JP2023109992A - Viral expression construct comprising fibroblast growth factor 21 (fgf21) coding sequence - Google Patents

Abstract

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

Description

The present invention is for use in the treatment of metabolic disorders in mammals, particularly in humans, for use in the treatment of liver inflammation and/or fibrosis, for use in the treatment of cancer, and/or The field of medicine includes gene therapy compositions for use in extending healthy life expectancy.

The prevalence of diabetes is increasing at an alarming rate and is a major global health problem. Obesity is strongly associated with insulin resistance and type 2 diabetes (T2D) (Moller,
D. E. , and Flier, J.; S. , 1991. N. Engl. J. Med.
325: 938-948 ). Moreover, obesity increases mortality risk (Peeters,
A. et al. , 2003. Ann. Intern. Med. 138 :24-32
), and is also a significant risk factor for heart disease, immune dysfunction, hypertension, arthritis, neurodegenerative diseases and certain types of cancer (Roberst, DL et al., 2010).
. Annu. Rev. Med. 61 :301-316; Spiegelman, B.;
M. et al. , 1993. J. Biol. Chem. 268 :6823-68
26; Whitmer, R.; A. , 2007. Curr. Alzheimer Res
. 4 :117-122). Despite the clinical importance of T2D and obesity, there are no effective treatments available. Therefore, there is an urgent need for new 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, D.L. et al.
al. , 2010. Annu. Rev. Med. 61 :301-316). Given the recent obesity epidemic, obesity-related cancer risks are a clinically important concern for which novel and safer approaches are urgently needed. 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 life expectancy. 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, new and safe anti-fibrotic therapeutic agents are needed.

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

Overexpression of FGF21 in transgenic mice protects those mice from diet-induced obesity (Kharitonenkov, A. et al., 2005. J. Cli
n. Invest 115 :1627-1635), FGF21 into ob/ob, db/db or high fat diet (HFD) fed mice or into obese ZDF rats.
administration led to a strong reduction in adiposity, significantly lowered blood glucose and triglycerides, decreased fasting insulin levels, and improved insulin sensitivity (Coskun, T. et al., 2008. Endocrinology
149 :6018-6027; Kharitonenkov, A.; et al. , 2
005. J. Clin. Invest 115 :1627-1635; Xu, J.; e
tal. , 2009. Diabetes 58 :250-259; Adams, A.;
C. et al. , 2012. PLoS. One. 7 : e38438. ;Berg
lund, E. D. et al. , 2009. Endocrinology 150
: 4084-4093). Moreover, administration of FGF21 to obese diabetic rhesus monkeys
It dramatically reduced fasting plasma glucose, fructosamine, triglyceride, insulin and glucagon levels and induced a small but significant weight loss (Kharit
onenkov, A.; et al. , 2007. Endocrinology 14
8 :774-781).

The 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. 34
6(2) :270-80; So, W.; Y. and Leung, P.; S. 2016.
Med Res Rev. 36(4) :672-704; Zhang, J.; and
Li, Y.; 2015. Front Endocrinol (Lausanne)
. 6 :168). To extend the half-life and improve the stability and solubility of FGF21,
Various engineering approaches have been developed. Currently, two artificial FGF21 mimetics (LY
2405319 and PF-05231023) have been tested in humans. Nonetheless, these FGF21 mimetics require multiple administrations and impose a significant burden on the patient. Moreover, artificial FGF21 mimetics/analogues may also be at a higher risk of exhibiting immunogenicity than native FGF21, for example patients treated with LY2405319 may experience injection site reactions, anti-drug antibodies and severe produced hypersensitivity reactions (Gaich, G. et al.
al. , 2013. Cell Metab. 18(3) :333-40).

Therefore, new treatments for diabetes and/or obesity and/or liver inflammation and/or fibrosis and/or cancer and/or extension of healthy life expectancy without all the drawbacks of existing treatments. There is still a need for effective treatment.

Moller, D.; E. , and Flier, J.; S. , 1991. N. Engl. J. Med. 325:938-948 Peeters, A.; et al. , 2003. Ann. Intern. Med. 138:24-32 Roberst, D.; L. et al. , 2010. Annu. Rev. Med. 61:301-316 Spiegelman, B.; M. et al. , 1993. J. Biol. Chem. 268: 6823-6826 Whitmer, R. A. , 2007. Curr. Alzheimer Res. 4:117-122 Muise, E. S. et al. , 2008. Mol. Pharmacol. 74:403-412 Coskun, T.; et al. , 2008. Endocrinology 149:6018-6027 Fisher, F.; M. 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 Adams, A.; C. et al. , 2012. PLoS. One. 7: e38438. Berglund, E.; D. et al. , 2009. Endocrinology 150:4084-4093 Kharitonenkov, A.; et al. , 2007. Endocrinology 148:774-781 Huang, J.; et al. , 2013. J Pharmacol Exp Ther. 346(2):270-80 So, W. Y. and Leung, P.; S. 2016. Med Res Rev. 36(4):672-704 Zhang, J.; and Li, Y.; 2015. Front Endocrinol (Lausanne). 6:168 Gaich, G.; et al. , 2013. Cell Metab. 18(3):333-40

The present inventors have investigated adeno-associated virus (AAV) vector-mediated F-mediated induction into liver, adipose tissue and/or skeletal muscle to combat metabolic disorders, preferably diabetes and/or obesity.
An improved gene therapy strategy based on GF21 gene transfer was designed. Gene therapy of the present invention may also be used to combat liver inflammation and/or fibrosis. In addition, the gene therapy of the present invention can also be used for extending healthy life expectancy by combating age-related metabolic disorders, preferably diabetes and/or obesity. Additionally, the gene therapy of the present invention can also be used to combat cancer, preferably liver cancer.

Creation of a single vector gene construct allows in vivo production of native FGF21, which results in reduced risk of immunogenicity or other toxicity.

However, those skilled in the art are aware that native FGF21 may be susceptible to proteolysis in vivo and/or have a rapid clearance rate in vivo. All vectors tested in the experimental department showed stable introduction of native FGF21 into the bloodstream.
It was found that the long-lasting secretion of Even a single administration of the gene transfer vector remains efficacious.

Therefore, the generation of such AAV vectors for in vivo production of native FGF21 is not routine for those skilled in the art, as demonstrated in the experimental part.

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

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

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

A more preferred nucleotide sequence encoding human FGF21 present in a viral expression construct of the invention is SEQ ID NO: 4, 5, 6 or 7 and at least 60%, 65%
, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100
have % identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions".
SEQ ID NO: 4 is the nucleotide sequence encoding human FGF21. SEQ ID NO: 5 is the codon-optimized nucleotide sequence encoding 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 are
They encode the same human FGF21 protein and were obtained by different codon optimization algorithms. Another preferred nucleotide sequence encoding murine FGF21 present in a viral expression construct of the invention is SEQ ID NO: 8 or 9 and at least 60%, 6
5%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 1
00% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions". SEQ ID NO:8 is the nucleotide sequence encoding mouse FGF21. SEQ ID NO:9
is a nucleotide sequence encoding codon-optimized murine FGF21. Another preferred nucleotide sequence encoding canine FGF21 present in a viral expression construct of the invention is SEQ ID NO: 10 or 11 and at least 60%, 65%, 70%, 7
5%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity, as explained in the section entitled "General Definitions,"
It can be assessed over the entire SEQ ID NO or over a portion thereof. SEQ ID NO: 10
is the nucleotide sequence encoding canine FGF21. SEQ ID NO: 11 is the nucleotide sequence encoding codon-optimized canine FGF21. The nucleotide sequence encoding FGF21 may be derived from any FGF21 gene or FGF21 coding sequence, preferably human, mouse or dog; or a mutated FGF21 gene or FGF21, preferably derived from human, mouse or dog. It may be a coding sequence, or a codon-optimized FGF21 gene or FGF21 coding sequence.

As used herein, FGF21 exerts at least a detectable level 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 in the experimental part, preferably using an insulin tolerance test, as described in Examples 8 or 9.

In certain embodiments, a 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) SEQ ID NO: 4, 5, 6, 7, 8, 9 , a nucleotide sequence having at least 60% sequence identity with 10 or 11 nucleotide sequences, (c) a nucleotide sequence that differs in sequence from the sequence of nucleotide sequence (b) due to the degeneracy of the genetic code. , the viral expression constructs described above are provided.

A preferred nucleotide sequence encoding FGF21 suitable for expression in a mammal is
SEQ ID NO: 1, 2 or 3 with at least 60%, 65%, 70%, 75%, 80%, 85%
, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description 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.

In one embodiment, a nucleotide sequence encoding FGF21 suitable for expression in a mammal and elements a), b), c), d) and e):
(a) a liver-specific promoter (b) an adipose tissue-specific promoter (c) a ubiquitous promoter, at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the liver, and a target sequence of a microRNA expressed in the heart (d) a skeletal muscle promoter and (e) a ubiquitous promoter and adeno-associated virus (AAV ) viral expression constructs as described above in combination with vector sequences, comprising at least one of said combinations, which allows for specific expression in skeletal muscle.

"target sequence of microRNA expressed in the liver" or "m
The iRNA target sequence"or"binding site of the microRNA expressed in the liver"is
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 heart-expressed microRNA" or "target sequence of a heart-expressed miRNA" or "binding site of a heart-expressed microRNA" refers to at least a portion of a heart-expressed microRNA A nucleotide sequence that is complementary or partially complementary to. The part of the microRNA expressed in the liver or the part of the microRNA expressed in the heart as defined herein means at least 5 or at least 6 of said microRNAs
means a nucleotide sequence of consecutive nucleotides. A 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 occur. . Alternatively, the binding site sequence can be partially complementary to at least part of the expressed microRNA, meaning that 1 mismatch/5, 6 consecutive nucleotides can occur. . A partially complementary binding site preferably contains perfect or near-perfect complementarity with the seed region of the microRNA, which is free of mismatches between the seed region of the microRNA and its binding site. (perfect complementarity) or 1 mismatch/5, 6 consecutive nucleotides (near perfect complementarity) can occur. Micro RN
The seed region of A consists of the 5' region of the microRNA, which is from about nucleotide 2 to about nucleotide 8 of the microRNA (ie, 6 nucleotides). The part defined herein is preferably the seed region of said microRNA. Degradation of messenger RNAs (mRNAs) containing target sequences for microRNAs expressed in the liver or in the heart can be through the RNA interference pathway, or
It may be through direct translational control (inhibition) of RNA. The present invention is not limited by the pathway ultimately utilized by the miRNA in inhibiting transgene or encoded protein expression.

In the context of the present invention, nucleotide sequences encoding target sequences for microRNAs expressed in the liver are defined by nucleotide sequences comprising nucleotide sequences having at least 60% sequence identity or similarity to SEQ ID NOs: 12 or 14-23. can be replaced.
A more preferred nucleotide sequence is SEQ ID NO: 12 or 14-23 and at least 60%
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,
Has 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions". In one embodiment, the nucleotide sequence encoding the target sequence of the microRNA expressed in the liver can be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity with SEQ ID NO:12. More preferred nucleotide sequences are SEQ ID NO: 12 and at least 60%, 65%, 70%, 75%, 80
%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. In a further embodiment, encoding a target sequence for a microRNA expressed in the liver;
At least one copy of the nucleotide sequence set forth in SEQ ID NOs: 12 or 14-23 is present in the viral expression constructs of the invention. In a further embodiment,
2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequences defined in SEQ ID NOS: 12 or 14-23, which encode the liver-specific microRNA target sequences, are included in the viral expression constructs of the invention. exist. In a preferred embodiment, miRT122
1, 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence encoding a (SEQ ID NO: 12) are present in the viral expression constructs of the invention.

As used herein, a liver-expressed microRNA target sequence exerts at least a detectable level the activity of a liver-expressed microRNA target sequence known to those of skill in the art. The activity of the target sequence of a microRNA expressed in the liver is to bind to its cognate microRNA expressed in the liver and to detarget transgene expression in the liver when operably linked to the transgene. is to mediate This activity can be assessed by measuring transgene expression levels in the liver by qPCR, as described in the experimental part.

In the context of the present invention, nucleotide sequences encoding target sequences for microRNAs expressed in the heart are defined by nucleotide sequences comprising nucleotide sequences having at least 60% sequence identity or similarity to SEQ ID NOs: 13 or 23-30. can be replaced.
Preferred nucleotide sequences are SEQ ID NO: 13 or 23-30 and at least 60%, 65
%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 10
have 0% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions". In one embodiment, the nucleotide sequence encoding the target sequence of the microRNA expressed in the heart can be replaced by a nucleotide sequence comprising a nucleotide sequence with at least 60% sequence identity or similarity to SEQ ID NO:13. Preferred nucleotide sequences are SEQ ID NO: 13 and at least 60%, 65%, 70%, 75%, 80%, 85%
%, 90%, 95%, 97%, 98%, 99%, 100% identity. In a further embodiment, at least one copy of the nucleotide sequence defined in SEQ ID NOS: 13 or 23-30, which encodes a target sequence for microRNAs expressed in the heart, is present in the 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: 13 or 23-30 encoding the target sequence of a cardiac-specific microRNA are of the present invention. Present in viral expression constructs. In a preferred embodiment, miRT1 (SEQ ID NO: 1
1, 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence encoding 3) are present in the viral expression construct of the invention.

The activity of the target sequence of a microRNA expressed in the heart is to bind to its cognate microRNA expressed in the heart and to detarget transgene expression in the heart when operably linked to a transgene. is to mediate This activity can be assessed by measuring transgene expression levels in the heart by qPCR, as described in the experimental section.

In certain embodiments, the nucleotide sequence encoding the target sequence of the microRNA expressed in the liver and the nucleotide sequence encoding the target sequence of the microRNA expressed in the heart consist of the sequences of SEQ ID NOs: 12 to 30 and/or combinations thereof. A viral expression construct as described above is provided which is selected from the group.

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 for a microRNA expressed in the liver and a sequence encoding a target sequence for a microRNA expressed in the heart. number thirteen
Alternatively, at least one copy of the nucleotide sequences defined in 23-30 is present in the viral expression constructs 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 for a microRNA expressed in the liver and expressed in the heart 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence defined in SEQ ID NOs: 13 or 23-30 encoding the target sequence of the microRNA that controls the viral expression construct of the present invention. In a further embodiment, miRT122
1, 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence encoding a (SEQ ID NO: 12) and 1, 2 of the nucleotide sequence encoding miRT1 (SEQ ID NO: 13),
3, 4, 5, 6, 7 or 8 copies are combined in the viral expression constructs 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 the viral expression construct of the invention.

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

A preferred ubiquitous promoter is the CAG promoter.

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 with SEQ ID NO:44. Preferred nucleotide sequences are SEQ ID NO: 44 and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,
99%, 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions".

Another preferred ubiquitous promoter is the cytomegalovirus (CMV) promoter.

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 with SEQ ID NO:45. Preferred nucleotide sequences are SEQ ID NO: 45 and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,
99%, 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions".

Preferably, said CMV promoter is used together with intronic sequences. In this regard, an 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:43. Preferred nucleotide sequences are SEQ ID NO: 43 and at least 60%, 65%, 70%, 75%, 80
%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions".

A preferred liver-specific promoter is the human α1-antitrypsin (hAAT) promoter.

In the context of the present invention, the nucleotide sequence of the hAAT promoter is SEQ ID NO: 47
can be replaced by a nucleotide sequence that includes a nucleotide sequence that has at least 60% sequence identity or similarity with Preferred nucleotide sequences are SEQ ID NO: 47 and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%
, 99% and 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions".

Preferably, the hAAT promoter is used together with intronic sequences. A preferred intron sequence is the hepatocyte control region (HCR) enhancer from apolipoprotein E. A most preferred intron sequence is the HCR enhancer from apolipoprotein E defined in SEQ ID NO:53. In this connection, intronic sequences are
It can be replaced by a nucleotide sequence that includes a nucleotide sequence that has at least 60% sequence identity or similarity with SEQ ID NO:53. A preferred nucleotide sequence is SEQ ID NO:5
3 and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 9
7%, 98%, 99%, 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions". In certain embodiments, the hAAT promoter is used with 1, 2, 3, 4 or 5 copies of intronic sequences. In a preferred embodiment, the hAAT promoter is used with 1, 2, 3, 4 or 5 copies of the HCR enhancer from apolipoprotein E defined in SEQ ID NO:53.

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

Adipose tissue-specific promoters include adipocyte protein 2 (aP2, also known as fatty acid binding protein 4 (FABP4)) promoter, PPARy promoter, adiponectin promoter, phosphoenolpyruvate carboxykinase (PEPCK)
promoter, promoter derived from human aromatase cytochrome p450 (p450arom) mini/aP2 promoter (composed of adipose-specific aP2 enhancer and basal aP2 promoter), uncoupling protein 1 (UCP1) promoter, mini/UCP1 promoter (fat (composed of a specific UCP1 enhancer and the basal UCP1 promoter), the adipsin promoter, the leptin promoter or the Foxa-2 promoter. A preferred adipose tissue-specific promoter is
mini/aP2 promoter (SEQ ID NO:54) and mini/UCP1 promoter (SEQ ID NO:55). In this regard, the adipose tissue-specific promoter 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 or SEQ ID NO:54. Preferred nucleotide sequences are SEQ ID NO: 53 or SEQ ID NO: 54 and at least 60%, 65%, 70%, 75%, 8
0%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions".

Preferred skeletal muscle promoters include 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;
Enh358MCK promoter (a combination of the MCK enhancer and the 358 bp proximal promoter of the MCK gene). A most preferred skeletal muscle promoter is SEQ ID NO:5
6, the C5-12 promoter. In this regard, the skeletal muscle promoter sequence may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity with SEQ ID NO:56. Preferred nucleotide sequences are SEQ ID NO: 56 and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%
%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed over the entire SEQ ID NO or over portions thereof as explained in the description section entitled "General Definitions".

A promoter as used herein (especially when its promoter sequence is defined as having a minimal percentage identity with a given SEQ ID NO) exhibits at least the activity of a promoter known to those skilled in the art. It is a thing. For a definition of such activity see "
See section entitled "General Definitions". Preferably, a promoter defined as having minimal percent identity to a given SEQ ID NO has a nucleotide sequence operably linked thereto (i.e. It regulates transcription of FGF21-encoding nucleotide sequence). In the context of the present invention, said promoter is operably linked to the FGF21 nucleotide sequence defined above. In one embodiment, the promoter is cell- and/or tissue-specific, preferably liver, adipose tissue and/or skeletal muscle specific.

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 elements b) and c);
A viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and comprising the nucleotide sequences of elements e) and c).

In certain embodiments, the liver-specific promoter is human α1-antitrypsin (hA
AT) promoter and/or an adipose tissue-specific promoter is mini/
ap2 promoter and/or mini/UCP1 promoter and/or skeletal muscle promoter is C5-12 promoter and/or ubiquitous promoter is cytomegalovirus (CMV) promoter and/or CA
A viral expression construct described herein is provided that is a G promoter.

In certain embodiments, viral expression constructs are included comprising 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).

In a preferred embodiment, a viral expression construct is included, comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element a), which is AAV8-hAAT-moFGF21. This construct is for example IT in FIG. 6A.
It contains a viral expression construct depicted as R2-hAAT-moFGF21-polyA-ITR2; the sequence of this expression construct is contained in SEQ ID NO:34.

For this construct, Example 3 surprisingly reveals high and stable liver-specific expression after intravenous administration. Expression was shown to be stable for up to 1 year (Example 12). Widespread beneficial therapeutic effects for reversal and treatment of obesity and diabetes were observed 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 improvement in fatty liver, hepatitis and liver fibrosis. Example 15 demonstrates amelioration of WAT inflammation associated with obesity. 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 I diabetes.

In certain embodiments, the nucleotide sequence encoding FGF21 suitable for expression in a mammal and element b) comprises the adipose tissue-specific promoter is the mini/aP2 promoter (SEQ ID NO: 54) and/or the mini/UCP1 promoter (SEQ ID NO: 54) and/or the mini/UCP1 promoter (SEQ ID NO: 5
5), viral expression constructs are included.

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

In a preferred embodiment, AAV9-CAG-moFGF21-dmiRT comprises a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element c).
or a viral expression construct that is AAV8-CAG-moFGF21-dmiRT. The notations dmiRT and doublemiRT are equivalent. These constructs are for example ITR2-CAG-moFGF21-4x miR in FIG. 1A.
It contains a viral expression construct depicted as T122a-4x miRT1-polyA-ITR2; the sequence of this expression construct is contained in SEQ ID NO:32.

For these constructs, Examples 1-2 surprisingly reveal high and stable adipose-specific expression after intra-eWAT administration. A wide range of beneficial therapeutic effects for the prevention, amelioration and treatment of obesity and diabetes were demonstrated in normal mice (Example 1) and ob/o
b Shown in mice (Examples 2 and 10). Example 10 also demonstrates significant improvement in fatty liver.

In certain embodiments, viral expression constructs are included comprising 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).

In certain embodiments, a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element e), wherein the ubiquitous promoter is the CMV promoter (SEQ ID NO: 45) and the AAV serotype is AAV1 is included.

In a preferred embodiment, a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element e) is included, which is AAV1-CMV-moFGF21. This construct is shown in FIG. 11A, for example, IT
It contains a viral expression construct depicted as R2-CMV-moFGF21-polyA-ITR2; the sequence of this expression construct is contained in SEQ ID NO:36.

For this construct, Example 20 demonstrates high and stable skeletal muscle-specific expression after intramuscular administration. A wide range of beneficial therapeutic effects for the prevention, amelioration and treatment of obesity and diabetes are demonstrated in HFD-fed mice (Examples 6 and 21). Example 20 demonstrates the beneficial health-span-extending effects of preventing obesity and diabetes.

In one embodiment, the nucleotide sequence encoding FGF21 suitable for expression in a mammal and the nucleotide sequences of elements b) and c), wherein the adipose tissue-specific promoter is the mini/aP2 promoter (SEQ ID NO: 54) and/or mini
A viral expression construct is included that is the /UCP1 promoter (SEQ ID NO:55).

In one embodiment, the nucleotide sequence encoding FGF21 suitable for expression in a mammal and the nucleotide sequences of elements e) and c) are included, the ubiquitous promoter is the CMV promoter (SEQ ID NO: 45), and the AAV serotype is AAV
1, viral expression constructs are included.

All constructs of the present invention are those of the prior art, eg Zhang et al.
, EBioMedicine 15 (2017) 173-183, in particular element a) which is a liver-specific promoter, preferably hAAT, and/or at least one encoding a ubiquitous promoter and a target sequence of a microRNA expressed in the liver. a combination of two nucleotide sequences, preferably miRT122a, and at least one nucleotide sequence encoding a target sequence for a microRNA expressed in the heart, preferably miRT1, the combination allowing specific expression in adipose tissue. c) and/or a combination of a ubiquitous promoter, preferably CMV, and an adeno-associated virus (AAV) vector sequence, preferably AAV1, a combination that allows specific expression in skeletal muscle. More attractive than anything else. Zhang et al. contains the wild-type mouse FGF21 coding sequence (EF1a-mFGF21
) (Zhang et al., EBioMedicine 15 (20
17) 173-183). This construct was compared to the construct of the present invention in Examples 23 and 24. In all in vitro and in vivo experiments,
All of the expression cassettes and AAV vectors of the invention mediate greater expression of FGF21 in target tissues or cell types and less expression of FGF21 in off-target tissues, which indicates that the expression cassettes and AAV vectors of the invention It demonstrates higher efficiency of AAV vectors as well as higher tissue specificity. In addition, the construct CMV
-moFGF21 and CAG-moFGF21-double miRTs are also EF
It mediated greater protein production and secretion into the culture medium in HEK293 cells compared to 1a-mFGF21. Moreover, hAAT-moFGF21 and AAV8-h
AAT-moFGF21 is also EF1a-mFGF21 and AAV8-EF1a-m
It mediated the secretion of more FGF21 into the bloodstream than FGF21.

Additional sequences may be present in the viral expression constructs of the invention, as detailed in the section of the description entitled "General Definitions." Preferred additional sequences include inverted terminal sequence (ITR), SV40 polyadenylation signal (SEQ ID NO:50), rabbit β-globin polyadenylation signal (SEQ ID NO:51), CMV enhancer sequence (SEQ ID NO:46) and apolipoprotein E and the HCR enhancer (SEQ ID NO: 53) from Within the context of the present invention, "ITR" means one 5'ITR and one 3'ITR
It is intended to encompass the TRs, each derived from the genome of AAV. A preferred ITR is A
from AV2, SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR
). Included within the context of the present invention is the use of the CMV enhancer sequence (SEQ ID NO:46) and the CMV promoter sequence (SEQ ID NO:45) as two separate sequences or as a single sequence (SEQ ID NO:52).

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

In certain embodiments, the above-defined nucleotide sequence encoding FGF21 suitable for expression in a mammal and of elements a) and/or b) and/or c) and/or d) and/or e) A viral expression construct comprising at least one of:
- 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 or HCR enhancer sequence located 5' to the nucleotide sequence encoding FGF21.

In a preferred embodiment, a FGF2 suitable for expression in mammals as defined above.
1 and elements a) and/or b) and/or c
) and/or d) and/or e) 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. good.

These sequences have been used in several constructs identified herein in the experimental department.

Therefore, in one embodiment, for each of these preferred viral expression constructs defined above, the ITR, the SV40 polyadenylation signal, the rabbit β-globin polyadenylation signal, the CMV enhancer sequence, the HCR enhancer from apolipoprotein E Additional sequences selected from the group consisting of sequences may be present.

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

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

Other preferred viral expression constructs comprise elements a) and/or b) and/or c) and/or d) and/or e) such that the expression cassette is flanked by the 5'ITR and 3'ITR. ing. Additionally, an SV40 polyadenylation signal is present.

Other preferred viral expression constructs comprise elements a) and/or b) and/or c) and/or d) and/or e) such that the expression cassette is flanked by the 5'ITR and 3'ITR. ing. Additionally, a rabbit β-globin polyadenylation signal is present.

Other preferred viral expression constructs comprise elements a) and/or b) and/or c) and/or d) and/or e) such that the expression cassette is flanked by the 5'ITR and 3'ITR. ing. Additionally, a CMV enhancer sequence is present.

Other preferred viral expression constructs comprise elements a) and/or b) and/or c) and/or d) and/or e) such that the expression cassette is flanked by the 5'ITR and 3'ITR. ing. In addition, HCR from apolipoprotein E
Enhancer sequences are present.

The most preferred designed viral expression constructs are:
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 (2 represented by a nucleotide sequence comprising SEQ ID NO: 4)
are mentioned.

As those skilled in the art will appreciate, each of these viral expression constructs contains two ITRs from AAV2, namely SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR).
R)) already included.

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

Throughout this application, as set forth in the general section entitled "General Definitions", the SEQ ID NOs of specific nucleotide sequences representing the preferred constructs designed herein (for example SEQ ID NO: A, picking up B or C), this:
i. a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity with SEQ ID NO: A, B or C;
ii. Due to the degeneracy of the genetic code, the sequence of the nucleic acid molecule of (i) may be replaced by a different nucleotide sequence.

its percentage identity (at least 60%) with a given nucleotide sequence, respectively
Each nucleotide sequence described herein by is, in a further preferred embodiment, a given nucleotide and at least 65%, 70%, 75%, 80%, 8
5%, 90%, 95%, 97%, 98%, 99% or more identity. In preferred embodiments, sequence identity is determined by comparing the full length of the sequences identified herein. Unless otherwise indicated herein, identity to a given SEQ ID is
Identity or similarity based on the full length (ie, over its entire length or in its entirety) of the sequence.

its minimal identity to a given SEQ ID NO specified above (i.e. at least 60%
) means that this construct or a viral expression construct or a viral vector comprising this construct or a composition comprising this construct or vector is expressed in cells, preferably hepatocytes, adipose tissue cells or skeletal muscle cells is included within the scope of the present invention when the expression of FGF21 can be induced in Expression of FGF21 can be assessed using techniques known to those of skill in the art.
In a preferred embodiment, said expression is assessed as performed in the laboratory.

In a preferred embodiment, the viral expression construct is SEQ ID NO: 4, 5, 6, 7
, 8, 9, 10 or 11 or SEQ ID NO: 4, 5, 6, 7, 8
, 9, 10 or 11 or SEQ ID NOs: 4, 5,
6, 7, 8, 9, 10 or 11 and at least 60%, 65%, 70%, 75%, 80
%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity.

Viral Vector In a further aspect there is provided a viral vector comprising a viral expression construct as defined above, wherein said viral vector is an adenoviral vector, an adeno-associated viral vector, a retroviral vector or a lentiviral vector, preferably Adeno-associated virus 1 (AAV1) vector, adeno-associated virus 8 (AAV8
) vectors and adeno-associated virus 9 (AAV9) vectors.

A "viral vector" and an "adeno-associated viral vector (AAV vector)" are
Further defined in the section entitled "General Definitions".

In certain embodiments, an AAV vector is used that comprises a recombinant AAV (rAAV)-based genome comprising each of the elements defined herein above and an ITR or portion thereof.
Preferred ITRs are those of AAV2 represented by SEQ ID NO: 48 (5'ITR) and SEQ ID NO: 49 (3'ITR).

Preferably, said AAV vector is an AAV1 vector, an AAV8 vector or an AAV
V9 vector.

The viral expression constructs and viral vectors of the invention are preferably for use as medicaments. The medicament is preferably for preventing, retarding, curing, ameliorating and/or treating metabolic disorders, preferably diabetes and/or obesity. Diabetes can be type 1 diabetes, type 2 diabetes or monogenic diabetes. In another preferred embodiment, the medicament is for preventing, retarding, curing, ameliorating and/or treating liver inflammation and/or fibrosis. In yet another preferred embodiment, the medicament preferably prevents, delays, cures, reverses and/or treats age-related metabolic disorders, preferably diabetes and/or obesity, to prolong healthy life expectancy. belongs to. In yet another preferred embodiment, the medicament is for preventing, delaying, curing, ameliorating and/or treating cancer, preferably liver cancer.

The subject to be treated can be a higher mammal, such as a cat, rodent (preferably mice, rats, gerbils and guinea pigs, more preferably mice and rats), or dogs, or humans.

In a further embodiment the nucleic acid molecule is suitable for expression in a mammal and
Alternatively, nucleic acid molecules represented by nucleotide sequences encoding mammalian codon-optimized FGF21 expressed in skeletal muscle are provided.

A definition of "codon-optimized" is provided in the description section entitled "General Definitions".

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

In a further aspect of the composition , the viral expression construct as defined above and/or the viral vector as defined above and/or the nucleic acid molecule as defined above are combined with one or more pharmaceutically acceptable additives or vehicles. Compositions comprising the two are provided.

This composition is preferably referred to as a gene therapy composition. Preferably, the composition is a pharmaceutical composition comprising pharmaceutically acceptable carriers, adjuvants, diluents, solubilizers, excipients, preservatives and/or additives.

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

In a preferred embodiment, said composition is preferably for preventing, delaying, curing, ameliorating and/or treating metabolic disorders, preferably diabetes and/or obesity. Diabetes can be type 1 diabetes, type 2 diabetes or monogenic diabetes. In another preferred embodiment, the medicament is for preventing, retarding, curing, ameliorating and/or treating liver inflammation and/or fibrosis. In yet another preferred embodiment, the medicament preferably prevents age-related metabolic disorders, preferably diabetes and/or obesity,
To prolong healthy life expectancy by delaying, healing, recovering and/or treating. In yet another preferred embodiment, the medicament prevents cancer, preferably liver cancer,
To delay, cure, restore and/or treat. The subject to be treated can be a higher mammal, such as a cat, rodent (preferably mice, rats, gerbils and guinea pigs, more preferably mice and rats), or dogs, or humans.

said viral expression constructs, viral vectors and/or nucleic acid molecules and/or
or when the 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 associated with metabolic disorders, preferably diabetes and/or or prevent, delay, or prevent obesity
It is said that it can be used for healing, recovery and/or treatment.

said viral expression constructs, viral vectors and/or nucleic acid molecules and/or
or when the composition is capable of exhibiting an anti-fibrotic effect, said viral expression construct, viral vector and/or nucleic acid molecule and/or composition preferably prevents, delays, cures liver inflammation and/or fibrosis. , is said to be able to be used for recovery and/or treatment.

said viral expression constructs, viral vectors and/or nucleic acid molecules and/or
or said viral expression construct, viral vector and/or nucleic acid molecule and/or composition is preferably anti-diabetic and/or anti-obesity effect upon aging. associated metabolic disorders, preferably diabetes and/or
Or it can be used for prolonging healthy life expectancy by preventing, delaying, curing, reversing and/or treating obesity.

said viral expression constructs, viral vectors and/or nucleic acid molecules and/or
or when the composition is capable of exhibiting an anti-cancer effect, said viral expression construct, viral vector and/or nucleic acid molecule and/or composition preferably prevents, delays, delays cancer, preferably liver cancer. It is said that it can be used for healing, recovery and/or treatment.

Anti-diabetic effects may be achieved when blood glucose handling is improved and/or when glucose tolerance is improved and/or when insulin sensitivity is improved. this is,
It 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 connection:
Improvement (respectively "improvement") refers to assays using assays known to the person skilled in the art or performed in the laboratory, such as blood glucose, blood insulin measurements and/or insulin tolerance tests and/or glucose tolerance tests means at least a detectable improvement (each a detectable improvement), such as with the implementation of The improvement is in assays such as blood glucose,
at least 5%, at least 10%, at least 20%, such as by measuring blood insulin and/or performing an insulin tolerance test and/or a glucose tolerance test;
It can be an improvement of 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%.

An anti-obesity effect can be achieved when body weight, weight gain and/or body fat percentage is reduced. Anti-obesity effects can also be achieved when body mass index (BMI), waist circumference, waist-to-hip ratio (WHR) and/or waist-to-height ratio (WHtR) are decreased. This can be assessed using techniques known to those skilled in the art or as done in the laboratory. In this context, "reduction" (respectively "improvement") means at least a detectable reduction (respectively a detectable improvement) using assays known to those skilled in the art or using assays performed in the laboratory. ). Anti-obesity effects include both prevention of obesity and amelioration of obesity as assessed by measurements of body weight, BMI and/or tissue weight of an individual.

Anti-inflammatory effects in the liver can be achieved by reducing macrophage infiltration, reducing pro-inflammatory cytokines. This can be assessed using techniques known to those skilled in the art or as done in the laboratory. In this context, "reduction" (respectively "improvement") means at least a detectable reduction (respectively a detectable improvement) using assays known to those skilled in the art or using assays performed in the laboratory. ).

Antifibrotic effects in the liver are associated with decreased extracellular matrix protein accumulation, blood markers such as type III collagen N-terminal propeptide in plasma, hyaluronic acid, tissue inhibitor of metalloproteinase type 1 (TIMP-1), YKL- 40, levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT)). Anti-fibrotic effects can also be achieved by modification of fibrosis scoring systems such as Metavir or Ishak. This can be assessed using techniques known to those skilled in the art or as done in the laboratory. In this context, "reduction" (respectively "improvement") means at least a detectable reduction (respectively a detectable improvement) using assays known to those skilled in the art or using assays performed in the laboratory. ).

The healthy life expectancy extending effect is defined hereinbefore as the anti-diabetic and/or anti-obesity effect prevents, delays, cures the onset or progression of age-related metabolic disorders, preferably diabetes and/or obesity. , can be reached when used to heal or treat. The effect of extending healthy life expectancy may also be achieved by increasing healthy life expectancy, wherein symptoms associated with metabolic disorders, preferably diabetes and/or obesity, are absent or reduced. The effect of extending healthy life expectancy is also
Improved coordination and balance (as assessed by the Rotarod test), memory (as assessed by the Object Recognition test) and/or neuromuscular coordination (as assessed by the Tightrope test), reduced mitochondrial and metabolic deterioration (assessed by measuring expression levels of genes involved in metabolic 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 done in the laboratory. In this context, "enhancement" (respectively "improvement") means, using assays known to those skilled in the art,
or at least a detectable increase (respectively a detectable improvement) using an assay performed in the laboratory.

Anti-cancer effects can be achieved by reducing the cumulative incidence of cancer over a lifetime. this is,
It can be assessed using techniques known to those skilled in the art or as performed in the laboratory. In this context, "reduction" (respectively "improvement") means at least a detectable reduction (respectively a detectable improvement) using assays known to those skilled in the art or using assays performed in the laboratory. ).

Anti-diabetic and/or anti-obesity effects are also associated with typical symptoms assessed by physicians (
It can also be seen when the progression of insulitis, beta-cell loss, weight gain, for example, slows down. A reduction in typical symptoms can mean a slowing of the progression of symptom development or a complete disappearance of symptoms. Symptoms and therefore also reduction of symptoms can be assessed using a variety of methods, most of which are the same methods used in the diagnosis of diabetes and/or obesity, including clinical examination. examination) and routine laboratory tests (l
Aboratory test). Such methods include both macroscopic and microscopic methods, as well as molecular methods, X-ray, biochemistry, immunohistochemistry and others.

Anti-inflammatory effects in the liver are also associated with typical symptoms assessed by physicians (e.g. fatigue,
cold-like symptoms, dark urine, white stools, abdominal pain, decreased appetite, unexplained weight loss, jaundice) may also be seen when progression slows. A reduction in typical symptoms can mean a slowing of the progression of symptom development or complete disappearance of symptoms. Symptoms, and therefore also reduction of symptoms, can be assessed using a variety of methods, most of which are the same methods 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, ultrasonography, angiography), biochemistry, immunohistochemistry. chemistry and others.

Antifibrotic effects in the liver are also associated with typical symptoms assessed by physicians, such as liver stiffness, jaundice, decreased appetite, difficulty in thinking clearly, fluid retention in the limbs or stomach,
nausea, unexplained weight loss, weakness) may also be seen when progression slows. A reduction in typical symptoms can mean a slowing of the progression of symptom development or a complete disappearance of symptoms. Symptoms, and therefore also reduction of symptoms, can be assessed using a variety of methods, most of which are the same methods used in the diagnosis of liver fibrosis, including clinical examination.
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, ultrasonography, angiography), biochemistry, immunohistochemistry. chemistry and others.

A healthy lifespan extension effect can also be seen when the progression of typical symptoms of age-related metabolic disorders (eg, insulin resistance, glucose intolerance, weight gain) as assessed by physicians slows down. A reduction in typical symptoms can mean a slowing of the progression of symptom development or a complete disappearance of symptoms. Symptoms and therefore also symptom reduction can be assessed using a variety of methods,
The methods are largely the same methods used in the diagnosis of diabetes and/or obesity and include clinical examinations and routine laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular methods, X-ray, biochemistry, immunohistochemistry and others.

Anticancer efficacy is also assessed by typical symptoms assessed by physicians (e.g., tumor size, unexplained weight loss, decreased appetite, feeling very full after eating small meals, nausea or vomiting, enlarged liver, enlarged spleen). , pain in the abdomen or near the right shoulder blade, swelling or fluid in the abdomen, pruritus,
jaundice) may also be seen when progression slows. A reduction in typical symptoms can mean a slowing of the progression of symptom development or a complete disappearance of symptoms. Symptoms and therefore also reduction of symptoms can be assessed using a variety of methods, most of which are the same methods used in cancer diagnosis, including clinical examination and Includes routine laboratory tests. Such methods include both macroscopic and microscopic methods, as well as molecular methods, imaging methods (X-ray, MRI, CT, ultrasonography, angiography), biochemistry, immunohistochemistry and others. are listed.

after treatment with a viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition of the invention for at least 1 week, 1 month, 6 months, 1 year or longer, as defined above If a symptom or trait is diminished (e.g. no longer detectable or slowed), an agent (viral expression construct, viral vector, nucleic acid molecule, composition) as defined herein is preferably A symptom or a characteristic of a patient or a cell, tissue or organ of said patient can be alleviated.

Viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions as defined herein for use according to the present invention may be used in patients with metabolic disorders such as diabetes and/or obesity, liver inflammation and/or or suitable for in vivo administration to cells, tissues and/or organs of individuals suffering from or at risk of developing fibrosis, age-related metabolic disorders, and/or cancer, etc. and can be administered in vivo, ex vivo or in vitro. Said viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions are useful for metabolic disorders such as diabetes and/or obesity, liver inflammation and/or fibrosis, age-related metabolic disorders, and/or or can be directly or indirectly administered in vivo to cells, tissues and/or organs of an individual suffering from or at risk of developing cancer or the like, in vivo, ex vivo or in vitro, directly or indirectly can be administered on a regular basis. Modes of administration include intravenous, subcutaneous, intramuscular, intrathecal, intraarticular, intracerebroventricular, intraperitoneal, intrafatty, via inhalation, oral, intranasal, intrahepatic, intravisceral, intraocular, intraaural. , topic
) and/or retrograde intraductal pancreatic administration
through tal pancreatic administration). Preferred modes of administration are intramuscular, intravenous or intra-adipose, as described in the "General Procedures for Examples" section of this application.

Viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions of the invention can be administered directly or indirectly by using any suitable means known in the art. In view of the advances already achieved to date, methods for supplying an individual or a cell, tissue, organ of said individual with a viral expression construct and/or a viral vector and/or a nucleic acid molecule and/or a composition according to the invention. Means improvement is expected. Such future improvements may, of course, be incorporated in order to achieve the stated advantages of the invention. Viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions can be delivered intact to an individual, a cell, tissue or organ of said individual. Depending on the disease or condition, said individual's cell, tissue or organ may be as previously defined herein. When viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions of the invention are administered, such viral expression constructs and/or vectors and/or nucleic acids and/or compositions are compatible with delivery methods. It is preferably dissolved in a diluted solution.

As encompassed herein, therapeutically effective doses of the above-described viral expression constructs, vectors, nucleic acid molecules and/or compositions are preferably administered in single and unique doses, thus repeated periodic administrations. Avoid administration. More preferably, a single dose to skeletal muscle,
It is administered into adipose tissue or intravenously.

Additional compounds may be present in the compositions of the invention. Said compounds may assist in the delivery of the composition. A list of suitable compounds is provided below: Complexes that complex or entrap each component defined herein in vesicles or liposomes for delivery across cell membranes;
Compounds capable of forming nanoparticles, micelles and/or liposomes. Many of these compounds are known in the art. A preferred compound is polyethyleneimine (PEI
), or similar cationic polymers, including polypropyleneimine or polyethyleneimine copolymers (PEC) and derivatives, synthetic amphiphiles (SAINT-18),
Lipofectin™, including DOTAP.

Depending on their identity, those skilled in the art will understand which formulation type is most suitable for the compositions defined herein.

Methods/Uses In a further aspect, 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 nucleic acid molecule as defined above for use as a medicament. A composition is provided.

In certain embodiments, said viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions are 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.

In another embodiment, said viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions are 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.

In yet another embodiment, said viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition preferably prevents, delays, delays, age-related metabolic disorders, preferably diabetes and/or obesity. It is provided for use in extending healthy life expectancy by healing, recovery and/or treatment.

In yet another embodiment, said viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions are provided for use in the treatment and/or prevention of cancer, preferably liver cancer. . Complications of cancer may also be included.

In a further aspect a metabolic disorder 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, Methods are provided for preferably preventing, delaying, reversing, curing and/or treating diabetes and/or obesity and their complications.

Such methods preferably comprise in an individual one or more symptom(s) such as 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, the method comprising: administration of viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions as defined herein.

In a further aspect liver inflammation 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 and/or methods of preventing, delaying, ameliorating, curing and/or treating fibrosis and their complications.

Such methods are 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 ameliorating one or more characteristic(s) or symptom(s) of a cell, tissue or organ of a cell, tissue or organ, the method comprising administering to said individual a viral expression construct as defined herein and/or This includes administering viral vectors and/or nucleic acid molecules and/or compositions.

In a further aspect comprises 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, preferably Methods are provided to extend healthy life expectancy by preventing, delaying, curing, ameliorating and/or treating age-related metabolic disorders, preferably diabetes and/or obesity.

Such methods are preferably for alleviating in an individual one or more symptom(s) of age-related metabolic disorders such as diabetes and/or obesity in cells, tissues or organs 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 a viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition.

In a further aspect a cancer 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, Methods are provided to preferably prevent, delay, reverse, cure and/or treat liver cancer and their complications.

Such methods are preferably for alleviating in an individual one or more symptom(s) such as cancer, for example liver cancer, in a cell, tissue or organ of said individual, or ameliorating one or more characteristic(s) or symptom(s) of a cell, tissue or organ of a viral expression construct as defined herein and/or This includes administering viral vectors and/or nucleic acid molecules and/or compositions.

In the context of the present invention, a viral expression construct as defined herein for the manufacture of a medicament for preventing, delaying, ameliorating, curing and/or treating metabolic disorders, preferably diabetes and/or obesity and/or use of viral vectors and/or nucleic acid molecules and/or compositions are provided.

In the context of the present invention, a viral expression construct as defined herein and/or for the manufacture of a medicament for preventing, retarding, curing, ameliorating and/or treating liver inflammation and/or fibrosis Alternatively, use of viral vectors and/or nucleic acid molecules and/or compositions is provided.

In the context of the present invention, preferably manufacture of a medicament for prolonging healthy life expectancy by preventing, delaying, curing, ameliorating and/or treating age-related metabolic disorders, preferably diabetes and/or obesity Use of the viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions as defined herein is provided for.

In the context of the present invention, a viral expression construct as defined herein and/or for the manufacture of a medicament for preventing, delaying, reversing, curing and/or treating cancer, preferably liver cancer. Alternatively, use of viral vectors and/or nucleic acid molecules and/or compositions is provided.

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), nonalcoholic steatohepatitis (N
ASH), coronary heart disease (CHD), hyperlipidemia, atherosclerosis, endocrine disease (e
ndocrinophaties), osteosarcopenic obesity syndrome (OSO), diabetic nephropathy, chronic nephropathy (CKD), cardiac hypertrophy,
Diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, arthritis, sepsis, ocular neovascularization, neurodegeneration, dementia and may also include depression, adenoma, carcinoma.

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, lipotrophic diabetes, latent autoimmune diabetes in adults (LADA).

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

Metabolic disorders, diabetes, obesity and the type of subject to be treated are defined earlier herein.

Inflammation and/or fibrosis of the liver include autoimmune hepatitis, viral hepatitis including hepatitis A, B, C, D and E, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH). ) and liver cirrhosis.

Cancers include astrocytoma, glioma, leukemia, lymphoma, melanoma, myeloma, neuroblastoma, sarcoma (chondrosarcoma, fibrosarcoma, rhabdomyoscaroma and osteosarcoma). ), schwannoma, seminioma, and carcinomas of the bladder, breast, cervix, colon, endometrium, esophagus, gallbladder, kidney, liver, lung, ovary, prostate, pancreas, rectum, skin, stomach, and thyroid. mentioned. A preferred cancer is liver cancer, preferably hepatocellular carcinoma. 1
In one embodiment, the method or use is performed in vitro, eg, using cell culture. Preferably said method or use is in vivo. Features of each of these methods/uses have already been defined herein.

In certain methods of the invention, viral expression constructs and/or vectors and/or nucleic acid molecules and/or compositions are known to be used to treat metabolic disorders, preferably diabetes and/or obesity in individuals. It can be combined with additional compounds that are

In another method of the invention, the viral expression constructs and/or vectors and/or nucleic acid molecules and/or compositions are known to be used to treat liver inflammation and/or fibrosis. can be combined with other compounds.

In yet another method of the invention, the viral expression construct and/or vector and/or nucleic acid molecule and/or composition is combined with additional compounds known to be used to extend healthy lifespan. obtain.

In yet another method of the invention, the viral expression constructs and/or vectors and/or nucleic acid molecules and/or compositions are known to be used to treat cancer, preferably liver cancer. can be combined with a specific compound.

In preferred embodiments, the use or treatment in the method according to the invention need not be repeated. Alternatively, in the use or method according to the invention, said administration of the viral expression construct or said composition may be repeated annually or every 2, 3, 4, 5, 6 years.

General definition
Identity/Similarity In the context of the present invention, protein fragments or polypeptides or peptides or derivative peptides as fibroblast growth factor 21 (FGF21) are represented by amino acid sequences.

In the context of the present invention, a nucleic acid molecule as a nucleic acid molecule encoding FGF21 is represented by a nucleic acid or nucleotide sequence encoding a protein fragment or polypeptide or peptide or derivative peptide. Nucleic acid molecules can include regulatory regions.

Given Sequence Identity Number (SEQ ID NO:
Each nucleic acid molecule or protein fragment or polypeptide or peptide or derivative peptide or construct identified herein by SEQ ID NO)) is
It is understood that there is no limitation to the specific sequences disclosed. Each coding sequence identified herein encodes a given protein fragment or polypeptide or peptide or derivative peptide or construct, or is itself a protein fragment or polypeptide or construct or peptide or derivative peptide. be. Throughout this application, whenever a reference is made to the SEQ ID NO of a specific nucleotide sequence encoding a given protein fragment or polypeptide or peptide or derivative peptide (taking 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 with SEQ ID NO:X;
ii. a nucleotide sequence that differs in sequence from the sequence of the nucleic acid molecule of (i) due to the degeneracy of the genetic code; or iii. It can be replaced by a nucleotide sequence encoding an amino acid sequence that has at least 60% amino acid identity or similarity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:X.

Throughout this application, whenever we refer to a specific amino acid sequence SEQ ID NO (taking SEQ ID NO Y as an example), this:
It can be replaced by 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.

Each nucleotide sequence or amino acid sequence described herein by its percentage identity or similarity (at least 60%) with a given nucleotide sequence or amino acid sequence, respectively, is in a further preferred embodiment a given and at least 65%, 70%, 75%, 80%, 85%, 90%,
95%, 97%, 98%, 99% or more identity or similarity. 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 refers to identity or similarity based on said sequence in full length (i.e. over its entire length or in its entirety). means.

Each non-coding (i.e. promoter or another regulatory region) nucleotide sequence has at least 60% sequence identity or similarity with a specific nucleotide sequence SEQ ID NO (take SEQ ID NO:A as an example) It can be replaced by any nucleotide sequence, including any nucleotide sequence. Preferred nucleotide sequences are SEQ ID NO: A and at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%
%, 99%, 100% identity. Identity can be assessed over the entire SEQ ID NO, or over 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, such as the activity of promoters, known to those skilled in the art.

"Sequence identity" is 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 defined herein as the relationship of In preferred embodiments, sequence identity is calculated on the basis of the full length of two given SEQ ID NOs, or on parts thereof. The portion preferably includes at least 10 of both SEQ ID NOs.
%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%
means In the art, "identity" also refers, as the case may be, to the degree of sequence relatedness between amino acid or nucleic acid sequences as determined by matches between strings of such sequences.

"Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and conservative amino acid substitutions thereof 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, Computational Molecular
Biology, Lesk, A.; M. , ed. , Oxford University
Press, New York, 1988;
rmatics and Genome Projects, Smith, D.; W. , e
d. , Academic Press, New York, 1993;
r Analysis of Sequence Data, Part I, Griff
in, A.I. M. , and Griffin, H.; G. , eds. , Humana Pre
ss, New Jersey, 1994;
Molecular Biology, von Heine, G.; , Academic
Press, 1987; and Sequence Analysis Prime
r, Gribskov, M.; and Devereux, J.; , eds. , M Sto
Ckton Press, New York, 1991; and Carillo, H.
. , and Lipman, D.; , SIAMJ. Applied Math. , 48:
1073 (1988).

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, for example, the GCG program package (Devereux, J., et al., Nucleic Acids Re
search 12(1):387 (1984)), BestFit, BLASTP, B.
LASTN and FASTA (Altschul, SF 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; , et al. , J. Mol. Biol
. 215:403-410 (1990). The well-known Smith Waterman algorithm can also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the Algorithm:
Needleman and Wunsch,J. Mol. Biol. 48:443-
453 (1970); Hentikoff and Hentikoff, Proc. N.
atl. Acad. Sci. USA. 89:10915-10919 (1992) Comparison matrix: BLOSSUM62; Gap Penalty
: 12; and Gap Length Penalty: 4. A useful program with these parameters is Genetics
It is published as the "Ogap" program by the Computer Group. The preceding parameters are the default parameters for amino acid comparisons (with no penalties for end gaps).

Preferred parameters for nucleic acid comparison include Algorithm: Needle
Mann and Wunsch,J. Mol. Biol. 48:443-453 (19
70); Comparison matrix: matches=+10, mismat
ch=0; Gap Penalty: 50; Gap Length Penalty: 3
is mentioned. Genetics Computer G in Madison, Wis.
It is available as a Gap program from roup. Given above are the default parameters for nucleic acid comparisons.

In determining the degree of amino acid similarity, those skilled in the art may consider so-called "conservative" amino acid substitutions, which are apparent to those skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, groups of amino acids with aliphatic side chains are glycine, alanine, valine, leucine and isoleucine; groups of amino acids with aliphatic-hydroxyl side chains are serine and threonine; groups are asparagine and glutamine; groups of amino acids with aromatic side chains are phenylalanine, tyrosine and tryptophan; groups of amino acids with basic side chains are lysine, arginine and histidine; The groups of amino acids it has are 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 sequence has been removed and a different residue inserted in its place. Preferably, the amino acid changes are conservative. A preferred conservative substitution for each naturally occurring amino acid is Al
a to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu;
Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pr
o; His to Asn or Gln; Ile to Leu or Val; Leu to Ile
or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile
Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Tr
p to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

A gene or coding sequence "gene" or "coding sequence" or "nucleic acid" or "nucleotide sequence" or "nucleic" refers to a region of DNA or RNA that "encodes" a particular protein, such as FGF21 ( transcription region). The coding sequence is transcribed (DNA) and translated into a polypeptide (RNA) when placed under the control of appropriate control regions, such as promoters. A gene can include several operably linked segments such as a promoter, a 5' leader sequence, an intron, a coding sequence and a 3' untranslated sequence or 3' untranslated region (3'UTR). contains a polyadenylation site or signal sequence. A chimeric or recombinant gene (such as the FGF21 gene) is
Genes not normally found in nature, such as genes whose promoters are 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 into an active protein.

Promoter As used herein, the term "promoter" refers to a gene (or coding sequence) that functions to control the transcription of a gene or genes (or coding sequences) upstream relative to the direction of transcription of the transcription start site of that gene. A nucleic acid fragment located in a DNA-dependent RNA polymerase binding site, a transcription initiation site, as well as, but not limited to, a transcription factor binding site,
Any other DNA sequence containing repressor and activator protein binding sites, and any other nucleotide known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Structurally specified by the presence of a sequence. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated depending on 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- and tissue-specific promoters regulate the expression of one or more genes (or coding sequences) primarily in one organ or tissue, but at detectable levels ("leakage") in other organs or tissues as well. ) can be tolerated. Leaky expression in other organs or tissues is relative to organ- or tissue-specific expression as assessed by standard assays known to those skilled in the art (e.g. PCR, Western blot analysis, ELISA). It means 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 but 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 capable of initiating transcription in adipose tissue, but still having some leaky expression in other (up to 5, 6, 7 or 8) organs or parts of the body. allow. Transcription in adipose tissue can be detected in adipose tissue and adipocytes such as white adipocytes, brown adipocytes, beige adipocytes, preadipocytes, stromal vascular cells, and the like. 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 parts of the body. . 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 permitting some leaky expression in other (up to 5, 6, 7 or 8) organs or parts of the body. . Transcription in skeletal muscle can be detected in skeletal muscle cells, such as muscle cells, myoblasts, satellite cells, and the like.

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

A suitable promoter for organ-specific and/or tissue-specific expression of a nucleotide sequence encoding FGF21 includes the human α1-antitrypsin promoter in combination with the hepatocyte control region (HCR) enhancer from apolipoprotein E. α1-
antitrypsin promoter, 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 , phenylalanine hydroxylase promoter, adipocyte protein 2 (aP2, also known as fatty acid binding protein 4 (FABP4)) promoter, PPARy promoter, adiponectin promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, human aromatase cytochrome p
450 (p450arom)-derived promoter mini/aP2 promoter (
(composed of adipose-specific aP2 enhancer and basal aP2 promoter), uncoupling protein 1 (UCP1) promoter, mini/UCP1 promoter (composed of adipose-specific UCP1 enhancer and basal UCP1 promoter), adipsin promoter, leptin promoter, Foxa-2 promoter, myosin light chain promoter, myosin heavy chain promoter, desmin promoter, C5-12 promoter, muscle creatine kinase (MCK) promoter, smooth muscle alpha-actin promoter, CK6 promoter, Unc-45 myosin chaperone B promoter, MC
The basal MCK promoter, Enh358MCK, combined with a copy of the K enhancer
Promoters (MCK enhancer in combination with the 358 bp proximal promoter of the MCK gene).

" Operably linked " means that a regulatory sequence, such as a promoter sequence or regulatory sequence, is preferably linked to a nucleotide sequence of interest encoding FGF21, its promoter or regulatory or regulatory sequence. is preferably FGF2 in a cell and/or subject
1 are defined herein as appropriately positioned constructs that direct or influence the transcription and/or production or expression of a nucleotide sequence of interest that encodes 1. For example, a promoter is operably linked to a coding sequence if the promoter is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is "under the control of" the promoter. be understood. Once 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", once said If nucleotide sequences are 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 target nucleotide sequence. understood as a thing.

Viral Expression Constructs Expression constructs carry genomes that are stable in cells and can remain episomal. Within the context of the present invention, cells may be meant to include cells used to make constructs or cells to which constructs are administered. Alternatively, the construct is capable of integrating into the cell's genome, eg, through homologous recombination or otherwise. A particularly preferred expression construct is an FGF21-encoding nucleotide sequence as defined herein is operably linked to a promoter as defined herein, said promoter being activated in a cell by said nucleotide sequence (i.e. sequence) is capable of directing expression. Preferably, said promoter directs expression of said nucleotide sequence in at least one cell of a specific organ and/or specific tissue. Preferably, said promoter directs expression of said nucleotide sequence in at least one cell of liver, adipose tissue and/or skeletal muscle. Preferably, said promoter is expressed in at least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80% of liver, adipose tissue and/or skeletal muscle cells.
, indicates expression at 90% or 100%. In the context of the present invention, FGF21 expressed in liver, adipose tissue or skeletal muscle refers to liver, liver, adipose tissue or skeletal muscle as compared to other organs or tissues.
Preferential or predominant (at least 10% more, at least 20% more, at least 30% more, at least 40% more, at least 5
0% more, at least 60% more, at least 70% more, at least 80% more, at least 90% more, at least 100% more, at least 150% more, at least 20
0% or more) expression. Throughout this application, when liver-specific or adipose-specific or skeletal muscle-specific is mentioned in the context of expression, the cell-type-specific of the cell type(s) comprising liver, adipose tissue or skeletal muscle respectively expression is also expected.

The viral expression constructs of the present invention contain nucleotide sequences in a form "suitable for expression in a mammal", which means that the viral expression construct is selected based on the mammalian host cell in which it will be used for expression. It is meant to include one or more regulatory sequences operably linked to the nucleotide sequence that permits. Preferably, said mammalian host cells used for expression are human, murine or canine cells.

Viral expression constructs of the invention include nucleotide sequences that are expressed in liver, adipose tissue and/or skeletal muscle.

As used herein, "adipose tissue" refers to mature adipocytes (i.e. adipocytes)
and a tissue composed of a combination of small blood vessels, nerve tissue, lymph nodes and stromal vascular cells (SVF). SVF is composed of endothelial cells, fibroblasts, adipocyte precursors (ie, 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 depot (depo
t) includes, but is not limited to, epidydymal WAT (
eWAT), inguinal WAT (iWAT), retroperitoneal WAT (rWAT), mesenteric WAT (m
WAT), interscapular BAT (iBAT).

As used herein, "skeletal muscle" refers to tissue composed of muscle fibers. A muscle fiber, also known as a myofiber, is a single multinucleated or syncytial cell resulting from the fusion of hundreds of myoblasts, some of which are satellites. It remains in mature muscle as undifferentiated cells known as cells. Individual muscle fibers are surrounded by connective tissue called endomysium. Approximately 10 to 100 muscle fibers form a fascicle or bundle, which are themselves surrounded by another layer of connective tissue called the perimysium. Ultimately, skeletal muscle is formed by a group of fiber bundles surrounded by another layer of connective tissue 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 aponeurosis, dense connective tissue structures that mediate the attachment of muscle to the periosteum or other connective tissue of muscle.

As used herein, "liver" refers to tissue composed of hepatocytes. Hepatocytes represent approximately 50-70% of the cells in the liver. In addition to hepatocytes, the liver also contains endothelial cells,
It is composed of parasinusoidal cells, oval cells, Kupffer cells and astrocytes (Ito cells). When activated by Kupffer cells, astrocytes transform into myofibroblasts.
A central vein and a portal track (portal triad) containing the anterior terminal branch of the hepatic artery, the hepatic portal vein, bile canaliculi and lymphatics are also found in the liver.

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 and is operably linked to a promoter capable of directing the expression of said nucleotide sequence. In certain embodiments, the expression cassette used herein is FG
comprising or consisting of a nucleotide sequence encoding F21, 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 cassettes are microRNAs expressed in the liver that are fully complementary to their cognate microRNAs.
and/or contain nucleotide sequences encoding target sequences of microRNAs expressed in the heart. In another embodiment, the described expression cassette comprises one or more nucleotide sequence(s) encoding microRNA binding sites that are imperfectly complementary (1 mismatch/5 consecutive nucleotides). ). In yet another embodiment, the expression cassette may contain both nucleotide sequences encoding complete and incomplete microRNA binding sites. The expression cassette is therefore a single complete microRNA
target site, multiple complete microRNA target sites, single incomplete microRNA target site, multiple incomplete microRNA target sites, or complete and incomplete microRNA
Nucleotide sequences encoding a combination of target sites can be used to tailor the level of regulation. In addition, nucleotide sequences encoding different microRNA target sites can be used, thus allowing a gene to be regulated by multiple microRNAs. A preferred position for the nucleotide sequence encoding the target sequence of the microRNA is the 3'UTR. However, the coding sequence or 5'UT
A nucleotide sequence (encoding the target sequence) inserted into either of the R sequences can also be used.

The choice of nucleotide sequence encoding the microRNA target sequence is determined by the desired expression pattern. The presence of an endogenous microRNA in a cell inhibits expression of a gene or coding sequence from an expression construct containing a nucleotide sequence encoding the target sequence of said microRNA. For expression of a gene or coding sequence of interest to be inhibited in a given cell type, a nucleotide sequence is chosen that encodes a target sequence recognized by microRNAs present in that cell type.

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 herein below The expression construct disclosed herein contains a nucleotide sequence encoding said FGF21 in a suitable cell, e.g. It can be prepared using recombinant techniques for expression in cultured cells or cells of multicellular organisms, see, for example, Ausubel et al. , “
Current Protocols in Molecular Biology",
Greene Publishing and Wiley-Interscience
, New York (1987) and Sambrook and Russell (2
001, supra); and others; both documents are incorporated herein by reference in their entireties. Kunkel (1985) Proc. Natl. Acad. Sci. 82
:488 (describing site-directed mutagenesis) and Roberts et al. (
1987) Nature 328:731-734 or Wells, J.; A. ,et
al. (1985) Gene 34:315 (describing cassette mutagenesis).

Typically, a nucleic acid or nucleotide sequence encoding FGF21 is used in an expression construct or vector. The phrases "expression vector" or "vector" generally refer to a nucleotide sequence capable of directing expression of a gene or coding sequence in a host compatible with that gene or coding sequence. These expression vectors typically include at least suitable promoter sequences and, optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression, as described herein, can also be used. A nucleic acid or DNA or nucleotide sequence encoding FGF21 is incorporated into a DNA construct capable of introduction and expression into in vitro cell culture. Specifically, the DNA construct may be used in prokaryotic hosts such as bacteria,
As an example, E. It can be introduced into mammalian, plant, insect (eg Sf9), yeast, fungal or other eukaryotic cell lines that are suitable for replication in E. coli or the like, or cultured.

A DNA construct prepared for introduction into a particular host is operably linked to a replication system recognized by the host, the intended DNA segment encoding the desired polypeptide, and the segment encoding the polypeptide. can include transcription and translation initiation and termination regulatory sequences. The term "operably linked" has already been defined herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates transcription of the coding sequence. DN of signal sequence
A is operably linked to DNA encoding a polypeptide if it is expressed as a protein precursor that participates in the secretion of the polypeptide. Generally, DNA sequences that are operably linked 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 at alternatively inserted adapters or linkers, or by gene synthesis.

Selection of an appropriate promoter sequence generally depends on the host cell chosen for expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters that are well known in the art (eg, Sambrook
K and Russell, 2001). Transcriptional regulatory sequences typically include heterologous enhancers or promoters that are recognized by the host. The selection of an appropriate promoter sequence is host dependent, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (eg Sambrook and Russell, 20, supra).
01). Expression vectors include a replication system and transcriptional and translational regulatory sequences, along with a site for insertion of a segment encoding a polypeptide. In most cases, the replication system is the cell (bacterial cell such as E. coli) used to make the vector.
Only works inside. Most plasmids and vectors do not replicate in the cells they infect. An example of a viable cell line and expression vector combination is Sambroo
K and Russell (2001, supra) and Metzger et al. (
1988) Nature 334 :31-36. For example, suitable expression vectors are yeast, such as S. cerevisiae. cerevisiae, e. g. , insect cells, e.g. S
f9 cells, mammalian cells such as CHO cells, and bacterial cells such as E. coli
can be expressed in The cell may therefore be a prokaryotic or eukaryotic host cell. The cells can be cells suitable for culture in liquid media or on solid media.

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

Viral vectors Viral vectors or viral gene therapy vectors are vectors comprising a viral expression construct as defined above.

Viral vectors or viral gene therapy vectors are vectors suitable for gene therapy. Vectors suitable for gene therapy are described in Anderson 1998, Nature
392 :25-30; Walther and Stein, 2000, Drugs 6
0 :249-71; Kay et al. , 2001, Nat. Med. 7 :33-4
0; Russell, 2000,J. Gen. Virol. 81 :2573-604;
Amado and Chen, 1999, Science 285 :674-6;
derico, 1999, Curr. Opin. Biotechnol. 10 :448-
53; Vigna and Naldini, 2000, J. Am. 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. Am.
Gen. Virol. 80 :3049-64; Reiser, 2000, Gene T.
her. 7 :910-3; and in the references cited therein.

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

Another suitable gene therapy vector is a retroviral vector. A preferred retroviral vector for use in the present invention is a lentivirus-based expression construct. Lentiviral vectors have the ability to infect dividing and non-dividing cells and integrate stably into their genome (Amado and Chen,
1999 Science 285:674-6). Methods for the construction and use of lentiviral-based expression constructs are described in US Pat. Nos. 6,165,782, 6,
207,455, 6,218,181, 6,277,633 and 6,32
3,031, and Federico (1999, Curr Opin Biote
chnol 10:448-53) and Vigna et al. (2000, J.G.
ene Med 2000;2:308-16).

Other suitable gene therapy vectors include adenoviral, herpes viral, polyoma viral or vaccinia viral vectors.

The gene therapy vector comprises a nucleotide sequence encoding FGF21 to be expressed,
Said nucleotide sequence is thereby operably linked to the appropriate regulatory sequences. Such regulatory sequences include at least promoter sequences. FGF21 from gene therapy vectors
Suitable promoters for the expression of a nucleotide sequence encoding include, for example, the CMV promoter, such as the viral long terminal repeat promoter (LTR) from mouse Moloney leukemia virus (MMLV), Rous sarcoma virus or HTLV-1, simian Virus 40 (SV40) early promoter, CAG promoter, α1-antitrypsin promoter, mini/aP2 promoter, mini/UCP1 promoter, C5-12 promoter and herpes simplex virus thymidine kinase promoter.

It has been described that some inducible promoter systems can be induced by administration of organic or inorganic low-molecular-weight compounds. Such inducible promoters include those controlled by heavy metals, such as the metallothionine promoter (
Brinster et al. 1982 Nature 296 :39-42;
Yo et al. 1982 Cell 29 :99-108), RU-486 (
regulated by progesterone antagonists (Wang et al. 19
94 Proc. Natl. Acad. Sci. USA 91 :8180-8184),
those controlled by steroids (Mader and White, 1993 Pro
c. Natl. Acad. Sci. USA 90 :5603-5607), those regulated by tetracyclines (Gossen and Bujard 1992 Proc
. Natl. Acad. Sci. USA 89 :5547-5551; US Patent Nos. 5,4
64,758; Furth et al. 1994 Proc. Natl. Acad
. Sci. USA 91 :9302-9306; Howe et al. 1995 J.
. Biol. Chem. 270 :14168-14174;
al. 1994 Mol. Cell. Biol. 14 :1669-1679;
Ockett et al. 1995 Proc. Natl. Acad. Sci. US
A 92 :6522-6526), and multiple 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, US6,432,705).
Those controlled by

Gene therapy vectors may further comprise nucleotide sequences encoding additional polypeptides.

Gene therapy vectors are 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 hereinbefore defined.

Adeno-associated virus vector (AAV vector)
A preferred viral vector or preferred gene therapy vector is an AAV vector. AAV vectors used herein are preferably recombinant AAV vectors (r
AAV vectors). As used herein, a "rAAV vector" is a recombinant vector containing a portion of the AAV genome encapsidated within a protein shell of capsid proteins derived from the AAV serotypes described herein. A vector. A portion of the AAV genome includes adeno-associated virus serotypes such as AAV1, AAV2, AAV3, AAV4, A
It may contain inverted terminal sequences (ITRs), such as from AV5. Preferred ITRs are those of AAV2 represented by sequences comprising or consisting of SEQ ID NO:48 (5'ITR) and SEQ ID NO:49 (3'ITR). The present invention also preferably provides SEQ ID NO: 48 as the 5'ITR and at least 80% (or at least 81%, 82%, 83%, 84%
, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%
, 94%, 95%, 96%, 97%, 98%, 99% or 100%) and sequences with at least 80% identity to SEQ ID NO: 49 as the 3'ITR. contain.

The protein shell containing the capsid proteins is isolated from AAV serotypes such as AAV1, AA
It can be derived from V2, AAV3, AAV4, AAV5, and the like. Preferred AAV capsids are those of AAV1, AAV3, AAV8, AAV9. Preferred ITR
is from AAV2. A protein shell may also be termed a capsid protein shell. A rAAV vector may have one, or preferably all, wild-type AAV genes deleted, but may still contain functional ITR nucleic acid sequences. Functional ITR sequences are required for replication, rescue and packaging of AAV virions. The ITR sequence can be a wild-type sequence or at least 80%, 85%, 90%,
They may have 95%, 97%, 98%, 99% or 100% sequence identity or may vary, for example, by insertion, mutation, deletion or substitution of nucleotides so long as they remain functional. Functionality, in this context, refers to the ability of the genome to be packaged directly into the capsid shell and then to allow expression 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.

A nucleic acid molecule represented by a selected nucleic acid sequence is preferably a rAAV
Inserted between genomic or ITR sequences, which are, for example, expression constructs comprising expression regulatory elements and 3' terminal sequences operably linked to a coding sequence. Said nucleic acid molecule is sometimes referred to as a transgene.

"AAV helper functions" generally refer to the r
Refers to the corresponding AAV functions required for AAV replication and packaging. AAV helper functions complement lost AAV functions in rAAV vectors, whereas AAV IT
Lacks R (that provided by the rAAV vector genome). AAV helper functions encompass the two major ORFs of AAV, the rep coding region and the cap coding region, or sequences that are substantially functionally identical thereto. Rep and Cap
Regions are well known in the art, for example Chiorini et al. (1999
, J. of Virology, Vol 73(2): 1309-1319) or U.
S5,139,941, which documents are incorporated herein by reference. AAV helper functions can be provided on the AAV helper construct. Introduction of the helper construct into the host cell occurs, for example by transformation, transfection or transduction, prior to or concurrently with introduction of the rAAV genome present in the rAAV vector identified herein. can be done. The AAV helper constructs of the present invention are therefore of the desired serotype on the one hand for the capsid protein shell of the rAAV vector and on the other hand for the replication and packaging of the rAAV genome present in said rAAV vector. It can be chosen to produce a combination.

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 types 1 and 2) and vaccinia virus. Additional functions provided by this helper virus are also described in US Pat. No. 6,531,
456, which is incorporated herein by reference.

A "transgene" is a gene or coding sequence or nucleic acid molecule (i.e., a molecule encoding FGF21) newly introduced into a cell, i. defined herein as a gene that may only be expressed in In this context, "insufficient" means that although said FGF21 is expressed in cells, the symptoms and/or diseases defined herein can still develop. In this case, the present invention allows overexpression of FGF21.
A transgene can contain sequences that are native to the cell, sequences that do not naturally occur in the cell,
This may include a combination of both. The transgene comprises FGF21 and/or additional proteins identified hereinabove, which can be operably linked to appropriate regulatory sequences for expression of the FGF21-encoding sequence in a cell. It may contain a coding sequence. Preferably, the transgene is not integrated into the genome of the host cell.

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

Production of AAV vectors Production of recombinant AAV (rAAV) to vectorize transgenes has been previously described. Ayuso E, et al. , Curr. Gene Ther. 2010
10:423-436, Okada T, et al. , Hum. Gene Ther
. 2009;20:1013-1021, Zhang H, et al. , Hum. G.
ene Ther. 2009;20:922-929 and Virag T, et al.
l. , Hum. Gene Ther. 2009;20:807-817. These protocols are used or adapted to generate the AAVs of the invention. In one embodiment, the production cell line encodes a polynucleotide of the invention (comprising an expression cassette flanked by ITRs) and rep and cap proteins,
Construct(s) providing helper functions are transiently transfected.
In another embodiment, the cell line stably supplies helper functions and a polynucleotide of the invention (including an expression cassette flanked by ITRs) and construct(s) encoding rep and cap proteins are Transiently transfected. 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 polynucleotides of the invention and polynucleotides encoding helper functions. In yet another embodiment, the cell line is a polynucleotide of the invention, re
Stably supplies p and cap proteins and helper functions. Methods of making and using these and other AAV production systems have been described in the art. Muz
Yczka N, et al. , US 5,139,941; Zhou X, et al.
, US 5,741,683; Samulski R, et al. , US 6,057, 1
52, Samulski R, et al. , US 6,204,059, Samulsk
iR, et al. , US 6,268,213, Rabinowitz J, et al.
l. , US 6,491,907; Zolotukhin S, et al. , US6,
660, 514, Shenk T, et al. , US 6,951,753, Snyde
rR, et al. , US 7,094,604, Rabinowitz J, et al.
l. , US 7,172,893; Monahan P, et al. , US 7,201,
898, Samulski R, et al. , US 7,229,823 and Ferr
ari F, et al. , US 7,439,065.

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

Whole genomes and corresponding ITRs of several AAV serotypes have been sequenced (
Chiorini et al. 1999,J. of Virology Vol.
73, No. 2, pp 1309-1319). These can be cloned or sold by Applied Biosystems Inc., for example. (Fosters, Calif.)
can be made by chemical synthesis known in the art using an oligonucleotide synthesizer supplied by Co., Inc., USA), or by standard molecular biology techniques.
ITRs can be cloned from the AAV viral genome or AAV IT
It can be excised from a vector containing R. The ITR nucleotide sequences can be ligated at either end to a nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or the AAV sequence between the ITRs can be ligated to the desired nucleotide sequence. You can replace it with an array.

Preferably, the rAAV genome present in the rAAV vector does not include any nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or cap (capsid) genes. This rAAV genome is detectable and /or genes encoding selectable products (eg, lacZ, aph, etc.), and the like.

The rAAV genome present in said rAAV vector further comprises a promoter sequence operably linked to the nucleotide sequence encoding FGF21. Preferred promoter sequences are promoters that confer expression in skeletal muscle cells and/or muscle, in liver cells and/or liver, and in adipocytes and/or adipose tissue. Examples of such promoters include CMV, CAG, m
ini/aP2, mini/UCP1, C5-12 and hAAT promoters.

A suitable 3' untranslated sequence can also be operably linked to the nucleotide sequence encoding FGF21. A suitable 3' untranslated region may be that which is naturally associated with the nucleotide sequence or may be derived from a different gene, such as SV4
0 polyadenylation signal (SEQ ID NO:50) and rabbit β-globin polyadenylation signal (SEQ ID NO:51).

Optionally, additional nucleotide sequences may be operably linked to the FGF21-encoding nucleotide sequence(s), such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, and the like.

Codon optimization "Codon optimization" as used herein means to modify an existing coding sequence or to design a coding sequence, e.g. Refers to the process used to improve translation in an expression host cell or organism or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, coding sequences to match the codon preferences of the expression host organism, e.g., to match the codon preferences of a mammalian, preferably murine, canine or human expression host. Processes involving selecting codons for are included. Codon optimization may also include 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 structural or instability motifs) are removed.

In this document, and in the claims thereto, the verb "to com
"prise" and variations thereof are used in a non-limiting sense to mean that items following the term are included, but do not exclude items not specifically mentioned.
In addition, the verb ``to consist'' can be changed to ``to consist''.
consist essentially of), which may be replaced by additional viral expression constructs, viral vectors, compositions, gene therapy compositions defined herein other than those specifically identified. Constituent(s) may be included, said additional constituent(s) not altering the inherent properties of the invention.

In addition, references to an element by the indefinite articles "a" or "an" indicate that there may be more than one element unless the context clearly requires that the element be unique. do not exclude The indefinite article "a" or "an" is therefore usually used for "at least one"
means

The word "approximately" or "about" when used in connection with a numerical value (approximately 10, about 10)
, preferably means that the value is 1% around a given value of 10.

All patents and references cited herein are hereby incorporated by reference in their entirety. Each embodiment identified in this specification may be combined unless otherwise indicated.

The invention is further illustrated in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

Figure legend:
Figure 1. Prevention of obesity by intra-eWAT administration of AAV9-CAG-moFGF21-dmiRT vector in C57Bl6 mice. (A) AAV-CAG-moFGF21-d
Schematic representation of the oublemiRT vector. The expression cassette contained the CAG promoter, a codon-optimized mouse FGF21 coding sequence, and four tandem repeats of miRT122a and four tandem repeats of miRT1 sequences cloned into the 3′ untranslated region of the expression cassette. . ITRs from AAV2 flanked the expression cassette. Diagrams are not to scale. CAG: chicken β-actin promoter/CMV enhancer; pA: polyA. (B) Expression levels of FGF21 in metabolic tissues. C57B
Codon-optimized mouse F in eWAT, iWAT, iBAT and liver of l6 mice
Expression levels of the GF21 coding sequence were measured by RTqPCR and normalized by Rplp0 values (n=8-11 animals/group). (C) Circulating levels of FGF21 (n=8-11 animals/group). (DE) Expression levels of FGF21R1 (D) and β-Klotho (E) in metabolic tissues. FGF in eWAT, iWAT, iBAT and liver of C57Bl6 mice
21 receptor 1 (FGF21R1) and β-Klotho expression levels were measured by RTqPCR and normalized by Rplp0 values (n=7 animals/group). (F) Weight evolution. Body weights were measured weekly (n=8-11 animals/group). (G) Representative images of animals. (H) Tissue weight. Chow-fed C57Bl6 mice treated with AAV vectors in eWAT and HF
eWAT, iWAT, rWAT, mWAT, iBAT and liver weights of D-fed C57B16 mice (n=8-11 animals/group). Analysis was performed with 10 ¹² vg of AAV9-CAG-mo
eWA of FGF21-doublemiRT or AAV9-CAG-null vectors
It was performed 14 weeks after intra-T administration. Results are expressed as mean ± SEM. ND, not detected. HFD, high fat diet; AU, arbitrary units. eWAT, epididymal white adipose tissue; iWAT,
Inguinal white adipose tissue. rWAT, retroperitoneal white adipose tissue; mWAT, mesenteric white adipose tissue;
iBAT Interscapular brown adipose tissue. *p<0.05 vs AAV9-CAG-null
l chow, **p<0.01 vs AAV9-CAG-null chow, *
**p<0.001 vs AAV9-CAG-null chow, $p<0.0
5 vs AAV9-CAG-null HFD, $$ p<0.01 vs AAV9
-CAG-null HFD, $$ p<0.001 vs AAV9-CAG-nu
ll HFD.

Figure 2. The AAV9-CAG-moFGF21-doublemiRT vector was eWAT
Histological analysis of adipose tissue and liver of C57Bl6 mice treated i.v. (A) AAV9-C
Chow-fed C57Bl6 mice and HFD-fed C57Bl treated with AG-moFGF21-doublemiRT or AAV9-CAG-null vector in eWAT
Representative images of sections of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), interscapular brown adipose tissue (iBAT) and liver from 6 mice stained with hematoxylin and eosin. Original magnification x 100. (B) Average area of white adipocytes in eWAT (n
= 4 animals/group). (C) Frequency distribution of white adipocyte area in eWAT (n=4 animals/group).
Analyzes were performed 14 weeks after intra-eWAT administration of 10 ¹² 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. AAV
9-CAG-null chow, *** p<0.001 vs AAV9-CAG-
null chow, $$ p<0.01 vs AAV9-CAG-null HFD
, $$ p<0.001 vs AAV9-CAG-null HFD.

Figure 3. The AAV9-CAG-moFGF21-doublemiRT vector was eWAT
Enhanced energy expenditure and insulin sensitivity in intra-treated C57Bl6 mice. (AB) Expression levels of UCP1 (A) and Dio2 (B). UCP1 in iWAT
and Dio2 expression levels were measured by RTqPCR and normalized with Rplp0 values (
n=7 animals/group). (C) Energy metabolism. Energy expenditure (EE) was measured with an indirect open circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were acquired during the light (basal) and dark (active) cycles after 9 weeks of AAV administration 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, assays were performed with 10 ¹² vg of AAV9-CA
It was performed 14 weeks after intra-eWAT administration of G-moFGF21-doublemiRT or AAV9-CAG-null vector. Results are expressed as mean ± SEM. HFD
, a high-fat diet. TG, triglyceride; Chol, cholesterol; *p<0.05v
s AAV9-CAG-null chow, **p<0.01 vs AAV9-C
AG-null chow, ***p<0.001 vs AAV9-CAG-nul
l Chow, $p<0.05 vs AAV9-CAG-null HFD, $$
p<0.01 vs AAV9-CAG-null HFD, $$ p<0.001
vs AAV9-CAG-null HFD.

Figure 4. Obesity reversal 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 Rplp0
normalized by value. (B) Circulating levels of FGF21. (CD) Evolution of body weight (C) and weight gain (D). Body weight was measured weekly. (E) Tissue weight. eWAT, iWAT, rWAT, mWAT, iBAT and liver weights of ob/ob mice treated with AAV vectors into eWAT. The assay was 10 ¹⁰ vg, 5 x 10 ¹⁰ vg, 2 x 10 ¹¹ vg or 10
¹² vg of AAV8-CAG-moFGF21-doublemiRT or 10 ¹² v
g of AAV8-CAG-null vector in eWAT 16 weeks after administration. 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 A
AV8-CAG-null, ***p<0.001 vs AAV8-CAG-null
l.

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

Figure 6. Intravenous administration of AAV8-hAAT-moFGF21-vector reverses obesity and improves glucose metabolism in ob/ob mice. (A) AAV-hAAT-mo
Schematic representation of the FGF21 vector. The expression cassette is human α1-antitrypsin (hAAT)
It contained a promoter- and codon-optimized murine FGF21 coding sequence.
ITRs from AAV2 flanked the expression cassette. Diagrams are not to scale. pA:p
oly A. (B) Expression levels of FGF21. The expression level of the codon-optimized mouse FGF21 coding sequence in the liver of ob/ob mice was measured by RTqPCR and Rp
Normalized by the lp0 value. (C) Circulating levels of FGF21. (DE) Evolution of body weight (C) and weight gain (D). Body weight was measured weekly. (F) Representative images of animals. (G) Tissue weight. eWAT, iWAT, rW in ob/ob mice treated intravenously with AAV vectors
AT, mWAT, iBAT and liver weights. (H) Intraperitoneal insulin tolerance test. ob/
ob mice were given an intraperitoneal injection of 0.75 U insulin/kg body weight and blood glucose levels were measured at the indicated time points. The study was performed 9 weeks after AAV administration. (I
) Fasting circulating insulin levels 3 months after AAV administration. Unless otherwise indicated, the analysis was performed in 10
¹¹ vg or 5×10 ¹¹ vg of AAV8-hAAT-moFGF21 or 5×1
It was performed 20 weeks after intravenous administration of 0 ¹¹ vg of AAV8-hAAT-null vector.
Results are expressed as mean±SEM. n=9-10 animals/group. ND, not detected. A.
U, arbitrary units. eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue; rW
AT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue; iBAT Interscapular brown adipose tissue. *p<0.05 vs AAV8-hAAT-null, **p<0.01
vs AAV8-hAAT-null, ***p<0.001 vs AAV8-h
AAT-null.

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. (BC)
Evolution of body weight (C) and weight gain (D). Body weight was measured weekly. Analysis was performed using 10 ¹⁰ vg or 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 or 5×10 ¹⁰ vg
52 weeks after intravenous administration of the AAV8-hAAT-null vector. Results are expressed as mean±SEM, n=9-12 animals/group. ***p<0.001v
s AAV8-hAAT-null chow, $$ p<0.01 vs AAV8-
hAAT-null HFD, $$p<0.001 vs AAV8-hAAT-n
ull HFD.

Figure 8. Long-term enhancement of energy expenditure and insulin sensitivity by intravenous administration of AAV-hAAT-moFGF21 vector in HFD-fed C57B16 mice. (A) Energy metabolism. Energy expenditure (EE) was measured with 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. 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 study was performed 7 weeks after AAV administration. (C) Fasting and fed insulin circulating levels. Results are expressed as mean±SEM, n=9-12 animals/group. HFD, high fat diet; * p < 0
. 05 vs AAV8-hAAT-null chow, **p<0.01 vs.
AAV8-hAAT-null chow, ***p<0.001 vs AAV8-
hAAT-null chow, $ p<0.05 vs AAV8-hAAT-null
l HFD, $$ p<0.01 vs AAV8-hAAT-null HFD, $$
$p<0.001 vs AAV8-hAAT-null HFD.

Figure 9. Obesity reversal by intravenous administration of AAV-hAAT-moFGF21 vector in aged HFD-fed mice. (A) Circulating levels of FGF21. (BC) Evolution of body weight (B) and weight gain (C). Body weight was measured weekly. Analysis is 10 ¹⁰ vg, 2 x 10 ¹⁰ v
g or 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 or 5×10 ¹⁰
It was performed 21 weeks after intravenous administration of the vg AAV8-hAAT-null vector. Results are expressed as mean±SEM, n=7-8 animals/group. HFD, high fat diet; ＊＊＊
p<0.05 vs AAV8-hAAT-null chow, $ p<0.05
vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8-
hAAT-null HFD. $$p<0.001 vs AAV8-hAAT-n
ull HFD.

Figure 10. Improving energy expenditure and insulin sensitivity by intravenous administration of AAV-hAAT-moFGF21 vectors in aged HFD-fed mice. (A) Energy metabolism. Energy expenditure (EE) was measured with an indirect open circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were obtained during the light cycle (basal state) and dark cycle (active phase) 6 weeks after AAV administration 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 study was performed 9 weeks after AAV administration. (C) Fasting and fed insulin circulating levels. Results are mean ± SEM
, n=7-8 animals/group. HFD, high fat diet; **p<0.01
vs AAV8-hAAT-null chow, *** p<0.001 vs AA
V8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT-
null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD
, $$ p<0.001 vs AAV8-hAAT-null HFD.

Figure 11. Weight loss by intramuscular administration of AAV-CMV-moFGF21 vector in C57Bl6 mice. (A) Schematic representation of the AAV-CMV-moFGF21 vector. The expression cassette contained the cytomegalovirus (CMV) promoter and codon-optimized murine FGF21 coding sequence. ITRs from AAV2 flanked the expression cassette. Diagrams are not to scale. pA: polyA. (B) Circulating FGF21 levels. (
CD) Evolution of body weight (C) and weight gain (D). Body weight was measured weekly. Results are mean ±
Expressed as SEM. n=6-7 animals/group. *p<0.05 vs AAV1-C
MV-null, **p<0.01 vs AAV1-CMV-null. FG in the figure
Labeling of F21 refers to moFGF21 according to the legend of this figure.

Figure 12. FGF2 by Codon Optimization of the Nucleotide Sequence Encoding Human FGF21
1 increased protein production. (A) Wild-type hFGF21 or codon-optimized human FG
hFGF21 protein levels in the culture medium of HEK293 cells transfected with three different versions of the F21 sequence. Results are expressed as mean ± SEM. n=3
well/group. ND, not detected. *p<0.05 vs non-transfected cells.

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

(A) eWAT and (B) obtained from ob/ob animals injected intra-eWAT at all doses tested with either null or an AAV8 vector encoding FGF21.
) Representative images of hematoxylin-eosin staining of liver tissue sections. Scale bar: eW
100 μm for AT, 200 μm for liver.

C Blood glucose in the fed state.

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

The FGF21 label in the figure refers to moFGF21.

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

Figure 14. Effect of FGF21 gene transfer on eWAT in ob/ob mice.

A AAV8-CAG-null vector or AAV8-CAG-moF at 11 weeks of age
Four different doses of either GF21-dmiRT vector (1×10 ¹⁰ , 5×10
¹⁰ , 2×10 ¹¹ , 1×10 ¹² vg/mouse) at 25 weeks of age injected intra-eWAT
Serum adiponectin levels in b/ob animals.

B qRT-PC of the macrophage marker F4/80 of the same animal as in (A)
Expression quantification by R. Representative images of immunostaining for the macrophage-specific marker Mac2 of 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 intra-eWAT treatment groups.

E, F Liver triglyceride and cholesterol content in the fed state in the same cohort as in (A).

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data information: All values are expressed as mean±SEM. n in (A, B, D)
= 4-8 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus null injected ob/ob group.

Figure 15. Reduced obesity and improved insulin sensitivity in ob/ob mice treated with AAV8-hAAT-moFGF21 vector.

Representative images of hematoxylin-eosin staining of eWAT tissue sections obtained from ob/ob animals injected with either A null or AAV vectors encoding FGF21 at 1×10 ¹¹ or 5×10 ¹¹ vg/mouse.

B Serum adiponectin levels in all groups.

Representative images of hematoxylin-eosin staining of liver tissue sections obtained from ob/ob animals injected with either C null or AAV vectors encoding FGF21 at 1×10 ¹¹ or 5×10 ¹¹ vg/mouse.

D Feeding blood glucose levels.

E Feeding serum insulin levels at 5 months post AAV.

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data information: All data are expressed as mean±SEM. n=9-10 animals/group in (AC, E, GH). *P<0.05, **P<0.01 and ***P<0.
001 vs null injected ob/ob group.

Figure 16. Effect of FGF21 liver gene transfer on ob/ob mice.

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

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

D, E Weights (D) and representative images (E) of livers obtained from animals belonging to the same experimental groups as in (A).

F, G Liver triglyceride and cholesterol content in the fed state in the same cohort as in (A).

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

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 null injected ob/ob group.

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 AAV8-hAAT-null vector or AAV8-hAAT- at 2 months of age
Liver PEPCs in ob/ob mice injected with either moFGF21 vector
Expression quantification by qRT-PCR of K and G6Pase.

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

G Relative expression levels of UCP1 in iBAT in the same cohort as in (A).

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data Information: All values are expressed as mean±SEM. n = 9 in (AG)
~10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 vs.
null injection ob/ob group.

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

A AAV8-hAAT-moFG as young adults (upper panel) or adults (lower panel)
Epididymal (eWAT), inguinal (iWA
T) and retroperitoneal (rWAT) white adipose tissue depot, liver and quadriceps weights.

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

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

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

Figure 19. FGF21 gene transfer into the liver counteracts HFD-induced obesity.

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

C Several doses of AAV8-hAAT-mo as young adults (left) or adults (right)
Representative images of epididymal white adipose (eWAT) pads obtained at sacrifice from animals treated with FGF21.

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

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

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data information: All values are expressed as mean±SEM. n = 7 to 1 in (E)
0 animals/group. HFD, high fat diet; ND, not detected.

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

A Assessment of locomotor activity through the open field test in animals (young adults) subjected to HFD feeding from approximately 2 months of age and treated 2 months later with either null or a vector encoding FGF21.

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 Expression quantification by qRT-PCR of Phospho1 in iWAT in young adults or in groups of animals that started HFD feeding as adults and received FGF21 vector.

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data Information: All values are expressed as mean±SEM. n = 7 in (AC)
~10 animals/group. In (E) n=4 animals/group. In (G) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus chow fed N
ull injection group. ^#P <0.05 and ^### P<0.001 versus HFD-fed Null injection group. HFD, high fat diet;

Figure 21. Energy consumption in October after gene transfer to the liver.

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

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

C Relative expression levels of Serca2b and RyR2 in iWAT in young adults or groups of animals that started HFD feeding as adults and received FGF21 vectors.

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

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

Figure 22. Restoration of AAV8-hAAT-moFGF21-mediated islet hyperplasia.

A Fasting glucagon levels in a group of animals that started on HFD feeding as young adults and received the FGF21 vector.

B, β-cell mass in a group of animals that began HFD feeding as adults and received the FGF21 vector.

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

D Duplicates for insulin (dark grey) and glucagon (light grey) in pancreatic sections from animals receiving 5×10 ¹⁰ vg/mouse of AAV8-hAAT-moFGF21 as young adults (top panels) or adults (bottom panels). Representative images of immunostaining. Scale bar: 100 μm.

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data Information: All values are expressed as mean±SEM. n = 7 in (AC)
~10 animals/group. In (D) n=4-5 animals/group. *P<0.05, **P<0.0
1 and ***P<0.001 vs chow-fed Null injection group. ^#P <0.05, ^#
#P <0.01 and ^### P<0.001 versus HFD-fed Null injection group. HFDs,
high-fat diet.

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 groups of mice that received FGF21 vector starting on HFD feeding as young adults.

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

Data information: All data are expressed as mean±SEM. n = 7 in (AD)
~10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 vs.
Chow-fed Null injection group. ^#P <0.05, ^## P<0.01 and ^### P<0.
001 vs. HFD-fed Null-injected group. HFD, high fat diet;

Figure 24. Reversal of WAT hypertrophy and inflammation by AAV8-hAAT-moFGF21 treatment.

A AAV8-hAAT-null or 5×10 ¹⁰ vg/mouse of AAV8-hAAT fed chow or HFD as young adults (left panel) or adults (right panel)
-Hematoxylin of eWAT from animals administered either of the moFGF21 vectors-
Representative images of eosin staining. 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.

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

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

E Immunohistochemistry for the macrophage-specific marker Mac2 in eWAT sections from animals receiving 5×10 ¹⁰ vg/mouse AAV8-hAAT-moFGF21 as adults. Micrographs show the presence of crown-like structures in eWAT of HFD-fed, null-injected animals (arrows and insets), but not in eWAT of HFD-fed, FGF21-treated mice. Scale bar: 200 μm and 50 μm (inset).

FH qRT-PC of inflammatory markers F4/80 (F), IL1-β (G) and TNF-α (H) in a group of animals that started HFD feeding as adults and received FGF21 vector
Expression quantification by R.

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data information: All values are expressed as mean±SEM. In (B) n=4 animals/group. In (FH) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^#P <0.05, ^## P
<0.01 and ^### P<0.001 versus HFD-fed Null-injected group. HFD, high fat diet;

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

A AAV8-hAAT-null or 5×10 ¹⁰ vg/mouse of AAV8-hA starting on chow or HFD feeding as young adults (top graph) or adults (bottom graph)
Frequency distribution of adipocyte area in groups of animals that received either AT-moFGF21 vector.

B, Mac2 immunohistochemistry in eWAT of animals beginning the study as young adults. HF
Crown-like structures formed by macrophage infiltration in eWAT of D-fed null-injected mice are indicated by arrows. Scale bar: 200 μm and 50 μm (inset)
.

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

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data Information: All values are expressed as mean±SEM. n=4 animals/group in (A). In (CE) n=7-10 animals/group. ***P<0.001 vs chow-fed Null-injected group. ^### P<0.001 vs HFD-fed Null-injected group. HFDs,
high-fat diet.

Figure 26. Treatment with vectors encoding FGF21 reverses fatty liver and liver inflammation.

A AAV8-hAAT-null or 5×10 ¹⁰ cells fed chow or HFD
Representative images of hematoxylin-eosin staining of liver sections obtained from animals treated with either vg/mouse AAV8-hAAT-moFGF21 vectors. HFD clearly induced lipid droplet accumulation in the liver, which is consistent with AAV in both young adults and adults.
8-hAAT-moFGF21 treatment recovered. Scale bar: 100 μm.

B, C Liver triglyceride and cholesterol content at feeding in the same animal cohort.

D Immunostaining for the macrophage-specific marker Mac-2 of liver sections from animals fed HFD and receiving AAV8-hAAT-null or 5×10 ¹⁰ vg/mouse of AAV8-hAAT-moFGF21 vector. Arrows point to the presence of crown-like structures. Scale bar: 200 μm and 50 μm (inset).

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

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

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

5×10 ¹⁰ vg/mouse of AAV8-hAAT-null or AAV8-hAAT
- Analysis of liver fibrosis through Masson's trichrome staining in HFD-fed animals that received either moFGF21 vector. AAV8-hAAT-moFGF21 treatment (right panel) significantly reduced detection of readily detectable collagen fibers (blue) in null vector treated animals (left panel). Scale bar: 50 μm. FGF2 in the figure
The label of 1 refers to moFGF21 according to the legend of this figure.

Figure 28. AAV8-hAAT-moFGF21 treatment ameliorates liver fibrosis.

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

B, C Expression quantification by qRT-PCR of Collagen 1 in liver in groups of animals that received FGF21 vector starting on HFD feeding as young adults (B) or adults (C).

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data Information: All values are expressed as mean±SEM. n = 7 in (BC)
~10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 vs.
Chow-fed Null injection group. ^#P <0.05 and ^### P<0.001 versus HFD
Feed Null injection group. HFD, high fat diet;

Figure 29. No bone abnormalities were observed in AAV8-hAAT-moFGF21 treated animals. Long-term effects of FGF21 gene transfer on bone were evaluated in HFD-fed mice treated as young adults or with the highest dose (5×10 ¹⁰ vg/mouse) of AAV8-hAAT-moFGF21 vector as adults versus chow or HFD null injections. Tested by comparison with fed animals.

A Full length from nose tip to ridge.

B tibia length.

H administered either C—O null or an AAV vector encoding FGF21
Epiphyses of tibiae (CJ
) and diaphysis (KO) microcomputed tomography (μCT) analysis.

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

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data information: All data are expressed as mean±SEM. n in (A, PQ)
= 7-10 animals/group. n=4 animals/group in (BO). **P<0.01 and **
*P<0.001 vs chow-fed Null injection group. 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; N, trabecular bone number; Tb. Th, trabecular width; Tb. Sp
, trabecular spacing.

Figure 30. Analysis of glycemic profiles in C57B16 mice treated with AAV8-hAAT-moFGF21 vector. Blood glucose levels were assessed under fed conditions. A.
AV, 5×10 ¹⁰ vg or 2×10 ¹¹ vg of AAV8-hAAT-moFGF21
(n=13 and 15, respectively) or IV administration of 2×10 ¹¹ vg of AAV8-null vector (n=15). STZ, treatment with streptozotocin (5 x 50 mg/kg)
. Results are mean + SEM. *p<0.05;***p<0.001 vs A
AV8-hAAT-Null. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

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

A 3×10 ¹¹ vg/mouse of AAV1-CMV-Null or AAV1-CMV-
Circulating levels of FGF21 measured 40 weeks after injection of either 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 vectors.

C Evolution of body weight during the 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 Feeding serum insulin levels.

Insulin sensitivity assessed through intraperitoneal injection of H insulin (0.75 units/kg body weight) and expressed as a percentage of starting blood glucose.

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data Information: All values are expressed as mean±SEM. n = 5 in (AH)
-7 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus N
ull injection group.

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

A, B Evolution of body weight (A) and weight gain (B) in animals treated with AAV1-CMV-moFGF21. C57Bl6 mice were fed HFD for approximately 12 weeks and then 3 × 10 ¹¹
vg/mouse of AAV1-CMV-moFGF21 vector was administered. Control obese mice and control chow-fed mice received 3×10 ¹¹ vg of AAV1-C
Received MV-null.

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

D,E Levels of fasting blood glucose (D) and fed serum insulin (E) 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 starting blood glucose level.

Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure.

Data Information: All values are expressed as mean±SEM. HFD in (AF)
Fed mice n=10 animals/group; chow-fed mice n=5 animals/group. ***P<0.
001 vs. HFD-fed null-injected group.

Figure 33. FGF2 by Codon Optimization of the Nucleotide Sequence Encoding Human FGF21
1 circulation level increase in vivo. wild-type hFGF21 or codon-optimized human F
Circulating levels of hFGF21 in C57B16 mice hydrodynamically administered plasmids encoding three different variants of the GF21 sequence. Results are mean ± SEM
is expressed as n=9-10 mice/group. ND, not detected. Negative control, untreated mice. *p<0.05 vs untreated mice.

Figure 34. hAAT-moFGF21, CAG-moFGF21-doublemiRT
and in vitro increase in FGF21 expression levels by the CMV-moFGF21 expression cassette. (A) WT mouse FGF21 coding sequence under control of EF1a promoter (EF1a-mFGF21) or codon-optimized mouse FGF21 coding sequence under control of CMV promoter (CMV-moFGF21) or miRT
A plasmid encoding a codon-optimized murine FGF21 coding sequence under the control of the CAG promoter (CAG-moFGF21-doublemiRT) with four tandem repeats of the 122a sequence and four tandem repeats of the miRT1 sequence was transfected. Expression level of FGF21 in transfected HEK293 cells. (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) Plasmids encoding WT mouse FGF21 coding sequence under control of EF1a promoter (EF1a-mFGF21) or codon-optimized mouse FGF21 coding sequence under control of CMV promoter (CMV-moFGF21) were transfected. C2C1
Expression level of FGF21 in 2 cells. (E) W under the control of the EF1a promoter
mouse FGF21 coding sequence of T (EF1a-mFGF21) or codon-optimized mouse FGF21 under the control of the hAAT promoter (hAAT-moFGF2
1) FGF21 in HepG2 cells transfected with a plasmid encoding
expression level. qPCR was performed using primers that detect both the wt and codon-optimized FGF21 coding sequences. Results are expressed as mean ± SEM. n=3 wells/group. ND, not detected. *p<0.05 vs control. #
## p<0.001 vs EF1a-mFGF21.

Figure 35. In vivo increase in hepatic FGF21 expression and circulating FGF21 levels by hAAT-moFGF21 and CMV-moFGF21 expression cassettes. (A) elongation factor 1
WT mouse FGF21 coding sequence under the control of the a(EF1a) promoter (
EF1a-mFGF21) or a codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21) or a codon-optimized mouse FGF21 coding sequence under the control of the hAAT promoter (hAAT-
C57Bl6 hydrodynamically administered with a plasmid encoding moFGF21)
Expression levels of FGF21 in mouse liver. qPCR was performed using primers that detect both the wt and codon-optimized FGF21 coding sequences. (B) (
FGF21 circulating levels in the same cohort as in A). Results are mean ± SEM
is expressed as n=5 mice/group. **p<0.01 vs. EF1a-mFG
F21
Figure 36. Hepatic FGF21 expression and FGF by AAV8-hAAT-moFGF21
21 In vivo increase in circulation levels. ( ^A ) AAV8 vectors ^{( AAV8} -EF1a- m
FGF21) or codon-optimized mouse FG under the control of the hAAT promoter
FGF21 expression levels in the liver of C57Bl6 mice intravenously administered F21 (AAV8-hAAT-moFGF21). qPCR of wt and codon-optimized FGF21
It was performed using primers that detect both coding sequences. (B) FGF21 circulating levels in the same cohort as in (A). Analysis was performed 2 weeks after 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-mFG
F21
Figure 37. In vivo increase in adipose FGF21 expression by AAV8-CAG-moFGF21-dmiRT. (AB) 2×10 ¹⁰ vg, 5×10 ¹⁰ vg or 1×10 ¹¹
vg, WT mouse FGF21 under control of the elongation factor 1a (EF1a) promoter
AAV8 vector encoding coding sequence (AAV8-EF1a-mFGF21)
or an AAV8 vector encoding a codon-optimized murine FGF21 coding sequence under control of the CAG promoter with four tandem repeats of miRT122a sequence and four tandem repeats of miRT1 sequence (AAV8-CAG-moF
Expression levels of FGF21 in eWAT (A) or liver (B) of C57B16 mice intra-eWAT with either GF21-doublemiRT). qPCR is wt
and codon-optimized FGF21 coding sequences. Analysis was performed 2 weeks after AAV. Results are expressed as mean ± SEM. n=4-5 mice/group. Control, untreated mice. eWAT, epididymis (epi
dydimal) White adipose tissue. *p<0.05, **p<0.01, and FIG. In vivo increase of FGF21 expression in skeletal muscle by AAV1-CMV-moFGF21. (AB) 5×10 ¹⁰ vg, 1×10 ¹¹ vg or 3×10 ¹¹ v
g, AAV8 vector (AAV8-EF1a-mFGF21) encoding WT mouse FGF21 coding sequence under control of elongation factor 1a (EF1a) promoter or codon-optimized mouse FGF21 coding sequence under control of CMV promoter Expression levels of FGF21 in quadriceps muscle (A) or liver (B) of C57B16 mice intramuscularly injected with either AAV1 vector encoding (AAV1-CMV-FGF21). qPCR was performed using primers that detect both the wt and codon-optimized FGF21 coding sequences. Analysis was performed 2 weeks after AAV. Results are expressed as mean ± SEM. n=4-5 mice/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.

General Procedures of the Examples Subject Characteristics Male C57B1/6J mice and B6. V-Lep ^ob /OlaHsd(ob/ob
) using mice. Mice were fed a standard diet (2018S Teklad Global Die
ts®, Harlan Labs. , Inc. , Madison, WI, US
) or high fat diet (TD.88137 Harlan Teklad Madison,
WI, US) were fed ad libitum and maintained under a 12 hour light-dark cycle (lights on at 8:00 am) and a stable temperature (22°C ± 2). For tissue sampling, mice were treated with the inhaled anesthetic Isoflurane (IsoFlo®, Abbott Laboratories).
ies, Abbott Park, Ill., US) and decapitated. Tissues of interest were excised and stored at −80° C. or using formalin until analysis. All experimental procedures were followed by the Ethic
s Committee for Animal and Human Experiments
It is approved by the entation.

Recombinant AAV Vectors Single-stranded AAV vectors of serotype 1, 8 or 9 were generated by triple transfection of HEK293 cells according to standard methods (Ayuso, E. et al.
. , 2010. Curr Gene Ther. 10(6) :423-36). Cells were placed in 10 roller bottles (850 cm ² , flat; Corning™, Sig
Ma-Aldrich Co. , Saint Louis, MO, US).
Cultured to 80% confluence in 10% FBS, plasmids carrying expression cassettes flanked by AAV2 ITRs, helper plasmids carrying AAV2 rep genes and cap genes of AAV serotypes 1, 8 or 9, and adenovirus helpers Functional plasmids were co-transfected by the calcium phosphate method. The transgenes used were: 1) miRT12 cloned into the 3' untranslated region of the expression cassette
2a sequence (5'CAAACACCATTGTCACACTCCA3') (SEQ ID NO: 12)
4 tandem repeats of and the miRT1 sequence (5′TTACATACTTCTTTA
CATTCCA 3′) (SEQ ID NO: 13) appended with four tandem repeats of cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter; 2) CMV promoter; or 3) human α1-antitrypsin promoter (
codon-optimized mouse, canine or human FGF2 driven by hAAT)
1 or wt FGF21. Null vectors were generated using non-coding plasmids carrying CAG, hAAT or CMV promoters. AAV was purified using an optimized method based on a polyethylene glycol precipitation step and two sequential cesium chloride (CsCl) gradients. This second-generation CsCl-based protocol dramatically reduced empty AAV capsids and DNA and protein impurities (Ayuso, E. et al., 2010. Cu
rr Gene Ther. 10(6) :423-36). PB the purified AAV vector
It was dialyzed against S, filtered and stored at -80°C. Viral genome titers were determined by quantitative PCR following the protocol described for AAV2 reference standards using linearized plasmid DNA as a standard curve (Lock M, et al., Hum.
ens Ther. 2010;21:1273-1285). The vector was constructed according to molecular biology techniques well known in the art.

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. 0.
AA in PBS containing 001% Pluronic® F68 (Gibco)
The V vector was resuspended and injected directly into the epididymal fat pad. Two injections of 50 μL of AAV solution were made into each epididymal fat pad (one injection near the testicle and one injection in the middle of the fat pad). The abdomen was rinsed with sterile saline solution and closed using a two layer approach.

Systemic Administration of AAV Vectors An appropriate volume of AAV solution was diluted in 200 μL of PBS containing 0.001% Pluronic® and injected manually into the lateral tail vein without pressure at the moment of infusion. . Prior to injection, animals were placed in a 250W infrared heat lamp (Philips NV, Amsterdam).
, NL) for several minutes to dilate the vessels and make the tail vein more visible and easier to access. Plastic retainer (Harvard Apparatus, Holli
Ston, Mass., US) was used to immobilize the animals for injection. No anesthesia was used as appropriate retention devices were utilized. Animals were injected using a 30 gauge needle.

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 by intramuscular injection divided into 6 injection sites placed in the quadriceps, gastrocnemius and tibialis cranealis muscles of each hind limb.

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) using a Nikon Eclipse E800 microscope (12 images per animal) connected to a video camera with monitor.
The area of adipocytes in hematoxylin/eosin WAT images acquired at 10× was determined using a Nikon, Tokyo, Japan), and the area of each adipocyte was quantified in μm ² . The average adipocyte area was calculated for each experimental group and the distribution of adipocytes according to size classification was represented by histograms. At least 25 per animal with 4 animals per group
0 adipocytes were analyzed.

Immunohistochemistry Tissues were fixed in 10% formalin for 12-24 hours, embedded in paraffin and sectioned. Sections were treated with rat anti-Mac2 antibody (1:50; CL8942AP; Cedarlane)
, guinea pig anti-insulin antibody (1:100; I-8510; Sigma-Aldric
h) or rabbit anti-glucagon antibody (1:100; 219-01; Signet Lab
s) overnight at 4°C. 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 a rabbit anti-guinea pig antibody conjugated to peroxidase (1:300; P0141; Dako) was used as secondary antibody. ABC
Sections were counterstained in Mayer's hematoxylin using a peroxidase kit (Pierce) for immunodetection. Hoechst (B2261; Sigma-Aldrich)
was used for nuclear counterstaining of fluorescent specimens. Fibrosis was assessed using picrosirius red staining and Masson's trichrome staining. The percentage of β-cell area in the pancreas,
Two insulin-stained sections separated by 200 μm were analyzed by dividing the area of insulin+ cells in one section by the total pancreatic area of that section. β-cell mass was calculated by multiplying the pancreas weight by the β-cell area percentage as previously described (Jimenez et al, 2011).

RNA analysis QIAzol Lysis Reagent (Q
iagen NV, Venlo, NL) or Triple isolation r
reagent (Roche Diagnostics Corp., Indianapo)
lis, IN, US), and RNeasy Lipid Tissue Miniki
Total RNA was obtained by using t (Qiagen NV, Venlo, NL). To remove residual viral genome, total RNA was treated with DNAseI (Qiagen NV, Ve
nlo, NL). For RT-PCR, 1 μg of RNA sample was transcr
IPTOR First Strand cDNA Synthesis Kit (04
379012001, Roche, California, USA). Real-time quantitative PCR was performed using the SmartCycler II® (Cephe
id, Sunnyvale, USA) in EXPRESS SYBRGreen qP
CR supermix (Invitrogen™, Life Technolo
gies Corp. , Carslbad, Calif., US). data to R
Normalized by plp0 values and analyzed as previously described (Pfaffl, M., N
Ucleic Acids Res. 2001;29(9):e45).

Hormone and Metabolite Assays Blood glucose levels were measured using a Glucometer Elite™ Analyzer (
Bayer, Leverkusen, Germany). FGF21
circulating levels were determined by the quantitative sandwich enzyme immunoassay Mouse/Rat FGF-
21 ELISA kit (MF2100, R&D systems, Abingdon, U.S.A.
K). 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 tissue, approximately 100 mg of frozen sample was weighed and added to 15 ml of chloroform:methanol (2:1).
homogenized inside. The lipid and aqueous phases were then separated by adding 3 ml of H ₂ SO ₄ 0.05% and letting them stand at 4° C. overnight. Once the phases were separated, the upper aqueous phase was removed using a Pasteur pipette and 1 ml of the lower lipid phase was recovered in the glass tube. 1 ml of chloroform and a 1% solution of Triton X-100 was added to the glass tube, which was incubated at 90° C. in a bath to evaporate the chloroform. Any remaining aqueous particles were removed from the lipid phase by using a mixture of chloroform and Triton X-100. After evaporation, the sample was concentrated by flushing the walls of the tube with chloroform, which was again warmed to 90° C. to evaporate the chloroform. Once the sediment was completely dry and concentrated, it was resuspended by adding 500 μl H20 miliQ at 37°C. Finally, the amount of triglycerides was determined by the commercial product GPO-PAP (Roch
e Diagnostics, Basel, Switzerland). Serum triglycerides and cholesterol were measured using an enzymatic assay kit (Horiba-A
BX, Montpellier, France) was quantified spectrophotometrically. All biochemical parameters were analyzed with a Pentra 400 Analyzer (Horiba-ABX
) was determined using

Blood glucose was determined using a Glucometer Elite™ (Bayer). Glucagon levels were measured by glucagon Radioimmunoassay (#GL-32K, EMD
Millipore). adiponectin, leptin, IGFBP1
and IGF1 were obtained from the Mouse Adiponectin ELISA kit (
80569, Crystal Chem), Mouse Leptin ELISA kit (90030, Crystal Chem), IGFBP1 (Mouse) ELISA
kit (KA3054, Abnova) and m/r IGF-I-ELISA kit (
E25, Mediagnost).

Insulin Tolerance Test For insulin tolerance test, insulin (0.75 IU/kg body weight; Humulin
Regular; Eli Lilly, Indianapolis, Ind.) 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.

Glucose Tolerance Test Awake mice were fasted overnight (16 hours) and administered glucose (2 g/kg body weight) by intraperitoneal injection. Blood glucose was measured in tail vein blood samples at the indicated time points. At the same time point, venous blood was collected via tail vein via tube (Microvette® CB 300).
, SARSTEDT) and immediately centrifuged to separate serum, which was used to measure insulin levels.

Oxygen Measurements 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 individualized and acclimated to 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 from light and dark cycles and adjusted for body weight. Met provided by the manufacturer to calculate energy consumption
Abolism software was used.

Transfection of HEK293, C2C12 and HepG2 Cells Cells were cultured in 24-well plates and 0.8 μg of DNA/well was
ctamine 2000 was used to transfect according to the manufacturer's instructions (T
Hermo Fisher Scientific).

Bone Analysis Bone volume and architecture were assessed by μCT. Mouse tibia was treated with neutral buffered formalin (10%
) and placed in an eXplore Locus CT scanner (General Ele
ctric) at a resolution of 27 microns. Trabeculae in 1 mm3 of proximal tibial epiphysis and 1.8 mm3 of cortical tibial diaphysis were analyzed in 4 mice/group. Bone parameters were analyzed using MicroView 3D Image Viewer & Analyzes
Calculated using is Tool. The length of the tibia is measured by the intercondylar eminence
measured from the ar eminence to the medial malleolus.

Western Blot Analysis iWAT and iBAT were treated with QIAzol Lysis Reagent (Qiagen
) and the protein fraction was separated from the organic phase according to the manufacturer's instructions. Proteins were separated by 12% SDS-PAGE and analyzed by rabbit polyclonal anti-UCP1.
It was analyzed by immunoblotting using an antibody (ab10983; Abcam) and a rabbit polyclonal anti-α-tubulin antibody (ab4074; Abcam). Detection was performed using ECL Plus detection reagents (Amersham Biosciences).

Open Field Testing Open field testing was conducted from 9:00 am to 1:00 pm as previously reported.
(Haurigot et al, 2013). Briefly, animals were placed in a brightly lit chamber intersected by two bundles of light beams that detect horizontal and vertical movement (
41 x 41 x 30 cm) (LE 8811; Panlab). Motor activity and exploratory behavior were assessed during the first 6 minutes. Total distance covered was assessed using a video tracking system (SMART Junior; Panlab).

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.

Examples Example 1. AAV-CAG-moFGF21-dmiRT in C57Bl6 mice
Prevention of obesity and diabetes by intra-eWAT administration of vectors. bottom. 10 ¹
CAG encompassing miR122 and miR1 target sites of ² viral genomes (vg)
An AAV9 vector encoding a codon-optimized mouse FGF21 coding sequence under the control of a ubiquitous promoter (AAV9-CAG-moFGF21-double
emiRT) (Fig. 1A) intra-eWAT (eWAT: epididymal white adipose tissue) administration
Adipose-specific overexpression of F21 (Fig. 1B) was similarly associated with high secretion of the protein into the blood stream (Fig. 1).
C) was mediated. AAV9-CAG-moFGF21-doublemiRT treated mice also showed lower levels of FGF21 receptor 1 (FGF2
1R1) (Fig. 1D) and overexpression of β-Klotho (FGF21 co-receptor) in adipose tissue and liver (Fig. 1E). CAG-moFGF21-double miR
The T construct is contained in SEQ ID NO:32 and the CAG-null construct is contained in SEQ ID NO:31.

Following AAV-mediated FGF21 gene transfer into eWAT, chow-fed mice exhibited weight loss (FIGS. 1F and 1G). When challenged with a high-fat diet (HFD),
Animals that overexpressed FGF21 in adipose tissue remained lean for the duration of the experiment, whereas AAV
9-CAG-null treated mice became progressively obese (FIGS. 1F and 1G). In response to their lower body weight, chow-fed AAV9-CAG-moFGF21-double
emiRT-treated mice and HFD-fed AAV9-CAG-moFGF21-double
All emiRT-treated mice showed decreased fat depot and liver weight (Fig. 1H).

Histological analysis of white adipose tissue by hematoxylin-eosin staining eWAT and iW
Reduction of White Adipocyte Size in AT (iWAT: Inguinal White Adipose Tissue) and iWAT
revealed multiple multilocular adipocytes within, suggesting that browning of this depot had occurred (Fig. 2A). Morphometric analysis showed AAV9-CAG-moFGF21-double
We further confirmed the reduction in the average area of white adipocytes in emiRT-treated mice (Fig. 2B
). The frequency distribution of white adipocyte area also differed between groups. Chow-fed AAV9
-CAG-moFGF21-doublemiRT treated mice and HFD-fed AAV9
-CAG-moFGF21-doublemiRT treated mice displayed an increase in the number of small adipocytes and fewer large adipocytes (Fig. 2C). Remarkably, HFD feeding A
The frequency distribution of white adipocyte area in AV9-CAG-moFGF21-doublemiRT-treated mice was almost identical to that of chow-fed AAV9-CAG-null-treated animals (Fig. 2C). Therefore, the HFD-induced hypertrophy of adipocytes observed in AAV9-CAG-null treated mice was prevented in mice overexpressing FGF21. Overexpression of UCP1 and Dio2 in iWAT (FIGS. 3A and 3B) further confirmed browning of iWAT in chow-fed AAV9-CAG-moFGF21-doublemiRT- and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice. .

Histological analysis of iBAT (iBAT: interscapular brown adipose tissue) showed that lipid accumulation in this depot was significantly higher in chow-fed AAV9-CAG-m compared to AAV9-CAG-null mice.
oFGF21-doublemiRT-treated mice and HFD-fed AAV9-CAG-m
showed less in oFGF21-doublemiRT treated mice (Fig. 2).
A). Depending on this result and browning of iWAT, HF during light and dark cycles
The energy expenditure of D-fed AAV9-CAG-moFGF21-doublemiRT treated mice (Fig. 3C) was higher than that of HFD-fed AAV9-CAG-null mice. Overall, these data suggest that AAV9-CAG-moFGF21-double
Suggesting that miRT-treated mice had enhanced thermogenic activity.

Liver tissue sections under either chow or HFD showed AAV9-CAG-nul
We showed reduced lipid accumulation in hepatocytes of mice that overexpressed FGF21 compared to l-treated mice (Fig. 2A). Therefore, HFD-fed AAV9-CAG-moFGF21-do
In ublemiRT-treated mice, their hepatic triglyceride (TG) content was normalized (Fig. 3D). In parallel, TG, total cholesterol, HDL-cholesterol and L
Circulating levels of DL-cholesterol were normalized in HFD-fed mice that overexpressed FGF21 (FIGS. 3E and 3F).

HFD-fed mice that overexpressed FGF21 were more insulin-sensitive than HFD-fed AAV9-null-treated mice (Fig. 3G), chow-fed AAV9-CAG-moFG
F21-doublemiRT-treated mice and HFD-fed AAV9-CAG-moFG
All F21-doublemiRT-treated mice showed their AAV9-CAG-nu
showed a decrease in circulating insulin levels compared to ll-treated counterparts (Fig. 3H).

Example 2. Intra-eWAT administration of AAV-CAG-moFGF21-dmiRT vector in ob/ob mice reverses obesity and improves glucose metabolism. The anti-diabetic and anti-obesogenic therapeutic potential of AAV-mediated genetic engineering was evaluated.
This mouse is deficient in leptin signaling and is a widely used genetic model of obesity and diabetes. To this end, a dose-response study was performed. ob/ob mice were injected with four different doses (10 ¹⁰ vg, 5×10 ¹⁰ vg, 2×10 ¹¹ vg or 10 ¹² vg) of the AAV8-CAG-moFGF21-doublemiRT vector (
FIG. 1A) was administered locally during eWAT. As a control, 10 ¹² in ob/ob animals
vg of AAV8-CAG-null vector was administered in eWAT.

Intra-eWAT administration of the AAV8-CAG-moFGF21-doublemiRT vector dose-dependently mediated specific overexpression of FGF21 in white adipose tissue, as well as high secretion of protein into the bloodstream (FIGS. 4A and 4B). . Specifically, AAV8-CAG-moFGF21-doublemiRT vector at a dose of 10 ¹² vg mediated very strong overexpression of FGF21 in eWAT and iWAT (Fig. 4A) and achieved the highest circulating FGF21 levels (Fig. 4A). 4B). In contrast, the lowest administered dose of 10 ¹⁰
vg AAV8-CAG-moFGF21-doublemiRT vector is eWAT
(Fig. 4A), animals treated with this dose showed no difference in serum FGF21 levels compared to AAV8-CAG-null treated animals (Fig. 4A). 4B), probably because FGF21 acted in a paracrine-autocrine manner. Thus, AAV8-CAG-null treated animals progressively gained weight, whereas AAV8-CAG-moFGF21-doublemi
Animals treated with the RT vector showed a dose-proportional decrease in body weight gain (FIGS. 4C and 4D). Strikingly, 10 ¹² vg of AAV8-CAG-moFGF
Animals treated with the 21-doublemiRT vector lost approximately 15% of their weight during the first two weeks after AAV administration and then gained weight until reaching starting weights (Fig. 4C).
and 4D). Therefore, 10 ¹² vg of AAV8-CAG-moFGF21-dou
Animals receiving intra-eWAT administration of the blemiRT vector showed a 40% difference in total body weight at the end of the experiment compared to AAV8-CAG-null treated animals (Fig. 4D). in agreement, 10
Animals treated with ¹² vg of AAV8-CAG-moFGF21-doublemiRT vector showed a marked reduction in steatosis and a 60% reduction in liver weight (Fig. 4E). iB
AT weight was increased in this cohort of mice, probably due to enhanced thermogenic activity (Fig. 4E).

5×10 ¹⁰ vg, 2×10 ¹¹ vg or 10 ¹² vg of AAV8-CAG-moF
Animals treated with the GF21-doublemiRT vector showed AAV8-CAG-nu
exhibited improved insulin sensitivity compared to ll-treated mice (Fig. 5A). 2 x 10 ¹¹
vg or 10 ¹² vg of AAV8-CAG-moFGF21-doublemiRT vector-treated animals also showed ob
/ob mice exhibited lower circulating insulin levels (Fig. 5B).

Example 3. Intravenous administration of AAV-hAAT-moFGF21 vectors in ob/ob mice reverses obesity and improves glucose metabolism. Anti-diabetic and anti-obesogenic effects mediated by increased circulating levels of FGF21 used were evaluated. Ob/ob mice were transfected with AAV8 ^{vectors (} AAV8- hA
AT-moFGF21) (Fig. 6A) was administered intravenously (IV). As a control, o
b/ob animals were administered 5×10 ¹¹ vg of AAV8-hAAT-null vector IV. The hAAT-moFGF21 construct is contained in SEQ ID NO: 34, hAAT-n
The ull construct is contained in SEQ ID NO:33.

Intravenous administration of AAV8-hAAT-moFGF21 vector increases FGF2
Specific overexpression of 1 also dose-dependently mediated high secretion of the protein into the bloodstream (
6B and 6C). Specifically, a dose of 5×10 ¹¹ vg of AAV8-hAAT-mo
The FGF21 vector mediated very strong overexpression of FGF21 in the liver (Fig. 6B
), which achieved the highest circulating FGF21 levels (Fig. 6C). AAV8-hAA at this dose
The body weight of animals treated with the T-moFGF21 vector decreased by approximately 7% during the first two weeks after AAV administration and increased slightly thereafter, whereas AAV8-hAAT-null treated mice progressively lost weight. Weight was increased (Figs. 6D, 6E and 6F). AAV8 at 10 ¹¹ vg
- Mice administered hAAT-moFGF21 vector are AAV8-hAAT-null
The l-treated animals gained significantly less weight (Figs. 6D, 6E and 6F).
Specifically, AAV8-hAAT-null animals were tested with 10 ¹¹ vg of AAV8-hAAT
-moFGF21 vector-treated animals showed a 50% weight gain at the end of the experiment compared to a 10% weight gain (Fig. 6E). FG in the liver in response to their lower body weight
Animals that overexpressed F21 showed a significant reduction in steatosis, especially in animals treated with the highest dose of vector, and showed a reduction in liver weight of approximately 60% (Fig. 6G).
iBAT weight increased similarly in both AAV8-hAAT-moFGF21-treated mouse groups (Fig. 6G), probably due to higher thermogenic activity in these animals compared to mice receiving the AAV8-hAAT-null vector. would be for

Animals treated with the AAV8-hAAT-moFGF21 vector showed no AAV8-hAA
They showed improved insulin sensitivity and decreased circulating insulin levels compared to T-null treated mice (FIGS. 6H and 6I).

Example 4. Long-term recovery from obesity and diabetes by intravenous administration of AAV-hAAT-moFGF21 vectors in HFD-fed mice. antidiabetic and antiobesogenic effects mediated by Nine week old male C57B16 mice (young adults) were fed HFD for 9 weeks and then IV administered 10 ¹⁰ vg or 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector (FIG. 6A). After AAV administration, AAV8-hAAT-moFGF21 treated mice were maintained on HFD for 52 weeks. As a control, 5×10 ¹⁰ vg of AAV8-
hAAT-null was administered IV to chow-fed C57B16 and HFD-fed C57B16 mice. These latter two cohorts of mice were then maintained on a chow diet or HFD.

Intravenous administration of AAV8-hAAT-moFGF21 vector in HFD-fed mice dose-dependently mediated high secretion of FGF21 into the bloodstream (Fig. 7A).

HFD-fed AAV8-null treated mice and 10 ¹⁰ vg AAV8-hAAT-mo
No difference in body weight was observed between HFD-fed animals receiving FGF21 vectors (FIGS. 7B and 7C). However, 5×10 ¹⁰ vg of AAV8-hAAT-moFGF
HFD-fed animals treated with 21 vector initially lost 20% in body weight after AAV administration and then progressively gained weight similar to chow-fed AAV8-hAAT-null-treated mice (FIGS. 7B and 7C). ). Strikingly, after 9 weeks post AAV administration, 5×10 ¹⁰ v
There were no statistically significant differences in overall body weight and body weight gain between HFD-fed animals administered 2.5 g of AAV8-hAAT-moFGF21 vector and chow-fed AAV8-hAAT-null treated mice (FIGS. 7B and 7C). .

HFD treated with 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector
Energy expenditure during light and dark cycles in chow-fed AAV
8-hAAT-null and HFD-fed AAV8-hAAT-null mice (Fig. 8A). Chow-fed AAV8-hAAT-null-treated animals and HFD-fed AAV8-hAAT-null-treated animals and 10 ¹⁰ vg of AAV8-
No difference in energy expenditure was observed between mice treated with the hAAT-moFGF21 vector (Fig. 8A). Overall, these data indicate that 5×10 ¹⁰ vg of AAV8-
It suggests that mice treated with hAAT-moFGF21 had enhanced thermogenic activity.

Animals treated with 10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector showed A
displayed improved insulin sensitivity compared to HFD-fed mice administered the AV8-hAAT-null vector, and their insulin sensitivity was similar to that of chow-fed mice treated with the AAV8-hAAT-null vector ( Figure 8B). Remarkably,
Animals receiving 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector showed A
Displayed improved insulin sensitivity compared to chow-fed mice administered AV8-hAAT-null vector, indicating normalization of circulating insulin levels (FIGS. 8B and 8C)
.

Example 5. Obesity and diabetes reversal by intravenous administration of AAV-hAAT-moFGF21 vectors in aged HFD-fed mice We also evaluated the anti-diabetic and anti-obesogenic effects of FGF21 in obese aged (adult) C57B16 mice. 7.5 months old male C57B16 mice were given HFD
weekly feeding followed by 10 ¹⁰ vg, 2 x 10 ¹⁰ vg or 5 x 10 ¹⁰ vg of AAV8
- hAAT-moFGF21 vector was administered IV (Fig. 6A). After AAV administration, AAV
8-hAAT-moFGF21 treated mice were maintained on HFD for 22 weeks. As a control, 5×10 ¹⁰ vg of AAV8-hAAT-null was administered to chow-fed aged C57Bl.
6 mice and HFD-fed aged C57B16 mice were administered IV. These latter two cohorts of mice were then maintained on a chow diet or HFD.

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

HFD-fed AAV8-null treated mice and 10 ¹⁰ vg AAV8-hAAT-mo
No difference in body weight was observed between HFD-fed animals receiving FGF21 vectors (FIGS. 9B and 9C). However, HFD-fed animals treated with either 2×10 ¹⁰ vg or 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector showed no AAV
Body weight was initially reduced by 15% and 20%, respectively, after dosing (Figures 9B and 9C). Subsequently, animals treated with 2×10 ¹⁰ vg AAV8-hAAT-moFGF21 vector progressively gained weight similar to chow-fed AAV8-hAAT-null treated mice, whereas 5×10 ¹⁰ vg AAV8- No significant changes in body weight were observed in animals treated with the hAAT-moFGF21 vector (Figures 9B and 9C). Notably, AAV
After 3 weeks post-dosing, there was no statistically significant difference in total body weight and body weight gain between HFD-fed animals receiving 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector and chow-fed AAV8-hAAT-null-treated mice. was not observed (Figs. 9B and 9C).

HFD treated with 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector
Energy expenditure during light and dark cycles in chow-fed AAV
8-hAAT-null and HFD-fed AAV8-hAAT-null mice (FIG. 10A). 2×10 ¹⁰ vg of AAV8-hAAT-moFGF
Animals treated with the 21 vector showed increased energy expenditure during the light cycle and a trend towards increased energy expenditure during the dark cycle (Fig. 10A). 10 ¹⁰ vg of AAV8
Animals treated with the -hAAT-moFGF21 vector showed increased energy expenditure during the dark cycle (Fig. 10A). Chow-fed AAV8-hAAT-null-treated animals and HFD-fed AAV8-hAAT-null-treated animals and 10 ¹⁰ vg of AAV8
No differences were observed between mice treated with the -hAAT-moFGF21 vector (Fig. 10A). Altogether, these data suggest that aged mice treated with AAV8-hAAT-moFGF21 had enhanced thermogenic activity.

Animals treated with 10 ¹⁰ vg or 2×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector displayed improved insulin sensitivity compared to HFD-fed mice receiving AAV8-hAAT-null vector and their insulin Susceptibility is AAV8-
It was similar to that of chow-fed mice treated with the hAAT-null vector (Fig. 10B). Remarkably, animals administered 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector displayed improved insulin sensitivity compared to chow-fed mice administered AAV8-hAAT-null vector (FIG. 10A). . 10 ¹⁰ vg, 2x
Animals treated with 10 ¹⁰ vg or 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector showed lower fasting and fed insulin circulating levels than HFD-fed AAV8-hAAT-null treated mice (FIG. 10C). . Remarkably, 2×10 ¹
There was no difference in circulating insulin levels at feeding between aged animals dosed IV with ⁰ vg or 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 vector and chow-fed AAV8-hAAT-null treated mice (Fig. 10C).

Example 6. Evaluation of Weight Loss by Intramuscular Administration of AAV-CMV-moFGF21 Vectors in C57Bl6 Mice We also evaluated the therapeutic potential of increasing circulating FGF21 levels by AAV-mediated genetic engineering of skeletal muscle in C57Bl6 mice. bottom. The CMV promoter and AAV1 serotype were chosen to target skeletal muscle. Although the CMV promoter is a ubiquitous promoter, its use with the AAV1 capsid allows very efficient targeting of skeletal muscle without transducing the liver, as previously published. (Mas et al., Diabetes 2006; Callejas et al.
al. , Diabetes 2013).

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

Intramuscular administration of the AAV1-CMV-moFGF21 vector mediated high secretion of FGF21 into the blood stream (Fig. 11B). Animals treated with the AAV1-CMV-moFGF21 vector showed reduced body weight and weight gain compared to AAV1-CMV-null treated mice (FIGS. 11C and 11D).

Example 7. Increased Protein Production with Codon-Optimized Human FGF21 Nucleotide Sequences Three different codon-optimized human FGF21 nucleotide sequences were placed in HEK293 cells to assess whether codon optimization can mediate increased FGF21 protein production. (SEQ ID NOS: 40-42) were transfected. As controls, non-transfected cells and cells transduced with the wild-type hFGF21 coding sequence were used. Expression of the three codon-optimized human FGF21 sequences and the WT 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 showed higher levels of human FGF2 compared to wild-type or codon-optimized FGF21 variant 2.
1 was able to be secreted into the culture medium (Fig. 12), thus demonstrating increased FGF21 protein production by codon optimization of variants 1 and 3.

Example 8. Resilience of Obesity and Diabetes in Mice by Administration of AAV Vectors Encoding Human FGF21 (In Vivo Experiments Demonstrating the Activity of FGF21)
HFD-fed mice are treated with an AAV vector encoding human FGF21. As a control, the same dose of AAV-null vector is administered to chow-fed and HFD-fed mice.

To assess 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 conducted:
- weekly measurement of body weight and food and fluid intake - measurement of body temperature - measurement of energy expenditure and respiratory quotient by indirect calorimetry - measurement of blood glucose - assessment of whole body glucose disposal by intraperitoneal glucose tolerance test - intraperitoneal insulin Assessment of insulin sensitivity by challenge testing - analysis in tissue and serum samples, including - examination of human FGF21 overexpression levels in target tissues and bloodstream - morphological and histological analysis.

- determination of circulating levels of hormones and cytokines - 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 classical Evaluation of browning capacity by gene expression of markers of white, brown and beige adipocytes Example 9: In vitro assays to assess FGF21 activity FGF21 increases glucose uptake and GLUT1 expression in 3T3-L1 cells (Kharitonenkov, A. et al., 200
5. J. Clin. Invest 115 :1627-1635).

Example 10. AAV8-CAG-moFGF21-dmiR in ob/ob mice
Intra-eWAT administration of T vectors reverses obesity and improves glucose metabolism: further findings.
The anti-diabetic and anti-obesogenic therapeutic potential of AV-mediated genetic engineering was evaluated (Example 2
(see ).

received intra-eWAT injection of AAV8-CAG-moFGF21-dmirT vector o
b/ob mice showed reduced white adipocyte size in the epididymal pad (Fig. 13A).
Circulating adiponectin levels also increased with dose (Fig. 14A). eWAT inflammation, assessed through Mac2 staining, was also reduced as a function of vector dose, as was the expression of the macrophage marker F4/80 (FIGS. 14B and C).

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

Example 11. Intravenous administration of AAV8-hAAT-moFGF21 vectors in ob/ob mice reverses obesity and improves glucose metabolism: further findings
The anti-diabetic and anti-obesogenic therapeutic potential of intravenous administration of 1 vector was evaluated (see Example 3).

Consistent with their lower body weight, ob/ob animals that overexpressed FGF21 in the liver, especially those treated with 5×10 ¹¹ vg, showed a significant reduction in the size of white adipocytes (FIG. 15A). ). This was evidenced by a decrease in Mac2 staining and expression of F4/80 and TNF-α in eWAT (FIGS. 16A-C), a dose-dependent increase in circulating adiponectin levels (FIG. 15B) and a decrease in WAT inflammation and was parallel. Strikingly, ob/ob mice treated with 5×10 ¹¹ vg exhibited a “crown-like”
It showed a significant reduction in structure (Fig. 16A).

7-month-old ob/ob mice showed marked hepatic steatosis, whereas the livers of FGF21-treated ob/ob mice showed no lipid accumulation in hepatocytes (FIG. 15C). This was associated with a 60% reduction in organ weights in ob/ob mice that received therapeutic vectors (FIGS. 16D and E),
Similarly a significant reduction in total liver triglyceride and cholesterol content (Fig. 16F and G
). ob/o treated with both doses of AAV8-hAAT-moFGF21
b Animals also showed reduced fed blood glucose, and their blood insulin in the fed state was reduced by approximately 70% (FIGS. 15D and E).

We investigated 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 phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase) expression was assessed by measuring it by qPCR. P.
Altered expression of these enzymes was observed in livers of AAV8-hAAT-moFGF21-treated ob/ob mice, except for animals treated with 1×10 ¹¹ vg of AAV8-hAAT-moFGF21 that showed increased EPCK expression. no (FIGS. 17A and B). These results suggest that AAV-mediated long-term FGF21 expression in the liver and subsequent increase in circulating FGF21 did not lower glucose by inhibiting hepatic glucose production.

The glucose-lowering effect of FGF21 has also been attributed to increased glucose uptake and enhanced energy expenditure by adipocytes (Xu J. et al., 2009.
AJP Endocrinol. Metab. 297 : E1105-E1114;
ingX. et al. , 2012. Cell Metab. 16 :387-3
93; Camacho R.; C. et al. , 2013. Eur. J. Pharm
acol. 715 :41-45; Emanuelli B.; et al. , 2014
. Clin. Invest. 124 :515-527;
A. et al. , 2005. Endocrinology 148 :774-7
81; Hondares E.; et al. , 2010. Cell Metab.
11 :206-212; Samms R.; J. et al. , 2015. cell
Rep. 11 :991-999). Therefore, we have identified key components of the glucose uptake machinery, such as the glucose transporters Glut1 and Glut4, the glucose kinase hexokinase I and II (HKI), in different pad adipose tissues (iWAT, eWAT and iBAT). and HKI), and in the case of iBAT, UCP1, were assessed by qPCR. AAV8-FGF21 treated ob/
In ob mice, Glut1 expression was increased in iWAT and iBAT (Fig. 17C
), Glut4 expression was increased in eWAT, iWAT and iBAT (Fig. 17D).
HKI and HKII were upregulated only in iBAT (FIGS. 17E and F). Moreover, UCP1 expression was increased in iBAT of ob/ob mice treated with high dose AAV8-hAAT-moFGF21 vector (FIG. 17G). Altogether, these results suggest that the long-term reversal of blood glucose 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 increased thermogenesis in iBAT. suggesting that it is due to enhancement.

Example 12. AAV8-hAA in HFD-fed and HFD-fed aged mice
Long-term recovery from obesity and diabetes by intravenous administration of T-moFGF21 vectors: tissue weight reduction and stable expression for up to 1 year. Representative images of animals belonging to all experimental groups are shown in Figures 19A-B.

Obesity reversal by AAV8-hAAT-moFGF21 treatment affected major white adipose tissue (WAT) depots, such as epididymis (eWAT), inguinal (iWAT), in animals treated either as young adults or as adults. and retroperitoneal (rWAT) fat pads, paralleling dose-dependent decreases in weight (FIGS. 18A and 19C). HF of liver weight
The D-induced increase was completely normalized by FGF21 gene transfer at the highest dose of vector used, whereas quadriceps weight was unchanged by diet or AAV delivery (FIGS. 18A and 19D).

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

Example 13. AAV8-hAA in HFD-fed and HFD-fed aged mice
Long-term recovery from obesity and diabetes by intravenous administration of T-moFGF21 vectors: enhancement of locomotor activity and investigation of thermogenesis mechanisms.
It was also seen in animals treated as young adults 10 months after moFGF21 delivery (Fig. 21A).

This finding was consistent with AAV8-hAAT-moFGF21-mediated effects on locomotor activity. 5×10 ¹⁰ vg as young adults, in contrast to the hypoactivity observed during the open field test in HFD-fed animals that received the AAV8-null vector.
Mice treated with AAV8-hAAT-moFGF21 exhibited spontaneous locomotor activity comparable to chow-fed null-injected animals. As shown in FIG. 20A, AAV
One year after 8-hAAT-moFGF21 delivery, treated animals traveled longer distances, rested less, and spent more time on slow and fast exercise than untreated HFD-fed controls. .

Believing that changes in energy expenditure may reflect changes in thermogenesis, we assessed the degree of activation of brown adipose tissue (BAT). 5×10 ¹⁰ vg as a young adult or adult
Both mice treated with AAV8-hAAT-moFGF21 showed decreased lipid deposition in iBAT (FIG. 20B). UCP1 protein content in BAT was dose-dependently increased in mice treated with the AAV8-hAAT-moFGF21 vector as young adults (Fig. 20C), suggesting that the non-shivering thermogenesis induced by FGF21 gene transfer to the liver was increased. coincided with the increase.

Browning of subcutaneous WAT is characterized by the appearance of beige adipocytes and is also associated with increased energy expenditure (Harms & Seale, 2013). browning is AAV8-hA
Histological evaluation of iWAT was performed to assess whether it could explain the increased energy expenditure observed after AT-moFGF21 treatment. Consistent with this pad weight reduction (Fig. 18
A), Adipocytes in HFD-fed AAV8-hAAT-moFGF21-treated animals were smaller than those in HFD-fed null-injected animals (Fig. 20D). Nevertheless, AAV8
Treatment with the -hAAT-moFGF21 vector did not result in increased detection of multilocular beige adipocytes in iWAT at any dose tested in either young adults or animals treated as adults (Fig. 20D). Therefore, iW between HFD-fed groups
There was no statistically significant difference in UCP1 protein levels in AT (Fig. 21B).

Creatinine-driven substrate cycle and sarcoplasmic reticulum Ca2+-ATPase 2b (Se
rca2b)-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. Me
d. 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-fold when compared to age-matched chow-fed and HFD-fed control groups.
was observed in the iWAT of HFD-fed mice treated with ×10 ¹⁰ vg AAV8-hAAT-moFGF21 (FIG. 20E), suggesting that creatinine-driven cycle activity was increased, likely as a result of FGF21 gene transfer. Chow-fed null-treated animals or HFD-fed null
No differences in Serca2b expression levels in iWAT of animals treated with the AAV8-hAAT-moFGF21 vector were detected when compared to l-treated animals (FIG. 21C).
. On the other hand, the iWA of another enzyme involved in the same cycle, ryanodine receptor 2 (RyR2)
T expression was increased by HFD feeding in both null- and AAV8-hAAT-moFGF21-treated mice (Fig. 21C). Overall, these results
We suggest that calcium cycle-dependent thermogenic mechanisms are not involved in the observed improvements in whole-body energy homeostasis after AAV-FGF21 treatment.

Example 14. AAV8-hAA in HFD-fed and HFD-fed aged mice
Long-term recovery from obesity and diabetes by intravenous administration of T-moFGF21 vectors: glucagon levels, islet hyperplasia and glucose tolerance. They showed decreased circulating levels of glucagon compared to treated mice (Fig. 22A).

AAV8-null-treated mice developed islet hyperplasia as a result of HFD feeding, whereas AAV8-hAAT-FGF21 vectors (2×10 ¹⁰ or 5×10 ¹⁰ vg/
β-cell mass in treated animals) was similar to that of control mice fed a chow diet (FIGS. 22B and C). HFD-fed AAV8-hAAT-moF
Double immunostaining for insulin and glucagon of pancreatic sections from GF21-treated mice showed a normal distribution of α- and β-cells in the islets of these animals, with glucagon-expressing cells in the periphery of the islets and in the center. Insulin-expressing cells were localized in the (Fig. 22D).

To assess glucose tolerance in FGF21-treated mice, an intraperitoneal glucose tolerance test (G
TT) (2 g glucose/kg body weight) was performed 10 weeks after AAV administration. HFD-fed animals injected with either null or a vector encoding FGF21 at a dose of 1×10 ¹⁰ vg/mouse were glucose intolerant and showed marked increases in circulating insulin levels during the GTT (FIGS. 23A and 23A). B). In contrast, 5×10 ¹⁰ vg/mouse of AAV
Animals treated with 8-hAAT-moFGF21 showed improved glucose clearance when compared to chow-fed control mice (FIG. 23A). Insulin levels were indistinguishable between these two experimental groups (Fig. 23B). These results are 5×10 ¹⁰
We further confirmed improved insulin sensitivity in HFD-fed mice treated with vg/mouse AAV8-hAAT-moFGF21.

Example 15. HFD by intravenous administration of AAV8-hAAT-moFGF21 vector
Reversal of WAT hypertrophy and inflammation associated with HFD feeding induces an increase in WAT adipocyte size (Sattar N. & Gi
llJ. M. R. , 2014. BMC Med. 12 :123). Administration of a vector encoding FGF21 counteracted this increase (Fig. 24A). Morphometric analysis of WAT was
The area of white adipocytes of animals treated with 1×10 ¹⁰ or 5×10 ¹⁰ vg of vector as young adults and of mice treated with 2×10 ¹⁰ or 5×10 ¹⁰ vg of vector as adults was It was found to be similar to that of animals fed a chow diet (Fig. 24B). In both FGF21-treated animal groups, there was a redistribution of adipocyte size, with a higher proportion of smaller adipocytes (FIG. 25A). Consistent with reduction in adiposity and restoration of WAT hypertrophy, the highest dose of AAV8-hAAT-moFG regardless of treatment initiation age
Adiponectin and leptin levels in animals treated with the F21 vector were also normalized (FIGS. 24C and D).

Obesity also causes WAT inflammation (Hajer GR et al., 20
08. Eur. HeartJ. 29 :2959-2571). We therefore 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 animals treated with 5 x 10 ¹⁰ vg AAV8-hAAT-moFGF21 as young adults or adults had no macrophages. There were no signs of invasion (Figures 24E and 25B). this is,
Macrophage markers F480 and CD68 and the pro-inflammatory cytokine TNF
In parallel with the normalization of α and IL-1β expression (FIGS. 24F-H and 25C-
E), indicating that FGF21 expression counteracted obesity-associated WAT inflammation.

Example 16. Intravenous Administration of AAV8-hAAT-moFGF21 Vector Restores Fatty Liver, Liver Inflammation and Fibrosis Liver tissue analysis showed that all null-treated animals fed HFD had significant hepatic steatosis at sacrifice. (FIGS. 26A-D). In contrast, 5 × 10 as young adults or adults
HFD-fed mice receiving ¹⁰ vg AAV8-hAAT-moFGF21 demonstrated reversal of this pathological lipid deposition (Fig. 26A). These histological findings were 5×10 ¹⁰ v
g Paralleled the marked reduction in total liver triglyceride and cholesterol content of AAV8-hAAT-moFGF21-treated animals (FIGS. 26B and C). In addition, 5×10 ¹⁰ vg AAV8-hAAT-moFGF2 in young adults or HFD-fed adults
Animals treated with 1 vector showed no signs of liver inflammation as evidenced by the lack of staining for Mac2, which indicated increased presence of macrophages in the liver of null-treated HFD-fed mice. revealed (Fig. 26D). Finally, FGF21 gene transfer to the liver reversed liver fibrosis. Collagen fibrils were readily detectable after picrosirius red or Masson's trichrome staining of liver sections from animals fed the HFD and injected with the control null vector, whereas AAV
It was not detectable in the liver of 8-hAAT-moFGF21 treated mice (Figures 28A and 27). These mice also showed a marked reduction in hepatic collagen 1 expression (Fig. 28).
B and C). Altogether, these findings indicated that AAV8-hAAT-moFGF21 treatment protected against the development of HFD-induced non-alcoholic steatohepatitis (NASH).

Example 17. Long-Term Safety of Liver-Directed AAV-FGF21 Treatment Pharmacological treatment or transgenic overexpression with FGF21 is associated with perturbation of bone homeostasis through enhanced bone resorption, which leads to bone loss. There is (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;
S. et al. , 2016. Cell Metab. 23 :427-440;
im A. M. 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. AAV8 at 9 or 29 weeks of age at sacrifice (approximately 16.5 months of age)
The snout-to-tail length and tibia length were normal in animals that received the -hAAT-moFGF21 vector (Figures 29A and B). We then examined bone structure by microcomputed tomography (μCT). Analysis of the proximal epiphysis of the tibia
Demonstrating no significant differences in trabecular and cortical bone in HFD-fed mice treated with 5×10 ¹⁰ vg AAV8-hAAT-moFGF21 compared to age-matched mice treated with null vector bottom. Specifically, bone mineral density (BMD) (Fig. 29C
), bone mineral content (BMC) (Fig. 29D), bone volume (BV) (Fig. 29E), bone volume/tissue volume ratio (BV/TV) (Fig. 29F), bone surface/bone volume ratio (BS/BV). (Fig. 29G),
Trabecular number (Tb.N) (Fig. 29H), trabecular width (Tb.Th) (Fig. 29I) or trabecular spacing (
Tb. Sp) (Fig. 29J) was not recorded. Similarly, analysis of compact bone in the tibia diaphysis showed that BMC, BMD
, BV, BV/TV or BS/BV (FIGS. 29K-O).

The pathological effects of FGF21 have been reported to be 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 reduced circulating IGFBP1 in animals treated as early as 12 months (young adults) or 6 months (adults) when compared to null-injected HFD-fed mice. It did not lead to an increase in protein levels (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 liver for bone tissue.

Example 18. HFD by intravenous administration of AAV8-hAAT-moFGF21 vector
Prevention of Induced Liver Tumors Prolonged HFD feeding (>60 weeks) is associated with increased development of liver neoplasms in C57BL/6J mice (Hill-Baskin AE et al., 2009.
Hum. Mol. Genet. 18 :2975-2988; Nakagawa H.; ，
2015. WorldJ. Hepatol. 7 :2110). In our study, in animals that started HFD as young adults and maintained it for 60 weeks, we found that 66.7% (6/9) of the animals injected with the null vector developed liver tumors. Found it.
Animals treated with the AAV8-hAAT-moFGF21 vector were protected from HFD-induced hepatic neoplastic development: 0% (0/8) of animals treated with vectors encoding 5×10 ¹⁰ vg of FGF21 The incidence was 40% (4/10) in the cohort that exhibited tumors and was treated with the lowest dose (1×10 ¹⁰ vg). Chow-fed mice (0/11)
none developed tumors during the same period (Table 1).

Table 1. Liver Tumor Development in Young Adults

Example 19. Reversal of STZ-induced hyperglycemia by AAV8-mediated liver-specific FGF21 overexpression Materials and Methods Animals We used 9-week-old male C57bl6 mice. Mice were fed standard chow ad libitum and maintained under a 12 hour light/dark cycle (lights on at 08:00). For diabetes induction, mice received five intraperitoneal injections of streptozotocin (50 mg/kg) dissolved in 0.1 mol/l citrate buffer (pH 4.5) on consecutive days. Blood glucose levels were measured with an analyzer (Glucometer Elite; Bayer, Leve
rkusen, Germany). Animal care and laboratory procedures are
Ethics C of the iversitat Autonoma de Barcelona
ommittee for Animal and Human Experiments
ation approved.

In Vivo Administration of AAV Vectors For systemic administration, AAV vectors were added to 0.001% F68 Pl in 200 μl of PBS.
Diluted in uronic® (Gibco) and injected via the tail vein.

Results To test the protective potential of AAV-derived FGF21 against type 1 diabetes, 5x
AAV8 vectors (AAV8 ^- hAAT- ^moF
GF21) was administered IV to male 9-week-old C57B16 mice. Control mice received 2×10 ¹¹ vg of AAV8-hAAT-Null vector. 2 of AAV administration
Weeks later, all animals were treated with streptozotocin (STZ) (5 doses of 50 mg/kg;
day) caused the diabetic process.

Analysis of blood glucose levels revealed that animals treated with AAV8 vectors encoding moFGF21 exhibited lower circulating glucose levels than C57B16 mice treated with AAV8-hAAT-Null vectors (FIG. 30).

Example 20. Intramuscular administration of AAV-CMV-moFGF21 vectors in C57B16 mice extends healthy lifespan by preventing age-related weight gain and insulin resistance Skeletal muscle (Skm) is an easily accessible tissue that secretes It has been used for the production of potential therapeutic proteins (Haurigot V. et al., 2010. J.
. Clin. Invest. 123 :3254-3271; Callejas D.;
et al. , 2013. Diabetes 62 :1718-1729;
M. L. et al. , 2017. Mol. Ther. Methods Clin.
Dev. 6 :1-7). To investigate whether Skm can serve as a viable source of circulating FGF21, a serotype 1 AAV vector with high tropism for Skm carrying optimized murine FGF21 under the control of the CMV promoter. (Chao L. et al.
l. , 2000. J. Clin. Invest. 106 :1221-1228;
Z. et al. , 2006. J. Virol. 80 : 9093-9103;
isowski L. et al. , 2015. Curr. Opin. Pharma
col. 24 :59-67) was used (AAV1-CMV-moFGF21). Vector was injected at a dose of 5×10 ¹⁰ vg/muscle into the quadriceps, gastrocnemius and tibialis anterior muscles of both limbs of 8-week-old C57B16 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 normal chow-fed healthy mice further allowed us to assess the long-term safety of FGF21 gene therapy.

11-month-old animals injected with a vector encoding FGF21 at 8 weeks of age 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 route of administration did not lead to transgene expression in the liver (Fig. 31B).

At the end of the approximately 10 month follow-up period, mice injected intramuscularly with AAV1-CMV-moFGF21 maintained their body weight at the beginning of the study, and were approximately 38% lower than controls, whose weight steadily increased as the animals aged. % lean (Fig. 31C). Muscle weight was largely unaffected by FGF21 gene transfer, whereas white and brown depot weights, as well as liver weights, were significantly reduced (FIG. 31D). Indeed, the weight of the analyzed WAT pads was reduced by >50% (Fig. 31D). Moreover, mice treated with AAV1-CMV-moFGF21 showed a marked reduction in liver total triglyceride content (FIG. 31E). No change in liver cholesterol levels was observed (Fig. 31F). In contrast to null injected animals, AAV1-CMV-m
Animals treated with oFGF21 exhibited normoglycemia (data not shown) and reduced blood insulin (FIG. 31G) when they were approximately 1 year of age. Thus, FGF21-treated mice exhibited significantly improved insulin sensitivity at the end of the study (Fig. 31H). as a whole,
This study demonstrates that administration of AAV vectors leading to 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. to demonstrate.

Example 21. AAV1-CMV-moFGF2 in HFD-fed C57Bl6 mice
Obesity and diabetes reversal by intramuscular administration of 1 vector.

We next evaluated whether im administration of the AAV1-CMV-moFGF21 vector could also reverse obesity and insulin resistance. To this end,
Two-month-old C57B16 mice were fed either chow or HFD for 12 weeks. During these first 3-month follow-ups, chow-fed animals gained 20% in weight, while HFD-fed animals became obese (95% weight gain) (FIGS. 32A and B). Vector was then injected at a dose of 5×10 ¹⁰ vg/muscle into the quadriceps, gastrocnemius and tibialis anterior muscles of both limbs of obese C57B16 mice (total dose, 3×10 ¹¹ vg/mouse). As controls, another cohort of obese mice and a cohort of chow-fed mice received 3×10 ¹¹ vg
of the non-coding null vector (AAV1-CMV-null). AAV
After delivery, mice were maintained on a chow or HFD diet. AAV1-CMV-moFGF
Animals treated with 21 experienced progressive weight loss (Figures 32A and B). AAV1
The reversal of obesity by -CMV-FGF21 treatment paralleled the increase in circulating FGF21 levels (Fig. 32C).

Null-treated mice fed HFD showed normal feeding blood glucose (Fig. 32D) but were hyperinsulinemia (Fig. 32E), indicating that these mice had developed insulin resistance. Suggest. In contrast, HFD-fed mice treated with AAV1-CMV-moFGF21 were normoglycemic and normo-insulin by the end of the study (Fig. 3).
2D and E). Moreover, AAV1-CMV-moFGF21-dosed animals exhibited greater insulin sensitivity than their HFD-fed controls (FIG. 32F).

Example 22. Increased FGF21 circulating levels by codon-optimized human FGF21 nucleotide sequences.

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

Materials and Methods In Vivo Delivery of Plasmids to Mice by Hydrodynamic Tail Vein Injection Plasmid DNA was diluted in saline to a volume (ml) equal to approximately 10% of the animal's average body weight (grams) and injected into the lateral tail vein. Injected manually in less tan. Prior to injection, animals were placed under a 250 W infrared heat lamp (Philips) for several minutes to dilate the blood vessels and make the tail vein more visible and easily accessible. Animals were immobilized for injection using plastic restrainers (Harvard Apparatus).
No anesthesia was used as it was not necessary as long as an appropriate restraint system was used. The inventors 2
Animals were injected using a 6G 3/8 inch gauge hypodermic needle (BD), which is the largest feasible needle gauge that fits snugly into the vein being accessed.

Results Mice treated with either codon-optimized human FGF21 version 2 or 3 secrete higher levels of human FGF21 into circulation compared to wild-type or codon-optimized FGF21 variant 1. (Fig. 33, thus demonstrating increased FGF21 protein production by codon optimization of variants 2 and 3).

Example 23. hAAT-moFGF21, CAG-moFGF21-doublemi
In vitro and in vivo increase in FGF21 expression and protein production levels by RT and CMV-moFGF21 expression cassette Materials and methods In vivo delivery of plasmid to mice by hydrodynamic tail vein injection gram) and injected manually into the lateral tail vein in less tan. Prior to injection, animals were placed under a 250 W infrared heat lamp (Philips) for several minutes to dilate the blood vessels and make the tail vein more visible and easier to access. Animals were immobilized for injection using plastic restrainers (Harvard Apparatus).
No anesthesia was used as it was not necessary as long as a suitable restraint system was used. The inventors 2
Animals were injected using a 6G 3/8 inch gauge hypodermic needle (BD), which is the largest feasible needle gauge that fits snugly into the vein being accessed.

Results In vitro HEK293 cells were transfected with the WT 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)
or codon-optimized mouse FGF21 coding sequence (CMV-moFGF21) under control of CMV promoter or under control of CAG promoter with 4 tandem repeats of miRT122a sequence and 4 tandem repeats of miRT1 sequence A codon-optimized mouse FGF21 coding sequence (CAG-moFGF
21-doublemiRT) was transfected. As a control, non-transfected cells were used. CAG-moFGF21-double
HEK293 cells transduced with emiRT expressed higher levels of FGF21 compared to cells transduced with EF1a-mFGF21 or non-transduced cells (FIG. 34A).
. Moreover, HEK29 transduced with CAG-moFGF21-doublemiRT
3 cells also showed higher intracellular FGF21 protein content and higher F in the culture medium
GF21 protein levels were shown (FIGS. 34B and C). HEK293 cells transduced with EF1a-mFGF21 or CMV-moFGF21 show similar levels of FGF21
(FIG. 34A), HEK293 cells transduced with CMV-moFGF21 exhibited higher intracellular FGF21 protein content and higher FGF21 protein levels in the culture medium (FIGS. 34B and C).

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

HepG2 cells were transfected with the WT mouse FGF21 coding sequence (EF1a-mFGF21) under the control of the EF1a promoter (Zhang et al., EBioMed
icine 15 (2017) 173-183) or a plasmid encoding codon-optimized murine FGF21 under the control of the hAAT promoter (hAAT-moF
GF21) were transfected. As a control, non-transfected cells were used. HepG2 cells transduced with hAAT-moFGF21 are EF1a-mFGF
Higher levels of FGF21 were expressed compared to cells transduced with 21 or non-transduced cells (Fig. 34E).

In vivo 8-week-old male C57B16 mice were injected with 5 μg of the WT mouse FGF21 coding sequence (EF1a-mFGF21) under the control of the elongation factor 1a (EF1a) promoter.
(Zhang et al., EBioMedicine 15 (2017) 173-
183) or codon-optimized mouse FGF21 under the control of the CMV promoter
The coding sequence (CMV-moFGF21) or the codon-optimized mouse FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF21)
was hydrodynamically administered with a plasmid encoding FGF21 expression level analysis in liver 24 hours after plasmid administration showed that hAAT-moFGF21 or CMV-
We found that animals treated with moFGF21 expressed much higher levels of FGF21 than animals that received EF1a-mFGF21 (FIG. 35A). In addition, hAAT-
Animals treated with moFGF21 or CMV-moFGF21 showed EF1a-mFGF
showed higher circulating FGF21 levels than animals receiving 21 (Fig. 35B).

Example 24. AAV8-hAAT-mo compared to AAV8-Ef1a-mFGF21
FGF21, AAV8-CAG-moFGF21-doublemiRT and AAV1
- Increased FGF21 expression in target tissues and circulating levels of FGF21 in vivo by CMC-moFGF21 Liver expression Male C57B16 mice received 1 x 10 ¹⁰ vg, 2 x 10 ¹⁰ vg or 5 x 10 ¹⁰
AAV8 vector (AAV8-EF1a-mFGF21) encoding WT mouse FGF21 coding sequence under control of vg elongation factor 1a (EF1a) promoter or codon-optimized mouse FGF2 under control of liver-specific hAAT promoter
1 coding sequence (AAV8-hAAT-moFGF2
1) was administered intravenously. Two weeks after AAV administration, animals treated with AAV8-hAAT-moFGF21 had higher expression levels of FGF21 in the liver and higher FGF21 expression than animals treated with AAV8-EF1a-mFGF21, regardless of vector dose. Both circulating levels were shown (FIGS. 36A and B).

Expression in Fat Male C57B16 mice received 2×10 ¹⁰ vg, 5×10 ¹⁰ vg or 1×10 ¹¹
vg, WT mouse FGF21 under control of the elongation factor 1a (EF1a) promoter
AAV8 vector encoding coding sequence (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 miRT122a sequence and four tandem repeats of miRT1 sequence (AAV8-CAG-moF
GF21-doublemiRT) were administered intra-eWAT. Two weeks after AAV administration, animals treated with AAV8-CAG-moFGF21-doublemiRT showed A
Animals that received AV8-EF1a-mFGF21 showed higher expression levels of FGF21 in WAT (FIG. 37A). Moreover, AAV8-CAG-moFGF21-dou
Animals treated with blemiRT showed much lower expression of FGF21 in the liver than animals treated with AAV8-EF1a-mFGF21 (FIG. 37B), indicating that
We demonstrate that intra-eWAT administration of the AAV8-CAG-moFGF21-doublemiRT vector efficiently eliminates transgene expression in off-target tissues.

Expression in Skeletal Muscle Male C57B16 mice were injected with 5×10 ¹⁰ vg, 1×10 ¹¹ vg or 3×10 ¹¹
vg, WT mouse FGF21 under control of the elongation factor 1a (EF1a) promoter
AAV8 vector encoding coding sequence (AAV8-EF1a-mFGF21)
or an AAV1 vector encoding a codon-optimized murine FGF21 coding sequence under control of the CMV promoter (AAV1-CMV-FGF21) was administered intramuscularly. Two weeks after AAV administration, animals treated with AAV1-CMV-FGF21 had a much higher F in skeletal muscle than animals administered AAV8-EF1a-mFGF21.
GF21 expression levels were shown (Fig. 38A). Moreover, AAV8-EF1a-mFGF
Animals treated with 21 showed high expression of FGF21 in the liver, whereas AAV1-CMV-F
Intramuscular administration of GF21 vector efficiently abolished transgene expression in the liver (Fig. 38B).
).

SEQ ID NO: Sequence type 1 Homo sapiens FGF21 amino acid sequence 2 Mus musculus FGF21 amino acid sequence 3 Canis lupus familiaris
ris) Amino acid sequence 4 of FGF21 Nucleotide sequence 5 of Homo sapiens FGF21 Codon-optimized Homo sapiens FGF2
1—nucleotide sequence of variant 1 6 codon-optimized Homo sapiens FGF2
1-Variant 2 nucleotide sequence 7 codon-optimized Homo sapiens FGF2
1 - Nucleotide sequence of variant 3 8 Nucleotide sequence of Mus musculus FGF21 9 Codon-optimized Mus musculus FGF2
Nucleotide sequence of 1 10 Canis lupus familiaris
ris) nucleotide sequence of FGF21 11 codon optimized canis lupus familiaris
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 Encoding miRT215 18 a nucleotide sequence encoding miRT192 19 a nucleotide sequence encoding miRT148a 20 a nucleotide sequence encoding miRT194 21 a nucleotide sequence encoding miRT124 22 a nucleotide sequence encoding miRT216 23 a nucleotide sequence encoding miRT125 24 a nucleotide sequence encoding miRT133a 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 chimeric intron composed of introns from human β-globin and immunoglobulin heavy chain genes 44 nucleotide sequence of CAG promoter 45 nucleotide sequence of CMV promoter 46 nucleotide sequence of CMV enhancer 47 nucleotide sequence of 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

Homo sapiens FGF21 amino acid sequence (SEQ ID NO: 1
)
MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQF
GGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESL
LQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEA
CSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRG
PARFLPLPGLLPPALPEPPGILAPQPPDVGSSDPLSMVGPS
QGRSP YAS

Homo sapiens FGF21 nucleotide sequence (SEQ ID NO: 4)
ATGGACTCGGACGAGAGACCGGGTTCGAGCACTCAGGACTGT
GGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCA
GGCACACCCCATCCCTGACTCCAGTCCTCTCCCTGCAATTC
GGGGGCCAAGTCCGGCAGCGGTACCTCTCACACAGATGATG
CCCAGCAGACAGAAGCCCCACCTGGAGATCAGGGAGGATGG
GACGGTGGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTC
CTGCAGCTGAAGCCTTGAAGCCGGGAGTTATTCAAATCT
TGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGA
TGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCC
TGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATG
TTTACCAGTCCGAAGCCCCACGGCCTCCCCGCTGCACCTGCC
AGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGA
CCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCCGCAC
TCCCGGAGCCACCCGGAATCCTGGCCCCCCCAGCCCCCCGA
TGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCC
CAGGGCCGAAGCCCCAGCTACGCTTCCTGA

Codon-optimized Homo sapiens FGF21-variant 1 nucleotide sequence (SEQ ID NO: 5)
ATGGATTCTGATGAGACAGGCTTCGAGCACAGCGGCCTGT
GGGTTTCAGTTCTGGCTGGACTGCTGCTGGGGAGCCTGTCA
GGCACACCCTATTCCAGATAGCAGCCCCTCTGCTGCAGTTC
GGCGGACAAGTGCGGCAGAGATACCTGTACACCGACGACG
CCCAGCAGACAGAAGCCCCACCTGGAAAATCAGAGAGGATGG
CACAGTTGGGCGGAGCCGCCGATCAGTCTCCTGAATCTCTG
CTCCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCC
TGGGCGTGAAAACCAGCCGGTTCCTGTGCCAAAGACCTGA
CGGCGCCCTGTATGGCAGCCTGCACTTTGATCCTGAGGCC
TGCAGCTTCAGAGAGCTGCTGCTTGAGGACGGCTACAACG
TGTACCAGTCTGAGGCCCATGGCCTGCCTCTGCATCTGCC
TGGAAACAAGAGCCCCTCACAGAGATCCCCGCTCCTAGAGGC
CCTGCCAGATTTCTGCCTCTTCCTGGATTGCCTCCTGCTC
TGCCAGAGCCTCCTGGAATTCTGGCTCTCTCAGCCTCCTGA
TGTGGGCAGCTCTGATCCCTCTGAGCATGGTCGGACCTAGC
CAGGGCAGATCTCCTAGCTACGCTCTTGA

Codon-optimized Homo sapiens FGF21-Variant 2 nucleotide sequence (SEQ ID NO: 6)
ATGGACAGCGATGAAAACCGGGTTCGAGCACAGCGGTCTGT
GGGTGTCCGTGCTGGCCGGACTGCTCCTGGGAGCCTGTCA
GGCGCACCCCATCCCTGACTCCTCGCCGCTGCTGCAATTC
GGCGGACAAGTCCGCCAGAGATACCTGTACACCGACGACG
CCCAGCAGACCGAAGCCCCACCTGGAAATTCGGGAGGACGG
GACTGTGGGAGGCGCTGCAGATCAGTCACCCGAGTCCCTC
CTCCAACTGAAGGCCTTGAAGCCCCGGCGTGATTCAGATCC
TGGGCGTGAAAACTTCCCGCTTCCTTTGCCAACGGCCGGA
TGGAGCTCTCTGTACGGATCCCTGCACTTCGACCCCGAAGCC
TGCTCATTCGCGAGCTGCTCCTTGAGGACGGCTATAACG
TGTACCAGTCTGAGGCCCATGGACTCCCCCTGCCATCTGCC
CGGCAACAAGTCCCCTCACCGGGATCCTGCCCCAAGAGGC
CCAGCTCGGTTTCTGCCTCTGCCGGGGACTGCCTCCAGCGT
TGCCCGAACCCCCCTGGTATCCTGGCCCCGCAACCACCTGA
CGTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAGC
CAGGGAAGGTCCCCGTCCTACGCATCCTGA

Codon-optimized Homo sapiens FGF21-variant 3 nucleotide sequence (SEQ ID NO: 7)
ATGGATTCCGACGAAAACTGGATTTGAACATTCAGGGGCTGT
GGGTCTCTGTGCTGGCTGGACTGCTGCTGGGGGCTTGTCA
GGCTCACCCCATCCCTGACAGCTCCCCCTCTGCTGCAGTTTC
GGAGGACAGGTGCGGCAGAGATACCTGTATACCGACGATG
CCCAGCAGACAGAGGCACACCTGGAGATCAGGGAGGACGG
AACCGTGGGGAGGAGCAGCCGATCAGTCTCCCGAGAGCTG
CTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCC
TGGGCGTGAAGACATCTCGGTTTCTGTGCCAGCGGCCCGA
CGGCGCCCCTGTACGGCTCCCTGCACTTCGATCCCGAGGCC
TGTTCTTTTAGGGAGCTGCTGCTGGGAGGACGGCTACAACG
TGTATCAGAGCGAGGCACACGGCCTGCCACTGCACCTGCC
TGGCAATAAGTCCCCCTCACCGCGATCCAGCACCCCAGGGGC
CCAGCACGCTTCCTGCCTCTGCCAGGCCTGCCCCCTGCCC
TGCCAGAGCCACCCGGCATCCTGGCCCCCCAGCCCTCCAGA
TGTGGGCTCCAGCGATCCTCTGTCAATGGTGGGGCCAAGT
CAGGGGCGGAGTCCTTCATACGCATCATAA

Nucleotide sequence encoding miRT122a (target sequence of microRNA 122a) (SEQ ID NO: 12)
5'CAAACACCATTGTCACACTCCA 3'

Nucleotide sequence encoding miRT1 (target sequence of microRNA 1) (SEQ ID NO: 13)
5' TTACATACTTCTTTACATTCCA 3'

Nucleotide sequence encoding miRT152 (target sequence of microRNA 152) (
SEQ ID NO: 14)
5' CCAAGTTCTGTCATGCACTGA 3'

Nucleotide sequence encoding miRT199a-5p (target 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' TCACAGGTTAAAAGGGTTCTCAGGGA 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 (cardiac-specific microRNA 4
99 target sequence) (SEQ ID NO: 30)
5'AAACATCACTGCAAGTCTTAA 3'

Nucleotide sequence of CAG promoter (SEQ ID NO:44)
GACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC
GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT
ACATAACTTACGGTAAATGGCCCGCCCTGGCTGACCGCCCA
ACGACCCCGCCCCATTGACGTCAATAATGACGTATGTTCC
CATAGTAACGCCAATAGGGGACTTTCCATTGACGTCAATGG
GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATTC
AAGTGTATCATATGCCAAGTACGCCCCCCTATTGACGTCAA
TGACGGTAAATGGCCCGCCTGGCATTATGCCCCAGTACATG
ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT
TAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTT
CTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCCACCCCCA
ATTTTGTATTTTATTTAATTTTTTTAATTATTTTGTGCAGCGA
TGGGGGCGGGGGGGGGGGGGGGCGCGCGCGCCAGGCGGGGGC
GGGGCGGGGGCGAGGGGCGGGGCGGGGGCGAGGCGGAGAGGT
GCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAAGTTTTC
CTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAA
GCGAAGCGCGCGCGCGGGCGGGAGTCGCTGCGTTTGCCTTCG
CCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCC
GGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGGCG
GGACGGCCCTTCTCCCTCCGGCTGTAATTAGCGCTTGGGTT
TAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCC
TTGAGGGGCTCCGGGAGGGCCCTTGTGCGGGGGGAGCGGG
CTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCC
GCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCG
GGCGCGGGCGCGGGGGCTTTGTGCGCTCCGCAGTGTGCGCGA
GGGGAGCGCGGCGGGGGGCGGTGCCCCGCGGTGCGGGGGG
CTGCGAGGGGACAAAGGCTGCGTGCGGGGTGTGTGTGCGTG
GGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGC
AACCCCCCCTGCACCCCCCCTCCCCGAGTTGCTGAGCACGG
CCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGGCGCG
GGGCTCCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGG
TGCCGGCGGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTC
GGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGT
CGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT
CGTGCGAGAGGCGCGCAGGACTTCCTTTGTCCCCAAATCTG
TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCT
AGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGG
AAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCGCGCCG
TCCCCTTCTCCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGG
GACGGCTGCCTTCGGGGGGGACGGGGCAGGGGCGGGGTTCG
GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA
CCATGTTCATGCCTTCTTCTTTTTCCTACAG

Nucleotide sequence of CMV promoter (SEQ ID NO:45)
GTGATGCGGTTTTTGGCAGTACACCAATGGGCGTGGATAGC
GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGA
CGTCAATGGGAGTTTGTTTTGGCACCAAATCAACGGGAC
TTTCCAAAATGTCGTAACAACTGCGATCGCCCCGCCCCGTT
GACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTAT
ATAAGCAGAGCT

Nucleotide sequence of CMV enhancer (SEQ ID NO:46)
GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTAC
GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT
ACATAACTTACGGTAAATGGCCCGCCCTGGCTGACCGCCCA
ACGACCCCGCCCCATTGACGTCAATAATGACGTATGTTCC
CATAGTAACGCCAATAGGGGACTTTCCATTGACGTCAATGG
GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATTC
AAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAA
TGACGGTAAATGGCCCGCCTGGCATTATGCCCCAGTACATG
ACCTTACGGACTTTCCTACTTGGCAGTACATCTACGTAT
TAGTCATCGCTATTACCATG

Nucleotide sequence of hAAT promoter (SEQ ID NO:47)
GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAG
TGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGAC
TGTCTGACTCACGCCACCCCCTCCACCTTTGGACACAGGAC
GCTGTGGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTA
AGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCCG
GGCAGCGTAGGCGGCGCGACTCAGATCCCAGCCAGTGGACT
TAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGG
TTAATATTCACCAGCAGCCTCCCCCCGTTGCCCCTCTCGGAT
CCACTGCTTAAATACGGACGAGGACAGGGCCCTGTTCTCCT
CAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT

Truncated AAV2 5'ITR (SEQ ID NO: 48)
GCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAG
C.
CCGGGCGTCG GGCGACCTTTT GGTCGCCCGG CCTCAGT
GAGCGAGCGAGCG
CGCAGAGAGG GAGTGGCCAA CTCCATCACT AGGGGTT
CCT

Truncated AAV2 3'ITR (SEQ ID NO: 49)
AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTCT
GCG
CGCTCGCTCCG CTCACTGAGG CCGGCGCGACC AAAGGTC
GCC CGACGCCCCGG GCTTTGCCCCG GGCGGCCTCA GTG
AGCGAGCGAGCGCGC

SV40 polyadenylation signal (SEQ ID NO:50)
TAAGATACATTGATGAGTTTGGACAAAACCACAACTAGAAT
GCAGTGAAAAAAAATGCTTATTTGTGAAATTTGTGGATGCT
ATTGCTTTATTTGTAACCATTATAAGCTGCAATAAAACAAG
TT

Rabbit β-globin polyadenylation signal (SEQ ID NO:51)
GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGA
AGCCCCTTGAGCATCTGACTTCTGGCTAATAAAAGGAAAATT
TATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTTCTC
TCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAAC
ATCAGAATGAGTATTTGGGTTTAGAGTTTGGCAACATATGC
CCATATGCTGGCTGCCATGAACAAAGGGTTGGCTATAAAAGA
GGTCATCAGTATATGAAACAGCCCCCCTGCTGTCCATTCCT
TATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTT
TATATTTTGTTTTTGTGTTTATTTTTTTCTTTAACATCCCTA
AAATTTTCCTTACATGTTTTACTAGGCCAGAATTTTTCCTCC
TCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTT
ATGGAGATC

CMV promoter and CMV enhancer sequences (SEQ ID NO:52)
GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTAC
GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT
ACATAACTTACGGTAAATGGCCCGCCCTGGCTGACCGCCCA
ACGACCCCGCCCCATTGACGTCAATAATGACGTATGTTCC
CATAGTAACGCCAATAGGGGACTTTCCATTGACGTCAATGG
GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATTC
AAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAA
TGACGGTAAATGGCCCGCCTGGCATTATGCCCCAGTACATG
ACCTTACGGACTTTCCTACTTGGCAGTACATCTACGTAT
TAGTCATCGCTATTACCATGGTGATGCGGTTTTTGGCAGTA
CACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT
CCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTT
GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
CTGCGATCGCCCGCCCCCGTTGACGCAAATGGGCGGTAGGC
GTGTACGGTGGGAGGTCTATATAAGCAGAGCT

hepatocyte control region (HCR) enhancer from apolipoprotein E (SEQ ID NO: 53)
CAGAGAGGTCTCTGACCTCTGCCCCAGCTCCAAGGTCAGC
AGGCAGGGAGGGCTGTGTGTTTGCTGTTTGCTGCTTGCAA
TGTTTGCCCATTTTAGGGACATGAGTAGGCTGAAGTTTGT
TCAGTGTGGGACTTCAGAGGCAGCACACAAAACAGC

miniaP2 promoter (SEQ ID NO: 54)
GATTA
ACCCGCCATG CTACTTATCT ACTCGACATT GATTATT
GAC TAGGGGAATT
CCAGCAGGAA TCAGGTAGCT GGAGAATTCGC ACAGAGGC
CAT GCGATTCTTG
GCAAGCCATG CGACAAAGGC AGAAATGCAC ATTTCAC
CCA GAGAGAAGGG
ATTGATGTCA GCAGGAAGTC ACCACCCAGA GAGCAAA
TGG AGTTCCCAGA
TGCCTGACAT TTGCCTTCTT ACTGGATCAG AGTTCAC
TAG TGGAAGTGTC
ACAGCCCCAAA CACTCCCCCA AAGCTCAGCC CTTCCTT
GCC TTGTAACAAT
CAAGCCGCTC CTGGATGAAC TGCTCCGCCC TCTGTCT
CTT TGGCAGGGTT
GGAGCCCACT GTGGCCTGAG CGACTTCTAT GGCTCCC
TTT TCTGTGATTT
TCATGGTTTC TGAGCTCTTT TCCCCCGCTT TATGATT
TTC TCTTTTTGTC
TCTCTCTTGC TAAACCTCCT TCGTATATAT GCCCTCT
CAG GTTTCATTTC
TGAATCATCT ACTGTGAACT ATTCCCATTG TTTGCCA
GAA GCCCCCTGGT
TCTTCCTTCT AGACACCAGG CAAGGGGCAG GAGGTAA
GAGGCAGGAGTCC
ATAAACAGC CCTGAGAGCC TGCTGGGTCA GTGCCTG
CTG TCAGAA

miniUCP1 promoter (SEQ ID NO: 55)
GACGTCACAG TGGGTCAGTC ACCCTTGATC ACACTGC
ACC AGTC TTC ACC
TTTCCACGCT TCCTGCCAGA GCATGAATCA GGCTCTC
TGG GGATAC GGC
CTCACCCCTA CTGAGGCAAA CTTTCTCCCA CTTCTCA
GAG GCTCTGAGGG
CAGCAAGGTC AGCCCTTTCT TTGGAATCTA GAACCAC
TCC CTGTCTTGAG
CTGACATCAC AGGGCAGGCA GATGCAGCAG GGAAGGGG
CCT GGGACTGGGA
CGTTCATCCT ACAAGAAAAGC TGTGGAACTT TTCAGCA
ACA TCTCAGAAAT
CAGATCGCAC TTATTCAAAG GAGCCAGGCC CTGCTCT
GCG CCCTG GTGGA
GGCTCCTCAT GTGAAGAGTG ACAAAAGGCCA CCATGTT
GTG GATACGGGGGC
GAAGCCCCTCCGGTGTGTCCTCCAGGCATTCATCAGGA
ACT AGTGCCAAAG
CAGAGGTGCT GGCCAGGGCT TTGGGAGTGA CGCG CGT
CTG GGAGGCTTGT
GCGCCCCAGGG CACGCCCCTG CCGATTCCCA CTAGCAG
GTC TTGGGGGGACC
TGGGCCGGCT CTGCCCCTCC TCCAGCAATC GGGCTAT
AAA GCTCTTCCAA
GTCAGGGCGC AGAAGTGCCG GGCGATCCGG GCTTAAA
GAGCGAGAGGAAG
GGACGCTCAC CTTTGAGCTC CTCCACAAAAT AGCCCTG
GTG GCTGCCACAG
AAGTTCGAAGTTGAGAGTTC GG

C5-12 promoter (SEQ ID NO:56)
CGGCCGTCCG CCTTCGGCAC CATCCTCACCG ACACCCA
AAT ATGGCGACGGGTGAGGAATG
GTGGGGAGTT ATTTTTAGAG CGGTGAGGAA GGTGGGGC
AGG CAGCAGGTGT TGGCGCTCTA
AAAATA ACTC CCGGGAGTTA TTTTTAGAGC GGAGGAA
TGG TGGACACCCA AATATGGCGA
CGGTTCCTCA CCCGTCGCCA TATTTGGGTG TCCGCCC
TCG GCCGGGGGCCGCATTCCTGGG
GGCCGGGCGG TGCTCCCGCC CGCCTCGATA AAAGGCT
CCG GGGCCGGCGG CGGCCCACGA
GCTACCCGGA GGAGCGGGAG GCGCCA

pAAV-EF1a-mmFGF21-pA (SEQ ID NO: 57)

CCTGCAGGCAGCTGCGCGCTTCGCTCGCTCACTGAGGCCGC
CCGGGCAAAGCCCCGGGCGTCGGGCGACCTTTGGTCGCCCG
GCCTCAGTGAGCGCGCGCGAGCGCGCAGAGAGGGGAGTGGCCA
ACTCCATCACTAGGGGTTCCTGCGGCCCGCGGCTCCGGTGC
CCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAG
AAGTTGGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGA
GAAGGTGGCGCGGGGTAAAACTGGGAAAAGTGATGTCGTGTA
CTGGCTCCGCCTTTTTTCCCGAGGGTGGGGGAGAACCGTAT
ATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACG
GGTTTGGCCGCCAGAACACAGGTAAGTGCCCGTGTGTGGGTTC
CCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTG
CCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGA
TCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGG
CCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTG
AGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTG
GTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCT
CTAGCCATTTAAAATTTTTTGATGACCTGCTGCGACGCTTT
TTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCT
GCACACTGGTATTTCGGTTTTTGGGGGCCGCGGGGCGGCGAC
GGGGCCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGG
GCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTC
TCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCG
CCGTGTATCGCCCCGCCCTGGGCGGCCAAGGCTGGCCCGGT
CGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGG
CCCTGCTGCAGGGAGCTCAAATGGAGGACGCGGCGCTCG
GGAGAGCGGGGCGGGTGAGTCACCCACACAAAGGAAAAGGG
CCTTTCCGTCCCTCAGCCGTCGCTTCATGTGACTCCACGGA
GTACCGGCGCGCCGTCCAGGCACCTCGATTAGTTTCTCGAGC
TTTTGGAGTACGTCGTCTTTAGGTTTGGGGGGGAGGGGTTTT
ATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGA
AGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAA
TTTGCCCTTTTTGAGTTTGGATCTTGGGTTCATTCTCAAGC
CTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGT
GTCGTGAGGAATTTCGACTGCTAGCACGCGTGATATCAAT
GGAATGGATGAGATCTAGAGTTGGGACCCTGGGGACTGTGG
GTCCGACTGCTGCTGGCTGTCTTCCTGCTGGGGGTCTACC
AAGCATACCCCATCCCTGACTCCAGCCCCCTCCTCCAGTT
TGGGGGTCAAGTCCGGCAGAGGTACCTCTCACACAGATGAC
GACCAAGACACTGAAGCCCCACCTGGAGATCAGGGAGGATG
GAACAGTGGGTAGGCGCAGCACACCGCAGTCCAGAAAGTCT
CCTGGAGCTCAAAGCCCTTGAAGCCAGGGGTCATTCAAATC
CTGGGTGTCAAAGCCCTCTAGGTTTCTTTGCCCAACAGCCCAG
ATGGAGCTCTCTATGGATCGCCTCACTTTGATCCTGAGGC
CTGCAGCTTCAGAGAACTGCTGCTGGGAGGACGGTTACAAT
GTGTACCAGTCTGAAGCCCATGGCCTGCCCCTGCGTCTGC
CTCAGAAGGACTCCCCAAAACCAGGATGCAACATCCTGGGGG
ACCTGTGCGCTTCCTGCCCATGCCAGGCCTGCTCCACGAG
CCCCAAGACCAAGCAGGATTCCTGCCCCCAGAGCCCCCAG
ATGTGGGCTCCTCTGACCCCCTGAGCATGGTAGAGCCTTTT
ACAGGGCCGAAGCCCCAGCTATGCGTCCTGAGATATCAAA
GAATTCTAAGCTTTGTCGACGAATGCAATTGTTGTTAATTA
ATTGTTAACTTGTTTTATTGCAGCTTATAATGGTTACAAAT
AAAGCAATAGCATCACAAATTTCACAAAAAAAGCATTTTT
TTCACTGCATTCTAGTTGTGGTTTGTCCAAAACTCATCAAT
GTATCTTAGTCGAGTTAATTAACGGCGGCCGCAGGAACCC
CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCCT
CGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC
GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC
AGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTTCTCCTTA
CGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAA
CCATAGTACGCGCCCTGTAGCGGCCGCATTAAGCGCGGCGG
GTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAG
CGCCCTAGCGCCCCGCTCCTTTCGCTTTCTTCCCCTTCCTTT
CTCGCCACGTTCGCCGCTTTCCCCGTCAAGCTCTAAATC
GGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA
CCTCGACCCCAAAAAAACTTGATTTGGGGTGATGGTTCACGT
AGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTTGA
CGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCA
AACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTT
GATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAA
AAAATGAGCTGATTTAACAAAAAAATTTAACGCGAATTTTAA
CAAATATTAACGTTTTACAATTTTATGGTGCACTCTCAGT
ACAATCTGCTCTCGATGCCGCATAGTTAAGCCAGCCCCGAC
ACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCT
GCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCC
GGGAGCTGCATGTGTCAGAGGTTTTTCACCGTCATCACCGA
AACGCGCGAGACGAAAGGGGCCTCGTGATACGCCTATTTTTT
ATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCA
GGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCCTATTT
GTTTATTTTTCTAAAACATTCAAATATGTATCCGCTCAT
GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAA
AGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTA
TTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA
CCCAGAAACGCTGGTGAAAGTAAAGATGCTGAAGATCAG
TTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACA
GCGGTAAGATCCTTGAGAGTTTTCGCCCCCGAAGAACGTTT
TCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCG
GTATTATCCCGTATTGACGCCGGGCAAGAGCCAACTCGGGTC
GCCGCATACACTATTCTCAGAATGACTTGGGTTGAGTACTC
ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTA
AGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACA
CTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA
GGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTA
ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCA
TACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAAT
GGCAACAACGTTGCGCAAAACTATTAACTGGCGAACTACTT
ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGGAGG
CGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCC
GGCTGGCTGGTTTTATTGCTGATAAATCTGGAGCCGGTGAG
CGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG
GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGGAG
TCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAG
ATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACC
AAGTTTACTCATATATACTTTTAGATTGATTTAAAAACTTCA
TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGAT
AATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC
ACTGAGCGTCAGACCCCGTAGAAAGATCAAAGGATCTTC
TTGAGATCCTTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA
ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTTGCCGG
ATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT
CAGCAGAGCGCAGATACCAAATACTGTCCTTTCTAGTGTAG
CCGTAGTTAGGCCCACCACTTCAAGAACTCTGTAGCACCGC
CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC
TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA
AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA
CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGAC
CTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA
AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATC
CGGTAAGCGGCAGGGTCGGAACAGGGAGAGCGCACGAGGGA
GCTTCCAGGGGGAAACGCCTGGTATCTTTTATAGTCCTGTC
GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGAT
GCTCGTCAGGGGGGCGGAGCCTATGGAAAAAACGCCAGCAA
CGCGGCCCTTTTTACGGTTCCTGGCCTTTTGGCTGGCCTTTTT
GCTCACATGT

Elongation factor 1 alpha promoter: 150 to 1327 (1178 bp)
From Mus musculus FGF21:1359 (633 bp
)
SEQ ID NO: 57 also contains truncated AAV2 5' and 3' ITRs and SV40 polyA (SEQ ID NOS: 48, 49 and 50, already included in the Sequence Listing).

Figure 1. Prevention of obesity by intra-eWAT administration of AAV9-CAG-moFGF21-dmiRT vector in C57Bl6 mice. (A) Schematic representation of the AAV-CAG-moFGF21-doublemiRT vector. The expression cassette contained the CAG promoter, a codon-optimized mouse FGF21 coding sequence, and four tandem repeats of miRT122a and four tandem repeats of miRT1 sequences cloned into the 3′ untranslated region of the expression cassette. . ITRs from AAV2 flanked the expression cassette. Diagrams are not to scale. CAG: chicken β-actin promoter/CMV enhancer; pA: polyA. (B) Expression levels of FGF21 in metabolic tissues. Expression levels of codon-optimized mouse FGF21 coding sequences in eWAT, iWAT, iBAT and liver of C57B16 mice were measured by RTqPCR and normalized by Rplp0 values (n=8-11 animals/group). (C) Circulating levels of FGF21 (n=8-11 animals/group). (DE) Expression levels of FGF21R1 (D) and β-Klotho (E) in metabolic tissues. Expression levels of FGF21 receptor 1 (FGF21R1) and β-Klotho in eWAT, iWAT, iBAT and liver of C57B16 mice were measured by RTqPCR and normalized by Rplp0 values (n=7 animals/group). (F) Weight evolution. Body weights were measured weekly (n=8-11 animals/group). (G) Representative images of animals. (H) Tissue weight. eWAT, iWAT, rWAT, mWAT, iBAT and liver weights of chow-fed C57B16 and HFD-fed C57B16 mice treated with AAV vectors into eWAT (n=8-11 animals/group). Analyzes were performed 14 weeks after intra-eWAT administration of 10 ¹² 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. Figure 2. Histological analysis of adipose tissue and liver of C57Bl6 mice treated with AAV9-CAG-moFGF21-doublemiRT vector in eWAT. (A) Epididymal white adipose tissue (eWAT), inguinal white adipose tissue (eWAT) of chow-fed and HFD-fed C57Bl6 mice treated with AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vector in eWAT iWAT) Representative images of interscapular brown adipose tissue (iBAT) and liver sections stained with hematoxylin and eosin. Original magnification x 100. (B) Mean area of white adipocytes in eWAT (n=4 animals/group). (C) Frequency distribution of white adipocyte area in eWAT (n=4 animals/group). Analyzes were performed 14 weeks after intra-eWAT administration of 10 ¹² 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. Figure 3. Enhanced energy expenditure and insulin sensitivity in C57Bl6 mice treated with AAV9-CAG-moFGF21-doublemiRT vector in eWAT. (AB) Expression levels of UCP1 (A) and Dio2 (B). Expression levels of UCP1 and Dio2 in iWAT were measured by RTqPCR and normalized with Rplp0 values (n=7 animals/group). (C) Energy metabolism. Energy expenditure (EE) was measured with an indirect open circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were acquired during the light (basal) and dark (active) cycles after 9 weeks of AAV administration 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, analyzes were performed 14 weeks after intra-eWAT administration of 10 ¹² vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vector. Results are expressed as mean ± SEM. HFD, high fat diet; TG, triglyceride; 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. Obesity reversal 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 codon-optimized mouse FGF21 coding sequences in eWAT, iWAT, iBAT and liver of ob/ob mice were measured by RTqPCR and normalized by Rplp0 values. (B) Circulating levels of FGF21. (CD) Evolution of body weight (C) and weight gain (D). Body weight was measured weekly. (E) Tissue weight. eWAT, iWAT, rWAT, mWAT, iBAT and liver weights of ob/ob mice treated with AAV vectors into eWAT. Analyzes were performed 16 weeks after intra-eWAT administration of 10 ¹⁰ vg, 5×10 ¹⁰ vg, 2×10 ¹¹ vg or 10 ¹² vg of AAV8-CAG-moFGF21-doublemiRT or 10 ¹² 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. Figure 5. Improvement of insulin sensitivity in ob/ob mice treated with 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 study was performed 9 weeks after AAV administration. (B) Fasting circulating insulin levels two 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. Intravenous administration of AAV8-hAAT-moFGF21-vector reverses obesity and improves glucose metabolism in ob/ob mice. (A) Schematic representation of the AAV-hAAT-moFGF21 vector. The expression cassette contained the human α1-antitrypsin (hAAT) promoter and codon-optimized mouse FGF21 coding sequence. ITRs from AAV2 flanked the expression cassette. Diagrams are not to scale. pA: polyA. (B) Expression levels of FGF21. Expression levels of the codon-optimized mouse FGF21 coding sequence in livers of ob/ob mice were measured by RTqPCR and normalized by Rplp0 values. (C) Circulating levels of FGF21. (DE) Evolution of body weight (C) and weight gain (D). Body weight was measured weekly. (F) Representative images of animals. (G) Tissue weight. eWAT, iWAT, rWAT, mWAT, iBAT and liver weights of ob/ob mice treated intravenously 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 study was performed 9 weeks after AAV administration. (I) Fasting circulating insulin levels 3 months after AAV administration. Unless otherwise indicated, analyzes were performed 20 weeks after intravenous administration of 10 ¹¹ vg or 5×10 ¹¹ vg of AAV8-hAAT-moFGF21 or 5×10 ¹¹ 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. 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. (BC) Evolution of body weight (C) and weight gain (D). Body weight was measured weekly. Analyzes were performed 52 weeks after intravenous administration of 10 ¹⁰ vg or 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 or 5×10 ¹⁰ 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. Figure 8. Long-term enhancement of energy expenditure and insulin sensitivity by intravenous administration of AAV-hAAT-moFGF21 vector in HFD-fed C57B16 mice. (A) Energy metabolism. Energy expenditure (EE) was measured with 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. 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 study was performed 7 weeks after AAV administration. (C) Fasting and fed insulin circulating levels. Results are expressed as mean±SEM, n=9-12 animals/group. HFD, high fat diet; *p<0.05 vs AAV8-hAAT-null chow, **p<0.01 vs AAV8-hAAT-null chow, ***p<0.001 vs AAV8-hAAT-null chow, $p <0.05 vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD, $$ p<0.001 vs AAV8-hAAT-null HFD. Figure 9. Obesity reversal by intravenous administration of AAV-hAAT-moFGF21 vector in aged HFD-fed mice. (A) Circulating levels of FGF21. (BC) Evolution of body weight (B) and weight gain (C). Body weight was measured weekly. Analyzes were performed 21 weeks after intravenous administration of 10 ¹⁰ vg, 2×10 ¹⁰ vg or 5×10 ¹⁰ vg of AAV8-hAAT-moFGF21 or 5×10 ¹⁰ vg of AAV8-hAAT-null vector. Results are expressed as mean±SEM, n=7-8 animals/group. HFD, high fat diet; *** p<0.05 vs AAV8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD. $$p<0.001 vs AAV8-hAAT-null HFD. Figure 10. Improving energy expenditure and insulin sensitivity by intravenous administration of AAV-hAAT-moFGF21 vectors in aged HFD-fed mice. (A) Energy metabolism. Energy expenditure (EE) was measured with an indirect open circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were obtained during the light cycle (basal state) and dark cycle (active phase) 6 weeks after AAV administration 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 study was performed 9 weeks after AAV administration. (C) Fasting and fed insulin circulating levels. Results are expressed as mean±SEM, n=7-8 animals/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. Weight loss by intramuscular administration of AAV-CMV-moFGF21 vector in C57Bl6 mice. (A) Schematic representation of the AAV-CMV-moFGF21 vector. The expression cassette contained the cytomegalovirus (CMV) promoter and codon-optimized murine FGF21 coding sequence. ITRs from AAV2 flanked the expression cassette. Diagrams are not to scale. pA: polyA. (B) Circulating FGF21 levels. (CD) Evolution 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. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. 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. Intra-eWAT administration of AAV8-CAG-moFGF21-dmiRT vector in ob/ob mice. A, B. Hematoxylin-eosin staining of (A) eWAT and (B) liver tissue sections obtained from ob/ob animals injected intra-eWAT at all doses testing either null or an AAV8 vector encoding FGF21. representative image. Scale bar: 100 μm for eWAT, 200 μm for liver. C Blood glucose in the fed state. Blood insulin in the fed state 3 months after D AAV. The FGF21 label in the figure refers to moFGF21. Data Information: All values are expressed as mean±SEM. n=6-9 animals/group in (A, B). n=4-8 animals/group in (CH). In (I) n=6-8 animals/group. *P<0.05, **P<0.01 and ***P<0.001 vs Null injection group. Figure 14. Effect of FGF21 gene transfer on eWAT in ob/ob mice. A At 11 weeks of age, either AAV8-CAG-null vector or AAV8-CAG-moFGF21-dmiRT vector was administered at four different doses (1×10 ¹⁰ , 5×10 ¹⁰ , 2×10 ¹¹ , 1×10 ¹² vg/day). Serum adiponectin levels in 25-week-old ob/ob animals injected intra-eWAT in mice). B. Expression quantification by qRT-PCR of the macrophage marker F4/80 in the same animals as in (A) C. eWAT sections from ob/ob mice that received the AAV8-CAG-moFGF21-dmiRT vector for the macrophage-specific marker Mac2. Representative images of immunostaining of . n=4-8/group. Scale bar: 200 μm. D Liver weights in all intra-eWAT treatment groups. E, F Liver triglyceride and cholesterol content in the fed state in the same cohort as in (A). Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. 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 versus null injected ob/ob group. Figure 15. Reduced obesity and improved insulin sensitivity in ob/ob mice treated with AAV8-hAAT-moFGF21 vector. Representative images of hematoxylin-eosin staining of eWAT tissue sections obtained from ob/ob animals injected with either A null or AAV vectors encoding FGF21 at 1×10 ¹¹ or 5×10 ¹¹ vg/mouse. B Serum adiponectin levels in all groups. Representative images of hematoxylin-eosin staining of liver tissue sections obtained from ob/ob animals injected with either C null or AAV vectors encoding FGF21 at 1×10 ¹¹ or 5×10 ¹¹ vg/mouse. D Feeding blood glucose levels. E Feeding serum insulin levels at 5 months post AAV. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data information: All data are expressed as mean±SEM. n=9-10 animals/group in (AC, E, GH). *P<0.05, **P<0.01 and ***P<0.001 versus null injected ob/ob group. Figure 16. Effect of FGF21 liver gene transfer on ob/ob mice. 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. B, C Expression quantification by qRT-PCR of inflammatory markers F4/80 (B) and TNF-α (C) in the same cohort of mice. D, E Weights (D) and representative images (E) of livers obtained from animals belonging to the same experimental group as in (A). F, G Liver triglyceride and cholesterol content in the fed state in the same cohort as in (A). Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. 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 versus null injected ob/ob group. 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 Expression quantification by qRT-PCR of hepatic PEPCK and G6Pase in ob/ob mice injected with either AAV8-hAAT-null vector or AAV8-hAAT-moFGF21 vector at 2 months of age. CF Expression quantification by qRT-PCR 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 levels of UCP1 in iBAT in the same cohort as in (A). Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. n=9-10 animals/group in (AG). *P<0.05, **P<0.01 and ***P<0.001 versus null injected ob/ob group. Figure 18. AAV8-mediated hepatic FGF21 gene transfer counteracts HFD-induced obesity. A Epididymal (eWAT), inguinal (iWAT) and retroperitoneal (rWAT) white adipose tissue obtained from mice treated with the AAV8-hAAT-moFGF21 vector as young adults (top panels) or adults (bottom panels). Weight of depot, liver and quadriceps. B Circulating levels of FGF21 at different time points after vector administration. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. n=7-10 animals/group in (AD). *P<0.05, **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^#P <0.05, ^## P<0.01 and ^### P<0.001 versus HFD-fed Null injection group. HFD, high fat diet; Figure 19. FGF21 gene transfer into the liver counteracts HFD-induced obesity. A, B Representative images of animals belonging to all experimental groups of studies performed in young adults (A) or adults (B). C Representative images of epididymal white adipose (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 livers of young adults or animals treated as adults. qPCR was performed using primers that specifically detect the coding sequence of codon-optimized murine FGF21 (coFGF21). Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. In (E) n=7-10 animals/group. HFD, high fat diet; ND, not detected. Figure 20. AAV8-hAAT-moFGF21-mediated increased energy expenditure and decreased fat accumulation in iBAT and iWAT. A Assessment of locomotor activity through the open field test in animals (young adults) subjected to HFD feeding from approximately 2 months of age and treated 2 months later with either null or a vector encoding FGF21. 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 Expression quantification by qRT-PCR of Phospho1 in iWAT in young adults or groups of animals that started HFD feeding as adults and received the FGF21 vector. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. n=7-10 animals/group in (AC). In (E) n=4 animals/group. In (G) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^#P <0.05 and ^### P<0.001 versus HFD-fed Null injection group. HFD, high fat diet; Figure 21. Energy consumption in October after gene transfer to the liver. A Energy expenditure in cohorts of animals that started 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. B Western blot analysis of UCP1 content in iWAT from the same animal cohort. A representative immunoblot is shown (left). The graph shows the densitometric analysis of two different immunoblots (right). C Relative expression levels of Serca2b and RyR2 in iWAT in young adults or groups of animals that started HFD feeding as adults and received the FGF21 vector. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. In (A) n=7-10 animals/group. In (B) n=4 animals/group. In (C) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^### P<0.001 vs HFD-fed Null-injected group. HFD, high fat diet; Figure 22. Restoration of AAV8-hAAT-moFGF21-mediated islet hyperplasia. A Fasting glucagon levels in a group of animals that started on HFD feeding as young adults and received the FGF21 vector. B, β-cell mass in a group of animals that began HFD feeding as adults and received the FGF21 vector. C Representative images of immunostaining for insulin in pancreatic sections from animals that received 5×10 ¹⁰ vg/mouse of AAV8-hAAT-moFGF21 as adults. Scale bar: 400 μm. Inset scale bar: 100 μm. D Duplicates for insulin (dark grey) and glucagon (light grey) in pancreatic sections from animals receiving 5×10 ¹⁰ vg/mouse of AAV8-hAAT-moFGF21 as young adults (top panels) or adults (bottom panels). Representative images of immunostaining. Scale bar: 100 μm. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. n=7-10 animals/group in (AC). In (D) n=4-5 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^#P <0.05, ^## P<0.01 and ^### P<0.001 versus HFD-fed Null injection group. HFD, high fat diet; 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 groups of mice that received FGF21 vector starting on HFD feeding as young adults. B Serum insulin levels during the glucose tolerance test shown in (A). Data information: All data are expressed as mean±SEM. n=7-10 animals/group in (AD). *P<0.05, **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^#P <0.05, ^## P<0.01 and ^### P<0.001 versus HFD-fed Null injection group. HFD, high fat diet; Figure 24. Reversal of WAT hypertrophy and inflammation by AAV8-hAAT-moFGF21 treatment. A From animals fed chow or HFD as young adults (left panel) or adults (right panel) and administered either AAV8-hAAT-null or 5×10 ¹⁰ vg/mouse of AAV8-hAAT-moFGF21 vector. Representative images of hematoxylin-eosin staining of eWAT from . 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. B Morphometric analysis of WAT adipocyte area in young adults or animals treated as adults. C, D Circulating levels of adiponectin (C) and leptin (D). E Immunohistochemistry for the macrophage-specific marker Mac2 in eWAT sections from animals receiving 5×10 ¹⁰ vg/mouse AAV8-hAAT-moFGF21 as adults. Micrographs show the presence of crown-like structures in eWAT of HFD-fed, null-injected animals (arrows and insets), but not in eWAT of HFD-fed, FGF21-treated mice. Scale bar: 200 μm and 50 μm (inset). FH Expression quantification by qRT-PCR of inflammatory markers F4/80 (F), IL1-β (G) and TNF-α (H) in a group of animals that started HFD feeding as adults and received FGF21 vector. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. In (B) n=4 animals/group. In (FH) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^#P <0.05, ^## P<0.01 and ^### P<0.001 versus HFD-fed Null injection group. HFD, high fat diet; Figure 25. Adipocyte size and inflammation in AAV8-hAAT-moFGF21 treated animals. A Animals receiving either AAV8-hAAT-null or 5×10 ¹⁰ vg/mouse of AAV8-hAAT-moFGF21 vector starting on chow or HFD feeding as young adults (top graph) or adults (bottom graph). Frequency distribution of adipocyte area in groups. B, Mac2 immunohistochemistry in eWAT of animals beginning the study as young adults. Crown-like structures formed by macrophage infiltration in eWAT of HFD-fed null-injected mice are indicated by arrows. Scale bar: 200 μm and 50 μm (inset). CE Relative expression levels by qRT-PCR of inflammatory markers F4/80, CD68 and TNF-α in the same animal cohort as in (B). Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. n=4 animals/group in (A). In (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. Treatment with vectors encoding FGF21 reverses fatty liver and liver inflammation. A. Representative hematoxylin-eosin staining of liver sections obtained from animals fed chow or HFD and administered either AAV8-hAAT-null or 5×10 ¹⁰ vg/mouse of AAV8-hAAT-moFGF21 vector. image. 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 Liver triglyceride and cholesterol content at feeding in the same animal cohort. D Immunostaining for the macrophage-specific marker Mac-2 of liver sections from animals fed HFD and receiving AAV8-hAAT-null or 5×10 ¹⁰ vg/mouse of AAV8-hAAT-moFGF21 vector. Arrows point to the presence of crown-like structures. Scale bar: 200 μm and 50 μm (inset). Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. In (BC) n=7-10 animals/group. **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^{# #} P<0.01 vs HFD-fed Null-injected group. HFD, high fat diet; Figure 27. AAV8-hAAT-moFGF21-mediated reversal of liver fibrosis. Analysis of liver fibrosis through Masson's Trichrome staining in HFD-fed animals receiving either 5×10 ¹⁰ vg/mouse of AAV8-hAAT-null or AAV8-hAAT-moFGF21 vectors. AAV8-hAAT-moFGF21 treatment (right panel) significantly reduced detection of readily detectable collagen fibers (blue) in null vector treated animals (left panel). Scale bar: 50 μm. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. 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 5×10 ¹⁰ vg/mouse. AAV8-hAAT-moFGF21 treatment (right panel) significantly reduced detection of readily detectable collagen fibers (black) in null vector treated animals (left panel). Scale bar: 50 μm. B, C Expression quantification by qRT-PCR of Collagen 1 in liver in groups of animals that received FGF21 vector starting on HFD feeding as young adults (B) or adults (C). Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. In (BC) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus chow-fed Null injection group. ^#P <0.05 and ^### P<0.001 versus HFD-fed Null injection group. HFD, high fat diet; Figure 29. No bone abnormalities were observed in AAV8-hAAT-moFGF21 treated animals. Long-term effects of FGF21 gene transfer on bone were evaluated in HFD-fed mice treated as young adults or with the highest dose (5×10 ¹⁰ vg/mouse) of AAV8-hAAT-moFGF21 vector as adults versus chow or HFD null injections. Tested by comparison with fed animals. A Full length from nose tip to ridge. B tibia length. Epiphysis (CJ) and diaphysis (KO) of tibiae obtained from HFD-fed mice administered either C-O null or an AAV vector encoding FGF21 at sacrifice, i.e., when animals were 18 months of age. microcomputed tomography (μCT) analysis of . P, Q Levels of circulating IGFBP1 (P) and IGF1 (Q) measured by ELISA. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data information: All data are expressed as mean±SEM. n=7-10 animals/group in (A, PQ). n=4 animals/group in (BO). **P<0.01 and ***P<0.001 versus chow-fed Null injection group. 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; N, trabecular bone number; Tb. Th, trabecular width; Tb. Sp, trabecular spacing; Figure 30. Analysis of glycemic profiles in C57B16 mice treated with AAV8-hAAT-moFGF21 vector. Blood glucose levels were assessed under fed conditions. IV administration of AAV, 5×10 ¹⁰ vg or 2×10 ¹¹ vg of AAV8-hAAT-moFGF21 (n=13 and 15, respectively) or 2×10 ¹¹ vg of AAV8-null vector (n=15). STZ, treatment with streptozotocin (5 x 50 mg/kg). Results are mean + SEM. *p<0.05;***p<0.001 vs AAV8-hAAT-Null. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Figure 31. Gene transfer of FGF21 into skeletal muscle of healthy animals. A Circulating levels of FGF21 measured 40 weeks after injection of either AAV1-CMV-Null or AAV1-CMV-moFGF21 vectors of 3×10 ¹¹ vg/mouse 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 vectors. C Evolution of body weight during the 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 Feeding serum insulin levels. Insulin sensitivity assessed through intraperitoneal injection of H insulin (0.75 units/kg body weight) and expressed as a percentage of starting blood glucose. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. Data Information: All values are expressed as mean±SEM. n=5-7 animals/group in (AH). *P<0.05, **P<0.01 and ***P<0.001 vs Null injection group. Figure 32. AAV1-mediated skeletal muscle FGF21 gene transfer counteracts HFD-induced obesity and insulin resistance. A, B Evolution of body weight (A) and weight gain (B) in animals treated with AAV1-CMV-moFGF21. C57B16 mice were fed HFD for approximately 12 weeks and then administered 3×10 ¹¹ vg/mouse of AAV1-CMV-moFGF21 vector. Control obese mice and control chow-fed mice received 3×10 ¹¹ vg of AAV1-CMV-null. C Circulating levels of FGF21 at different time points after vector administration. D, E Levels of fasting blood glucose (D) and fed serum insulin (E) 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 starting blood glucose levels. Labeling of FGF21 in the figure refers to moFGF21 according to the legend of this figure. 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. In vivo increase in FGF21 circulating levels by codon optimization of the nucleotide sequence encoding human FGF21. Circulating levels of hFGF21 in C57B16 mice hydrodynamically administered with 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. Increased FGF21 expression levels in vitro by hAAT-moFGF21, CAG-moFGF21-doublemiRT and CMV-moFGF21 expression cassettes. (A) WT mouse FGF21 coding sequence under control of EF1a promoter (EF1a-mFGF21) or codon-optimized mouse FGF21 coding sequence under control of CMV promoter (CMV-moFGF21) or four sequences of miRT122a FGF21 in HEK293 cells transfected with a plasmid encoding a codon-optimized mouse FGF21 coding sequence under the control of the CAG promoter (CAG-moFGF21-doublemiRT) with a tandem repeat and four tandem repeats of the miRT1 sequence. expression level. (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) Plasmids encoding WT mouse FGF21 coding sequence under control of EF1a promoter (EF1a-mFGF21) or codon-optimized mouse FGF21 coding sequence under control of CMV promoter (CMV-moFGF21) were transfected. Expression level of FGF21 in C2C12 cells. (E) HepG2 transfected with plasmids encoding WT mouse FGF21 coding sequence under control of EF1a promoter (EF1a-mFGF21) or codon-optimized mouse FGF21 under control of hAAT promoter (hAAT-moFGF21). Expression level of FGF21 in cells. qPCR was performed using primers that detect both the wt and codon-optimized FGF21 coding sequences. Results are expressed as mean ± SEM. n=3 wells/group. ND, not detected. *p<0.05 vs control. ### p<0.001 vs EF1a-mFGF21. Figure 35. In vivo increase in hepatic FGF21 expression and circulating FGF21 levels by hAAT-moFGF21 and CMV-moFGF21 expression cassettes. (A) WT mouse FGF21 coding sequence under control of elongation factor 1a (EF1a) promoter (EF1a-mFGF21) or codon-optimized mouse FGF21 coding sequence under control of CMV promoter (CMV-moFGF21) or hAAT Expression levels of FGF21 in the liver of C57Bl6 mice hydrodynamically administered with a plasmid encoding a codon-optimized murine FGF21 coding sequence (hAAT-moFGF21) under the control of a promoter. qPCR was performed using primers that detect both the wt and codon-optimized FGF21 coding sequences. (B) FGF21 circulating 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. In vivo increase in hepatic FGF21 expression and circulating FGF21 levels by AAV8-hAAT-moFGF21. ( ^A ) AAV8 vectors ^{( AAV8} -EF1a- FGF21 expression levels in the liver of C57Bl6 mice intravenously injected with either mFGF21) or codon-optimized mouse FGF21 under the control of the hAAT promoter (AAV8-hAAT-moFGF21). qPCR was performed using primers that detect both the wt and codon-optimized FGF21 coding sequences. (B) FGF21 circulating levels in the same cohort as in (A). Analysis was performed 2 weeks after 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. In vivo increase in adipose FGF21 expression by AAV8-CAG-moFGF21-dmiRT. (AB) AAV8 vectors encoding 2×10 ¹⁰ vg, 5×10 ¹⁰ vg or 1×10 ¹¹ vg of the WT 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 murine FGF21 coding sequence under control of a CAG promoter with four tandem repeats of miRT122a sequences and four tandem repeats of miRT1 sequences (AAV8- Expression levels of FGF21 in eWAT (A) or liver (B) of C57B16 mice intra-eWAT with either CAG-moFGF21-doublemiRT). qPCR was performed using primers that detect both the wt and codon-optimized FGF21 coding sequences. Analysis was performed 2 weeks after AAV. Results are expressed as mean±SEM. n=4-5 mice/group. Control, untreated mice. eWAT, epidydimal 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) AAV8 vectors encoding 5×10 ¹⁰ vg, 1×10 ¹¹ vg or 3×10 ¹¹ vg of the WT mouse FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8- Quadriceps (A) of C57Bl6 mice intramuscularly injected with either EF1a-mFGF21) or an AAV1 vector encoding a codon-optimized mouse FGF21 coding sequence under the control of the CMV promoter (AAV1-CMV-FGF21). Or the expression level of FGF21 in the liver (B). qPCR was performed using primers that detect both the wt and codon-optimized FGF21 coding sequences. Analysis was performed 2 weeks after AAV. Results are expressed as mean ± SEM. n=4-5 mice/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 (28)

A viral expression construct suitable for mammalian expression and comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF21) expressed in liver, adipose tissue and/or skeletal muscle. A nucleotide sequence encoding FGF21 suitable for expression in a mammal and elements a), b), c), d) and e):
(a) a liver-specific promoter (b) an adipose tissue-specific promoter (c) a ubiquitous promoter, at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the liver, and a target sequence of a microRNA expressed in the heart (d) a skeletal muscle promoter and (e) a ubiquitous promoter and adeno-associated virus (AAV 4.) The viral expression construct of claim 1, comprising at least one of said combinations in combination with vector sequences, which allows specific expression in skeletal muscle. A 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) SEQ ID NO: 4, 5, 6, 7, 8, 9 , a nucleotide sequence having at least 60% sequence identity with 10 or 11 nucleotide sequences, (c) a nucleotide sequence that differs in sequence from the sequence of nucleotide sequence (b) due to the degeneracy of the genetic code. , the viral expression construct of claim 1 or 2. The nucleotide sequence encoding the target sequence of the microRNA expressed in the liver and the nucleotide sequence encoding the target sequence of the microRNA expressed in the heart are selected from the group consisting of sequences of SEQ ID NOS: 12 to 30 and/or combinations thereof , the viral expression construct of claim 2 or 3. the liver-specific promoter is the human α1-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 C5-12 is a promoter and/or the ubiquitous promoter is the cytomegalovirus (CMV) promoter and/or the CAG promoter,
5. The viral expression construct of any one of claims 2-4. A viral vector comprising a viral expression construct according to any one of claims 1 to 5, which is an adenoviral vector, an adeno-associated viral vector, a retroviral vector or a lentiviral vector, preferably adeno-associated virus 1 ( A.
The viral vector, which is an adeno-associated viral vector selected from the group consisting of an AV1) vector, an adeno-associated virus 8 (AAV8) vector and an adeno-associated virus 9 (AAV9) vector. A nucleic acid molecule that is suitable for expression in a mammal and is represented by a mammalian codon-optimized nucleotide sequence encoding FGF21 that is expressed in liver, adipose tissue and/or skeletal muscle. 8. The nucleic acid molecule of claim 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. The viral expression construct as defined in any one of claims 1 to 5 and/or the viral vector as defined in claim 6 and/or the nucleic acid molecule as defined in claim 7 or 8 are combined with one or more pharmaceutical agents. composition together with a legally acceptable additive or vehicle. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 and/or a nucleic acid as defined in claim 7 or 8 for use as a medicament A molecule and/or composition as defined in claim 9. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 and/or a viral vector as defined in claim 7 or 7 for use in the treatment and/or prevention of metabolic disorders. A nucleic acid molecule as defined in 8 and/or a composition as defined in claim 9. 12. The viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition of claim 11, wherein the metabolic disorder is diabetes and/or obesity. 12. The viral expression construct and/or of claim 11, wherein the metabolic disorder is NASH
or viral vectors and/or nucleic acid molecules and/or compositions. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 for use in the treatment and/or prevention of inflammation and/or fibrosis of the liver and /or a nucleic acid molecule as defined in claim 7 or 8 and/or a composition as defined in claim 9. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 and/or a viral vector as defined in claim 7 or 8 for use in extending healthy life expectancy and/or a composition as defined in claim 9. for use in the treatment and/or prevention of cancer, preferably liver cancer, according to claim 1
A viral expression construct as defined in any one of 5 to 5 and/or claim 6
and/or a nucleic acid molecule as defined in claim 7 or 8 and/or a composition as defined in claim 9. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 and/or a nucleic acid molecule as defined in claim 7 or 8 and/or a nucleic acid molecule as defined in claim 9 prevention of metabolic disorders, including the use of prescribed compositions;
Methods of delaying, relieving, curing and/or treating. 18. The method of claim 17, wherein the metabolic disorder is diabetes and/or obesity. 18. The method of claim 17, wherein the metabolic disorder is NASH. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 and/or a nucleic acid molecule as defined in claim 7 or 8 and/or a nucleic acid molecule as defined in claim 9 A method of preventing, retarding, reversing, curing and/or treating liver inflammation and/or fibrosis comprising the use of the defined compositions. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 and/or a nucleic acid molecule as defined in claim 7 or 8 and/or a nucleic acid molecule as defined in claim 9 A method of extending healthy life expectancy comprising the use of a defined composition. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 and/or a nucleic acid molecule as defined in claim 7 or 8 and/or a nucleic acid molecule as defined in claim 9 A method of preventing, delaying, reversing, curing and/or treating cancer, preferably liver cancer, comprising the use of the defined composition. A viral expression construct as defined in any one of claims 1 to 5 and/or and/or a nucleic acid molecule as defined in claim 7 or 8 and/or a composition as defined in claim 9. 24. Use according to claim 23, wherein the metabolic disorder is diabetes and/or obesity. 24. Use according to claim 23, wherein the metabolic disorder is NASH. A viral expression construct and/or as defined in any one of claims 1 to 5 for the manufacture of a medicament for preventing, delaying, ameliorating, curing and/or treating liver inflammation and/or fibrosis Use of a viral vector as defined in claim 6 and/or a nucleic acid molecule as defined in claim 7 or 8 and/or a composition as defined in claim 9. A viral expression construct as defined in any one of claims 1 to 5 and/or a viral vector as defined in claim 6 and/or a viral vector as defined in claim 7 and/or claim 7 or Use of a nucleic acid molecule as defined in 8 and/or a composition as defined in claim 9. A viral expression construct and/or as defined in any one of claims 1 to 5 for the manufacture of a medicament for preventing, delaying, reversing, curing and/or treating cancer, preferably liver cancer Use of a viral vector as defined in claim 6 and/or a nucleic acid molecule as defined in claim 7 or 8 and/or a composition as defined in claim 9.

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