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

Abstract

The present invention relates to viral expression constructs and related viral vectors and nucleic acid molecules and compositions, and uses thereof, wherein the constructs and vectors are suitable for expression in mammals and comprise a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) that is expressed in the liver, adipose tissue and / or skeletal muscle.

Description

[Technical Field]

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

[0002] The prevalence of diabetes is increasing at an alarming rate and is a major health problem worldwide. Obesity is strongly associated with insulin resistance and type 2 diabetes (T2D) (Moller, DE, and Flier, JS, 1991. N. Engl. J. Med. 325: 938 -948). Furthermore, obesity increases the risk of death (Peeters, A. et al., 2003. Ann. Intern. Med. 138 :24-32), and is also a significant risk factor for heart disease, immune dysfunction, high blood pressure, arthritis, neurodegenerative diseases, and certain types of cancer (Roberst, DL et al., 2010. Annu. Rev. Med. 61 :301-316;Spiegelman,BM et al.,1993. J.Biol.Chem. 268 :6823-6826;Whitmer,RA,2007. Curr.Alzheimer Res. 4 :117-122). Despite the clinical importance of T2D and obesity, no effective treatments are available. Therefore, there is an urgent need for novel and safe approaches to prevent and combat the T2D-obesity epidemic. In recent years, it has become widely accepted that obesity is an important risk factor for cancer (Roberst, DL et al., 2010. Annu. Rev. Med. 61:301-316). Given the recent obesity epidemic, obesity-related cancer risk is a significant clinical concern that urgently requires novel and safe approaches. Increased body weight and insulin resistance are also associated with aging. Therefore, novel and safe approaches to prevent and reverse obesity and diabetes are needed to extend healthy lifespan. Liver fibrosis is the excessive accumulation of extracellular matrix proteins, such as collagen, and is primarily caused by chronic liver inflammation. Advanced liver fibrosis leads to cirrhosis, portal hypertension, and liver failure. Therefore, novel and safe antifibrotic therapeutic agents are needed.

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

[0004] Overexpression of FGF21 in transgenic mice protects them from diet-induced obesity (Kharitonenkov, A. et al., 2005. J. Clin. Invest 115 :1627-1635), administration of FGF21 to ob / ob mice, db / db mice, or high-fat diet (HFD)-fed mice, or to obese ZDF rats, led to a strong reduction in adiposity, significantly lowered blood glucose and triglycerides, reduced fasting insulin levels, and improved insulin sensitivity (Coskun, T. et al., 2008. Endocrinology 149 :6018-6027;Kharitonenkov,A. et al.,2005. J.Clin.Invest 115 :1627-1635;Xu,J. et al.,2009. Diabetes 58 :250-259;Adams,AC et al.,2012.PLoS.One. 7 :e38438.;Berglund,ED et al.,2009. Endocrinology 150 Furthermore, administration of FGF21 to obese diabetic rhesus monkeys dramatically reduced fasting plasma glucose, fructosamine, triglycerides, insulin, and glucagon levels, and induced a small but significant weight loss (Kharitonenkov, A. et al., 2007. Endocrinology 148 :774-781).

[0005] Native FGF21 protein exhibits poor pharmacokinetic properties: it has a short half-life, is prone to proteolysis in vivo, and is prone to aggregation in vitro (Huang, J. et al., 2013. J Pharmacol Exp Ther. 346(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). Various engineering approaches have been developed to extend the half-life of FGF21 and improve its stability and solubility. Currently, two artificial FGF21 mimetics (LY2405319 and PF-05231023) are being tested in humans. Nevertheless, these FGF21 mimetics require multiple administrations, imposing a significant burden on patients. Furthermore, artificial FGF21 mimetics / analogs may have a higher risk of immunogenicity than natural FGF21; for example, patients treated with LY2405319 developed injection site reactions, anti-drug antibodies, and severe hypersensitivity reactions (Gaich, G. et al., 2013. Cell Metab. 18(3) :333-40).

[0006] Therefore, there remains a need for new treatments for diabetes, and / or obesity, and / or liver inflammation and / or fibrosis, and / or cancer, and / or extending healthy lifespan that do not have all the drawbacks of existing treatments. [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-licensed Document 5] Whitmer, RA, 2007. Curr. Alzheimer Res. 4:117-122 [Non-licensed Document 6] Muise,ES et al.,2008. Mol.Pharmacol. 74:403-412 [Non-licensed Document 7] Coskun,T. et al.,2008. Endocrinology 149:6018-6027 [Non-licensed Document 8] Fisher, FM et al., 2012. Genes Dev. 26:271-281 [Non-licensed Document 9] Kharitonenkov,A. et al.,2005. J.Clin.Invest 115:1627-1635 [Non-licensed Document 10] Konishi,M. et al.,2000. J. Biol. Chem. 275:12119-12122 [Non-licensed Document 11] Tomlinson,E. et al.,2002. Endocrinology 143:1741-1747 [Non-licensed Document 12] Xu,J. et al.,2009. Diabetes 58:250-259 [Non-licensed Document 13] Adams,AC et al.,2012. PLoS.One. 7:e38438. [Non-licensed Document 14] Berglund, ED et al., 2009. Endocrinology 150:4084-4093 [Non-licensed Document 15] Kharitonenkov,A. et al.,2007. Endocrinology 148:774-781 [Non-licensed Document 16] Huang, J. et al.,2013. J Pharmacol Exp Ther. 346(2):270-80 [Non-Patent Document 17] So,WY and Leung,PS 2016. Med Res Rev. 36(4):672-704 [Non-Patent Document 18] Zhang, J. and Li, Y. 2015. Front Endocrinol (Lausanne). 6:168 [Non-Patent Document 19] Gaich, G. et al., 2013. Cell Metab. 18(3):333-40 Summary of the Invention [Means for solving the problem]

[0008] The present inventors have designed an improved gene therapy strategy based on adeno-associated virus (AAV) vector-mediated FGF21 gene transfer into the liver, adipose tissue, and / or skeletal muscle to combat metabolic disorders, preferably diabetes and / or obesity.The gene therapy of the present invention can also be used to combat liver inflammation and / or fibrosis.In addition, the gene therapy of the present invention can also be used to extend healthy lifespan by combating age-related metabolic disorders, preferably diabetes and / or obesity.In addition, the gene therapy of the present invention can also be used to combat cancer, preferably liver cancer.

[0009] The creation of a single vector gene construct allows for the in vivo production of native FGF21, which poses a reduced risk of immunogenicity or other toxicities.

[0010] However, those skilled in the art know that native FGF21 may be prone to proteolysis in vivo and / or have a rapid clearance rate in vivo.All vectors tested in the experimental section were found to be able to enable stable, long-term secretion of native FGF21 into the bloodstream.Even a single administration of gene transfer vectors maintains efficacy.

[0011] Therefore, the generation of such an AAV vector for the in vivo production of native FGF21 is not routine for those skilled in the art, as demonstrated in the experimental section. In one aspect, the present invention provides the following. [Item 1] A viral expression construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21), suitable for expression in a mammal and expressed in the liver, adipose tissue and / or skeletal muscle. [Item 2] A nucleotide sequence encoding FGF21 suitable for expression in a mammal, and elements a), b), c), d) and e): (a) Liver-specific promoter (b) Adipose tissue-specific promoter (c) a combination of a ubiquitous promoter, at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the liver, and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the heart, wherein the combination enables specific expression in adipose tissue. (d) skeletal muscle promoter, and (e) a combination of a ubiquitous promoter and an adeno-associated virus (AAV) vector sequence that allows specific expression in skeletal muscle; 2. The viral expression construct of item 1, comprising at least one of: [Item 3] A nucleotide sequence encoding FGF21 suitable for mammalian expression is: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, or 3 (b) a nucleotide sequence having at least 60% sequence identity with the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11 (c) a nucleotide sequence that differs in sequence from the sequence in nucleotide sequence (b) due to the degeneracy of the genetic code 3. The viral expression construct of item 1 or 2, selected from the group consisting of: [Item 4] 4. The viral expression construct of item 2 or 3, wherein the nucleotide sequence encoding the target sequence of a microRNA expressed in the liver and the nucleotide sequence encoding the target sequence of a microRNA expressed in the heart are selected from the group consisting of SEQ ID NOs: 12 to 30 and / or combinations thereof. [Item 5] 5. The viral expression construct of any one of items 2 to 4, wherein the liver-specific promoter is a human alpha1-antitrypsin (hAAT) promoter, and / or the adipose tissue-specific promoter is a mini / ap2 promoter and / or a mini / UCP1 promoter, and / or the skeletal muscle promoter is a C5-12 promoter, and / or the ubiquitous promoter is a cytomegalovirus (CMV) promoter and / or a CAG promoter. [Item 6] A viral vector comprising the viral expression construct according to any one of Items 1 to 5, wherein the viral vector is an adenoviral vector, an adeno-associated viral vector, a retroviral vector, or a lentiviral vector, and is preferably an adeno-associated viral vector selected from the group consisting of adeno-associated virus 1 (AAV1) vector, adeno-associated virus 8 (AAV8) vector, and adeno-associated virus 9 (AAV9) vector. [Item 7] A nucleic acid molecule represented by a nucleotide sequence encoding mammalian codon-optimized FGF21, suitable for expression in mammals and expressed in liver, adipose tissue and / or skeletal muscle. [Item 8] 8. The nucleic acid molecule of item 7, wherein the nucleotide sequence has at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11. [Item 9] A composition comprising a viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8, together with one or more pharmaceutically acceptable excipients or vehicles. [Item 10] A viral expression construct as defined in any one of items 1 to 5 and / or a viral vector as defined in item 6 and / or a nucleic acid molecule as defined in item 7 or 8 and / or a composition as defined in item 9 for use as a medicament. [Item 11] A viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9 for use in the treatment and / or prevention of a metabolic disorder. [Item 12] 12. The viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition according to item 11, wherein the metabolic disorder is diabetes and / or obesity. [Item 13] 12. The viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition according to item 11, wherein the metabolic disorder is NASH. [Item 14] A viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9 for use in the treatment and / or prevention of liver inflammation and / or fibrosis. [Item 15] A viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9 for use in extending healthspan. [Item 16] A viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9 for use in the treatment and / or prevention of cancer, preferably liver cancer. [Item 17] A method for preventing, delaying, ameliorating, curing and / or treating a metabolic disorder comprising the use of a viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9. [Item 18] 18. The method according to item 17, wherein the metabolic disorder is diabetes and / or obesity. [Item 19] 18. The method of item 17, wherein the metabolic disorder is NASH. [Item 20] A method for preventing, delaying, ameliorating, curing and / or treating liver inflammation and / or fibrosis, comprising the use of a viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9. [Item 21] A method for extending healthy lifespan, comprising the use of a viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9. [Item 22] A method for preventing, delaying, ameliorating, curing and / or treating cancer, preferably liver cancer, comprising the use of a viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9. [Item 23] Use of a viral expression construct as defined in any one of items 1 to 5 and / or a viral vector as defined in item 6 and / or a nucleic acid molecule as defined in item 7 or 8 and / or a composition as defined in item 9 for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating a metabolic disorder. [Item 24] 24. The use according to item 23, wherein the metabolic disorder is diabetes and / or obesity. [Item 25] 24. The use according to item 23, wherein the metabolic disorder is NASH. [Item 26] Use of a viral expression construct as defined in any one of items 1 to 5 and / or a viral vector as defined in item 6 and / or a nucleic acid molecule as defined in item 7 or 8 and / or a composition as defined in item 9 for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating liver inflammation and / or fibrosis. [Item 27] Use of a viral expression construct defined in any one of items 1 to 5 and / or a viral vector defined in item 6 and / or a nucleic acid molecule defined in item 7 or 8 and / or a composition defined in item 9 for the manufacture of a medicament for extending healthy lifespan. [Item 28] Use of a viral expression construct as defined in any one of items 1 to 5 and / or a viral vector as defined in item 6 and / or a nucleic acid molecule as defined in item 7 or 8 and / or a composition as defined in item 9 for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating cancer, preferably liver cancer. DETAILED DESCRIPTION OF THE INVENTION

[0012] Viral expression constructs In a first aspect, a viral expression construct is provided that is suitable for expression in a mammal and comprises a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) that is expressed in the liver, adipose tissue and / or skeletal muscle.

[0013] Definitions of "viral expression construct," "suitable for expression in mammals," "liver," "adipose tissue," and "skeletal muscle" are provided in the description section entitled "General Definitions."

[0014] Preferred nucleotide sequences encoding FGF21 present in viral expression constructs of the invention have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity to SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11. Identity may be assessed across the entire SEQ ID NO, or across portions thereof, as explained in the section entitled "General Definitions."

[0015] More preferred nucleotide sequences encoding human FGF21 present in viral expression constructs of the invention have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 4, 5, 6, or 7. Identity can be assessed across the entire SEQ ID NO: or across portions thereof, as explained in the section entitled "General Definitions." SEQ ID NO: 4 is a nucleotide sequence encoding human FGF21. SEQ ID NO: 5 is a nucleotide sequence encoding codon-optimized human FGF21, variant 1. SEQ ID NO: 6 is a nucleotide sequence encoding codon-optimized human FGF21, variant 2. SEQ ID NO: 7 is a nucleotide sequence encoding codon-optimized human FGF21, variant 3. Variant 1, variant 2, and variant 3 encode the same human FGF21 protein and were obtained by different codon optimization algorithms. Another preferred nucleotide sequence encoding mouse FGF21 present in a viral expression construct of the invention has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8 or 9. Identity can be assessed across the entire SEQ ID NO: 8 or 9, or across portions thereof, as explained in the section entitled "General Definitions." SEQ ID NO: 8 is a nucleotide sequence encoding mouse FGF21. SEQ ID NO: 9 is a nucleotide sequence encoding codon-optimized mouse FGF21. Another preferred nucleotide sequence encoding canine FGF21 present in a viral expression construct of the invention has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10 or 11. Identity can be assessed across the entire SEQ ID NO: 10 or across portions thereof, as explained in the description section entitled "General Definitions." SEQ ID NO: 10 is a nucleotide sequence encoding canine FGF21. SEQ ID NO: 11 is a nucleotide sequence encoding codon-optimized canine FGF21.The nucleotide sequence encoding FGF21 may be any FGF21 gene or FGF21 coding sequence, preferably derived from a human, mouse, or dog; or may be a mutated FGF21 gene or FGF21 coding sequence, or a codon-optimized FGF21 gene or FGF21 coding sequence, preferably derived from a human, mouse, or dog.

[0016] FGF21 as used herein exhibits at least detectable levels of the activity of FGF21 known to those skilled in the art. The activity of FGF21 is to improve insulin sensitivity. This activity can be assessed using an insulin tolerance test as described in the experimental section, preferably in Example 8 or 9.

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

[0018] Preferred nucleotide sequences encoding FGF21 suitable for expression in mammals encode polypeptides comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1, 2, or 3. Identity can be assessed across the entire SEQ ID NO, or across portions thereof, as explained in the section entitled "General Definitions." 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.

[0019] In one embodiment, a nucleotide sequence encoding FGF21 suitable for expression in a mammal and elements a), b), c), d) and e): (a) Liver-specific promoter (b) Adipose tissue-specific promoter (c) a combination of a ubiquitous promoter, at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the liver, and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the heart, wherein the combination enables specific expression in adipose tissue. (d) skeletal muscle promoter, and (e) a combination of a ubiquitous promoter and an adeno-associated virus (AAV) vector sequence that allows specific expression in skeletal muscle; The above viral expression construct is provided, comprising at least one of:

[0020] A "target sequence of a microRNA expressed in the liver" or a "target sequence of a miRNA expressed in the liver" or a "binding site of a microRNA expressed in the liver" refers to a nucleotide sequence that is complementary or partially complementary to at least a portion of a microRNA expressed in the liver. Similarly, a "target sequence of a microRNA expressed in the heart" or a "target sequence of a miRNA expressed in the heart" or a "binding site of a microRNA expressed in the heart" refers to a nucleotide sequence that is complementary or partially complementary to at least a portion of a microRNA expressed in the heart. A portion of a microRNA expressed in the liver or a portion of a microRNA expressed in the heart, as defined herein, refers to a nucleotide sequence of at least 5 or at least 6 consecutive nucleotides of the microRNA. The binding site sequence can be perfectly complementary to at least a portion of the expressed microRNA, meaning that the sequence can be a perfect match and no mismatches can occur. Alternatively, the binding site sequence can be partially complementary to at least a portion of the expressed microRNA, meaning that one mismatch per 5 or 6 consecutive nucleotides can occur. A partially complementary binding site preferably contains perfect or near-perfect complementarity with the seed region of a microRNA, meaning that there can be no mismatch (perfect complementarity) or one mismatch per 5 or 6 consecutive nucleotides (near-perfect complementarity) between the seed region of a microRNA and its binding site. The seed region of a microRNA consists of the 5' region of the microRNA (i.e., 6 nucleotides) from about nucleotide 2 to about nucleotide 8 of the microRNA. The portion defined herein is preferably the seed region of the microRNA. Degradation of messenger RNA (mRNA) containing the target sequence of a microRNA expressed in the liver or a microRNA expressed in the heart can be via the RNA interference pathway or direct translational control (inhibition) of the mRNA.The present invention is not limited by the pathway ultimately utilized by the miRNA in inhibiting expression of the transgene or its encoded protein.

[0021] In the context of the present invention, a nucleotide sequence encoding a target sequence of a microRNA expressed in the liver may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 12 or 14-23. More preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12 or 14-23. Identity may be assessed across the entire SEQ ID NO or across portions thereof, as explained in the section entitled "General Definitions." In one embodiment, a nucleotide sequence encoding a target sequence of a microRNA expressed in the liver may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 12. More preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12. In a further embodiment, at least one copy of a nucleotide sequence set forth in SEQ ID NO: 12 or 14-23 encoding a target sequence of a microRNA expressed in the liver is present in a viral expression construct of the invention. In a further embodiment, 2, 3, 4, 5, 6, 7, or 8 copies of a nucleotide sequence set forth in SEQ ID NO: 12 or 14-23 encoding a target sequence of a liver-specific microRNA are present in a viral expression construct of the invention. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7, or 8 copies of a nucleotide sequence encoding miRT122a (SEQ ID NO: 12) are present in a viral expression construct of the invention.

[0022] The target sequence of the microRNA expressed in the liver used herein exerts the activity of the target sequence of the microRNA expressed in the liver known to those skilled in the art at least at detectable level.The activity of the target sequence of the microRNA expressed in the liver is to bind to its homologous microRNA expressed in the liver, and when operatively linked to transgene, mediate the detargeting of transgene expression in the liver.This activity can be assessed by measuring the expression level of transgene in the liver by qPCR, as described in the experimental section.

[0023] In the context of the present invention, a nucleotide sequence encoding a target sequence of a microRNA expressed in the heart may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 13 or 23-30. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13 or 23-30. Identity may be assessed across the entire SEQ ID NO or across portions thereof, as explained in the section entitled "General Definitions." In one embodiment, a nucleotide sequence encoding a target sequence of a microRNA expressed in the heart may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 13. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In a further embodiment, at least one copy of a nucleotide sequence set forth in SEQ ID NOs: 13 or 23-30 encoding a target sequence of a microRNA expressed in the heart is present in a viral expression construct of the invention. In a further embodiment, 2, 3, 4, 5, 6, 7, or 8 copies of a nucleotide sequence set forth in SEQ ID NOs: 13 or 23-30 encoding a target sequence of a cardiac-specific microRNA are present in a viral expression construct of the invention. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7, or 8 copies of a nucleotide sequence encoding miRT1 (SEQ ID NO: 13) are present in a viral expression construct of the invention.

[0024] The activity of the target sequence of the microRNA expressed in the heart is to bind to its cognate microRNA expressed in the heart, and when operatively linked to transgene, mediate the detargeting of transgene expression in the heart.This activity can be assessed by measuring the expression level of transgene in the heart by qPCR, as described in the experimental section.

[0025] In one embodiment, the above-mentioned viral expression construct is provided, wherein the nucleotide sequence encoding the target sequence of a microRNA expressed in the liver and the nucleotide sequence encoding the target sequence of a microRNA expressed in the heart are selected from the group consisting of SEQ ID NOs: 12 to 30 and / or combinations thereof.

[0026] In one embodiment, at least one copy of the nucleotide sequence set forth in SEQ ID NOs: 12 or 14-23 encoding a target sequence of a microRNA expressed in the liver and at least one copy of the nucleotide sequence set forth in SEQ ID NOs: 13 or 23-30 encoding a target sequence of a microRNA expressed in the heart are present in a viral expression construct of the invention. In a further embodiment, 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence set forth in SEQ ID NOs: 12 or 14-23 encoding a target sequence of a microRNA expressed in the liver and 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence set forth in SEQ ID NOs: 13 or 23-30 encoding a target sequence of a microRNA expressed in the heart are present in a viral expression construct of the invention. In a further embodiment, 1, 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence encoding miRT122a (SEQ ID NO: 12) and 1, 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence encoding miRT1 (SEQ ID NO: 13) are combined in a viral expression construct of the invention. In a further embodiment, 4 copies of the nucleotide sequence encoding miRT122a (SEQ ID NO: 12) and 4 copies of the nucleotide sequence encoding miRT1 (SEQ ID NO: 13) are combined in a viral expression construct of the invention.

[0027] The definitions "promoter," "liver-specific promoter," "adipose tissue-specific promoter," "ubiquitous promoter," and "skeletal muscle promoter" are provided in the description section entitled "General Definitions."

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

[0029] In the context of the present invention, the nucleotide sequence of the CAG promoter may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 44. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity to SEQ ID NO: 44. Identity may be assessed across the entire SEQ ID NO, or across portions thereof, as explained in the section entitled "General Definitions."

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

[0031] In the context of the present invention, the nucleotide sequence of the CMV promoter may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 45. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity to SEQ ID NO: 45. Identity may be assessed across the entire SEQ ID NO, or across portions thereof, as explained in the section entitled "General Definitions."

[0032] Preferably, the CMV promoter is used in conjunction with an intron sequence. In this regard, the intron sequence may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 43. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 43. Identity may be assessed across the entire SEQ ID NO, or across portions thereof, as explained in the section entitled "General Definitions."

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

[0034] In the context of the present invention, the nucleotide sequence of the hAAT promoter may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 47. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity to SEQ ID NO: 47. Identity may be assessed across the entire SEQ ID NO, or across portions thereof, as explained in the section entitled "General Definitions."

[0035] Preferably, the hAAT promoter is used together with an intron sequence. A preferred intron sequence is the hepatocyte control region (HCR) enhancer from apolipoprotein E. The most preferred intron sequence is the HCR enhancer from apolipoprotein E as defined in SEQ ID NO: 53. In this context, the intron sequence may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity with SEQ ID NO: 53. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 53. Identity may be assessed across the entire SEQ ID NO or across portions thereof, as explained in the section entitled "General Definitions." In one embodiment, the hAAT promoter is used together with 1, 2, 3, 4, or 5 copies of an intron sequence. In a preferred embodiment, the hAAT promoter is used in conjunction with 1, 2, 3, 4 or 5 copies of the HCR enhancer from apolipoprotein E as defined in SEQ ID NO:53.

[0036] Other liver-specific promoters are the albumin promoter, major urinary protein promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, liver-enriched protein activator promoter, transthyretin promoter, thyroxine-binding globulin promoter, apolipoprotein A1 promoter, liver fatty acid-binding protein promoter, and phenylalanine hydroxylase promoter.

[0037] Examples of adipose tissue-specific promoters include the adipocyte protein 2 (aP2, also known as fatty acid-binding protein 4 (FABP4)) promoter, the PPARy promoter, the adiponectin promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, the promoter derived from human aromatase cytochrome p450 (p450arom), the mini / aP2 promoter (composed of the adipose-specific aP2 enhancer and the basal aP2 promoter), the uncoupling protein 1 (UCP1) promoter, the mini / UCP1 promoter (composed of the adipose-specific UCP1 enhancer and the basal UCP1 promoter), the adipsin promoter, the leptin promoter, and the Foxa-2 promoter. Preferred adipose tissue-specific promoters are the mini / aP2 promoter (SEQ ID NO: 54) and the mini / UCP1 promoter (SEQ ID NO: 55). In this context, the adipose tissue-specific promoter sequence may be replaced by a nucleotide sequence containing a nucleotide sequence having at least 60% sequence identity or similarity with SEQ ID NO: 53 or SEQ ID NO: 54. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity to SEQ ID NO: 53 or SEQ ID NO: 54. Identity can be assessed across the entire SEQ ID NO, or across portions thereof, as explained in the section entitled "General Definitions."

[0038] Preferred skeletal muscle promoters are the myosin light chain promoter, myosin heavy chain promoter, desmin promoter, muscle creatine kinase (MCK) promoter, smooth muscle alpha-actin promoter, CK6 promoter, Unc-45 myosin chaperone B promoter, the basal MCK promoter combined with a copy of the MCK enhancer, and the Enh358MCK promoter (the MCK enhancer combined with the 358-bp proximal promoter of the MCK gene). The most preferred skeletal muscle promoter is the C5-12 promoter set forth in SEQ ID NO: 56. In this regard, the skeletal muscle promoter sequence may be replaced by a nucleotide sequence containing a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 56. Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 56. Identity can be assessed across the entire SEQ ID NO, or across portions thereof, as explained in the section entitled "General Definitions."

[0039] As used herein, a promoter (especially when the promoter sequence is defined as having a minimal percentage identity with a given SEQ ID NO) is one that exhibits at least the activity of a promoter known to those skilled in the art. For a definition of such activity, see the section entitled "General Definitions." Preferably, a promoter defined as having a minimal percentage identity with a given SEQ ID NO controls the transcription of an operably linked nucleotide sequence (i.e., a nucleotide sequence encoding FGF21) as assessed in an assay known to those skilled in the art. In the context of the present invention, the promoter is operably linked to the FGF21 nucleotide sequence defined above. In one embodiment, the promoter is cell-specific and / or tissue-specific, preferably liver-, adipose tissue-, and / or skeletal muscle-specific.

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

[0041] In one embodiment, a viral expression construct described herein is provided wherein the liver-specific promoter is the human alpha-1-antitrypsin (hAAT) promoter, and / or the adipose tissue-specific promoter is the mini / ap2 promoter and / or the mini / UCP1 promoter, and / or the skeletal muscle promoter is the C5-12 promoter, and / or the ubiquitous promoter is the cytomegalovirus (CMV) promoter and / or the CAG promoter.

[0042] In one embodiment, a viral expression construct is included that comprises a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element a), wherein the liver-specific promoter is the hAAT promoter (SEQ ID NO: 47).

[0043] In a preferred embodiment, a viral expression construct is included that includes a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element a), which is AAV8-hAAT-moFGF21. This construct contains, for example, the viral expression construct depicted in Figure 6A as ITR2-hAAT-moFGF21-polyA-ITR2; the sequence of this expression construct is included in SEQ ID NO:34.

[0044] For this construct, Example 3 surprisingly demonstrates high and stable liver-specific expression after intravenous administration. Expression was shown to be stable for up to one year (Example 12). Widespread beneficial therapeutic effects for the reversal and treatment of obesity and diabetes are demonstrated in ob / ob mice (Examples 3 and 11), high-fat diet (HFD)-fed mice (Examples 4, 12-14), and aged HFD-fed mice (Examples 5, 12-14). Examples 11 and 16 also demonstrate significant improvements in hepatic steatosis, hepatitis, and liver fibrosis. Example 15 demonstrates improvements in obesity-associated WAT inflammation. Example 17 indicates long-term safety of the treatment. Example 18 demonstrates beneficial effects in preventing liver tumors. Example 19 demonstrates therapeutic potential in a model of type 1 diabetes.

[0045] In one embodiment, a viral expression construct is included that comprises a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element b), wherein the adipose tissue-specific promoter is the mini / aP2 promoter (sequence number 54) and / or the mini / UCP1 promoter (sequence number 55).

[0046] In one embodiment, a viral expression construct is included, comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element c), wherein the ubiquitous promoter is a CAG promoter (SEQ ID NO: 44), at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the liver is selected from the group consisting of SEQ ID NOs: 12 or 14-23, and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the heart is selected from the group consisting of SEQ ID NOs: 13 or 23-30.

[0047] In a preferred embodiment, a viral expression construct is included that contains a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element c), and is designated AAV9-CAG-moFGF21-dmiRT or AAV8-CAG-moFGF21-dmiRT. The terms dmiRT and doublemiRT are equivalent. These constructs include, for example, the viral expression construct depicted in Figure 1A as ITR2-CAG-moFGF21-4x miRT122a-4x miRT1-polyA-ITR2; the sequence of this expression construct is contained in SEQ ID NO: 32.

[0048] For these constructs, Examples 1-2 surprisingly demonstrate high and stable adipose-specific expression after administration into eWAT. Wide-ranging beneficial therapeutic effects for the prevention, reversal, and treatment of obesity and diabetes are demonstrated in normal mice (Example 1) and ob / ob mice (Examples 2 and 10). Example 10 also demonstrates significant improvement in hepatic steatosis.

[0049] In one embodiment, a viral expression construct is included that comprises a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element d), wherein the skeletal muscle promoter is the C5-12 promoter (SEQ ID NO: 56).

[0050] In one embodiment, a viral expression construct is included that comprises a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element e), wherein the ubiquitous promoter is a CMV promoter (SEQ ID NO: 45), and the AAV serotype is AAV1.

[0051] In a preferred embodiment, a viral expression construct is included that includes a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element e), which is AAV1-CMV-moFGF21, e.g., the viral expression construct depicted in Figure 11A as ITR2-CMV-moFGF21-polyA-ITR2; the sequence of this expression construct is included in SEQ ID NO:36.

[0052] For this construct, Example 20 demonstrates high and stable skeletal muscle-specific expression after intramuscular administration. Wide-ranging beneficial therapeutic effects for the prevention, reversal, and treatment of obesity and diabetes are demonstrated in HFD-fed mice (Examples 6 and 21). Example 20 demonstrates the beneficial health-span extension effects of preventing obesity and diabetes.

[0053] In one embodiment, a viral expression construct is included, which comprises a nucleotide sequence encoding FGF21 suitable for expression in a mammal and the nucleotide sequences of elements b) and c), and the adipose tissue-specific promoter is the mini / aP2 promoter (sequence number 54) and / or the mini / UCP1 promoter (sequence number 55).

[0054] In one embodiment, a viral expression construct is included that includes a nucleotide sequence encoding FGF21 suitable for expression in a mammal and the nucleotide sequences of element e) and element c), wherein the ubiquitous promoter is a CMV promoter (SEQ ID NO: 45), and the AAV serotype is AAV1.

[0055] All of the constructs of the present invention are more attractive than those of the prior art, such as those disclosed in Zhang et al., EBioMedicine 15 (2017) 173-183, in particular those comprising element a) a liver-specific promoter, preferably hAAT, and / or element c) a combination of a ubiquitous promoter and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the liver, preferably miRT122a, and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the heart, preferably miRT1, which combination enables specific expression in adipose tissue, and / or element e) a combination of a ubiquitous promoter, preferably CMV, and an adeno-associated virus (AAV) vector sequence, preferably AAV1, which combination enables specific expression in skeletal muscle. Zhang et al. disclosed a wild-type mouse FGF21 coding sequence (EF1a-mFGF21) under the control of the elongation factor 1a (EF1a) promoter (Zhang et al., EBioMedicine 15 (2017) 173-183). This construct was compared to the constructs of the present invention in Examples 23 and 24. In all in vitro and in vivo experiments, all expression cassettes and AAV vectors of the present invention mediated greater expression of FGF21 in target tissues or cell types and less expression of FGF21 in off-target tissues, demonstrating the higher efficiency and tissue specificity of the expression cassettes and AAV vectors of the present invention. In addition, the constructs CMV-moFGF21 and CAG-moFGF21-double miRT also mediated greater protein production and secretion into the culture medium in HEK293 cells compared to EF1a-mFGF21. Moreover, hAAT-moFGF21 and AAV8-hAAT-moFGF21 also mediated more secretion of FGF21 into the bloodstream than EF1a-mFGF21 and AAV8-EF1a-mFGF21.

[0056] As explained in 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 an inverted terminal repeat (ITR), an SV40 polyadenylation signal (SEQ ID NO: 50), a rabbit β-globin polyadenylation signal (SEQ ID NO: 51), a CMV enhancer sequence (SEQ ID NO: 46), and an HCR enhancer from apolipoprotein E (SEQ ID NO: 53). Within the context of the present invention, "ITR" is intended to encompass one 5'ITR and one 3'ITR, each derived from the AAV genome. Preferred ITRs are from AAV2 and are represented by SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR). Within the context of the present invention, the use of a CMV enhancer sequence (SEQ ID NO: 46) and a CMV promoter sequence (SEQ ID NO: 45) as two separate sequences or as a single sequence (SEQ ID NO: 52) is encompassed.

[0057] Each of these additional sequences can be present in a viral expression construct of the invention (eg, as depicted in Figures 1, 2, 3, 4, 5, 6, 7, 8 and 9, and as depicted in Figures 11, 31 and 32).

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

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

[0060] These sequences were used in the experimental section and in several constructs identified herein.

[0061] Thus, in one embodiment, for each of these preferred viral expression constructs defined above, additional sequences selected from the group consisting of ITRs, SV40 polyadenylation signals, rabbit β-globin polyadenylation signals, CMV enhancer sequences, and HCR enhancer sequences from apolipoprotein E may be present.

[0062] In a preferred embodiment, the viral expression construct comprises a nucleotide sequence encoding FGF21 suitable for expression in a mammal and at least one of elements a) and / or b) and / or c) and / or d) and / or e), wherein additional sequences selected from the group consisting of ITRs, SV40 polyadenylation signal, rabbit β-globin polyadenylation signal, CMV enhancer sequence, and HCR enhancer sequence are present. Preferred ITRs are those of AAV2 represented by SEQ ID NO:48 (5'ITR) and SEQ ID NO:49 (3'ITR).

[0063] A preferred viral expression construct comprises elements a) and / or b) and / or c) and / or d) and / or e), such that the expression cassette is flanked by 5' and 3' ITRs.

[0064] Other preferred viral expression constructs contain elements a) and / or b) and / or c) and / or d) and / or e) such that the expression cassette is flanked by 5' and 3' ITRs, in addition an SV40 polyadenylation signal is present.

[0065] Other preferred viral expression constructs contain elements a) and / or b) and / or c) and / or d) and / or e) such that the expression cassette is flanked by 5' and 3' ITRs. In addition, a rabbit β-globin polyadenylation signal is present.

[0066] Other preferred viral expression constructs contain elements a) and / or b) and / or c) and / or d) and / or e) such that the expression cassette is flanked by 5' and 3' ITRs, and in addition, a CMV enhancer sequence is present.

[0067] Other preferred viral expression constructs contain elements a) and / or b) and / or c) and / or d) and / or e) such that the expression cassette is flanked by the 5' and 3' ITRs. In addition, the HCR enhancer sequence from apolipoprotein E is 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 containing SEQ ID NO: 4) Examples include:

[0069] As one of skill in the art will appreciate, each of these viral expression constructs already contains two ITRs from AAV2, namely SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR).

[0070] Constructs B and G contain a rabbit β-globin polyadenylation signal. Construct F contains an SV40 polyadenylation signal, a CMV enhancer sequence, and a chimeric intron nucleotide sequence (composed of introns from human β-globin and immunoglobulin heavy chain genes). Constructs D, H, and L contain an SV40 polyadenylation signal, an HCR enhancer sequence, and a chimeric intron nucleotide sequence (composed of introns from human β-globin and immunoglobulin heavy chain genes).

[0071] As explained in the general section entitled "General Definitions," throughout this application, whenever reference is made to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: A, B or C as examples) that represents a preferred construct designed herein, this means: i. a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: A, B, or C; ii. a nucleotide sequence that differs in sequence from the sequence of the nucleic acid molecule in (i) due to the degeneracy of the genetic code can be replaced by

[0072] Each nucleotide sequence described herein by its percentage identity (at least 60%) with each given nucleotide sequence has, in a more preferred embodiment, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity with each given nucleotide sequence. In a preferred embodiment, sequence identity is determined by comparing the entire length of the sequences identified herein. Unless otherwise indicated herein, identity with a given SEQ ID NO refers to identity or similarity based on the full-length (i.e., over its entire length or as a whole) sequence.

[0073] A construct defined by its minimum identity (i.e., at least 60%) to a given SEQ ID NO identified above is within the scope of the present invention if this construct, or a viral expression construct, or a viral vector containing this construct, or a composition containing this construct or vector, can induce the expression of FGF21 in cells, preferably in hepatocytes, adipose tissue cells, or skeletal muscle cells. FGF21 expression can be assessed using techniques known to those skilled in the art. In a preferred embodiment, the expression is assessed as performed in the experimental section.

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

[0075] viral vectors In a further embodiment, there is provided a viral vector comprising the viral expression construct defined above, wherein said viral vector is an adenoviral vector, an adeno-associated viral vector, a retroviral vector or a lentiviral vector, preferably an adeno-associated viral vector selected from the group consisting of an adeno-associated viral 1 (AAV1) vector, an adeno-associated viral 8 (AAV8) vector and an adeno-associated viral 9 (AAV9) vector.

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

[0077] In one embodiment, an AAV vector is used that comprises a genome based on a recombinant AAV (rAAV) that includes the elements and ITRs or portions thereof defined herein above. Preferred ITRs are those of AAV2, represented by SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR).

[0078] Preferably, the AAV vector is an AAV1 vector, an AAV8 vector, or an AAV9 vector.

[0079] The viral expression constructs and viral vectors of the present invention are preferably for use as pharmaceuticals. The pharmaceutical is preferably for preventing, delaying, curing, ameliorating, and / or treating metabolic disorders, preferably diabetes and / or obesity. Diabetes may be type 1 diabetes, type 2 diabetes, or monogenic diabetes. In another preferred embodiment, the pharmaceutical is for preventing, delaying, curing, ameliorating, and / or treating liver inflammation and / or fibrosis. In yet another preferred embodiment, the pharmaceutical is preferably for extending healthy lifespan by preventing, delaying, curing, ameliorating, and / or treating age-related metabolic disorders, preferably diabetes and / or obesity. In yet another preferred embodiment, the pharmaceutical is for preventing, delaying, curing, ameliorating, and / or treating cancer, preferably liver cancer.

[0080] The subject to be treated may be a higher mammal, such as a cat, a rodent (preferably a mouse, rat, gerbil and guinea pig, more preferably a mouse and rat), or a dog, or a human.

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

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

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

[0084] composition In a further aspect, there is provided a composition comprising a viral expression construct as defined above and / or a viral vector as defined above and / or a nucleic acid molecule as defined above together with one or more pharmaceutically acceptable excipients or vehicles.

[0085] This composition is preferably referred to as a gene therapy composition. Preferably, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluent, solubilizer, excipient, preservative and / or additive.

[0086] Such pharmaceutically acceptable carriers, excipients, preservatives, solubilizers, diluents and / or additives can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.

[0087] In a preferred embodiment, the composition is preferably for preventing, delaying, curing, reversing, and / or treating metabolic disorders, preferably diabetes and / or obesity. Diabetes may be type 1 diabetes, type 2 diabetes, or monogenic diabetes. In another preferred embodiment, the medicament is for preventing, delaying, curing, reversing, and / or treating liver inflammation and / or fibrosis. In yet another preferred embodiment, the medicament is preferably for extending healthy lifespan by preventing, delaying, curing, reversing, and / or treating age-related metabolic disorders, preferably diabetes and / or obesity. In yet another preferred embodiment, the medicament is for preventing, delaying, curing, reversing, and / or treating cancer, preferably liver cancer. The subject to be treated may be a higher mammal, for example, a cat, a rodent (preferably a mouse, rat, gerbil, and guinea pig, more preferably a mouse and rat), or a dog, or a human.

[0088] When said viral expression construct, viral vector and / or nucleic acid molecule and / or composition is capable of exhibiting an anti-diabetic effect and / or an anti-obesity effect, said viral expression construct, viral vector and / or nucleic acid molecule and / or composition is preferably said to be able to be used for preventing, delaying, curing, ameliorating and / or treating metabolic disorders, preferably diabetes and / or obesity.

[0089] When said viral expression construct, viral vector and / or nucleic acid molecule and / or composition is capable of exhibiting an anti-fibrotic effect, said viral expression construct, viral vector and / or nucleic acid molecule and / or composition is preferably said to be able to be used to prevent, delay, heal, restore and / or treat liver inflammation and / or fibrosis.

[0090] When said viral expression construct, viral vector and / or nucleic acid molecule and / or composition is capable of exhibiting an anti-diabetic effect and / or an anti-obesity effect during ageing, said viral expression construct, viral vector and / or nucleic acid molecule and / or composition is said to be preferably usable for extending healthy lifespan by preventing, delaying, curing, ameliorating and / or treating age-related metabolic disorders, preferably diabetes and / or obesity.

[0091] When said viral expression construct, viral vector and / or nucleic acid molecule and / or composition is capable of exhibiting an anti-cancer effect, said viral expression construct, viral vector and / or nucleic acid molecule and / or composition is preferably said to be able to be used for preventing, delaying, curing, ameliorating and / or treating cancer, preferably liver cancer.

[0092] An antidiabetic effect can be achieved when blood glucose processing is improved, and / or glucose tolerance is improved, and / or insulin sensitivity is improved. This can be assessed using techniques known to those skilled in the art or as performed in the experimental part, preferably as assessed in Examples 8 or 9. In this context, improvement (respectively, "improvement") means at least a detectable improvement (respectively, a detectable improvement) using an assay known to those skilled in the art or performed in the experimental part, such as measuring blood glucose, blood insulin, and / or performing an insulin tolerance test and / or a glucose tolerance test. The improvement can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% improvement using an assay such as measuring blood glucose, blood insulin, and / or performing an insulin tolerance test and / or a glucose tolerance test.

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

[0094] Anti-inflammatory effect in liver can be achieved by reducing macrophage infiltration, reducing pro-inflammatory cytokines.This can be assessed by using techniques known to those skilled in the art or by using the techniques carried out in the experimental section.In this context, "reduction" (respectively "improvement") means at least a detectable reduction (respectively a detectable improvement) by using the assay known to those skilled in the art or by using the assay carried out in the experimental section.

[0095] Anti-fibrotic effects in the liver can be achieved by reducing extracellular matrix protein accumulation, blood markers (e.g., plasma levels of type III collagen N-terminal propeptide, hyaluronic acid, tissue inhibitor of metalloproteinase type 1 (TIMP-1), YKL-40, serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT)). Anti-fibrotic effects can also be achieved by improving fibrosis scoring systems, such as Metavir or Ishak. This can be assessed using techniques known to those skilled in the art or as performed in the experimental section. In this context, "reduction" (respectively, "improvement") means at least a detectable reduction (respectively, a detectable improvement) using an assay known to those skilled in the art or as performed in the experimental section.

[0096] The healthspan extension effect can be achieved when the anti-diabetic and / or anti-obesity effect defined herein above is used to prevent, delay, cure, ameliorate or treat the onset or progression of age-related metabolic disorders, preferably diabetes and / or obesity.The healthspan extension effect can also be achieved by an increase in healthspan, where symptoms associated with metabolic disorders, preferably diabetes and / or obesity, are absent or reduced.The healthspan extension effect can also be achieved by improving coordination and balance (assessed by the rotarod test), memory (assessed by the object recognition test) and / or neuromuscular coherence (assessed by the tight rope test), and reducing the deterioration of mitochondria and metabolism (assessed by measuring the expression levels of genes involved in metabolism and mitochondrial function, such as PGC-1 alpha, ATP synthase and ERR alpha).This can be assessed using techniques known to those skilled in the art or as performed in the experimental section.In this context, "increase" (respectively "improvement") means at least a detectable increase (respectively a detectable improvement) using an assay known to those skilled in the art or using an assay performed in the experimental section.

[0097] An anti-cancer effect can be achieved by a reduction in the cumulative incidence of cancer over a lifetime. This can be assessed using techniques known to those skilled in the art or as performed in the experimental section. In this context, "reduction" (respectively, "improvement") means at least a detectable reduction (respectively, a detectable improvement) using an assay known to those skilled in the art or as performed in the experimental section.

[0098] The anti-diabetic effect and / or anti-obesity effect can also be observed when the progression of typical symptoms assessed by a physician (e.g., insulitis, beta cell loss, weight gain) slows down. A reduction in typical symptoms can mean a slowdown in the progression of symptom onset or a complete disappearance of symptoms. Symptoms and therefore their reduction can be assessed using various methods, most of which are the same as those used in the diagnosis of diabetes and / or obesity, including clinical examinations and routine laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular methods, X-rays, biochemistry, immunohistochemistry, and others.

[0099] The anti-inflammatory effect on the liver can also be observed when the progression of typical symptoms assessed by a physician (for example, fatigue, flu-like symptoms, dark urine, pale stool, abdominal pain, loss of appetite, unexplained weight loss, jaundice) slows down.The reduction of typical symptoms can mean the slowing down of the progression of symptom development or the complete disappearance of symptoms.Symptoms and therefore the reduction of symptoms can be assessed using various methods, most of which are the same as those used in the diagnosis of liver fibrosis, including clinical examination and routine laboratory tests.Such methods include both macroscopic and microscopic methods, as well as molecular methods, imaging methods (elastic imaging, X-ray, MRI, CT, ultrasound, angiography), biochemistry, immunohistochemistry and others.

[0100] The anti-fibrotic effect in the liver can also be recognized by the slowing down of the progression of typical symptoms assessed by doctors (for example, liver stiffness, jaundice, loss of appetite, difficulty in clear thinking, fluid retention in the limbs or stomach, nausea, unexplained weight loss, weakness).The reduction of typical symptoms can mean the slowing down of the progression of symptom development or the complete disappearance of symptoms.Symptoms and therefore the reduction of symptoms can be assessed using various methods, most of which are the same as those used in the diagnosis of liver fibrosis, including clinical examination and routine laboratory tests.Such methods include both macroscopic and microscopic methods, as well as molecular methods, imaging methods (elastic imaging, X-ray, MRI, CT, ultrasound, angiography), biochemistry, immunohistochemistry and others.

[0101] The effect of extending healthy lifespan can also be observed when the progression of typical symptoms of age-related metabolic disorders (e.g., insulin resistance, impaired glucose tolerance, weight gain) assessed by a physician slows down. A reduction in typical symptoms can mean a slowdown in the progression of the onset of symptoms or a complete disappearance of symptoms. Symptoms and therefore their reduction can be assessed using various methods, most of which are the same as those used in the diagnosis of diabetes and / or obesity, including clinical examinations and routine laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular methods, X-rays, biochemistry, immunohistochemistry, and others.

[0102] Anti-cancer effects can also be observed when the progression of typical symptoms assessed by a physician (e.g., tumor size, unexplained weight loss, decreased appetite, feeling very full after eating a small meal, nausea or vomiting, liver enlargement, spleen enlargement, pain in the abdomen or near the right shoulder blade, abdominal swelling or fluid retention, itching, jaundice) slows. A reduction in typical symptoms can mean a slowdown in the progression of symptom development or a complete disappearance of symptoms. Symptoms and therefore symptom reduction can be assessed using various methods, most of which are the same as those used in cancer diagnosis, including clinical examinations and routine laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular methods, imaging methods (X-ray, MRI, CT, ultrasound, angiography), biochemistry, immunohistochemistry, and others.

[0103] The agents defined herein (viral expression constructs, viral vectors, nucleic acid molecules, compositions) are preferably capable of alleviating one symptom or one characteristic of a patient or a cell, tissue or organ of said patient if said symptom or characteristic defined above is reduced (e.g. no longer detectable or slowed down) after at least one week, one month, six months, one year or longer of treatment with a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition of the invention.

[0104] The viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions defined herein for use in accordance with the present invention may be suitable for administration in vivo to cells, tissues and / or organs of an individual suffering from or at risk of developing a metabolic disorder, such as diabetes and / or obesity, liver inflammation and / or fibrosis, age-related metabolic disorders, and / or cancer, and may be administered in vivo, ex vivo or in vitro. The viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions may be administered in vivo directly or indirectly to cells, tissues and / or organs of an individual suffering from or at risk of developing a metabolic disorder, such as diabetes and / or obesity, liver inflammation and / or fibrosis, age-related metabolic disorders, and / or cancer, and may be administered in vivo, ex vivo or in vitro directly or indirectly. The mode of administration can be intravenous, subcutaneous, intramuscular, intrathecal, intraarticular, intracerebroventricular, intraperitoneal, intraadipose, via inhalation, oral, intranasal, intrahepatic, intravisceral, intraocular, intraauditory, topical and / or via retrograde intraductal pancreatic administration. Preferred modes of administration are intramuscular, intravenous or intraadipose, as described in the "General Procedures for the Examples" section that is part of this application.

[0105] The viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions of the present invention can be administered directly or indirectly by using suitable means known in the art. Given the advances already achieved to date, improvements in the means for delivering the viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions of the present invention to an individual or the cells, tissues, or organs of the individual are expected. Such future improvements can, of course, be incorporated to achieve the effects of the present invention. The viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions can be delivered directly to an individual or the cells, tissues, or organs of the individual. Depending on the disease or condition, the cells, tissues, or organs of the individual may be as defined herein above. When the viral expression constructs and / or viral vectors and / or nucleic acid molecules and / or compositions of the present invention are administered, they are preferably dissolved in a solution compatible with the delivery method.

[0106] As encompassed herein, a therapeutically effective dose of the above-mentioned viral expression constructs, vectors, nucleic acid molecules and / or compositions is preferably administered in a single and unique dose, thus avoiding repeated periodic administration. More preferably, a single dose is administered into skeletal muscle, into adipose tissue or intravenously.

[0107] Additional compounds may be present in the compositions of the present invention. Such compounds may aid in the delivery of the composition. The following is a list of suitable compounds: compounds capable of forming complexes, nanoparticles, micelles and / or liposomes that complex or entrap each of the components defined herein into vesicles or liposomes for delivery across cell membranes. Many of these compounds are known in the art. Suitable compounds include polyethyleneimine (PEI) or similar cationic polymers, including polypropyleneimine or polyethyleneimine copolymers (PEC) and derivatives, synthetic amphiphiles (SAINT-18), Lipofectin™, DOTAP.

[0108] Depending on their identity, the skilled artisan will understand which formulation type is most suitable for the compositions defined herein.

[0109] Method / Usage In a further aspect there is provided a 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 a composition as defined above for use as a medicament.

[0110] In one embodiment, the viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition is provided for use in the treatment and / or prevention of metabolic disorders, preferably diabetes and / or obesity. Complications of metabolic disorders may also be included.

[0111] In another embodiment, the viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition is provided for use in the treatment and / or prevention of liver inflammation and / or fibrosis. Complications of liver inflammation and / or fibrosis may also be included.

[0112] In yet another embodiment, said viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition is provided for use in extending healthy lifespan, preferably by preventing, delaying, curing, ameliorating and / or treating age-related metabolic disorders, preferably diabetes and / or obesity.

[0113] In yet another embodiment, the viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition is provided for use in the treatment and / or prevention of cancer, preferably liver cancer. Complications of cancer may also be included.

[0114] In a further aspect there is provided a method of preventing, delaying, ameliorating, curing and / or treating metabolic disorders, preferably diabetes and / or obesity and their complications, comprising the use of a 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 a composition as defined above.

[0115] Such a method is preferably for alleviating in an individual one or more symptom(s) of a metabolic disorder, such as diabetes and / or obesity, in a cell, tissue or organ of said individual, or for alleviating one or more characteristic(s) or symptom(s) of a cell, tissue or organ of said individual, which method comprises administering to said individual a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition as defined herein.

[0116] In a further aspect there is provided a method of preventing, delaying, ameliorating, curing and / or treating liver inflammation and / or fibrosis and their complications comprising the use of a 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 a composition as defined above.

[0117] Such a method is preferably for alleviating in an individual one or more symptom(s) of liver inflammation and / or fibrosis in a cell, tissue or organ of said individual, or alleviating one or more characteristic(s) or symptom(s) of a cell, tissue or organ of said individual, the method comprising administering to said individual a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition as defined herein.

[0118] In a further aspect there is provided a method of extending healthy lifespan by preferably preventing, delaying, curing, ameliorating and / or treating age-related metabolic disorders, preferably diabetes and / or obesity, comprising the use of a 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 a composition as defined above.

[0119] Such a method is preferably for alleviating in an individual one or more symptom(s) of age-related metabolic disorders, such as diabetes and / or obesity, in a cell, tissue or organ of said individual, or for alleviating one or more characteristic(s) or symptom(s) of a cell, tissue or organ of said individual, which method comprises administering to said individual a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition as defined herein.

[0120] In a further aspect there is provided a method of preventing, delaying, ameliorating, curing and / or treating cancer, preferably liver cancer and complications thereof, comprising the use of a 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 a composition as defined above.

[0121] Such a method is preferably for alleviating in an individual one or more symptom(s) of cancer, such as liver cancer, in a cell, tissue or organ of said individual, or for alleviating one or more characteristic(s) or symptom(s) of a cell, tissue or organ of said individual, which method comprises administering to said individual a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition as defined herein.

[0122] In the context of the present invention there is provided the use of a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition as defined herein for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating a metabolic disorder, preferably diabetes and / or obesity.

[0123] In the context of the present invention there is provided the use of a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition as defined herein for the manufacture of a medicament for preventing, delaying, curing, ameliorating and / or treating liver inflammation and / or fibrosis.

[0124] In the context of the present invention, there is preferably provided the use of a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition as defined herein for the manufacture of a medicament for preventing, delaying, curing, ameliorating and / or treating age-related metabolic disorders, preferably diabetes and / or obesity, thereby extending healthy lifespan.

[0125] In the context of the present invention there is provided the use of a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition as defined herein for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating cancer, preferably liver cancer.

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

[0127] Diabetes includes prediabetes, hyperglycemia, type 1 diabetes, type 2 diabetes, maturity-onset diabetes of the young (MODY), monogenic diabetes, neonatal diabetes, gestational diabetes, unstable diabetes, idiopathic diabetes, drug- or chemical-induced diabetes, stiff-man syndrome, lipoatrophic diabetes, and latent autoimmune diabetes of adults (LADA).

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

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

[0130] Liver inflammation and / or fibrosis includes autoimmune hepatitis, viral hepatitis including hepatitis A, B, C, D and E, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH) and cirrhosis.

[0131] Cancers include astrocytoma, glioma, leukemia, lymphoma, melanoma, myeloma, neuroblastoma, sarcoma (including chondrosarcoma, fibrosarcoma, rhabdomyoscaroma, and osteosarcoma), schwannoma, seminoma, and carcinoma of the bladder, breast, cervix, colon, endometrium, esophagus, gallbladder, kidney, liver, lung, ovary, prostate, pancreas, rectum, skin, stomach, and thyroid gland. A preferred cancer is liver cancer, preferably hepatocellular carcinoma. In one embodiment, the method or use is performed in vitro, for example, using cell culture. Preferably, the method or use is in vivo. The characteristics of each of these methods / uses have been previously defined herein.

[0132] In certain methods of the invention, the viral expression construct and / or vector and / or nucleic acid molecule and / or composition may be combined with additional compounds known to be used to treat metabolic disorders, preferably diabetes and / or obesity, in individuals.

[0133] In another method of the invention, the viral expression construct and / or vector and / or nucleic acid molecule and / or composition may be combined with additional compounds known to be used to treat liver inflammation and / or fibrosis.

[0134] In yet another method of the present invention, the viral expression construct and / or vector and / or nucleic acid molecule and / or composition may be combined with additional compounds known to be used to extend healthspan.

[0135] In yet another method of the present invention, the viral expression construct and / or vector and / or nucleic acid molecule and / or composition may be combined with additional compounds known to be used to treat cancer, preferably liver cancer.

[0136] In a preferred embodiment, the treatment in the uses or methods according to the present invention does not need to be repeated. Alternatively, in the uses or methods according to the present invention, said administration of the viral expression construct or said composition may be repeated every year or every 2, 3, 4, 5 or 6 years.

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

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

[0139] It is to be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derived peptide or construct identified herein by a given Sequence Identity Number (SEQ ID NO) is not limited to the specific sequence disclosed. Each coding sequence identified herein encodes a given protein fragment or polypeptide or peptide or derived peptide or construct, or is itself a protein fragment or polypeptide or construct or peptide or derived peptide. Throughout this application, whenever reference is made to the SEQ ID NO of a specific nucleotide sequence encoding a given protein fragment or polypeptide or peptide or derived peptide (take SEQ ID NO:X as an example), this means: i. a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO:X; ii. a nucleotide sequence that differs in sequence from the sequence of the nucleic acid molecule in (i) due to the degeneracy of the genetic code; or iii. A nucleotide sequence encoding an amino acid sequence having at least 60% amino acid identity or similarity with the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:X. can be replaced by

[0140] Throughout this application, whenever a specific amino acid sequence SEQ ID NO (take SEQ ID NO:Y as an example), this means: A polypeptide comprising an amino acid sequence having at least 60% sequence identity or similarity with the amino acid sequence of SEQ ID NO: Y. can be replaced by

[0141] Each nucleotide or amino acid sequence described herein by its percentage identity or similarity (at least 60%) to a given nucleotide or amino acid sequence, respectively, in more preferred embodiments has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity or similarity to the given nucleotide or amino acid sequence, respectively. In preferred embodiments, sequence identity or similarity is determined by comparing the full length of the sequences identified herein. Unless otherwise indicated herein, identity or similarity to a given SEQ ID NO refers to identity or similarity based on the full length (i.e., over its entire length or as a whole) of said sequence.

[0142] Each non-coding (i.e., promoter or other regulatory region) nucleotide sequence can be replaced with a nucleotide sequence containing a nucleotide sequence having at least 60% sequence identity or similarity to a specific nucleotide sequence SEQ ID NO: A (take SEQ ID NO: A as an example). Preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: A. Identity can be assessed across the entire SEQ ID NO, or across portions thereof, as described herein. In preferred embodiments, such non-coding nucleotide sequences, such as promoters, exhibit or exert at least the activity of such non-coding nucleotide sequences known to those of skill in the art, such as promoter activity.

[0143] "Sequence identity" is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences, or between two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In preferred embodiments, sequence identity is calculated based on the full length of the two given SEQ ID NOs, or on a portion thereof, where that portion preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NOs. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.

[0144] "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conservative amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be easily calculated by known methods, including, but not limited to, those described in "Computational Molecular Biology," Lesk, AM, ed., Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects," Smith, DW, ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data," Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; "Sequence Analysis in Molecular Biology," von Heine, G., Academic Press, 1987; and "Sequence Analysis Primer," Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0145] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, by way of example, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410(1990)). The BLAST X program 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 may also be used to determine identity.

[0146] Preferred parameters for polypeptide sequence comparison include: Comparison matrix: BLOSSUM62 from Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A useful program with these parameters is available from the Genetics Computer Group, Madison, WI, as the "Ogap" program. The aforementioned parameters are the default parameters for amino acid comparisons (no penalty for end gaps).

[0147] Preferred parameters for nucleic acid comparison include: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches = +10, mismatch = 0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from the Genetics Computer Group in Madison, Wis. The above are the default parameters for nucleic acid comparison.

[0148] When determining the degree of amino acid similarity, those skilled in the art may consider so-called "conservative" amino acid substitutions that are apparent to those skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each naturally occurring amino acid are Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

[0149] Genes or coding sequences "Gene" or "coding sequence" or "nucleic acid" or "nucleotide sequence" or "nucleic" refers to a region of DNA or RNA (transcribed region) that "encodes" a particular protein, such as FGF21. A coding sequence, when placed under the control of an appropriate regulatory region, such as a promoter, is transcribed (DNA) and translated (RNA) into a polypeptide. A gene can include several operably linked segments, such as a promoter, a 5' leader sequence, introns, a coding sequence, and a 3' untranslated sequence or region (3' UTR), which may contain a polyadenylation site or signal sequence. A chimeric or recombinant gene (such as the FGF21 gene) is a gene not normally found in nature, such as a gene whose promoter is not naturally associated with some or all of the transcribed DNA region. "Gene expression" refers to the process by which a gene is transcribed into RNA and / or an active protein.

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

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

[0152] Suitable promoters for organ-specific and / or tissue-specific expression of a nucleotide sequence encoding FGF21 include the human α1-antitrypsin promoter, the α1-antitrypsin promoter in combination with the hepatocyte control region (HCR) enhancer from apolipoprotein E, the albumin promoter, the major urinary protein promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, the liver-enriched protein activator promoter, the transthyretin promoter, the thyroxine-binding globulin promoter, the apolipoprotein A1 promoter, the liver fatty acid-binding protein promoter, the phenylalanine hydroxylase promoter, the adipocyte protein 2 (aP2, also known as fatty acid-binding protein 4 (FABP4)) promoter, the PPARy promoter, the adiponectin promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, and the promoter derived from human aromatase cytochrome p450 (p450arom). These include the mini / aP2 promoter (composed of the adipose-specific aP2 enhancer and the basal aP2 promoter), the uncoupling protein 1 (UCP1) promoter, the mini / UCP1 promoter (composed of the adipose-specific UCP1 enhancer and the basal UCP1 promoter), the adipsin promoter, the leptin promoter, the Foxa-2 promoter, the myosin light chain promoter, the myosin heavy chain promoter, the desmin promoter, the C5-12 promoter, the muscle creatine kinase (MCK) promoter, the smooth muscle alpha-actin promoter, the CK6 promoter, the Unc-45 myosin chaperone B promoter, the basal MCK promoter combined with a copy of the MCK enhancer, and the Enh358MCK promoter (combination of the MCK enhancer and the 358-bp proximal promoter of the MCK gene).

[0153] operably linked "Operably linked" is defined herein as a configuration in which a control sequence, such as a promoter sequence or regulatory sequence, is appropriately positioned relative to a nucleotide sequence of interest, preferably encoding FGF21, such that the promoter or control or regulatory sequence directs or affects the transcription and / or production or expression of the nucleotide sequence of interest, preferably encoding FGF21, in a cell and / or subject. For example, a promoter is operably linked to a coding sequence if it is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is understood to be "under the control of" the promoter. When one or more nucleotide sequences and / or elements contained within a construct are defined herein as "configured to be operably linked to an optional nucleotide sequence of interest," it is understood that once the nucleotide sequence of interest is present in the construct, the nucleotide sequences and / or elements are configured within the construct such that these nucleotide sequences and / or elements are all operably linked to the nucleotide sequence of interest.

[0154] Viral expression constructs An expression construct possesses a genome that is stable and capable of remaining episomal within a cell. Within the context of the present invention, a cell may be meant to include a cell used to generate the construct or a cell to which the construct is administered. Alternatively, the construct may be capable of being integrated into the genome of a cell, for example, through homologous recombination or otherwise. Particularly preferred expression constructs comprise a nucleotide sequence encoding FGF21 as defined herein operably linked to a promoter as defined herein, wherein the promoter is capable of directing expression of the nucleotide sequence (i.e., coding sequence) in a cell. Preferably, the promoter directs expression of the nucleotide sequence in at least one cell of a specific organ and / or tissue. Preferably, the promoter directs expression of the nucleotide sequence in at least one cell of the liver, adipose tissue, and / or skeletal muscle. Preferably, the promoter directs expression in at least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80%, 90% or 100% of the cells in the liver, adipose tissue and / or skeletal muscle. In the context of the present invention, FGF21 expressed in the liver, adipose tissue or skeletal muscle refers to preferential or predominant (at least 10% more, at least 20% more, at least 30% more, at least 40% more, at least 50% more, at least 60% more, at least 70% more, at least 80% more, at least 90% more, at least 100% more, at least 150% more, at least 200% or more) expression of FGF21 in the liver, adipose tissue or skeletal muscle compared to other organs or tissues. Throughout this application, when liver-specific, adipose-specific or skeletal muscle-specific is mentioned in relation to expression, cell type-specific expression of the cell type(s) that make up the liver, adipose tissue or skeletal muscle, respectively, is also expected.

[0155] The viral expression constructs of the present invention comprise nucleotide sequences in a form "suitable for mammalian expression," meaning that the viral expression construct includes one or more regulatory sequences operably linked to the nucleotide sequence to be expressed, selected based on the mammalian host cell to be used for expression. Preferably, the mammalian host cell used for expression is a human, murine, or canine cell.

[0156] The viral expression constructs of the present invention comprise nucleotide sequences that are expressed in the liver, adipose tissue and / or skeletal muscle.

[0157] As used herein, "adipose tissue" refers to tissue composed of a combination of mature adipocytes (i.e., adipocytes) and small blood vessels, neural tissue, lymph nodes, and stromal vascular fluid (SVF). SVF is composed of endothelial cells, fibroblasts, adipocyte precursor cells (i.e., preadipocytes), and immune cells, such as macrophages and T cells. In mammals, two different types of adipose tissue are conventionally distinguished: white adipose tissue (WAT) and brown adipose tissue (BAT). In mammals, adipose tissue is contained in a multi-depot organ. Fat depots include, but are not limited to, epididymal WAT (eWAT), inguinal WAT (iWAT), retroperitoneal WAT (rWAT), mesenteric WAT (mWAT), and interscapular BAT (iBAT).

[0158] As used herein, "skeletal muscle" refers to tissue composed of muscle fibers. Muscle fibers, also known as myofibers, are single, multinucleated or syncytial cells resulting from the fusion of hundreds of myoblasts, some of which remain within mature muscles as undifferentiated cells known as satellite cells. Individual muscle fibers are surrounded by connective tissue called endomysium. Approximately 10 to 100 muscle fibers form fascicles or bundles, which are themselves surrounded by another connective tissue layer called the perimysium. Finally, skeletal muscle is formed by groups of fascicles surrounded by another connective tissue layer called the epimysium. In addition to muscle fibers, skeletal muscle is also composed of numerous blood vessels and nerves. The ends of the muscle converge toward tendons and aponeuroses, dense connective tissue structures that mediate muscle attachment to the periosteum or other muscle connective tissues.

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

[0160] Such preferred expression constructs are said to comprise an expression cassette. As used herein, an expression cassette comprises or consists of a nucleotide sequence encoding FGF21, operably linked to a promoter capable of directing expression of the nucleotide sequence. In certain embodiments, an expression cassette as used herein comprises or consists of a nucleotide sequence encoding FGF21, a promoter, and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the liver and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the heart. In one embodiment, the described expression cassette contains nucleotide sequences encoding a microRNA expressed in the liver and / or a target sequence of a microRNA expressed in the heart that are perfectly complementary to their cognate microRNAs. In another embodiment, the described expression cassette contains one or more nucleotide sequence(s) encoding microRNA-binding sites that are imperfectly complementary (one mismatch per five consecutive nucleotides). In yet another embodiment, the expression cassette may contain nucleotide sequences encoding both perfect and imperfect microRNA-binding sites. Therefore, expression cassettes can be tailored to achieve different levels of regulation by using nucleotide sequences encoding a single complete microRNA target site, multiple complete microRNA target sites, a single incomplete microRNA target site, multiple incomplete microRNA target sites, or a combination of complete and incomplete microRNA target sites. Furthermore, nucleotide sequences encoding target sites for different microRNAs can be used, thereby enabling genes to be regulated by multiple microRNAs. The preferred location of the nucleotide sequence encoding the microRNA target sequence is the 3'UTR. However, nucleotide sequences (encoding target sequences) inserted into either the coding sequence or the 5'UTR sequence can also be used.

[0161] The selection of the nucleotide sequence encoding the target sequence of microRNA is determined by the desired expression pattern.The presence of endogenous microRNA in cells inhibits the expression of the gene or coding sequence from the expression construct containing the nucleotide sequence encoding the target sequence of said microRNA.For the expression of the gene or coding sequence of interest to be inhibited in a given cell type, the nucleotide sequence encoding the target sequence recognized by the microRNA present in that cell type is selected.

[0162] A viral expression construct is an expression construct intended for use in gene therapy. It is designed to contain a portion of the viral genome as defined later in this specification. The expression constructs disclosed herein can be prepared using recombinant techniques to express the nucleotide sequence encoding FGF21 in suitable cells, such as cultured cells or cells of multicellular organisms, as described, for example, in Ausubel et al., "Current Protocols in Molecular Biology," Greene Publishing and Wiley-Interscience, New York (1987) and Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entireties. See also Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site-directed mutagenesis) and Roberts et al. (1987) Nature 328:731-734 or Wells, JA, et al. (1985) Gene 34:315 (describing cassette mutagenesis).

[0163] Typically, nucleic acids or nucleotide sequences encoding FGF21 are used in expression constructs or expression vectors. The phrase "expression vector" or "vector" generally refers to a nucleotide sequence capable of directing the expression of a gene or coding sequence in a host compatible with the gene or coding sequence. These expression vectors typically include at least a suitable promoter sequence and optionally include a transcription termination signal. Additional factors necessary or helpful for expression, as described herein, may also be used. Nucleic acids or DNA or nucleotide sequences encoding FGF21 are incorporated into DNA constructs capable of introduction and expression in in vitro cell culture. Specifically, the DNA constructs are suitable for replication in prokaryotic hosts, such as bacteria, e.g., E. coli, or can be introduced into cultured mammalian, plant, insect (e.g., Sf9), yeast, fungal, or other eukaryotic cell lines.

[0164] A DNA construct prepared for introduction into a particular host may include a replication system recognized by the host, a DNA segment intended to encode the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term "operably linked" has been defined herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates transcription of the coding sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide. Generally, operably linked DNA sequences are contiguous, and in the case of a signal sequence, contiguous and in reading frame. However, enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites, or with adapters or linkers inserted in place thereof, or by gene synthesis.

[0165] The selection of an appropriate promoter sequence generally depends on the host cell selected for expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters well known in the art (see, for example, Sambrook and Russell, 2001, supra). The transcriptional regulatory sequence typically includes a heterologous enhancer or promoter recognized by the host. The selection of an appropriate promoter sequence depends on the host, but promoters such as trp, lac, and phage promoters, tRNA promoters, and glycolytic enzyme promoters are known and available (see, for example, Sambrook and Russell, 2001, supra). The expression vector contains a replication system and transcriptional and translational regulatory sequences, along with an insertion site for the polypeptide-encoding segment. In most cases, the replication system functions only in the cells (bacterial cells such as E. coli) used to produce the vector. Most plasmids and vectors do not replicate in the cells they infect. Examples of viable cell line and expression vector combinations are described in Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature 334 :31-36. For example, suitable expression vectors can be expressed in yeast, e.g., S. cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells, and bacterial cells, e.g., E. coli. The cells can therefore be prokaryotic or eukaryotic host cells. The cells can be cells suitable for culture in liquid medium or on solid medium.

[0166] Alternatively, the host cell is a cell that is part of a multicellular organism, such as a transgenic plant or animal.

[0167] viral vectors A viral vector or viral gene therapy vector is a vector that contains a viral expression construct as defined above.

[0168] Viral vectors or viral gene therapy vectors are suitable vectors for gene therapy. Suitable vectors for gene therapy are described in Anderson 1998, Nature 392 :25-30;Walther and Stein,2000,Drugs 60 :249-71;Kay et al.,2001, Nat.Med. 7 :33-40;Russell,2000,J.Gen.Virol. 81 :2573-604;Amado and Chen,1999,Science 285 :674-6;Federico,1999,Curr.Opin.Biotechnol. 10 :448-53;Vigna and Naldini,2000,J.Gene Med. 2 :308-16;Marin et al.,1997,Mol.Med.Today 3 :396-403;Peng and Russell,1999,Curr.Opin.Biotechnol. 10 :454-7;Sommerfelt,1999,J.Gen.Virol. 80 :3049-64;Reiser,2000,Gene Ther. 7 :910-3; and in the references cited therein.

[0169] Particularly suitable gene therapy vectors include adenovirus and adeno-associated virus (AAV) vectors.These vectors infect a wide range of dividing and non-dividing cell types, including synovial cells and liver cells.As indicated above, the episomal nature of adenovirus and AAV vectors after cell entry makes these vectors suitable for therapeutic use (Russell, 2000, J.Gen.Virol. 81:2573-2604; Goncalves, 2005, Virol J. 2(1):43). AAV vectors are even more preferred because they are known to provide very stable, long-term expression of transgenes (up to 9 years in dogs (Niemeyer et al, Blood. 2009 Jan 22;113(4):797-806) and approximately 10 years in humans (Buchlis, G. et al., Blood. 2012 Mar 29;119(13):3038-41). Preferred adenoviral vectors are modified to reduce the host response, as reviewed by Russell (2000, supra). Gene therapy methods using AAV vectors have been described in Wang et al., 2005, J Gene Med. March 9 (Epub ahead of print), Mandel et al., 2004, Curr Opin Mol Ther. 6(5):482-90 and Martin et al., 2004, Eye 18(11):1049-55, Nathwani et al. al, N Engl J Med. 2011 Dec 22;365(25):2357-65, Apparailly et al, Hum Gene Ther. 2005 Apr;16(4):426-34.

[0170] Another suitable gene therapy vector is a retroviral vector. A preferred retroviral vector for use in the present invention is a lentiviral-based expression construct. Lentiviral vectors have the ability to infect dividing and non-dividing cells and stably integrate into their genomes (Amado and Chen, 1999 Science 285:674-6). Methods for the construction and use of lentiviral-based expression constructs are described in U.S. Patent Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633, and 6,323,031, as well as Federico (1999, Curr Opin Biotechnol 10:448-53) and Vigna et al. (2000, J Gene Med 2000;2:308-16).

[0171] Other suitable gene therapy vectors include adenovirus vectors, herpesvirus vectors, polyomavirus vectors or vaccinia virus vectors.

[0172] The gene therapy vector comprises a nucleotide sequence encoding the FGF21 to be expressed, whereby the nucleotide sequence is operably linked to an appropriate regulatory sequence. Such regulatory sequence includes at least a promoter sequence. Examples of promoters suitable for expressing the nucleotide sequence encoding FGF21 from the gene therapy vector include the CMV promoter, viral long terminal repeat promoters (LTRs) from, for example, murine Moloney leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1, the simian virus 40 (SV40) early promoter, the CAG promoter, the α1-antitrypsin promoter, the mini / aP2 promoter, the mini / UCP1 promoter, the C5-12 promoter, and the herpes simplex virus thymidine kinase promoter.

[0173] Several inducible promoter systems have been described that can be induced by the administration of organic or inorganic small molecule compounds. Such inducible promoters include those regulated by heavy metals, such as the metallothionine promoter (Brinster et al. 1982 Nature 1999). 296 :39-42;Mayo et al. 1982 Cell 29 :99-108), and those regulated by RU-486 (progesterone antagonist) (Wang et al. 1994 Proc. Natl. Acad. Sci. USA 91 :8180-8184), which is regulated by steroids (Mader and White, 1993 Proc. Natl. Acad. Sci. USA 90 :5603-5607), which is regulated by tetracycline (Gossen and Bujard 1992 Proc. Natl. Acad. Sci. USA 89 :5547-5551; U.S. Patent No. 5,464,758; 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;Shockett et al. 1995 Proc.Natl.Acad.Sci.USA 92 :6522-6526), ​​and several chimeric transactivators composed of the tetR polypeptide as the VP16 activation domain and the ligand binding domain of the estrogen receptor (Yee et al., 2002, US Pat. No. 6,432,705).

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

[0175] The gene therapy vector is preferably formulated in a composition or pharmaceutical composition as defined herein. In this regard, the composition or pharmaceutical composition may comprise a suitable pharmaceutical carrier as defined herein above.

[0176] Adeno-associated virus vector (AAV vector) A preferred viral vector or gene therapy vector is an AAV vector. As used herein, the AAV vector preferably includes a recombinant AAV vector (rAAV vector). As used herein, "rAAV vector" refers to a recombinant vector that includes a portion of the AAV genome encapsidated in a protein shell of capsid proteins derived from an AAV serotype as described herein. The portion of the AAV genome may include inverted terminal repeats (ITRs) derived from adeno-associated virus serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, etc. Preferred ITRs are those of AAV2, represented by the sequence comprising or consisting of SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR). The present invention also preferably encompasses the use of a sequence having 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 to SEQ ID NO: 48 as the 5' ITR and a sequence having at least 80% identity to SEQ ID NO: 49 as the 3' ITR.

[0177] The protein shell containing the capsid protein can be derived from an AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, etc. Preferred AAV capsids are those of AAV1, AAV3, AAV8, and AAV9. Preferred ITRs are from AAV2. The protein shell can also be referred to as a capsid protein shell. rAAV vectors can be deleted for one, or preferably all, wild-type AAV genes, but can still contain functional ITR nucleic acid sequences. Functional ITR sequences are necessary for replication, rescue, and packaging of AAV virions. The ITR sequences can be wild-type sequences or can have at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with the wild-type sequence, or can be altered, for example, by nucleotide insertion, mutation, deletion, or substitution, as long as they remain functional. In this context, functionality refers to the ability of the genome to be directly packaged into a capsid shell and subsequently express in infected host or target cells. In the context of the present invention, the capsid protein shell may be of a different serotype than the rAAV vector genome ITRs.

[0178] The nucleic acid molecule represented by the selected nucleic acid sequence is preferably inserted between the rAAV genome or ITR sequences identified above, e.g., an expression construct comprising expression regulatory elements operably linked to a coding sequence and a 3' terminal sequence. The nucleic acid molecule may also be referred to as a transgene.

[0179] "AAV helper functions" generally refer to the corresponding AAV functions required for rAAV replication and packaging, which are provided in trans to the rAAV vector. AAV helper functions supplement the AAV functions missing in the rAAV vector, but lack the AAV ITRs (provided by the rAAV vector genome). AAV helper functions encompass the two major AAV ORFs, namely, the rep coding region and the cap coding region, or sequences functionally substantially identical thereto. The Rep and Cap regions are well known in the art; see, for example, Chiorini et al. (1999, J. of Virology, Vol. 73(2):1309-1319) or US Pat. No. 5,139,941, which are incorporated herein by reference. AAV helper functions can be provided on an AAV helper construct. Introduction of the helper construct into the host cell can occur prior to or simultaneously with the introduction of the rAAV genome present in the rAAV vector identified herein, for example, by transformation, transfection, or transduction. The AAV helper constructs of the invention can therefore be selected to produce a combination of serotypes desired for the capsid protein shell of the rAAV vector, on the one hand, and for replication and packaging of the rAAV genome present in the rAAV vector, on the other hand.

[0180] An "AAV helper virus" provides additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenovirus, herpes simplex virus (such as HSV type 1 and type 2), and vaccinia virus. The additional functions provided by the helper virus can also be introduced into host cells via a plasmid, as described in US Pat. No. 6,531,456, which is incorporated herein by reference.

[0181] A "transgene" is defined herein as a gene or coding sequence or nucleic acid molecule (i.e., a molecule encoding FGF21) newly introduced into a cell, i.e., a gene that may be present in the cell but not normally expressed or expressed at an insufficient level. In this context, "insufficient" means that the FGF21 is expressed in the cell, but the symptoms and / or diseases defined herein can still occur. In this case, the present invention allows for overexpression of FGF21. A transgene may include a sequence that is native to the cell, a sequence that does not naturally occur in the cell, or a combination of both. A transgene may contain a sequence encoding FGF21 and / or an additional protein as specified herein above, which may be operably linked to appropriate regulatory sequences for expression of the FGF21-encoding sequence in the cell. Preferably, the transgene is not integrated into the genome of the host cell.

[0182] "Transduction" refers to the delivery of FGF21 to recipient host cells by a viral vector.For example, the transduction of target cells by the rAAV vector of the present invention leads to the introduction of the rAAV genome contained in the vector into the transduced cells."Host cell" or "target cell" refers to the cell into which DNA delivery occurs, such as the muscle cells of a subject.AAV vectors can transduce both dividing and non-dividing cells.

[0183] AAV vector production The production of recombinant AAV (rAAV) for vectoring transgenes has been previously described. See Ayuso E, et al., Curr. Gene Ther. 2010;10:423-436; Okada T, et al., Hum. Gene Ther. 2009;20:1013-1021; Zhang H, et al., Hum. Gene Ther. 2009;20:922-929; and Virag T, et al., Hum. Gene Ther. 2009;20:807-817. These protocols can be used or adapted to produce the AAV of the present invention. In one embodiment, a production cell line is transiently transfected with a polynucleotide of the present invention (comprising an expression cassette flanked by ITRs) and construct(s) encoding rep and cap proteins and providing helper functions. In another embodiment, the cell line stably supplies helper functions and is transiently transfected with a polynucleotide of the invention (comprising an expression cassette flanked by ITRs) and a construct(s) encoding rep and cap proteins. In another embodiment, the cell line stably supplies rep and cap proteins and helper functions and is transiently transfected with a polynucleotide of the invention. In another embodiment, the cell line stably supplies rep and cap proteins and is transiently transfected with a polynucleotide of the invention and a polynucleotide encoding helper functions. In yet another embodiment, the cell line stably supplies a polynucleotide of the invention, rep and cap proteins and helper functions. Methods for making and using these and other AAV production systems are described in the art.Muzyczka N,et al.,US5,139,941,Zhou 6,491,907, Zolotukhin S,et al.,US6,660,514, Shenk T,et al.,US6,951,753, Snyder R,et al.,US7,094,604, Rabinowitz J,et al.,US7,172,893, Monahan P,et al.,US7,201,898, Samulski R,et al. See, e.g., US 7,229,823 and Ferrari F, et al., US 7,439,065.

[0184] The rAAV genome present in the rAAV vector comprises at least the nucleotide sequence of the inverted terminal repeat region (ITR) of one of the AAV serotypes (preferably that of serotype AAV2 disclosed previously herein), or a nucleotide sequence substantially identical thereto, or a nucleotide sequence having at least 60% identity thereto, and a nucleotide sequence encoding FGF21 (under the control of suitable regulatory elements) inserted between the two ITRs. The vector genome requires the use of flanking 5' and 3' ITR sequences to enable efficient packaging of the vector genome into the rAAV capsid.

[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, p1309-1319). They can be cloned or prepared by chemical synthesis known in the art, for example, using an oligonucleotide synthesizer supplied by Applied Biosystems Inc. (Fosters, CA, USA), or by standard molecular biology techniques. ITRs can be cloned from the AAV viral genome or excised from a vector containing AAV ITRs. The ITR nucleotide sequences can be ligated at either end to nucleotide sequences encoding one or more therapeutic proteins, or the AAV sequences between the ITRs can be replaced with desired nucleotide sequences, using standard molecular biology techniques.

[0186] Preferably, the rAAV genome present in the rAAV vector does not contain any nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or cap (capsid) genes, etc. The rAAV genome may further contain a marker or reporter gene, such as an antibiotic resistance gene, a gene encoding a fluorescent protein (e.g., gfp), or a gene encoding a product detectable and / or selectable by chemical, enzymatic, or other methods known in the art (e.g., lacZ, aph, etc.).

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

[0188] Suitable 3' untranslated sequences can also be operably linked to the nucleotide sequence encoding FGF21. Suitable 3' untranslated regions can be those naturally associated with the nucleotide sequence or can be derived from different genes, such as the SV40 polyadenylation signal (SEQ ID NO: 50) and the rabbit β-globin polyadenylation signal (SEQ ID NO: 51).

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

[0190] Codon optimization As used herein, "codon optimization" refers to a process used to modify an existing coding sequence or design a coding sequence, for example, to improve translation of a transcript RNA molecule transcribed from the coding sequence in an expression host cell or organism, or to improve transcription of the coding sequence. Codon optimization includes, but is not limited to, processes that involve selecting codons for a coding sequence to match the codon preferences of the expression host organism, for example, to match the codon preferences of a mammalian, preferably mouse, dog, or human expression host. Codon optimization also removes elements that potentially negatively affect RNA stability and / or translation, such as termination sequences, TATA boxes, splice sites, ribosome entry sites, repeat and / or GC-rich sequences, and RNA secondary structure or instability motifs.

[0191] In this document and in the claims, the verb "to comprise" and its conjugations are used in an open-ended sense to mean that the items following the word are inclusive, but items not specifically mentioned are not excluded. Additionally, the verb "to consist" may be replaced by "to consist essentially of," meaning that the viral expression constructs, viral vectors, compositions, and gene therapy compositions defined herein may contain additional component(s) other than those specifically identified, where said additional component(s) do not alter the inherent characteristics of the invention.

[0192] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that the element be one and only one. The indefinite article "a" or "an" therefore normally means "at least one."

[0193] The words "approximately" or "about," when used in connection with a numerical value (approximately 10, about 10), preferably mean that the value is within 1% of the given value of 10.

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

[0195] The present invention will be further illustrated in the following examples, which are not intended to limit the scope of the invention but merely serve to clarify it.

[0196] Figure legend: 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 from chow-fed and HFD-fed C57Bl6 mice treated with AAV vectors in eWAT (n = 8–11 animals / group). 12The study was performed 14 weeks after intraeWAT administration of vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vector. 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.

[0197] Figure 2. Histological analysis of adipose tissue and liver from C57Bl6 mice treated with AAV9-CAG-moFGF21-doublemiRT vector in eWAT. (A) Representative images stained with hematoxylin and eosin of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), interscapular brown adipose tissue (iBAT), and liver sections from chow-fed and HFD-fed C57Bl6 mice treated with 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 on 10 12 The study was performed 14 weeks after intraeWAT administration of vg 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.

[0198] 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). The study was performed 11 weeks after AAV administration. (H) Fasting insulin circulating levels. Unless otherwise indicated, analyses were performed at 10 12 The study was performed 14 weeks after intraeWAT administration of vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vector. 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.

[0199] 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 at 10 10 vg, 5×10 10 vg, 2 × 10 11 vg or 10 12 vg of AAV8-CAG-moFGF21-doublemiRT or 10 12 The study was performed 16 weeks after intraeWAT administration of vg of AAV8-CAG-null vector. Results are 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, 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.

[0200] 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.

[0201] 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) 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 insulin circulating levels 3 months after AAV administration. Unless otherwise indicated, analyses were performed at 10 11 vg or 5×10 11 vg of AAV8-hAAT-moFGF21 or 5 × 10 11The study was performed 20 weeks after intravenous administration of vg of AAV8-hAAT-null vector. Results are expressed as mean ± SEM. n=9–10 animals / 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-hAAT-null; ** p<0.01 vs. AAV8-hAAT-null; *** p<0.001 vs. AAV8-hAAT-null.

[0202] 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 on 10 10 vg or 5×10 10 vg of AAV8-hAAT-moFGF21 or 5 × 10 10 The study was performed 52 weeks after intravenous administration of vg of AAV8-hAAT-null vector. Results are expressed as mean ± SEM, n = 9–12 animals / 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.

[0203] 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.

[0204] 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 on 10 10 vg, 2 × 10 10 vg or 5×10 10 vg of AAV8-hAAT-moFGF21 or 5 × 10 10The study was performed 21 weeks after intravenous administration of 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.

[0205] 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.

[0206] 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.

[0207] 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.

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

[0209] A, B Representative images of hematoxylin-eosin staining of (A) eWAT and (B) liver tissue sections obtained from ob / ob animals injected intra-eWAT with either null or AAV8 vectors encoding FGF21 at all doses tested. Scale bar: 100 μm for eWAT, 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-injected group.

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

[0215] A At 11 weeks of age, mice were injected with either the AAV8-CAG-null vector or the AAV8-CAG-moFGF21-dmiRT vector at four different doses (1 × 10 10 , 5×10 10 , 2 × 10 11 , 1×10 12 Serum adiponectin levels in 25-week-old ob / ob animals injected intra-eWAT with 1000 mg / mouse (vg / mouse).

[0216] B Quantification of the expression of the macrophage marker F4 / 80 by qRT-PCR in the same animals as in (A). C. Representative image of immunostaining for the macrophage-specific marker Mac2 in eWAT sections from ob / ob mice receiving the AAV8-CAG-moFGF21-dmiRT vector. n = 4–8 per group. Scale bar: 200 μm.

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

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

[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. 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.

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

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

[0223] B Serum adiponectin levels in all groups.

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

[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. the null-injected ob / ob group.

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

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

[0231] B, C Quantification of the expression of inflammatory markers F4 / 80 (B) and TNF-α (C) by qRT-PCR in the same mouse cohort.

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

[0233] F, G Liver triglyceride and cholesterol contents in the fed state in the same cohort as in (A).

[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. n = 9-10 animals / group in (B, DF, HI). *P<0.05, **P<0.01, and ***P<0.001 vs. the null-injected ob / ob group.

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

[0237] A, B Quantification of hepatic PEPCK and G6Pase expression by qRT-PCR in ob / ob mice injected with either the AAV8-hAAT-null vector or the AAV8-hAAT-moFGF21 vector at 2 months of age.

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

[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. the null-injected ob / ob group.

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

[0243] 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).

[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 Representative images of animals belonging to all experimental groups for studies performed in young adults (A) or adults (B).

[0249] C Representative images of epididymal white fat (eWAT) pads obtained at sacrifice from animals treated with several doses of AAV8-hAAT-moFGF21 as young adults (left) or adults (right).

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

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

[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. In (E), n = 7–10 animals / group. HFD, high-fat diet. ND, not detected.

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

[0255] A Assessment of locomotor activity through 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 vector encoding FGF21.

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

[0257] C. Western blot analysis of UCP1 content in iBAT from the same animal cohort as in (A). A representative immunoblot is shown (left). The histogram depicts the densitometric analysis of two different immunoblots (right).

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

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

[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. (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.

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

[0263] A. Energy expenditure in a cohort of animals that began HFD feeding at 2 months of age was measured 10 months after AAV8-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 representative immunoblot is shown (left). The graph shows a densitometric analysis of two different immunoblots (right).

[0265] C Relative expression levels of Serca2b and RyR2 in iWAT in animals that started HFD feeding as young adults or adults and received FGF21 vector.

[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 = 7-10 animals / group. (B) n = 4 animals / group. (C) n = 7-10 animals / group. *P<0.05, **P<0.01, and ***P<0.001 vs. chow-fed null-injected 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 Fasting glucagon levels in the group of animals that received the FGF21 vector starting on HFD feeding as young adults.

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

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

[0272] D 5 × 10 as young adults (upper panel) or adults (lower panel) 10 Representative image of double immunostaining for insulin (dark gray) and glucagon (light gray) in pancreatic sections from animals that received AAV8-hAAT-moFGF21 in vg / mouse. Scale 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. (D) n = 4-5 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.

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

[0276] A Glucose tolerance was tested after intraperitoneal injection of glucose (2 g / kg body weight) in a group of mice that started HFD feeding as young adults and received the FGF21 vector.

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

[0278] Data information: All data 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.

[0279] Figure 24. Amelioration of WAT hypertrophy and inflammation by AAV8-hAAT-moFGF21 treatment.

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

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

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

[0283] E 5 x 10 as adults 10 Immunohistochemistry for the macrophage-specific marker Mac2 in eWAT sections from animals receiving AAV8-hAAT-moFGF21 in vg / mice. 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).

[0284] Quantification by qRT-PCR of the expression of inflammatory markers F4 / 80 (F), IL1-β (G), and TNF-α (H) in animals receiving the FGF21 vector starting on a HFD as FH adults.

[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. (B) n = 4 animals / group. (FH) 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.

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

[0288] A. Young adults (top graph) or adults (bottom graph) were fed chow or HFD and administered AAV8-hAAT-null or 5 × 10 10 Frequency distribution of adipocyte area in animal groups that received either the AAV8-hAAT-moFGF21 vector in vg / mice.

[0289] 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).

[0290] Relative expression levels of the inflammatory markers F4 / 80, CD68, and TNF-α by qRT-PCR in the same animal cohort as in CE (B).

[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. (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.

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

[0294] A. Chow or HFD-fed mice were infected with AAV8-hAAT-null or 5 × 10 10Representative images of hematoxylin-eosin stained liver sections from animals administered either the AAV8-hAAT-moFGF21 vector or the AAV8-hAAT-moFGF21 vector in vg / mouse. HFD clearly induced lipid droplet accumulation in the liver, which was reversed by AAV8-hAAT-moFGF21 treatment in both young and adult mice. Scale bar: 100 μm.

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

[0296] D HFD fed with AAV8-hAAT-null or 5 × 10 10 Immunostaining for the macrophage-specific marker Mac-2 in liver sections from animals that received the AAV8-hAAT-moFGF21 vector in vg / mice. 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. n=7-10 animals / group in (BC). **P<0.01 and ***P<0.001 vs. chow-fed null-injected 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 10Analysis of liver fibrosis via Masson's trichrome staining in HFD-fed animals receiving either the AAV8-hAAT-null or AAV8-hAAT-moFGF21 vectors in vg / mice. 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.

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

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

[0303] B, C Quantification of collagen 1 expression by qRT-PCR in the liver in groups of animals that received the FGF21 vector starting HFD feeding as young adults (B) or adults (C).

[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-fed Null-injected group. HFD, high-fat diet.

[0306] 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 using the highest dose (5x10) of FGF21 administered to young adults or adults. 10 vg / mouse) compared with HFD-fed mice treated with the AAV8-hAAT-moFGF21 vector and null-injected chow- or HFD-fed animals.

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

[0308] B Tibial length.

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

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

[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) 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.

[0313] Figure 30. Analysis of glycemic profiles in C57B16 mice treated with AAV8-hAAT-moFGF21 vectors. Blood glucose levels were assessed under fed conditions. AAV, 5x10 10 vg or 2×10 11vg of AAV8-hAAT-moFGF21 (n = 13 and 15, respectively) or 2 × 10 11 IV administration of vg 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.

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

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

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

[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 Insulin sensitivity assessed through intraperitoneal injection of insulin (0.75 units / kg body weight) and expressed as a percentage of starting 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. Null-injected group.

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

[0325] A, B Body weight (A) and weight gain (B) evolution in animals treated with AAV1-CMV-moFGF21. C57Bl6 mice were fed a HFD for approximately 12 weeks and then treated with 3 × 10 11 vg / mouse of the AAV1-CMV-moFGF21 vector. Control obese mice and control chow-fed mice received 3 × 10 11 vg received AAV1-CMV-null.

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

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

[0328] 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.

[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-injected group.

[0331] 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.

[0332] 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) in the same cells as in (A). (D) FGF21 expression levels in C2C12 cells transfected with plasmids encoding either the wild-type mouse FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or the codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21). (E) FGF21 expression levels in HepG2 cells transfected with plasmids encoding either the wild-type mouse FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or the codon-optimized mouse FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF21). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. Results are expressed as mean ± SEM. n = 3 wells per group. ND, not detected. * p < 0.05 vs. control. ### p < 0.001 vs. EF1a-mFGF21.

[0333] Figure 35. Increase in hepatic FGF21 expression and circulating FGF21 levels in vivo with hAAT-moFGF21 and CMV-moFGF21 expression cassettes. (A) FGF21 expression levels in the liver of C57B16 mice hydrodynamically administered with plasmids encoding the wild-type murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (EF1a-mFGF21), the codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21), or the codon-optimized murine FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF21). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. (B) Circulating FGF21 levels in the same cohort as in (A). Results are expressed as mean ± SEM. n = 5 mice / group. ** p < 0.01 vs. EF1a-mFGF21 Figure 36. AAV8-hAAT-moFGF21 increases hepatic FGF21 expression and circulating FGF21 levels in vivo. (A) 1 x 10 10 vg, 2 × 10 10 vg or 5×10 10 (B) FGF21 expression levels in the liver of C57Bl6 mice intravenously administered with an AAV8 vector encoding wild-type murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or codon-optimized murine FGF21 under the control of the hAAT promoter (AAV8-hAAT-moFGF21). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. (B) Circulating FGF21 levels in the same cohort as in (A). Analysis was performed 2 weeks post-AAV. Results are expressed as mean ± SEM. n=4-5 mice / group. Control, untreated mice. **p<0.01 and ***p<0.001 vs. control. ##p<0.01 and ###p<0.001 vs. AAV8-EF1a-mFGF21. Figure 37. Increased adipose FGF21 expression in vivo by AAV8-CAG-moFGF21-dmiRT. (AB) 2 x 10 10 vg, 5×10 10 vg or 1×10 11 FGF21 expression levels in the eWAT (A) or liver (B) of C57Bl6 mice administered intraeWAT with either an AAV8 vector encoding wild-type murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or an AAV8 vector encoding a codon-optimized murine 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 (AAV8-CAG-moFGF21-doublemiRT). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. Analysis was performed 2 weeks post-AAV administration. Results are expressed as mean ± SEM. n = 4–5 mice per group. Control, untreated mice. eWAT, epididymal white adipose tissue. * p<0.05, ** p<0.01, and Figure 38. In vivo increase of FGF21 expression in skeletal muscle by AAV1-CMV-moFGF21. (AB) 5x10 10 vg, 1×10 11 vg or 3×10 11FGF21 expression levels in the quadriceps (A) or liver (B) of C57Bl6 mice intramuscularly administered either an AAV8 vector encoding the wild-type murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or an AAV1 vector encoding a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (AAV1-CMV-FGF21). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. Analysis was performed 2 weeks post-AAV. Results are expressed as mean ± SEM. n = 4–5 mice per group. Control, untreated mice. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control. # 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 obWe used OlaHsd / OlaHsd (ob / ob) mice. Mice were fed a standard diet (2018S Teklad Global Diets®, Harlan Labs., Inc., Madison, WI, US) or a high-fat diet (TD.88137 Harlan Teklad, Madison, WI, US) ad libitum and maintained under a 12-h light / dark cycle (lights on at 8:00 am) and a stable temperature (22°C ± 2°C). For tissue sampling, mice were anesthetized with the inhalant anesthetic isoflurane (IsoFlo®, Abbott Laboratories, Abbott Park, IL, US) and decapitated. Tissues of interest were excised and stored at -80°C or in formalin until analysis. All experimental procedures were approved by the Ethics Committee for Animal and Human Experimentation at the Universitat Autónoma de Barcelona.

[0335] Recombinant AAV vectors Single-stranded AAV vectors of serotype 1, 8, or 9 were produced by triple transfection of HEK293 cells according to standard methods (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6) :423-36). The cells were placed in 10 roller bottles (850 cm 2Cells were cultured to 80% confluence in DMEM 10% FBS (flat; Corning™, Sigma-Aldrich Co., Saint Louis, MO, US) and cotransfected by the calcium phosphate method with a plasmid carrying an expression cassette flanked by AAV2 ITRs, a helper plasmid carrying the AAV2 rep gene and the cap gene of AAV serotype 1, 8, or 9, and a plasmid carrying adenovirus helper functions. The transgenes used were: 1) a cytomegalovirus (CMV) early enhancer / chicken beta-actin (CAG) promoter with four tandem repeats of the miRT122a sequence (5'CAAACACCATTGTCACACTCCA3') (SEQ ID NO: 12) and four tandem repeats of the miRT1 sequence (5'TTACATACTTCTTTACATTCCA3') (SEQ ID NO: 13) cloned into the 3' untranslated region of the expression cassette; 2) a CMV promoter; or 3) a sequence encoding codon-optimized mouse, canine, or human FGF21 or wt FGF21 driven by the human α1-antitrypsin promoter (hAAT). Null vectors were generated using non-coding plasmids carrying the CAG, hAAT, or CMV promoter. AAV was purified using an optimized method based on polyethylene glycol precipitation steps and two consecutive cesium chloride (CsCl) gradients. This second-generation CsCl-based protocol dramatically reduced empty AAV capsids as well as DNA and protein impurities (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6) :423-36). The purified AAV vector was dialyzed against PBS, filtered, and stored at -80°C. The viral genome titer was determined by quantitative PCR according to the protocol described for the AAV2 reference standard using linear plasmid DNA as a standard curve (Lock M, et al., Hum. Gens Ther. 2010;21:1273-1285). The vector was constructed according to molecular biology techniques well known in the art.

[0336] In vivo eWAT administration of AAV vectors Mice were anesthetized using an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). A laparotomy was performed to remove the epididymal white adipose tissue. The AAV vector was resuspended in PBS containing 0.001% Pluronic® F68 (Gibco) and injected directly into the epididymal fat pad. Each epididymal fat pad was injected twice with 50 μL of AAV solution (one injection near the testicle and one injection into the center of the fat pad). The abdomen was rinsed with sterile saline solution and closed using a two-layer approach.

[0337] Systemic administration of AAV vectors The appropriate amount of AAV solution was diluted in 200 μL of PBS containing 0.001% Pluronic® and injected manually into the lateral tail vein without applying pressure at the moment of injection. Prior to injection, the animals were placed under a 250 W infrared heat lamp (Philips NV, Amsterdam, NL) for several minutes to dilate the blood vessels and allow for easier visualization and access of the tail vein. The animals were immobilized for injection using a plastic restrainer (Harvard Apparatus, Holliston, MA, US). No anesthesia was required, as appropriate restraining devices were utilized. Animals were injected using a 30-gauge needle.

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

[0339] Immunohistochemical and morphometric analysis Tissues were fixed in formalin (Panreac Quimica) for 24 hours, embedded in paraffin, and sectioned. Tissue samples were stained with hematoxylin-eosin. Image analysis software (analySIS 3.0; Soft Imaging System, Center Valley, PA, EEUU) was used to determine the area of ​​adipocytes in 12 hematoxylin / eosin WAT images per animal acquired at 10× using a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) connected to a video camera with a monitor. The area of ​​each adipocyte was expressed in μm 2 The mean adipocyte area was calculated for each experimental group, and the distribution of adipocytes according to size categories was represented by histograms. Four animals were used per group, and at least 250 adipocytes per animal were analyzed.

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

[0341] RNA analysis Total RNA was obtained from fat depots or liver using QIAzol Lysis Reagent (Qiagen NV, Venlo, NL) or Tripure isolation reagent (Roche Diagnostics Corp., Indianapolis, IN, US), and the RNeasy Lipid Tissue Minikit (Qiagen NV, Venlo, NL), respectively. To remove residual viral genomes, total RNA was treated with DNAse I (Qiagen NV, Venlo, NL). For RT-PCR, 1 μg of RNA sample was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (04379012001, Roche, California, USA). Real-time quantitative PCR was performed in a SmartCycler II® (Cepheid, Sunnyvale, USA) using EXPRESS SYBRGreen qPCR supermix (Invitrogen™, Life Technologies Corp., Carlsbad, CA, US). Data were normalized to Rplp0 values ​​and analyzed as previously described (Pfaffl, M., Nucleic Acids Res. 2001;29(9):e45).

[0342] Hormone and metabolite assays Blood glucose levels were measured using a Glucometer Elite™ analyzer (Bayer, Leverkusen, Germany). Circulating FGF21 levels were determined using a quantitative sandwich enzyme immunoassay Mouse / Rat FGF-21 ELISA kit (MF2100, R&D systems, Abingdon, UK). Serum insulin concentrations were determined using a Rat Insulin ELISA sandwich assay (90010, Crystal Chem INC. Downers Grove, IL 60515, USA). To extract lipids from tissues, approximately 100 mg of frozen sample was weighed and homogenized in 15 ml of chloroform:methanol (2:1). The lipid and aqueous phases were then separated by adding 3 ml of 0.05% H2SO4, and the mixture was left overnight at 4°C. Once the phases were separated, the upper aqueous phase was removed using a Pasteur pipette, and 1 ml of the lower lipid phase was reconstituted in a glass tube. One ml of chloroform and a 1% solution of Triton X-100 were added to a glass tube, which was then incubated in a bath at 90°C to evaporate the chloroform. Any remaining aqueous particles were removed from the lipid phase using a mixture of chloroform and Triton X-100. After evaporation, the sample was concentrated by rinsing the walls of the tube with chloroform, and the tube was again heated at 90°C to evaporate the chloroform. Once the sediment was completely dry and concentrated, it was resuspended by adding 500 μl of H20 miliQ at 37°C. Finally, triglyceride content was determined using a commercially available GPO-PAP (Roche Diagnostics, Basel, Switzerland). Serum triglycerides and cholesterol were quantified spectrophotometrically using an enzymatic assay kit (Horiba-ABX, Montpellier, France). All biochemical parameters were determined using a Pentra 400 Analyzer (Horiba-ABX).

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

[0344] Insulin tolerance test For the insulin tolerance test, insulin (0.75 IU / kg body weight; Humulin Regular; Eli Lilly, Indianapolis, IN) was injected intraperitoneally into awake, fed mice. Glucose concentrations were determined in blood samples obtained from the tail vein at the indicated time points after insulin injection.

[0345] Glucose tolerance test Conscious mice were fasted overnight (16 hours) and administered glucose (2 g / kg body weight) intraperitoneally. Blood glucose was measured in tail vein blood samples at the indicated time points. Venous blood was collected from the tail vein into tubes (Microvette® CB 300, SARSTEDT) and immediately centrifuged to separate serum, which was used to measure insulin levels.

[0346] Oxygen measurement An indirect open-circuit calorimeter (Oxylet, Panlab, Cornella, Spain) was used to simultaneously monitor oxygen consumption and carbon dioxide production in eight metabolic chambers. Mice were individually housed and acclimated to the metabolic chambers for 24 hours, and data were collected for 3 minutes every 15 minutes in each cage for an additional 24 hours. Data were obtained over light and dark cycles and adjusted for body weight. Energy expenditure was calculated using the Metabolism software provided by the manufacturer.

[0347] Transfection of HEK293, C2C12 and HepG2 cells Cells were cultured in 24-well plates and transfected with 0.8 μg of DNA / well using Lipofectamine 2000 according to the manufacturer's instructions (Thermo Fisher Scientific).

[0348] bone analysis Bone volume and structure were assessed by μCT. Mouse tibias were fixed in neutral buffered formalin (10%) and scanned using an eXplore Locus CT scanner (General Electric) at a resolution of 27 microns. In four mice per group, 1 mm3 of the proximal tibial epiphysis and 1.8 mm3 of the cortical tibial diaphysis were analyzed for trabecular bone. Bone parameters were calculated using the MicroView 3D Image Viewer & Analysis Tool. Tibia length was measured from the intercondilar eminence to the medial malleolus.

[0349] Western blot analysis iWAT and iBAT were homogenized in QIAzol Lysis Reagent (Qiagen), and the protein fraction was isolated from the organic phase according to the manufacturer's instructions. Proteins were separated by 12% SDS-PAGE and analyzed by immunoblotting using rabbit polyclonal anti-UCP1 antibody (ab10983; Abcam) and rabbit polyclonal anti-α-tubulin antibody (ab4074; Abcam). Detection was performed using ECL Plus detection reagents (Amersham Biosciences).

[0350] Open field test The open field test was performed between 9:00 AM and 1:00 PM, as previously reported (Haurigot et al., 2013). Briefly, animals were placed in the center of a brightly lit chamber (41 × 41 × 30 cm) (LE 8811; Panlab) intersected by two bundles of light beams that detected horizontal and vertical movement. Locomotor activity and exploratory behavior were assessed during the first 6 min. Total distance traveled was assessed using a video tracking system (SMART Junior; Panlab).

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

[0352] Example Example 1. Prevention of obesity and diabetes by intra-eWAT administration of AAV-CAG-moFGF21-dmiRT vector in C57Bl6 mice We evaluated the therapeutic potential of AAV-mediated genetic engineering of adipose tissue with FGF21 to prevent obesity and diabetes in 8-week-old male C57B16 mice. 12Intraepididymal white adipose tissue (eWAT) administration of an AAV9 vector (AAV9-CAG-moFGF21-doublemiRT) encoding a codon-optimized murine FGF21 coding sequence under the control of a CAG ubiquitous promoter encompassing the miR122 and miR1 target sites (Figure 1A) mediated adipose-specific overexpression of FGF21 (Figure 1B) as well as high secretion of the protein into the bloodstream (Figure 1C). AAV9-CAG-moFGF21-doublemiRT-treated mice also showed overexpression of FGF21 receptor 1 (FGF21R1) in eWAT (Figure 1D) and β-Klotho (FGF21 coreceptor) in adipose tissue and liver (Figure 1E) compared with AAV9-CAG-null vector (a vector with comparable infectivity but no transgene encoding). The CAG-moFGF21-double miRT construct is contained in SEQ ID NO: 32, and the CAG-null construct is contained in SEQ ID NO: 31.

[0353] After AAV-mediated FGF21 gene transfer into eWAT, mice fed a chow diet exhibited weight loss (Figures 1F and 1G). When challenged with a high-fat diet (HFD), animals overexpressing FGF21 in adipose tissue remained lean throughout the experiment, whereas AAV9-CAG-null-treated mice became progressively obese (Figures 1F and 1G). In accordance with their lower body weight, both chow-fed and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice exhibited reduced fat depot and liver weights (Figure 1H).

[0354] Histological analysis of white adipose tissue by hematoxylin-eosin staining revealed a decrease in white adipocyte size in eWAT and iWAT (iWAT: inguinal white adipose tissue) and multiple multilocular adipocytes in iWAT, suggesting browning of this depot (Figure 2A). Morphometric analysis further confirmed a decrease in the average area of ​​white adipocytes in AAV9-CAG-moFGF21-doublemiRT-treated mice (Figure 2B). The frequency distribution of white adipocyte area also differed between groups. Chow-fed and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice presented with an increased number of small adipocytes and fewer large adipocytes (Figure 2C). Strikingly, the frequency distribution of white adipocyte area in HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice was nearly identical to that in chow-fed AAV9-CAG-null-treated animals (Figure 2C). Therefore, the HFD-induced hypertrophy of adipocytes observed in AAV9-CAG-null-treated mice was prevented in FGF21-overexpressing mice. Overexpression of UCP1 and Dio2 in iWAT (Figures 3A and 3B) further confirmed the browning of iWAT in chow-fed and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice.

[0355] Histological analysis of iBAT (interscapular brown adipose tissue) showed that lipid accumulation in this depot was lower in chow-fed AAV9-CAG-moFGF21-doublemiRT-treated mice and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice compared with AAV9-CAG-null mice (Figure 2A). In accordance with this result and the browning of iBAT, the energy expenditure of HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice during the light and dark cycles (Figure 3C) was higher than that of HFD-fed AAV9-CAG-null mice. Overall, these data suggest that AAV9-CAG-moFGF21-doublemiRT-treated mice had enhanced thermogenic activity.

[0356] Liver tissue sections showed reduced lipid accumulation in hepatocytes of mice overexpressing FGF21 compared with AAV9-CAG-null-treated mice, either under chow or HFD (Figure 2A). Accordingly, in HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice, their hepatic triglyceride (TG) content was normalized (Figure 3D). In parallel, circulating levels of TG, total cholesterol, HDL-cholesterol, and LDL-cholesterol were normalized in HFD-fed mice overexpressing FGF21 (Figures 3E and 3F).

[0357] HFD-fed mice overexpressing FGF21 were more insulin sensitive than HFD-fed AAV9-null-treated mice (Figure 3G), and both chow-fed AAV9-CAG-moFGF21-doublemiRT-treated mice and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice showed reduced circulating insulin levels compared with their AAV9-CAG-null-treated counterparts (Figure 3H).

[0358] Example 2. Reversal of obesity and improvement of glucose metabolism by intra-eWAT administration of AAV-CAG-moFGF21-dmiRT vector in ob / ob mice We evaluated the antidiabetic and antiobesogenic therapeutic potential of AAV-mediated genetic engineering of adipose tissue with FGF21 in 11-week-old male ob / ob mice, which are deficient in leptin signaling and are a widely used genetic model of obesity and diabetes. To this end, a dose-response study was performed. Ob / ob mice were administered four different doses (10 10 vg, 5×10 10 vg, 2 × 10 11 vg or 10 12 The AAV8-CAG-moFGF21-double miRT vector (vg) was administered topically into the eWAT of ob / ob animals as a control. 12 vg of AAV8-CAG-null vector was administered into eWAT.

[0359] Intraepithelial administration of the AAV8-CAG-moFGF21-doublemiRT vector mediated specific overexpression of FGF21 in white adipose tissue as well as high secretion of the protein into the bloodstream in a dose-dependent manner (Figures 4A and 4B). 12 The AAV8-CAG-moFGF21-double miRT vector at a dose of 10 vg mediated very strong overexpression of FGF21 in eWAT and iWAT (Figure 4A) and achieved the highest circulating FGF21 levels (Figure 4B). In contrast, the lowest administered dose, 10 10 The vg AAV8-CAG-moFGF21-double miRT vector produced only a very moderate overexpression of FGF21 in eWAT (Figure 4A), and animals treated with this dose showed no difference in serum FGF21 levels compared to AAV8-CAG-null-treated animals (Figure 4B), likely because FGF21 acted in a paracrine-autocrine manner. Accordingly, AAV8-CAG-null-treated animals progressively gained weight, whereas animals treated with the AAV8-CAG-moFGF21-double miRT vector showed a decrease in weight gain proportional to the administered dose of the vector (Figures 4C and 4D). Remarkably, 10 12Animals treated with 1000 mg of the AAV8-CAG-moFGF21-doublemiRT vector lost approximately 15% of their body weight during the first 2 weeks after AAV administration, and then regained weight until they reached their starting weight (Figures 4C and 4D). 12 Animals receiving vg of the AAV8-CAG-moFGF21-doublemiRT vector in eWAT showed a 40% difference in total body weight at the end of the experiment compared to AAV8-CAG-null treated animals (Figure 4D). 12 Animals treated with the vg AAV8-CAG-moFGF21-double miRT vector showed a significant reduction in steatosis and a 60% reduction in liver weight (Figure 4E). iBAT 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 Animals treated with vg of the AAV8-CAG-moFGF21-doublemiRT vector exhibited improved insulin sensitivity compared to AAV8-CAG-null treated mice (Figure 5A). 11 vg or 10 12 Animals treated with the vg AAV8-CAG-moFGF21-doublemiRT vector also exhibited lower circulating insulin levels than ob / ob mice treated with the AAV8-CAG-null vector (Fig. 5B ).

[0361] Example 3. Reversal of obesity and improvement of glucose metabolism by intravenous administration of AAV-hAAT-moFGF21 vector in ob / ob mice We also evaluated the antidiabetic and antiobesogenic effects mediated by increasing circulating levels of FGF21 using AAV-mediated genetic engineering of the liver in 8-week-old male ob / ob mice. 11 vg or 5×10 11An AAV8 vector encoding a codon-optimized murine FGF21 coding sequence under the control of the liver-specific human α1-antitrypsin (hAAT) promoter (AAV8-hAAT-moFGF21) (Figure 6A) was administered intravenously (IV) to ob / ob animals as controls. 11 vg of AAV8-hAAT-null vector was administered IV. The hAAT-moFGF21 construct is contained in SEQ ID NO: 34, and the hAAT-null construct is contained in SEQ ID NO: 33.

[0362] Intravenous administration of the AAV8-hAAT-moFGF21 vector mediated specific overexpression of FGF21 in the liver as well as high secretion of the protein into the bloodstream in a dose-dependent manner (Figures 6B and 6C). 11 A dose of 1000 mg of AAV8-hAAT-moFGF21 vector mediated very strong overexpression of FGF21 in the liver (Figure 6B) and achieved the highest circulating FGF21 levels (Figure 6C). The body weight of animals treated with this dose of AAV8-hAAT-moFGF21 vector decreased by approximately 7% during the first 2 weeks after AAV administration and then increased slightly, whereas AAV8-hAAT-null-treated mice progressively gained weight (Figures 6D, 6E, and 6F). 11 Mice administered with 10 vg of the AAV8-hAAT-moFGF21 vector gained significantly less weight than AAV8-hAAT-null treated animals (Figures 6D, 6E, and 6F). 11At the end of the experiment, animals treated with the AAV8-hAAT-moFGF21 vector showed a 50% weight gain compared to a 10% weight gain in animals treated with the AAV8-hAAT-moFGF21 vector (Figure 6E). In accordance with their lower body weights, animals overexpressing FGF21 in the liver, particularly those treated with the highest dose of vector, 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 (Figure 6G), likely due to higher thermogenic activity in these animals compared to mice administered the AAV8-hAAT-null vector.

[0363] Animals treated with the AAV8-hAAT-moFGF21 vector showed improved insulin sensitivity and reduced circulating insulin levels compared to AAV8-hAAT-null treated mice (Figures 6H and 6I).

[0364] Example 4. Long-term reversal of obesity and diabetes by intravenous administration of AAV-hAAT-moFGF21 vector in HFD-fed mice We also evaluated the antidiabetic and antiobesogenic effects mediated by increasing circulating levels of FGF21 using AAV-mediated genetic engineering of the liver in obese C57B16 mice. Nine-week-old male C57B16 mice (young adults) were fed a HFD for 9 weeks, then 10 days after birth. 10 vg or 5×10 10 vg of the AAV8-hAAT-moFGF21 vector was administered intravenously (Figure 6A). After AAV administration, AAV8-hAAT-moFGF21-treated mice were maintained on a HFD for 52 weeks. As a control, 5 × 10 10 vg of AAV8-hAAT-null was administered IV to chow-fed and HFD-fed C57Bl6 mice, and these latter two mouse cohorts were subsequently maintained on either a chow diet or an HFD.

[0365] Intravenous administration of the 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 No difference in body weight was observed between HFD-fed animals administered 5 × 10 vg of the AAV8-hAAT-moFGF21 vector and those administered 5 × 10 vg of the AAV8-hAAT-moFGF21 vector (Figures 7B and 7C). 10 HFD-fed animals treated with vg of the AAV8-hAAT-moFGF21 vector initially lost 20% of their body weight after AAV administration, then progressively gained weight similar to chow-fed AAV8-hAAT-null-treated mice (Figures 7B and 7C). Strikingly, from week 9 onwards after AAV administration, 5 × 10 10 No statistically significant differences in total body weight and weight gain were observed between HFD-fed animals administered vg of the AAV8-hAAT-moFGF21 vector and chow-fed AAV8-hAAT-null-treated mice (Figures 7B and 7C).

[0367] 5×10 10 The energy expenditure during the light and dark cycles of HFD-fed mice treated with vg of the AAV8-hAAT-moFGF21 vector was higher than that of chow-fed AAV8-hAAT-null mice and HFD-fed AAV8-hAAT-null mice (Figure 8A). 10 No difference in energy expenditure was observed between mice administered 5 × 10 vg of the AAV8-hAAT-moFGF21 vector (Figure 8A). Overall, these data suggest that 10 This suggests that mice treated with vg AAV8-hAAT-moFGF21 had improved thermogenic activity.

[0368] 10 10Animals treated with 5 × 10 AAV8-hAAT-moFGF21 vectors exhibited improved insulin sensitivity compared with HFD-fed mice administered with the AAV8-hAAT-null vector, and their insulin sensitivity was similar to that of chow-fed mice treated with the AAV8-hAAT-null vector (Figure 8B). 10 Animals administered the AAV8-hAAT-moFGF21 vector exhibited improved insulin sensitivity and normalized circulating insulin levels compared to chow-fed mice administered the AAV8-hAAT-null vector (Figures 8B and 8C).

[0369] Example 5. Reversal of obesity and diabetes by intravenous administration of AAV-hAAT-moFGF21 vector in aged HFD-fed mice We also evaluated the antidiabetic and antiobesogenic effects of FGF21 in obese aged (adult) C57B16 mice. Male C57B16 mice aged 7.5 months were fed a HFD for 8 weeks, then 10 days after birth. 10 vg, 2 × 10 10 vg or 5×10 10 vg of the AAV8-hAAT-moFGF21 vector was administered intravenously (Figure 6A). After AAV administration, AAV8-hAAT-moFGF21-treated mice were maintained on a HFD for 22 weeks. As a control, 5 × 10 10 vg of AAV8-hAAT-null was administered intravenously to chow-fed and HFD-fed aged C57Bl6 mice, and these latter two mouse cohorts were subsequently maintained on either a chow diet or an HFD.

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

[0371] HFD-fed AAV8-null treated mice and 10 10No difference in body weight was observed between HFD-fed animals administered 2 × 10 vg of the AAV8-hAAT-moFGF21 vector and those administered 2 × 10 vg of the AAV8-hAAT-moFGF21 vector (Figures 9B and 9C). 10 vg or 5×10 10 HFD-fed animals treated with either AAV8-hAAT-moFGF21 vector at 2 × 10 mg / kg / day initially lost 15% and 20% of their body weight, respectively, after AAV administration (Figures 9B and 9C). 10 vg AAV8-hAAT-moFGF21 vector-treated animals progressively gained weight similar to chow-fed AAV8-hAAT-null-treated mice, but by 5 × 10 10 No significant changes in body weight were observed in animals treated with 5×10 vg of the AAV8-hAAT-moFGF21 vector (Figures 9B and 9C). Remarkably, from 3 weeks after AAV administration, 5×10 10 No statistically significant differences in total body weight and weight gain were observed between HFD-fed animals administered vg of the AAV8-hAAT-moFGF21 vector and chow-fed AAV8-hAAT-null-treated mice (Figures 9B and 9C).

[0372] 5×10 10 The energy expenditure during the light and dark cycles of HFD-fed mice treated with vg of the AAV8-hAAT-moFGF21 vector was higher than that of chow-fed and HFD-fed AAV8-hAAT-null mice (Figure 10A). 10 Animals treated with the AAV8-hAAT-moFGF21 vector showed increased energy expenditure during the light cycle and a trend toward increased energy expenditure during the dark cycle (Figure 10A). 10 Animals treated with 10 vg of the AAV8-hAAT-moFGF21 vector showed increased energy expenditure during the dark cycle (Figure 10A). 10No difference was observed between mice administered either the vg or 100 AAV8-hAAT-moFGF21 vectors (Figure 10A). Overall, these data suggest that aged mice treated with AAV8-hAAT-moFGF21 had enhanced thermogenic activity.

[0373] 10 10 vg or 2×10 10 Animals treated with 5 × 10 AAV8-hAAT-moFGF21 vectors exhibited improved insulin sensitivity compared with HFD-fed mice administered with the AAV8-hAAT-null vector, and their insulin sensitivity was similar to that of chow-fed mice treated with the AAV8-hAAT-null vector (Fig. 10B). 10 Animals administered with the AAV8-hAAT-moFGF21 vector showed improved insulin sensitivity compared to chow-fed mice administered with the AAV8-hAAT-null vector (Figure 10A). 10 vg, 2 × 10 10 vg or 5×10 10 Animals treated with vg of the AAV8-hAAT-moFGF21 vector exhibited lower fasting and fed circulating insulin levels than HFD-fed AAV8-hAAT-null-treated mice (Figure 10C). 10 vg or 5×10 10 No difference in circulating fed insulin levels was observed between old animals administered IV with vg of the AAV8-hAAT-moFGF21 vector and chow-fed AAV8-hAAT-null treated mice (Fig. 10C).

[0374] Example 6. Evaluation of weight loss due to intramuscular administration of AAV-CMV-moFGF21 vector in C57B16 mice We also evaluated the therapeutic potential of increasing circulating FGF21 levels through AAV-mediated genetic engineering of skeletal muscle in C57B16 mice. To target skeletal muscle, we chose the CMV promoter and AAV1 serotype. While the CMV promoter is a ubiquitous promoter, its use with the AAV1 capsid allows for highly efficient targeting of skeletal muscle without transducing the liver, as previously reported (Mas et al., Diabetes 2006; Callejas et al., Diabetes 2013).

[0375] 3×10 11 A dose of 5 × 10 vg of an AAV1 vector encoding a codon-optimized murine FGF21 coding sequence under the control of the ubiquitous CMV promoter (AAV1-CMV-moFGF21) (Figure 11A) was injected intramuscularly (IM) into the quadriceps, gastrocnemius, and tibialis cranealis muscles of each hind limb of 6- to 12-week-old male C57BL6 mice. 10 As a control, age-matched C57B16 animals were administered 3 × 10 11 vg of AAV1-CMV-null vector (5 × 10 10 The CMV-moFGF21 construct is contained in SEQ ID NO: 36, and the CMV-null construct is contained in SEQ ID NO: 35.

[0376] Intramuscular administration of the AAV1-CMV-moFGF21 vector mediated high secretion of FGF21 into the bloodstream (Fig. 11B). Animals treated with the AAV1-CMV-moFGF21 vector showed reduced body weight and weight gain compared with AAV1-CMV-null-treated mice (Figs. 11C and 11D).

[0377] Example 7. Increased protein production with codon-optimized human FGF21 nucleotide sequences To evaluate whether codon optimization can mediate increased FGF21 protein production, HEK293 cells were transfected with plasmids encoding three different codon-optimized human FGF21 nucleotide sequences (SEQ ID NOs: 40-42). Non-transfected cells and cells transduced with the wild-type hFGF21 coding sequence served as controls. Expression of the three codon-optimized human FGF21 sequences and the wild-type human FGF21 sequence was under the control of the hAAT promoter (SEQ ID NO: 47). Cells transduced with codon-optimized human FGF21 version 1 or 3 were able to secrete higher levels of human FGF21 into the culture medium compared to wild-type or codon-optimized FGF21 variant 2 (Figure 12), thus demonstrating increased FGF21 protein production by codon optimization of variants 1 and 3.

[0378] Example 8. Reversal of obesity and diabetes in mice by administration of an AAV vector encoding human FGF21 (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 an AAV-null vector was administered to chow-fed and HFD-fed mice.

[0379] To evaluate the ability of human FGF21 to induce browning of WAT and thermogenic activity of BAT, increase energy expenditure, and improve glucose and energy metabolism, the following studies are performed: - 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 - Determination of serum metabolic parameters, such as free fatty acids, glycerol, triglycerides, cholesterol and ketone bodies - Examination of the presence of beige adipocytes in the inguinal fat pad by immunohistochemistry and assessment of browning capacity by gene expression of classical white, brown and beige adipocyte markers Example 9: In vitro assay to assess FGF21 activity FGF21 is expected to increase glucose uptake and GLUT1 expression in 3T3-L1 cells (Kharitonenkov, A. et al., 2005. J. Clin. Invest. 115 :1627-1635).

[0381] Example 10. Reversal of obesity and improvement of glucose metabolism by intra-eWAT administration of AAV8-CAG-moFGF21-dmiRT vector in ob / ob mice: Further findings We further evaluated the antidiabetic and antiobesogenic therapeutic potential of AAV-mediated genetic engineering of adipose tissue with FGF21 in ob / ob mice (see Example 2).

[0382] Ob / ob mice receiving intraeWAT injections of the AAV8-CAG-moFGF21-dmirT vector showed reduced white adipocyte size in the epididymal pad (Figure 13A). Circulating adiponectin levels also increased with dose (Figure 14A). Inflammation in eWAT, as assessed by Mac2 staining, also decreased as a function of vector dose, as did expression of the macrophage marker F4 / 80 (Figures 14B and C).

[0383] Livers from ob / ob mice injected with the null vector or the lowest dose of AAV8-CAG-moFGF21-dmirT showed accumulation of lipid droplets in hepatocytes (Figure 13B). 10 Administration of a vector encoding FGF21 at 5×10 vg / mouse or higher doses completely prevented the development of fatty liver (FIG. 13B), which correlated with the organ weight (FIG. 14D) and its total triglyceride and cholesterol content (FIGS. 14E and F). 10 Further evidence that the 1 × 10 vg / mouse dose represents a threshold for therapeutic efficacy came from analyses of blood glucose and blood insulin. 10 vg / mouse dose did not modify blood glucose levels in the fed state and only partially reduced insulin levels, whereas 5 × 10 10 Doses of 1000 mg / mouse or higher completely normalized blood glucose and blood insulin (FIGS. 13C and D). Overall, this example confirms the therapeutic potential of overexpressing FGF21 in adipose tissue.

[0384] Example 11. Reversal of obesity and improvement of glucose metabolism by intravenous administration of AAV8-hAAT-moFGF21 vector in ob / ob mice: Further findings We further evaluated the antidiabetic and antiobesogenic therapeutic potential of intravenous administration of the AAV8-hAAT-moFGF21 vector in ob / ob mice (see Example 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 reduction in the size of white adipocytes (Fig. 15A). This was paralleled by a dose-dependent increase in circulating adiponectin levels (Fig. 15B) and a reduction in WAT inflammation, as evidenced by decreased Mac2 staining and expression of F4 / 80 and TNF-α in eWAT (Fig. 16A-C). Remarkably, 5 × 10 11 vg-treated ob / ob mice showed a significant reduction in "crown-like" structures in eWAT (FIG. 16A).

[0386] Although 7-month-old ob / ob mice exhibited significant hepatic steatosis, the livers of FGF21-treated ob / ob mice showed no lipid accumulation in hepatocytes (Fig. 15C). This was consistent with a 60% reduction in organ weight in ob / ob mice receiving the therapeutic vector (Fig. 16D and E), as well as a significant reduction in total liver triglyceride and cholesterol content (Fig. 16F and G). ob / ob animals treated with both doses of AAV8-hAAT-moFGF21 also showed reduced fed blood glucose, and their blood insulin in the fed state was reduced by approximately 70% (Fig. 15D and E).

[0387] We evaluated whether the decrease in circulating glucose levels observed in ob / ob mice after AAV8-hAAT-moFGF21 treatment was due to suppression of hepatic gluconeogenesis by measuring the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) by qPCR. A 1 × 10 increase in PEPCK expression was observed. 11 Except for animals treated with vg of AAV8-hAAT-moFGF21, no changes in the expression of these enzymes were observed in the livers of AAV8-hAAT-moFGF21-treated ob / ob mice (Figures 17A and 17B). These results suggest that AAV-mediated long-term expression of FGF21 in the liver and the subsequent increase in circulating FGF21 did not lower glucose by inhibiting hepatic glucose production.

[0388] The glucose-lowering effect of FGF21 is also attributed to increased glucose uptake and enhanced energy expenditure by adipocytes (Xu J. et al., 2009. AJP Endocrinol. Metab. 297 :E1105-E1114;Ding X. et al.,2012. Cell Metab. 16 :387-393;Camacho RC et al.,2013. Eur.J.Pharmacol. 715:41-45;Emanuelli B. et al.,2014. Clin.Invest. 124 :515-527;Kharitonenkov A. et al.,2005. Endocrinology 148 :774-781;Hondares E. et al.,2010. Cell Metab. 11 :206-212;Samms RJ et al.,2015. Cell Rep. 11 :991-999). Therefore, we assessed the expression of key components of the glucose uptake machinery, such as the glucose transporters Glut1 and Glut4, the glucose phosphorylating enzymes hexokinase I and II (HKI and HKI), and UCP1 in iBAT, in the adipose tissues of different pads (iWAT, eWAT, and iBAT) by qPCR. In AAV8-FGF21-treated ob / ob mice, the expression of Glut1 increased in iWAT and iBAT (Fig. 17C), and the expression of Glut4 increased in eWAT, iWAT, and iBAT (Fig. 17D). HKI and HKII were upregulated only in iBAT (Fig. 17E and F). Moreover, UCP1 expression increased in iBAT of ob / ob mice treated with a high dose of the AAV8-hAAT-moFGF21 vector (Fig. 17G). Overall, these results suggest that the long-term glycemic improvement observed in ob / ob mice after treatment with the AAV8-hAAT-moFGF21 vector is likely due to increased glucose uptake by white and brown adipocytes and enhanced thermogenesis in iBAT.

[0389] Example 12. Long-term reversal of obesity and diabetes by intravenous administration of AAV8-hAAT-moFGF21 vector in HFD-fed and HFD-fed aged mice: reduced tissue weight and stable expression for up to 1 year Representative images of animals from all experimental groups from studies conducted in young adults or adults (see Examples 4 and 5) are shown in Figures 19A-B.

[0390] Reversal of obesity by AAV8-hAAT-moFGF21 treatment was paralleled by a dose-dependent reduction in the weight of major white adipose tissue (WAT) depots, including the epididymal (eWAT), inguinal (iWAT), and retroperitoneal (rWAT) fat pads, in both young and adult animals (Figures 18A and 19C). HFD-induced increases in liver weight were completely normalized by FGF21 gene transfer with the highest dose of vector used, whereas quadriceps muscle weight was unchanged by diet or AAV delivery (Figures 18A and 19D).

[0391] AAV8-hAAT-moFGF21-treated mice of both ages showed specific overexpression of codon-optimized FGF21 in the liver (Figure 19E), which led to dose-dependent secretion of FGF21 into the bloodstream in both groups of mice at levels that remained stable for up to 1 year after a single administration of the vector (Figure 18B).

[0392] Example 13. Long-term recovery from obesity and diabetes by intravenous administration of AAV8-hAAT-moFGF21 vector in HFD-fed and HFD-fed aged mice: Improvement of locomotor activity and investigation of the mechanism of thermogenic activity Increased energy expenditure (see Examples 4 and 5) was also seen in animals treated as young adults 10 months after AAV8-hAAT-moFGF21 delivery (FIG. 21A).

[0393] This finding was consistent with the AAV8-hAAT-moFGF21-mediated effect on locomotor activity. In contrast to the reduced activity observed during the open field test in HFD-fed animals receiving the AAV8-null vector, the 5 × 10 10Mice treated with vg AAV8-hAAT-moFGF21 exhibited spontaneous locomotor activity comparable to that of chow-fed, null-injected animals. As shown in Figure 20A, one year after AAV8-hAAT-moFGF21 delivery, treated animals traveled greater distances, rested less, and spent more time engaged in slow and fast movements than untreated, HFD-fed controls.

[0394] Considering that changes in energy expenditure may reflect changes in thermogenesis, we assessed the degree of brown adipose tissue (BAT) activation. 10 Both mice treated with vg AAV8-hAAT-moFGF21 showed reduced lipid deposition in iBAT (Fig. 20B). UCP1 protein content in BAT was increased dose-dependently in mice treated with the AAV8-hAAT-moFGF21 vector as young adults (Fig. 20C), consistent with increased non-shivering thermogenesis induced by FGF21 gene transfer to the liver.

[0395] Browning of subcutaneous WAT is characterized by the appearance of beige adipocytes and is associated with increased energy expenditure (Harms & Seale, 2013). To assess whether browning could explain the enhanced energy expenditure observed after AAV8-hAAT-moFGF21 treatment, we performed histological evaluation of iWAT. Consistent with the weight loss of this pad (Figure 18A), adipocytes in HFD-fed AAV8-hAAT-moFGF21-treated animals were smaller than those in HFD-fed null-injected animals (Figure 20D). Nevertheless, treatment with the AAV8-hAAT-moFGF21 vector did not result in increased detection of multilocular beige adipocytes in iWAT at any tested dose, either in animals treated as young adults or adults (Figure 20D). Thus, there was no statistically significant difference in UCP1 protein levels in iWAT between HFD-fed groups (Figure 21B).

[0396] Creatinine-driven substrate cycling and sarcoplasmic reticulum Ca2+-ATPase 2b (Serca2b)-mediated calcium cycling can increase thermogenesis in iWAT independently of UCP1 (Kazak L. et al., 2015. Cell 163 :643-655;Ikeda K. et al.,2017. Nat.Med. 23 :1454-1465). Higher levels of expression of phosphatase orphan 1 (Phospho1), an enzyme involved in the creatinine-driven substrate cycle, were observed in 5 × 10 mice compared with age-matched chow-fed and HFD-fed controls. 10 vg of creatinine-driven thermogenic cycle activity was observed in the iWAT of HFD-fed mice treated with AAV8-hAAT-moFGF21 (Figure 20E), suggesting that the activity of the creatinine-driven thermogenic cycle was likely increased as a result of FGF21 gene transfer. Regarding calcium cycling-dependent thermogenic mechanisms, no difference in Serca2b expression levels was detected in the iWAT of animals treated with the AAV8-hAAT-moFGF21 vector compared with chow-fed or HFD-fed null-treated animals (Figure 21C). On the other hand, iWAT expression of ryanodine receptor 2 (RyR2), another enzyme involved in the same cycle, was increased by HFD feeding in both null-treated and AAV8-hAAT-moFGF21-treated mice (Figure 21C). Overall, these results suggest that calcium cycling-dependent thermogenic mechanisms are not involved in the improvement in whole-body energy homeostasis observed after AAV-FGF21 treatment.

[0397] Example 14. Long-term reversal of obesity and diabetes by intravenous administration of AAV8-hAAT-moFGF21 vector in HFD-fed and HFD-fed aged mice: glucagon levels, islet hyperplasia, and glucose tolerance Moreover, HFD-fed animals treated with the AAV8-hAAT-moFGF21 vector as young adults showed decreased circulating levels of glucagon compared to HFD-fed null-treated mice (FIG. 22A).

[0398] AAV8-null treated mice developed islet hyperplasia as a result of HFD feeding, whereas AAV8-hAAT-FGF21 vector (2 × 10 10 or 5×10 10 The β-cell mass of animals treated with HFD-fed AAV8-hAAT-moFGF21 (at a dose of 10 ...

[0399] To assess glucose tolerance in FGF21-treated mice, an intraperitoneal glucose tolerance test (GTT) (2 g glucose / kg body weight) was performed 10 weeks after AAV administration. Either null or FGF21-encoding vectors were administered at 1 × 10 10 HFD-fed animals injected with a dose of 5×10 vg / mouse were glucose intolerant and showed a significant increase in circulating insulin levels during the GTT (FIGS. 23A and B). In contrast, 5×10 10 Animals treated with AAV8-hAAT-moFGF21 in vg / mouse showed improved glucose clearance when compared to chow-fed control mice (Figure 23A). Insulin levels were indistinguishable between the two experimental groups (Figure 23B). These results are consistent with a 5x10 10 We further confirmed the improvement in insulin sensitivity in HFD-fed mice treated with AAV8-hAAT-moFGF21 in vg / mouse.

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

[0401] Obesity also induces inflammation in WAT (Hajer GR et al., 2008. Eur. Heart J. 29 Therefore, we analyzed inflammation in this tissue through immunostaining for the macrophage-specific marker Mac2 and the expression of pro-inflammatory molecules. HFD-fed mice showed an increased presence of macrophages, represented as "crown-like" structures, in eWAT, whereas young adult or adult mice had 5 × 10 10 Animals treated with vg AAV8-hAAT-moFGF21 had no signs of macrophage infiltration (Figures 24E and 25B), which paralleled normalized expression of macrophage markers F480 and CD68 and the pro-inflammatory cytokines TNFα and IL-1β (Figures 24F-H and 25C-E), indicating that FGF21 expression counteracted obesity-associated WAT inflammation.

[0402] Example 16. Amelioration of fatty liver, liver inflammation, and fibrosis by intravenous administration of AAV8-hAAT-moFGF21 vector Liver histology analysis showed that all null-treated animals fed a HFD had significant hepatic steatosis at the time of sacrifice (Figures 26A-D). In contrast, 5x10 young adults or adults 10 HFD-fed mice receiving vg AAV8-hAAT-moFGF21 demonstrated reversal of this pathological lipid deposition (Figure 26A). These histological findings were consistent with a 5x10 10 This was paralleled by a significant reduction in total liver triglyceride and cholesterol content in vg AAV8-hAAT-moFGF21-treated animals (Figures 26B and C). In addition, 5x10 10 Animals treated with the vg AAV8-hAAT-moFGF21 vector showed no signs of liver inflammation, as evidenced by the lack of staining for Mac2, which revealed increased presence of macrophages in the livers of null-treated, HFD-fed mice (Figure 26D). Finally, hepatic FGF21 gene transfer reversed liver fibrosis. Collagen fibers were readily detectable after picrosirius red or Masson's trichrome staining of liver sections from HFD-fed animals injected with the control null vector, whereas they were undetectable in the livers of AAV8-hAAT-moFGF21-treated mice (Figures 28A and 27). These mice also showed a significant reduction in hepatic collagen 1 expression (Figures 28B and 28C). Overall, these findings indicated that AAV8-hAAT-moFGF21 treatment protects against the development of HFD-induced nonalcoholic steatohepatitis (NASH).

[0403] Example 17. Long-term safety of liver-directed AAV-FGF21 treatment Pharmacological treatment or transgenic overexpression of FGF21 is associated with the disruption of bone homeostasis through enhanced bone resorption, which can lead to bone loss (Wei W. et 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; Kim AM et al., 2017. Diabetes, Obes. Metab. Given the therapeutic potential of AAV8-hAAT-moFGF21 for the treatment of obesity and diabetes, we evaluated the long-term effects of gene transfer on bone in animals treated with the highest dose of vector. At the time of sacrifice (approximately 16.5 months of age), snout-to-tail length and tibia length were normal in animals administered the AAV8-hAAT-moFGF21 vector at 9 or 29 weeks of age (Figures 29A and B). We then examined bone structure by micro-computed tomography (μCT). Analysis of the proximal tibia epiphysis was performed in 5 × 10 mice fed an HFD. 10 We found no significant differences in trabecular and cortical bone in mice administered vg AAV8-hAAT-moFGF21 compared with age-matched mice treated with the null vector. Specifically, no differences were noted in bone mineral density (BMD) (Figure 29C), bone mineral content (BMC) (Figure 29D), bone volume (BV) (Figure 29E), bone volume / tissue volume ratio (BV / TV) (Figure 29F), bone surface / bone volume ratio (BS / BV) (Figure 29G), trabecular number (Tb.N) (Figure 29H), trabecular width (Tb.Th) (Figure 29I), or trabecular spacing (Tb.Sp) (Figure 29J). Similarly, analysis of compact bone in the tibial diaphysis showed no differences in BMC, BMD, BV, BV / TV, or BS / BV between HFD-fed, null-injected, or FGF21-treated groups (Figures 29K-O).

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

[0405] Example 18. Prevention of HFD-induced liver tumors by intravenous administration of AAV8-hAAT-moFGF21 vector Long-term HFD feeding (>60 weeks) is associated with increased incidence of liver neoplasia in C57BL / 6J mice (Hill-Baskin AE et al., 2009. Hum. Mol. Genet. 18 :2975-2988;Nakagawa H.,2015. World J.Hepatol. 7 In our study, in animals that started on a HFD as young adults and maintained it for 60 weeks, we found liver tumors in 66.7% (6 / 9) of animals injected with the null vector. Animals treated with the AAV8-hAAT-moFGF21 vector were protected from HFD-induced liver neoplasia development: 5 × 10 10 0% (0 / 8) of animals treated with the vector encoding FGF21 at the lowest dose (1 × 10 10 The incidence in the cohort treated with IFN-glucose (vg) was 40% (4 / 10). None of the chow-fed mice (0 / 11) 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 reverses STZ-induced hyperglycemia material and method animal We used 9-week-old male C57bl6 mice. Mice were fed a standard diet ad libitum and maintained under a 12-h light-dark cycle (lights on at 08:00). To induce diabetes, mice received five daily intraperitoneal injections of streptozotocin (50 mg / kg) dissolved in 0.1 mol / L citrate buffer (pH 4.5). Blood glucose levels were assessed using a Glucometer Elite analyzer (Bayer, Leverkusen, Germany). Animal care and experimental procedures were approved by the Ethics Committee for Animal and Human Experimentation of the Universitat Autónoma de Barcelona.

[0408] In vivo administration of AAV vectors For systemic administration, AAV vectors were diluted in 200 μl of 0.001% F68 Pluronic® (Gibco) in PBS 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 An AAV8 vector encoding codon-optimized murine FGF21 under the control of the hAAT promoter (AAV8-hAAT-moFGF21) was administered intravenously to male 9-week-old C57B16 mice. Control mice received 2 × 10 11 Two weeks after AAV administration, all animals were treated with streptozotocin (STZ) (5 doses of 50 mg / kg; 1 dose per day) to induce the diabetic process.

[0410] Analysis of blood glucose levels revealed that animals treated with the AAV8 vector encoding moFGF21 exhibited lower circulating glucose levels than C57B16 mice treated with the AAV8-hAAT-Null vector (Figure 30).

[0411] Example 20. Intramuscular administration of AAV-CMV-moFGF21 vector extends healthy lifespan by preventing age-related weight gain and insulin resistance in C57B16 mice. Skeletal muscle (Skm) is an easily accessible tissue and has been used for the production of secretable therapeutic proteins (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). To investigate whether Skm can serve as a viable source of circulating FGF21, we used an AAV vector of serotype 1 carrying optimized mouse FGF21 under the control of a CMV promoter, which has high tropism for Skm (Chao L. et al., 2000. J. Clin. Invest. 106 :1221-1228;Wu Z. et al.,2006. J. Virol. 80 :9093-9103;Lisowski L. et al.,2015. Curr.Opin.Pharmacol. 24 :59-67) was used (AAV1-CMV-moFGF21). The vector was 5 × 10 10 vg / muscle into the quadriceps, gastrocnemius, and tibialis anterior muscles of both limbs of 8-week-old C57Bl6 mice (total dose, 3 × 10 11 vg / mouse). Control animals were injected with the AAV1-CMV-Null vector at the same dose. The use of healthy mice fed a standard diet further enabled us to evaluate the long-term safety of FGF21 gene therapy.

[0412] Eleven-month-old animals injected at 8 weeks of age with a vector encoding FGF21 showed a significant increase in circulating FGF21 (Fig. 31A), which paralleled high expression levels of vector-derived FGF21 in the three injected muscles (Fig. 31B). Consistent with previous reports, this combination of vector serotype, promoter, and administration route did not lead to transgene expression in the liver (Fig. 31B).

[0413] At the end of the approximately 10-month follow-up period, mice intramuscularly injected with AAV1-CMV-moFGF21 maintained their baseline weight and were approximately 38% leaner than controls, whose weight steadily increased as the animals aged (Figure 31C). While muscle weight was largely unaffected by FGF21 gene transfer, the weights of the white and brown depots, as well as the liver, were significantly reduced (Figure 31D). Indeed, the weight of the analyzed WAT pad was reduced by >50% (Figure 31D). Furthermore, mice treated with AAV1-CMV-moFGF21 exhibited a significant reduction in liver total triglyceride content (Figure 31E). No changes were observed in liver cholesterol levels (Figure 31F). In contrast to null-injected animals, animals treated with AAV1-CMV-moFGF21 exhibited normoglycemia (data not shown) and reduced blood insulin at approximately 1 year of age (Figure 31G). Accordingly, FGF21-treated mice showed significantly improved insulin sensitivity at the end of the study (Figure 31H). Overall, this study demonstrates that administration of AAV vectors that deliver therapeutically relevant levels of circulating FGF21 is safe for long-term health and can be used to reverse age-related increases in body weight and insulin resistance.

[0414] Example 21. Reversal of obesity and diabetes by intramuscular administration of AAV1-CMV-moFGF21 vector in HFD-fed C57B16 mice.

[0415] We next evaluated whether im administration of the AAV1-CMV-moFGF21 vector could also reverse obesity and insulin resistance. To this end, 2-month-old C57B16 mice were fed either chow or HFD for 12 weeks. During these first 3 months of follow-up, chow-fed animals gained 20% weight, while HFD-fed animals became obese (95% weight gain) (Figures 32A and B). The vector was then administered intravenously at 5x10 10 vg / muscle into the quadriceps, gastrocnemius, and tibialis anterior muscles of both limbs of obese C57Bl6 mice (total dose, 3 × 10 11 As controls, a separate cohort of obese mice and a cohort of chow-fed mice were administered at 3 × 10 11 The mice received a non-coding null vector (AAV1-CMV-null) of moFGF21. After AAV delivery, mice were maintained on chow or HFD. Animals treated with AAV1-CMV-moFGF21 experienced progressive weight loss (Figures 32A and B). The reversal of obesity by AAV1-CMV-FGF21 treatment was paralleled by an increase in circulating levels of FGF21 (Figure 32C).

[0416] Null-treated mice fed a HFD exhibited normal fed blood glucose (Fig. 32D) but were hyperinsulinemic (Fig. 32E), suggesting that these mice had developed insulin resistance. In contrast, HFD-fed mice treated with AAV1-CMV-moFGF21 were normoglycemic and normoinsulinic by the end of the study (Fig. 32D and E). Moreover, animals administered AAV1-CMV-moFGF21 exhibited greater insulin sensitivity than their HFD-fed counterparts (Fig. 32F).

[0417] Example 22. Increasing circulating levels of FGF21 with codon-optimized human FGF21 nucleotide sequences.

[0418] To assess whether codon optimization can mediate increased circulating levels of FGF21, 8-week-old male C57B16 mice were hydrodynamically injected with plasmids encoding three different codon-optimized human FGF21 nucleotide sequences (SEQ ID NOS: 40-42) under the control of the hAAT promoter. Untreated mice and mice hydrodynamically injected with a plasmid encoding the wild-type hFGF21 coding sequence under the control of the hAAT promoter served as controls.

[0419] material and method In vivo delivery of plasmids into mice by hydrodynamic tail vein injection Plasmid DNA was diluted in saline to a volume (ml) equivalent to approximately 10% of the animal's average body weight (grams) and manually injected into the lateral tail vein in less than 5 seconds. Prior to injection, animals were placed under a 250W infrared heat lamp (Philips) for several minutes to dilate the blood vessels and allow for easier visualization and access of the tail vein. Animals were immobilized for injection using a plastic restrainer (Harvard Apparatus). No anesthesia was used, as it was not necessary as long as appropriate restraining devices were utilized. We injected animals using a 26G 3 / 8-inch gauge hypodermic needle (BD), the largest feasible needle gauge that fit snugly into the vein to be accessed.

[0420] result Mice treated with either codon-optimized human FGF21 version 2 or 3 were able to secrete higher levels of human FGF21 into the circulation compared to wild-type or codon-optimized FGF21 variant 1 (Figure 33), thus demonstrating increased FGF21 protein production by codon-optimizing variants 2 and 3.

[0421] Example 23. Increased FGF21 expression and protein production levels in vitro and in vivo by hAAT-moFGF21, CAG-moFGF21-double miRT, and CMV-moFGF21 expression cassettes material and method In vivo delivery of plasmids into mice by hydrodynamic tail vein injection Plasmid DNA was diluted in saline to a volume (ml) equivalent to approximately 10% of the animal's average body weight (grams) and manually injected into the lateral tail vein in less than 5 seconds. Prior to injection, animals were placed under a 250W infrared heat lamp (Philips) for several minutes to dilate the blood vessels and allow for easier visualization and access of the tail vein. Animals were immobilized for injection using a plastic restrainer (Harvard Apparatus). No anesthesia was used, as it was not necessary as long as appropriate restraining devices were utilized. We injected animals using a 26G 3 / 8-inch gauge hypodermic needle (BD), the largest feasible needle gauge that fit snugly into the vein to be accessed.

[0422] result In vitro HEK293 cells were transfected with plasmids encoding the wild-type mouse FGF21 coding sequence (EF1a-mFGF21) under the control of the elongation factor 1a (EF1a) promoter (Zhang et al., EBioMedicine 15 (2017) 173-183) (SEQ ID NO: 57), 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-doublemiRT). Non-transfected cells served as controls. HEK293 cells transduced with CAG-moFGF21-double miRT expressed higher levels of FGF21 compared with cells transduced with EF1a-mFGF21 or non-transduced cells (Figure 34A). Moreover, HEK293 cells transduced with CAG-moFGF21-double miRT also showed higher intracellular FGF21 protein content and higher FGF21 protein levels in the culture medium (Figures 34B and C). HEK293 cells transduced with EF1a-mFGF21 or CMV-moFGF21 expressed similar levels of FGF21 (Figure 34A), whereas HEK293 cells transduced with CMV-moFGF21 showed higher intracellular FGF21 protein content and higher FGF21 protein levels in the culture medium (Figures 34B and C).

[0423] C2C12 cells were transfected with plasmids encoding either the wild-type mouse FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) (Zhang et al., EBioMedicine 15 (2017) 173-183) or a codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21). Non-transfected cells served as controls. C2C12 cells transduced with CMV-moFGF21 expressed higher levels of FGF21 compared with cells transduced with EF1a-mFGF21 or non-transduced cells (Figure 34D).

[0424] HepG2 cells were transfected with a plasmid encoding either the wild-type mouse FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) (Zhang et al., EBioMedicine 15 (2017) 173-183) or a codon-optimized mouse FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF21). Non-transfected cells served as controls. HepG2 cells transduced with hAAT-moFGF21 expressed higher levels of FGF21 than either EF1a-mFGF21-transduced or non-transduced cells (Figure 34E).

[0425] In vivo Eight-week-old male C57Bl6 mice were hydrodynamically administered 5 μg of plasmids encoding wild-type mouse FGF21 coding sequences under the control of the elongation factor 1a (EF1a) promoter (EF1a-mFGF21) (Zhang et al., EBioMedicine 15 (2017) 173-183), codon-optimized mouse FGF21 coding sequences under the control of the CMV promoter (CMV-moFGF21), or codon-optimized mouse FGF21 coding sequences under the control of the hAAT promoter (hAAT-moFGF21). Analysis of FGF21 expression levels in the liver 24 hours after plasmid administration revealed that animals treated with hAAT-moFGF21 or CMV-moFGF21 expressed significantly higher levels of FGF21 than animals receiving EF1a-mFGF21 (Figure 35A). In addition, animals treated with hAAT-moFGF21 or CMV-moFGF21 exhibited higher circulating levels of FGF21 than animals receiving EF1a-mFGF21 (FIG. 35B).

[0426] Example 24. Increase in FGF21 expression in target tissues and circulating FGF21 levels in vivo by AAV8-hAAT-moFGF21, AAV8-CAG-moFGF21-doublemiRT, and AAV1-CMC-moFGF21 compared to AAV8-Ef1a-mFGF21 Liver expression Male C57Bl6 mice were given 1 × 10 10 vg, 2 × 10 10 vg or 5×10 10An AAV8 vector encoding a wild-type mouse FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or an AAV8 vector encoding a codon-optimized mouse FGF21 coding sequence under the control of the liver-specific hAAT promoter (AAV8-hAAT-moFGF21) was intravenously administered to mice. Two weeks after AAV administration, animals treated with AAV8-hAAT-moFGF21 showed both higher FGF21 expression levels in the liver and higher circulating FGF21 levels than animals treated with AAV8-EF1a-mFGF21, regardless of vector dose (Figures 36A and 36B).

[0427] Expression in fat Male C57Bl6 mice were treated with 2 × 10 10 vg, 5×10 10 vg or 1×10 11 We administered either an AAV8 vector encoding wild-type mouse FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or an AAV8 vector encoding a 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 (AAV8-CAG-moFGF21-doublemiRT) into the eWAT of mice. Two weeks after AAV administration, animals treated with AAV8-CAG-moFGF21-doublemiRT showed higher FGF21 expression levels in WAT than animals treated with AAV8-EF1a-mFGF21 (Figure 37A). Moreover, animals treated with AAV8-CAG-moFGF21-doublemiRT showed much lower FGF21 expression in the liver than animals administered AAV8-EF1a-mFGF21 (Figure 37B), demonstrating that intra-eWAT administration of the AAV8-CAG-moFGF21-doublemiRT vector efficiently eliminates 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 We intramuscularly administered either an AAV8 vector (AAV8-EF1a-mFGF21) encoding the wild-type mouse FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter or an AAV1 vector (AAV1-CMV-FGF21) encoding a codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter. Two weeks after AAV administration, animals treated with AAV1-CMV-FGF21 showed significantly higher FGF21 expression levels in skeletal muscle than animals treated with AAV8-EF1a-mFGF21 (Figure 38A). Furthermore, although animals treated with AAV8-EF1a-mFGF21 showed high expression of FGF21 in the liver, intramuscular administration of the AAV1-CMV-FGF21 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. Amino acid sequence of Canis lupus familiaris FGF21 4. Nucleotide sequence of Homo sapiens FGF21 5. Nucleotide sequence of codon-optimized Homo sapiens FGF21-variant 1 6. Nucleotide sequence of codon-optimized Homo sapiens FGF21-variant 2 7. Nucleotide sequence of codon-optimized Homo sapiens FGF21-variant 3 8. Nucleotide sequence of Mus musculus FGF21 9. Nucleotide sequence of codon-optimized Mus musculus FGF21 10 Nucleotide sequence of Canis lupus familiaris FGF21 Nucleotide sequence of 11 codon-optimized Canis lupus familiaris FGF21 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 Nucleotide sequence of a chimeric intron composed of introns from human β-globin and immunoglobulin heavy chain genes 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 Amino acid sequence of Homo sapiens FGF21 (SEQ ID NO: 1) MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS Nucleotide sequence of Homo sapiens FGF21 (SEQ ID NO: 4) ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTCCTGA Nucleotide sequence of codon-optimized Homo sapiens FGF21-variant 1 (SEQ ID NO: 5) ATGGATTCTGATGAGACAGGCTTCGAGCACAGCGGCCTGTGGGTTTCAGTTCTGGCTGGACTGCTGCTGGGAGCCTGTCAGGCACACCCTATTCCAGATAGCAGCCCTCTGCTGCAGTTCGGCGGACAAGTGCGGCAGAGATACCTGTACACCGACG ACGCCCAGCAGACAGAAGCCCACCTGGAAATCAGAGAGGATGGCACAGTTGGCGGAGCCGCCGATCAGTCTCCTGAATCTCTGCTCCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAAACCAGCCGGTTCCTGTGCCAAAGA CCTGACGGCGCCCTGTATGGCAGCCTGCACTTTGATCCTGAGGCCTGCAGCTTCAGAGAGCTGCTGCTTGAGGACGGCTACAACGTGTACCAGTCTGAGGGCCCATGGCCTGCCTCTGCATCTGCCTGGAAACAAGAGCCCTCACAGAGATCCCGCTC CTAGAGGCCCTGCCAGATTTCTGCCCTTCCTGGATTGCCTCCTGCTCTGCCAGAGCCTCCTGGAATTCTGGCTCCTCAGCCTCCTGATGTGGGCAGCTCTGATCCTCTGAGCATGGTCGGACCTAGCCAGGGCAGATCTCCTAGCTACGCCTCTTGA Nucleotide sequence of codon-optimized Homo sapiens FGF21-variant 2 (SEQ ID NO: 6) ATGGACAGCGATGAAACCGGGTTCGAGCACAGCGGTCTGTGGGTGTCCGTGCTGGCCGGACTGCTCCTGGGAGCCTGTCAGGCGCACCCCATCCCTGACTCCTCGCCGCTGCTGCAATTCGGCGGACAAGTCCGCCAGAGATACCTGTACACCGACG ACGCCCAGCAGACCGAAGCCCACCTGGAAATTCGGGAGGACGGGACTGTGGGAGGCGCTGCAGATCAGTCACCCGAGTCCCTCCTCCAACTGAAGGCCTTGAAGCCCGGCGTGATTCAGATCCTGGGCGTGAAAACTTCCCGCTTCCTTTGCCAACGG CCGGATGGAGCTCTGTACGGATCCCTGCACTTCGACCCCGAAGCCTGCTCATTCCGCGAGCTGCTCCTTGAGGACGGCTATAACGTGTACCAGTCTGAGGGCCCATGGACTCCCCTGCATCTGCCCGGCAACAAGTCCCCTCACCGGGATCCTGCCC CAAGAGGCCCAGCTCGGTTTCTGCCTCTGCCGGGACTGCCTCCAGCGTTGCCCGAACCCCCTGGTATCCTGGCCCCGCAACCACCTGACGTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAGCCAGGGAAGGTCCCCGTCCTACGCATCCTGA Nucleotide sequence of codon-optimized Homo sapiens FGF21-variant 3 (SEQ ID NO: 7) ATGGATTCCGACGAAACTGGATTTGAACATTCAGGGCTGTGGGTCTCTGTGCTGGCTGGACTGCTGCTGGGGGCTTGTCAGGCTCACCCCATCCCTGACAGCTCCCCTCTGCTGCAGTTCGGAGGACAGGTGCGGCAGAGATACCTGTATACCGACG ATGCCCAGCAGACAGAGGCACACCTGGAGATCAGGGAGGACGGAACCGTGGGAGGAGCAGCCGATCAGTCTCCCGAGAGCCTGCTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAGACATCTCGGTTTCTGTGCCAGCGG CCCGACGGCGCCCTGTACGGCTCCCTGCACTTCGATCCCGAGGCCTGTTCTTTTAGGAGGCTGCTGCTGGAGGACGGCTACAACGTGTATCAGAGCGAGGCACACGGCCTGCCACTGCACCTGCCTGGCAATAAGTCCCTCACCGCGATCCAGCAC CCAGGGGCCCAGCACGCTTCCTGCCTCTGCCAGGCCTGCCCCCTGCCCTGCCAGAGCCACCCGGCATCCTGGCCCCCCAGCCTCCAGATGTGGGCTCCAGCGATCCTCTGTCAATGGTGGGGCCAAGTCAGGGGCGGAGTCCTTCATACGCATCATAA 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 sequence of microRNA 199a) (SEQ ID NO: 15) 5' GAACAGGTAGTCTGAACACTGGG 3' Nucleotide sequence encoding miRT199a-3p (target sequence of microRNA 199a) (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) (SEQ ID NO: 27) 5' CACAAGATCGGATCTACGGGTT 3' Nucleotide sequence encoding miRT208-5p (target sequence of microRNA 208a) (SEQ ID NO: 28) 5' GTATAACCCGGGCCAAAAGCTC 3' Nucleotide sequence encoding miRT208a-3p (target sequence of microRNA 208a) (SEQ ID NO: 29) 5' ACAAGCTTTTTGCTCGTCTTAT 3' Nucleotide sequence encoding miRT499-5p (target sequence of cardiac-specific microRNA 499) (SEQ ID NO: 30) 5'AAACATCACTGCAAGTCTTAA 3' Nucleotide sequence of the CAG promoter (SEQ ID NO: 44) Nucleotide sequence of the CMV promoter (SEQ ID NO: 45) GTGATGCGGTTTTGGCAGTAACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT Nucleotide sequence of CMV enhancer (SEQ ID NO: 46) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACCGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG Nucleotide sequence of the hAAT promoter (SEQ ID NO: 47) GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT Shortened AAV2 5’ ITR (SEQ ID NO: 48) GCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT AGGGGTTCCT Shortened AAV2 3’ ITR (SEQ ID NO: 49) AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGC SV40 polyadenylation signal (SEQ ID NO: 50) TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT Rabbit β-globin polyadenylation signal (SEQ ID NO: 51) GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAA AGGTTGGCTATAAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC CMV promoter and CMV enhancer sequence (SEQ ID NO: 52) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAAT GACGTATGTTCCCATAGTAACGCCAATGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCC GCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT Hepatocyte control region (HCR) enhancer from apolipoprotein E (SEQ ID NO: 53) CAGAGAGGTCTCTGACCTCTGCCCCAGCTCCAAGGTCAGCAGGCAGGGAGGGCTGTGTGTTTGCTGTTTGCTGCTTGCAATGTTTGCCCATTTTAGGGACATGAGTAGGCTGAAGTTTGTTCAGTGTGGACTTCAGAGGCAGCACACAAACAGC miniaP2 promoter (SEQ ID NO: 54) GATTA ACCCGCCATG CTACTTATCT ACTCGACATT GATTATTGAC TAGGGGAATT CCAGCAGGAA TCAGGTAGCT GGAGAATCGC ACAGAGCCAT GCGATTCTTG GCAAGCCATG CGACAAAGGC AGAAATGCAC ATTTCACCCA GAGAGAAGGG ATTGATGTCA GCAGGAAGTC ACCACCCAGA GAGCAAATGG AGTTCCCAGA TGCCTGACAT TTGCCTTCTT ACTGGATCAG AGTTCACTAG TGGAAGTGTC ACAGCCCAAA CACTCCCCCA AAGCTCAGCC CTTCCTTGCC TTGTAACAAT CAAGCCGCTC CTGGATGAAC TGCTCCGCCC TCTGTCTCTT TGGCAGGGTT GGAGCCCACT GTGGCCTGAG CGACTTCTAT GGCTCCCTTT TCTGTGATTT TCATGGTTTC TGAGCTCTTT TCCCCCGCTT TATGATTTTC TCTTTTTGTC TCTCTCTTGC TAAACCTCCT TCGTATATAT GCCCTCTCAG GTTTCATTTC TGAATCATCT ACTGTGAACT ATTCCCATTG TTTGCCAGAA GCCCCCTGGT TCTTCCTTCT AGACACCAGG CAAGGGGCAG GAGGTAAGAG GCAGGAGTCC ATAAAACAGC CCTGAGAGCC TGCTGGGTCA GTGCCTGCTG TCAGAA miniUCP1 promoter (SEQ ID NO: 55) GACGTCACAG TGGGTCAGTC ACCCTTGATC ACACTGCACC AGTCTTCACC TTTCCACGCT TCCTGCCAGA GCATGAATCA GGCTCTCTGG GGATACCGGC CTCACCCCTA CTGAGGCAAA CTTTCTCCCA CTTCTCAGAG GCTCTGAGGG CAGCAAGGTC AGCCCTTTCT TTGGAATCTA GAACCACTCC CTGTCTTGAG CTGACATCAC AGGGCAGGCA GATGCAGCAG GGAAGGGCCT GGGACTGGGA CGTTCATCCT ACAAGAAAGC TGTGGAACTT TTCAGCAACA TCTCAGAAAT CAGATCGCAC TTATTCAAAG GAGCCAGGCC CTGCTCTGCG CCCTGGTGGA GGCTCCTCAT GTGAAGAGTG ACAAAAGGCA CCATGTTGTG GATACGGGGC GAAGCCCCTC CGGTGTGTCC TCCAGGCATC ATCAGGAACT AGTGCCAAAG CAGAGGTGCT GGCCAGGGCT TTGGGAGTGA CGCGCGTCTG GGAGGCTTGT GCGCCCAGGG CACGCCCCTG CCGATTCCCA CTAGCAGGTC TTGGGGGACC TGGGCCGGCT CTGCCCCTCC TCCAGCAATC GGGCTATAAA GCTCTTCCAA GTCAGGGCGC AGAAGTGCCG GGCGATCCGG GCTTAAAGAG CGAGAGGAAG GGACGCTCAC CTTTGAGCTC CTCCACAAAT AGCCCTGGTG GCTGCCACAG AAGTTCGAAG TTGAGAGTTC GG C5-12 promoter (SEQ ID NO: 56) CGGCCGTCCG CCTTCGGCAC CATCCTCACG ACACCCAAAT ATGGCGACGG GTGAGGAATG GTGGGGAGTT ATTTTTAGAG CGGTGAGGAA GGTGGGCAGG CAGCAGGTGT TGGCGCTCTA AAAATAACTC CCGGGAGTTA TTTTTAGAGC GGAGGAATGG TGGACACCCA AATATGGCGA CGGTTCCTCA CCCGTCGCCA TATTTGGGTG TCCGCCCTCG GCCGGGGCCG CATTCCTGGG GGCCGGGCGG TGCTCCCGCC CGCCTCGATA AAAGGCTCCG GGGCCGGCGG CGGCCCACGA GCTACCCGGA GGAGCGGGAG GCGCCA pAAV-EF1a-mmFGF21-pA (SEQ ID NO: 57) 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 (already included in the sequence listing, SEQ ID NOs: 48, 49 and 50). [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) in the same cells as in (A). (D) FGF21 expression levels in C2C12 cells transfected with plasmids encoding either the wild-type mouse FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or the codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21). (E) FGF21 expression levels in HepG2 cells transfected with plasmids encoding either the wild-type mouse FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or the codon-optimized mouse FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF21). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. Results are expressed as mean ± SEM. n = 3 wells per group. ND, not detected. * p < 0.05 vs. control. ### p < 0.001 vs. EF1a-mFGF21. [Figure 35]Figure 35. Increase in hepatic FGF21 expression and circulating FGF21 levels in vivo with hAAT-moFGF21 and CMV-moFGF21 expression cassettes. (A) FGF21 expression levels in the liver of C57B16 mice hydrodynamically administered with plasmids encoding the wild-type murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (EF1a-mFGF21), the codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21), or the codon-optimized murine FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF21). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. (B) Circulating FGF21 levels in the same cohort as in (A). Results are expressed as mean ± SEM. n = 5 mice / group. ** p < 0.01 vs. EF1a-mFGF21 [Figure 36] Figure 36. AAV8-hAAT-moFGF21 increases hepatic FGF21 expression and circulating FGF21 levels in vivo. (A) FGF21 expression levels in the liver of C57B16 mice intravenously administered 1 × 10 10 vg, 2 × 10 10 vg, or 5 × 10 10 vg of an AAV8 vector encoding wild-type murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or codon-optimized murine FGF21 under the control of the hAAT promoter (AAV8-hAAT-moFGF21). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. (B) Circulating FGF21 levels in the same cohort as in (A). Analysis was performed 2 weeks post-AAV administration. 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-mFGF21 [Figure 37]Figure 37. Increased adipose FGF21 expression in vivo by AAV8-CAG-moFGF21-dmiRT. (AB) FGF21 expression levels in the eWAT (A) or liver (B) of C57Bl6 mice administered 2 × 10 vg, 5 × 10 vg, or 1 × 10 vg of either an AAV8 vector encoding wild-type mouse FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or an AAV8 vector encoding 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 (AAV8-CAG-moFGF21-doublemiRT) into the eWAT. qPCR was performed using primers detecting both the wild-type and codon-optimized FGF21 coding sequences. Analysis was performed 2 weeks post-AAV. Results are expressed as mean ± SEM. n=4-5 mice / group. Control, untreated mice. eWAT, epididymal white adipose tissue. *p<0.05, **p<0.01, and [Figure 38]Figure 38. In vivo increase in FGF21 expression in skeletal muscle by AAV1-CMV-moFGF21. (AB) FGF21 expression levels in quadriceps (A) or liver (B) of C57B16 mice intramuscularly injected with 5 × 10 vg, 1 × 10 vg, or 3 × 10 vg of either an AAV8 vector encoding the wild-type murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or an AAV1 vector encoding the codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (AAV1-CMV-FGF21). qPCR was performed using primers that detect both the wild-type and codon-optimized FGF21 coding sequences. Analysis was performed 2 weeks post-AAV. Results are expressed as mean ± SEM. n = 4–5 mice per group. Control, untreated mice. * p<0.05, ** p<0.01, and *** p<0.001 vs. control. # p<0.05, ## p<0.01, and ### p<0.001 vs. AAV8-EF1a-mFGF21.

Claims

1. 1. An adeno-associated virus 1 (AAV1) vector comprising a viral expression construct for expression in a mammal, wherein the viral expression construct comprises a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) to be expressed in skeletal muscle and a ubiquitous promoter.

2. A nucleic acid molecule represented by a nucleotide sequence encoding mammalian codon-optimized FGF21 for expression in a mammal, which is expressed in the liver, adipose tissue and / or skeletal muscle, wherein the nucleotide sequence has at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6 or 7.

3. A composition comprising an AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2, together with one or more pharmaceutically acceptable excipients or vehicles.

4. 10. An AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for use as a medicament.

5. 10. An AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for use in the treatment and / or prevention of metabolic disorders.

6. 6. The AAV1 vector and / or nucleic acid molecule and / or composition for use according to claim 5, wherein the metabolic disorder is diabetes and / or obesity.

7. 6. The AAV1 vector and / or nucleic acid molecule and / or composition for use according to claim 5, wherein the metabolic disorder is NASH.

8. An AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for use in the treatment and / or prevention of liver inflammation and / or fibrosis.

9. 10. An AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for use in extending healthspan.

10. 10. An AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for use in the treatment and / or prevention of cancer.

11. An AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for use in the treatment and / or prevention of liver cancer.

12. Use of an AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating a metabolic disorder.

13. 13. The use according to claim 12, wherein the metabolic disorder is diabetes and / or obesity.

14. 13. The use according to claim 12, wherein the metabolic disorder is NASH.

15. Use of an AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating liver inflammation and / or fibrosis.

16. Use of an AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for the manufacture of a medicament for extending healthy lifespan.

17. Use of an AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating cancer.

18. Use of an AAV1 vector as defined in claim 1 and / or a nucleic acid molecule as defined in claim 2 and / or a composition as defined in claim 3 for the manufacture of a medicament for preventing, delaying, ameliorating, curing and / or treating liver cancer.

19. A nucleic acid molecule as defined in claim 2, wherein the nucleotide sequence has at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6 or 7.

20. A nucleic acid molecule as defined in claim 2, wherein the nucleotide sequence has at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6 or 7.

21. A nucleic acid molecule as defined in claim 2, wherein the nucleotide sequence has 100% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6 or 7.

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