CN110913886A - Viral expression construct comprising fibroblast growth factor 21(FGF21) coding sequence - Google Patents

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

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CN110913886A
CN110913886A CN201880047871.5A CN201880047871A CN110913886A CN 110913886 A CN110913886 A CN 110913886A CN 201880047871 A CN201880047871 A CN 201880047871A CN 110913886 A CN110913886 A CN 110913886A
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vector
fgf21
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nucleotide sequence
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韦罗尼卡·希门尼斯·森萨诺
法蒂玛·博希·图贝特
克劳迪娅·汉布里纳·帕利亚雷斯
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Universitat Autonoma de Barcelona UAB
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Abstract

The present 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 fibroblast growth factor 21(FGF21) to be expressed in liver, adipose tissue and/or skeletal muscle.

Description

Viral expression construct comprising fibroblast growth factor 21(FGF21) coding sequence
Technical Field
The present invention relates to the field of medicine, including gene therapy compositions for the treatment of metabolic disorders, for the treatment of liver inflammation and/or fibrosis, for the treatment of cancer and/or for the extension of the health life of mammals, especially humans.
Background
The prevalence of diabetes is increasing at an alarming rate and is a major health problem worldwide. Obesity is closely related to insulin resistance and type 2 diabetes (T2D) (Moller, d.e., and Flier, j.s.,1991.n.engl.j.med.325:938-948). Furthermore, obesity increases the risk of death (Peeters, a.et al, 2003.ann.13824-32) and also important risk factors for heart disease, immune dysfunction, hypertension, arthritis, neurodegenerative diseases and certain types of cancer (Roberst, d.l.et., 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). Despite the clinical significance of T2D and obesity, no effective treatment is available. Therefore, there is an urgent need for novel and safe methods to prevent and address the current T2D-obesity epidemic. Recently, obesity has been widely recognized as a significant risk factor for cancer (Roberst, d.l.et al.,2010. annu.rev.med).61:301-316). Given the current obesity epidemic, the risk of obesity-related cancer is a clinically significant concern, for which novel and safe approaches are urgently needed. Weight gain and insulin resistance are also associated with aging. Therefore, there is a need for a novel and safe method of preventing and reversing obesity and diabetes that extends the health life. Liver fibrosis is an excessive accumulation of extracellular matrix proteins (e.g., collagen), primarily due to chronic hepatitis. Advanced liver fibrosis can lead to cirrhosis, portal hypertension, and liver failure. Therefore, there is a need for novel and safe anti-fibrotic therapeutics.
Fibroblast growth factor 21(FGF21) has been shown to be secreted primarily by the liver, but also by adipose tissue and the pancreas (Muise, e.s.et al, 2008.mol.pharmacol.74403-Growth of colored adipose tissue (BAT) and expression of thermogenic genes in BAT and White Adipose Tissue (WAT), stimulation of energy expenditure (costun, t.et., 2008. Endocrinology)149:6018-6027；Fisher,F.M.et al.,2012.Genes Dev.26:271-281；Kharitonenkov,A.et al.,2005.J.Clin.Invest115:1627-1635；Konishi,M.et al.,2000.J.Biol.Chem.275:12119-12122；Tomlinson,E.et al.,2002.Endocrinology143:1741-1747；Xu,J.et al.,2009.Diabetes58:250-259). Overexpression of FGF21 in transgenic mice protected them from diet-induced obesity (kharitonnkov, a.et al, 2005. j.clin.invest)1151627-149:6018-6027；Kharitonenkov,A.etal.,2005.J.Clin.Invest115:1627-1635；Xu,J.et al.,2009.Diabetes58:250-259；Adams,A.C.et al.,2012.PLoS.One.7:e38438.；Berglund,E.D.et al.,2009.Endocrinology150:4084-4093). Furthermore, administration of FGF21 to obese diabetic rhesus monkeys significantly reduced fasting plasma glucose, fructosamine, triglycerides, insulin and glucagon levels and caused small but significant weight loss (kharitonnkov, a.et al, 2007148:774-781)。
Native FGF21 protein exhibits poor pharmacokinetic profiles. It has a short half-life and is prone to proteolytic degradation in vivo and aggregation in vitro (Huang, j.et al, 2013.J Pharmacol Exp Ther.346(2):270-80；So,W.Y.andLeung,P.S.2016.Med Res Rev.36(4):672-704；Zhang,J.and Li,Y.2015.Front Endocrinol(Lausanne).6:168). Various engineering approaches have been developed to extend half-life and improve the stability and solubility of FGF 21. Currently, two engineered FGF21 mimetics (LY2405319 and PF-05231023) are being tested in humans. However, those FGF21 mimetics require multiple administrations, which places a significant burden on the patient. In addition, the engineered FGF21 mimetics/analogs may be listedPatients who exhibit a higher risk of immunogenicity, exhibit injection site reactions, anti-drug antibodies and severe hypersensitivity than native FGF21, e.g., patients receiving treatment with LY2405319 (Gaich, g.et al, 2013.Cell Metab).18(3):333-40)。
Thus, there remains a need for new therapies for diabetes and/or obesity and/or liver inflammation and/or fibrosis and/or cancer and/or extending the health life that do not have all the disadvantages of existing therapies.
Disclosure of Invention
The present inventors have devised improved gene therapy strategies based on adeno-associated virus (AAV) vector-mediated transfer of the FGF21 gene into the liver, adipose tissue and/or skeletal muscle to combat metabolic disorders, preferably diabetes and/or obesity. The gene therapy of the 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 to extend the health life by combating age-related metabolic disorders, preferably diabetes and/or obesity. In addition, the gene therapy of the present invention can also be used against cancer, preferably liver cancer.
The generation of a single vector gene construct allows for the production of native FGF21 in vivo, which would result in a reduced risk of immunogenicity or other toxicity.
However, the skilled person is aware that native FGF21 may be susceptible to in vivo proteolytic degradation and/or have a rapid in vivo clearance rate. All vectors tested in the experimental section were found to be capable of long-term secretion of stable native FGF21 into the bloodstream. The efficacy can be maintained even with a single administration of the gene transfer vector.
Thus, as demonstrated in the experimental section, the generation of such AAV vectors for the in vivo production of native FGF21 is not routine for those skilled in the art.
Viral expression constructs
In a first aspect, there is provided a viral expression construct suitable for expression in a mammal and comprising a nucleotide sequence encoding fibroblast growth factor 21(FGF21) to be expressed in liver, adipose tissue and/or skeletal muscle.
Definitions of "viral expression constructs", "suitable for expression in mammals", "liver", "adipose tissue" and "skeletal muscle" are provided in the section description entitled "general definitions".
The preferred nucleotide sequence encoding FGF21 present in the viral expression construct of the present invention is identical to SEQ ID NO: 4.5, 6, 7, 8, 9, 10 or 11 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. The assessment of identity can be performed throughout SEQ ID NOs or parts thereof according to the heading "general definition".
A more preferred nucleotide sequence encoding human FGF21 present in the viral expression constructs of the present invention shares sequence identity with seq id NO: 4.5, 6 or 7 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout seq id NOs or portions thereof as set forth in the section of the specification entitled "general definitions". SEQ ID NO: 4 is a nucleotide sequence encoding human FGF 21. SEQ ID NO: 5 is a codon optimized nucleotide sequence encoding human FGF21, variant 1. SEQ ID NO: 6 is a codon optimized nucleotide sequence encoding human FGF21, variant 2. SEQ ID NO: 7 is a codon optimized nucleotide sequence encoding human FGF21, variant 3. Variant 1, variant 2 and variant 3 encode the same human FGF21 protein and were obtained by different algorithms of codon optimization. Another preferred nucleotide sequence encoding mouse FGF21 present in the viral expression construct of the present invention is identical to SEQ ID NO: 8 or 9 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout SEQ ID NOs or portions thereof as set forth in the section of the specification entitled "general definitions". SEQ ID NO: and 8 is a nucleotide sequence encoding mouse FGF 21. SEQ ID NO: 9 is a codon optimized nucleotide sequence encoding mouse FGF 21. Another preferred nucleotide sequence encoding canine FGF21 present in the viral expression construct of the present invention is identical to SEQ ID NO: 10 or 11 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity, which can be evaluated in the whole SEQ ID NO or parts thereof as explained in the section of the specification entitled "general definitions". SEQ ID NO: 10 is a nucleotide sequence encoding canine FGF 21. SEQ ID NO: 11 is a codon optimized nucleotide sequence encoding canine FGF 21. The nucleotide sequence encoding FGF21 can be derived from any FGF21 gene or FGF21 coding sequence, preferably from human, mouse or dog; or a mutated FGF21 gene or FGF21 coding sequence, or a codon-optimized FGF21 gene or FGF21 coding sequence, preferably derived from human, mouse or dog.
As known to those skilled in the art, FGF21 used herein exerts at least detectable levels of FGF21 activity. The activity of FGF21 is to increase insulin sensitivity. This activity can be assessed using the insulin resistance test, as described in the experimental section, preferably as in examples 8 or 9.
In one embodiment, there is provided a viral expression construct as described above, wherein the nucleotide sequence encoding FGF21 suitable for expression in a mammal is selected from the group consisting of:
(a) encoding a polypeptide comprising a sequence identical to SEQ ID NO: 1. 2 or 3 has at least 60% sequence identity to the amino acid sequence of the polypeptide.
(b) And SEQ ID NO: 4.5, 6, 7, 8, 9, 10, or 11 has a nucleotide sequence with at least 60% sequence identity.
(c) A nucleotide sequence whose sequence differs from that of the nucleotide sequence of (b) due to the degeneracy of the genetic code.
A preferred nucleotide sequence encoding FGF21 suitable for expression in a mammal encodes a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1. 2 or 3, having an amino acid sequence of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout SEQ ID NOs or portions thereof as set forth in the section of the specification entitled "general definitions". SEQ ID NO: 1 is the amino acid sequence of human FGF 21. SEQ ID NO: 2 is the amino acid sequence of murine FGF 21. SEQ ID NO: 3 is the amino acid sequence of canine FGF 21.
In one embodiment, there is provided a viral expression construct as described above comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and at least one of the elements (elements) a), b), c), d), and e):
(a) liver-specific promoters
(b) Adipose tissue specific promoters
(c) A combination of a ubiquitous promoter (ubiquitin 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, wherein the combination can be specifically expressed in adipose tissue
(d) Skeletal muscle promoter and
(e) a combination of a ubiquitous promoter and an adeno-associated virus (AAV) vector sequence, wherein said combination is specifically expressible in skeletal muscle.
By "target sequence of microrna expressed in the liver" or "target sequence of miRNA expressed in the liver" or "binding site of microrna expressed in the liver" is meant a nucleotide sequence that is complementary or partially complementary to at least a portion of microrna expressed in the liver. Similarly, "a target sequence of a microrna expressed in the heart" or "a target sequence of a miRNA expressed in the heart" or "a binding site of a microrna expressed in the heart" refers to a nucleotide sequence that is complementary or partially complementary to at least a portion of a microrna expressed in the heart. As defined herein, a portion of a microrna expressed in the liver or a portion of a microrna expressed in the heart refers to a nucleotide sequence of at least five or at least six consecutive nucleotides of the microrna. The binding site sequence may have perfect complementarity to at least a portion of the expressed microrna, meaning that the sequence may be a perfect match, with no mismatches. Alternatively, the binding site sequence may be complementary to at least a portion of the expressed microrna, meaning that one mismatch/five, six consecutive nucleotides may occur. A partially complementary binding site preferably comprises perfect or near perfect complementarity to the seed region of the microrna, which means that no mismatches (perfect complementarity) or one mismatch/five, six consecutive nucleotides (near perfect complementarity) occur between the seed region of the microrna and its binding site. The seed region of the microrna consists of the 5' region of the microrna from about nucleotide 2 to about nucleotide 8 (i.e., 6 nucleotides) of the microrna. The portion as defined herein is preferably a seed region of the microrna. Degradation of messenger RNA (mRNA) comprising the target sequence of micrornas expressed in the liver or in the heart can be controlled (inhibited) by RNA interference pathways or via direct translation of mRNA. The present invention is not limited by the way in which mirnas are ultimately utilized in inhibiting transgene or encoding protein expression.
In the context of the present invention, the nucleotide sequence encoding the target sequence of the micro RNA expressed in the liver may be comprised of a nucleotide sequence identical to SEQ ID NO: 12 or 14-23, or a nucleotide sequence substitution of a nucleotide sequence having at least 60% sequence identity or similarity. More preferred nucleotide sequences are identical to SEQ ID NO: 12 or 14-23 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout SEQ ID NOs or portions thereof as set forth in the section of the specification entitled "general definitions". In one embodiment, the nucleotide sequence encoding the target sequence of the microrna expressed in the liver may be comprised of a nucleotide sequence identical to SEQ ID NO: 12 nucleotide sequence substitutions of nucleotide sequences having at least 60% sequence identity or similarity. More preferred nucleotide sequences are identical to SEQ ID NO: 12 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. In a further embodiment, at least one copy of a nucleotide sequence encoding a target sequence of a microrna expressed in the liver, such as seq id NO: 12 or 14-23, in a viral expression construct of the invention. In a further embodiment, the nucleic acid sequence encoding SEQ ID NO: 12 or 14-23, two, three, four, five, six, seven or eight copies of the nucleotide sequence of the target sequence of the liver-specific microrna defined in the viral expression construct of the invention. In preferred embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence encoding miRT122a (SEQ ID NO: 12) are present in the viral expression constructs of the invention.
As used herein, a target sequence of a microrna expressed in the liver exerts at least a detectable level of activity of a target sequence of a microrna expressed in the liver as known to the skilled person. The activity of the target sequence of the microrna expressed in the liver is binding to its homologous microrna expressed in the liver and, when operably linked to a transgene, mediates off-target expression of the transgene in the liver. This activity can be assessed by measuring the level of transgene expression in the liver by qPCR, as described in the experimental section.
In the context of the present invention, the nucleotide sequence encoding the target sequence of the microrna expressed in the heart may be comprised of a nucleotide sequence identical to SEQ ID NO: 13 or 23-30 nucleotide sequence substitutions of nucleotide sequences having at least 60% sequence identity or similarity. Preferred nucleotide sequences are similar to SEQ ID NO: 13 or SEQ ID NO: 23-30 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout SEQ ID NOs or portions thereof as set forth in the section of the specification entitled "general definitions". In one embodiment, the nucleotide sequence encoding the target sequence of the microrna expressed in the heart may be comprised of a nucleotide sequence identical to SEQ ID NO: 13 nucleotide sequence substitutions of nucleotide sequences having at least 60% sequence identity or similarity. Preferred nucleotide sequences are similar to SEQ id no: 13 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. In further embodiments, as set forth in SEQ ID NO: 13 or 23-30 is present in the viral expression construct of the invention. In a further embodiment, in SEQ ID NO: 13 or 23-30, two, three, four, five, six, seven or eight copies of the nucleotide sequence encoding the target sequence of the heart-specific microrna are present in the viral expression construct of the invention. In preferred embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 copies of the nucleotide sequence encoding miRT1(SEQ ID NO: 13) are present in the viral expression constructs of the invention.
The activity of the target sequence of microrna expressed in the heart is to bind to its homologue, the microrna expressed in the heart, and when operably linked to a transgene, mediate off-target of transgene expression in the heart. This activity can be assessed by measuring transgene expression levels in the heart using qPCR, as described in the experimental section.
In one embodiment, there is provided a viral expression construct as described above, wherein 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 the sequences SEQ ID NO: 12 to 30 and/or combinations thereof.
In one embodiment, the nucleic acid sequence as set forth in SEQ ID NO: 12 or 14-23, and at least one copy of a nucleotide sequence encoding a target sequence of a microrna expressed in the liver, as defined in SEQ ID NO: 13 or 23-30 is present in the viral expression construct of the invention. In further embodiments, the nucleic acid sequence as set forth in SEQ ID NO: 12 or 14-23, and two, three, four, five, six, seven or eight copies of a nucleotide sequence encoding a target sequence of a microrna expressed in the liver as defined in SEQ ID NO: 13 or 23-30, two, three, four, five, six, seven or eight copies of the nucleotide sequence encoding the target sequence of the microrna expressed in the heart are present in the viral construct of the invention. In a further embodiment, 1, 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence encoding miRT122a (SEQ ID NO: 12) and 1, 2, 3, 4, 5, 6, 7 or 8 copies of the nucleotide sequence encoding miRT1(SEQ ID NO: 13) are combined in a viral expression construct of the invention. In a further embodiment, four copies of the nucleotide sequence encoding miRT122a (SEQ ID NO: 12) and four copies of the nucleotide sequence encoding miRT1(SEQ ID NO: 13) are combined in a viral expression construct of the invention.
Definitions "promoter", "liver-specific promoter", "adipose tissue-specific promoter", "ubiquitous promoter", "skeletal muscle promoter" are provided in the part of the specification 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 comprised of a nucleotide sequence identical to SEQ ID NO: 44 nucleotide sequence substitutions of a nucleotide sequence having at least 60% sequence identity or similarity. Preferred nucleotide sequences are similar to SEQ ID NO: 44 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% sequence identity. Identity can be assessed throughout seq id NOs or portions thereof as set forth in the section of the specification 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 consist of a nucleotide sequence comprising a nucleotide sequence identical to SEQ ID NO: 45 nucleotide sequence substitutions of nucleotide sequences having at least 60% sequence identity or similarity. Preferred nucleotide sequences are similar to seq id NO: 45 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout SEQ ID NOs or portions thereof as set forth in the section of the specification entitled "general definitions".
Preferably, the CMV promoter is used with an intron sequence. In this context, an intron sequence may be comprised of a sequence identical to SEQ ID NO: 43 nucleotide sequence substitutions of nucleotide sequences having at least 60% sequence identity or similarity. Preferred nucleotide sequences are similar to SEQ ID NO: 43 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout SEQ ID NOs or portions thereof as set forth in the section of the specification entitled "general definitions".
A preferred liver-specific promoter is the human α 1-antitrypsin (hAAT) promoter.
In the context of the present invention, the nucleotide sequence of the hAAT promoter may be comprised of a nucleotide sequence identical to SEQ ID NO: 47 nucleotide sequence substitutions of nucleotide sequences having at least 60% sequence identity or similarity. Preferred nucleotide sequences are similar to SEQ ID NO: 47 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout seq id NOs or portions thereof as set forth in the section of the specification entitled "general definitions".
Preferably, the hAAT promoter is used with an intron sequence. A preferred intron sequence is the Hepatocyte Control Region (HCR) enhancer of apolipoprotein E. The most preferred intron sequences are the HCR enhancers from apolipoprotein E, such as seq id NO: 53 as defined in. Herein, the intron sequence may be comprised of a sequence identical to SEQ ID NO: 53 nucleotide sequence substitutions having at least 60% sequence identity or similarity. Preferred nucleotide sequences are similar to SEQ ID NO: 53 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout SEQ ID NOs or portions thereof as set forth in the section of the specification entitled "general definitions". In one embodiment, the hAAT promoter is used with 1, 2, 3, 4, or 5 copies of an intron sequence. In a preferred embodiment, the hAAT promoter is operably linked to the promoter as set forth in SEQ ID NO: 1, 2, 3, 4 or 5 copies of the HCR enhancer from apolipoprotein E as defined by 53 are used together.
Other liver-specific promoters are the albumin promoter, the major urinary protein promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, the liver Rich protein activator promoter, the transthyretin promoter, the thyroxine-binding globulin promoter, the apolipoprotein A1 promoter, the liver fatty acid-binding protein promoter, and the phenylalanine hydroxylase promoter.
The adipose tissue specific promoter is the adipocyte protein 2(aP2, also known as fatty acid binding protein 4(FABP4)) promoter, the PPARy promoter, the adiponectin promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, the promoter derived from the human aromatase cytochrome p450(p450arom), the mini/aP2 promoter (consisting of the fat specific aP2 enhancer and the basal aP2 promoter), the uncoupling protein 1(UCP1) promoter, the mini/UCP1 promoter (consisting of the fat specific UCP1 and the basal UCP1 promoter), the lipase promoter, the leptin promoter or the Foxa-2 promoter. Preferred adipose tissue-specific promoters are the mini/aP2 promoter (SEQ ID NO: 54) and the mini/UCP1 promoter (SEQ ID NO: 55). Herein, the adipose tissue-specific promoter sequence may be comprised of a sequence identical to SEQ ID NO: 53 or SEQ ID NO: 54, or a nucleotide sequence substitution of a nucleotide sequence having at least 60% sequence identity or similarity. Preferred nucleotide sequences are similar to SEQ id no: 53 or SEQ ID NO: 54 have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. Identity can be assessed throughout SEQ ID NOs or portions thereof as set forth in the section of the specification entitled "general definitions".
Preferred skeletal muscle promoters are the myosin light chain promoter, myosin heavy chain promoter, desmin promoter, Muscle Creatine Kinase (MCK) promoter, smooth muscle α -actin promoter, CK6 promoter, Unc-45 myosin partner B promoter, the basal MCK promoter in combination with a copy of the MCK enhancer, the Enh358MCK promoter (combination of the MCK enhancer with the 358bp proximal promoter of the MCK gene). The most preferred skeletal muscle promoter is the C5-12 promoter as defined in SEQ ID NO: 56. herein, the skeletal muscle promoter sequence may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to SEQ ID NO: 56. preferred nucleotide sequences have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity with SEQ ID NO: 56. the identity may be assessed throughout SEQ ID NO or in parts thereof as set forth in the section of the specification entitled "general definitions".
The promoter used herein (especially when the promoter sequence is defined as having the smallest percentage of identity to a given SEQ ID NO) should exert at least promoter activity known to those skilled in the art. For a definition of such activity, reference is made to the part of the description entitled "general definitions". Preferably, the promoter defined as having the smallest percent identity to a given seq id NO should control transcription of the nucleotide sequence to which it is operably linked (i.e., the nucleotide sequence encoding FGF21), as assessed in analytical tests known to the skilled artisan. In the context of the present invention, the promoter is operably linked to the FGF21 nucleotide sequence defined above. In one embodiment, the promoter is cell-specific and/or tissue-specific, preferably liver, adipose tissue and/or skeletal muscle-specific.
Thus, the present invention includes several viral expression constructs:
a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and comprising element a),
a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and comprising element b),
a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and comprising element c),
a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and comprising element d),
a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and comprising element e),
a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and comprising the nucleotide sequences of element b) and element c),
a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and comprising the nucleotide sequences of element e) and element c).
In one embodiment, a viral expression construct as described herein is provided, wherein the liver-specific promoter is human α 1-antitrypsin (hAAT) promoter and/or the adipose tissue-specific promoter is mini/ap2 promoter and/or mini/UCP1 promoter and/or the skeletal muscle promoter is C5-12 promoter and/or the ubiquitous promoter is Cytomegalovirus (CMV) promoter and/or CAG promoter.
In one embodiment, a viral expression construct is encompassed comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element a), wherein the liver-specific promoter is a hAAT promoter (SEQ ID NO: 47).
In a preferred embodiment, a viral expression construct is contemplated comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element a), wherein the construct is AAV8-hAAT-moFGF 21. This construct, for example, comprises the viral expression construct shown in figure 6A: ITR2-hAAT-moFGF21-polyA-ITR 2; the sequence of the expression construct is contained in SEQ ID NO: 34, respectively. For this construct, example 3 reveals, inter alia, a high and stable liver-specific expression after intravenous administration. Expression was confirmed to be stable for up to 1 year (example 12). Broadly beneficial therapeutic effects on the reversal and treatment of obesity and diabetes were demonstrated in ob/ob mice (examples 3 and 11), High Fat Diet (HFD) fed mice (examples 4, 12-14) and geriatric HFD fed mice (examples 5, 12-14). Examples 11 and 16 also reveal significant improvements in hepatic steatosis, hepatitis and liver fibrosis. Example 15 demonstrates the improvement of WAT inflammation associated with obesity. Example 17 indicates the long-term safety of the therapy. Example 18 reveals the beneficial effect of preventing liver tumors. Example 19 shows therapeutic potential in a type I diabetes model.
In one embodiment, a viral expression construct is contemplated comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element b), wherein the adipose tissue specific promoter is the mini/aP2 promoter (seq id NO: 54) and/or the mini/UCP1 promoter (SEQ ID NO 55).
In one embodiment, a viral expression construct is contemplated comprising a 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) and wherein the at least one nucleotide sequence encoding a target sequence of a microrna expressed in the liver is selected from the group consisting of SEQ ID NO: 12 or 14-23, and at least one nucleotide sequence encoding a target sequence of a microrna expressed in the heart is selected from the group consisting of seq id NO: 13 or 23-30.
In a preferred embodiment, a viral expression construct is contemplated comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element c), wherein the construct is AAV9-CAG-moFGF21-dmiRT or AAV8-CAG-moFGF 21-dmiRT. The expressions dmiRT and double mirT are equivalent. These constructs, for example, comprise the viral expression construct shown in FIG. 1A: ITR2-CAG-moFGF21-4x mirT122a-4x mirT1-polyA-ITR 2; the sequence of the expression construct is contained in SEQ ID NO: 32 (c).
For these constructs, examples 1-2 unexpectedly revealed high and stable fat-specific expression after administration in eWAT. A broad beneficial therapeutic effect in preventing, reversing and treating obesity and diabetes has been demonstrated in normal mice (example 1) and ob/ob mice (examples 2 and 10). Example 10 also reveals improvement in hepatic steatosis.
In one embodiment, a viral expression construct is contemplated 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 one embodiment, a viral expression construct is contemplated comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element e), wherein the ubiquitous promoter is a CMV promoter (SEQ ID NO: 45) and the AAV serotype is AAV 1.
In a preferred embodiment, a viral expression construct is contemplated comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element e), wherein the construct is AAV1-CMV-moFGF 21. This construct, for example, comprises the viral expression construct shown in figure 11A: ITR2-CMV-moFGF21-polyA-ITR 2; the sequence of the expression construct is contained in SEQ ID NO: 36 of (a). For this construct, example 20 reveals high and stable skeletal muscle specific expression following intramuscular administration. A broad beneficial therapeutic effect in the prevention, reversal and treatment of obesity and diabetes was demonstrated in HFD fed mice (examples 6 and 21). Example 20 demonstrates the beneficial effects in extending the healthy life by preventing obesity and diabetes.
In one embodiment, a viral expression construct is contemplated comprising a 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 the mini/UCP1 promoter (SEQ ID NO 55).
In one embodiment, a viral expression construct is contemplated comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and element e) and element c), wherein the ubiquitous promoter is a CMV promoter (SEQ ID NO: 45) and the AAV serotype is AAV 1.
All constructs of the invention are more attractive than the constructs disclosed in the prior art, such as Zhang et al. EBioMedicine 15(2017)173-183, in particular those comprising: element a) which is a liver-specific promoter, preferably hAAT, and/or element c) which is a combination of a ubiquitous promoter and at least one nucleotide sequence encoding a target sequence of a microrna expressed in the liver, preferably miRT122a, and at least one nucleotide sequence encoding a target sequence of a microrna expressed in the heart, preferably miRT1, wherein the combination is capable of specific expression in adipose tissue, and/or element e) which is a combination of a ubiquitous promoter, preferably CMV, and an adeno-associated virus (AAV) vector sequence, preferably AAV1, wherein the combination can be specifically expressed in skeletal muscle. Zhang et al discloses the wild type murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (EF1a-mFGF21) (Zhang et al, EBiomedicine 15(2017) 173-. In examples 23 and 24, this construct was compared to the construct of the present invention. In all in vitro and in vivo experiments, all expression cassettes and AAV vectors of the invention mediated higher expression of FGF21 in target tissues or cell types and lower expression of FGF21 in off-target tissues, demonstrating higher potency and higher tissue specificity of the expression cassettes and AAV vectors of the invention. In addition, the constructs CMV-moFGF21 and CAG-moFGF 21-bis miRT construct mediated higher protein production and secretion into the culture medium in HEK293 cells compared to EF1a-mFGF 21. Furthermore, hAAT-moFGF21 and AAV8-hAAT-moFGF21 mediated altered secretion of FGF21 into the bloodstream over EF1a-mFGF21 and AAV8-EF1a-mFGF 21.
Preferred additional sequences include Inverted Terminal Repeats (ITRs), SV40 polyadenylation signal (SEQ ID NO: 50), rabbit β -globulin polyadenylation signal (SEQ ID NO: 51), CMV enhancer sequence (SEQ ID NO: 46), and HCR enhancer from apolipoprotein E (SEQ ID NO: 53). in the context of the present invention, "ITR" is intended to encompass one 5'ITR and one 3' ITR, each derived from the genome of an AAV.A preferred ITR is from AAV2 and is expressed by SEQ ID NO: 48(5'ITR) and SEQ ID NO: 49(3' ITR). in the context of the present invention, it is contemplated to use 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 a viral expression construct of the invention (see, e.g., the descriptions in figures 1, 2, 3, 4, 5, 6, 7, 8, and 9 and in figures 11, 31, and).
In one embodiment, a viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and at least one of the elements a) and/or b) and/or c) and/or d) and/or e) as defined previously further comprises:
-ITRs flanking the expression cassette of the construct,
-SV 40 or rabbit β -globulin polyadenylation signal located 3' to the nucleotide sequence encoding FGF21, and/or
A CMV enhancer sequence or HCR enhancer sequence located 5' to the nucleotide sequence encoding FGF 21.
In a preferred embodiment, the viral expression construct comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and at least one of the elements a) and/or b) and/or c) and/or d) and/or e) as defined previously further comprises ITRs flanked by expression cassettes of said construct and optionally ITRs flanked by expression cassettes of said construct
-SV 40 or rabbit β -globulin polyadenylation signal and/or located 3' of the nucleotide sequence coding for FGF21
A CMV enhancer sequence or HCR enhancer sequence located 5' to the nucleotide sequence encoding FGF 21.
These sequences were used in the experimental part of some of the constructs identified herein.
Thus, in one embodiment, for each of these preferred viral expression constructs as defined above, additional sequences selected from the group consisting of ITR, SV40 polyadenylation signal, rabbit β -globulin polyadenylation signal, CMV enhancer sequence, HCR enhancer sequence from apolipoprotein E may be present.
In a preferred embodiment, the viral expression construct comprises a nucleotide sequence encoding FGF21 suitable for expression in a mammal and at least one of the elements a) and/or b) and/or c) and/or d) and/or e), wherein additional sequences present are selected from the group consisting of ITR, SV40 polyadenylation signal, rabbit β -globin polyadenylation signal, CMV enhancer sequence, HCR enhancer sequence preferred ITR is that of AAV2 represented by SEQ ID NO 48(5'ITR) and SEQ ID NO 49(3' ITR).
Preferred viral expression constructs comprise the elements a) and/or b) and/or c) and/or d) and/or e) and have the expression cassette flanked by a 5'ITR and a3' ITR.
Other preferred viral expression constructs comprise the elements a) and/or b) and/or c) and/or d) and/or e) and have the expression cassette flanked by a 5'ITR and a3' ITR. In addition, an SV40 polyadenylation signal is also present.
Other preferred viral expression constructs comprise elements a) and/or b) and/or c) and/or d) and/or e) and are such that the expression cassette is flanked by a 5'ITR and a3' ITR in addition, a rabbit β -globulin polyadenylation signal is also present.
Other preferred viral expression constructs comprise the elements a) and/or b) and/or c) and/or d) and/or e) and have the expression cassette flanked by a 5'ITR and a3' ITR. In addition, a CMV enhancer sequence is also present.
Other preferred viral expression constructs comprise the elements a) and/or b) and/or c) and/or d) and/or e) and have the expression cassette flanked by a 5'ITR and a3' ITR. In addition, an HCR enhancer sequence from apolipoprotein E is also present.
Most preferred contemplated viral expression constructs include:
construct B (represented by the nucleotide sequence comprising SEQ ID NO: 32),
construct D (represented by the nucleotide sequence comprising SEQ ID NO: 34),
construct F (represented by the nucleotide sequence comprising SEQ ID NO: 36),
construct G (represented by the nucleotide sequence comprising SEQ ID NO: 37),
construct H (represented by the nucleotide sequence comprising SEQ ID NO: 38),
construct I (represented by the nucleotide sequence comprising SEQ ID NO: 39),
construct J (represented by the nucleotide sequence comprising SEQ ID NO: 40),
construct K (represented by the nucleotide sequence comprising SEQ ID NO: 41).
Construct L (represented by the nucleotide sequence comprising SEQ ID NO: 42).
As will be appreciated by those skilled in the art, each of these viral expression constructs already comprises two ITRs from AAV2 (i.e., SEQ ID NO: 48(5'ITR) and SEQ ID NO: 49(3' ITR)).
Construct B and G contained rabbit β -globin polyadenylation signal construct F contained the SV40 polyadenylation signal, the CMV enhancer sequence and the nucleotide sequence of the chimeric intron (consisting of the intron of human β -globin and the immunoglobulin heavy chain gene). construct D, H-L contained the SV40 polyadenylation signal, the HCR enhancer sequence and the nucleotide sequence of the chimeric intron (consisting of the intron of human β -globin and the immunoglobulin heavy chain gene).
As explained in the general section entitled "general definitions" throughout this application, whenever reference is made to a particular nucleotide sequence SEQ ID NO (taken as SEQ ID NO: A, B or C) that represents a preferred construct designed herein, it can be replaced by:
i. comprises a nucleotide sequence substantially identical to SEQ ID NO: a nucleotide sequence of a nucleotide sequence having at least 60% sequence identity or similarity to a, B or C;
a nucleotide sequence which differs in sequence from the sequence of the nucleic acid molecule of (i) due to the degeneracy of the genetic code.
In another preferred embodiment, each nucleotide sequence described herein has an identity of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity to a given nucleotide, respectively, by virtue of its percent identity (at least 60%) to the given nucleotide sequence. 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 NO refers to identity or similarity based on the full length of the sequence (i.e., over its full length or its entirety).
A construct defined by the lowest identity (i.e., at least 60%) to a given SEQ ID NO identified above is encompassed within the scope of the present invention when the construct or viral expression construct or viral vector comprising the construct or composition comprising the construct can induce expression of FGF21 in a cell, preferably in a hepatocyte, adipose tissue cell or skeletal muscle cell. Expression of FGF21 can be assessed using techniques known to the skilled artisan. In a preferred embodiment, expression is assessed as performed in the experimental section.
In a preferred embodiment, the viral expression construct is such that the construct consists of a nucleic acid comprising SEQ ID NO: 4.5, 6, 7, 8, 9, 10 or 11 or a nucleotide sequence identical to SEQ ID NO: 4.5, 6, 7, 8, 9, 10 or 11 or a sequence having at least 60% identity to SEQ ID NO: 4.5, 6, 7, 8, 9, 10 or 11, or a sequence representation having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity.
Viral vectors
In a further aspect, there is provided a viral vector comprising a viral expression construct as defined above, wherein the viral vector is an adenoviral, adeno-associated viral, retroviral or lentiviral vector, preferably an adeno-associated viral vector selected from the group consisting of an adeno-associated viral 1(AAV1) vector, an adeno-associated viral 8(AAV8) vector and an adeno-associated viral 9(AAV9) vector.
"viral vectors" and "adeno-associated viral vectors (AAV vectors)" are further defined in the section of the specification entitled "general definitions".
In one embodiment, an AAV vector is used comprising each element as defined herein before and a recombinant AAV (raav) -based genome comprising an ITR or a portion thereof. Preferred ITRs are those consisting of SEQ ID NO: 48(5' ITR) and SEQ ID NO: 49(3' ITR) of AAV 2.
Preferably, the AAV vector is an AAV1 vector, an AAV8 vector or an AAV9 vector.
The viral expression constructs and viral vectors of the invention are preferably used as medicaments. The medicament is preferably used for the prevention, delay of progression, cure, reversal and/or treatment of metabolic disorders, preferably diabetes and/or obesity. The diabetes may be type 1 diabetes, type 2 diabetes or monogenic diabetes. In another preferred embodiment, the medicament is for the prevention, delay, cure, reversal and/or treatment of liver inflammation and/or fibrosis. In a further preferred embodiment, the medicament is for prolonging the healthy life, preferably by preventing, delaying, curing, reversing and/or treating metabolic disorders associated with aging, preferably diabetes and/or obesity. In a further preferred embodiment, the medicament is for the prevention, delay, cure, reversal and/or treatment of cancer, preferably liver cancer.
The subject to be treated may be a higher mammal, e.g., a cat, a rodent (preferably a mouse, a rat, a gerbil and a guinea pig, more preferably a mouse and a rat), or a dog, or a human.
Nucleic acid molecules
In a further aspect, there is provided a nucleic acid molecule suitable for expression in a mammal and represented by a mammalian codon-optimized nucleotide sequence encoding FGF21 to be expressed in liver, adipose tissue and/or skeletal muscle.
The definition of "codon optimization" has been provided in the section of the specification entitled "common definitions".
In one embodiment, a nucleic acid molecule as above is encompassed, wherein the nucleotide sequence is identical to SEQ ID NO: 4.5, 6, 7, 8, 9, 10, or 11 has at least 60% sequence identity. Preferred nucleotide sequences are similar to SEQ ID NO: 4.5, 6, 7, 8, 9, 10, or 11 have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity.
Composition comprising a metal oxide and a metal oxide
In a further aspect, there is provided a composition comprising a viral expression construct as defined above and/or a viral vector as defined above and/or a nucleic acid molecule as defined above, and one or more pharmaceutically acceptable excipients or vectors.
The composition is preferably referred to as a gene therapy composition. Preferably the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluent, solubilizer, filler, preservative and/or excipient.
Such pharmaceutical carriers, fillers, preservatives, solubilizers, diluents and/or excipients are referred to, for example, Remington: The Science and Practice of Pharmacy,20th edition.Baltimore, MD: Lippincott Williams & Wilkins, 2000.
In a preferred embodiment, the composition is for use as a medicament, preferably for the prevention, delay of progression, cure, reversal and/or treatment of metabolic disorders, preferably diabetes and/or obesity. The diabetes may be type 1 diabetes, type 2 diabetes or monogenic diabetes. In another preferred embodiment, the medicament is for the prevention, delay of progression, cure, reversal and/or treatment of liver inflammation and/or fibrosis. In a further preferred embodiment, the medicament is for extending the health life, preferably by preventing, delaying, curing, reversing and/or treating metabolic disorders associated with aging, preferably diabetes and/or obesity. In another preferred embodiment, the medicament is for the prevention, delay of progression, cure, reversal and/or treatment of cancer, preferably liver cancer. The subject to be treated may be a higher mammal, e.g., a cat, a rodent (preferably a mouse, a rat, a gerbil and a guinea pig, more preferably a mouse and a rat), or a dog, or a human.
When the viral expression construct, viral vector and/or nucleic acid molecule and/or composition is capable of exhibiting an anti-diabetic effect and/or an anti-obesity effect, said viral expression construct, viral vector and/or nucleic acid molecule and/or composition is preferably said to be useful for preventing, delaying, reversing, curing and/or treating a metabolic disorder, preferably diabetes and/or obesity.
When the viral expression construct, viral vector and/or nucleic acid molecule and/or composition is capable of exhibiting an anti-fibrotic effect, the viral expression construct, viral vector and/or nucleic acid molecule and/or composition is preferably said to be capable of being used for preventing, delaying, curing, reversing and/or treating liver inflammation and/or fibrosis.
When said viral expression construct, viral vector and/or nucleic acid molecule and/or composition is capable of exhibiting an anti-diabetic effect and/or an anti-obesity effect during aging, said viral expression construct, viral vector and/or nucleic acid molecule and/or composition is preferably said to be capable of being used for prolonging the health life, preferably by preventing, delaying, curing, reversing and/or treating metabolic disorders associated with aging, preferably diabetes and/or obesity.
When the viral expression construct, viral vector and/or nucleic acid molecule and/or composition may exhibit an anti-cancer effect, the viral expression construct, viral vector and/or nucleic acid molecule and/or composition is preferably said to be capable of being used for the prevention, delay, cure, reversal and/or treatment of cancer, preferably liver cancer.
An anti-diabetic effect may be achieved when glucose clearance in the blood is increased and/or glucose tolerance is improved and/or insulin sensitivity is increased. This can be assessed using techniques known to the skilled person or as performed in the experimental part, preferably following the techniques assessed in examples 8 or 9. In this context, "increase" (respectively "improvement") means at least a detectable increase (respectively a detectable improvement) of the performance of the assay using assays known to the skilled person or using assays as performed in the experimental part, such as a measurement of the performance of a blood glucose, blood insulin and/or insulin resistance test and/or a glucose tolerance test. An increase may be an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% as measured by the performance of an analytical test, such as a blood glucose, blood insulin and/or insulin resistance test and/or a glucose tolerance test.
Anti-obesity effects are achieved when body weight, weight gain and/or percent body fat are reduced. Anti-obesity effects may also be achieved when Body Mass Index (BMI), waist circumference, waist-to-hip ratio (WHR) and/or waist height ratio (WHtR) are reduced. This can be assessed using techniques known to the skilled person or as assessed in the experimental part. In this context, "decrease" (respectively "improvement") means at least a detectable amount of decrease (respectively a detectable improvement) using analytical tests known to the skilled person or using analytical tests performed in the experimental part. Anti-obesity effects include obesity prevention and reversal of obesity, as assessed by measurement of the individual's body weight, BMI of the tissue, and/or weight.
The anti-inflammatory effect of the liver can be achieved through the reduction of macrophage infiltration and the reduction of proinflammatory cytokines. This can be evaluated using techniques known to the skilled person or following the techniques evaluated in the experimental part. In this context, "decrease" (respectively "improvement") means at least a detectable amount of decrease (respectively a detectable improvement) using analytical tests known to the skilled person or using analytical tests performed in the experimental part.
Anti-fibrotic effects in the liver can be achieved by reducing the levels of deposited extracellular matrix proteins, blood markers (e.g., including the N-terminal propeptide of type III collagen in plasma, hyaluronic acid, tissue inhibitor of type 1 metalloprotease (TIMP-1), YKL-40, Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT)). Anti-fibrotic effects can also be achieved by improving fibrosis scoring systems such as Metavir or Ishak. This can be evaluated using techniques known to the skilled person or following the techniques evaluated in the experimental part. In this context, "decrease" (respectively "improvement") means at least a detectable amount of decrease (respectively a detectable improvement) using analytical tests known to the skilled person or using analytical tests performed in the experimental part.
By increasing health life, there may also be achieved an effect of increasing health life, wherein symptoms associated with metabolic disorders, preferably diabetes and/or obesity, are absent or reduced, by an increase in health life, wherein reduced mitochondria and metabolic degradation (assessed by measuring the expression levels of genes involved in metabolism and mitochondrial function, such as PGC-1 α synthase and ERR α), are also achieved by a cordage Test (Tightrope Test), reduced mitochondria and metabolic degradation (assessed by measuring the expression levels of PGC-1 α synthase and ERR α), or a health life prolonging effect may be achieved.
The anti-diabetic effect and/or anti-obesity effect may also be observed when the progression of typical symptoms (e.g., insulitis, β cell loss, weight gain) has slowed as assessed by a physician.
The anti-inflammatory effect of the liver can also be observed when the progression of typical symptoms (e.g., fatigue, flu-like symptoms, dark urine, pale stools, abdominal pain, loss of appetite, unexplained weight loss, jaundice) is slowed as assessed by the physician. Alleviation of typical symptoms may mean a slowing of the progression of symptom development or a complete disappearance of symptoms. These methods, using a variety of methods, are largely the same as those used to diagnose liver fibrosis, including clinical examination and routine laboratory testing, can assess symptoms, and thus symptom relief. Such methods include macroscopic and microscopic methods, as well as molecular methods, imaging methods (elastography, X-ray, MRI, CT, ultrasound, angiography), biochemistry, immunohistochemistry, and the like.
Antifibrotic action of the liver is also observed when the progression of typical symptoms (e.g. liver stiffness, jaundice, loss of appetite, difficulty in clear thinking, hydroleg or stomach, nausea, unexplained weight loss, weakness) is slowed as assessed by the physician. Alleviation of typical symptoms may mean a slowing of the progression of symptom development or a complete disappearance of symptoms. Symptoms, and thus symptom reduction, can be assessed using a variety of methods, largely identical to those used to diagnose liver fibrosis, including clinical examinations and routine laboratory tests. Such methods include macroscopic and microscopic methods, as well as molecular methods, imaging methods (elastography, X-ray, MRI, CT, ultrasound, angiography), biochemistry, immunohistochemistry, and the like.
An effect of extending the healthy life may also be observed when the progression of typical symptoms of metabolic disorders associated with aging (e.g. insulin resistance, glucose intolerance, weight gain) has been slowed as assessed by the physician. Alleviation of typical symptoms may mean a slowing of the progression of symptom development or a complete disappearance of symptoms. Symptoms, and thus reduction of symptoms, can be assessed using a variety of methods, largely identical to those used to diagnose diabetes and/or obesity, including clinical examination and routine laboratory testing. Such methods include macroscopic and microscopic methods, as well as molecular methods, X-ray, biochemistry, immunohistochemistry, and the like.
Anticancer effects may also be observed when the progression of typical symptoms (e.g., tumor size, unexplained weight loss, loss of appetite, feeling very full after a small meal, nausea or vomiting, enlarged liver, enlarged spleen, pain near the abdomen or right scapula, abdominal swelling or fluid accumulation, itching, jaundice) has been reduced as assessed by the physician. Alleviation of typical symptoms may mean a slowing of the progression of symptom development or a complete disappearance of symptoms. Symptoms, and thus symptom relief, can be assessed using a variety of methods, largely identical to those used for cancer diagnosis, including clinical examination and routine laboratory testing. Such methods include macroscopic and microscopic methods, as well as molecular methods, imaging methods (X-ray, MRI, CT, ultrasound, angiography), biochemistry, immunohistochemistry, and the like.
A medicament (viral expression construct, viral vector, nucleic acid molecule, composition) as defined herein preferably may alleviate a symptom or a property of a patient or of a patient's cells, tissues or organs if the symptom or property has been alleviated (e.g., no longer detectable or has been alleviated) as defined above after at least one week, one month, six months, one year or more of treatment with the viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition of the invention.
The viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions as defined herein for use according to the invention may be suitable for administration to cells, tissues and/or organs in vivo of individuals affected by or at risk of developing metabolic disorders such as diabetes and/or obesity, hepatitis and/or fibrosis, metabolic disorders associated with aging and/or cancer, and may be administered in vivo, ex vivo or in vitro. The viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition may be administered directly or indirectly to cells, tissues and/or organs in an individual affected by or at risk of developing a metabolic disorder such as diabetes and/or obesity, hepatitis and/or fibrosis, an aging-related metabolic disorder and/or cancer, and may be administered directly or indirectly in vivo, ex vivo or in vitro. The mode of administration may be intravenous, subcutaneous, intramuscular, intrathecal, intraarticular, intraventricular, intraperitoneal, intraadipose tissue, by inhalation, oral, intranasal, intrahepatic, intravisceral, intraocular, intraaural, topical and/or retrograde pancreatic duct administration. Preferred modes of administration are intramuscular, intravenous or intraadipose tissue, as described in the section "general schemes for examples" of the present application.
The viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions of the invention may be administered directly or indirectly using suitable methods known in the art. In view of the advances that have been achieved to date, improvements in the manner in which the viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions of the invention are provided to an individual or cells, tissues, organs of said individual would be expected. Such future modifications may of course be introduced to achieve the above-described effects of the invention. The viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition may be delivered to the individual, cells, tissues or organs of the individual as such. Depending on the disease or disorder, the cells, tissues or organs of the individual may be as previously defined herein. When administering the viral expression constructs and/or viral vectors and/or nucleic acid molecules and/or compositions of the present invention, it is preferred that such viral expression constructs and/or vectors and/or nucleic acids and/or compositions are dissolved in a solution compatible with the delivery method.
As contemplated herein, a therapeutically effective dose of a viral expression construct, vector, nucleic acid molecule and/or composition as above is preferably administered in a single and distinct dose, thereby avoiding repeated periodic administrations. More preferably, a single dose is administered to skeletal muscle, adipose tissue, or intravenously.
Additional compounds may be present in the compositions of the present invention. The compounds may aid in the delivery of the composition. The following provides a list of suitable compounds: a compound capable of forming a complex, nanoparticle, micelle and/or liposome delivering each component defined herein, a compound complexed or entrapped in a vesicle or liposome through the cell membrane. Many of these compounds are known in the art. Suitable compounds include Polyethyleneimine (PEI), or similar cationic polymers including polypropyleneimine or polyethyleneimine copolymers (PEC) and derivatives thereof, synthetic amphiphiles (SAINT-18), lipofectinsTM、DOTAP。
Depending on their identity, the skilled person will know which type of formulation is most suitable for the composition defined herein.
Method/use
In a further aspect, there is provided a viral expression construct as defined above and/or a viral vector as defined above and/or a nucleic acid molecule as defined above and/or a composition as defined above for use as a medicament.
In one embodiment, the viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition is provided for the treatment and/or prevention of a metabolic disorder, preferably diabetes and/or obesity. Complications of metabolic disorders may also be included.
In another embodiment, the viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition is provided for 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, the viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition is provided for extending the health life, preferably by preventing, delaying, curing, reversing and/or treating metabolic disorders associated with aging, preferably diabetes and/or obesity.
In yet another embodiment, the viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition is provided for the treatment and/or prevention of cancer, preferably liver cancer. Complications of cancer may also be included.
In a further aspect, there is provided a method of preventing, delaying, reversing, curing and/or treating a metabolic disorder, preferably diabetes and/or obesity and complications thereof, comprising the use of a viral expression construct as defined above and/or a viral vector as defined above and/or a nucleic acid molecule as defined above and/or a composition as defined above.
Such a method is preferably for alleviating one or more symptoms of a metabolic disorder, such as diabetes and/or obesity, in a cell, tissue or organ of an individual, or alleviating one or more characteristics or symptoms of a cell, tissue or organ of an individual, the method comprising administering to the individual a viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition as defined herein.
In a further aspect, there is provided a method of preventing, delaying, reversing, curing and/or treating liver inflammation and/or fibrosis and complications thereof, comprising the use of a viral expression construct as defined above and/or a viral vector as defined above and/or a nucleic acid molecule as defined above and/or a composition as defined above.
Such a method is preferably for alleviating one or more symptoms of liver inflammation and/or fibrosis in a cell, tissue or organ of an individual, or alleviating one or more characteristics or symptoms of a cell, tissue or organ of an individual, the method comprising administering to the individual a viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition as defined herein.
In a further aspect, there is provided a method of prolonging the health life, preferably by preventing, delaying, curing, reversing and/or treating metabolic disorders associated with aging, preferably diabetes and/or obesity, comprising the use of a viral expression construct as defined above and/or a viral vector as defined above and/or a nucleic acid molecule as defined above and/or a composition as defined above.
Such a method is preferably for alleviating one or more symptoms of, or alleviating one or more characteristics or symptoms of, a metabolic disorder associated with aging, such as diabetes and/or obesity, in a cell, tissue or organ of an individual, the method comprising administering to the individual a viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition as defined herein.
In a further aspect, there is provided a method of preventing, delaying, reversing, curing and/or treating cancer, preferably liver cancer and its complications, comprising the use of a viral expression construct as defined above and/or a viral vector as defined above and/or a nucleic acid molecule as defined above and/or a composition as defined above.
Such a method is preferably for alleviating one or more symptoms of a cancer, such as liver cancer, in a cell, tissue or organ of an individual, or alleviating one or more characteristics or symptoms in a cell, tissue or organ of an individual, the method comprising administering to said individual a viral expression construct and/or a viral vector and/or a nucleic acid molecule and/or a composition as defined herein.
In the context of the present invention, there is provided the use of a viral expression construct and/or a viral vector and/or a nucleic acid molecule and/or a composition as defined herein for the preparation of a medicament for the prevention, delay of progression, reversal, cure and/or treatment of a metabolic disorder, preferably diabetes and/or obesity.
In the context of the present invention, there is provided the use of a viral expression construct and/or a viral vector and/or a nucleic acid molecule and/or a composition as defined herein in the manufacture of a medicament for the prevention, delay, cure, reversal and/or treatment of liver inflammation and/or fibrosis.
In the context of the present invention, there is provided the use of a viral expression construct and/or a viral vector and/or a nucleic acid molecule and/or a composition as defined herein for the manufacture of a medicament for extending the health life, preferably by preventing, delaying, curing, reversing and/or treating metabolic disorders associated with aging, preferably diabetes and/or obesity.
In the context of the present invention, there is provided the use of a viral expression construct and/or a viral vector and/or a nucleic acid molecule and/or a composition as defined herein for the manufacture of a medicament for the prevention, delay, reversal, cure and/or treatment of cancer, preferably liver cancer.
Metabolic disorders include metabolic syndrome, diabetes, obesity, complications associated with diabetes, hyperglycemia, insulin resistance, glucose intolerance, hepatic steatosis, Alcoholic Liver Disease (ALD), Non Alcoholic Fatty Liver Disease (NAFLD), Non Alcoholic Steatohepatitis (NASH), Coronary Heart Disease (CHD), hyperlipidemia, atherosclerosis, endocrine disorders, osteopenia-sarcopenia Obesity Syndrome (OSO), diabetic nephropathy, Chronic Kidney Disease (CKD), cardiac hypertrophy, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, arthritis, sepsis, ocular neovascularization, neurodegeneration, dementia, and may also include depression, adenoma, cancer.
Diabetes includes prediabetes, hyperglycemia, type 1 diabetes, type 2 diabetes, juvenile adult onset diabetes (MODY), monogenic diabetes, neonatal diabetes, gestational diabetes, fragile-onset diabetes, idiopathic diabetes, drug-or chemically-induced diabetes, stiff person syndrome, lipodystrophy diabetes, adult delayed autoimmune diabetes (LADA).
Obesity includes overweight, intermediate/upper obesity, peripheral/lower obesity, morbid obesity, osteopenia-sarcopenia Obesity Syndrome (OSO), pediatric obesity, mendelian (single gene) syndrome obesity, mendelian non-syndrome obesity, multigenic obesity. Metabolic disorders, diabetes, obesity and the type of subject treated have been previously defined herein.
Liver inflammation and/or fibrosis includes autoimmune hepatitis, viral hepatitis including hepatitis a, B, C, D and E, alcoholic hepatitis, nonalcoholic steatohepatitis (NASH) and cirrhosis.
Cancers include astrocytomas, gliomas, leukemias, lymphomas, melanomas, myelomas, neuroblastomas, sarcomas (including chondrosarcomas, fibrosarcomas, rhabdomyomas, and osteosarcomas), schwannoma, seminomas, and cancers of the bladder, breast, cervix, colon, endometrium, esophagus, kidney, liver, lung, ovary, prostate, pancreas, rectum, skin, stomach, and thyroid. Preferred cancer is liver cancer, preferably hepatocellular carcinoma. In one embodiment, the method or use is performed in vitro, e.g., using cell culture. Preferably the method or use is in vivo. Each feature of these methods/uses has been defined herein.
In the methods of the invention, the viral expression construct and/or vector and/or nucleic acid molecule and/or composition may be combined with other compounds known for the treatment of metabolic disorders, preferably diabetes and/or obesity, in an individual.
In another method of the invention, the viral expression construct and/or vector and/or nucleic acid molecule and/or composition may be combined with other compounds known for the treatment of liver inflammation and/or fibrosis.
In another method of the invention, the viral expression construct and/or vector and/or nucleic acid molecule and/or composition may be combined with other compounds known to be useful for extending the health life.
In another method of the invention, the viral expression construct and/or vector and/or nucleic acid molecule and/or composition may be combined with other compounds known for the treatment of cancer, preferably liver cancer.
In a preferred embodiment, the treatment in the use or method according to the invention is not necessarily repeated. Alternatively, in a use or method according to the invention, administration of the viral expression construct or composition may be repeated every year or every 2, 3, 4, 5, 6 years.
General definition
Consistency/similarity
In the context of the present invention, a protein fragment or polypeptide or peptide or derived peptide as fibroblast growth factor 21(FGF21) is represented by an amino acid sequence.
In the context of the present invention, a nucleic acid molecule which is a nucleic acid molecule encoding FGF21 is represented by a nucleic acid or nucleotide sequence encoding a protein fragment or polypeptide or peptide or derived peptide. The nucleic acid molecule may comprise a regulatory region.
It is to be understood that each nucleic acid molecule or protein fragment or polypeptide or peptide or derivatized peptide or construct identified herein by a given sequence identity number (SEQ ID NO) is not limited to the particular sequence disclosed. Each coding sequence identified herein will encode a given protein fragment or polypeptide or peptide or derived peptide or construct, or will itself be a protein fragment or polypeptide or construct or peptide or derived peptide. Throughout this application, each reference to a particular nucleotide sequence of SEQ ID NO (exemplified by SEQ ID NO: X) encoding a given protein fragment or polypeptide or peptide or derived peptide may be substituted with:
i. comprises a nucleotide sequence substantially identical to SEQ ID NO: x a nucleotide sequence of a nucleotide sequence having at least 60% sequence identity or similarity;
a nucleotide sequence whose sequence differs from the sequence of the nucleic acid molecule of (i) due to the degeneracy of the genetic code; or the like, or, alternatively,
encoding or consisting of the nucleotide sequence of SEQ ID NO: x encodes an amino acid sequence having an amino acid identity or similarity of at least 60%.
In the present application, each reference to a particular amino acid sequence SEQ ID NO (exemplified by SEQ ID NO: Y) may be made with the following substitutions: comprising a sequence identical to the amino acid sequence of SEQ ID NO: y has an amino acid sequence having at least 60% sequence identity or similarity.
In a further preferred embodiment, each nucleotide or amino acid sequence described herein has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity or similarity to a given nucleotide or amino acid sequence, respectively, by virtue of its percent identity or similarity (at least 60%) to the given nucleotide or amino acid sequence, respectively. In preferred embodiments, sequence identity or similarity is determined by aligning the full length of the sequences identified herein. Unless otherwise indicated herein, identity or similarity to a given SEQ ID NO refers to identity or similarity based on the full length of the sequence (i.e., over its entire length or the entirety).
Each non-coding nucleotide sequence (i.e., the nucleotide sequence of a promoter or another regulatory region) may be replaced by a nucleotide sequence comprising a nucleotide sequence having at least 60% sequence identity or similarity to the particular nucleotide sequence SEQ ID NO (exemplified by SEQ ID NO: A). Preferred nucleotide sequences are similar to SEQ ID NO: a has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% identity. As described herein, identity can be assessed within the entire SEQ ID NO or a portion thereof. In a preferred embodiment, such non-coding nucleotide sequence, e.g., a promoter, exhibits or exerts at least the activity of the non-coding nucleotide sequence, e.g., the activity of a promoter, as known to those skilled in the art.
"sequence identity" is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences as determined by aligning the sequences. In a preferred embodiment, the sequence identity is calculated based on the full length of two given SEQ ID NOs or parts thereof. A portion thereof preferably means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the two SEQ ID NOs. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences as the case may be, depending on the match between strings of such sequences.
The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutions to the sequence of a second polypeptide. "identity" and "similarity" are readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, a.m., ed., oxford university Press, New York, 1988; biocontrol, information and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; computer Analysis of sequence data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, m.and deveux, j., eds., M Stockton Press, new york, 1991; and Carillo, H., and Lipman, D., SIAM J.applied Math.,48:1073 (1988).
The preferred method of determining identity is designed to provide the largest match between the tested sequences. Methods for determining consistency and similarity have been programmed into 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 Research12(1):387(1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S.F.et al., J.mol.biol.215: 403-. The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J.mol.biol.215: 403-. The well-known Smith Waterman algorithm may also be used to determine consistency.
Preferred parameters for polypeptide sequence alignment include the following: algorithm, Needleman and Wunsch, J.mol.biol.48: 443-; company matrix: BLOSSUM62from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA.89:10915-10919 (1992); gap Penalty:12 and Gap Length Penalty: 4. Programs with these parameters are publicly available as "Ogap" programs from genetics computer Group, located in madison, wisconsin. The foregoing parameters are the default parameters for amino acid alignments (no penalty for end gaps).
Preferred parameters for nucleic acid alignment include the following: algorithm, Needleman and Wunsch, J.mol.biol.48: 443-; match matrix, matches ═ 10, mismatch ═ 0; 50 is GapPenalty; gap Length Penalty: 3. Available as the Gap program from genetics computer Group, located in madison, wisconsin. Given above are default parameters for nucleic acid alignment.
Alternatively, the skilled person may also consider so-called "conservative" amino acid substitutions when determining the degree of amino acid similarity, as will be apparent to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; one group of amino acids having aliphatic hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; one group of amino acids with aromatic side chains is phenylalanine, tyrosine and tryptophan; one group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chains 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. Preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala-Ser; Arg-Lys; Asn-Gln or His; Asp-Glu; Cys-Ser or Ala; Gln-Asn; Glu-Asp; Gly-Pro; His-Asn or Gln; Ile-Leu or Val; Leu-Ile or Val; Lys-Arg; gln or Glu; Met-Leu or Ile; Phe-Met, Leu or Tyr; Ser-Thr; Thr-Ser; Trp-Tyr; Tyr-Trp or Phe; and Val-Ile or Leu.
Genes or coding sequences
"Gene" or "coding sequence" or "nucleic acid" or "nucleotide sequence" or "nucleic acid" refers to a region of DNA or RNA (transcribed region) that "encodes" a particular protein, such as FGF 21. When placed under the control of appropriate regulatory regions, such as a promoter, the coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide. A gene may comprise several operably linked segments, such as a promoter, a 5 'leader sequence, an intron, a coding sequence and a3' untranslated sequence or a3 'untranslated region (3' UTR), including a polyadenylation site or a signal sequence. Chimeric or recombinant genes (e.g., FGF21 genes) are genes that are not normally found in nature, such as genes in which a promoter is not associated in nature with some or all of the transcribed DNA region. "expression of a gene" refers to a process in which a gene is transcribed into RNA and/or translated into active protein.
Promoters
As used herein, the term "promoter" refers to a nucleic acid fragment having a sequence that controls the transcription of one or more genes (or coding sequences), located upstream in the direction of transcription relative to the transcription start site of the gene, and structurally recognized by the presence of a binding site for a DNA-dependent RNA polymerase, the transcription start site, and any other DNA sequences, including but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art, to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated according to physiological or developmental conditions. An "organ-specific" or "tissue-specific" promoter is a promoter that is active in a particular type of organ or tissue, respectively. Organ-specific and tissue-specific promoters primarily regulate the expression of one or more genes (or coding sequences) in one organ or tissue, but may also allow detectable levels of ("leaky") expression in other organs or tissues. Leaky expression in other organs or tissues refers to expression that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold or at least 5-fold lower than organ-specific or "tissue-specific" expression, but still detectable, as assessed by standard tests known to the skilled person (e.g., PCR, western blot analysis, ELISA). The maximum number of organs or tissues from which leakage expression can be detected is five, six, seven or eight. An "adipose tissue-specific promoter" is a promoter that can initiate transcription in adipose tissue while still allowing any leaky expression to occur in other (up to five, six, seven or eight) organs and parts of the body. Transcription in adipose tissue can be detected in adipose tissue and adipocytes, such as white adipocytes, brown adipocytes, beige adipocytes, preadipocytes, stromal vascular cells. A "liver-specific promoter" is a promoter that can initiate transcription in the liver while still allowing any leaky expression to occur in other (up to five, six, seven or eight) organs and parts of the body. Transcription in the liver can be detected in liver tissue and in liver cells, such as hepatocytes, kupffer cells, and/or oval cells. Similarly, a "skeletal muscle promoter" is a promoter that can initiate transcription in skeletal muscle while still allowing any leaky expression to occur in other (up to five, six, seven or eight) organs and parts of the body. Transcription in skeletal muscle can be detected in skeletal muscle cells, such as muscle cells, myoblasts, satellite cells.
A "ubiquitous promoter" is active in substantially all tissues, organs and cells of an organism.
Suitable promoters for organ-specific and/or tissue-specific expression of the nucleotide sequence encoding FGF21 include the human α -antitrypsin promoter, α -antitrypsin promoter combined with the Hepatocyte Control Region (HCR) promoter from apolipoprotein E, the albumin promoter, the major urinary protein promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, the hepatic rich protein activator promoter, the transthyretin promoter, the thyroxine binding globulin promoter, the apolipoprotein a1 promoter, the hepatic fatty acid binding protein promoter, the phenylalanine hydroxylase promoter, adipocyte protein 2(aP2, also known as fatty acid binding protein 4(FABP4)) promoter, the PPARy promoter, the adiponectin promoter, the promoter derived from the human aromatase cytochrome p450(p450arom), the mini/aP2 promoter (consisting of the fat-specific aP2 and the basal aP2 promoters), the disintegrin 1(UCP 3) promoter, the mini/p 1 promoter (consisting of the basal oxap and the basal oxap gene promoter), the myoglobin promoter combined with the basal protein promoter of the muscle protein gene, the creatine gene promoter, the basal promoter of mcxa 6342, the basal promoter, the myoglobin promoter, the basal promoter of the protein promoter, the protein promoter of the protein apo gene, the protein 12, the protein promoter of the gene, the protein promoter of the protein 12, the protein promoter of the gene, the gene of the gene, the gene promoter of the gene, the gene of the gene encoding FGF 6346 bp-12, the.
Operable connection
"operably linked" is defined herein as a configuration in which a control sequence, such as a promoter sequence or regulatory sequence, is suitably placed at a position relative to a nucleotide sequence of interest, preferably encoding FGF21, such that the promoter or control or regulatory sequence directs or affects the transcription and/or production or expression of the nucleotide sequence of interest, preferably the nucleotide sequence encoding FGF21, in a cell and/or subject. For example, a promoter is operably linked to a coding sequence if it is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is understood to be "under the control" of the promoter. When one or more nucleotide sequences and/or elements contained within a construct are defined herein as being "configured to be operably linked to an optional nucleotide sequence of interest," a nucleotide sequence and/or element should be understood as being configured in the construct in such a way that these nucleotide sequences and/or elements are all operably linked to the nucleotide sequence of interest once the nucleotide sequence of interest is present in the construct.
Viral expression constructs
The expression construct carries a genome that can be stable and maintain episomal form in the cell. In the context of the present invention, a cell may be meant to encompass a cell used to prepare the construct or a cell to which the construct is to be administered. Alternatively, the construct may be integrated into the genome of the cell, for example, by homologous recombination or otherwise. A particularly preferred expression construct is one in which a nucleotide sequence encoding FGF21, as defined herein, is operably linked to a promoter, as defined herein, wherein the promoter is capable of directing expression of the nucleotide sequence (i.e., the coding sequence) in a cell. Preferably, the promoter directs expression of the nucleotide sequence in at least one cell of a specific organ and/or a specific tissue. Preferably, the promoter directs expression of the nucleotide sequence in at least one cell of the liver, adipose tissue and/or skeletal muscle. Preferably, the promoter directs expression in at least 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100% of the cells of the liver, adipose tissue and/or skeletal muscle. In the context of the present invention, FGF21 to be expressed in liver, adipose tissue, or skeletal muscle refers to preferential or predominant (at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, or more) expression of FGF21 in liver, adipose tissue, or skeletal muscle as compared to other organs or tissues. Throughout the application, where liver-specific, or adipose-specific or skeletal muscle-specific is mentioned in the context of expression, specific expression of cell types of the cell types constituting liver, adipose tissue or skeletal muscle, respectively, is also envisaged.
The viral expression construct of the invention comprises a nucleotide sequence in a form "suitable for expression in a mammal", which means that the viral expression construct comprises one or more regulatory sequences selected on the basis of the mammalian host cell to be used for expression, i.e. the mammalian host cell to which the nucleotide sequence to be expressed is operably linked. Preferred mammalian host cells for expression are human, murine or canine cells.
The viral expression constructs of the invention comprise nucleotide sequences to be expressed in liver, adipose tissue and/or skeletal muscle.
As used herein, "adipose tissue" refers to tissue composed of mature adipocytes (adipocyte), i.e., fat cells (fatcell), and a combination of small blood vessels, nerve tissue, lymph nodes, and Stromal Vascular Fraction (SVF). SVF is composed of endothelial cells, fibroblasts, adipocyte precursor cells (i.e., preadipocytes), and immune cells such as macrophages and T cells. In mammals, two different types of adipose tissue are traditionally distinguished: white Adipose Tissue (WAT) and Brown Adipose Tissue (BAT). In mammals, adipose tissue is contained in multiple storage organs (multi-depot organ). Adipose stores include, but are not limited to, epididymal WAT (eWAT), Inguinal WAT (iWAT), retroperitoneal WAT (rWAT), mesenteric WAT (mWAT), Interscapular BAT (iBAT).
As used herein, "skeletal muscle" refers to tissue composed of muscle fibers. Muscle fibers, also known as muscle fibers, are single multinucleated or syncytial cells resulting from the fusion of hundreds of myoblasts, some of which remain in mature muscle as undifferentiated cells (known as satellite cells). The individual muscle fibers are surrounded by connective tissue called the endomysial membrane. About 10 to 100 muscle fibers form a fiber bundle, or fascicle, which is itself surrounded by another layer of connective tissue known as the muscle capsule. Finally, skeletal muscle is formed of a group of muscle fascicles surrounded by another layer of connective tissue, also known as the periosteum. In addition to muscle fibers, skeletal muscle is also made up of many blood vessels and nerves. The ends of the muscles converge in a dense connective tissue structure, tendons and aponeurosis that mediate the attachment of the muscle to the periosteum of the skeleton or other connective tissue of the muscle.
As used herein, "liver" refers to a tissue consisting of hepatocytes. Hepatocytes account for approximately 50% to 70% of the cells within the liver. In addition to hepatocytes, the liver is composed of endothelial cells, pericytes, oval cells, Kupffer (Kupffer) cells, and stellate cells (Ito cells). When activated by kupffer cells, the stellate cells are transformed into myofibroblasts. There is also a central vein and portal vein (portal triple) in the liver containing the anterior branch of the hepatic artery, the hepatic portal vein, the bile duct and the lymphatic vessel.
This preferred expression construct is said to comprise an expression cassette. An expression cassette as used herein comprises or consists of a nucleotide sequence encoding FGF21 operably linked to a promoter, wherein the promoter can direct expression of the nucleotide sequence. In one embodiment, the expression cassette used herein comprises or consists of a nucleotide sequence encoding FGF21, a promoter and at least one nucleotide sequence encoding a target sequence of a microrna expressed in the liver and at least one nucleotide sequence encoding a target sequence of a microrna expressed in the heart. In one embodiment, the described expression cassette comprises a nucleotide sequence encoding a target sequence of micro RNAs expressed in liver and/or micro RNAs expressed in heart, which has perfect complementarity to their homologous micro RNAs. In another embodiment, the described expression cassette comprises one or more nucleotide sequences encoding microrna binding sites with imperfect complementarity (one mismatch per five consecutive nucleotides). In yet another embodiment, the expression cassette may comprise both nucleotide sequences encoding perfect and imperfect microrna binding sites. Thus, by using nucleotide sequences encoding single perfect, multiple perfect, single imperfect, multiple imperfect, or a combination of perfect and imperfect target sites of micrornas, expression cassettes can be customized to produce different levels of regulation. In addition, nucleotide sequences encoding target sites for different micrornas can be used, thus allowing the gene to be regulated by multiple micrornas. A preferred position of the nucleotide sequence encoding the microRNA target sequence is the 3' UTR. However, nucleotide sequences inserted into the coding sequence or 5' UTR sequence (coding target sequences) may also be used.
The choice of nucleotide sequence encoding the microRNA target sequence depends on the desired expression pattern. The presence of endogenous micrornas in a cell will inhibit the expression of a gene or coding sequence from an expression construct comprising a nucleotide sequence encoding a microrna target sequence. To express a gene or coding sequence of interest to be inhibited in a given cell type, a nucleotide sequence is selected that encodes a target sequence recognized by a microrna present in that cell type.
Viral expression constructs are expression constructs intended for gene therapy. It is designed to comprise part of the viral genome, as defined later herein.
The expression constructs disclosed herein can be prepared using recombinant techniques in which the nucleotide sequence encoding FGF21 is expressed in suitable cells, e.g., cells in culture or in cells of multicellular organisms, such as those described in the documents Ausubel et al, "Current Protocols in molecular biology", Greene Publishing and Wiley-Interscience, New York (1987) and Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entirety. See also Kunkel (1985) Proc. Natl. Acad. Sci.82:488 (describing site-directed mutagenesis) and Roberts et al (1987) Nature 328:731-734 or Wells, J.A., et al (1985) Gene34:315 (describing cassette mutagenesis).
Typically, a nucleic acid or nucleotide sequence encoding FGF21 will be used in an expression construct or expression vector. The phrase "expression vector" or "vector" generally refers to a nucleotide sequence capable of effecting expression of a gene or coding sequence in a host compatible with such sequence. These expression vectors typically include at least a suitable promoter sequence and optionally a transcription termination signal. Other factors necessary or helpful for affecting expression may also be used, as described herein. The nucleic acid or DNA or nucleotide sequence encoding FGF21 is introduced into a DNA construct capable of being introduced and expressed in cell culture in vitro. In particular, the DNA construct is suitable for replication in a prokaryotic host, such as a bacterium, e.g.E.coli, or may be introduced into a cultured mammalian, plant, insect (e.g.Sf 9), yeast, fungal or other eukaryotic cell line. Preparation of a DNA construct for introduction into a particular host may include a replication system recognized by the host, the desired DNA segment encoding the desired polypeptide, and transcriptional and translational initiation and termination control sequences operably linked to the segment encoding the polypeptide. The term "operably linked" has been defined herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates transcription of the sequence. If the DNA for the signal sequence is expressed as a preprotein involved in the secretion of the polypeptide, the DNA for the signal sequence is operably linked to the DNA encoding the polypeptide. Generally, operably linked DNA sequences are contiguous and, in the case of a signal sequence, are both contiguous and in reading frame. However, enhancers do not have to be contiguous with the coding sequence that they control their transcription. Ligation may be accomplished by ligation at convenient restriction sites or adaptors or linkers inserted in place of them or by gene synthesis.
The choice of a suitable promoter sequence will generally depend on the host cell chosen for expression of the DNA fragment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters well known in the art (see, e.g., Sambrook and russell,2001, supra). Transcriptional regulatory sequences typically include heterologous enhancers or promoters that are recognized by the host.The choice of suitable promoters depends on the host, but promoters such as trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell,2001, supra). Expression vectors include replication systems, transcriptional and translational regulatory sequences, and insertion sites for polypeptide-encoding segments. In most cases, the replication system is only functional in the cells used for the preparation of the vector (bacterial cells, such as E.coli). Most plasmids and vectors do not replicate in cells infected with the vector. Examples of possible combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, supra) and Metzger et al, (1988) Nature334: 31-36. For example, suitable expression vectors may be expressed in yeast, e.g., saccharomyces cerevisiae (s.cerevisiae), e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells, and bacterial cells, e.g., escherichia coli. Thus, the cell may be a prokaryotic or eukaryotic host cell. The cells may be cells suitable for culture on liquid or 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
A viral vector or viral gene therapy vector is a vector comprising a viral expression construct as defined above.
Viral vectors or viral gene therapy vectors are suitable vectors for gene therapy. Vectors suitable for gene therapy are described in Anderson 1998, Nature392:25-30；Walther and Stein,2000,Drugs60:249-71；Kay et al.,2001,Nat.Med.7:33-40；Russell,2000,J.Gen.Virol.81:2573-604；Amadoand Chen,1999,Science285:674-6；Federico,1999,Curr.Opin.Biotechnol.10:448-53；Vigna and Naldini,2000,J.Gene Med.2：308-16；Marin et al.,1997,Mol.Med.Today3:396-403；Peng and Russell,1999,Curr.Opin.Biotechnol.10:454-7；Sommerfelt,1999,J.Gen.Virol.80:3049-64；Reiser,2000,Gene Ther.7910-3; and the references cited therein.
Particularly suitable gene therapy vectors include adenovirus and adeno-associated virus (AAV) vectors. These vectors infect most dividing and non-dividing cell types, including synoviocytes and hepatocytes. The episomal nature of adenovirus and AAV vectors after entry into cells makes these vectors suitable for therapeutic applications (Russell, 2000, J.Gen.Virol.81: 2573-. AAV vectors are even more preferred because they are known to result in very stable long-term expression of transgene expression (up to 9 years in dogs (Niemeyer et al, blood.2009 Jan 22; 113 (4): 797-. Preferred adenoviral vectors are modified to reduce host response as reviewed in Russell (2000, supra). Methods of Gene therapy using AAV vectors are described in Wang et al, 2005, J Gene med. march 9 (prepress electronic edition), mantel 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.2011dec 22; 2357 (25), 2357-65, Appliaily et al, Hum Gene ther.2005 Apr; 16(4) 426-34.
Another suitable gene therapy vector includes retroviral vectors. Preferred retroviral vectors for use in the present invention are lentivirus-based expression constructs. Lentiviral vectors have the ability to infect and stably integrate into the genome of dividing and non-dividing cells (Amado and Chen, 1999 Science 285: 674-6). Methods for lentiviral expression construct construction and use are described in U.S. Pat. Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633 and 6,323,031, and Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al (2000, J Gene Med 2000; 2: 308-16).
Other suitable gene therapy vectors include adenoviral vectors, herpesvirus vectors, polyoma virus vectors or vaccinia virus vectors.
Suitable promoters for expressing a nucleotide sequence encoding FGF21 from a gene therapy vector include, for example, CMV promoters, viral long terminal repeat promoters (LTRs), such as those from Murine Monocytic Leukemia Virus (MMLV) rous sarcoma virus or HTLV-1, simian virus 40(SV 40) early promoters, CAG promoters, α -antitrypsin promoters, mini/aP2 promoters, mini/UCP1 promoters, C5-12 promoters and herpes simplex virus thymidine kinase promoters.
Several inducible promoter systems have been described that can be induced by administration of small organic or inorganic compounds. Such inducible promoters include those under the control of heavy metals, such as the metallothionein (metallothionein) promoter (Brinster et al 1982Nature)296:39-42；Mayo et al.1982 Cell2999-108), RU-486 (progesterone antagonist) (Wang et al 1994 Proc. Natl. Acad. Sci. USA)918180-89:5547-5551；U.S.Pat.No.5,464,758；Furth etal.1994Proc.Natl.Acad.Sci.USA91:9302-9306；Howe et al.1995J.Biol.Chem.270:14168-14174；Resnitzky et al.1994Mol.Cell.Biol.14:1669-1679；Shockett etal.1995Proc.Natl.Acad.Sci.USA926522-6526) and the tTAER system based on a multiple chimeric transactivator consisting of a tetR polypeptide as the activation domain of VP16 and the ligand binding domain of the estrogen receptor (yeee et al, 2002, US6,432,705).
The gene therapy vector may optionally comprise a further nucleotide sequence encoding a further polypeptide.
The gene therapy vector is preferably formulated in a composition or pharmaceutical composition as defined herein. In this context, the composition or pharmaceutical composition may comprise a suitable pharmaceutical carrier as previously defined herein.
Adeno-associated virus vector (AAV vector)
Preferred viral vectors or preferred gene therapy vectors are AAV vectors. The AAV vector used herein preferably comprises a recombinant AAV vector (rAAV vector). As used herein, "rAAV vector" refers to a recombinant vector comprising a portion of an AAV genome encapsidated in a protein shell derived from the capsid proteins of the AAV serotypes as explained herein. Portions of the AAV genome may comprise Inverted Terminal Repeats (ITRs) derived from adeno-associated virus serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, and the like. Preferred ITRs are encoded by a polynucleotide comprising SEQ ID NO: 48(5' ITR) and SEQ ID NO: 49(3' ITRs) or those of AAV2 represented by the sequences consisting thereof. The invention also preferably encompasses polypeptides that are homologous to SEQ ID NO: 48 and SEQ ID NO: 49 with at least 80% identity.
The protein shell consisting of capsid proteins may be derived from AAV serotypes such as AAV1, 2, 3, 4, 5, and the like. Preferred AAV capsids are AAV1, AAV3, AAV8, AAV9 capsids. Preferred ITRs are from AAV 2. The protein shell may also be referred to as a capsid protein shell. The rAAV vector may be deleted for one or preferably all of the wild-type AAV genes, but may still comprise functional ITR nucleic acid sequences. Functional ITR sequences are necessary for replication, rescue and packaging of AAV virions. An ITR sequence may be a wild-type sequence, or may have at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to a wild-type sequence, or may be modified by, for example, insertion, mutation, deletion or substitution of nucleotides, as long as the ITR sequence remains functional. In this context, functional refers to the ability to introduce genomic packaging into the capsid and subsequently allow its expression in the host cell or target cell to be infected. In the context of the present invention, the capsid protein shell may be of a different serotype than the rAAV vector genomic ITRs.
The nucleic acid molecule represented by the selected nucleic acid sequence is preferably inserted between the rAAV genome or ITR sequences identified above, e.g., an expression construct comprising an expression regulatory element operably linked to a coding sequence and a3' termination sequence. Nucleic acid molecules may also be referred to as transgenes.
"AAV helper functions" generally refer to the corresponding AAV functions provided for in-trans replication and packaging of rAAV vectors. AAV helper functions complement AAV functions that are absent in rAAV vectors, but they lack AAV ITRs (which are provided by the rAAV vector genome). AAV helper functions include the two major ORFs of AAV, i.e., the rep and cap coding regions or sequences that are essentially functionally identical. Rep and Cap regions are well known in the art, see, e.g., Chiori et al (1999, J. of virology, Vol 73(2): 1309-. AAV helper functions can be provided on AAV helper constructs. Introduction of the helper construct into the host cell can occur, for example, by transformation, transfection, or transduction prior to or concurrent with introduction of the rAAV genome present in the rAAV vectors identified herein. Thus, the AAV helper constructs of the invention may be selected such that they produce a desired combination of serotypes of the capsid protein shell of the rAAV vector on the one hand, and of the rAAV genome present in the rAAV vector replication and packaging on the other hand.
An "AAV helper virus" provides other functions required for AAV replication and packaging. Suitable AAV helper viruses include adenovirus, herpes simplex virus (e.g., HSV type 1 and 2), and vaccinia virus. Other functions provided by helper viruses may also be introduced into host cells by plasmids, as in US6,531,456, which is incorporated herein by reference.
A "transgene" is defined herein as a gene or coding sequence or nucleic acid molecule (i.e., a molecule encoding FGF21) that is newly introduced into a cell, i.e., a gene that may be present but may not be expressed or expressed at an insufficient level in general. In this context, "insufficient" means that despite the expression of FGF21 in a cell, the condition and/or disease as defined herein can develop. In this case, the present invention allows overexpression of FGF 21. The transgene may comprise a sequence native to the cell, a sequence not naturally found in the cell, and may comprise a combination of both. The transgene may comprise sequences encoding FGF21 and/or other proteins as previously identified herein, which may be operably linked to suitable regulatory sequences to express the sequence encoding FGF21 in a cell. Preferably, the transgene is not integrated into the genome of the host cell.
By "transduction" is meant the delivery of FGF21 to a recipient host cell by a viral vector. For example, transduction of a target cell by a rAAV vector of the invention results in transfer of the rAAV genome contained in the vector into the transduced cell. "host cell" or "target cell" refers to a cell that is subjected to DNA delivery, such as a muscle cell of a subject. AAV vectors can transduce both dividing and non-dividing cells.
Production of AAV vectors
The production of recombinant aav (raav) for re-staging transgenes has been previously described. See Ayuso E, et al, curr. gene ther.2010; 423-; 20: 1013-; 922-; 20:807-817. These protocols can be used or adapted to produce the AAV of the invention. In one embodiment, the producer cell line is transiently transfected with a polynucleotide of the invention (comprising an ITR-flanked expression cassette) and a construct that encodes rep and cap proteins and provides ancillary functions. In another embodiment, the cell line stably provides helper functions and is transiently transfected with the polynucleotide of the invention (comprising an ITR-flanked expression cassette) and a construct encoding rep and cap proteins. In another embodiment, the cell line stably supplies rep and cap proteins as well as helper functions and is transiently transfected with a polynucleotide of the invention. In another embodiment, the cell line stably supplies rep and cap proteins and is transiently transfected with a polynucleotide of the present invention and a polynucleotide encoding helper functions. In yet another embodiment, the cell line stably supplies the polynucleotides, rep and cap proteins of the invention, as well as helper functions. Methods of making and using these and other AAV production systems have been described in the art. See, muzyzka N, et al, US 5,139,941, Zhou X, et al, US 5,741,683, Samulski R, et al, US6,057,152, Samulski R, et al, US6,204,059, Samulski R, et al, US6,268,213, Rabinowitz J, et al, US6,491,907, Zolotukhin S, et al, US6,660,514, Shenk T, et al, US6,951,753, Snyder R, et al, US 7,094,604, raowitz J, et al, US 7,172,893, Monahan P, et al, US 7,201,898, Samulski R, et al, US 7,229,823, and Ferrari F, et al, US 7,439,065.
The rAAV genome present in the rAAV vector comprises at least the nucleotide sequence of the Inverted Terminal Repeat (ITRs) of one AAV serotype, preferably those of serotype AAV2 as previously disclosed herein, or a nucleotide sequence substantially identical thereto or at least 60% identical thereto, and a nucleotide sequence encoding FGF21 inserted between the two ITRs (under the control of suitable regulatory elements). The vector genome requires the use of 5 'and 3' ITR flanking sequences for efficient packaging of the vector genome in the rAAV capsid.
The complete genome of several AAV serotypes and corresponding ITRs has been sequenced (Chiorini et al 1999, J.of Virology Vol.73, No.2, p 1309-1319). They may be cloned or prepared by chemical synthesis as known in the art, using, for example, an oligonucleotide synthesizer, supplied by Applied Biosystems inc. (Fosters, CA, USA), or standard molecular biology techniques. The ITRs may be cloned from the AAV viral genome or spliced by a vector comprising AAV ITRs. The ITR nucleotide sequences may be linked at either end to a nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or the AAV sequences between the ITRs may be replaced with the desired nucleotide sequences.
Preferably, the rAAV genome present in the rAAV vector does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV. The rAAV genome may further comprise a marker or reporter gene, such as, for example, a gene encoding an antibiotic resistance gene, a fluorescent protein (e.g., gfp), or a gene encoding a chemical, enzymatic, or otherwise detectable and/or selectable product known in the art (e.g., lacZ, aph, etc.).
The rAAV genome present in the rAAV vector further comprises a promoter sequence operably linked to the nucleotide sequence encoding FGF 21. Preferred promoter sequences are promoters which confer expression in skeletal muscle cells and/or in skeletal muscle, liver cells and/or in the liver and fat cells and/or in adipose tissue. Examples of such promoters include the CMV, CAG, mini/aP2, mini/UCP1, C5-12 and hAAT promoters as previously defined herein.
Suitable 3' untranslated regions may be those that are naturally associated with the nucleotide sequence, or may be derived from different genes, such as the SV40 polyadenylation signal (SEQ ID NO: 50) and the rabbit β -globin polyadenylation signal (SEQ ID NO: 51).
Alternatively, other nucleotide sequences may be operably linked to the nucleotide sequence encoding FGF21, such as nucleotide sequences encoding a signal sequence, a nuclear localization signal, an expression enhancer, and the like.
Codon optimization
As used herein, "codon optimization" refers to a process for modifying an existing coding sequence or designing a coding sequence, e.g., to improve translation or improve transcription of a coding sequence in a host cell or organism that expresses a transcribed RNA molecule transcribed from the coding sequence. Codon optimization includes, but is not limited to, including the selection of codons for the coding sequence to adapt to the codon preference of the expression host organism. For example, codon optimization also eliminates elements (e.g., termination sequences, TATA boxes, splice sites, ribosome entry sites, repetitive and/or GC-rich sequences and RNA secondary structure or instability motifs) that potentially negatively impact RNA stability and/or translation in order to adapt to the codon bias of a mammalian, preferably murine, canine, or human, expression host.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb "to consist of … …" may be replaced by the word "consisting essentially of … …" means that the viral expression construct, viral vector, composition, gene therapy composition as defined herein may comprise other components in addition to the specifically identified components, which do not alter the unique characteristics of the present invention.
In addition, the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that only one of the elements is present. Thus, the indefinite article "a" or "an" usually means "at least one".
When the word "about" or "approximately" is used in conjunction with a numerical value (about 10), it is preferred to mean that the value may be about 10% more or 10% less than the given value.
All patent and literature references cited in this specification are incorporated herein by reference in their entirety. Each of the embodiments identified herein may be combined together, unless otherwise indicated.
The invention is further explained in the following examples. These examples do not limit the scope of the present invention, but are merely illustrative thereof.
Drawings
Figure 1. schematic representation of prevention of obesity by administration of AAV9-CAG-moFGF21-dmiRT vector within eWAT in C57B16 mice (a) four tandem repeats of AAV-CAG-moFGF 21-double miRT vector the expression cassette comprises a CAG promoter, a murine codon-optimized FGF21 coding sequence and a miRT122a sequence, and four tandem repeats of miRT1 sequence cloned in the 3' untranslated region of the expression cassette the ITR from AAV2 is flanked by the expression cassette the schematic representation is not drawn to scale CAG: chicken β -actin promoter/CMV enhancer; papa: expression level of polyA a (B) FGF 8 in metabolic tissue, measurement of expression level of murine codon-optimized FGF 6 in RTqPCR in eWAT, iWAT, ivot 686 and liver of C57B16 mice, and normalization of body weight with rpp 0 value (bln) to bllo 11 mice, expression level of rat C11-mow 7, ivot 468 in C57B16 mice (5) and measurement of cycle expression level of AAV 11, ivot 468 in AAV 7, wat-wat 468, and liver-5, aatr 468, and expression level of AAV 7, and expression level of mouse C11 mice (5)Weight of the zang (n-8 to 11 animals/group). Giving 10 within eWAT12Analysis was carried out 14 weeks after vg AAV9-CAG-moFGF 21-dimIRT or AAV 9-CAG-empty vector. Results are expressed as mean ± SEM. ND, not detected. HFD, high fat diet. AU, arbitrary unit. eWAT, white adipose tissue of epididymis. iWAT, groin white adipose tissue. rWAT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue. iBAT, interscapular brown adipose tissue. P<0.05 vs. AAV 9-CAG-empty general diet (chow).; p<0.01 vs. AAV 9-CAG-empty general diet<0.001 vs. AAV 9-CAG-empty general diet, $ p<0.05 relative to AAV 9-CAG-null HFD, $ $ p<0.01 vs. AAV 9-CAG-empty HFD, $ $ p<0.01 relative to AAV 9-CAG-null HFD.
FIG. 2 histological analysis of adipose tissue and liver of C57B16 mice treated with AAV9-CAG-MOFGF 21-dimirT vector eWAT. (A) Representative images of epididymal white adipose tissue (eWAT), Inguinal White Adipose Tissue (iWAT), Interscapular Brown Adipose Tissue (iBAT) and liver sections stained with hematoxylin and eosin were treated with C57Bl6 mice fed with plain diet and HFD with AAV9-CAG-moFGF 21-double miRT or AAV 9-CAG-empty vector eWAT. Original magnification x 100. (B) Mean area of white adipocytes in eWAT (n-4 animals/group). (C) Frequency distribution of white adipocyte area of eWAT (n-4 animals/group). Giving 10 within eWAT12Analysis was carried out 14 weeks after vg AAV9-CAG-moFGF 21-dimIRT or AAV 9-CAG-empty vector. Results are expressed as mean ± SEM. HFD, high fat diet. P<0.01 vs. AAV 9-CAG-empty general diet<0.001 vs. AAV 9-CAG-empty general diet, $ $ p<0.01 vs. AAV 9-CAG-empty HFD, $ $ p<0.001 vs. AAV 9-CAG-null HFD.
FIG. 3 increase in energy expenditure and insulin sensitivity in C57B16 mice treated with AAV9-CAG-MOFGF 21-Dual miRT vector eWAT. (A-B) expression levels of UCP1(A) and Dio2 (B). The expression levels of UCP1 and Dio2 in iWAT were measured by RTqPCR and normalized with the Rplp0 value (n-7 animals/group). (C) Energy metabolism. Energy consumption (EE) was measured with an indirect open calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Bright cycle (basal state) and 9 weeks after AAV administrationData were collected during the dark cycle (active phase) and adjusted for body weight (n-8-11 animals/group). (D) Hepatic triglyceride content (n-8 to 10 animals/group). (E-F) serum triglyceride (E) and cholesterol (F) levels (n-8 to 11 animals/group). (G) Intraperitoneal insulin resistance test. Mice were injected intraperitoneally with 0.75U insulin/kg body weight and blood glucose levels were measured at the indicated time points (n ═ 6 to 11 animals/group). Testing was performed 11 weeks after AAV administration. (H) Fasting insulin circulating levels. Unless otherwise stated, 10 is given within the eWAT12Analysis was carried out 14 weeks after vg AAV9-CAG-moFGF 21-dimIRT or AAV 9-CAG-empty vector. Results are expressed as mean ± SEM. HFD, high fat diet. TG, triglycerides. Chol, cholesterol. P<0.05 relative to AAV 9-CAG-empty general diet<0.01 vs. AAV 9-CAG-empty general diet<0.001 vs. AAV 9-CAG-empty general diet, $ p<0.05 relative to AAV 9-CAG-null HFD, $ $ p<0.01 vs. AAV 9-CAG-empty HFD, $ $ p<0.001 vs. AAV 9-CAG-null HFD.
FIG. 4 reversal of obesity in ob/ob mice by intraeWAT administration of AAV8-CAG-moFGF21-dmiRT vector. (A) Expression level of FGF21 in metabolic tissues. Expression levels of eWAT, iWAT, iBAT and murine codon optimized FGF21 coding sequence in liver of ob/ob mice were measured by RTqPCR and normalized with Rplp0 values. (B) Circulating levels of FGF 21. (C-D) body weight (C) and weight gain (D) evolution. Body weight was measured weekly. (E) The weight of the tissue. eWAT, iWAT, rWAT, mWAT, iBAT and liver weight of ob/ob mice treated with AAV vector eWAT. Giving 10 within eWAT10vg、5×1010vg、2×1011vg or 1012vg AAV8-CAG-MOFGF 21-dimirT or 1012Analysis was performed 16 weeks after vg AAV 8-CAG-empty vector. Results are expressed as mean ± SEM. n-7 to 8 animals/group. ND, not detected. AU, arbitrary unit. eWAT, white adipose tissue of epididymis. iWAT, groin white adipose tissue. rWAT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue. iBAT, interscapular brown adipose tissue. P<0.05 vs. AAV 8-CAG-null, ap<0.01 vs. AAV 8-CAG-null<0.001 vs. AAV 8-CAG-null.
FIG. 5 insulin sensitivity enhancement of ob/ob mice treated with AAV8-CAG-MOFGF 21-dimirT vector eWAT. (A) Intraperitoneal insulin resistance test. ob/ob mice were injected intraperitoneally with 0.75U insulin/kg body weight and blood glucose levels were measured at the indicated time points. Testing was performed 9 weeks after AAV administration. (B) Fasting insulin circulating levels 2 months after AAV administration. Results are expressed as mean ± SEM, n-7 to 8 animals/group. P <0.05 relative to AAV 8-CAG-null, # p <0.01 relative to AAV 8-CAG-null, # p <0.001 relative to AAV 8-CAG-null.
Figure 6. reversal of obesity and improvement of glucose metabolism by intravenous administration of AAV8-hAAT-moFGF 21-vector in ob/ob mice (a) schematic of AAV-hAAT-moFGF21 vector expression cassette comprises human α 1-antitrypsin (hAAT) promoter and murine codon optimized FGF21 coding sequence ITR from AAV2 flanked by ITR from the expression cassette not to scale expression levels of pA: polyA. (B) FGF21 expression levels, measurement of expression levels of murine codon optimized FGF21 coding sequence in the liver of ob/ob mice by rtqppcr and normalization of rpp 0 values to (C) circulating levels of FGF 21. (D-E) body weight (C) and weight gain (D) evolution, measurement of body weight per week (F) representative image of animal, (G) weight of tissue, treatment of mice with AAV vector intravenous mouse, treatment of waot, waot t, measurement of insulin resistance per week, measurement of insulin resistance at 0 g.75 g. intraperitoneal injection after intravenous administration of AAV vector11vg or 5X 1011vg AAV8-hAAT-moFGF21 or 5X 1011Analysis was performed 20 weeks after vg AAV 8-hAAT-empty vector. Results are expressed as mean ± SEM. n-9 to 10 animals/group. ND, not detected. AU, arbitrary unit. eWAT, white adipose tissue of epididymis. iWAT, groin white adipose tissue. rWAT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue. iBAT, interscapular brown adipose tissue. P<0.05 vs. AAV 8-hAAT-void,. p<0.01 vs. AAV 8-hAAT-void,. times.p<0.001 vs. AAV 8-hAAT-null.
FIG. 7 Long-term reversal of obesity by intravenous administration of AAV-hAAT-MoFGF21 vector in C57b16 mice fed HFD. (A) Circulating levels of FGF 21. (B-C) body weight (C) and weight gain (D) evolution. Body weight was measured weekly. Administration 10 intravenously10vg or 5X 1010vg AAV8-hAAT-moFGF21 or 5X 1010Analysis was performed 52 weeks after vg AAV 8-hAAT-empty vector. Results are expressed as mean ± SEM, n-9 to 12 animals/group. P<0.001 vs. AAV 8-hAAT-empty general diet, $ $ p<0.01 vs. AAV 8-hAAT-empty HFD, $ $ p<0.001 vs. AAV 8-hAAT-null HFD.
FIG. 8 Long-term increases in energy expenditure and insulin sensitivity of AAV-hAAT-moFGF21 administered intravenously to C57B16 mice fed HFD. (A) Energy metabolism. Energy consumption (EE) was measured with an indirect open calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were collected during light (basal) and dark (active) cycles in 4 weeks after AAV administration and adjusted for body weight. (B) Intraperitoneal insulin resistance test. C57B16 mice were injected intraperitoneally with 0.75U insulin/kg body weight and blood glucose levels were measured at the indicated time points. Testing was performed 7 weeks after AAV administration. (C) Fasting and fed insulin circulating levels. Results are expressed as mean ± SEM, n-9 to 12 animals/group. HFD, high fat diet. P <0.05 relative to AAV 8-hAAT-empty general diet, # p <0.01 relative to AAV 8-hAAT-empty general diet, # p <0.001 relative to AAV 8-hAAT-empty general diet, $ p <0.05 relative to AAV 8-hAAT-empty general diet, $ $ p <0.01 relative to AAV 8-hAAT-empty HFD, $ $ p <0.001 relative to AAV 8-hAAT-empty HFD.
FIG. 9 obesity reversal by intravenous administration of AAV-hAAT-moFGF21 vector in old HFD fed mice. (A) Circulating levels of FGF 21. (B-C) body weight (B) and weight gain (C) evolves. Body weight was measured weekly. Administration 10 intravenously10vg，2×1010vg or 5X 1010vg AAV8-hAAT-moFGF21 or 5X 1010Analysis was performed 21 weeks after vg AAV 8-hAAT-empty vector. Results are expressed as mean ± SEM, n-7 to 8 animals/group. HFD, high fat diet. P<0.05 relative to AAV 8-hAAT-empty HFD, $ p<0.05 $ p relative to AAV 8-hAAT-null HFD<0.01 relative to AAV8-hAAT-empty HFD, $ $ p<0.001 vs. AAV 8-hAAT-null HFD.
FIG. 10 increase in energy expenditure and insulin sensitivity by intravenous administration of AAV-hAAT-moFGF21 vector in old HFD fed mice. (A) Energy metabolism. Energy consumption (EE) was measured with an indirect open calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were collected during light (basal) and dark (active) cycles 6 weeks after AAV administration and adjusted for body weight. (B) Intraperitoneal insulin resistance test. Old C57B16 mice were injected intraperitoneally with 0.75U insulin/kg body weight and blood glucose levels were measured at the indicated time points. Testing was performed 9 weeks after AAV administration. (C) Fasting and fed insulin circulating levels. Results are expressed as mean ± SEM, n-7 to 8 animals/group. HFD, high fat diet. P <0.01 vs AAV 8-hAAT-empty general diet, $ p <0.001 vs AAV 8-hAAT-empty general diet, $ p <0.05 vs AAV 8-hAAT-empty HFD, $ p <0.01 vs AAV 8-hAAT-empty HFD, $ $ p <0.001 vs AAV 8-hAAT-empty HFD.
FIG. 11 weight loss by intramuscular administration of AAV-CMV-MOFGF21 vector in C57B16 mice. (A) Schematic representation of AAV-CMV-moFGF21 vector. The expression cassette comprises a Cytomegalovirus (CMV) promoter and a murine codon-optimized FGF21 coding sequence. The ITRs from AAV2 are flanked by expression cassettes. The schematic drawings are not to scale. pA: and poly A. (B) FGF21 level in circulation. (C-D) body weight (C) and weight gain (D) evolution. Body weight was measured weekly. Results are expressed as mean ± SEM. n-6 to 7 animals per group. P <0.05 relative to AAV 1-CMV-null, p <0.01 relative to AAV 1-CMV-null. According to this legend, the FGF21 label in the figure refers to moFGF 21.
FIG. 12. increase in production of FGF21 protein by codon optimization of the nucleotide sequence encoding human FGF 21. (A) The level of hFGF21 protein 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 is3 wells/set. ND, not detected. P <0.05 vs untransfected cells.
FIG. 13. eWAT in ob/ob mice was administered internally with AAV8-CAG-moFGF21-dmiRT vector.
Representative images of hematoxylin-eosin staining of sections of (a) eWAT and (B) liver tissue obtained from ob/ob animals injected with empty or FGF 21-encoded AAV8 vector within eWAT at all doses tested. Scale bar: the eWAT was 100 μm and the liver 200 μm.
Blood glucose level in C fed state.
D AAV blood insulin in fed state 3 months post.
The FGF21 label in the figure refers to MOFGF 21.
Data information: all values are expressed as mean ± SEM. In (a, B), n is 6 to 9 animals per group. In (C-H), n is 4 to 8 animals per group. In (I), n ═ 6 to 8 animals per group. Relative to the empty-injected group, P <0.05, P <0.01 and P < 0.001.
FIG. 14 Effect of FGF21 gene transfer on eWAT in ob/ob mice.
A4 different doses (1X 10) were injected at 11 weeks of age10、5×1010、2×1011、1×1012vg/mouse) or AAV8-CAG-moFGF21-dmiRT vector at 25 weeks of age in ob/ob animals.
B expression of macrophage marker F4/80 in the same group of animals as (A) was quantified by qRT-PCR.
Representative image of the immunostaining of macrophage-specific marker Mac2 by eWAT sections of ob/ob mice that received AAV8-CAG-moFGF21-dmiRT vector. n is 4 to 8/group. Scale bar: 200 μm.
D liver weights within treatment groups within all ewats.
E, F triglyceride and cholesterol levels in the same group as in (A) fed
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a, B, D), n is 4 to 8 animals per group. Relative to the ob/ob group of empty-injections, P <0.05, P <0.01 and P < 0.001.
FIG. 15 reduction of obesity and increased insulin sensitivity in ob/ob mice treated with AAV8-hAAT-moFGF21 vector.
A is taken from 1X 1011Or 5X 1011Representative images of hematoxylin-eosin staining of eWAT tissue sections of ob/ob animals injected with empty or FGF 21-encoded AAV vector in vg/mouse.
Serum adiponectin levels in all groups B.
C is taken from 1X 1011Or 5X 1011Representative images of hematoxylin-eosin staining of liver tissue sections of ob/ob animals injected with empty or FGF 21-encoded AAV vector in vg/mouse.
D eating blood glucose levels.
Fed serum insulin levels 5 months after E AAV.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all data represent mean ± SEM. In (a-C, E, G-H), n is 9-10 animals per group. P <0.05, P <0.01 and P <0.001 compared to the non-injected ob/ob group.
FIG. 16 Effect of FGF21 liver gene transfer on ob/ob mice.
Immunohistochemistry for macrophage-specific marker Mac2 in eWAT sections of ob/ob mice that received AAV8-hAAT-moFGF21 vector. Scale bar: 500 μm.
B, C expression of the inflammatory markers F4/80(B) and TNF- α (C) in the same group of mice quantified by qRT-PCR.
D, E weight of liver obtained from animals of the same experimental group in (a) (D) and representative image (E).
Fed hepatic triglyceride and cholesterol levels in the same groups as in G (A)
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (B, D-F, H-1), n is 9 to 10 animals per group. Relative to the injection-empty ob/ob group, P <0.05, P <0.01 and P < 0.001.
FIG. 17 AAV8-hAAT-moFGF21 treatment increased expression of genes involved in glucose uptake and thermogenesis in adipose tissue of ob/ob mice.
A, B liver PEPCK and G6Pase expression in ob/ob mice injected with 2 months old AAV 8-hAAT-empty vector or AAV8-hAAT-moFGF21 vector by qRT-PCR.
C, F quantification by qRT-PCR of GLUT1(C), GLUT4(D), HKI (E) and HKII (F) expression in the same animals as in (A)
G relative expression of UCP1 in iBAT in the same group as in (A).
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a-G), n is 9 to 10 animals per group. Relative to the injection-empty ob/ob group, P <0.05, P <0.01 and P < 0.001.
Figure 18 AAV 8-mediated hepatic gene transfer of FGF21 counteracted HFD-induced obesity.
A weight of epididymis (iWAT), Inguinal (iWAT), and intraperitoneal (rWAT) white adipose tissue depots, liver, and quadriceps femoris muscle obtained from mice treated with AAV8-hAAT-moFGF21 vector as young adults (top panel) or adults (bottom panel).
B circulating levels of FGF21 at various time points after vehicle administration.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a-D), n is 7 to 10 animals per group. P <0.05, P <0.01 and P <0.001, relative to the normal diet-fed empty injection group. # P <0.05, # P <0.01 and # P <0.001, relative to the empty injection group fed with HFD. HFD, high fat diet.
FIG. 19 transfer of FGF21 gene to liver counteracted HFD-induced obesity.
Representative images of animals belonging to all experimental groups of the study carried out in young adults (a) or adults (B).
C as representative images of epididymal white fat (eWAT) pads obtained at the time of sacrifice from animals treated with multiple doses of AAV8-hAAT-moFGF21 in young adults (left) or adults (right).
D representative images of liver obtained from animals treated as young adults (left) or adults (right).
Expression of E AAV-derived FGF21 in the liver of animals treated as young adults or adults. qPCR was performed with primers that specifically detect the codon optimized murine FGF21(coFGF21) coding sequence.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (E), n is 7 to 10 animals per group. HFD, high fat diet. ND, not detected.
FIG. 20 AAV8-hAAT-moFGF 21-mediated energy expenditure and decreased fat deposition in iBAT and iWAT.
A, B received HFD feeding since-2 months of age and evaluation of autonomic activity by the open field test in animals treated 2 months later (young adults) with either empty vector or FGF 21-encoding vector.
B haematoxylin-eosin staining of iBAT tissue sections taken from animals as young adult (left) or adult (right).
Western blot analysis of the content of UCP1 in iBAT of the same group of animals as in (A). Representative immunoblots are shown (left). The histograms depict the optical density analysis of two different immunoblots (right).
D haematoxylin-eosin staining taken from iWAT tissue sections taken as young adult (left) or adult (right) animals.
E quantification of Phospho1 expression in iWAT by qRT-PCR in groups of animals starting HFD feeding and receiving FGF21 vector at young or adult age.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a-C), n is 7 to 10 animals per group. In (E), n is 4 animals per group. In (G), n is 7-10 animals per group. P <0.05, P <0.01 and P <0.001, relative to the empty injected group fed on the normal diet. # P <0.05 and # P <0.001, relative to the empty injection group fed HFD. HFD, high fat diet.
FIG. 21 energy expenditure 10 months after gene transfer to liver.
A energy expenditure in HFD-fed animals was initiated at 2 months of age measured 10 months after AAV 8-hAAT-empty or AAV8-hAAT-moFGF21 vector delivery. Data is acquired during light and dark cycles.
B western blot analysis of UCP1 content in iWAT from the same group of animals. Representative immunoblots are shown (left). The figure shows the optical density analysis of two different immunoblots (right).
Relative expression of C Serca2b and RyR2 in iWAT in animal groups starting HFD feeding and receiving FGF21 vector in young or adult adults
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a), n is 7 to 10 animals per group. In (B), n is 4 animals per group. In (C), n is 7 to 10 animals per group. P <0.05, P <0.01 and P <0.001, compared to the empty injected group fed on the normal diet. # P <0.001, relative to the empty injection group fed HFD. HFD, high fat diet.
FIG. 22 reversal of islet proliferation mediated by AAV8-hAAT-moFGF 21.
Fasting glucagon levels in the group of animals that started HFD feeding and received FGF21 carrier after adulthood.
B β cell mass in the group of animals initially fed HFD and received FGF21 vector after adulthood.
C received 5X 10 adult-onset10Representative image of immunostaining for insulin in pancreatic sections of animals of AAV8-hAAT-moFGF21 of vg/mouse. Scale bar: 400 μm. Drawing scale insertion: 100 μm.
D received 5X 10 in young adults (upper panel) or adults (lower panel)10Representative images of double immunostaining for insulin (dark grey) and glucagon (light grey) in pancreatic sections of animals of vg/mouse AAV8-hAAT-moFGF 21. Scale bar: 100 μm.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a-C), n is 7 to 10 animals per group. In (D), n is 4 to 5 animals per group. P <0.05, P <0.01 and P <0.001, compared to the group of empty injections fed with normal diet. # P <0.05, # P <0.01 and # P <0.001, relative to the empty injection group fed HFD. HFD, high fat diet.
FIG. 23 treatment with AAV8-hAAT-moFGF21 improves glucose tolerance.
A glucose tolerance after intraperitoneal injection of glucose (2g/kg body weight) was studied in a group of mice that started HFD feeding and received FGF21 carrier as young adults.
Serum insulin levels during the glucose tolerance test shown in b (a).
Data information: all data represent mean ± SEM. In (a-D), n is 7 to 10 animals per group. P <0.05, P <0.01 and P <0.001, compared to the normal diet-fed empty-injected group. # P <0.05, # P <0.01 and # P <0.001, relative to the empty-injection group fed HFD. HFD, high fat diet.
FIG. 24 reversal of WAT hypertrophy and inflammation by treatment with AAV8-hAAT-moFGF 21.
A was from the diet of normal diet or HFD and was administered AAV 8-hAAT-null or 5X 10 as a young adult (left panel) or adult (right panel)10Representative images of hematoxylin-eosin staining of eWAT in animals of AAV8-hAAT-moFGF21 vector in vg/mouse. Despite HFD feeding, the empty injected mice had larger adipocytes, whereas the adipocytes of the HFD fed FGF21 treated animals were reduced in size. Scale bar: 100 μm.
B as a morphometric analysis of WAT adipocyte area in young adult or adult treated animals.
Circulating levels of C, D adiponectin (C) and leptin (D).
E as adult acceptance 5X 1010Immunohistochemistry for macrophage specific marker Mac2 in eWAT sections of adult animals of AAV8-hAAT-moFGF21 in vg/mouse. Micrographs graphically illustrate the presence of coronaries in the eWAT of HFD-fed, empty injected animals (arrows and inset), but not in the eWAT of HFD-fed FGF 21-treated mice. Ratio ofExample and measurement: 200 μm and 50 μm (inset).
F-H was quantified by qRT-PCR to initiate HFD feeding and as expression of the inflammatory markers F4/80(F), IL1- β (G) and TNF- α (H) in the adult group of animals that received the FGF21 vector.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (B), n is 4 animals per group. In (F-H), n is 7 to 10 animals per group. P <0.05, P <0.01 and P <0.001, relative to the empty injected group fed on the normal diet. # P <0.05, # P <0.01 and # P <0.001, relative to the HFD-fed null-injection group. HFD, high fat diet.
FIG. 25 adipocytes size and inflammation in AAV8-hAAT-moFGF21 treated animals.
A started on the plain diet or HFD and received AAV 8-hAAT-empty or 5X 10 as young adults (top panel) or adults (bottom panel)10Frequency distribution of adipocyte area in animal group of vg/mouse AAV8-hAAT-moFGF21 vector.
B where Mac2 immunohistochemistry was studied in eWAT animals starting from young adults. Arrows indicate the coronary structures formed by infiltration of macrophages in the eWAT of empty-injected mice fed HFD. Scale bar: 200 μm and 50 μm (inset).
C-E relative expression by qRT-PCR of markers for inflammation F4/80, CD68 and TNF- α in the same group of animals as in (B).
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a), n is 4 animals per group. In (C-E), n is 7 to 10 animals per group. P <0.001 vs normal diet-fed empty-injected group. # P <0.001, relative to the empty-injection group fed HFD. HFD, high fat diet.
Figure 26 treatment with a vector encoding FGF21 reversed hepatic steatosis and inflammation.
A is taken from a diet fed with normal diet or HFD and administered AAV 8-hAAT-null or 5X 1010Animal liver section of vg/mouse AAV8-hAAT-moFGF21 vectorRepresentative images of hematoxylin-eosin staining of the sheets. HFD clearly induced lipid droplet deposition in the liver and AAV8-hAAT-moFGF21 treatment reversed this, whether in young adults or adults. Scale bar: 100 μm.
B, C fed hepatic triglyceride and cholesterol levels in the same group of animals.
D received AAV 8-hAAT-null or 5X 1010Macrophage-specific marker Mac-2 immunostaining of liver sections from animals fed HFD of AAV8-hAAT-moFGF21 vector in vg/mouse. Arrows indicate the presence of coronal structures. Scale bar: 200 μm and 50 μm (inset).
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (B-C), n is 7 to 10 animals per group. P <0.01 and P <0.001, compared to the normal diet-fed empty-injected group. # P <0.01 vs. the empty-injected group fed HFD. HFD, high fat diet.
FIG. 27 improvement of liver fibrosis mediated by AAV8-hAAT-moFGF 21.
Receive 5X 1010Analysis of liver fibrosis by Masson trichrome staining in animals fed with AAV 8-hAAT-null or AAV8-hAAT-moFGF21 vector in HFD at vg/mouse. Treatment with AAV8-hAAT-moFGF21 (right panel) significantly reduced the detection of collagen fibers (blue) that could be readily detected in animals treated with empty vector (left panel). Scale bar: 50 μm. According to this legend, the FGF21 label in the figure refers to moFGF 21.
FIG. 28 treatment with AAV8-hAAT-moFGF21 ameliorates liver fibrosis.
A received 5X 1010Analysis of liver fibrosis by Picrorspirius staining in HFD-fed animals of AAV 8-hAAT-null or AAV8-hAAT-moFGF21 vector at vg/mouse. Treatment with AAV8-hAAT-moFGF21 (right panel) significantly reduced the detection of collagen fibers (black) that were readily detectable in the group of animals treated with empty vector (left panel). Scale bar: 50 μm.
B, C expression of collagen 1 in the liver was quantified by qRT-PCR in groups of animals starting HFD feeding and receiving FGF21 vector as young adults (B) or adults (C).
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (B-C), n is 7 to 10 animals per group. P <0.05, P <0.01 and P <0.001 compared to the empty injected group fed on the normal diet. # P <0.05 and # P <0.001, relative to the empty injection group fed HFD. HFD, high fat diet.
FIG. 29 No bone abnormalities were observed in animals treated with AAV8-hAAT-moFGF 21. By young or adult adults at high doses (5X 10)10vg/mouse) AAV8-hAAT-moFGF21 vector treated HFD fed mice were compared to air-injected normal diet or HFD fed animals, and the long-term effects of FGF21 gene transfer on bone were studied.
A nose to anus length.
B tibial length.
Microcomputerized tomography (μ CT) analysis (C-J) and bone analysis (K-O) of the tibia at C-O sacrifice (i.e. at an animal age of 18 months) obtained from HFD-fed mice administered either empty or an AAV vector encoding FGF 21.
P, Q circulating IGFBP1(P) and IGF1(Q) levels determined by ELISA.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all data represent mean ± SEM. In (a, P-Q), n is 7 to 10 animals per group. In (B-0), n is 4 animals per group. P <0.01 and P <0.001, relative to the normal diet-fed empty-injected group. HFD, high fat diet; BMD, bone mineral density; BMC, bone mineral content; BV, bone mass; BV/TV, bone volume/tissue volume ratio; BS/BV, bone surface/bone volume ratio; tb.n, trabecular number; th, trabecular thickness; sp, trabecular detachment.
FIG. 30 analysis of blood glucose distribution in C57B16 mice treated with AAV8-hAAT-moFGF21 vector. Blood glucose levels were evaluated under fed conditions. AAV, IV administration 5X 1010vg or 2X 1011vg of AAV8-hAAT-moFGF21 (n ═ 13 and 15, respectively) or 2X 1011vg AAV 8-empty vector (n 15). STZ, with streptozotocin(5X 50 mg/kg). Results shown are mean + SEM. P<0.05；***p<0.001 vs. AAV 8-hAAT-null. According to this legend, the FGF21 label in the figure refers to moFGF 21.
FIG. 31 Gene transfer of FGF21 to skeletal muscle of healthy animals.
A injection of 3X 10 into skeletal muscle of healthy animals fed on a regular diet11vg/circulating level of FGF21 measured 40 weeks after AAV 1-CMV-empty or AAV1-CMV-moFGF21 vector.
B expression of AAV-derived FGF21 in muscle and liver of healthy animals injected intramuscularly with AAV 1-CMV-null or AAV1-CMV-moFGF21 vector.
C change in body weight over the follow-up period of 40 weeks.
D different wet tissue weights of muscle, fat pad and liver.
E, hepatic triglyceride and cholesterol levels of F in the fed state.
G serum insulin levels fed.
Insulin sensitivity was assessed by intraperitoneal injection of insulin (0.75 units/kg body weight) and expressed as a percentage of initial blood glucose.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a-H), n is 5 to 7 animals per group. Relative to the empty injection group, P <0.05, P <0.01 and P < 0.001.
Figure 32 AAV 1-mediated skeletal muscle gene transfer of FGF21 counteracted HFD-induced obesity and insulin resistance.
Evolution of body weight (A) and body weight gain (B) in animals treated with AAV1-CMV-MoFGF 21. C57B16 mice were fed HFD for about 12 weeks and then given 3X 1011vg/mouse AAV1-CMV-moFGF21 vector. Control obese mice and control plain diet-fed mice received 3X 1011vg AAV 1-CMV-null.
C circulating levels of FGF21 at various time points after vector administration.
D, E fasting plasma glucose (D) and fed serum insulin (E) levels in the same group of animals as (A, B).
Insulin sensitivity was determined in all experimental groups after intraperitoneal injection of insulin (0.75 units/kg body weight). Results were calculated as a percentage of the initial blood glucose level.
According to this legend, the FGF21 label in the figure refers to moFGF 21.
Data information: all values are expressed as mean ± SEM. In (a-F) HFD fed mice, n ═ 10 animals/group; general diet-fed animals n ═ 5 animals per group. P <0.001 was fed to the empty injection group relative to HFD.
FIG. 33. increased circulating levels of FGF21 in vivo by codon optimization of the nucleotide sequence encoding human FGF 21. Circulating levels of hFGF21 in C57B16 mice hydrodynamically administered using a plasmid encoding wild-type hFGF21 or three different variants of codon optimized human FGF21 sequence. Results are expressed as mean ± SEM. n-9 to 10 mice/group. ND, not detected. Negative control, untreated mice. P <0.05 vs untreated mice.
FIG. 34 increased in vitro FGF21 expression levels by hAAT-moFGF21, CAG-moFGF 21-dimIRT and CMV-moFGF21 expression cassettes. (A) Expression levels of FGF21 in HEK293 cells transfected with plasmids encoding the coding sequence of WT murine FGF21 under the control of the EF1a promoter (EF1a-mFGF21) or the coding sequence of WT murine FGF21 under the control of the CMV promoter (CMV-moFGF21) or the CAG promoter in combination with the four tandem repeats of the miRT122a sequence and the four tandem repeats of the miRT1 sequence (CAG-moFGF 21-dimirt). (B and C) intracellular FGF21 protein content (B) and FGF21 protein level in medium (C) in the same cells as in (A). (D) Expression level of FGF21 in C2C12 cells transfected with a plasmid encoding the WT murine FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or the codon optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF 21). (E) Expression level of FGF21 in HepG2 cells transfected with plasmids encoding either the WT murine FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or the codon optimized murine FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF 21). qPCR was performed with primers that detect the wt and codon optimized FGF21 coding sequences. Results are expressed as mean ± SEM. n is3 wells/set. ND, not detected. P <0.05, relative to control. # p <0.001 vs EF1a-mFGF 21.
FIG. 35 in vivo increased circulating levels of hepatic FGF21 and FGF21 by hAAT-moFGF21 and CMV-moFGF21 expression cassettes. (A) Expression levels of FGF21 in the liver of C57B16 mice hydrodynamically administered with plasmids encoding either the WT murine FGF21 coding sequence (EF1a-mFGF21) under the control of the elongation factor 1a (EF1a) promoter or the codon optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21) or hAAT promoter (hAAT-moFGF 21). qPCR was performed with primers that detect the wt and codon optimized FGF21 coding sequences. (B) Circulating levels of FGF21 in the same group as (a). Results are expressed as mean ± SEM. n-5 mice/group. P <0.01 vs EF1a-mFGF 21.
FIG. 36. in vivo hepatic FGF21 expression and circulating levels of FGF21 increased by AAV8-hAAT-moFGF 21. (A) By 1X 1010vg，2×1010vg or 5X 1010vg AAV8 vector encoding WT murine FGF21 coding sequence under the control of elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or AAV8 vector encoding codon optimized murine FGF21 coding sequence under the control of hAAT promoter (AAV8-hAAT-moFGF21) expression levels of FGF21 in the liver of C57B16 mice injected intravenously. qPCR was performed with primers that simultaneously detected the wt and codon optimized FGF21 coding sequences. (B) Circulating levels of FGF21 in the same group as (a). Analysis was performed two weeks after AAV. Results are expressed as mean ± SEM. n-4 to 5 mice/group. Control, untreated mice. P<0.01 and<0.001 vs control. # p<0.01 and # # # p<0.001 relative to AAV8-EF1a-mFGF 21.
FIG. 37. in vivo increased expression of fatty FGF21 by AAV8-CAG-moFGF 21-dmiRT. 2X 10 for (A-B)10vg、5×1010vg or 1X 1011vg of AAV8 vector encoding WT murine FGF21 coding sequence under the control of elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or AAV8 vector encoding codon optimized murine FGF21 coding sequence under the control of CAG promoter binding with four tandem repeats of miRT122a sequence together with four tandem repeats of miRT1 sequence (AAV8-CAG-moFGF 21-double miRT) eWAT injected in C57B16 mouse eWAT(A) Or the expression level of FGF21 in liver (B). qPCR was performed with primers that simultaneously detected the wt and codon optimized FGF21 coding sequences. Analysis was performed two weeks after AAV. Results are expressed as mean ± SEM. n-4 to 5 mice/group. Control, untreated mice. eWAT, white adipose tissue of epididymis. P<0.05，**p<0.01 and
FIG. 38 expression of FGF21 in vivo skeletal muscle by AAV1-CMV-moFGF 21. (A-B) with 5X 1010vg、1×1011vg or 3X 1011vg AAV8 vector encoding WT murine FGF21 coding sequence under the control of elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or AAV1 vector encoding codon optimized murine FGF21 coding sequence under the control of CMV promoter (AAV1-CMV-FGF21) FGF21 expression levels in the quadriceps (a) or liver (B) of C57B16 mice administered intramuscularly. qPCR was performed with primers that simultaneously detected the wt and codon optimized FGF21 coding sequences. Analysis was performed two weeks after AAV. Results are expressed as mean ± SEM. n-4 to 5 mice/group. Control, untreated mice. P<0.05，**p<0.01 and<0.001 vs control. # p<0.05，##p<0.01 and # # # p<0.001 relative to AAV8-EF1a-mFGF 21.
Detailed Description
Examples
General scheme of the various embodiments
Subject characteristics
Male C57Bl/6J mice and B6.V-Lep were usedobOlaHsd (ob/ob) mice. Mice were fed a standard diet (2018S Teklad Global) ad libitum
Figure BDA0002371788690000591
Harlan labs, inc, Madison, WI, US) or a high fat diet (td.88137 Harlan Teklad Madison, WI, US) and maintained under a 12 hour dark cycle (lights on at 8:00 am) and a steady temperature (22 ℃ ± 2). For tissue sampling, mice were treated with isoflurane (Iso) by inhalation of the anesthetic
Figure BDA0002371788690000592
Abbott laboratories, Abbott Park, IL, US) anesthetized and decapitated. Excising the group of interestTissues were kept at-80 ℃ or preserved in formalin until analysis. All experimental protocols were approved by the animal and human experimental ethics committee of the autonomic university of barcelona.
Recombinant AAV vectors
According to standard methods (Ayuso, e.et al, 2010.Curr Gene Ther.10(6)423-36) producing single-stranded AAV vectors of serotype 1,8 or 9 by triple transfection of HEK293 cells. Cells were cultured in 10 roller bottles (850 cm) in DMEM 10% FBS2Flat; corningTMSigma-Aldrich Co., Saint Louis, MO, US) fused up to 80% and co-transfected by the calcium phosphate method with a plasmid with an expression cassette flanked by AAV2 ITRs, helper plasmids of AAV with AAV2 rep and serotype 1,8 or 9cap genes, and a plasmid with adenoviral helper functions the transgenes used were murine, canine or human codon optimized or wt FGF21 coding sequences, four tandem repeats of 1) Cytomegalovirus (CMV) early enhancer/chicken β actin (CAG) promoter and mirT122a sequence (5' CAAACACCATTGTCACACTCCA3') (SEQ ID NO: 12) and four tandem repeats of mirT1 sequence (5' TTACATACTTCTTTACATTCCA3') (SEQ ID NO: 13) cloned in the 3' untranslated region of the expression cassette, 2) CMV promoter, or 3) human α 1-antitrypsin promoter (hAAT) driven by the non-coding sequence of the GeneHA, hAAT or CMV promoter for the production of the non-coding plasmid with the CAG, hAAT, for the precipitation of the AAV, the protein purification of the AAV, the purification of the AAV, the protein using the continuous precipitation and purification of the AAV based on the two steps of the AAV empty DNA.10(6):423-36). The purified AAV vectors were dialyzed against PBS, filtered and stored at-80 ℃. The titer of the viral genome was determined by quantitative PCR following the protocol described for AAV2 with reference to standard substances, using linearized plasmid DNA as a standard curve (Lock M, et al, hum. Gene Ther.2010; 21: 1273-1285). Vectors are constructed according to molecular biology techniques well known in the art.
eWAT in vivo administration of AAV vectors
Mice were anesthetized with intraperitoneal injections of ketamine (100mg/kg) and xylazine (10 mg/kg). Performing laparotomy to expose epididymal whiteColored adipose tissue. AAV vector 0.001%
Figure BDA0002371788690000601
F68(Gibco) was resuspended in PBS and injected directly into the epididymal fat pad. Each epididymal fat pad was injected twice with 50 μ L AAV solution (one injection near the testes and the other injection in the middle of the fat pad). The abdomen was rinsed with sterile saline solution and closed by the double layer method.
Systemic administration of AAV vectors
Adding appropriate amount of AAV solution to 0.001%
Figure BDA0002371788690000602
Diluted in 200 μ L PBS and manually injected into the lateral tail vein without pressure at the time of delivery. Prior to injection, animals were placed under a 250W infrared heating lamp (Philips NV, Amsterdam, NL) for a few minutes to dilate the vessels and facilitate visualization and easier access to the tail vein. Injected animals were fixed using a plastic restraint (Harvard Apparatus, Holliston, MA, US). Anesthesia is not required due to the use of a suitable restriction device. Animals were injected with a30 gauge needle.
Intramuscular administration of AAV vectors
Mice were anesthetized with intraperitoneal injections of ketamine (100mg/kg) and xylazine (10 mg/kg). The hind limbs were shaved and the vehicle was administered by intramuscular injection in six injection sites, with a total of 180 μ Ι _ volume distributed in the quadriceps femoris, gastrocnemius and cranial tibialis of each hind limb.
Immunohistochemistry and morphometric analysis
Tissues were fixed in formalin (Panreac quinimica) for 24 hours, embedded in paraffin, and then sectioned. Tissue samples were stained with hematoxylin-eosin. Adipocyte area was determined in images taken 10-fold using a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) connected to a video camera with a monitor (analysis3.0; Soft Imaging System, Center Valley, PA, EEUU) with image analysis software, and each adipocyte area was measured in μm2And (4) quantifying. Calculate each factThe mean adipocyte area of the group was examined and the distribution of adipocytes according to size class was represented in the histogram. Four animals were used per group and at least 250 adipocytes were analyzed per animal.
Immunohistochemistry
Tissues were fixed in 10% formalin for 12 to 24 hours, embedded in paraffin and sectioned, sections were incubated overnight at 4 ℃ with rat anti-Mac 2(1: 50; CL8942 AP; cedarrane), guinea pig anti-insulin (1: 100; I-8510; Sigma-Aldrich) or rabbit anti-glucagon (1: 100; 219-01; sight Labs), biotinylated rabbit anti-rat (1: 300; E0467; Dako), goat anti-rabbit IgG (Alexa Fluor 568-conjugated) (1: 200; a 11011; ThermoFisher), goat anti-IgG (Alexa Fluor 488-conjugated) (1: 300; a 11073; ThermoFisher) or peroxidase-coupled rabbit anti-guinea pig (1: 300; P0141; Dako) were used as a second generation ABC peroxidase kit (Pierce section) for immunodetection and sections were counterstained with trypsin cells in hoyer sections (hoyer) for total area staining with two areas of insulin cells in a specimen, as described by mass staining with two areas of pancreas in a 10% formalin, total staining with trypsin, calculated by a percentage of area of pancreas cells in a specimen stained with macr-12, macr-aldicar, and counterstaining with total area of pancreas cells in a trichromatic stain with a staining with a.
RNA analysis
Total RNA was obtained from fat banks or liver by using QIAzol lysis reagent (Qiagen NV, Venlo, NL) or Tripure isolation reagent (Roche Diagnostics Corp., Indianapolis, IN, US) and RNeasy Lipid Tissue Minikit (Qiagen NV, Venlo, NL), respectively. To eliminate residual viral genome, total RNA was treated with DNAseI (Qiagen NV, Venlo, NL). For RT-PCR, 1. mu.g of RNA samples were reverse transcribed using the Transcriptor First StrandcDNA Synthesis kit (04379012001, Roche, California, USA). EXPRESS SYBRGreen qPCR supermix (Invitrogen) was usedTMLife Technologies Corp., Carslbad, Calif., US) at Smart
Figure BDA0002371788690000621
Real-time quantitative PCR was performed in (Cepheid, Sunnyvale, USA). The data were normalized with the value of Rplp0 and analyzed as previously described (Pfaffl, M., Nucleic Acids Res.2001; 29(9): e 45).
Hormone and metabolite testing
Using a Glucometer EliteTMThe analyzer (Bayer, Leverkusen, Germany) measures blood glucose levels. Circulating levels of FGF21 murine/rabbit FGF-21ELISA kit by quantitative Sandwich enzyme immunoassay (MF2100, R)&Dsystems, Abingdon, UK). Serum insulin concentrations were determined by a rat insulin ELISA sandwich assay (90010, Crystal chemlinc. downers Grove, IL 60515, usa). To extract lipids from tissues, approximately 100mg of frozen samples were weighed and homogenized in 15mL chloroform methanol (2: 1). Then, by adding 3mL of 0.05% H2SO4And left overnight at 4 ℃ to separate the lipid and aqueous phases. After separation of the phases, the upper aqueous phase was removed using a pasteur pipette and 1mL of the lower lipid phase was recovered in a glass tube. 1mL of chloroform and 1% Triton X-100 solution were added to a glass tube and incubated in a bath at 90 ℃ to evaporate the chloroform. Any residual aqueous phase particles were removed from the lipid phase by using chloroform and Triton X-100 mixture. After evaporation, chloroform was washed onto the tube wall to concentrate the sample and then heated again to 90 ℃ to evaporate the chloroform. Once the pellet was completely dried and concentrated, it was prepared by adding 500. mu. L H at 37 deg.C2The OmiliQ was resuspended. The amount of triglycerides was finally determined using the commercial GPO-PAP (Roche Diagnostics, Basel, Switzerland). Serum triglycerides and cholesterol were quantitatively determined spectrophotometrically using an enzyme assay kit (Horiba-ABX, Montpellier, France). All biochemical parameters were determined using a Pentra 400 analyzer (Horiba-ABX).
Blood glucose was measured using a Glucometer EliteTM (Bayer). Glucagon levels were measured using a glucagon radioimmunoassay (# GL-32K, EMD Millipore). Adiponectin, leptin, IGFBP1, and IGF1 were measured using 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 resistance test
For the insulin resistance test, insulin (0.75IU/kg body weight; Humulin Regular; EliLilly, Indianapolis, IN) was injected intraperitoneally into conscious-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
Conscious mice were fasted overnight (16 hours) and given an intraperitoneal injection of glucose (2g/kg body weight). Blood glucose was measured in tail vein blood samples at the indicated time points. Collecting venous blood from the tail vein at the same time point in test tubes (
Figure BDA0002371788690000631
CB 300, SARSTEDT) and immediately centrifuged off serum, which was used to measure insulin levels.
Blood oxygen saturation
The oxygen consumption and carbon dioxide production in the eight metabolic chambers were monitored simultaneously using an indirect open calorimeter (Oxylet, Panlab, Cornell a, Spain). Mice were individualized and acclimated to the metabolic chambers for 24 hours, and 3 min data was collected in each cage every 15 min for 24 hours. Data were collected from the light and dark cycle and adjusted for body weight. To calculate the energy consumption, the metablism software supplied by the manufacturer was used.
Transfection of HEK293, C2C12 and HepG2 cells
Cells were cultured in 24-well plates according to the manufacturer's instructions (Thermo Fisher Scientific) and transfected using Lipofectamine 2000 at 0.8 μ g DNA/well.
Bone analysis
Bone volume and structure were assessed by μ CT. The mouse tibia was fixed in neutral buffered formalin (10%) and scanned at 27 micron resolution using an elxpore Locus CT scanner (General Electric). At 1mm, 4 mice/group3And proximal tibia of 1.8mm3The trabecular bone is analyzed in the tibial cortical bone. Bone parameters were calculated using a MicroView 3D ImageViewer and an analysis tool. The length of the tibia is measured from the intercondylar eminence to the medial malleolus.
Western blot analysis
iWAT and iBAT were homogenized in QIAzol lysis reagent (Qiagen) and the protein fraction was separated from the organic phase according to the manufacturer's instructions the proteins were separated by 12% SDS-PAGE and analyzed by immunoblotting of rabbit polyclonal anti-UCP 1(ab 10983; Abcam) and rabbit polyclonal anti- α -tubulin (ab 4074; Abcam) antibodies using ECLPlus detection reagent (Amersham Biosciences).
Open field test
Open field tests were performed at 9:00am to 1:00pm as previously reported (Haurigot et al, 2013). Briefly, the animal was placed in the center of a bright chamber (41X 30cm) crossed by two beams (LE 8811; Panlab) detecting horizontal and vertical motion. Exercise and exploration activities were evaluated during the first 6 minutes. The total distance covered was evaluated using a video tracking system (SMARTJunior; Panlab).
Statistical analysis
All values are expressed as mean ± SEM. Differences between the two groups were compared by student t-test. p <0.05 was considered significant.
Examples
Example 1 prevention of obesity and diabetes by En-WAT administration of AAV-CAG-moFGF21-dmiRT vector in C57Bl6 mice
We evaluated the therapeutic potential of AAV-mediated genetic engineering of adipose tissue using FGF21 to prevent obesity and diabetes in 8-week-old males C57B 16. eWAT (eWAT: white adipose tissue of epididymis) 1012AAV9 vector encoding mouse codon optimized FGF21 coding sequence under control of CAG ubiquitous promoter (AAV9-CAG-moFGF 21-double miRT) (fig. 1A) at target sites including miR122 and miR1 of the viral genome (vg) mediated FGF21 fat specific overexpression (fig. 1B) and high secretion of the protein into blood (fig. 1C). With AAV 9-CAG-empty vector (retaining the same infectivity but notVector encoding any transgene) AAV9-CAG-moFGF 21-double miRT treated mice also showed overexpression of FGF21 receptor 1(FGF21R1) in eWAT (fig. 1D) and β -Klotho (FGF21 co-receptor). CAG-moFGF 21-double miRT construct is contained in SEQ ID NO: 32, while CAG-null construct is contained in SEQ ID NO: 31.
Mice fed the normal diet lost weight following AAV-mediated gene transfer of FGF21 to eWAT (fig. 1F and 1G). When challenged with a High Fat Diet (HFD), animals overexpressing FGF21 in adipose tissue remained lean throughout the experiment, while AAV 9-CAG-null-treated mice became obese (fig. 1F and 1G). Based on their lower body weight, mice treated with both normal diet and HFD fed AAV9-CAG-moFGF 21-dimirt showed a reduction in fat pool and liver weight (fig. 1H).
Histological analysis of white adipose tissue by hematoxylin-eosin staining showed a reduction in white adipocyte size in the plurality of multivesicular adipocytes in eWAT and iWAT (iWAT: inguinal white adipose tissue) and iWAT, indicating that the depot had browned (fig. 2A). The morphometric analysis further confirmed that the average area of white adipocytes decreased in AAV9-CAG-moFGF 21-double miRT treated mice (fig. 2B). The frequency distribution of white adipocyte cell area also varied between groups. Mice treated with both normal diet and HFD fed AAV9-CAG-moFGF 21-dimirt showed an increase in the number of small adipocytes and a decrease in the number of large adipocytes (fig. 2C). Notably, the frequency distribution of white adipocyte area in AAV9-CAG-moFGF 21-double miRT-fed mice treated with HFD was nearly identical to that of normal diet-fed AAV 9-CAG-null-treated animals (fig. 2C). Thus, HFD induction of adipocyte hypertrophy observed in AAV 9-CAG-null treated mice was blocked in mice overexpressing FGF 21. Overexpression of UCP1 and Dio2 in iWAT (fig. 3A and 3B) further confirmed browning of iWAT in AAV9-CAG-moFGF 21-double miRT treated mice fed with normal diet and HFD.
Histological analysis of iBAT (iBAT: interscapular brown adipose tissue) compared to AAV 9-CAG-empty mice showed a reduction in lipid accumulation in this depot in AAV9-CAG-moFGF 21-double miRT treated mice fed normal diet and HFD (fig. 2A). According to this result and browning of iWAT, energy expenditure during light and dark cycles was higher in the AAV9-CAG-moFGF 21-double miRT-treated mice fed with HFD (fig. 3C) than in the AAV 9-CAG-empty mice fed with HFD. Taken together, these data indicate that AAV9-CAG-moFGF 21-double miRT treated mice have increased thermogenic activity.
Liver histological sections showed a reduction in lipid accumulation in hepatocytes of mice overexpressing FGF21 compared to AAV 9-CAG-null treated mice on either plain diet or HFD (fig. 2A). Thus, mice treated with AAV9-CAG-moFGF 21-dimirt fed HFD normalized their hepatic content of Triglycerides (TG) (fig. 3D). Meanwhile, circulating levels of TG, total cholesterol, HDL-cholesterol and LDL-cholesterol were normalized in HFD-fed mice overexpressing FGF21 (fig. 3E and 3F).
HFD-fed mice overexpressing FGF21 were more sensitive to insulin than HFD-fed AAV 9-null-treated mice (fig. 3G), and the normal diet and HFD-fed AAV9-CAG-moFGF 21-double miRT-treated mice had decreased circulating levels of insulin compared to their AAV 9-CAG-null-treated counterparts (fig. 3H).
Example 2 Reversal of obesity and improvement of glucose metabolism by En-WAT administration of AAV-CAG-moFGF21-dmiRT vector in ob/ob mice
We evaluated the therapeutic potential of adipose tissue for anti-diabetes and anti-obesity with AAV-mediated genetic engineering of FGF21 in 11-week-old male ob/ob mice, which have leptin signaling abnormalities and are widely used genetic models of obesity and diabetes. For this reason, dose response studies were performed. ob/ob mice were dosed at four different doses (10)10vg，5×1010vg，2×1011vg or 1012vg) AAV8-CAG-moFGF 21-double miRT vector was administered locally in eWAT (fig. 1A). As a control, the ob/ob animals were given 10 in eWAT12vg AAV 8-CAG-empty vector.
The eWAT internal administration of AAV8-CAG-moFGF 21-bis miRT vector mediated specific overexpression of FGF21 in white adipose tissue and high secretion of proteins into the bloodstream in a dose-dependent manner (fig. 4A and 4B). Specifically, the dose is 1012vg AAV8-CAG-moFGF 21-double miRT vector mediated very robust overexpression of FGF21 in eWAT and iWAT (fig. 4A) and achieved the highest circulating FGF21 level (fig. 4B). In contrast, 10 of the lowest dose administration10vgAAV8-CAG-moFGF 21-double miRT vector produced very little overexpression of FGF21 in eWAT only (fig. 4A), and animals treated with this dose had no difference in serum FGF21 levels compared to animals treated with AAV 8-CAG-null (fig. 4B), probably because FGF21 acted in a paracrine-autocrine fashion. Thus, while AAV 8-CAG-null treated animals gradually increased their body weight, animals treated with AAV8-CAG-moFGF 21-bis miRT vector showed a decrease in body weight gain proportional to the dose of vector administered (fig. 4C and 4D). Notably, 10 is used12The animals treated with vg AAV8-CAG-moFGF 21-double miRT vector lost about 15% of their weight in the first two weeks following AAV administration and thereafter gained their weight until they reached the initial weight (fig. 4C and 4D). Thus, 10 was used at the end of the experiment compared to AAV 8-CAG-empty-treated animals12vg AAV8-CAG-moFGF 21-double miRT vector eWAT administered animals showed a difference of 40% of total body weight (fig. 4D). In agreement, use 1012vg AAV8-CAG-moFGF 21-double miRT vector treated animals showed significant fat reduction and a 60% reduction in liver weight (fig. 4E). In this group of mice, iBAT increased in weight (fig. 4E), probably due to increased thermogenic activity.
Mice treated with 5X 10 compared to AAV 8-CAG-null10vg、2×1011vg or 1012vg AAV8-CAG-moFGF 21-double miRT vector treated animals showed improved insulin sensitivity (fig. 5A). By 2X 1011vg or 1012Animals treated with vgAAV8-CAG-MOFGF 21-double miRT vector also showed lower circulating levels of insulin than ob/ob mice treated with AAV 8-CAG-empty vector (FIG. 5B).
Example 3 Reversal of obesity and improvement of glucose metabolism by intravenous administration of AAV-hAAT-moFGF21 vector in ob/ob mice
We also evaluated the anti-diabetic and anti-obesity mediated increase in circulating levels of FGF21 by AAV-mediated genetic engineering of the liver in 8-week-old male ob/ob miceObesity action. Intravenous (IV) administration of 10 to ob/ob mice11vg or 5X 1011vg AAV8 vector encoding murine codon optimized FGF21 coding sequence under control of liver specific human α 1-antitrypsin (hAAT) promoter (AAV8-hAAT-moFGF21) (FIG. 6A.) As a control, ob/ob animals IV were given 5X 1011vg AAV 8-hAAT-empty vector. The hAAT-moFGF21 construct is contained in SEQ ID NO: 34 and the hAAT-null construct is contained in seq id NO: 33, respectively.
Intravenous administration of AAV8-hAAT-moFGF21 vector mediated specific overexpression of FGF21 in the liver and high secretion of this protein into the bloodstream in a dose-dependent manner (fig. 6B and 6C). Specifically, 5 × 1011AAV8-hAAT-moFGF21 vector at vg doses mediated very robust overexpression of FGF21 in the liver (fig. 6B) and reached the highest circulating FGF21 level (fig. 6C). The body weight of animals treated with this dose of AAV8-hAAT-moFGF21 vector decreased by about 7% during the first two weeks following AAV administration, and slightly increased thereafter, whereas AAV 8-hAAT-null treated mice gradually gained body weight (fig. 6D, 6E and 6F). Administration of 1011The body weight gain of mice with vg AAV8-hAAT-moFGF21 vector was significantly lower than that of AAV 8-hAAT-null treated animals (FIGS. 6D, 6E and 6F). Specifically, with 1011The 10% weight gain of the animals treated with vg AAV8-hAAT-moFGF21 vector increased 50% of the AAV 8-hAAT-null animals weight at the end of the experiment (FIG. 6E). Based on their lower body weight, animals overexpressing FGF21 in the liver showed significant fat loss, particularly in those treated with the highest dose of vehicle, a reduction in liver weight of about 60% (fig. 6G). Similar increases in iBAT weight were observed in both groups of mice treated with AAV8-hAAT-moFGF21 (fig. 6G), presumably due to higher thermogenic activity in these animals compared to mice administered AAV 8-hAAT-empty vector.
Animals treated with AAV8-hAAT-moFGF21 vector showed improved insulin sensitivity and decreased circulating levels of insulin compared to AAV 8-hAAT-null treated mice (FIGS. 6H and 6I).
Example 4 Long term reversal of obesity and diabetes in HFD fed mice by intravenous administration of AAV-hAAT-moFGF21 vector
We also assessed the anti-diabetic and anti-obesity effects mediated by increased circulating levels of FGF21 by AAV-mediated genetic engineering in the liver of obese C57B16 mice. Male C57B16 mice (young adults) 9 weeks old were fed HFD for 9 weeks and then injected intravenously with 1010vg or 5X 1010vg of AAV8-hAAT-moFGF21 vector (FIG. 6A). After AAV administration, AAV8-hAAT-moFGF21 treated mice were maintained for 52 weeks of HFD. As a control, IV was given 5X 1010vg AAV 8-hAAT-empty to normal diet and HFD fed C57B16 mice. The mice in the latter two groups were maintained on a regular diet or HFD.
Intravenous administration of AAV8-hAAT-moFGF21 vector mediated high secretion of FGF21 into the bloodstream in a dose-dependent manner in HFD-fed mice (fig. 7A).
AAV 8-null-treated mice on HFD feeding and administration of 1010No weight difference was observed between animals fed HFD with vg AAV8-hAAT-moFGF21 vector (FIGS. 7B and 7C). However, with 5X 1010Animals fed HFD treated with vg AAV8-hAAT-moFGF21 vector initially lost 20% of body weight following AAV administration, and then gained weight gradually, similar to AAV 8-hAAT-empty-treated mice fed with the normal diet (fig. 7B and 7C). Notably, starting at week 9 after AAV administration, at 5 × 1010No statistically significant differences in total body weight and weight gain were observed between the HFD-fed animals given the vg AAV8-hAAT-moFGF21 vector and the normal diet-fed AAV 8-hAAT-empty treated animals (fig. 7B and 7C).
During the light and dark cycles, 5X 10 is used10The energy expenditure in mice fed HFD treated with vg AAV8-hAAT-moFGF21 vector was higher than in mice fed normal diet and HFD treated with AAV 8-hAAT-null (fig. 8A). AAV 8-hAAT-null treated animals on plain diet and HFD feeding and administration of 1010No difference in energy expenditure was observed between mice with vg AAV8-hAAT-MoFGF21 vector (FIG. 8A). Taken together, these data show that the 5 × 10 is used10vg AAV8-hAAT-moFGF21 treated mice have increased thermogenic activity.
Compared to HFD-fed mice given AAV 8-hAAT-empty vector, 10 was used10Animals treated with vg AAV8-hAAT-moFGF21 vector exhibited higher insulin sensitivity, and their insulin sensitivity was similar to that of normal diet-fed mice treated with AAV 8-hAAT-empty vector (fig. 8B). It is evident that 5X 10 administration was compared to mice fed the normal diet given AAV 8-hAAT-empty vector10Animals with vg AAV8-hAAT-moFGF21 vector showed improved insulin sensitivity and normalized circulating levels of insulin (FIGS. 8B and 8C).
Example 5 Reversal of obesity and diabetes by intravenous administration of AAV-hAAT-moFGF21 vector in elderly HFD-fed mice
We also evaluated the anti-diabetic and anti-obesity effects of FGF21 in obese aged (adult) C57B16 mice. Seven and a half-month-old male C57B16 mice were fed HFD for 8 weeks, followed by 10 IV10vg、2×1010vg or 5X 1010vg AAV8-hAAT-moFGF21 vector (FIG. 6A). After AAV administration, AAV8-hAAT-moFGF21 treated mice were maintained with HFD for 22 weeks. As a control, aged C57B16 mice IV fed normal diet and HFD were given 5X 1010vg AAV 8-hAAT-null. The latter two groups of mice were thereafter maintained on a regular diet or HFD.
Intravenous administration of AAV8-hAAT-moFGF21 vector mediated high secretion of FGF21 into the bloodstream in a dose-dependent manner in aged HFD-fed mice (fig. 9A).
AAV 8-null-treated mice on HFD feeding and administration of 1010No body weight differences were observed between animals fed HFD with vg AAV8-hAAT-moFGF21 vector (FIGS. 9B and 9C). However, 2X 10 was used after AAV administration10vg or 5X 1010Animals fed the vector-treated HFD of vg AAV8-hAAT-moFGF21 initially lost 15% and 20% of body weight, respectively (FIGS. 9B and 9C). Thereafter, 2X 1010Animals treated with vg AAV8-hAAT-moFGF21 vector gained weight gradually in analogy to mice treated with AAV 8-hAAT-null diet fed with 5X 1010No significant weight changes were observed in animals treated with vg AAV8-hAAT-moFGF21 vector (fig. 9B and 9C). Notably, starting at week 3 after AAV administration, at 5 × 1010vg AAV8-hAAT-moFGFNo statistically significant differences in total body weight and weight gain were observed between HFD-fed animals given vector 21 and normal diet-fed AAV 8-hAAT-null-treated animals (FIGS. 9B and 9C).
During the light and dark cycles, 5X 10 is used10The energy expenditure in mice fed HFD treated with vg AAV8-hAAT-moFGF21 vector was higher than in normal diet and HFD-fed AAV 8-hAAT-empty mice (fig. 10A). By 2X 1010The vg AAV8-hAAT-moFGF21 vector treated animals showed an increase in energy expenditure during the light cycle and a trend of an increase in energy expenditure during the dark cycle (fig. 10A). By 1010The vg AAV8-hAAT-moFGF21 vector treated animals showed increased energy expenditure during dark cycles (FIG. 10A). AAV 8-hAAT-null treated animals on plain diet and HFD feeding and administration of 1010No difference was observed between mice with vg AAV8-hAAT-moFGF21 vector (FIG. 10A). Taken together, these data indicate that aged mice treated with AAV8-hAAT-moFGF21 have increased thermogenic activity.
Compared to mice fed HFD given AAV 8-hAAT-empty vector, mice fed 10F10vg or 2X 1010Animals treated with vg AAV8-hAAT-moFGF21 vector exhibited higher insulin sensitivity, and their insulin sensitivity was similar to mice fed the normal diet treated with AAV 8-hAAT-empty vector (fig. 10B). Notably, administration of 5X 10 was compared to mice fed the normal diet given AAV 8-hAAT-empty vector10Animals with vg AAV8-hAAT-moFGF21 vector showed improved insulin sensitivity (FIG. 10A). By 1010vg、2×1010vg or 5X 1010The vg AAV8-hAAT-moFGF21 vector treated animals exhibited fasting and fed circulating levels of insulin that were lower than that of HFD-fed AAV 8-hAAT-null treated mice (FIG. 10C). Notably, 2 × 10 was given in IV10vg or 5X 1010No difference in circulating levels of fed insulin was observed between old animals fed vg AAV8-hAAT-moFGF21 vector and normal diet fed AAV 8-hAAT-null treated mice (FIG. 10C).
Example 6 evaluation of weight loss in C57B16 mice by intramuscular administration of AAV-CMV-MoFGF21 vector
We also evaluated the therapeutic potential of C57B16 mice to increase circulating levels of FGF21 through AAV-mediated skeletal muscle genetic engineering. To target skeletal muscle, the CMV promoter and AAV1 serotypes were selected. Although the CMV promoter is a ubiquitous promoter, its use with AAV1 husks can very effectively target skeletal muscle without transduction of the liver, as previously published literature (Mas et al, Diabetes 2006; Callejas et al, Diabetes 2013).
Quadriceps femoris, gastrocnemius and cranial tibialis (5X 10) of each hindlimb of 6 to 12 week-old male C57BL6 mice by intramuscular injection10vg/muscle) to 3X 1011vg doses of AAV1 vector (AAV1-CMV-moFGF21) encoding murine codon-optimized FGF21 coding sequence under control of ubiquitous CMV promoter (fig. 11A). As a control, age-matched C57B16 animals were injected intramuscularly 3X 10 in the same muscle11vg AAV 1-CMV-empty vector (5X 10)10vg/muscle). The CMV-moFGF21 construct is contained in SEQ ID NO: 36 and the CMV-null construct is comprised in SEQ ID NO: 35, respectively.
Intramuscular administration of AAV1-CMV-moFGF21 vector mediated high secretion of FGF21 into the bloodstream (fig. 11B). Animals treated with AAV1-CMV-moFGF21 vector showed reduced body weight and weight gain compared to mice treated with AAV 1-CMV-null (FIGS. 11C and 11D).
Example 7 increase of protein production by codon optimized human FGF21 nucleotide sequence
To evaluate whether codon optimization could mediate increased production of FGF21 protein, HEK293 cells were transfected with plasmids encoding three different codon-optimized human FGF21 nucleotide sequences (SEQ ID NOS: 40-42). As controls, untransfected 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 could secrete higher levels of human FGF21 in the culture medium compared to wild-type or codon-optimized FGF21 variant 2 (fig. 12), thus confirming increased production of FGF21 protein by codon-optimization of variants 1 and 3.
Example 8 reversal of obesity and diabetes in mice by administration of AAV vector encoding human FGF21 (in vivo experiments demonstrating the Activity of FGF21)
HFD-fed mice were treated with AAV vector encoding human FGF 21. As a control, the same dose of AAV-empty vector was administered to mice fed normal diet and HFD.
To evaluate the ability of human FGF21 to induce WAT browning and BAT thermogenic activity, increase energy expenditure, and improve glucose and energy metabolism, the following experiments were performed:
weekly measurement of body weight and food and liquid intake
Body temperature measurement
-measuring energy expenditure and respiratory quotient by indirect calorimetry
-blood glucose measurement
Evaluation of systemic glucose exclusion by an intraperitoneal glucose tolerance test
Evaluation of insulin sensitivity by intraperitoneal insulin resistance test
-analyzing tissue and serum samples, including
Checking the overexpression level of human FGF21 in target tissues and into the 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
Evaluation of browning capacity by examination of the presence of beige adipocytes in the inguinal fat pad by immunohistochemistry and gene expression of the classical white, brown and beige adipocyte markers
Example 9: in vitro assay for evaluating FGF21 Activity
FGF21 is expected to increase glucose uptake and GLUT1 expression in 3T3-L1 cells (kharitonnkov, a.et al, 2005.j.clin.invest115:1627-1635)。
Example 10 reversal of obesity and improvement of glucose metabolism by intraewat administration of AAV8-CAG-moFGF21-dmiRT vector in ob/ob mice: further observations
We further evaluated the therapeutic potential of AAV-mediated adipose tissue genetic engineering of anti-diabetic and anti-obesity in ob/ob mice using FGF21 (see example 2).
Ob/ob mice receiving an interior avacat injection of AAV8-CAG-moFGF21-dmirT vector showed a reduction in white adipocyte size of the epididymal pad (fig. 13A). Circulating adiponectin levels also increased with dose (fig. 14A). The eWAT inflammation assessed by Mac2 staining also decreased as a function of vector dose, as did the expression of macrophage marker F4/80 (fig. 14B and C).
The livers of ob/ob mice injected with empty vector or lowest dose of AAV8-CAG-moFGF21-dmirT showed accumulation of lipid droplets in hepatocytes (FIG. 13B). Administration of 5X 1010The dose of FGF 21-encoding vector of vg/mouse or higher completely prevented the development of hepatic steatosis (fig. 13B), which was related to organ weight (fig. 14D) and its total triglyceride and cholesterol content (fig. 14E and F). 5X 1010Further evidence that the dose of vg/mouse represents a threshold for therapeutic efficacy comes from the analysis of blood glucose and blood insulin. Although 1X 1010The dose of vg/mouse did not alter the blood glucose level in the fed state, but only partially reduced the insulin level, but 5X 1010vg/mouse and higher doses completely normalized blood glucose and blood insulin (fig. 13C and D). In summary, this example demonstrates the therapeutic potential of overexpressing FGF21 in adipose tissue.
Example 11 reversal of obesity and improvement of glucose metabolism by intravenous administration of AAV8-hAAT-MOFGF21 vector in ob/ob mice: further observations
We further evaluated the anti-diabetic and anti-obesity therapeutic potential of intravenous administration of AAV8-hAAT-moFGF21 vector in ob/ob mice (see example 3).
Consistent with their lower body weight, ob/ob animals overexpressing FGF21 in the liver showed a significant reduction in white adipocyte size, particularly receiving 5 x 1011Those animals treated with vg (fig. 15A). This was associated with a dose-dependent increase in circulating adiponectin levels (FIG. 15B) and a decrease in WAT inflammationLow agreement, as evidenced by the reduction in staining by Mac2 and the expression of F4/80 and TNF- α in eWAT (FIGS. 16A-C)11vg-treated ob/ob mice showed a significant reduction in "corona" structure in eWAT (fig. 16A).
Although 7-month-old ob/ob mice exhibited significant hepatic steatosis, the livers of FGF 21-treated ob/ob mice did not exhibit lipid accumulation in hepatocytes (fig. 15C). This is consistent with a 60% reduction in the weight of this organ in ob/ob mice receiving the therapeutic vehicle (fig. 16D and E) and a significant reduction in total hepatic triglyceride and cholesterol levels (fig. 16F and G). Ob/ob animals treated with two doses of AAV8-hAAT-moFGF21 also showed a decrease in fed blood glucose and their blood insulin in the fed state was reduced to 70% (fig. 15D and E).
We assessed by qPCR the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) whether the reduction in circulating glucose levels observed in ob/ob mice following treatment with AAV8-hAAT-moFGF21 was due to inhibition of hepatic gluconeogenesis. No changes in the expression of these enzymes were observed in the liver of ob/ob mice treated with AAV8-hAAT-MoFGF21, but with 1X 1011vg AAV8-hAAT-MOFGF21 treated animals exhibited increased PEPCK expression (FIGS. 17A and B). These results indicate that AAV-mediated long-term expression of intrahepatic FGF21 and subsequent increase in circulating FGF21 did not reduce glucose by inhibiting hepatic glucose production.
The hypoglycemic effect of FGF21 is also attributed to increased glucose uptake and increased energy expenditure by adipocytes (Xu j.et al, 2009.AJP endocrinol.meta.297:E1105–E1114；Ding X.et al.,2012.CellMetab.16:387–393；Camacho R.C.et al.,2013.Eur.J.Pharmacol.715:41–45；EmanuelliB.et al.,2014.Clin.Invest.124:515–527；Kharitonenkov A.et al.,2005.Endocrinology148:774–781；Hondares E.et al.,2010.Cell Metab.11:206–212；Samms R.J.et al.,2015.Cell Rep.11:991-999). Therefore, we evaluated key components of the glucose uptake mechanism such as glucose transporters Glut1 and Glut4, glucose in different adipose tissue pads (iWAT, eWAT and iba) by qPCRExpression of the sugar phosphorylases hexokinase I and II (HKI and HKI) and UCP1 in the case of iBAT. In AAV8-FGF21 treated ob/ob mice, expression of Glut1 was increased in iWAT and iBAT (fig. 17C), and Glut4 was increased in eWAT, iWAT and iBAT (fig. 17D). HKI and HKII were only up-regulated in iBAT (fig. 17E and F). Furthermore, UCP1 expression was increased in iBAT of ob/ob mice treated with high dose AAV8-hAAT-moFGF21 vector (fig. 17G). Taken together, these results indicate that the observed long-term improvement in blood glucose in ob/ob mice after treatment with AAV8-hAAT-moFGF21 vector may be due to increased glucose uptake by white and brown adipocytes and increased heat production in iBAT.
Example 12 Long-term reversal of obesity and diabetes by intravenous administration of AAV8-hAAT-MoFGF21 vector in HFD-fed mice and HFD-fed aged mice: tissue weight reduction and stable expression for up to 1 year
Figures 19A-B show representative images of animals belonging to all experimental groups of studies conducted in young or adult adults (see, examples 4 and 5). In the surrounding young adult or adult-treated animals, obesity reversal by AAV8-hAAT-moFGF21 treatment was consistent with a dose-dependent decrease in the weight of the major White Adipose Tissue (WAT) pool, such as the epididymis (eWAT), Inguinal (iWAT), and retroperitoneal (rWAT) fat pads (fig. 18A and 19C). HFD-induced liver weight gain was completely normalized by FGF21 gene transfer at the highest dose of the vector used, while the weight of the quadriceps femoris was unchanged under diet or AAV delivery (fig. 18A and fig. 19D).
AAV8-hAAT-moFGF21 treated mice of both ages showed specific overexpression of codon optimized FGF21 in liver (fig. 19E), which resulted in dose dependent secretion of FGF21 into the bloodstream in both groups of mice, while the levels remained stable for up to 1 year following a single administration of the vector (fig. 18B).
Example 13 Long-term reversal of obesity and diabetes by intravenous administration of AAV8-hAAT-MoFGF21 vector in HFD-fed mice and HFD-fed aged mice: increase in exercise capacity and study of heat generation mechanism
Increased energy expenditure was also observed in animals treated as young adults 10 months after delivery of AAV8-hAAT-moFGF21 (fig. 21A) (see, examples 4 and 5).
This observation is consistent with the effect on locomotor activity mediated by AAV8-hAAT-moFGF 21. In contrast to the hypofunction observed in open field testing of animals fed HFD receiving AAV 8-empty vector, 5X 10 as a young adult10vg AAV8-hAAT-moFGF21 treated mice exhibited the same degree of spontaneous locomotor ability as empty injected animals fed the normal diet. As shown in figure 20A, 1 year after delivery of AAV8-hAAT-moFGF21, treated animals walked farther, had less time to rest, and took more time to perform slow and fast movements compared to untreated HFD-fed controls.
Given that changes in energy expenditure may reflect changes in thermogenesis, we evaluated the degree of activation of Brown Adipose Tissue (BAT). As adult light or adult 5X 1010Both vg AAV8-hAAT-moFGF21 treated mice showed reduced lipid deposition in iBAT (fig. 20B). In mice treated with AAV8-hAAT-moFGF21 vector as a young adult, the amount of UCP1 protein in BAT increased in a dose-dependent manner (fig. 20C), which was associated with increased non-tremor thermogenesis due to FGF21 gene transfer to liver.
Subcutaneous WAT browning, characterized by the appearance of beige adipocytes, was also associated with increased energy expenditure (Harms & seal, 2013). To assess whether browning could be due to the increase in energy expenditure observed after treatment with AAV8-hAAT-moFGF21, a histological evaluation of iWAT was performed. Consistent with the weight loss of the pad (fig. 18A), the adipocytes of the HFD-fed AAV8-hAAT-moFGF 21-treated animals were smaller than those of the HFD-fed, empty-injected animals (fig. 20D). However, treatment with AAV8-hAAT-moFGF21 vector did not result in increased detection of multi-limbed adipocytes in iWAT at any of the doses tested, whether as a young adult or adult animal (fig. 20D). Thus, there were no statistically significant differences in UCP1 protein levels in iWAT between HFD feeding groups (fig. 21B).
Creatine driven substrate circulation and muscular/endoplasmic reticulum (sarco/endoplasmic reticulum) Ca2+Calcium cycling mediated by ATPase 2b (Serca2b)Thermogenesis in iWAT can be enhanced independently of UCP1 (Kazak l.et al, 2015. Cell)163:643–655；Ikeda K.et al.,2017.Nat.Med.23:1454-1465). In comparison with age-matched normal diet and HFD fed control group (FIG. 20E), 5X 10 was used10Higher expression levels of the phosphatase orphan 1(Phospho 1), an enzyme involved in creatine-driven substrate cycling, were observed in iWAT fed mice with vg AAV8-hAAT-moFGF21 treated HFD, indicating that creatine-driven cycling activity may be increased due to FGF21 gene transfer. Regarding the calcium cycle-dependent thermogenesis mechanism, no difference in the level of Serca2b expression was detected in iWAT of animals treated with AAV8-hAAT-moFGF21 vector compared to normal diet or HFD fed null-treated animals (fig. 21C). In another aspect, increased iWAT expression of ryanodine receptor 2(RyR2), another enzyme involved in the same cycle, was increased by HFD feeding in mice treated with null and AAV8-hAAT-moFGF21 (fig. 21C). Taken together, these results indicate that the calcium cycle-dependent mechanism of thermogenesis does not participate in the improvement in systemic energy homeostasis observed after AAV-FGF21 treatment.
Example 14. reversal of obesity and long-term diabetes by intravenous administration of AAV8-hAAT-moFGF21 vector in HFD-fed mice and HFD-fed aged mice: glucagon levels, islet hyperplasia and glucose tolerance
In addition, HFD-fed animals as young adults treated with AAV8-hAAT-moFGF21 vector exhibited decreased glucagon circulating levels compared to HFD-fed null-treated mice (fig. 22A).
AAV8-hAAT-FGF21 vector (2X 10) although AAV 8-null-treated mice developed islet proliferation due to HFD feeding10Or 5X 1010Dose of vg/mouse) the β cell mass of treated animals was similar to that of normal diet fed control mice (fig. 22B and C). double immunostaining of insulin and glucagon from pancreas sections of AAV8-hAAT-moFGF 21-treated mice fed with HFD indicated that α and β cells were normally distributed in the islets of the animals, with glucagon-expressing cells surrounding insulin-expressing cells in the islets and nuclei (fig. 22D).
For evaluation of treatment with FGF21Was subjected to intraperitoneal Glucose Tolerance Test (GTT) (2g glucose/kg bw) 10 weeks after AAV administration. By air-injection or at 1X 1010Dose vg/mouse animals fed HFD injected with FGF 21-encoding vector were glucose intolerant and showed significant increases in circulating insulin levels during GTT (fig. 23A and B). In contrast, 5X 10 mice were fed on a regular diet compared to control mice10The AAV8-hAAT-moFGF21 treated animals at vg/mouse showed improved glucose clearance (fig. 23A). There was no difference in insulin levels between the two experimental groups (fig. 23B). These results further confirm the use of 5X 1010vg/mouse insulin sensitivity was improved in mice fed with AAV8-hAAT-moFGF21 treated HFD.
Example 15 Reversal of WAT hypertrophy and inflammation associated with HFD by intravenous administration of AAV8-hAAT-MoFGF21 vector
HFD feeding resulted in an increase in WAT adipocyte size (Sattar N.&Gill j.m.r., 2014. bmcmed.12: 123). Administration of the vector encoding FGF21 counteracted this increase (fig. 24A). Morphometric analysis of WAT showed that the use of 1X 1010Or 5X 1010vg vector as a young adult treated animal and 2X 1010Or 5X 1010The white adipocyte area of the vg vehicle treated mice as adults was similar to that of animals fed the normal diet (fig. 24B). In both groups of FGF 21-treated animals, there was a redistribution of adipocyte size with a greater proportion of smaller adipocytes (fig. 25A). Consistent with the reduction in obesity and reversal of WAT hypertrophy, adiponectin and leptin levels were also normalized in animals treated with the highest dose of AAV8-hAAT-moFGF21 vector, regardless of the age at which treatment began (fig. 24C and D).
Obesity also causes inflammation of WAT (Sattar N.&Gill J.M.R.,2014.BMC Med.12:123). We therefore analyzed inflammation in this tissue by immunostaining for macrophage-specific marker Mac2 and expression of pro-inflammatory molecules. Although HFD-fed mice show an increased presence of macrophages, as indicated by a "coronal" structure, in eWAT, 5X 10 is used10Vg AAV8-hAAT-moFGF 21-treated animals as young adults or adults had no evidence of macrophage infiltration (Fig. 24E and 25B), consistent with the expression of macrophage markers F480 and CD68 and the proinflammatory cytokines TNF α and IL-1 β (fig. 24F-H and 25C-E), indicating that FGF21 expression counteracts the obesity-associated inflammation of WAT.
EXAMPLE 16 reversing hepatic steatosis, inflammation and fibrosis by intravenous administration of AAV8-hAAT-MoFGF21 vector
Histological analysis of the liver showed that all of the animals left free to be treated with HFD had significant hepatic steatosis at sacrifice (fig. 26A-D). In contrast, 5X 10 was accepted as a young or adult10HFD-fed mice of vg AAV8-hAAT-moFGF21 demonstrated this reversal of pathological lipid deposition (fig. 26A). These histological findings were combined with 5X 1010The significant reduction in total triglyceride and cholesterol levels was consistent in the vgAAV8-hAAT-moFGF21 treated animals (fig. 26B and C). In addition, HFD was fed and administered at 5 x 10 when young adults or adults showed no signs of liver inflammation (as evidenced by the lack of Mac2 staining)10vg AAV8-hAAT-moFGF21 vector treated animals revealed increased presence of macrophages in the liver of mice fed empty-treated HFD (fig. 26D). Finally, the transfer of FGF21 gene to the liver reverses liver fibrosis. Collagen fibers were readily detected after picroririus Red staining or Masson trichrome staining of liver sections from animals fed HFD and injected with control empty vector, but not in the livers of AAV8-hAAT-moFGF21 treated mice (fig. 28A and fig. 27). These mice also showed a significant reduction in liver expression of collagen 1 (fig. 28B and C). Taken together, these findings indicate that treatment with AAV8-hAAT-moFGF21 can prevent the development of HFD-induced nonalcoholic steatohepatitis (NASH).
Example 17 Long term safety of liver Targeted AAV-FGF21 treatment
Pharmacological treatment of FGF21 or transgene overexpression is associated with interference of skeletal homeostasis by increasing bone resorption, which may lead to bone loss (Wei w.et al, 2012. proc.natl.acad.sci).109:3143–3148；Wang X.etal.,2015.Cell Metab.22:811–824；Charoenphandhu N.et al.,2017.J.BoneMiner.Metab.35:142–149；Talukdar S.et al.,2016.Cell Metab.23427-440; kima.m.et al, 2017.Diabetes, obes.meta). In view of the therapeutic potential of AAV8-hAAT-moFGF21 for the treatment of obesity and diabetes, we evaluated the long-term effects of gene transfer on the bones of animals treated with the highest dose of vector. Animals given AAV8-hAAT-moFGF21 vector at sacrifice (approximately 16.5 months of age), 9 or 29 weeks of age had normal nasal-anal and tibial lengths (fig. 29A and B). Then, we examined the bone structure by micro computed tomography (μ CT). Analysis of proximal tibial epiphyses showed that HFD was fed and administered 5 x 10 compared to age-matched mice treated with empty vehicle10There was no significant difference in trabecular and cortical bone in the mice of vgAAV8-hAAT-MoFGF 21. Specifically, no difference was recorded in 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 thickness (tb.th) (fig. 29I) or trabecular separation (tb.sp) (fig. 29J). Similarly, analysis of dense bone at the tibial stem bone showed no difference in BMC, BMD, BV/TV or BS/BV between the HFD-fed, empty-injected or FGF 21-treated groups (fig. 29K-O).
The pathological effects of FGF21 have been reported to be mediated, at least in part, by the liver increasing production of insulin-like growth factor binding protein 1(IGFPB1) (Wang x.et al, 2015.Cell Metab.22:811-824). Consistent with the absence of skeletal alterations, high dose AAV8-hAAT-moFGF21 treatment did not result in increased circulating IGFBP1 protein levels in animals treated 12 months earlier (young adults) or 6 months earlier (adults) compared to HFD-fed mice by empty injection (fig. 29P). Circulating IGF1 levels were also normal in all experimental groups (fig. 29Q). Taken together, these results support the safety of AAV-mediated transfer of FGF21 gene to liver for skeletal tissue.
Example 18 prevention of HFD-induced liver tumors by intravenous administration of AAV8-hAAT-moFGF21 vector
Long-term feeding HFD (>60 weeks) was associated with an increased incidence of liver tumors in C57BL/6J mice (Hill-baskin a.e.et al, 2009.hum.mol.genet.18:2975–2988；Nakagawa H.,2015.WorldJ.Hepatol.7:2110). In our countryIn the study of (1), as an animal starting HFD in a young adult and maintaining it for 60 weeks, we found 66.7% (6/9) with liver tumors in empty vector injected animals. Animals treated with AAV8-hAAT-moFGF21 vector were protected from HFD-induced liver tumor development: 5X 10100% (0/8) of the animals treated with vg FGF 21-encoding vector exhibited tumors, while the lowest dose (1X 10) was used10vg) was 40% (4/10). Mice fed without (0/11) the normal diet developed tumors during the same time period (table 1).
TABLE 1 incidence of liver tumors in young adults.
Group of Liver cancer Liver cancer (%)
Common diet AAV 8-empty 0/11 0％
HFD AAV 8-null 6/9 66.7％
HFD AAV8-FGF21(1×1010vg/mouse) 4/10 40％
HFD AAV8-FGF21(5×1010vg/mouse) 0/8 0％
Example 19 amelioration of STZ-induced hyperglycemia by liver-specific AAV 8-mediated FGF21 overexpression
Materials and methods
Animal(s) production
We used 9-week-old male C57bl6 mice. Mice were free to eat a standard diet and kept on a 12 hour light-dark cycle (lit at 08: 00). To induce diabetes, mice received five intraperitoneal injections of streptozotocin (50mg/kg) dissolved in 0.1mol/L citrate buffer (pH 4.5) for five consecutive days. Blood glucose levels were assessed using an analyzer (glucometertelite; Bayer, Leverkusen, Germany). Animal care and experimental protocols were approved by the institutional Animal and human laboratory Committee of the university of Barcelona (Ethics Committee in Animal and human experimentation of the university of the Barcelona).
In vivo administration of AAV vectors
For systemic administration, AAV vectors were diluted in 200. mu.L of 0.001% F68
Figure BDA0002371788690000801
(Gibco) in PBS and injected through the tail vein.
Results
To test the protective potential of AAV-derived FGF21 for type 1 diabetes, 5 × 10 was used10vg or 2X 1011vg of AAV8 vector encoding codon optimized murine FGF21 coding sequence under the control of hAAT promoter (AAV8-hAAT-moFGF21) IV was administered to male 9 week old C57B16 mice. Control mice received 2X 1011vg AAV 8-hAAT-empty vector. Two weeks after AAV administration, all animals were treated with Streptozotocin (STZ) (50mg/kg at 5 doses; 1 dose per day) to trigger the course of diabetes.
Analysis of blood glucose levels showed that animals treated with AAV8 vector encoding moFGF21 exhibited lower circulating glucose levels than C57B16 mice treated with AAV 8-hAAT-empty vector (fig. 30).
Example 20 the health life was extended by intramuscular administration of AAV-CMV-moFGF21 vector in C57B16 mice due to prevention of weight gain and age-related insulin resistance.
Skeletal muscle (Skm) is a readily available tissue and has been used to produce secretable therapeutic proteins (haurigotot v.et al, 2010. j.clin.invest).123:3254–3271；Callejas D.et al.,2013.Diabetes62:1718–1729；Jaén M.L.et al.,2017.Mol.Ther.Methods Clin.Dev.6:1-7). To explore whether Skm could represent a viable source of circulating FGF21, an AAV vector of serotype 1(AAV 1-CMV-moFGF21) was used, which showed high Skm tropism (Chao l.et al, 2000.j.clin.invest.106:1221–1228；Wu Z.et al.,2006.J.Virol.80:9093–9103；Lisowski L.et al.,2015.Curr.Opin.Pharmacol.2459-67), carrying murine optimized FGF21 under the control of the CMV promoter. The vector is added at 5X 1010Vg/muscle dose was injected into quadriceps, gastrocnemius and cranial tibialis muscles of both legs of 8-week-old C57B16 mice (total dose was 3X 10)11vg/mouse). Control animals were injected with the same dose of AAV 1-CMV-empty vector. The use of healthy mice fed a standard diet further allows us to evaluate the long-term safety of FGF21 gene therapy.
11-month-old animals injected with FGF 21-encoding vector at 8 weeks of age showed a significant increase in circulating FGF21 (fig. 31A), consistent with high expression levels of vector-derived FGF21 in 3 injected muscles (fig. 31B). Consistent with previous reports, this combination of vector serotype, promoter, and route of administration did not result in transgene expression in liver (fig. 31B).
At the end of the-10 month follow-up period, mice intramuscularly injected with AAV1-CMV-moFGF21 maintained the body weight at the beginning of the study, leaner to 38% to the control group, and steadily increased in body weight as the animals grew in age (fig. 31C). Although muscle weight was hardly affected by FGF21 gene transfer, the white and brown fat pools and the weight of liver were greatly reduced (fig. 31D). In fact, the weight of the analyzed WAT pad was reduced by > 50% (fig. 31D). In addition, mice treated with AAV1-CMV-moFGF21 showed a significant reduction in liver total triglyceride content (fig. 31E). No change in liver cholesterol levels was observed (fig. 31F). In contrast to the empty injected animals, animals treated with AAV1-CMV-moFGF21 exhibited normoglycemia (data not shown) and decreased blood insulin by about 1 year of age (fig. 31G). Thus, FGF 21-treated mice showed significantly improved insulin sensitivity at the end of the study (fig. 31H). In summary, this study demonstrates that AAV vector administration resulting in treatment-related levels of circulating FGF21 is safe in long-term health and can be used to reverse body weight gain and aging-related insulin resistance.
Example 21 reversal of obesity and diabetes by intramuscular administration of AAV1-CMV-moFGF21 vector in HFD fed C57B16 mice.
We next evaluated if im administration of AAV1-CMV-MOFGF21 vector can also reverse obesity and insulin resistance. For this purpose, two month old C57B16 mice were fed either plain diet or HFD for 12 weeks. During the first three months of follow-up, HFD-fed animals became obese (95% weight gain) despite a 20% weight gain in the normal diet-fed animals (fig. 32A and B). The vector was then incubated at 5X 1010Vg/muscle dose was injected into quadriceps, gastrocnemius and cranial tibialis of both thighs of obese C57B16 mice (total dose 3X 1011vg/mouse). As a control, another group of obese mice and normal diet-fed mice received 3X 1011vg non-coding empty vector (AAV 1-CMV-empty). Following AAV delivery, mice were kept on a normal diet or HFD feeding. Animals treated with AAV1-CMV-moFGF21 experienced a gradual loss of body weight (fig. 32A and B). Reversal of obesity by treatment with AAV1-CMV-FGF21 was consistent with increased circulating levels of FGF21 (fig. 32C).
The HFD-fed null-treated mice exhibited normal fed blood glucose (fig. 32D), but had hyperinsulinemia (fig. 32E), indicating that these mice have developed insulin resistance. In contrast, at the end of the study, the HFD treated with AAV1-CMV-moFGF21 was fed to mice but was normoglycemic and normoglycemic (fig. 32D and E). In addition, animals administered AAV1-CMV-moFGF21 exhibited higher insulin sensitivity compared to their HFD feeding controls (fig. 32F).
Example 22 circulating levels of FGF21 were increased by codon-optimized human FGF21 nucleotide sequence.
To evaluate whether codon optimization could mediate an increase in circulating levels of FGF21, 8-week-old male C57B16 mice were hydrodynamically injected with plasmids encoding three different codon-optimized human FGF21 nucleotide sequences (SEQ ID NOS: 40-42) under the control of the hAAT promoter. As controls, null-treated 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
Delivery of plasmids into mice by hydrodynamic tail vein injection
Plasmid DNA was diluted in saline at a volume (mL) of up to 10% of the average animal body weight (grams) and manually injected into the lateral tail vein in less than 5 seconds. Prior to injection, the animals were placed under a 250W infrared heating lamp (Philips) for a few minutes to dilate the vessels for easy visualization and easier access to the tail vein. The injected animals were fixed using a plastic restraint (Harvard Apparatus). Anesthesia is not necessary because of the use of appropriate fixation devices. We used a 26G 3/8 inch gauge hypodermic needle (BD), which is the largest needle gauge available for injecting animals against the vein contacted.
Results
Codon-optimized human FGF21, version 2 or 3 treated mice secreted 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 FGF21 expression and protein production levels were increased in vitro and in vivo by hAAT-moFGF21, CAG-moFGF 21-dimRT and CMV-moFGF21 expression cassettes
Materials and methods
Delivery of plasmids into mice by hydrodynamic tail vein injection
Plasmid DNA was diluted in saline at a volume (mL) of up to 10% of the average animal body weight (grams) and manually injected into the lateral tail vein in less than 5 seconds. Prior to injection, the animals were placed under a 250W infrared heating lamp (Philips) for a few minutes to dilate the vessels for easy visualization and easier access to the tail vein. The injected animals were fixed using a plastic restraint (Harvard Apparatus). Anesthesia is not necessary because of the use of appropriate fixation devices. We used a 26G 3/8 inch gauge hypodermic needle (BD), which is the largest needle gauge available for injecting animals against the vein contacted.
Results
In vitro
HEK293 cells were transfected with plasmids encoding the WT murine FGF21 coding sequence (EF1a-mFGF21) under the control of the elongation factor 1a (EF1a) promoter (Zhang et al, EBioMedine 15(2017)173-183) (SEQ ID NO: 57) or the codon optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21) or the codon optimized murine FGF21 coding sequence under the control of the CAG promoter in combination with four cascade repeats of the mirT122a sequence (CAG-moFGF 21-double mirT). As a control, untransfected cells were used. HEK293 cells transduced with CAG-moFGF 21-bis miRT expressed higher levels of FGF21 compared to cells transduced with EF1a-mFGF21 or untransduced cells (fig. 34A). Furthermore, HEK293 cells transduced with CAG-moFGF 21-bis miRT also showed higher intracellular FGF21 protein content and higher FGF21 protein levels in culture (fig. 34B and C). While HEK293 cells transduced with EF1a-mFGF21 or CMV-moFGF21 expressed similar FGF21 levels (fig. 34A), HEK293 cells transduced with CMV-moFGF21 showed higher intracellular FGF21 protein content and higher FGF21 protein levels in culture (fig. 34B and C).
C2C12 cells were transfected with plasmids encoding the WT murine FGF21 coding sequence (EF1a-mFGF21) (Zhang et al, EBiomedicine 15(2017)173-183) under the control of the EF1a promoter or the codon-optimized murine FGF21 coding sequence (CMV-moFGF21) under the control of the CMV promoter. As a control, untransfected cells were used. C2C12 cells transduced with CMV-moFGF21 expressed higher levels of FGF21 compared to cells transduced with EF1a-mFGF21 or untransduced cells (fig. 34D).
HepG2 cells were transfected with plasmids encoding the WT murine FGF21 coding sequence (EF1a-mFGF21) (Zhang et al, EBioMedicine 15(2017)173-183) under the control of the EF1a promoter or the codon optimized murine FGF21 coding sequence (hAAT-moFGF21) under the control of the hAAT promoter. As a control, untransfected cells were used. HepG2 cells transduced with hAAT-moFGF21 expressed higher levels of FGF21 compared to cells transduced with EF1a-mFGF21 or untransduced cells (fig. 34E).
In vivo
8 week old male C57Bl6 mice were hydrodynamically administered 5 μ g of a codon optimized murine FGF21 coding sequence either under the control of the elongation factor 1a (EF1a) promoter (EF1a-mFGF21) (Zhang et al, EBiomedicine 15(2017) 173-) 183) or under the control of a CMV promoter (CMV-moFGF21) or under the control of an hAAT promoter (hAAT-moFGF 21). Analysis of the expression level of FGF21 in the liver 24 hours after plasmid administration showed that animals treated with hAAT-moFGF21 or CMV-moFGF21 expressed much higher levels of FGF21 compared to animals receiving EF1a-mFGF21 (fig. 35A). In addition, animals treated with hAAT-moFGF21 or CMV-moFGF21 showed higher circulating levels of FGF21 than animals receiving EF1a-mFGF21 (FIG. 35B).
Example 24 AAV8-hAAT-moFGF21, AAV8-CAG-moFGF 21-dimIRT and AAV1-CMC-moFGF21 increase FGF21 expression and FGF21 circulating levels in target tissues in vivo compared to AAV8-Ef1a-mFGF 21.
Hepatic expression
Intravenous administration of 1X 10 to C57B16 Male mice10vg、2×1010vg or 5X 1010vg an AAV8 vector encoding the WT murine FGF21 coding sequence (AAV8-EF1a-mFGF21) under the control of the elongation factor 1a (EF1a) promoter or the codon optimized mouse FGF21 coding sequence (AAV8-hAAT-moFGF21) under the control of a liver-specific hAAT promoter. Two weeks after AAV administration, animals treated with AAV8-hAAT-moFGF21 showed higher expression levels of FGF21 and higher circulating levels of FGF21 in the liver than animals treated with AAV8-EF1a-mFGF21, regardless of vector dose (fig. 36A and fig. 36 AB).
Fat expression
2X 10 In-Male C57Bl6 mice eWAT10vg，5×1010vg or 1X 1011vg AAV8 vector encoding WT murine FGF21 coding sequence under the control of elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or binding of CAG promoter to four stages of mirT122a sequenceAAV8 vector (AAV8-CAG-moFGF 21-double miRT) encoding a codon optimized murine FGF21 coding sequence under the control of four tandem repeats of the tandem repeat and miRT1 sequences. Two weeks after AAV administration, AAV8-CAG-moFGF 21-double miRT treated animals showed higher expression levels of FGF21 in WAT compared to animals administered AAV8-EF1a-mFGF21 (fig. 37A). Furthermore, FGF21 expression in the liver was much lower in animals treated with AAV8-CAG-moFGF 21-dimirt than in animals administered with AAV8-EF1a-mFGF21 (fig. 37B), confirming that the intra-eWAT administration of AAV8-CAG-moFGF 21-dimirt vector effectively excluded expression of the transgene in off-target tissues.
Skeletal muscle expression
Male C57Bl6 mice were injected intramuscularly at 5X 1010vg、1×1011vg or 3X 1011vg an AAV8 vector encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or an AAV1 vector encoding the codon optimized murine FGF21 coding sequence under the control of the CMV promoter (AAV1-CMV-FGF 21). Two weeks after AAV administration, animals treated with AAV1-CMV-FGF21 had much higher expression levels of FGF21 in skeletal muscle than animals administered AAV8-EF1a-mFGF21 (fig. 38A). Furthermore, animals treated with AAV8-EF1a-mFGF21 showed high expression of FGF21 in the liver, whereas intramuscular administration of AAV1-CMV-FGF21 vector effectively abolished liver transgene expression (fig. 38B).
Sequence of
Figure BDA0002371788690000861
Figure BDA0002371788690000871
Figure BDA0002371788690000881
Amino acid sequence of homo sapiens FGF21 (SEQ ID NO: 1)
MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS
Nucleotide sequence of homo sapiens FGF21 (SEQ ID NO: 4)
ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGCTACGCTTCCTGA
Codon-optimized nucleotide sequence of homo sapiens FGF21 variant 1(SEQ ID NO: 5)
ATGGATTCTGATGAGACAGGCTTCGAGCACAGCGGCCTGTGGGTTTCAGTTCTGGCTGGACTGCTGCTGGGAGCCTGTCAGGCACACCCTATTCCAGATAGCAGCCCTCTGCTGCAGTTCGGCGGACAAGTGCGGCAGAGATACCTGTACACCGACGACGCCCAGCAGACAGAAGCCCACCTGGAAATCAGAGAGGATGGCACAGTTGGCGGAGCCGCCGATCAGTCTCCTGAATCTCTGCTCCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAAACCAGCCGGTTCCTGTGCCAAAGACCTGACGGCGCCCTGTATGGCAGCCTGCACTTTGATCCTGAGGCCTGCAGCTTCAGAGAGCTGCTGCTTGAGGACGGCTACAACGTGTACCAGTCTGAGGCCCATGGCCTGCCTCTGCATCTGCCTGGAAACAAGAGCCCTCACAGAGATCCCGCTCCTAGAGGCCCTGCCAGATTTCTGCCTCTTCCTGGATTGCCTCCTGCTCTGCCAGAGCCTCCTGGAATTCTGGCTCCTCAGCCTCCTGATGTGGGCAGCTCTGATCCTCTGAGCATGGTCGGACCTAGCCAGGGCAGATCTCCTAGCTACGCCTCTTGA
Codon-optimized nucleotide sequence of homo sapiens FGF21 variant 2(SEQ ID NO: 6)
ATGGACAGCGATGAAACCGGGTTCGAGCACAGCGGTCTGTGGGTGTCCGTGCTGGCCGGACTGCTCCTGGGAGCCTGTCAGGCGCACCCCATCCCTGACTCCTCGCCGCTGCTGCAATTCGGCGGACAAGTCCGCCAGAGATACCTGTACACCGACGACGCCCAGCAGACCGAAGCCCACCTGGAAATTCGGGAGGACGGGACTGTGGGAGGCGCTGCAGATCAGTCACCCGAGTCCCTCCTCCAACTGAAGGCCTTGAAGCCCGGCGTGATTCAGATCCTGGGCGTGAAAACTTCCCGCTTCCTTTGCCAACGGCCGGATGGAGCTCTGTACGGATCCCTGCACTTCGACCCCGAAGCCTGCTCATTCCGCGAGCTGCTCCTTGAGGACGGCTATAACGTGTACCAGTCTGAGGCCCATGGACTCCCCCTGCATCTGCCCGGCAACAAGTCCCCTCACCGGGATCCTGCCCCAAGAGGCCCAGCTCGGTTTCTGCCTCTGCCGGGACTGCCTCCAGCGTTGCCCGAACCCCCTGGTATCCTGGCCCCGCAACCACCTGACGTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAGCCAGGGAAGGTCCCCGTCCTACGCATCCTGA
Codon-optimized nucleotide sequence of homo sapiens FGF21 variant 3(SEQ ID NO: 7)
ATGGATTCCGACGAAACTGGATTTGAACATTCAGGGCTGTGGGTCTCTGTGCTGGCTGGACTGCTGCTGGGGGCTTGTCAGGCTCACCCCATCCCTGACAGCTCCCCTCTGCTGCAGTTCGGAGGACAGGTGCGGCAGAGATACCTGTATACCGACGATGCCCAGCAGACAGAGGCACACCTGGAGATCAGGGAGGACGGAACCGTGGGAGGAGCAGCCGATCAGTCTCCCGAGAGCCTGCTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAGACATCTCGGTTTCTGTGCCAGCGGCCCGACGGCGCCCTGTACGGCTCCCTGCACTTCGATCCCGAGGCCTGTTCTTTTAGGGAGCTGCTGCTGGAGGACGGCTACAACGTGTATCAGAGCGAGGCACACGGCCTGCCACTGCACCTGCCTGGCAATAAGTCCCCTCACCGCGATCCAGCACCCAGGGGCCCAGCACGCTTCCTGCCTCTGCCAGGCCTGCCCCCTGCCCTGCCAGAGCCACCCGGCATCCTGGCCCCCCAGCCTCCAGATGTGGGCTCCAGCGATCCTCTGTCAATGGTGGGGCCAAGTCAGGGGCGGAGTCCTTCATACGCATCATAA
Nucleotide sequence encoding miRT122a (target sequence of microrna 122 a) (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 199 a) (SEQ ID NO: 15)
5’GAACAGGTAGTCTGAACACTGGG 3’
Nucleotide sequence encoding mirT199a-3p (target sequence of microRNA 199 a) (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 for microrna 148 a) (SEQ ID NO: 19)
5’ACAAAGTTCTGTAGTGCACTGA 3’
Nucleotide sequence encoding miRT194 (target sequence of microrna 194) (SEQ ID NO: 20)
5’TCCACATGGAGTTGCTGTTACA 3’
Nucleotide sequence encoding mirT124 (target sequence of microRNA 124) (SEQ ID NO: 21)
5’GGCATTCACCGCGTGCCTTA 3’
Nucleotide sequence encoding mirT216 (target sequence of microRNA 216) (SEQ ID NO: 22)
5’TCACAGTTGCCAGCTGAGATTA 3’
Nucleotide sequence encoding mirT125 (target sequence of microRNA 125) (SEQ ID NO: 23)
5’TCACAGGTTAAAGGGTCTCAGGGA 3’
Nucleotide sequence encoding miRT133a (target sequence for microrna 133 a) (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 for microRNA 99) (SEQ ID NO: 27)
5’CACAAGATCGGATCTACGGGTT 3’
Nucleotide sequence encoding mirT208-5p (target sequence of microRNA 208 a) (SEQ ID NO: 28)
5’GTATAACCCGGGCCAAAAGCTC 3’
Nucleotide sequence encoding miRT208a-3p (target sequence of microrna 208 a) (SEQ ID NO: 29)
5’ACAAGCTTTTTGCTCGTCTTAT 3’
Nucleotide sequence encoding miRT499-5p (target sequence of heart-specific microRNA 499) (SEQ ID NO: 30)
5’AAACATCACTGCAAGTCTTAA 3’
Nucleotide sequence of CAG promoter (SEQ ID NO: 44)
GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAG
Nucleotide sequence of CMV promoter (SEQ ID NO: 45)
GTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT
Nucleotide sequence of CMV enhancer (SEQ ID NO: 46)
GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG
Nucleotide sequence of hAAT promoter (SEQ ID NO: 47)
GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT
Truncated AAV 25' ITR (SEQ ID NO: 48)
GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT
Truncated AAV 23' ITR (SEQ ID NO: 49)
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG GGCGGCCTCA GTGAGCGAGCGAGCGCGC
SV40 polyadenylation signal (SEQ ID NO: 50)
TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT
Rabbit β -globulin polyadenylation Signal (SEQ ID NO: 51)
GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC
CMV promoter and CMV enhancer sequence (SEQ ID NO: 52)
GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT
The Hepatocyte Control Region (HCR) enhancer of apolipoprotein E (SEQ ID NO: 53)
CAGAGAGGTCTCTGACCTCTGCCCCAGCTCCAAGGTCAGCAGGCAGGGAGGGCTGTGTGTTTGCTGTTTGCTGCTTGCAATGTTTGCCCATTTTAGGGACATGAGTAGGCTGAAGTTTGTTCAGTGTGGACTTCAGAGGCAGCACACAAACAGC
miniAP2 promoter (SEQ ID NO: 54)
Figure BDA0002371788690000951
Figure BDA0002371788690000961
MiniUCP1 promoter (SEQ ID NO: 55)
Figure BDA0002371788690000962
Figure BDA0002371788690000971
C5-12 promoter (SEQ ID NO: 56)
Figure BDA0002371788690000972
pAAV-EF1a-mmFGF21-pA(SEQ ID NO：57)
CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTTCGACTGCTAGCACGCGTGATATCAATGGAATGGATGAGATCTAGAGTTGGGACCCTGGGACTGTGGGTCCGACTGCTGCTGGCTGTCTTCCTGCTGGGGGTCTACCAAGCATACCCCATCCCTGACTCCAGCCCCCTCCTCCAGTTTGGGGGTCAAGTCCGGCAGAGGTACCTCTACACAGATGACGACCAAGACACTGAAGCCCACCTGGAGATCAGGGAGGATGGAACAGTGGTAGGCGCAGCACACCGCAGTCCAGAAAGTCTCCTGGAGCTCAAAGCCTTGAAGCCAGGGGTCATTCAAATCCTGGGTGTCAAAGCCTCTAGGTTTCTTTGCCAACAGCCAGATGGAGCTCTCTATGGATCGCCTCACTTTGATCCTGAGGCCTGCAGCTTCAGAGAACTGCTGCTGGAGGACGGTTACAATGTGTACCAGTCTGAAGCCCATGGCCTGCCCCTGCGTCTGCCTCAGAAGGACTCCCCAAACCAGGATGCAACATCCTGGGGACCTGTGCGCTTCCTGCCCATGCCAGGCCTGCTCCACGAGCCCCAAGACCAAGCAGGATTCCTGCCCCCAGAGCCCCCAGATGTGGGCTCCTCTGACCCCCTGAGCATGGTAGAGCCTTTACAGGGCCGAAGCCCCAGCTATGCGTCCTGAGATATCAAAGAATTCTAAGCTTGTCGACGAATGCAATTGTTGTTAATTAATTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAGTCGAGTTAATTAACGGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT
Elongation factor 1 α promoter 150 to 1327(1178bp)
Mouse FGF 21: 1359 to 1991(633bp)
SEQ ID NO: 57 also contains truncated AAV 25 'and 3' ITRs and SV40 polyA (already included in the sequence listing, SEQ ID NOS: 48, 49 and 50).
Sequence listing
<110> autonomy university of Barcelona
<120> viral expression construct comprising fibroblast growth factor 21(FGF21) coding sequence
<130>P6066689PCT
<150>EP17172818.1
<151>24-05-2017
<150>ES201700615
<151>24-05-2017
<160>57
<170>PatentIn version 3.5
<210>1
<211>209
<212>PRT
<213> Intelligent people
<400>1
Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser
1 5 10 15
Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro
20 25 30
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr
35 40 45
Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg
50 55 60
Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu
65 70 75 80
Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val
85 90 95
Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly
100 105 110
Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu
115 120 125
Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu
130 135 140
His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly
145 150 155 160
Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu
165 170 175
Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp
180 185 190
Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala
195 200 205
Ser
<210>2
<211>210
<212>PRT
<213> mice
<400>2
Met Glu Trp Met Arg Ser Arg Val Gly Thr Leu Gly Leu Trp Val Arg
1 5 10 15
Leu Leu Leu Ala Val Phe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile
20 25 30
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg
35 40 45
Tyr Leu Tyr Thr Asp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile
50 55 60
Arg Glu Asp Gly Thr Val Val Gly Ala Ala His Arg Ser Pro Glu Ser
65 70 75 80
Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly
85 90 95
Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr
100 105 110
Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu
115 120 125
Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro
130 135 140
Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp
145 150 155 160
Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln
165 170 175
Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser
180 185 190
Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr
195 200 205
Ala Ser
210
<210>3
<211>209
<212>PRT
<213> domestic dog
<400>3
Met Gly Trp Ala Glu Ala Gly Phe Glu His Leu Gly Leu Trp Val Pro
1 5 10 15
Val Leu Ala Val Leu Leu Leu Glu Ala Cys Arg Ala His Pro Ile Pro
20 25 30
Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr
35 40 45
Leu Tyr Thr Asp Asp Ala Gln Glu Thr Glu Ala His Leu Glu Ile Arg
50 55 60
Ala Asp Gly Thr Val Val Gly Ala Ala Arg Gln Ser Pro Glu Ser Leu
65 70 75 80
Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val
85 90 95
LysThr Ser Arg Phe Leu Cys Gln Gly Pro Asp Gly Thr Leu Tyr Gly
100 105 110
Ser Leu His Phe Asp Pro Val Ala Cys Ser Phe Arg Glu Leu Leu Leu
115 120 125
Glu Asp Gly Tyr Asn Ile Tyr His Ser Glu Thr Leu Gly Leu Pro Leu
130 135 140
Arg Leu Arg Pro His Asn Ser Ala Tyr Arg Asp Leu Ala Pro Arg Gly
145 150 155 160
Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Leu Pro Ala Pro Pro Glu
165 170 175
Pro Pro Gly Ile Leu Ala Pro Glu Pro Pro Asp Val Gly Ser Ser Asp
180 185 190
Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala
195 200 205
Ser
<210>4
<211>630
<212>DNA
<213> Intelligent people
<400>4
atggactcgg acgagaccgg gttcgagcac tcaggactgt gggtttctgt gctggctggt 60
cttctgctgg gagcctgcca ggcacacccc atccctgact ccagtcctct cctgcaattc 120
gggggccaag tccggcagcg gtacctctac acagatgatg cccagcagac agaagcccac 180
ctggagatca gggaggatgg gacggtgggg ggcgctgctg accagagccc cgaaagtctc 240
ctgcagctga aagccttgaa gccgggagtt attcaaatct tgggagtcaa gacatccagg 300
ttcctgtgcc agcggccaga tggggccctg tatggatcgc tccactttga ccctgaggcc 360
tgcagcttcc gggagctgct tcttgaggac ggatacaatg tttaccagtc cgaagcccac 420
ggcctcccgc tgcacctgcc agggaacaag tccccacacc gggaccctgc accccgagga 480
ccagctcgct tcctgccact accaggcctg ccccccgcac tcccggagcc acccggaatc 540
ctggcccccc agccccccga tgtgggctcc tcggaccctc tgagcatggt gggaccttcc 600
cagggccgaa gccccagcta cgcttcctga 630
<210>5
<211>630
<212>DNA
<213> Artificial sequence
<220>
<223> codon optimized homo sapiens FGF 21-variant 1
<400>5
atggattctg atgagacagg cttcgagcac agcggcctgt gggtttcagt tctggctgga 60
ctgctgctgg gagcctgtca ggcacaccct attccagata gcagccctct gctgcagttc 120
ggcggacaag tgcggcagag atacctgtac accgacgacg cccagcagac agaagcccac 180
ctggaaatca gagaggatgg cacagttggc ggagccgccg atcagtctcc tgaatctctg 240
ctccagctga aggccctgaa gcctggcgtg atccagatcc tgggcgtgaa aaccagccgg 300
ttcctgtgcc aaagacctga cggcgccctg tatggcagcc tgcactttga tcctgaggcc 360
tgcagcttca gagagctgct gcttgaggac ggctacaacg tgtaccagtc tgaggcccat 420
ggcctgcctc tgcatctgcc tggaaacaag agccctcaca gagatcccgc tcctagaggc 480
cctgccagat ttctgcctct tcctggattg cctcctgctc tgccagagcc tcctggaatt 540
ctggctcctc agcctcctga tgtgggcagc tctgatcctc tgagcatggt cggacctagc 600
cagggcagat ctcctagcta cgcctcttga 630
<210>6
<211>630
<212>DNA
<213> Artificial sequence
<220>
<223> codon optimized homo sapiens FGF 21-variant 2
<400>6
atggacagcg atgaaaccgg gttcgagcac agcggtctgt gggtgtccgt gctggccgga 60
ctgctcctgg gagcctgtca ggcgcacccc atccctgact cctcgccgct gctgcaattc 120
ggcggacaag tccgccagag atacctgtac accgacgacg cccagcagac cgaagcccac 180
ctggaaattc gggaggacgg gactgtggga ggcgctgcag atcagtcacc cgagtccctc 240
ctccaactga aggccttgaa gcccggcgtg attcagatcc tgggcgtgaa aacttcccgc 300
ttcctttgcc aacggccgga tggagctctg tacggatccc tgcacttcga ccccgaagcc 360
tgctcattcc gcgagctgct ccttgaggac ggctataacg tgtaccagtc tgaggcccat 420
ggactccccc tgcatctgcc cggcaacaag tcccctcacc gggatcctgc cccaagaggc 480
ccagctcggt ttctgcctct gccgggactg cctccagcgt tgcccgaacc ccctggtatc 540
ctggccccgc aaccacctga cgtcggttcg tcggacccgc tgagcatggt cggtccgagc 600
cagggaaggt ccccgtccta cgcatcctga 630
<210>7
<211>630
<212>DNA
<213> Artificial sequence
<220>
<223> codon optimized homo sapiens FGF 21-variant 3
<400>7
atggattccg acgaaactgg atttgaacat tcagggctgt gggtctctgt gctggctgga 60
ctgctgctgg gggcttgtca ggctcacccc atccctgaca gctcccctct gctgcagttc 120
ggaggacagg tgcggcagag atacctgtat accgacgatg cccagcagac agaggcacac 180
ctggagatca gggaggacgg aaccgtggga ggagcagccg atcagtctcc cgagagcctg 240
ctgcagctga aggccctgaa gcctggcgtg atccagatcc tgggcgtgaa gacatctcgg 300
tttctgtgcc agcggcccga cggcgccctg tacggctccc tgcacttcga tcccgaggcc 360
tgttctttta gggagctgct gctggaggac ggctacaacg tgtatcagag cgaggcacac 420
ggcctgccac tgcacctgcc tggcaataag tcccctcacc gcgatccagc acccaggggc 480
ccagcacgct tcctgcctct gccaggcctg ccccctgccc tgccagagcc acccggcatc 540
ctggcccccc agcctccaga tgtgggctcc agcgatcctc tgtcaatggt ggggccaagt 600
caggggcgga gtccttcata cgcatcataa 630
<210>8
<211>633
<212>DNA
<213> mice
<400>8
atggaatgga tgagatctag agttgggacc ctgggactgt gggtccgact gctgctggct 60
gtcttcctgc tgggggtcta ccaagcatac cccatccctg actccagccc cctcctccag 120
tttgggggtc aagtccggca gaggtacctc tacacagatg acgaccaaga cactgaagcc 180
cacctggaga tcagggagga tggaacagtg gtaggcgcag cacaccgcag tccagaaagt 240
ctcctggagc tcaaagcctt gaagccaggg gtcattcaaa tcctgggtgt caaagcctct 300
aggtttcttt gccaacagcc agatggagct ctctatggat cgcctcactt tgatcctgag 360
gcctgcagct tcagagaact gctgctggag gacggttaca atgtgtacca gtctgaagcc 420
catggcctgc ccctgcgtct gcctcagaag gactccccaa accaggatgc aacatcctgg 480
ggacctgtgc gcttcctgcc catgccaggc ctgctccacg agccccaaga ccaagcagga 540
ttcctgcccc cagagccccc agatgtgggc tcctctgacc ccctgagcat ggtagagcct 600
ttacagggcc gaagccccag ctatgcgtcc tga 633
<210>9
<211>633
<212>DNA
<213> Artificial sequence
<220>
<223> codon optimized mouse FGF21
<400>9
atggaatgga tgagaagcag agtgggcacc ctgggcctgt gggtgcgact gctgctggct 60
gtgtttctgc tgggcgtgta ccaggcctac cccatccctg actctagccc cctgctgcag 120
tttggcggac aagtgcggca gagatacctg tacaccgacg acgaccagga caccgaggcc 180
cacctggaaa tccgcgagga tggcacagtc gtgggcgctg ctcacagaag ccctgagagc 240
ctgctggaac tgaaggccct gaagcccggc gtgatccaga tcctgggcgt gaaggccagc 300
agattcctgt gccagcagcc tgacggcgcc ctgtacggct ctcctcactt cgatcctgag 360
gcctgcagct tcagagagct gctgctggag gacggctaca acgtgtacca gtctgaggcc 420
cacggcctgc ccctgagact gcctcagaag gacagcccta accaggacgc cacaagctgg 480
ggacctgtgc ggttcctgcc tatgcctgga ctgctgcacg agccccagga tcaggctggc 540
tttctgcctc ctgagcctcc agacgtgggc agcagcgacc ctctgagcat ggtggaacct 600
ctgcagggca gaagccccag ctacgcctct tga 633
<210>10
<211>629
<212>DNA
<213> domestic dog
<400>10
atgggctggg ccgaggccgg gttcgagcac ctgggactgt gggtccctgt gctggctgtg 60
cttttgctgg aagcctgccg ggcacatccg atccctgact ccagccccct cctacaattt 120
ggaggtcaag ttcgacagcg gtacctctac accgacgatg cccaggagac agaggcccac 180
ctagagatca gggccgatgg cacagggtgg gggctgcccg ccagagccct gaaagtctcc 240
tggagctgaa agccctaaag ccaggggtca ttcaaatctt gggagtcaaa acatccaggt 300
tcctgtgcca gggcccagat gggacactat atggctcgct ccatttcgac cctgtggcct 360
gcagtttccg agaactgctt cttgaggatg ggtacaacat ctaccactcc gagacccttg 420
gtctcccgct tcgcctgcgc ccccacaact ccgcataccg ggacttggca ccccgcgggc 480
ctgcccgctt cctgccactg ccaggcctgc ttccagcacc cccagagcct ccagggatcc 540
tggccccgga gcctcctgac gtgggctcct cggaccctct gagcatggtg gggccttcac 600
agggccggag tcccagctat gcttcctaa 629
<210>11
<211>630
<212>DNA
<213> Artificial sequence
<220>
<223> codon optimized Canine FGF21
<400>11
atgggatggg ctgaggctgg attcgaacac ctgggactct gggtgcccgt cctggccgtg 60
ctgctcctgg aggcttgcag ggctcatccc atccctgaca gctccccact cctgcagttt 120
ggaggacagg tgaggcagcg gtacctgtat accgacgatg cccaggagac agaagctcac 180
ctggaaattc gggctgatgg aacagtggtc ggagctgccc gacagtcccc agagtctctc 240
ctggaactga aggccctcaa acccggagtg atccagattc tgggcgtcaa gacttctaga 300
ttcctgtgcc agggaccaga cggcaccctg tacggcagcc tgcatttcga tcctgtggcc 360
tgttcctttc gagagctcct gctcgaagac ggctacaaca tctatcactc tgagaccctg 420
ggactcccac tgcgactcag acctcataat agtgcctatc gagatctggc tcccaggggc 480
ccagctaggt ttctgccact ccccggactg ctccctgctc cacctgagcc acccggcatt 540
ctggctccag aacctccaga cgtgggctct agtgatccac tgagtatggt cggcccctca 600
caggggaggt cacctagcta cgccagctga 630
<210>12
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT122a
<400>12
caaacaccat tgtcacactc ca 22
<210>13
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT1
<400>13
ttacatactt ctttacattc ca 22
<210>14
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT152
<400>14
ccaagttctg tcatgcactg a 21
<210>15
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mirT199a-5p
<400>15
gaacaggtag tctgaacact ggg 23
<210>16
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mirT199a-3p
<400>16
taaccaatgt gcagactact gt 22
<210>17
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT215
<400>17
gtctgtcaat tcataggtca t 21
<210>18
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT192
<400>18
ggctgtcaat tcataggtca g 21
<210>19
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT148a
<400>19
acaaagttct gtagtgcact ga 22
<210>20
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT194
<400>20
tccacatgga gttgctgtta ca 22
<210>21
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT124
<400>21
ggcattcacc gcgtgcctta 20
<210>22
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT216
<400>22
tcacagttgc cagctgagat ta 22
<210>23
<211>24
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT125
<400>23
tcacaggtta aagggtctca ggga 24
<210>24
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT133a
<400>24
cagctggttg aaggggacca aa 22
<210>25
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT206
<400>25
ccacacactt ccttacattc ca 22
<210>26
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT130
<400>26
atgccctttt aacattgcac tg 22
<210>27
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT99
<400>27
cacaagatcg gatctacggg tt 22
<210>28
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mirT208a-5p
<400>28
gtataacccg ggccaaaagc tc 22
<210>29
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding mirT208a-3p
<400>29
acaagctttt tgctcgtctt at 22
<210>30
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence encoding miRT499-5p
<400>30
aaacatcact gcaagtctta a 21
<210>31
<211>6343
<212>DNA
<213> Artificial sequence
<220>
<223> pAAV-CAG-null
<400>31
aattcgagct cggtacccgg gaatcaattc actcctcagg tgcaggctgc ctatcagaag 60
gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct 120
gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg 180
aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca 240
tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca 300
tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg 360
aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt 420
ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat 480
gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt 540
ctcttatgga gatccctcga cctgcagccc aagctgtaga taagtagcat ggcgggttaa 600
tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 660
cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 720
cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 780
tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 840
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 900
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 960
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 1020
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 1080
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 1140
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 1200
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 1260
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 1320
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 1380
gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 1440
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 1500
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 1560
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 1620
acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 1680
ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 1740
ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 1800
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 1860
tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 1920
gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 1980
tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 2040
tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 2100
ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 2160
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 2220
ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 2280
gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 2340
ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 2400
cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 2460
ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 2520
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 2580
gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 2640
ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 2700
gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 2760
aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg 2820
tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 2880
cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 2940
taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc 3000
gcacagatgc gtaaggagaa aataccgcat caggcgattc caacatccaa taaatcatac 3060
aggcaaggca aagaattagc aaaattaagc aataaagcct cagagcataa agctaaatcg 3120
gttgtaccaa aaacattatg accctgtaat acttttgcgg gagaagcctt tatttcaacg 3180
caaggataaa aatttttaga accctcatat attttaaatg caatgcctga gtaatgtgta 3240
ggtaaagatt caaacgggtg agaaaggccg gagacagtca aatcaccatc aatatgatat 3300
tcaaccgttc tagctgataa attcatgccg gagagggtag ctatttttga gaggtctcta 3360
caaaggctat caggtcattg cctgagagtc tggagcaaac aagagaatcg atgaacggta 3420
atcgtaaaac tagcatgtca atcatatgta ccccggttga taatcagaaa agccccaaaa 3480
acaggaagat tgtataagca aatatttaaa ttgtaagcgt taatattttg ttaaaattcg 3540
cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 3600
cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga 3660
gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 3720
atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag 3780
cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 3840
acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 3900
tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 3960
cgtactatgg ttgctttgac gagcacgtat aacgtgcttt cctcgttaga atcagagcgg 4020
gagctaaaca ggaggccgat taaagggatt ttagacagga acggtacgcc agaatcctga 4080
gaagtgtttt tataatcagt gaggccaccg agtaaaagag tctgtccatc acgcaaatta 4140
accgttgtcg caatacttct ttgattagta ataacatcac ttgcctgagt agaagaactc 4200
aaactatcgg ccttgctggt aatatccaga acaatattac cgccagccat tgcaacggaa 4260
tcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 4320
gctattacgc cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg 4380
tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc 4440
caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac 4500
tcgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 4560
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 4620
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 4680
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 4740
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 4800
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 4860
attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat 4920
ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 4980
gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 5040
gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 5100
tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 5160
gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 5220
cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 5280
cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 5340
gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg 5400
tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 5460
gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg 5520
gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt 5580
gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc 5640
cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg 5700
gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg 5760
ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg 5820
gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc 5880
agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc 5940
cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg 6000
gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc 6060
ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga 6120
ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc 6180
tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg attaattcga 6240
gcgaacgcgt cgagtcgctc ggtacgattt aaattgaatt ggcctcgagc gcaagcttga 6300
gctagctcga tatcggccta ggctggatcc gcgcggccgc aag 6343
<210>32
<211>7319
<212>DNA
<213> Artificial sequence
<220>
<223>pAAV-CAG-moFGF21-dmiRT
<400>32
agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 60
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 120
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 180
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 240
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 300
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 360
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 420
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 480
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 540
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 600
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 660
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 720
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 780
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 840
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 900
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 960
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1020
caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 1080
gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 1140
gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 1200
ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 1260
attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 1320
gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 1380
tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 1440
agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 1500
gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 1560
actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 1620
tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 1680
attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 1740
tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 1800
tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 1860
aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 1920
tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 1980
cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 2040
acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt 2100
gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 2160
gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt 2220
aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 2280
cacagatgcg taaggagaaa ataccgcatc aggcgattcc aacatccaat aaatcataca 2340
ggcaaggcaa agaattagca aaattaagca ataaagcctc agagcataaa gctaaatcgg 2400
ttgtaccaaa aacattatga ccctgtaata cttttgcggg agaagccttt atttcaacgc 2460
aaggataaaa atttttagaa ccctcatata ttttaaatgc aatgcctgag taatgtgtag 2520
gtaaagattc aaacgggtga gaaaggccgg agacagtcaa atcaccatca atatgatatt 2580
caaccgttct agctgataaa ttcatgccgg agagggtagc tatttttgag aggtctctac 2640
aaaggctatc aggtcattgc ctgagagtct ggagcaaaca agagaatcga tgaacggtaa 2700
tcgtaaaact agcatgtcaa tcatatgtac cccggttgat aatcagaaaa gccccaaaaa 2760
caggaagatt gtataagcaa atatttaaat tgtaagcgtt aatattttgt taaaattcgc 2820
gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc 2880
ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag 2940
tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 3000
tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc 3060
actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa 3120
cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 3180
agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 3240
gtactatggt tgctttgacg agcacgtata acgtgctttc ctcgttagaa tcagagcggg 3300
agctaaacag gaggccgatt aaagggattt tagacaggaa cggtacgcca gaatcctgag 3360
aagtgttttt ataatcagtg aggccaccga gtaaaagagt ctgtccatca cgcaaattaa 3420
ccgttgtcgc aatacttctt tgattagtaa taacatcact tgcctgagta gaagaactca 3480
aactatcggc cttgctggta atatccagaa caatattacc gccagccatt gcaacggaat 3540
cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcc 3600
actgaggccc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt 3660
cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc 3720
aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctact 3780
cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 3840
ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 3900
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 3960
actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 4020
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 4080
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 4140
ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc 4200
tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg 4260
atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg 4320
ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt 4380
ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg 4440
ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc 4500
cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 4560
ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc 4620
cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg 4680
tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc 4740
gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc 4800
ggtgccccgc ggtgcggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt 4860
gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc 4920
tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc 4980
ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc 5040
gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg 5100
tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg 5160
acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc 5220
tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 5280
tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg 5340
ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg 5400
cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg 5460
caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattgatta attcgagcga 5520
acgcgtcgag tcgctcggta cgatttaaat tgaattggcc tcgagcgcaa gcttgagcta 5580
gcgccaccat ggaatggatg agaagcagag tgggcaccct gggcctgtgg gtgcgactgc 5640
tgctggctgt gtttctgctg ggcgtgtacc aggcctaccc catccctgac tctagccccc 5700
tgctgcagtt tggcggacaa gtgcggcaga gatacctgta caccgacgac gaccaggaca 5760
ccgaggccca cctggaaatc cgcgaggatg gcacagtcgt gggcgctgct cacagaagcc 5820
ctgagagcct gctggaactg aaggccctga agcccggcgt gatccagatc ctgggcgtga 5880
aggccagcag attcctgtgc cagcagcctg acggcgccct gtacggctct cctcacttcg 5940
atcctgaggc ctgcagcttc agagagctgc tgctggagga cggctacaac gtgtaccagt 6000
ctgaggccca cggcctgccc ctgagactgc ctcagaagga cagccctaac caggacgcca 6060
caagctgggg acctgtgcgg ttcctgccta tgcctggact gctgcacgag ccccaggatc 6120
aggctggctt tctgcctcct gagcctccag acgtgggcag cagcgaccct ctgagcatgg 6180
tggaacctct gcagggcaga agccccagct acgcctcttg agaatgcggg cccggtaccc 6240
ccgacgcggc cgctaattct agatcgcgaa caaacaccat tgtcacactc cagtatacac 6300
aaacaccatt gtcacactcc agatatcaca aacaccattg tcacactcca aggcgaacaa 6360
acaccattgt cacactccaa ggctattcta gatcgcgaat tacatacttc tttacattcc 6420
agtatacatt acatacttct ttacattcca gatatcatta catacttctt tacattccaa 6480
ggcgaattac atacttcttt acattccaag gctacctgag gcccgggggt acctcttaat 6540
taactggcct catgggcctt ccgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 6600
cagtcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca 6660
caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct 6720
tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa 6780
ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa 6840
tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg 6900
ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca 6960
tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc 7020
tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg 7080
actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa 7140
gctgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag 7200
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 7260
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctggcgtaa 7319
<210>33
<211>5678
<212>DNA
<213> Artificial sequence
<220>
<223> pGG 2-hAAT-null
<400>33
cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60
acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 120
atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca 180
tgctctagac atggctcgac agatctgata tcatcgatga attcgagctc ggtacccggc 240
cgcagattta ggtgacacta tagaatatgc atcactagta agcttgcgaa ttccagtcta 300
cagagaggtc tctgacctct gccccagctc caaggtcagc aggcagggag ggctgtgtgt 360
ttgctgtttg ctgcttgcaa tgtttgccca ttttagggac atgagtaggc tgaagtttgt 420
tcagtgtgga cttcagaggc agcacacaaa cagcaagctt gcgaattcca gtctacagag 480
aggtctctga cctctgcccc agctccaagg tcagcaggca gggagggctg tgtgtttgct 540
gtttgctgct tgcaatgttt gcccatttta gggacatgag taggctgaag tttgttcagt 600
gtggacttca gaggcagcac acaaacagca agcttgcgaa ttccagtcta cagagaggtc 660
tctgacctct gccccagctc caaggtcagc aggcagggag ggctgtgtgt ttgctgtttg 720
ctgcttgcaa tgtttgccca ttttagggac atgagtaggc tgaagtttgt tcagtgtgga 780
cttcagaggc agcacacaaa cagcaagctt tgctctagac tggaattcgt cgacgagctc 840
cctatagtga gtcgtattag aggccgactg acccggtacc cggggatctt gctaccagtg 900
gaacagccac taaggattct gcagtgagag cagagggcca gctaagtggt actctcccag 960
agactgtctg actcacgcca ccccctccac cttggacaca ggacgctgtg gtttctgagc 1020
caggtacaat gactcctttc ggtaagtgca gtggaagctg tacactgccc aggcaaagcg 1080
tccgggcagc gtaggcgggc gactcagatc ccagccagtg gacttagccc ctgtttgctc 1140
ctccgataac tggggtgacc ttggttaata ttcaccagca gcctcccccg ttgcccctct 1200
ggatccactg cttaaatacg gacgaggaca gggccctgtc tcctcagctt caggcaccac 1260
cactgacctg ggacagtgaa tgtccccctg atctgcggcc gtgactctct taaggtagcc 1320
ttgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta 1380
aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc 1440
acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt 1500
caattacagc tcttaaggct agagtactta atacgactca ctataggcta gcctcgacct 1560
cgagacgcgt gatatcggat cccggccggc ggccgcttcc ctttagtgag ggttaatgct 1620
tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 1680
aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 1740
ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 1800
gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg 1860
gactagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac 1920
ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc 1980
gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 2040
gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 2100
ctgaatggcg aatggaattc cagacgattg agcgtcaaaa tgtaggtatt tccatgagcg 2160
tttttccgtt gcaatggctg gcggtaatat tgttctggat attaccagca aggccgatag 2220
tttgagttct tctactcagg caagtgatgt tattactaat caaagaagta ttgcgacaac 2280
ggttaatttg cgtgatggac agactctttt actcggtggc ctcactgatt ataaaaacac 2340
ttctcaggat tctggcgtac cgttcctgtc taaaatccct ttaatcggcc tcctgtttag 2400
ctcccgctct gattctaacg aggaaagcac gttatacgtg ctcgtcaaag caaccatagt 2460
acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 2520
ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 2580
cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 2640
gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 2700
catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 2760
gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 2820
aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 2880
acgcgaattt taacaaaata ttaacgtcta caatttaaat atttgcttat acaatcttcc 2940
tgtttttggg gcttttctga ttatcaaccg gggtacatat gattgacatg ctagttttac 3000
gattaccgtt catcgattct cttgtttgct ccagactctc aggcaatgac ctgatagcct 3060
ttgtagagac ctctcaaaaa tagctaccct ctccggcatg aatttatcag ctagaacggt 3120
tgaatatcat attgatggtg atttgactgt ctccggcctt tctcacccgt ttgaatcttt 3180
acctacacat tactcaggca ttgcatttaa aatatatgag ggttctaaaa atttttatcc 3240
ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag ggtcataatg tttttggtac 3300
aaccgattta gctttatgct ctgaggcttt attgcttaat tttgctaatt ctttgccttg 3360
cctgtatgat ttattggatg ttggaatcgc ctgatgcggt attttctcct tacgcatctg 3420
tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 3480
ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 3540
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 3600
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 3660
gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 3720
cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 3780
caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 3840
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 3900
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 3960
gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 4020
atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 4080
caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 4140
gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 4200
accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 4260
ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 4320
gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 4380
acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 4440
atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 4500
ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 4560
gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 4620
gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 4680
tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 4740
taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 4800
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 4860
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4920
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4980
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 5040
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 5100
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 5160
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 5220
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 5280
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 5340
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 5400
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 5460
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 5520
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 5580
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 5640
aaaccgcctc tccccgcgcg ttggccgatt cattaatg 5678
<210>34
<211>6363
<212>DNA
<213> Artificial sequence
<220>
<223>pGG2-hAAT-moFGF21
<400>34
cgcgtgatat cggatcccgg ccggcggccg cttcccttta gtgagggtta atgcttcgag 60
cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 120
aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 180
ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt 240
gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatccga taagggacta 300
gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta 360
gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca 420
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgccag 480
ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 540
tggcgaatgg aattccagac gattgagcgt caaaatgtag gtatttccat gagcgttttt 600
ccgttgcaat ggctggcggt aatattgttc tggatattac cagcaaggcc gatagtttga 660
gttcttctac tcaggcaagt gatgttatta ctaatcaaag aagtattgcg acaacggtta 720
atttgcgtga tggacagact cttttactcg gtggcctcac tgattataaa aacacttctc 780
aggattctgg cgtaccgttc ctgtctaaaa tccctttaat cggcctcctg tttagctccc 840
gctctgattc taacgaggaa agcacgttat acgtgctcgt caaagcaacc atagtacgcg 900
ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 960
cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 1020
gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 1080
ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 1140
ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 1200
ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 1260
attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 1320
aattttaaca aaatattaac gtctacaatt taaatatttg cttatacaat cttcctgttt 1380
ttggggcttt tctgattatc aaccggggta catatgattg acatgctagt tttacgatta 1440
ccgttcatcg attctcttgt ttgctccaga ctctcaggca atgacctgat agcctttgta 1500
gagacctctc aaaaatagct accctctccg gcatgaattt atcagctaga acggttgaat 1560
atcatattga tggtgatttg actgtctccg gcctttctca cccgtttgaa tctttaccta 1620
cacattactc aggcattgca tttaaaatat atgagggttc taaaaatttt tatccttgcg 1680
ttgaaataaa ggcttctccc gcaaaagtat tacagggtca taatgttttt ggtacaaccg 1740
atttagcttt atgctctgag gctttattgc ttaattttgc taattctttg ccttgcctgt 1800
atgatttatt ggatgttgga atcgcctgat gcggtatttt ctccttacgc atctgtgcgg 1860
tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag 1920
ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 1980
atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 2040
gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 2100
tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg 2160
aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 2220
accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 2280
tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 2340
gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 2400
ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 2460
gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 2520
gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 2580
agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 2640
gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 2700
cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 2760
gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 2820
gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 2880
ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 2940
gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 3000
ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 3060
tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 3120
actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 3180
taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 3240
gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 3300
tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 3360
ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 3420
gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 3480
tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 3540
cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 3600
gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 3660
actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 3720
ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 3780
gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 3840
atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 3900
tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 3960
tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 4020
aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 4080
gcctctcccc gcgcgttggc cgattcatta atgcagcagc tgcgcgctcg ctcgctcact 4140
gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc 4200
gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctt gtagttaatg 4260
attaacccgc catgctactt atctacgtag ccatgctcta gacatggctc gacagatctg 4320
atatcatcga tgaattcgag ctcggtaccc ggccgcagat ttaggtgaca ctatagaata 4380
tgcatcacta gtaagcttgc gaattccagt ctacagagag gtctctgacc tctgccccag 4440
ctccaaggtc agcaggcagg gagggctgtg tgtttgctgt ttgctgcttg caatgtttgc 4500
ccattttagg gacatgagta ggctgaagtt tgttcagtgt ggacttcaga ggcagcacac 4560
aaacagcaag cttgcgaatt ccagtctaca gagaggtctc tgacctctgc cccagctcca 4620
aggtcagcag gcagggaggg ctgtgtgttt gctgtttgct gcttgcaatg tttgcccatt 4680
ttagggacat gagtaggctg aagtttgttc agtgtggact tcagaggcag cacacaaaca 4740
gcaagcttgc gaattccagt ctacagagag gtctctgacc tctgccccag ctccaaggtc 4800
agcaggcagg gagggctgtg tgtttgctgt ttgctgcttg caatgtttgc ccattttagg 4860
gacatgagta ggctgaagtt tgttcagtgt ggacttcaga ggcagcacac aaacagcaag 4920
ctttgctcta gactggaatt cgtcgacgag ctccctatag tgagtcgtat tagaggccga 4980
ctgacccggt acccggggat cttgctacca gtggaacagc cactaaggat tctgcagtga 5040
gagcagaggg ccagctaagt ggtactctcc cagagactgt ctgactcacg ccaccccctc 5100
caccttggac acaggacgct gtggtttctg agccaggtac aatgactcct ttcggtaagt 5160
gcagtggaag ctgtacactg cccaggcaaa gcgtccgggc agcgtaggcg ggcgactcag 5220
atcccagcca gtggacttag cccctgtttg ctcctccgat aactggggtg accttggtta 5280
atattcacca gcagcctccc ccgttgcccc tctggatcca ctgcttaaat acggacgagg 5340
acagggccct gtctcctcag cttcaggcac caccactgac ctgggacagt gaatgtcccc 5400
ctgatctgcg gccgtgactc tcttaaggta gccttgcaga agttggtcgt gaggcactgg 5460
gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc 5520
gagacagaga agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt 5580
tgcctttctc tccacaggtg tccactccca gttcaattac agctcttaag gctagagtac 5640
ttaatacgac tcactatagg ctagcctcga cctcgagcgc aagcttgagc tagcgccacc 5700
atggaatgga tgagaagcag agtgggcacc ctgggcctgt gggtgcgact gctgctggct 5760
gtgtttctgc tgggcgtgta ccaggcctac cccatccctg actctagccc cctgctgcag 5820
tttggcggac aagtgcggca gagatacctg tacaccgacg acgaccagga caccgaggcc 5880
cacctggaaa tccgcgagga tggcacagtc gtgggcgctg ctcacagaag ccctgagagc 5940
ctgctggaac tgaaggccct gaagcccggc gtgatccaga tcctgggcgt gaaggccagc 6000
agattcctgt gccagcagcc tgacggcgcc ctgtacggct ctcctcactt cgatcctgag 6060
gcctgcagct tcagagagct gctgctggag gacggctaca acgtgtacca gtctgaggcc 6120
cacggcctgc ccctgagact gcctcagaag gacagcccta accaggacgc cacaagctgg 6180
ggacctgtgc ggttcctgcc tatgcctgga ctgctgcacg agccccagga tcaggctggc 6240
tttctgcctc ctgagcctcc agacgtgggc agcagcgacc ctctgagcat ggtggaacct 6300
ctgcagggca gaagccccag ctacgcctct tgagaatgcg ggcccggtac ccccgacgcg 6360
gcc 6363
<210>35
<211>4403
<212>DNA
<213> Artificial sequence
<220>
<223> pAAV-CMV-null
<400>35
cgacggtacc agcgctgtcg aggccgcttc gagcagacat gataagatac attgatgagt 60
ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg 120
ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca 180
ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc 240
tctacaaatg tggtaaaatc gattaggatc ttcctagagc atggctacct agacatggct 300
cgacagatca gcgctcatgc tctggaagat ctcgatttaa atgcggccgc aggaacccct 360
agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc 420
aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag 480
ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 540
ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt 600
gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 660
gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 720
gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 780
ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 840
ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 900
atctcgggct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 960
aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt 1020
ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac 1080
ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 1140
caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 1200
cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata 1260
atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 1320
ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 1380
cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 1440
cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 1500
aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 1560
ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 1620
gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 1680
cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 1740
acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 1800
gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 1860
aacatggggg atcatgtaac tcgccttgat cgttgggaaccggagctgaa tgaagccata 1920
ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 1980
ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 2040
gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 2100
aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 2160
aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 2220
aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 2280
gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 2340
gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 2400
tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 2460
gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 2520
caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 2580
actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 2640
acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 2700
cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 2760
gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 2820
cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 2880
gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 2940
tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 3000
tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 3060
gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact 3120
gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc 3180
gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcgat 3240
atctgtagtt aatgattaac ccgccatgct acttatctac agatctcaat attggccatt 3300
agccatatta ttcattggtt atatagcata aatcaatatt ggctattggc cattgcatac 3360
gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat gaccgccatg 3420
ttggcattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 3480
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 3540
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 3600
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 3660
tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 3720
ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt 3780
attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 3840
gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3900
ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactgcgatc gcccgccccg 3960
ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta 4020
gtgaaccgtc agatcactag gctagctatt gcggtagttt atcacagtta aattgctaac 4080
gcagtcagtg cttctgacac aacagtctcg aacttaagct gcagtgactc tcttaaggta 4140
gccttgcaga agttggtcgt gaggcactgg gcaggtaagt atcaaggtta caagacaggt 4200
ttaaggagac caatagaaac tgggcttgtc gagacagaga agactcttgc gtttctgata 4260
ggcacctatt ggtcttactg acatccactt tgcctttctc tccacaggtg tccactccca 4320
gttcaattac agctcttaag gctagagtac ttaatacgac tcactataga atacgactca 4380
ctatagggag acgctagcgt cga 4403
<210>36
<211>5073
<212>DNA
<213> Artificial sequence
<220>
<223>pAAV-CMV-moFGF21
<400>36
ggggctagcg ccaccatgga atggatgaga agcagagtgg gcaccctggg cctgtgggtg 60
cgactgctgc tggctgtgtt tctgctgggc gtgtaccagg cctaccccat ccctgactct 120
agccccctgc tgcagtttgg cggacaagtg cggcagagat acctgtacac cgacgacgac 180
caggacaccg aggcccacct ggaaatccgc gaggatggca cagtcgtggg cgctgctcac 240
agaagccctg agagcctgct ggaactgaag gccctgaagc ccggcgtgat ccagatcctg 300
ggcgtgaagg ccagcagatt cctgtgccag cagcctgacg gcgccctgta cggctctcct 360
cacttcgatc ctgaggcctg cagcttcaga gagctgctgc tggaggacgg ctacaacgtg 420
taccagtctg aggcccacgg cctgcccctg agactgcctc agaaggacag ccctaaccag 480
gacgccacaa gctggggacc tgtgcggttc ctgcctatgc ctggactgct gcacgagccc 540
caggatcagg ctggctttct gcctcctgag cctccagacg tgggcagcag cgaccctctg 600
agcatggtgg aacctctgca gggcagaagc cccagctacg cctcttgaga atgcgggccc 660
ggtaccccct cgacggtacc agcgctgtcg aggccgcttc gagcagacat gataagatac 720
attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 780
atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac 840
aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc 900
aagtaaaacc tctacaaatg tggtaaaatc gattaggatc ttcctagagc atggctacct 960
agacatggct cgacagatca gcgctcatgc tctggaagat ctcgatttaa atgcggccgc 1020
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 1080
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 1140
gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 1200
gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 1260
cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 1320
cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 1380
tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 1440
aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 1500
ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 1560
actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta 1620
ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 1680
gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 1740
gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 1800
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 1860
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 1920
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 1980
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 2040
ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 2100
cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 2160
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 2220
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 2280
cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 2340
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 2400
aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 2460
tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 2520
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 2580
tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 2640
gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 2700
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 2760
tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 2820
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 2880
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 2940
gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 3000
aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 3060
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 3120
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 3180
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 3240
gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt 3300
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 3360
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 3420
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 3480
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 3540
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 3600
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 3660
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 3720
acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg 3780
ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc 3840
ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg 3900
cggccgcgat atctgtagtt aatgattaac ccgccatgct acttatctac agatctcaat 3960
attggccatt agccatatta ttcattggtt atatagcata aatcaatatt ggctattggc 4020
cattgcatac gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat 4080
gaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt acggggtcat 4140
tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 4200
gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 4260
cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 4320
tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta 4380
aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt 4440
acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg 4500
ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 4560
ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactgcgatc 4620
gcccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 4680
agctcgttta gtgaaccgtc agatcactag gctagctatt gcggtagttt atcacagtta 4740
aattgctaac gcagtcagtg cttctgacac aacagtctcg aacttaagct gcagtgactc 4800
tcttaaggta gccttgcaga agttggtcgt gaggcactgg gcaggtaagt atcaaggtta 4860
caagacaggt ttaaggagac caatagaaac tgggcttgtc gagacagaga agactcttgc 4920
gtttctgata ggcacctatt ggtcttactg acatccactt tgcctttctc tccacaggtg 4980
tccactccca gttcaattac agctcttaag gctagagtac ttaatacgac tcactataga 5040
atacgactca ctatagggag acgctagcgt cga 5073
<210>37
<211>7292
<212>DNA
<213> Artificial sequence
<220>
<223>pAAV-CAG-cloFGF21-dmiRT
<400>37
ggccgctaat tctagatcgc gaacaaacac cattgtcaca ctccagtata cacaaacacc 60
attgtcacac tccagatatc acaaacacca ttgtcacact ccaaggcgaa caaacaccat 120
tgtcacactc caaggctatt ctagatcgcg aattacatac ttctttacat tccagtatac 180
attacatact tctttacatt ccagatatca ttacatactt ctttacattc caaggcgaat 240
tacatacttc tttacattcc aaggctacct gaggcccggg ggtacctctt aattaactgg 300
cctcatgggc cttccgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagtcag 360
gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac 420
cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat 480
ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg 540
tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat 600
ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta 660
taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa 720
gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca 780
tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc 840
ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc caagctgtag 900
ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 960
ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 1020
cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctggcgt aaagtgagcg 1080
agcgagcgcg cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 1140
tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 1200
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 1260
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 1320
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 1380
tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 1440
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 1500
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 1560
cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1620
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 1680
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 1740
gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 1800
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 1860
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 1920
agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 1980
gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 2040
aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 2100
aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 2160
ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 2220
gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 2280
aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 2340
ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 2400
tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 2460
ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 2520
cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 2580
agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 2640
gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 2700
gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 2760
acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 2820
acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 2880
agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 2940
aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 3000
gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 3060
tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa 3120
aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct 3180
ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag 3240
acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc 3300
ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg 3360
cgtaaggaga aaataccgca tcaggcgatt ccaacatcca ataaatcata caggcaaggc 3420
aaagaattag caaaattaag caataaagcc tcagagcata aagctaaatc ggttgtacca 3480
aaaacattat gaccctgtaa tacttttgcg ggagaagcct ttatttcaac gcaaggataa 3540
aaatttttag aaccctcata tattttaaat gcaatgcctg agtaatgtgt aggtaaagat 3600
tcaaacgggt gagaaaggcc ggagacagtc aaatcaccat caatatgata ttcaaccgtt 3660
ctagctgata aattcatgcc ggagagggta gctatttttg agaggtctct acaaaggcta 3720
tcaggtcatt gcctgagagt ctggagcaaa caagagaatc gatgaacggt aatcgtaaaa 3780
ctagcatgtc aatcatatgt accccggttg ataatcagaa aagccccaaa aacaggaaga 3840
ttgtataagc aaatatttaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt 3900
tttgttaaatcagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat 3960
caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat 4020
taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac 4080
tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc 4140
ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga 4200
gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 4260
cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtactatg 4320
gttgctttga cgagcacgta taacgtgctt tcctcgttag aatcagagcg ggagctaaac 4380
aggaggccga ttaaagggat tttagacagg aacggtacgc cagaatcctg agaagtgttt 4440
ttataatcag tgaggccacc gagtaaaaga gtctgtccat cacgcaaatt aaccgttgtc 4500
gcaatacttc tttgattagt aataacatca cttgcctgag tagaagaact caaactatcg 4560
gccttgctgg taatatccag aacaatatta ccgccagcca ttgcaacgga atcgccattc 4620
gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt ccactgaggc 4680
ccagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac 4740
ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat 4800
cactaggggt tccttgtagt taatgattaa cccgccatgc tacttatcta ctcgacattg 4860
attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 4920
ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 4980
ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 5040
ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 5100
tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 5160
tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 5220
cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc 5280
ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc 5340
gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 5400
gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 5460
ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 5520
tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc cccggctctg 5580
actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa 5640
ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg 5700
gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg 5760
cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg 5820
cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc 5880
gcggtgcggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt 5940
gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag 6000
ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg 6060
ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg 6120
ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg 6180
cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt 6240
tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc 6300
gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt 6360
cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct 6420
gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta 6480
gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc 6540
tggttattgt gctgtctcat cattttggca aagaattgat taattcgagc gaacgcgtcg 6600
agtcgctcgg tacgatttaa attgaattgg cctcgagcgc aagcttgagc tagcgccacc 6660
atgggatggg ctgaggctgg attcgaacac ctgggactct gggtgcccgt cctggccgtg 6720
ctgctcctgg aggcttgcag ggctcatccc atccctgaca gctccccact cctgcagttt 6780
ggaggacagg tgaggcagcg gtacctgtat accgacgatg cccaggagac agaagctcac 6840
ctggaaattc gggctgatgg aacagtggtc ggagctgccc gacagtcccc agagtctctc 6900
ctggaactga aggccctcaa acccggagtg atccagattc tgggcgtcaa gacttctaga 6960
ttcctgtgcc agggaccaga cggcaccctg tacggcagcc tgcatttcga tcctgtggcc 7020
tgttcctttc gagagctcct gctcgaagac ggctacaaca tctatcactc tgagaccctg 7080
ggactcccac tgcgactcag acctcataat agtgcctatc gagatctggc tcccaggggc 7140
ccagctaggt ttctgccact ccccggactg ctccctgctc cacctgagcc acccggcatt 7200
ctggctccag aacctccaga cgtgggctct agtgatccac tgagtatggt cggcccctca 7260
caggggaggt cacctagcta cgccagctga gc 7292
<210>38
<211>6278
<212>DNA
<213> Artificial sequence
<220>
<223>pGG2-hAAT-cloFGF21
<400>38
ggccgcttcc ctttagtgag ggttaatgct tcgagcagac atgataagat acattgatga 60
gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga 120
tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg 180
cattcatttt atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa 240
cctctacaaa tgtggtaaaa tccgataagg gactagagca tggctacgta gataagtagc 300
atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc 360
tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 420
cccgggcggc ctcagtgagc gagcgagcgc gccagctggc gtaatagcga agaggcccgc 480
accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaattc cagacgattg 540
agcgtcaaaa tgtaggtatt tccatgagcg tttttccgtt gcaatggctg gcggtaatat 600
tgttctggat attaccagca aggccgatag tttgagttct tctactcagg caagtgatgt 660
tattactaat caaagaagta ttgcgacaac ggttaatttg cgtgatggac agactctttt 720
actcggtggc ctcactgatt ataaaaacac ttctcaggat tctggcgtac cgttcctgtc 780
taaaatccct ttaatcggcc tcctgtttag ctcccgctct gattctaacg aggaaagcac 840
gttatacgtg ctcgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg 900
cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 960
ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 1020
atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 1080
ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 1140
tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 1200
accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 1260
taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgtcta 1320
caatttaaat atttgcttat acaatcttcc tgtttttggg gcttttctga ttatcaaccg 1380
gggtacatat gattgacatg ctagttttac gattaccgtt catcgattct cttgtttgct 1440
ccagactctc aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct 1500
ctccggcatg aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt 1560
ctccggcctt tctcacccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa 1620
aatatatgag ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa 1680
agtattacag ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt 1740
attgcttaat tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaatcgc 1800
ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact 1860
ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc 1920
gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 1980
gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga 2040
aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag 2100
acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 2160
atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 2220
tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 2280
gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 2340
gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 2400
gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 2460
ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 2520
tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 2580
acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 2640
cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 2700
catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 2760
cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 2820
ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 2880
ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataaatctggagcc 2940
ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 3000
atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 3060
gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 3120
atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 3180
tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 3240
cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 3300
ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 3360
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 3420
gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 3480
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 3540
gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 3600
acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 3660
tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 3720
gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 3780
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 3840
cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 3900
ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 3960
gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 4020
agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 4080
cattaatgca gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc 4140
gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg 4200
ccaactccat cactaggggt tccttgtagt taatgattaa cccgccatgc tacttatcta 4260
cgtagccatg ctctagacat ggctcgacag atctgatatc atcgatgaat tcgagctcgg 4320
tacccggccg cagatttagg tgacactata gaatatgcat cactagtaag cttgcgaatt 4380
ccagtctaca gagaggtctc tgacctctgc cccagctcca aggtcagcag gcagggaggg 4440
ctgtgtgttt gctgtttgct gcttgcaatg tttgcccatt ttagggacat gagtaggctg 4500
aagtttgttc agtgtggact tcagaggcag cacacaaaca gcaagcttgc gaattccagt 4560
ctacagagag gtctctgacc tctgccccag ctccaaggtc agcaggcagg gagggctgtg 4620
tgtttgctgt ttgctgcttg caatgtttgc ccattttagg gacatgagta ggctgaagtt 4680
tgttcagtgt ggacttcaga ggcagcacac aaacagcaag cttgcgaatt ccagtctaca 4740
gagaggtctc tgacctctgc cccagctcca aggtcagcag gcagggaggg ctgtgtgttt 4800
gctgtttgct gcttgcaatg tttgcccatt ttagggacat gagtaggctg aagtttgttc 4860
agtgtggact tcagaggcag cacacaaaca gcaagctttg ctctagactg gaattcgtcg 4920
acgagctccc tatagtgagt cgtattagag gccgactgac ccggtacccg gggatcttgc 4980
taccagtgga acagccacta aggattctgc agtgagagca gagggccagc taagtggtac 5040
tctcccagag actgtctgac tcacgccacc ccctccacct tggacacagg acgctgtggt 5100
ttctgagcca ggtacaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag 5160
gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct 5220
gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt 5280
gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca 5340
ggcaccacca ctgacctggg acagtgaatg tccccctgat ctgcggccgt gactctctta 5400
aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga 5460
caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc 5520
tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac 5580
tcccagttca attacagctc ttaaggctag agtacttaat acgactcact ataggctagc 5640
gccaccatgg gatgggctga ggctggattc gaacacctgg gactctgggt gcccgtcctg 5700
gccgtgctgc tcctggaggc ttgcagggct catcccatcc ctgacagctc cccactcctg 5760
cagtttggag gacaggtgag gcagcggtac ctgtataccg acgatgccca ggagacagaa 5820
gctcacctgg aaattcgggc tgatggaaca gtggtcggag ctgcccgaca gtccccagag 5880
tctctcctgg aactgaaggc cctcaaaccc ggagtgatcc agattctggg cgtcaagact 5940
tctagattcc tgtgccaggg accagacggc accctgtacg gcagcctgca tttcgatcct 6000
gtggcctgtt cctttcgaga gctcctgctc gaagacggct acaacatcta tcactctgag 6060
accctgggac tcccactgcg actcagacct cataatagtg cctatcgaga tctggctccc 6120
aggggcccag ctaggtttct gccactcccc ggactgctcc ctgctccacc tgagccaccc 6180
ggcattctgg ctccagaacc tccagacgtg ggctctagtg atccactgag tatggtcggc 6240
ccctcacagg ggaggtcacc tagctacgcc agctgagc 6278
<210>39
<211>6278
<212>DNA
<213> Artificial sequence
<220>
<223>pGG2-hAAT-hsFGF21
<400>39
gatctgatat catcgatgaa ttcgagctcg gtacccggcc gcagatttag gtgacactat 60
agaatatgca tcactagtaa gcttgcgaat tccagtctac agagaggtct ctgacctctg 120
ccccagctcc aaggtcagca ggcagggagg gctgtgtgtt tgctgtttgc tgcttgcaat 180
gtttgcccat tttagggaca tgagtaggct gaagtttgtt cagtgtggac ttcagaggca 240
gcacacaaac agcaagcttg cgaattccag tctacagaga ggtctctgac ctctgcccca 300
gctccaaggt cagcaggcag ggagggctgt gtgtttgctg tttgctgctt gcaatgtttg 360
cccattttag ggacatgagt aggctgaagt ttgttcagtg tggacttcag aggcagcaca 420
caaacagcaa gcttgcgaat tccagtctac agagaggtct ctgacctctg ccccagctcc 480
aaggtcagca ggcagggagg gctgtgtgtt tgctgtttgc tgcttgcaat gtttgcccat 540
tttagggaca tgagtaggct gaagtttgtt cagtgtggac ttcagaggca gcacacaaac 600
agcaagcttt gctctagact ggaattcgtc gacgagctcc ctatagtgag tcgtattaga 660
ggccgactga cccggtaccc ggggatcttg ctaccagtgg aacagccact aaggattctg 720
cagtgagagc agagggccag ctaagtggta ctctcccaga gactgtctga ctcacgccac 780
cccctccacc ttggacacag gacgctgtgg tttctgagcc aggtacaatg actcctttcg 840
gtaagtgcag tggaagctgt acactgccca ggcaaagcgt ccgggcagcg taggcgggcg 900
actcagatcc cagccagtgg acttagcccc tgtttgctcc tccgataact ggggtgacct 960
tggttaatat tcaccagcag cctcccccgt tgcccctctg gatccactgc ttaaatacgg 1020
acgaggacag ggccctgtct cctcagcttc aggcaccacc actgacctgg gacagtgaat 1080
gtccccctga tctgcggccg tgactctctt aaggtagcct tgcagaagtt ggtcgtgagg 1140
cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg 1200
cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat 1260
ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct cttaaggcta 1320
gagtacttaa tacgactcac tataggctag cgccaccatg gactcggacg agaccgggtt 1380
cgagcactca ggactgtggg tttctgtgct ggctggtctt ctgctgggag cctgccaggc 1440
acaccccatc cctgactcca gtcctctcct gcaattcggg ggccaagtcc ggcagcggta 1500
cctctacaca gatgatgccc agcagacaga agcccacctg gagatcaggg aggatgggac 1560
ggtggggggc gctgctgacc agagccccga aagtctcctg cagctgaaag ccttgaagcc 1620
gggagttatt caaatcttgg gagtcaagac atccaggttc ctgtgccagc ggccagatgg 1680
ggccctgtat ggatcgctcc actttgaccc tgaggcctgc agcttccggg agctgcttct 1740
tgaggacgga tacaatgttt accagtccga agcccacggc ctcccgctgc acctgccagg 1800
gaacaagtcc ccacaccggg accctgcacc ccgaggacca gctcgcttcc tgccactacc 1860
aggcctgccc cccgcactcc cggagccacc cggaatcctg gccccccagc cccccgatgt 1920
gggctcctcg gaccctctga gcatggtggg accttcccag ggccgaagcc ccagctacgc 1980
ttcctgagcg gccgcttccc tttagtgagg gttaatgctt cgagcagaca tgataagata 2040
cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 2100
aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 2160
caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag 2220
caagtaaaac ctctacaaat gtggtaaaat ccgataaggg actagagcat ggctacgtag 2280
ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 2340
ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 2400
cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg ccagctggcg taatagcgaa 2460
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaattcc 2520
agacgattga gcgtcaaaat gtaggtattt ccatgagcgt ttttccgttg caatggctgg 2580
cggtaatatt gttctggata ttaccagcaa ggccgatagt ttgagttctt ctactcaggc 2640
aagtgatgtt attactaatc aaagaagtat tgcgacaacg gttaatttgc gtgatggaca 2700
gactctttta ctcggtggcc tcactgatta taaaaacact tctcaggatt ctggcgtacc 2760
gttcctgtct aaaatccctt taatcggcct cctgtttagc tcccgctctg attctaacga 2820
ggaaagcacg ttatacgtgc tcgtcaaagc aaccatagta cgcgccctgt agcggcgcat 2880
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 2940
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 3000
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 3060
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 3120
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 3180
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 3240
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 3300
taacgtctac aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat 3360
tatcaaccgg ggtacatatg attgacatgc tagttttacg attaccgttc atcgattctc 3420
ttgtttgctc cagactctca ggcaatgacc tgatagcctt tgtagagacc tctcaaaaat 3480
agctaccctc tccggcatga atttatcagc tagaacggtt gaatatcata ttgatggtga 3540
tttgactgtc tccggccttt ctcacccgtt tgaatcttta cctacacatt actcaggcat 3600
tgcatttaaa atatatgagg gttctaaaaa tttttatcct tgcgttgaaa taaaggcttc 3660
tcccgcaaaa gtattacagg gtcataatgt ttttggtaca accgatttag ctttatgctc 3720
tgaggcttta ttgcttaatt ttgctaattc tttgccttgc ctgtatgatt tattggatgt 3780
tggaatcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 3840
tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 3900
ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 3960
gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 4020
gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 4080
gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 4140
tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 4200
caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 4260
ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 4320
gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 4380
aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 4440
ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 4500
atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 4560
gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 4620
gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 4680
atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 4740
aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 4800
actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 4860
aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 4920
tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 4980
ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 5040
agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 5100
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 5160
aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 5220
gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 5280
atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 5340
gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 5400
gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 5460
tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 5520
accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 5580
ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 5640
cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 5700
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 5760
ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 5820
tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 5880
ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 5940
cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 6000
gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 6060
tggccgattc attaatgcag cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa 6120
agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag 6180
agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct 6240
acttatctac gtagccatgc tctagacatg gctcgaca 6278
<210>40
<211>6278
<212>DNA
<213> Artificial sequence
<220>
<223> pGG2-hAAT-hsoFGF21 variant 1
<400>40
gatctgatat catcgatgaa ttcgagctcg gtacccggcc gcagatttag gtgacactat 60
agaatatgca tcactagtaa gcttgcgaat tccagtctac agagaggtct ctgacctctg 120
ccccagctcc aaggtcagca ggcagggagg gctgtgtgtt tgctgtttgc tgcttgcaat 180
gtttgcccat tttagggaca tgagtaggct gaagtttgtt cagtgtggac ttcagaggca 240
gcacacaaac agcaagcttg cgaattccag tctacagaga ggtctctgac ctctgcccca 300
gctccaaggt cagcaggcag ggagggctgt gtgtttgctg tttgctgctt gcaatgtttg 360
cccattttag ggacatgagt aggctgaagt ttgttcagtg tggacttcag aggcagcaca 420
caaacagcaa gcttgcgaat tccagtctac agagaggtct ctgacctctg ccccagctcc 480
aaggtcagca ggcagggagg gctgtgtgtt tgctgtttgc tgcttgcaat gtttgcccat 540
tttagggaca tgagtaggct gaagtttgtt cagtgtggac ttcagaggca gcacacaaac 600
agcaagcttt gctctagact ggaattcgtc gacgagctcc ctatagtgag tcgtattaga 660
ggccgactga cccggtaccc ggggatcttg ctaccagtgg aacagccact aaggattctg 720
cagtgagagc agagggccag ctaagtggta ctctcccaga gactgtctga ctcacgccac 780
cccctccacc ttggacacag gacgctgtgg tttctgagcc aggtacaatg actcctttcg 840
gtaagtgcag tggaagctgt acactgccca ggcaaagcgt ccgggcagcg taggcgggcg 900
actcagatcc cagccagtgg acttagcccc tgtttgctcc tccgataact ggggtgacct 960
tggttaatat tcaccagcag cctcccccgt tgcccctctg gatccactgc ttaaatacgg 1020
acgaggacag ggccctgtct cctcagcttc aggcaccacc actgacctgg gacagtgaat 1080
gtccccctga tctgcggccg tgactctctt aaggtagcct tgcagaagtt ggtcgtgagg 1140
cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg 1200
cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat 1260
ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct cttaaggcta 1320
gagtacttaa tacgactcac tataggctag cgccaccatg gattctgatg agacaggctt 1380
cgagcacagc ggcctgtggg tttcagttct ggctggactg ctgctgggag cctgtcaggc 1440
acaccctatt ccagatagca gccctctgct gcagttcggc ggacaagtgc ggcagagata 1500
cctgtacacc gacgacgccc agcagacaga agcccacctg gaaatcagag aggatggcac 1560
agttggcgga gccgccgatc agtctcctga atctctgctc cagctgaagg ccctgaagcc 1620
tggcgtgatc cagatcctgg gcgtgaaaac cagccggttc ctgtgccaaa gacctgacgg 1680
cgccctgtat ggcagcctgc actttgatcc tgaggcctgc agcttcagag agctgctgct 1740
tgaggacggc tacaacgtgt accagtctga ggcccatggc ctgcctctgc atctgcctgg 1800
aaacaagagc cctcacagag atcccgctcc tagaggccct gccagatttc tgcctcttcc 1860
tggattgcct cctgctctgc cagagcctcc tggaattctg gctcctcagc ctcctgatgt 1920
gggcagctct gatcctctga gcatggtcgg acctagccag ggcagatctc ctagctacgc 1980
ctcttgagcg gccgcttccc tttagtgagg gttaatgctt cgagcagaca tgataagata 2040
cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 2100
aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 2160
caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag 2220
caagtaaaac ctctacaaat gtggtaaaat ccgataaggg actagagcat ggctacgtag 2280
ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 2340
ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 2400
cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg ccagctggcg taatagcgaa 2460
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaattcc 2520
agacgattga gcgtcaaaat gtaggtattt ccatgagcgt ttttccgttg caatggctgg 2580
cggtaatatt gttctggata ttaccagcaa ggccgatagt ttgagttctt ctactcaggc 2640
aagtgatgtt attactaatc aaagaagtat tgcgacaacg gttaatttgc gtgatggaca 2700
gactctttta ctcggtggcc tcactgatta taaaaacact tctcaggatt ctggcgtacc 2760
gttcctgtct aaaatccctt taatcggcct cctgtttagc tcccgctctg attctaacga 2820
ggaaagcacg ttatacgtgc tcgtcaaagc aaccatagta cgcgccctgt agcggcgcat 2880
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 2940
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 3000
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 3060
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 3120
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 3180
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 3240
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 3300
taacgtctac aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat 3360
tatcaaccgg ggtacatatg attgacatgc tagttttacg attaccgttc atcgattctc 3420
ttgtttgctc cagactctca ggcaatgacc tgatagcctt tgtagagacc tctcaaaaat 3480
agctaccctc tccggcatga atttatcagc tagaacggtt gaatatcata ttgatggtga 3540
tttgactgtc tccggccttt ctcacccgtt tgaatcttta cctacacatt actcaggcat 3600
tgcatttaaa atatatgagg gttctaaaaa tttttatcct tgcgttgaaa taaaggcttc 3660
tcccgcaaaa gtattacagg gtcataatgt ttttggtaca accgatttag ctttatgctc 3720
tgaggcttta ttgcttaatt ttgctaattc tttgccttgc ctgtatgatt tattggatgt 3780
tggaatcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 3840
tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 3900
ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 3960
gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 4020
gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 4080
gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 4140
tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 4200
caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 4260
ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 4320
gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 4380
aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 4440
ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 4500
atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 4560
gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 4620
gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 4680
atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 4740
aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 4800
actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 4860
aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 4920
tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 4980
ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 5040
agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 5100
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 5160
aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 5220
gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 5280
atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 5340
gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 5400
gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 5460
tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 5520
accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 5580
ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 5640
cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 5700
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 5760
ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 5820
tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 5880
ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 5940
cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 6000
gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 6060
tggccgattc attaatgcag cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa 6120
agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag 6180
agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct 6240
acttatctac gtagccatgc tctagacatg gctcgaca 6278
<210>41
<211>6278
<212>DNA
<213> Artificial sequence
<220>
<223> pGG2-hAAT-hsoFGF21 variant 2
<400>41
cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60
acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 120
atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca 180
tgctctagac atggctcgac agatctgata tcatcgatga attcgagctc ggtacccggc 240
cgcagattta ggtgacacta tagaatatgc atcactagta agcttgcgaa ttccagtcta 300
cagagaggtc tctgacctct gccccagctc caaggtcagc aggcagggag ggctgtgtgt 360
ttgctgtttg ctgcttgcaa tgtttgccca ttttagggac atgagtaggc tgaagtttgt 420
tcagtgtgga cttcagaggc agcacacaaa cagcaagctt gcgaattcca gtctacagag 480
aggtctctga cctctgcccc agctccaagg tcagcaggca gggagggctg tgtgtttgct 540
gtttgctgct tgcaatgttt gcccatttta gggacatgag taggctgaag tttgttcagt 600
gtggacttca gaggcagcac acaaacagca agcttgcgaa ttccagtcta cagagaggtc 660
tctgacctct gccccagctc caaggtcagc aggcagggag ggctgtgtgt ttgctgtttg 720
ctgcttgcaa tgtttgccca ttttagggac atgagtaggc tgaagtttgt tcagtgtgga 780
cttcagaggc agcacacaaa cagcaagctt tgctctagac tggaattcgt cgacgagctc 840
cctatagtga gtcgtattag aggccgactg acccggtacc cggggatctt gctaccagtg 900
gaacagccac taaggattct gcagtgagag cagagggcca gctaagtggt actctcccag 960
agactgtctg actcacgcca ccccctccac cttggacaca ggacgctgtg gtttctgagc 1020
caggtacaat gactcctttc ggtaagtgca gtggaagctg tacactgccc aggcaaagcg 1080
tccgggcagc gtaggcgggc gactcagatc ccagccagtg gacttagccc ctgtttgctc 1140
ctccgataac tggggtgacc ttggttaata ttcaccagca gcctcccccg ttgcccctct 1200
ggatccactg cttaaatacg gacgaggaca gggccctgtc tcctcagctt caggcaccac 1260
cactgacctg ggacagtgaa tgtccccctg atctgcggcc gtgactctct taaggtagcc 1320
ttgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta 1380
aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc 1440
acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt 1500
caattacagc tcttaaggct agagtactta atacgactca ctataggcta gcgccaccat 1560
ggacagcgat gaaaccgggt tcgagcacag cggtctgtgg gtgtccgtgc tggccggact 1620
gctcctggga gcctgtcagg cgcaccccat ccctgactcc tcgccgctgc tgcaattcgg 1680
cggacaagtc cgccagagat acctgtacac cgacgacgcc cagcagaccg aagcccacct 1740
ggaaattcgg gaggacggga ctgtgggagg cgctgcagat cagtcacccg agtccctcct 1800
ccaactgaag gccttgaagc ccggcgtgat tcagatcctg ggcgtgaaaa cttcccgctt 1860
cctttgccaa cggccggatg gagctctgta cggatccctg cacttcgacc ccgaagcctg 1920
ctcattccgc gagctgctcc ttgaggacgg ctataacgtg taccagtctg aggcccatgg 1980
actccccctg catctgcccg gcaacaagtc ccctcaccgg gatcctgccc caagaggccc 2040
agctcggttt ctgcctctgc cgggactgcc tccagcgttg cccgaacccc ctggtatcct 2100
ggccccgcaa ccacctgacg tcggttcgtc ggacccgctg agcatggtcg gtccgagcca 2160
gggaaggtcc ccgtcctacg catcctgagc ggccgcttcc ctttagtgag ggttaatgct 2220
tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 2280
aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 2340
ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 2400
gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg 2460
gactagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac 2520
ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc 2580
gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 2640
gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 2700
ctgaatggcg aatggaattc cagacgattg agcgtcaaaa tgtaggtatt tccatgagcg 2760
tttttccgtt gcaatggctg gcggtaatat tgttctggat attaccagca aggccgatag 2820
tttgagttct tctactcagg caagtgatgt tattactaat caaagaagta ttgcgacaac 2880
ggttaatttg cgtgatggac agactctttt actcggtggc ctcactgatt ataaaaacac 2940
ttctcaggat tctggcgtac cgttcctgtc taaaatccct ttaatcggcc tcctgtttag 3000
ctcccgctct gattctaacg aggaaagcac gttatacgtg ctcgtcaaag caaccatagt 3060
acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 3120
ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 3180
cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 3240
gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 3300
catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 3360
gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 3420
aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 3480
acgcgaattt taacaaaata ttaacgtcta caatttaaat atttgcttat acaatcttcc 3540
tgtttttggg gcttttctga ttatcaaccg gggtacatat gattgacatg ctagttttac 3600
gattaccgtt catcgattct cttgtttgct ccagactctc aggcaatgac ctgatagcct 3660
ttgtagagac ctctcaaaaa tagctaccct ctccggcatg aatttatcag ctagaacggt 3720
tgaatatcat attgatggtg atttgactgt ctccggcctt tctcacccgt ttgaatcttt 3780
acctacacat tactcaggca ttgcatttaa aatatatgag ggttctaaaa atttttatcc 3840
ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag ggtcataatg tttttggtac 3900
aaccgattta gctttatgct ctgaggcttt attgcttaat tttgctaatt ctttgccttg 3960
cctgtatgat ttattggatg ttggaatcgc ctgatgcggt attttctcct tacgcatctg 4020
tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 4080
ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 4140
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 4200
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 4260
gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 4320
cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 4380
caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 4440
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 4500
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 4560
gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 4620
atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 4680
caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 4740
gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 4800
accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 4860
ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 4920
gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 4980
acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 5040
atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 5100
ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 5160
gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 5220
gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 5280
tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 5340
taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 5400
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 5460
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 5520
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 5580
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 5640
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 5700
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 5760
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 5820
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 5880
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 5940
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 6000
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 6060
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 6120
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 6180
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 6240
aaaccgcctc tccccgcgcg ttggccgatt cattaatg 6278
<210>42
<211>6278
<212>DNA
<213> Artificial sequence
<220>
<223> pGG2-hAAT-hsoFGF21 variant 3
<400>42
cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60
acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 120
atcactaggg gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca 180
tgctctagac atggctcgac agatctgata tcatcgatga attcgagctc ggtacccggc 240
cgcagattta ggtgacacta tagaatatgc atcactagta agcttgcgaa ttccagtcta 300
cagagaggtc tctgacctct gccccagctc caaggtcagc aggcagggag ggctgtgtgt 360
ttgctgtttg ctgcttgcaa tgtttgccca ttttagggac atgagtaggc tgaagtttgt 420
tcagtgtgga cttcagaggc agcacacaaa cagcaagctt gcgaattcca gtctacagag 480
aggtctctga cctctgcccc agctccaagg tcagcaggca gggagggctg tgtgtttgct 540
gtttgctgct tgcaatgttt gcccatttta gggacatgag taggctgaag tttgttcagt 600
gtggacttca gaggcagcac acaaacagca agcttgcgaa ttccagtcta cagagaggtc 660
tctgacctct gccccagctc caaggtcagc aggcagggag ggctgtgtgt ttgctgtttg 720
ctgcttgcaa tgtttgccca ttttagggac atgagtaggc tgaagtttgt tcagtgtgga 780
cttcagaggc agcacacaaa cagcaagctt tgctctagac tggaattcgt cgacgagctc 840
cctatagtga gtcgtattag aggccgactg acccggtacc cggggatctt gctaccagtg 900
gaacagccac taaggattct gcagtgagag cagagggcca gctaagtggt actctcccag 960
agactgtctg actcacgcca ccccctccac cttggacaca ggacgctgtg gtttctgagc 1020
caggtacaat gactcctttc ggtaagtgca gtggaagctg tacactgccc aggcaaagcg 1080
tccgggcagc gtaggcgggc gactcagatc ccagccagtg gacttagccc ctgtttgctc 1140
ctccgataac tggggtgacc ttggttaata ttcaccagca gcctcccccg ttgcccctct 1200
ggatccactg cttaaatacg gacgaggaca gggccctgtc tcctcagctt caggcaccac 1260
cactgacctg ggacagtgaa tgtccccctg atctgcggcc gtgactctct taaggtagcc 1320
ttgcagaagt tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta 1380
aggagaccaa tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc 1440
acctattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt 1500
caattacagc tcttaaggct agagtactta atacgactca ctataggcta gcgccaccat 1560
ggattccgac gaaactggat ttgaacattc agggctgtgg gtctctgtgc tggctggact 1620
gctgctgggg gcttgtcagg ctcaccccat ccctgacagc tcccctctgc tgcagttcgg 1680
aggacaggtg cggcagagat acctgtatac cgacgatgcc cagcagacag aggcacacct 1740
ggagatcagg gaggacggaa ccgtgggagg agcagccgat cagtctcccg agagcctgct 1800
gcagctgaag gccctgaagc ctggcgtgat ccagatcctg ggcgtgaaga catctcggtt 1860
tctgtgccag cggcccgacg gcgccctgta cggctccctg cacttcgatc ccgaggcctg 1920
ttcttttagg gagctgctgc tggaggacgg ctacaacgtg tatcagagcg aggcacacgg 1980
cctgccactg cacctgcctg gcaataagtc ccctcaccgc gatccagcac ccaggggccc 2040
agcacgcttc ctgcctctgc caggcctgcc ccctgccctg ccagagccac ccggcatcct 2100
ggccccccag cctccagatg tgggctccag cgatcctctg tcaatggtgg ggccaagtca 2160
ggggcggagt ccttcatacg catcataagc ggccgcttcc ctttagtgag ggttaatgct 2220
tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 2280
aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 2340
ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 2400
gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tccgataagg 2460
gactagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac 2520
ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc 2580
gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 2640
gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 2700
ctgaatggcg aatggaattc cagacgattg agcgtcaaaa tgtaggtatt tccatgagcg 2760
tttttccgtt gcaatggctg gcggtaatat tgttctggat attaccagca aggccgatag 2820
tttgagttct tctactcagg caagtgatgt tattactaat caaagaagta ttgcgacaac 2880
ggttaatttg cgtgatggac agactctttt actcggtggc ctcactgatt ataaaaacac 2940
ttctcaggat tctggcgtac cgttcctgtc taaaatccct ttaatcggcc tcctgtttag3000
ctcccgctct gattctaacg aggaaagcac gttatacgtg ctcgtcaaag caaccatagt 3060
acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 3120
ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 3180
cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 3240
gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 3300
catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 3360
gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 3420
aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 3480
acgcgaattt taacaaaata ttaacgtcta caatttaaat atttgcttat acaatcttcc 3540
tgtttttggg gcttttctga ttatcaaccg gggtacatat gattgacatg ctagttttac 3600
gattaccgtt catcgattct cttgtttgct ccagactctc aggcaatgac ctgatagcct 3660
ttgtagagac ctctcaaaaa tagctaccct ctccggcatg aatttatcag ctagaacggt 3720
tgaatatcat attgatggtg atttgactgt ctccggcctt tctcacccgt ttgaatcttt 3780
acctacacat tactcaggca ttgcatttaa aatatatgag ggttctaaaa atttttatcc 3840
ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag ggtcataatg tttttggtac 3900
aaccgattta gctttatgct ctgaggcttt attgcttaat tttgctaatt ctttgccttg 3960
cctgtatgat ttattggatg ttggaatcgc ctgatgcggt attttctcct tacgcatctg 4020
tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 4080
ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 4140
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 4200
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 4260
gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 4320
cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 4380
caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 4440
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 4500
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 4560
gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 4620
atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 4680
caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 4740
gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 4800
accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 4860
ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 4920
gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 4980
acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 5040
atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 5100
ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 5160
gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 5220
gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 5280
tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 5340
taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 5400
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 5460
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 5520
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 5580
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 5640
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 5700
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 5760
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 5820
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 5880
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 5940
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 6000
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 6060
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 6120
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 6180
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 6240
aaaccgcctc tccccgcgcg ttggccgatt cattaatg 6278
<210>43
<211>133
<212>DNA
<213> Artificial sequence
<220>
<223> chimeric intron consisting of intron of human β -globulin and immunoglobulin heavy chain gene
<400>43
gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60
cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120
tttctctcca cag 133
<210>44
<211>1671
<212>DNA
<213> Artificial sequence
<220>
<223> CAG promoter
<400>44
gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 420
cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 480
tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 540
gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 600
cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 660
gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc 720
ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 780
gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 840
ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt 900
gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg 960
ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg 1020
gtgccccgcg gtgcgggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg 1080
ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct 1140
ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg 1200
gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg 1260
cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt 1320
cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga 1380
cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct 1440
agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500
cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg 1560
gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 1620
ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca g 1671
<210>45
<211>212
<212>DNA
<213> Artificial sequence
<220>
<223> CMV promoter
<400>45
gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt 60
ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120
tttccaaaat gtcgtaacaa ctgcgatcgc ccgccccgtt gacgcaaatg ggcggtaggc 180
gtgtacggtg ggaggtctat ataagcagag ct 212
<210>46
<211>380
<212>DNA
<213> Artificial sequence
<220>
<223> CMV enhancer
<400>46
ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatcatatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg 380
<210>47
<211>397
<212>DNA
<213> Artificial sequence
<220>
<223> hAAT promoter
<400>47
gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga gggccagcta 60
agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg gacacaggac 120
gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg aagctgtaca 180
ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact 240
tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct 300
cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct 360
cagcttcagg caccaccact gacctgggac agtgaat 397
<210>48
<211>128
<212>DNA
<213> Artificial sequence
<220>
<223> truncation AAV 25' ITR
<400>48
gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60
tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaactccatcactag 120
gggttcct 128
<210>49
<211>128
<212>DNA
<213> Artificial sequence
<220>
<223> truncation AAV 23' ITR
<400>49
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120
gagcgcgc 128
<210>50
<211>122
<212>DNA
<213> Artificial sequence
<220>
<223> SV40 polyadenylation Signal
<400>50
taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 60
tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 120
tt 122
<210>51
<211>449
<212>DNA
<213> Artificial sequence
<220>
<223> Rabbit β -globulin polyadenylation Signal
<400>51
gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 60
tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 120
tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt 180
tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga 240
ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg 300
acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta 360
aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc 420
atagctgtcc ctcttctctt atggagatc 449
<210>52
<211>592
<212>DNA
<213> Artificial sequence
<220>
<223> CMV promoter and CMV enhancer
<400>52
ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc 420
ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480
ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctgcgatcgc ccgccccgtt 540
gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ct 592
<210>53
<211>154
<212>DNA
<213> Artificial sequence
<220>
<223> Hepatocyte Control Region (HCR) enhancer of apolipoprotein E
<400>53
cagagaggtc tctgacctct gccccagctc caaggtcagc aggcagggag ggctgtgtgt 60
ttgctgtttg ctgcttgcaa tgtttgccca ttttagggac atgagtaggc tgaagtttgt 120
tcagtgtgga cttcagaggc agcacacaaa cagc 154
<210>54
<211>651
<212>DNA
<213> Artificial sequence
<220>
<223> mini/aP2 promoter
<400>54
gattaacccg ccatgctact tatctactcg acattgatta ttgactaggg gaattccagc 60
aggaatcagg tagctggaga atcgcacaga gccatgcgat tcttggcaag ccatgcgaca 120
aaggcagaaa tgcacatttc acccagagag aagggattga tgtcagcagg aagtcaccac 180
ccagagagca aatggagttc ccagatgcct gacatttgcc ttcttactgg atcagagttc 240
actagtggaa gtgtcacagc ccaaacactc ccccaaagct cagcccttcc ttgccttgta 300
acaatcaagc cgctcctgga tgaactgctc cgccctctgt ctctttggca gggttggagc 360
ccactgtggc ctgagcgact tctatggctc ccttttctgt gattttcatg gtttctgagc 420
tcttttcccc cgctttatga ttttctcttt ttgtctctct cttgctaaac ctccttcgta 480
tatatgccct ctcaggtttc atttctgaat catctactgt gaactattcc cattgtttgc 540
cagaagcccc ctggttcttc cttctagaca ccaggcaagg ggcaggaggt aagaggcagg 600
agtccataaa acagccctga gagcctgctg ggtcagtgcc tgctgtcaga a 651
<210>55
<211>722
<212>DNA
<213> Artificial sequence
<220>
<223> mini/UCP1 promoter
<400>55
gacgtcacag tgggtcagtc acccttgatc acactgcacc agtcttcacc tttccacgct 60
tcctgccaga gcatgaatca ggctctctgg ggataccggc ctcaccccta ctgaggcaaa 120
ctttctccca cttctcagag gctctgaggg cagcaaggtc agccctttct ttggaatcta 180
gaaccactcc ctgtcttgag ctgacatcac agggcaggca gatgcagcag ggaagggcct 240
gggactggga cgttcatcct acaagaaagc tgtggaactt ttcagcaaca tctcagaaat 300
cagatcgcac ttattcaaag gagccaggcc ctgctctgcg ccctggtgga ggctcctcat 360
gtgaagagtg acaaaaggca ccatgttgtg gatacggggc gaagcccctc cggtgtgtcc 420
tccaggcatc atcaggaact agtgccaaag cagaggtgct ggccagggct ttgggagtga 480
cgcgcgtctg ggaggcttgt gcgcccaggg cacgcccctg ccgattccca ctagcaggtc 540
ttgggggacc tgggccggct ctgcccctcc tccagcaatc gggctataaa gctcttccaa 600
gtcagggcgc agaagtgccg ggcgatccgg gcttaaagag cgagaggaag ggacgctcac 660
ctttgagctc ctccacaaat agccctggtg gctgccacag aagttcgaag ttgagagttc 720
gg 722
<210>56
<211>326
<212>DNA
<213> Artificial sequence
<220>
<223> C5-12 promoter
<400>56
cggccgtccg ccttcggcac catcctcacg acacccaaat atggcgacgg gtgaggaatg 60
gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt tggcgctcta 120
aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca aatatggcga 180
cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg cattcctggg 240
ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg cggcccacga 300
gctacccgga ggagcgggag gcgcca 326
<210>57
<211>4930
<212>DNA
<213> Artificial sequence
<220>
<223>pAAV-EF1a-mmFGF21-pA
<400>57
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgcg gctccggtgc ccgtcagtgg gcagagcgca 180
catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga 240
gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg 300
agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg 360
ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta 420
cgggttatgg cccttgcgtg ccttgaatta cttccactgg ctgcagtacg tgattcttga 480
tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc 540
ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg 600
gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg 660
atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct 720
gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg 780
cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc 840
tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg 900
ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg 960
ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc 1020
acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga 1080
gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt 1140
aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga 1200
agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg 1260
atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt 1320
gtcgtgagga atttcgactg ctagcacgcg tgatatcaat ggaatggatg agatctagag 1380
ttgggaccct gggactgtgg gtccgactgc tgctggctgt cttcctgctg ggggtctacc 1440
aagcataccc catccctgac tccagccccc tcctccagtt tgggggtcaa gtccggcaga 1500
ggtacctcta cacagatgac gaccaagaca ctgaagccca cctggagatc agggaggatg 1560
gaacagtggt aggcgcagca caccgcagtc cagaaagtct cctggagctc aaagccttga 1620
agccaggggt cattcaaatc ctgggtgtca aagcctctag gtttctttgc caacagccag 1680
atggagctct ctatggatcg cctcactttg atcctgaggc ctgcagcttc agagaactgc 1740
tgctggagga cggttacaat gtgtaccagt ctgaagccca tggcctgccc ctgcgtctgc 1800
ctcagaagga ctccccaaac caggatgcaa catcctgggg acctgtgcgc ttcctgccca 1860
tgccaggcct gctccacgag ccccaagacc aagcaggatt cctgccccca gagcccccag 1920
atgtgggctc ctctgacccc ctgagcatgg tagagccttt acagggccga agccccagct 1980
atgcgtcctg agatatcaaa gaattctaag cttgtcgacg aatgcaattg ttgttaatta 2040
attgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 2100
ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 2160
gtatcttagt cgagttaatt aacggcggcc gcaggaaccc ctagtgatgg agttggccac 2220
tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 2280
gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct 2340
gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa 2400
ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 2460
gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 2520
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 2580
cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt 2640
agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 2700
aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt 2760
gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 2820
aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt 2880
acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac 2940
gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 3000
gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 3060
ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 3120
ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 3180
tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 3240
aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 3300
tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 3360
ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 3420
tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 3480
gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 3540
aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 3600
agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 3660
acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 3720
actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 3780
accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 3840
actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 3900
cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 3960
cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 4020
gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 4080
ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 4140
tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 4200
aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 4260
gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 4320
acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 4380
tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 4440
ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 4500
atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 4560
agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 4620
cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 4680
agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 4740
acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 4800
gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 4860
ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 4920
gctcacatgt 4930

Claims (28)

1. A viral expression construct suitable for expression in a mammal and comprising a nucleotide sequence encoding fibroblast growth factor 21(FGF21) to be expressed in liver, adipose tissue and/or skeletal muscle.
2. The viral expression construct of claim 1, comprising a nucleotide sequence encoding FGF21 suitable for expression in a mammal and at least one of elements a), b), c), d), and e):
(a) liver-specific promoters
(b) Adipose tissue specific promoters
(c) Combination of a ubiquitous promoter with at least one nucleotide sequence coding for a target sequence of a microRNA expressed in the liver and at least one nucleotide sequence coding for a target sequence of a microRNA expressed in the heart, wherein said combination is capable of specific expression in adipose tissue
(d) Skeletal muscle promoter and
(e) a combination of a ubiquitous promoter and an adeno-associated virus (AAV) vector sequence, wherein said combination is capable of specific expression in skeletal muscle.
3. The viral expression construct according to claim 1 or 2, wherein the nucleotide sequence encoding FGF21 suitable for expression in a mammal is selected from the group consisting of:
(a) encoding a polypeptide comprising a sequence identical to SEQ ID NO: 1. 2 or 3 has at least 60% sequence identity
(b) And SEQ ID NO: 4.5, 6, 7, 8, 9, 10 or 11 has at least 60% sequence identity of the nucleotide sequence
(c) A nucleotide sequence whose sequence differs from that of the nucleotide sequence of (b) due to the degeneracy of the genetic code.
4. The viral expression construct according to claim 2 or 3, wherein 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 the sequences SEQ ID NO: 12 to 30 and/or combinations thereof.
5. The viral expression construct according to any one of claims 2 to 4, wherein said liver-specific promoter is the human α 1-antitrypsin (hAAT) promoter and/or said adipose tissue-specific promoter is the mini/aP2 and/or mini/UCP1 promoter and/or said skeletal muscle promoter is the C5-12 promoter and/or said ubiquitous promoter is the Cytomegalovirus (CMV) promoter and/or the CAG promoter.
6. A viral vector comprising the viral expression construct according to any one of claims 1 to 5, wherein the viral vector is an adenoviral, adeno-associated viral, retroviral or lentiviral vector, preferably an adeno-associated viral vector selected from the group consisting of an adeno-associated virus 1(AAV1) vector, an adeno-associated virus 8(AAV8) vector and an adeno-associated virus 9(AAV9) vector.
7.A nucleic acid molecule suitable for expression in a mammal and represented by a mammalian codon-optimized nucleotide sequence encoding FGF21 to be expressed in liver, adipose tissue and/or skeletal muscle.
8. The nucleic acid molecule of claim 7, wherein the nucleotide sequence is identical to SEQ id no: 4.5, 6, 7, 8, 9, 10, or 11 has at least 70% sequence identity.
9.A composition comprising 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 one or more pharmaceutically acceptable excipients or vectors.
10.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 composition as defined in claim 9 for use as a medicament.
11. 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 composition as defined in claim 9 for use in the treatment and/or prevention of a metabolic disorder.
12. Viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition according to claim 11, wherein said metabolic disorder is diabetes and/or obesity.
13. The viral expression construct and/or viral vector and/or nucleic acid molecule and/or composition according to claim 11, wherein the metabolic disorder is NASH.
14. 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 composition as defined in claim 9 for use in the treatment and/or prevention of liver inflammation and/or fibrosis.
15. 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 composition as defined in claim 9 for use in extending the health life.
16. 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 composition as defined in claim 9 for use in the treatment and/or prevention of cancer, preferably liver cancer.
17.A method for preventing, delaying, reversing, curing and/or treating a metabolic disorder comprising the use of 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 composition as defined in claim 9.
18. The method of claim 17, wherein the metabolic disorder is diabetes and/or obesity.
19. The method of claim 17, wherein the metabolic disorder is NASH.
20. A method for preventing, delaying, reversing, curing and/or treating liver inflammation and/or fibrosis, comprising the use of 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 composition as defined in claim 9.
21. A method for extending the health life comprising the use of 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 composition as defined in claim 9.
22.A method for the prevention, delay, reversal, cure and/or treatment of cancer, preferably liver cancer, comprising the use of 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 composition as defined in claim 9.
23. Use of 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 composition as defined in claim 9 for the manufacture of a medicament for the prevention, delay, reversal, cure and/or treatment of a metabolic disorder.
24. The use according to claim 23, wherein the metabolic disorder is obesity and/or diabetes.
25. The use of claim 23, wherein the metabolic disorder is NASH.
26. Use of 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 composition as defined in claim 9 for the manufacture of a medicament for the prevention, delay, reversal, cure and/or treatment of liver inflammation and/or fibrosis.
27. Use of 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 composition as defined in claim 9 for the manufacture of a medicament for extending the health life.
28. Use of 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 composition as defined in claim 9 for the manufacture of a medicament for the prevention, delay, reversal, cure and/or treatment of cancer, preferably liver cancer.
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CN117247973B (en) * 2022-04-19 2024-05-10 康霖生物科技（杭州）有限公司 Nucleic acid construct for treating hereditary coagulation factor deficiency disease and application thereof

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