liver, and quadriceps muscle obtained from mice treated with the AAV8-hAAT-moFGF21 vector as young adults (upper panel) or adults (lower panel). B. Circulating levels of FGF21 at different time points after vector administration. The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data description: All values ​​are expressed as mean ± SEM. n = 7–10 animals / group in (A-D). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the chow-fed, null-injected group. #P<0.05, ##P<0.01, and ###P<0.001 versus the HFD-fed Null-injected group. HFD, high-fat diet. [Figure 19]Figure 19. Hepatic FGF21 gene transfer counteracts HFD-induced obesity. A, B Representative images of animals belonging to all experimental groups in studies conducted as young adults (A) or adults (B). C Representative images of epididymal white adipose tissue (eWAT) pads obtained at sacrifice from animals treated with several doses of AAV8-hAAT-moFGF21 as young adults (left) or adults (right). D Representative images of livers obtained from animals treated as young adults (left) or adults (right). E AAV-derived FGF21 expression in the livers of animals treated as young adults or adults. qPCR was performed using primers specifically detecting the coding sequence of codon-optimized murine FGF21 (coFGF21). The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data Description: All values ​​are expressed as mean ± SEM. In (E), n=7-10 animals / group. HFD, high-fat diet. ND, not detected. [Figure 20]Figure 20. AAV8-hAAT-moFGF21-mediated increased energy expenditure and decreased adiposity in iBAT and iWAT. A. Assessment of locomotor activity via the open field test in animals (young adults) placed on a HFD from approximately 2 months of age and treated 2 months later with either null or FGF21-encoding vectors. B. Hematoxylin-eosin staining of iBAT tissue sections from animals treated as young adults (left) or adults (right). C. Western blot analysis of UCP1 content in iBAT from the same animal cohort as in (A). A representative immunoblot is shown (left). Histograms depict densitometric analysis of two different immunoblots (right). D. Hematoxylin-eosin staining of iWAT tissue sections from animals treated as young adults (left) or adults (right). E Quantification of Phospho1 expression by qRT-PCR in iWAT in animals receiving FGF21 vector starting on an HFD diet as young adults or adults. The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data description: All values ​​are expressed as mean ± SEM. (AC) n = 7–10 animals / group. (E) n = 4 animals / group. (G) n = 7–10 animals / group. *P<0.05, **P<0.01, and ***P<0.001 vs. chow-fed Null-injected group. #P<0.05 and ###P<0.001 vs. HFD-fed Null-injected group. HFD, high-fat diet. [Figure 21]Figure 21. Energy expenditure 10 months after hepatic gene transfer. A. Energy expenditure was measured 10 months after AAV8-hAAT-null or AAV8-hAAT-moFGF21 vector delivery in cohorts of animals initiated on an HFD at 2 months of age. Data were acquired during the light and dark cycles. B. Western blot analysis of UCP1 content in iWAT from the same cohort of animals. A representative immunoblot is shown (left). The graph shows densitometric analysis of two different immunoblots (right). C. Relative expression of Serca2b and RyR2 in iWAT in groups of animals initiated on an HFD as young adults or adults and receiving FGF21 vectors. The FGF21 label in the figure refers to moFGF21, as per the figure legend. Data description: All values ​​are expressed as mean ± SEM. (A) n = 7–10 animals / group. (B) n = 4 animals / group. In (C), n=7-10 animals / group. *P<0.05, **P<0.01, and ***P<0.001 vs. the chow-fed Null-injected group. ###P<0.001 vs. the HFD-fed Null-injected group. HFD, high-fat diet. [Figure 22]Figure 22. AAV8-hAAT-moFGF21-mediated reversal of islet hyperplasia. A. Fasting glucagon levels in animals receiving FGF21 vectors and initiated on a HFD as young adults. B. Beta cell mass in animals receiving FGF21 vectors and initiated on a HFD as adults. C. Representative images of immunostaining for insulin in pancreatic sections from animals receiving 5 x 10 vg / mouse of AAV8-hAAT-moFGF21 as adults. Scale bar: 400 μm. Inset scale bar: 100 μm. D. Representative images of double immunostaining for insulin (dark gray) and glucagon (light gray) in pancreatic sections from animals receiving 5 x 10 vg / mouse of AAV8-hAAT-moFGF21 as young adults (top panel) or adults (bottom panel). Scale bar: 100 μm. The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data information: All values ​​are expressed as mean ± SEM. In (AC), n = 7-10 animals / group. In (D), n = 4-5 animals / group. *P<0.05, **P<0.01, and ***P<0.001 vs. the chow-fed Null-injected group. #P<0.05, ##P<0.01, and ###P<0.001 vs. the HFD-fed Null-injected group. HFD, high-fat diet. [Figure 23] Figure 23. Treatment with AAV8-hAAT-moFGF21 improves glucose tolerance. A. Glucose tolerance was tested after intraperitoneal injection of glucose (2 g / kg body weight) in mice that started on an HFD diet as young adults and received the FGF21 vector. B. Serum insulin levels during the glucose tolerance test shown in (A). Data presentation: All data are expressed as mean ± SEM. n=7-10 animals / group in (A-D). *P<0.05, **P<0.01, and ***P<0.001 vs. the chow-fed Null-injected group. #P<0.05, ##P<0.01, and ###P<0.001 vs. the HFD-fed Null-injected group. HFD, high-fat diet. [Figure 24]Figure 24. AAV8-hAAT-moFGF21 treatment reverses WAT hypertrophy and inflammation. A Representative images of hematoxylin-eosin staining of eWAT from animals fed chow or HFD as young adults (left panel) or adults (right panel) and administered either AAV8-hAAT-null or 5 x 1010 vg / mouse of AAV8-hAAT-moFGF21 vectors. Adipocytes in HFD-fed null-injected mice were larger, while adipocytes in HFD-fed FGF21-treated animals were reduced in size. Scale bar: 100 μm. B Morphometric analysis of WAT adipocyte area in animals treated as young adults or adults. C, D Circulating levels of adiponectin (C) and leptin (D). E. Immunohistochemistry for the macrophage-specific marker Mac2 in eWAT sections from animals that received 5 × 1010 vg / mouse of AAV8-hAAT-moFGF21 as adults. Photomicrographs show the presence of crown-like structures in the eWAT of HFD-fed null-injected animals (arrows and inset), but not in the eWAT of HFD-fed FGF21-treated mice. Scale bars: 200 μm and 50 μm (inset). FH. Quantification by qRT-PCR of the expression of inflammatory markers F4 / 80 (F), IL1-β (G), and TNF-α (H) in animals that received the FGF21 vector starting on an HFD diet as adults. The FGF21 label in the figure refers to moFGF21 as per the figure legend. Data presentation: All values ​​are expressed as mean ± SEM. (B) n = 4 animals / group. (FH) n = 7–10 animals / group. *P<0.05, **P<0.01, and ***P<0.001 vs. the chow-fed Null-injected group. #P<0.05, ##P<0.01, and ###P<0.001 vs. the HFD-fed Null-injected group. HFD, high-fat diet. [Figure 25]Figure 25. Adipocyte size and inflammation in AAV8-hAAT-moFGF21-treated animals. A. Frequency distribution of adipocyte area in groups of animals initiated as young adults (top graph) or adults (bottom graph) on chow or HFD and receiving either AAV8-hAAT-null or 5x1010vg / mouse AAV8-hAAT-moFGF21 vectors. B. Mac2 immunohistochemistry in eWAT of animals initiated as young adults. Arrows indicate crown-like structures formed by macrophage infiltration in the eWAT of HFD-fed, null-injected mice. Scale bars: 200 μm and 50 μm (inset). C. E. Relative expression of inflammatory markers F4 / 80, CD68, and TNF-α by qRT-PCR in the same cohort of animals as in (B). The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data information: All values ​​are expressed as mean ± SEM. (A) n = 4 animals / group. (CE) n = 7-10 animals / group. ***P < 0.001 vs. chow-fed null-injected group. ###P < 0.001 vs. HFD-fed null-injected group. HFD, high-fat diet. [Figure 26]Figure 26. Treatment with a vector encoding FGF21 ameliorates hepatic steatosis and liver inflammation. A. Representative images of hematoxylin-eosin staining of liver sections obtained from animals fed a chow or HFD and administered either AAV8-hAAT-null or 5×1010 vg / mouse of AAV8-hAAT-moFGF21 vector. HFD clearly induced lipid droplet accumulation in the liver, which was reversed by AAV8-hAAT-moFGF21 treatment in both young adults and adults. Scale bar: 100 μm. B, C. Hepatic triglyceride and cholesterol content upon feeding in the same animal cohort. D. Immunostaining for the macrophage-specific marker Mac-2 of liver sections from animals fed a HFD and receiving either AAV8-hAAT-null or 5×1010 vg / mouse of AAV8-hAAT-moFGF21 vector. Arrows indicate the presence of crown-like structures. Scale bars: 200 μm and 50 μm (inset). The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data information: All values ​​are expressed as mean ± SEM. n = 7–10 animals / group in (BC). **P<0.01 and ***P<0.001 vs. the chow-fed Null-injected group. # #P<0.01 vs. the HFD-fed Null-injected group. HFD, high-fat diet. [Figure 27] Figure 27. AAV8-hAAT-moFGF21-mediated reversal of liver fibrosis. Analysis of liver fibrosis via Masson's trichrome staining in HFD-fed animals receiving either AAV8-hAAT-null or AAV8-hAAT-moFGF21 vectors at 5 x 10 vg / mouse. AAV8-hAAT-moFGF21 treatment (right panel) significantly reduced the detection of collagen fibers (blue) readily detectable in animals treated with the null vector (left panel). Scale bar: 50 μm. The FGF21 label in the figure refers to moFGF21 according to the figure legend. [Figure 28]Figure 28. AAV8-hAAT-moFGF21 treatment ameliorates liver fibrosis. A. Analysis of liver fibrosis via Picrosirius staining in HFD-fed animals receiving either AAV8-hAAT-null or AAV8-hAAT-moFGF21 vectors at 5 x 10 vg / mouse. AAV8-hAAT-moFGF21 treatment (right panel) significantly reduced the detection of collagen fibers (black), which were readily detectable in animals treated with the null vector (left panel). Scale bar: 50 μm. B, C. Quantification of collagen 1 expression in the liver by qRT-PCR in animals receiving the FGF21 vector starting on a HFD diet as young adults (B) or adults (C). The FGF21 label in the figure refers to moFGF21, as indicated by the figure legend. Data presentation: All values ​​are expressed as mean ± SEM. n = 7–10 animals / group in (B-C). *P<0.05, **P<0.01, and ***P<0.001 vs. the chow-fed Null-injected group. #P<0.05 and ###P<0.001 vs. the HFD-fed Null-injected group. HFD, high-fat diet. [Figure 29]Figure 29. No bone abnormalities were observed in AAV8-hAAT-moFGF21-treated animals. The long-term effects of FGF21 gene transfer on bone were examined by comparing HFD-fed mice treated with the highest dose (5 × 10 vg / mouse) of AAV8-hAAT-moFGF21 vector as young adults or adults with null-injected chow- or HFD-fed animals. A. Total length from snout to tail base. B. Tibia length. CO. Microcomputed tomography (μCT) analysis of tibia epiphysis (CJ) and diaphysis (KO) obtained from HFD-fed mice administered either null or FGF21-encoding AAV vectors at sacrifice, when the animals were 18 months of age. P, Q. Levels of circulating IGFBP1 (P) and IGF1 (Q) measured by ELISA. The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data information: All data are expressed as mean ± SEM. (A, PQ) n = 7-10 animals / group. (BO) n = 4 animals / group. **P<0.01 and ***P<0.001 vs. chow-fed null-injected group. HFD, high-fat diet; BMD, bone mineral density; BMC, bone mineral content; BV, bone volume; BV / TV, bone volume / tissue volume ratio; BS / BV, bone surface / bone volume ratio; Tb.N, trabecular number; Tb.Th, trabecular width; Tb.Sp, trabecular spacing. [Figure 30] Figure 30. Analysis of glycemic profiles in C57B16 mice treated with AAV8-hAAT-moFGF21 vectors. Blood glucose levels were assessed under fed conditions. IV administration of AAV, 5x1010vg or 2x1011vg of AAV8-hAAT-moFGF21 (n=13 and 15, respectively), or 2x1011vg of AAV8-null vector (n=15). Treatment with STZ, streptozotocin (5x50mg / kg). Results are mean + SEM. *p<0.05; ***p<0.001 vs. AAV8-hAAT-Null. The FGF21 label in the figure refers to moFGF21 according to the figure legend. [Figure 31]Figure 31. Gene transfer of FGF21 into skeletal muscle of healthy animals. A. Circulating FGF21 levels measured 40 weeks after injection of 3 x 1011 vg / mouse of either AAV1-CMV-Null or AAV1-CMV-moFGF21 vector into skeletal muscle of healthy animals fed a chow diet. B. AAV-derived FGF21 expression in muscle and liver of healthy animals injected intramuscularly with AAV1-CMV-Null or AAV1-CMV-moFGF21 vector. C. Body weight evolution over a 40-week follow-up period. D. Tissue wet weights of different muscles, fat pads, and liver. E, F. Liver triglyceride and cholesterol content in the fed state. G. Fed serum insulin levels. H. Insulin sensitivity assessed via intraperitoneal injection of insulin (0.75 units / kg body weight) and expressed as a percentage of starting blood glucose. The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data information: All values ​​are expressed as mean ± SEM. n=5-7 animals / group in (A-H). *P<0.05, **P<0.01, and ***P<0.001 vs. Null-injected group. [Figure 32]Figure 32. AAV1-mediated skeletal muscle FGF21 gene transfer counteracts HFD-induced obesity and insulin resistance. A, B. Body weight (A) and weight gain (B) progression in animals treated with AAV1-CMV-moFGF21. C57B16 mice were fed an HFD for approximately 12 weeks and then administered 3 x 10 vg / mouse of the AAV1-CMV-moFGF21 vector. Control obese mice and control chow-fed mice received 3 x 10 vg of AAV1-CMV-null. C. Circulating FGF21 levels at different time points after vector administration. D, E. Fasting blood glucose (D) and fed serum insulin (E) levels in the same groups of animals as in (A, B). F. Insulin sensitivity was determined in all experimental groups after intraperitoneal injection of insulin (0.75 units / kg body weight). Results were calculated as a percentage of the initial blood glucose level. The FGF21 label in the figure refers to moFGF21 according to the figure legend. Data information: All values ​​are expressed as mean ± SEM. In (AF), HFD-fed mice n=10 animals / group; chow-fed mice n=5 animals / group. ***P<0.001 vs. HFD-fed null-injected group. [Figure 33] Figure 33. Codon optimization of the nucleotide sequence encoding human FGF21 increases circulating FGF21 levels in vivo. Circulating levels of hFGF21 in C57B16 mice hydrodynamically administered plasmids encoding wild-type hFGF21 or three different variants of the codon-optimized human FGF21 sequence. Results are expressed as mean ± SEM. n=9-10 mice / group. ND, not detected. Negative control, untreated mice. *p<0.05 vs. untreated mice. [Figure 34]Figure 34. Increase in FGF21 expression levels in vitro with hAAT-moFGF21, CAG-moFGF21-double miRT, and CMV-moFGF21 expression cassettes. (A) FGF21 expression levels in HEK293 cells transfected with plasmids encoding the wild-type mouse FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21), the codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21), or the codon-optimized mouse FGF21 coding sequence under the control of the CAG promoter with four tandem repeats of the miRT122a sequence and four tandem repeats of the miRT1 sequence (CAG-moFGF21-double miRT). (B and C) Intracellular FGF21 protein content (B) and FGF21 protein levels in the culture medium (C) ...

Claims

1. An adeno-associated virus 1 (AAV1) vector comprising a viral expression construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) that is expressed in skeletal muscle and a ubiquitous promoter.

2. The AAV1 vector described in claim 1, wherein the ubiquitous promoter is a cytomegalovirus (CMV) promoter or a CAG promoter.

3. The method of claim 1, wherein the nucleotide sequence encoding FGF21 is: (a) a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 5, 6, or 7; or (b) a nucleotide sequence that differs in sequence from the sequence of nucleotide sequence (a) due to the degeneracy of the genetic code.

3. The AAV1 vector of claim 1 or 2, selected from the group consisting of:

4. A nucleic acid molecule represented by a nucleotide sequence optimized for mammalian codons encoding FGF21, wherein the nucleotide sequence has at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6, or 7.

5. A composition comprising an AAV1 vector as defined in any one of claims 1 to 3 and / or a nucleic acid molecule as defined in claim 4, together with one or more pharmaceutically acceptable excipients or vehicles.

6. Use of an AAV1 vector defined in any one of claims 1 to 3 and / or a nucleic acid molecule defined in claim 4 and / or a composition defined in claim 5 in the manufacture of a medicament for the prevention, delay, cure, amelioration and / or treatment of diabetes and / or obesity.

7. Use of an AAV1 vector as defined in any one of claims 1 to 3 and / or a nucleic acid molecule as defined in claim 4 and / or a composition as defined in claim 5 in the manufacture of a medicament for the prevention, delay, cure, amelioration and / or treatment of NASH.

8. Use of an AAV1 vector as defined in any one of claims 1 to 3 and / or a nucleic acid molecule as defined in claim 4 and / or a composition as defined in claim 5 in the manufacture of a medicament for the prevention, delay, healing, amelioration and / or treatment of liver inflammation and / or fibrosis.

9. Use of an AAV1 vector as defined in any one of claims 1 to 3 and / or a nucleic acid molecule as defined in claim 4 and / or a composition as defined in claim 5 in the manufacture of a medicament for the prevention, delay, cure, amelioration and / or treatment of liver cancer.

10. The composition of claim 5 formulated for intramuscular injection.

11. The composition of any one of claims 6 to 9, wherein the AAV1 vector, nucleic acid molecule, and / or composition is formulated for intramuscular injection.

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