Fibroblast growth factor 21 (FGF-21) gene therapy

Abstract

FIELD: biotechnology.SUBSTANCE: embodiments of a recombinant vector based on rAAV for use in the treatment or prevention of diabetes and obesity. Said vector rAAV comprises (i) a gene construct comprising a nucleotide sequence encoding FGF-21 operably linked to a universal promoter and (ii) an AAV capsid protein, wherein said rAAV vector is introduced into the cerebrospinal fluid and FGF-21 is expressed in the CNS. Pharmaceutical compositions containing such a vector are also provided.EFFECT: reliable for therapy aimed at the brain.19 cl, 19 dwg, 4 ex

Description

[0001] Level of technology

[0002] The prevalence of diabetes is increasing at an alarming rate and represents a significant global public health problem. Obesity is significantly associated with insulin resistance and type 2 diabetes (T2D) (Moller, D. and Flier, J.S., 1991. Engl. J. Med. 325:938-948). Both T2DM and obesity increase the risk of mortality (Peeters, A. et al., 2003. Ann. Intern. Med. 138:24-32) and also increase the risk of chronic diseases with high morbidity, including cardiovascular disease, hypertension, and certain types of cancer (Haslam, DW et al., 2005, Lancet. 366, 1197-1209; Roberts, DL et al., 2010, Annu. Rev. Med. 61, 301-316). Insulin resistance and obesity-associated diseases are associated with subsequent reduction in life expectancy and worsening quality of life.

[0003] At this point, it is generally accepted that in obese conditions, chronic low-grade inflammation occurs in peripheral tissues such as adipose tissue, liver, or skeletal muscle, which may cause metabolic dysfunction, including the development of insulin resistance (Valdearcos, M. et al., 2015, Annu. Rev. Physiol. 77, 131-160; Hotamisligil, GS et al., 2017, Nature. 542, 177-185). Recently, a growing number of sources have demonstrated that obesity and insulin resistance are also associated with inflammation in the brain (Guillemot-Legris, O. et al., 2017, Trends Neurosci. 40, 237-253; Beilharz, JE et al., 2016, Behav. Brain Res. 306, 1-7). Moreover, obesity and insulin resistance are associated not only with neuroinflammation but also with cognitive impairment in animal models and humans (Guillemot-Legris, O. et al., 2017, Trends Neurosci. 40, 237-253).

[0004] Fibroblast growth factor 21 (FGF-21), a growth factor that is secreted predominantly in the liver but also in adipose tissue and pancreas (Muise, ES et al., 2008. Mol. Pharmacol. 74:403-412), has been shown to increase brown adipose tissue (BAT) growth and thermogenic gene expression in BAT and white adipose tissue (WAT), stimulating energy expenditure (Coskun, T. et al., 2008. Endocrinology 149:6018-6027; Fisher, F. M. et al., 2012. Genes Dev. 26:271-281; ​​Kharitonenkov, A. et al., 2005. J. Clin. Invest 115:1627-1635; Konishi, M. et al., 2000. J Biol. Chem. 275:12119-12122; Tomlinson, E. et al. 2002. Endocrinology 143:1741-1747; Xu, J. et al., 2009. Diabetes 58:250-259).

[0005] Native FGF-21 protein exhibits poor pharmacokinetic characteristics. 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, WY and Leung, PS 2016. Med Res Rev. 36(4):672-704; Zhang, J. and Li, Y. 2015. Front Endocrinol (Lausanne). 6:168). Various engineering approaches have been developed to increase the half-life and improve the stability and solubility of FGF-21. Currently, two engineered FGF-21 mimetics (LY2405319 and PF-05231023) are being tested in humans. However, these FGF-21 mimetics require multiple administrations, which places a significant burden on patients.Furthermore, engineered FGF-21 mimetics / analogues may exhibit a higher risk of immunogenicity than native FGF-21, for example, injection site reactions, drug antibodies, and severe hypersensitivity reactions have been reported in patients treated with LY2405319 (Gaich, G. et al., 2013. Cell Metab. 18(3):333-40). Thus, the long-term and efficient expression provided by a single administration of the vectors of the present invention represents a significant advantage over other therapies.

[0006] Given the important role that neuroinflammation appears to play in cognitive decline, as well as in whole-body energy and glucose metabolism observed in diabetes and obesity, new therapeutic approaches targeting central nervous system (CNS) inflammation may be of significant importance. Recent studies have demonstrated that the peripheral metabolic effects of FGF-21 may indeed be mediated by FGF-21 signaling in the CNS, particularly in the hypothalamus, which is the main brain region regulating energy metabolism at the whole-body level (DA Sarrufet al., Diabetes. 59, 1817–1824 (2010); AL Bookoutet al., Nat. Med. 19, 1147–1152 (2013); BM Owenet al., Cell Metab. 20, 670–677 (2014); N Douriset al., Endocrinology. 156, 2470–2481 (2015).

[0007] Technical field

[0008] The aspects described in this document relate to the field of medicine, including gene therapy compositions intended for use in the treatment of a metabolic disorder in mammals, in particular in humans.

[0009] Brief description of the invention

[0010] According to the first aspect, there is provided a gene construct that comprises a nucleotide sequence encoding fibroblast growth factor 21 (FGF-21) for use in therapy, wherein said therapy comprises expressing said gene construct in the central nervous system (CNS), preferably in the brain, more preferably in the hypothalamus. According to some embodiments, there is provided a gene construct that comprises a nucleotide sequence encoding fibroblast growth factor 21 (FGF-21) for use in treating a metabolic disorder, wherein said therapy comprises expressing said gene construct in the central nervous system (CNS), preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0011] Preferably, the nucleotide sequence encoding FGF-21 is operably linked to a universal promoter. In a preferred embodiment, the universal promoter is selected from the group consisting of the CAG (CMV early enhancer / chicken beta-actin promoter) and the CMV (cytomegalovirus, CMV) promoter, wherein the universal promoter is preferably the CAG promoter. Preferably, the nucleotide sequence encoding FGF-21 is operably linked to the universal promoter and at least one target sequence of microRNA expressed in the tissue in which it is desired to prevent the expression of FGF-21.

[0012] Preferably, at least one target sequence of the microRNA is selected from those target sequences that bind to microRNAs expressed in the heart and / or liver of a mammal.

[0013] More preferably, the nucleotide sequence that encodes FGF-21 is operably linked to a universal promoter and at least one target sequence of microRNA expressed in the liver, as well as at least one target sequence of microRNA expressed in the heart.

[0014] Preferably, the target sequence of the microRNA that is expressed in the heart is selected from SEQ ID NO: 13 and 21-25, and the target sequence of the microRNA that is expressed in the liver is selected from SEQ ID NO: 12 and 14-20.

[0015] More preferably, said gene construct comprises a target sequence of microRNA-122a and a target sequence of microRNA-1.

[0016] Preferably, the universal promoter is selected from the group consisting of a CAG promoter and a CMV promoter, wherein preferably the universal promoter is a CAG promoter.

[0017] Preferably, the nucleotide sequence encoding FGF-21 is selected from the group consisting of:

[0018] (a) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that has at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1, 2 or 3;

[0019] (b) a nucleotide sequence that has at least 60% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11; and

[0020] (c) a nucleotide sequence, the sequence of which differs from the sequence of the nucleotide sequence according to (b) due to the degeneracy of the genetic code.

[0021] According to the second aspect, there is provided an expression vector that comprises a gene construct as described in the first aspect, for use in therapy, wherein said therapy comprises expressing said gene construct in the CNS, preferably in the brain, more preferably in the hypothalamus. According to some embodiments, there is provided an expression vector that comprises a gene construct as described in the first aspect, for use in treating a metabolic disorder, wherein said therapy comprises expressing said gene construct in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus. Preferably, the expression vector is a viral vector.

[0022] The expression vector is preferably selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors and lentiviral vectors, wherein the expression vector is preferably an adeno-associated viral vector.

[0023] Preferably, the expression vector is an adeno-associated virus vector of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10, rh8, Cb4, rh74, DJ, 2 / 5, 2 / 1, 1 / 2 or Anc80, wherein preferably the expression vector is an adeno-associated virus vector of serotype 1, 2 or 9.

[0024] According to the third aspect, a pharmaceutical composition is provided that comprises a gene construct according to the description of the first aspect and / or an expression vector according to the description of the second aspect, together with one or more pharmaceutically acceptable ingredients, for use in therapy, wherein said therapy includes expression of said gene construct in the CNS and / or in the brain.According to some embodiments, there is provided a pharmaceutical composition that comprises a gene construct as described in the first aspect and / or an expression vector as described in the second aspect, together with one or more pharmaceutically acceptable ingredients, for use in the treatment of a metabolic disorder, wherein said therapy comprises expressing said gene construct in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0025] According to the fourth aspect, there is provided a gene construct for use according to the description of the first aspect, and / or an expression vector for use according to the description of the second aspect, and / or a pharmaceutical composition for use according to the description of the third aspect, wherein said gene construct and / or said expression vector and / or said pharmaceutical composition are administered by introduction into the CSF (cerebrospinal fluid).

[0026] According to the fifth aspect, there is provided a gene construct for use according to the description of the first aspect, and / or an expression vector for use according to the description of the second aspect, and / or a pharmaceutical composition for use according to the description of the third aspect, for use in the treatment and / or prevention of a metabolic disorder, wherein preferably said metabolic disorder is diabetes and / or obesity.

[0027] Description

[0028] The present inventors have developed an improved gene therapy strategy based on FGF-21 gene therapy targeting the central nervous system (CNS) to combat obesity and / or diabetes. Specifically, as detailed in the experimental section, the present inventors have discovered the following unexpected benefits of brain-targeted FGF-21 gene therapy:

[0029] • The gene constructs and vectors as described herein can provide robust and widespread overexpression in the brain (Examples 1, 2, 3 and 4)

[0030] • The gene constructs and vectors as described herein cause a decrease in adipocyte size, a decrease in fat accumulation in brown adipocytes, increased thermogenesis, a decrease in circulating triglycerides and free fatty acids, pancreatic health (increased islet number, improved beta cell mass), and a decrease in systemic inflammation (decreased levels of proinflammatory cytokines such as F4 / 80, IL6, TNF-alpha) (Example 1.1).

[0031] • In a widely used mouse model of obesity and diabetes, FGF-21 expression in the brain resulted in a marked reduction in body weight gain, fat deposition, and liver weight, as well as a complete normalization of postprandial glycemia (Example 1), improvement in insulin resistance, improvement in glucose tolerance, and a decrease in gluconeogenesis (Example 4).

[0032] • In a widely used mouse model of aging with age-related brain pathologies, FGF-21 expression in the brain resulted in a clear reduction in body weight gain and liver weight (Example 2).

[0033] • In both mouse models, hypothalamic inflammation is reduced (Examples 1, 2).

[0034] Thus, the aspects and embodiments of the present invention as described herein solve at least some of the problems and needs as discussed herein.

[0035] Gene construct

[0036] According to the first aspect, a gene construct is proposed that contains a nucleotide sequence encoding fibroblast growth factor 21 (FGF-21).

[0037] The term "gene construct" as used herein has the generally accepted, ordinary meaning known to those skilled in the art, taking into account the disclosure of the present invention. A "gene construct" may also be referred to as an "expression cassette" or an "expression construct" and refers to a gene or group of genes containing a gene that encodes a protein of interest, operably linked to a promoter that controls its expression. The portion of this application under the heading "general information" contains more details regarding the term "gene construct." The term "operably linked" as used herein is further described in the portion of this document under the heading "general information."

[0038] According to some embodiments, the gene construct as described herein is suitable for expression in mammals. As used herein, the term "suitable for expression in mammals" may mean that the gene construct comprises one or more regulatory sequences selected based on the mammalian host cells to be used for expression and operably linked to the nucleotide sequence to be expressed. Preferably, said mammalian host cells to be used for expression are human, mouse, or dog cells.

[0039] According to some embodiments, the gene construct as described herein is for use in therapy. According to a preferred embodiment, the gene construct as described herein is for use in the treatment and / or prevention of a metabolic disorder. According to a preferred embodiment, said therapy may mean expressing said gene construct in the CNS, preferably in the brain, more preferably in the hypothalamus. According to some embodiments, expressing the gene construct in the brain may mean expressing the gene construct in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb, preferably in the hypothalamus.Thus, expression of the gene construct in the brain may mean expression of the gene construct in at least one, or at least two, or at least three, or all brain regions selected from the group consisting of the hypothalamus, cerebral cortex, hippocampus, cerebellum, and olfactory bulb. According to a preferred embodiment, the therapy comprises expression of said gene construct in the hypothalamus. According to some embodiments, expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb may mean specific expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb.In an embodiment, the expression does not include expression in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. In some embodiments, the expression does not include expression in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, and heart. A description of CNS-specific expression and / or brain-specific expression and / or hypothalamus-specific expression and / or cerebral cortex-specific expression and / or hippocampus-specific expression and / or cerebellum-specific expression and / or olfactory bulb-specific expression is provided in the section entitled "General Information."

[0040] Expression can be assessed as described in the section titled "General Information." The terms "CNS," "brain," and "hypothalamus" are described in the section titled "General Information."

[0041] According to some embodiments, the gene construct as described herein is intended for use in therapy, wherein said gene construct is administered by administration into the CSF (cerebrospinal fluid) (via delivery into the cisterna magna, intrathecal or intraventricular delivery), by intraparenchymal administration or intranasal administration. The preferred route of administration is administration into the CSF.

[0042] The terms "administration into the CSF," "intranasal administration," "intraparenchymal administration," "administration into the cisterna magna," "intrathecal administration," and "intraventricular administration" are described in the portion of this application under the heading "general information" in this document.

[0043] According to some embodiments, the gene construct as described herein comprises a nucleotide sequence encoding FGF-21 for expression in the CNS, preferably in the brain, more preferably in the hypothalamus. According to some embodiments, the gene construct as described herein is suitable for expression in the CNS, preferably in the brain, more preferably in the hypothalamus. According to some embodiments, expression of the gene construct in the brain may mean expression of the gene construct in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb.Thus, expression of the gene construct in the brain may mean expression of the gene construct in at least one, or at least two, or at least three, or all brain regions selected from the group consisting of the hypothalamus, cerebral cortex, hippocampus, cerebellum, and olfactory bulb. Expression in the hypothalamus is most preferred. Expression can be assessed as described in the section entitled "General Information."

[0044] In the context of embodiments of the present invention, FGF-21 for expression in the CNS, and / or in the brain, and / or in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb; and a gene construct suitable for expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb refer to the preferential or predominant expression of FGF-21 (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) in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum,and / or in the olfactory bulb compared to other organs or tissues. The other organs or tissues may be the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, etc. Preferably, the other organs are the liver and / or heart. According to an embodiment, the expression is not detectable in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. According to some embodiments, the expression is not detectable in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen,stomach and testicle. Expression can be assessed as described in the section titled "General Information."

[0045] The nucleotide sequence encoding FGF-21 present in the gene construct according to the invention may be derived from any FGF-21 gene or FGF-21 coding sequence, preferably from an FGF-21 gene or FGF-21 coding sequence of a human, mouse or dog; or from a mutated FGF-21 gene or mutated FGF-21 coding sequence, preferably a human, mouse or dog; or from a codon-optimized FGF-21 gene or codon-optimized FGF-21 coding sequence, preferably a human, mouse or dog.

[0046] Thus, according to some embodiments, the preferred nucleotide sequence encoding FGF-21 encodes a polypeptide that comprises an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% identity or similarity to SEQ ID NO: 1, 2, or 3. SEQ ID NO: 1 is the amino acid sequence of human FGF-21. SEQ ID NO: 2 is the amino acid sequence of mouse FGF-21. SEQ ID NO: 3 is the amino acid sequence of canine FGF-21. According to some embodiments, the nucleotide sequence that encodes FGF-21 that is present in the gene construct according to the invention has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any sequence selected from the group consisting of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11.

[0047] The terms "identity" or "sequence identity" and "similarity" or "sequence similarity" are described in the section under the heading "general information".

[0048] According to some embodiments, the nucleotide sequence encoding human FGF-21 that is present in the gene construct of the invention has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4, 5, 6, or 7. SEQ ID NO: 4 is a nucleotide sequence that encodes human FGF-21. SEQ ID NO: 5 is a codon-optimized nucleotide sequence that encodes human FGF-21, variant 1. SEQ ID NO: 6 is a codon-optimized nucleotide sequence that encodes human FGF-21, variant 2. SEQ ID NO: 7 is a codon-optimized nucleotide sequence that encodes human FGF-21, variant 3. Variant 1, variant 2, and variant 3 encode the same human FGF-21 protein and were obtained using different codon optimization algorithms. A description of the term "codon optimization" is provided in the section under the heading "General Information".

[0049] According to some embodiments, the nucleotide sequence encoding mouse FGF-21 that is present in the gene construct of the invention has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 9. SEQ ID NO: 8 is a nucleotide sequence that encodes mouse FGF-21. SEQ ID NO: 9 is a codon-optimized nucleotide sequence that encodes mouse FGF-21.

[0050] According to some embodiments, the nucleotide sequence encoding canine FGF-21 that is present in the gene construct of the invention has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or 11. SEQ ID NO: 10 is a nucleotide sequence that encodes canine FGF-21. SEQ ID NO: 11 is a codon-optimized nucleotide sequence that encodes canine FGF-21.

[0051] According to some embodiments, a gene construct according to the present description is provided, wherein the nucleotide sequence encoding FGF-21 is selected from the group consisting of:

[0052] (a) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 99% or 100% identity or similarity to the amino acid sequence of SEQ ID NO: 1, 2 or 3.,

[0053] (b) a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11.

[0054] (c) a nucleotide sequence, the sequence of which differs from the sequence of the nucleotide sequence according to (b) due to the degeneracy of the genetic code.

[0055] According to a preferred embodiment, the nucleotide sequence that encodes FGF-21 is a codon-optimized nucleotide sequence, preferably a human codon-optimized nucleotide sequence, preferably selected from SEQ ID NO: 5, 6 and 7.

[0056] FGF-21 encoded by the nucleotide sequences described herein exhibits at least a detectable level of FGF-21 activity, as known to those skilled in the art. FGF-21 activity can manifest itself as an anti-obesity and / or anti-diabetic effect, as described in more detail below. FGF-21 activity can also include increasing insulin sensitivity. This activity can be assessed using methods known to those skilled in the art, for example, using an insulin tolerance test or a glucose tolerance test.

[0057] According to some embodiments, the nucleotide sequence encoding FGF-21 is operably linked to a universal promoter. A preferred universal promoter is selected from the CMV promoter and the CAG promoter, preferably the CAG promoter. According to some embodiments, the nucleotide sequence encoding FGF-21 is operably linked to a universal promoter and at least one target sequence of a microRNA expressed in a tissue in which it is desired to prevent FGF-21 expression.

[0058] The terms "universal promoter," "operably linked," and "microRNA" are described in the section titled "General Information." The term "microRNA target sequence expressed in tissue," or "target sequence that binds to microRNA expressed in tissue," or "microRNA binding site expressed in tissue" as used herein refers to a nucleotide sequence that is complementary or partially complementary to at least a portion of the microRNA expressed in said tissue, as described herein.

[0059] According to some embodiments, at least one target sequence of the microRNA is selected from those target sequences that bind to microRNAs expressed in the heart and / or liver of the mammal.

[0060] According to some embodiments, the nucleotide sequence that encodes FGF-21 is operably linked to a universal promoter and to at least one microRNA target sequence expressed in the liver, as well as to at least one microRNA target sequence expressed in the heart.

[0061] In the present description, the term "target sequence of a microRNA expressed in the liver," or "target sequence that binds to a microRNA expressed in the liver," or "binding site of a microRNA expressed in the liver" refers to a nucleotide sequence that is complementary or partially complementary to at least a portion of the microRNA expressed in the liver. Similarly, in the present description, the term "target sequence of a microRNA expressed in the heart," or "target sequence that binds to a microRNA expressed in the heart," or "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 the microRNA expressed in the heart.

[0062] A region of a microRNA expressed in the liver or a region of a microRNA expressed in the heart, according to the present description, means a nucleotide sequence of at least four, at least five, at least six, or at least seven consecutive nucleotides of said microRNA. The binding site sequence may have perfect complementarity with respect to at least the region of the expressed microRNA, that is, said sequences perfectly correspond to each other without any mismatches. Alternatively, the binding site sequence may be partially complementary to at least the region of the expressed microRNA, and this means the possibility of one mismatch in four, five, six, or seven consecutive nucleotides.Partially complementary binding sites preferably have perfect or nearly perfect complementarity with the seed region of the microRNA, i.e., there may be no mismatch between the seed region of the microRNA and its binding site (perfect complementarity) or there may be one mismatch per four, five, six, or seven consecutive nucleotides (nearly perfect complementarity). The seed region of the microRNA consists of the 5' region of the microRNA from approximately nucleotide 2 to approximately nucleotide 8 of said microRNA. The region according to the present description is preferably the seed region of said microRNA. Degradation of messenger RNA (mRNA), which contains the target sequence for microRNA expressed in the liver or for microRNA expressed in the heart, can be carried out through the RNA interference pathway or by direct control of translation (inhibition) of mRNA.The present invention is in no way limited by the pathway that the miRNA (miRNA) ultimately utilizes to inhibit the expression of the transgene or encoded protein.

[0063] In the context of the present invention, a target sequence that binds to microRNAs expressed in the liver can be replaced with a nucleotide sequence that comprises a nucleotide sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12 or 14-20.,

[0064] According to a preferred embodiment, the target sequence of the microRNA expressed in the liver can be replaced with a nucleotide sequence that comprises a nucleotide sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 12. According to another embodiment, at least one copy of the liver-expressed miRNA target sequence as described in SEQ ID NO: 12 or 14-20 is present in the gene construct of the present invention. According to another embodiment, two, three, four, five, six, seven or eight copies of the liver-expressed miRNA target sequence as described in SEQ ID NO: 12 or 14-20 are present in the gene construct of the present invention. According to a preferred embodiment, one, two, three, four, five, six, seven or eight copies of the miRT-122a sequence (SEQ ID NO: 12) are present in the gene construct of the present invention. The preferred copy number of the liver-expressed miRNA target sequence is four copies.

[0065] The liver-expressed microRNA target sequence according to the present disclosure exhibits at least a detectable level of liver-expressed microRNA target sequence activity known to those skilled in the art. The liver-expressed microRNA target sequence activity comprises binding to a cognate liver-expressed microRNA and, when functionally bound to a transgene, mediating the abolition of liver-expressed transgene targeting. This activity can be assessed by measuring liver-expressed transgene mRNA or protein levels using standard assays known to those skilled in the art, such as qPCR (quantitative polymerase chain reaction), Wernblot, or ELISA.

[0066] In the context of the present invention, a target sequence that binds to microRNAs expressed in the heart can be replaced with a nucleotide sequence that comprises a nucleotide sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 13 or 21-25.,

[0067] According to a preferred embodiment, the target sequence of the microRNA expressed in the heart can be replaced with a nucleotide sequence that comprises a nucleotide sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 13. According to another embodiment, at least one copy of the target sequence of the microRNA expressed in the heart as described in SEQ ID NO: 13 or 21-25 is present in the gene construct of the present invention. According to another embodiment, two, three, four, five, six, seven or eight copies of the target sequence of the microRNA expressed in the heart as described in 13 or 21-25 are present in the gene construct of the present invention. According to a preferred embodiment, one, two, three, four, five, six, seven or eight copies of the nucleotide sequence that encodes miRT-1 (SEQ ID NO: 13) are present in the gene construct of the present invention. The preferred copy number of the target sequence of the microRNA expressed in the heart is four copies.

[0068] The cardiac-expressed microRNA target sequence in the present disclosure exhibits at least a detectable level of cardiac-expressed microRNA target sequence activity, as known to those skilled in the art. The cardiac-expressed microRNA target sequence activity comprises binding to a cognate cardiac-expressed microRNA and, when functionally bound to a transgene, mediating the abolition of cardiac transgene expression targeting. This activity can be assessed by measuring cardiac transgene expression levels at the mRNA or protein level using standard assays known to those skilled in the art, such as qPCR, Wernblot, or ELISA.

[0069] According to some embodiments, the gene construct of the present invention comprises at least one copy of the target sequence of the microRNA expressed in the liver as described in SEQ ID NO: 12 or 14-20, and at least one copy of the target sequence of the microRNA expressed in the heart as described in SEQ ID NO: 13 or 21-25. According to a further embodiment, the gene construct of the present invention comprises two, three, four, five, six, seven or eight copies of the target sequence of the microRNA expressed in the liver as described in SEQ ID NO: 12 or 14-20, and two, three, four, five, six, seven or eight copies of the target sequence of the microRNA expressed in the heart as described in SEQ ID NO: 13 or 21-25.According to a further embodiment, the gene construct of the present invention combines one, two, three, four, five, six, seven or eight copies of the nucleotide sequence encoding miRT-122a (SEQ ID NO: 12), and one, two, three, four, five, six, seven or eight copies of the nucleotide sequence encoding miRT-1 (SEQ ID NO: 13). According to a further embodiment, the gene construct of the present invention combines four copies of the nucleotide sequence encoding miRT-122a (SEQ ID NO: 12) and four copies of the nucleotide sequence encoding miRT-1 (SEQ ID NO: 13).

[0070] According to some embodiments, there is provided a gene construct as described above, wherein the target sequence of the microRNA expressed in the liver and the target sequence of the microRNA expressed in the heart are selected from the group consisting of SEQ ID NOs: 12-25 and / or combinations thereof. According to some embodiments, there is provided a gene construct as described above, wherein the target sequence of the microRNA expressed in the heart is selected from SEQ ID NOs: 13 and 12-25, and the target sequence of the microRNA expressed in the liver is selected from SEQ ID NOs: 12 and 14-20. According to some embodiments, there is provided a gene construct as described above, wherein said gene construct comprises a target sequence of microRNA-122a and a target sequence of microRNA-1.

[0071] According to some embodiments, the universal promoter according to the present disclosure is selected from the group consisting of a CAG promoter, a CMV promoter, a minimal CMV promoter, a β-actin promoter, a Rous sarcoma virus (RSV) promoter, an elongation factor 1 alpha (EF1α) promoter, an early growth response factor 1 (Egr-1) promoter, a eukaryotic initiation factor 4A (elF4A) promoter, a ferritin heavy chain (FerH) gene promoter, a ferritin heavy / light chain (FerL) gene promoter, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, a GRP78 promoter, a GRP94 promoter, a heat shock protein 70 (hsp70) promoter, a ubiquitin B promoter, an SV40 promoter, beta kinesin, ROSA26 promoter and PGK-1 (phosphoglycerate kinase 1, PGK-1) promoter.

[0072] In a preferred embodiment, the universal promoter is a CAG promoter. CAG promoters, as demonstrated in the examples, are suitable for use in the gene construct according to the invention. In some embodiments, the CAG promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%,at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 27.

[0073] Another preferred universal promoter is the cytomegalovirus (CMV) promoter. In some embodiments, the CMV promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 28. Preferably, said CMV promoter is used together with an intron sequence. In some embodiments, the intron sequence comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 26.,

[0074] Another preferred universal promoter is the minimal CMV promoter. According to some embodiments, the minimal CMV promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 36.,

[0075] Another preferred universal promoter is the EF1α promoter. According to some embodiments, the EF1α promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 37.,

[0076] Another preferred universal promoter is the RSV promoter. In some embodiments, the RSV promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 38.,

[0077] According to some embodiments, the nucleotide sequence encoding FGF-21 is operably linked to a tissue-specific promoter. According to a preferred embodiment, the tissue-specific promoter is a CNS-specific promoter, more preferably a brain-specific promoter, most preferably a hypothalamus-specific promoter.

[0078] The term "tissue-specific promoter" is described in the section titled "General Information".

[0079] According to some embodiments, the CNS-specific promoter according to the present disclosure is selected from the group consisting of the synapsin-1 promoter, the neuron-specific enolase (NSE) promoter, the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, the tyrosine hydroxylase (TH) promoter, the Forkhead Box A2 (FOXA2) promoter, the alpha-internexin (INA) promoter, the nestin (NES) promoter, the glial fibrillary acidic protein (GFAP) promoter, the aldehyde dehydrogenase family member L1 1 (ALDH1L1) promoter, the myelin-associated oligodendrocyte basic protein (MOBP) promoter, the homeobox protein 9 (HB9) promoter, and the myelin basic protein (MBP) promoter.

[0080] According to some embodiments, the brain-specific promoter according to the present description is selected from the group consisting of the synapsin-1 promoter, the neuron-specific enolase (NSE) promoter, the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, the tyrosine hydroxylase (TH) promoter, the Forkhead Box A2 (FOXA2) promoter, the alpha-internexin (INA) promoter, the nestin (NES) promoter, the glial fibrillary acidic protein (GFAP) promoter, the aldehyde dehydrogenase family member L1 1 (ALDH1L1) promoter, the myelin-associated oligodendrocyte basic protein (MOBP) promoter, and the myelin basic protein (MBP) promoter.

[0081] In some embodiments, the hypothalamus-specific promoter may be a gonadotropin-releasing hormone (GnRH) promoter.

[0082] According to a preferred embodiment, the CNS and / or brain-specific promoter is the synapsin-1 promoter. In some embodiments, the synapsin-1 promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 39.,

[0083] Another preferred CNS and / or brain-specific promoter is the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter. In some embodiments, the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%,at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 40.,

[0084] Another preferred promoter specific to the CNS and / or brain is the glial fibrillary acidic protein (GFAP) promoter. According to some embodiments, the glial fibrillary acidic protein (GFAP) promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 41.,

[0085] Another preferred promoter specific to the CNS and / or brain is the nestin promoter. In some embodiments, the nestin promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 42.,

[0086] Another preferred CNS-specific promoter is the homeobox protein 9 (HB9) promoter. In some embodiments, the homeobox protein 9 (HB9) promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 43.,

[0087] Another preferred promoter specific to the CNS and / or brain is the tyrosine hydroxylase (TH) promoter. In some embodiments, the tyrosine hydroxylase (TH) promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 44.,

[0088] Another preferred promoter specific to the CNS and / or brain is the myelin basic protein (MBP) promoter. According to some embodiments, the myelin basic protein (MBP) promoter comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 45.,

[0089] According to some embodiments, the CNS, brain, and / or hypothalamus-specific promoters described herein direct the expression of said nucleotide sequence in at least one cell of the CNS and / or brain and / or hypothalamus. Preferably, said promoter directs expression in at least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80%, 90%, or 100% of the cells of the CNS and / or brain and / or hypothalamus. A CNS and / or brain-specific promoter as described herein also includes promoters that direct expression in a specific region or subpopulation of cells in the CNS and / or brain. Thus, the CNS and / or brain-specific promoters described herein may also direct expression in at least 10%, 20%, 30%, 40%, 40%, 60%, 70%, 80%, 90%, or 100% of the cells in the hippocampus, cerebellum, cerebral cortex, hypothalamus, and / or olfactory bulb.Expression can be assessed as described in the section titled "general information".

[0090] A promoter in the present description (in particular, when the promoter sequence is described as having a minimum percentage identity to a given SEQ ID NO) should exhibit at least the promoter activity as known to one skilled in the art. Preferably, a promoter that is described as having a minimum percentage identity to a given SEQ ID NO should control the transcription of a nucleotide sequence to which it is operably linked (for example, at least a nucleotide sequence that encodes FGF-21), as assessed in an assay known to one skilled in the art. For example, such an assay may include measuring transgene expression. Expression can be assessed as described in the section entitled "General Information."

[0091] Additional sequences may be present in the gene construct of the present invention. Examples of additional sequences suitable for use in the present invention include inverted terminal repeats (ITRs), SV40 polyadenylation signal (SEQ ID NO: 32), rabbit β-globin polyadenylation signal (SEQ ID NO: 33), and CMV enhancer sequence (SEQ ID NO: 29). In the context of the present invention, the term "ITR" includes one 5'-ITR and one 3'-ITR, each of which was isolated from the AAV (adeno-associated virus) genome. Preferred ITRs are derived from AAV2 and are represented by SEQ ID NO: 30 (5'-ITR) and SEQ ID NO: 31 (3'-ITR). In the context of the present invention, the use of a CMV enhancer sequence (SEQ ID NO: 29) and a CMV promoter sequence (SEQ ID NO: 28) as two separate sequences or as a single sequence (SEQ ID NO: 34) is provided.Each of these additional sequences may be present in the gene construct according to the said invention.

[0092] Additional nucleotide sequences, such as nucleotide sequences that encode signal sequences, nuclear localization signals, expression enhancers, etc., may optionally be operably linked to the nucleotide sequence(s) encoding FGF-21.

[0093] According to some embodiments, a gene construct is provided that comprises a nucleotide sequence encoding FGF-21, wherein optionally said gene construct does not comprise a target sequence of a microRNA expressed in the tissue in which it is desired to prevent the expression of FGF-21.

[0094] Expression vector

[0095] The gene constructs described herein can be placed in expression vectors. Thus, according to another aspect, an expression vector is provided that contains a gene construct as described in any of the preceding embodiments. A description of the term "expression vector" is provided in the section under the heading "general information."

[0096] According to some embodiments, the expression vector according to the present disclosure is for use in therapy. According to a preferred embodiment, the expression vector according to the present disclosure is for use in the treatment and / or prevention of a metabolic disorder. According to a preferred embodiment, said therapy comprises expressing the gene construct contained in the expression vector in the CNS, preferably in the brain, more preferably in the hypothalamus. According to some embodiments, expressing the gene construct in the brain may mean expressing the gene construct in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb, preferably in the hypothalamus.Thus, expression of the gene construct in the brain may mean expression of the gene construct in at least one, or at least two, or at least three, or all brain regions selected from the group consisting of the hypothalamus, cerebral cortex, hippocampus, cerebellum, and olfactory bulb. According to a preferred embodiment, the therapy comprises expression of said gene construct in the hypothalamus. According to some embodiments, expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb may mean specific expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb.In an embodiment, the expression does not include expression in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. In some embodiments, the expression does not include expression in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, and heart. A description of CNS-specific expression and / or brain-specific expression and / or hypothalamus-specific expression and / or cerebral cortex-specific expression and / or hippocampus-specific expression and / or cerebellum-specific expression and / or olfactory bulb-specific expression is provided in the section entitled "General Information."

[0097] Expression can be assessed as described in the section titled "General Information." The terms "CNS," "brain," and "hypothalamus" are described in the section titled "General Information."

[0098] According to some embodiments, the expression vector according to the present disclosure is intended for use in therapy, wherein said expression vector is administered by administration into the CSF (cerebrospinal fluid) (via delivery into the cisterna magna, intrathecal or intraventricular delivery), by intraparenchymal administration or intranasal administration. A preferred route of administration is administration into the CSF.

[0099] In this document, the terms "administration into CSF", "intranasal administration", "intraparenchymal administration", "administration into the cisterna magna", "intrathecal administration" and "intraventricular administration" are described in the part of this application under the heading "general information".

[0100] According to some embodiments, the expression vector is a viral expression vector. The term "viral expression vector" is described in the section under the heading "General Information."

[0101] The viral vector may be a viral vector selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors. An adenoviral vector is also known as a vector isolated from an adenovirus, an adeno-associated vector is also known as a vector derived from an adeno-associated virus, a retroviral vector is also known as a vector derived from a retrovirus, and a lentiviral vector is also known as a vector derived from a lentivirus. A preferred viral vector is an adeno-associated viral vector. The term "adeno-associated viral vector" is described in the section entitled "General Information."

[0102] According to some embodiments, the vector is an adeno-associated vector or an adeno-associated viral vector or a vector derived from an adeno-associated virus (AAV) selected from the group consisting of AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype rh10 (AAVrh10), AAV serotype rh8 (AAVrh8), AAV serotype Cb4 (AAVCb4), AAV serotype rh74 (AAVrh74), AAV serotype DJ (AAVDJ), AAV serotype 2 / 5 (AAV2 / 5), AAV serotype 2 / 1 (AAV2 / 1), AAV serotype 1 / 2 (AAV1 / 2), AAV serotype Anc80 (AAVAnc80).

[0103] According to a preferred embodiment, the vector is AAV serotype 1, 2, or 9 (AAV1, AAV2, or AAV9). These AAV serotypes, as demonstrated in the examples, are suitable for use as an expression vector in accordance with the invention.

[0104] According to a preferred embodiment, the expression vector is AAV1 or AAV2 or AAV9, preferably AAV9, and comprises a gene construct comprising a nucleotide sequence that encodes FGF-21. More preferably, such a gene construct comprises a CAG promoter that comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60% identity to SEQ ID NO:27. More preferably, such a gene construct comprises at least one target sequence of a microRNA expressed in the tissue in which it is desired to prevent the expression of FGF-21, according to the present description.

[0105] According to another preferred embodiment, the expression vector is AAV1 and comprises a gene construct comprising a nucleotide sequence encoding FGF-21, wherein optionally said gene construct does not comprise a target sequence of a microRNA. According to the embodiment, the gene construct does not comprise a target sequence of a microRNA expressed in the tissue in which it is desired to prevent the expression of FGF-21. More preferably, such a gene construct comprises a CAG promoter that comprises, consists essentially of, or consists of a nucleotide sequence that has at least 60% identity to SEQ ID NO:27.

[0106] Composition

[0107] According to another embodiment, a composition is provided that comprises a gene construct as described above and / or a viral vector as described above, together with one or more pharmaceutically acceptable ingredients.

[0108] Such a composition may be referred to as a gene therapy composition. In a preferred embodiment, said composition is a pharmaceutical composition.

[0109] In the present description, "pharmaceutically acceptable ingredients" include pharmaceutically acceptable carriers, fillers, preservatives, solvents, carrier media, diluents, and / or excipients. Accordingly, said one or more pharmaceutically acceptable ingredients can be selected from the group consisting of pharmaceutically acceptable carriers, fillers, preservatives, solvents, carrier media, diluents, and / or excipients. Such pharmaceutically acceptable carriers, fillers, preservatives, solvents, carrier media, diluents, and / or excipients can be found, for example, in the publication: Remington: The Science and Practice of Pharmacy, 22nd edition. Pharmaceutical Press (2013).

[0110] According to some embodiments, the composition according to the present description is intended for use in therapy. According to a preferred embodiment, the composition according to the present description is intended for use in the treatment and / or prevention of a metabolic disorder. According to a preferred embodiment, said therapy includes the expression of a gene construct contained in the composition in the CNS, preferably in the brain, more preferably in the hypothalamus. According to some embodiments, the expression of the gene construct in the brain may mean the expression of the gene construct in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb, preferably in the hypothalamus.Thus, expression of the gene construct in the brain may mean expression of the gene construct in at least one, or at least two, or at least three, or all brain regions selected from the group consisting of the hypothalamus, cerebral cortex, hippocampus, cerebellum, and olfactory bulb. According to a preferred embodiment, the therapy comprises expression of said gene construct in the hypothalamus. According to some embodiments, expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb may mean specific expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb.In an embodiment, the expression does not include expression in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. In some embodiments, the expression does not include expression in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, and heart. A description of CNS-specific expression and / or brain-specific expression and / or hypothalamus-specific expression and / or cerebral cortex-specific expression and / or hippocampus-specific expression and / or cerebellum-specific expression and / or olfactory bulb-specific expression is provided in the section entitled "General Information."

[0111] Expression can be assessed as described in the section titled "General Information." The terms "CNS," "brain," and "hypothalamus" are described in the section titled "General Information."

[0112] According to some embodiments, the gene construct according to the present description is intended for use in therapy, wherein said gene construct is administered by administration into the CSF (cerebrospinal fluid) (via delivery into the cisterna magna, intrathecal or intraventricular delivery), by intraparenchymal administration or intranasal administration. The preferred route of administration is administration into the CSF.

[0113] The terms "administration into CSF", "intranasal administration", "intraparenchymal administration", "administration into the cisterna magna", "intrathecal administration" and "intraventricular administration" as used herein are described in the part of this application under the heading "general information".

[0114] An additional compound may be present in the composition of the present invention. Said compound may assist in the delivery of the composition. Suitable compounds in this context are: compounds capable of forming complexes, nanoparticles, micelles and / or liposomes that deliver each moiety according to the present description, complexed or enclosed 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; synthetic amphiphiles (SAINT-18, 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium chloride); lipofectin™, DOTAP (DOTAP, 1,2-dioleoyloxy-3-(N,N,N-trimethylammonium)propane chloride).A person skilled in the art will know what type of formulation is most suitable for the composition according to the present description.

[0115] Method and application

[0116] According to another aspect, a gene construct according to the present description is provided for use in therapy, wherein said therapy comprises expressing said gene construct in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0117] Further provided is an expression vector according to the present description for use in therapy, wherein said therapy comprises expressing a gene construct contained in the expression vector in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0118] Further provided is a pharmaceutical composition according to the present description for use in therapy, wherein said therapy comprises the expression of a gene construct contained in the pharmaceutical composition in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0119] According to some embodiments, the gene construct according to the present description and / or the expression vector according to the present description and / or the pharmaceutical composition according to the present description are intended for use in the treatment and / or prevention of a metabolic disorder, preferably obesity and / or diabetes, wherein said therapy comprises the expression of said gene construct in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0120] According to another aspect, a method of treatment is provided that comprises administering a gene construct, an expression vector or a pharmaceutical composition according to the present description, wherein said method comprises expressing the gene construct according to the present description in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0121] According to some embodiments, administering a gene construct, expression vector, or pharmaceutical composition means administering to a subject in need thereof a therapeutically effective amount of the gene construct, expression vector, or pharmaceutical composition.

[0122] According to some embodiments, a method of treatment is provided that comprises administering a gene construct, an expression vector, or a pharmaceutical composition according to the present description, wherein said method is intended for treating and / or preventing a metabolic disorder and comprises expressing the gene construct in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0123] According to another aspect, the use of a gene construct, expression vector or pharmaceutical composition according to the present description for the preparation of a medicament is proposed, wherein said medicament provides for the expression of the gene construct according to the present description in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0124] According to some embodiments, there is provided the use of a gene construct, expression vector or pharmaceutical composition according to the present disclosure for the production of a medicament, wherein said medicament is intended for the treatment and / or prevention of a metabolic disorder and provides for the expression of the gene construct according to the present disclosure in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0125] According to a further aspect, there is provided the use of a gene construct, expression vector or pharmaceutical composition according to the present description for medical treatment, wherein said medical treatment comprises the expression of the gene construct according to the present description in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0126] According to some embodiments, there is provided the use of a gene construct, expression vector or pharmaceutical composition according to the present disclosure for medical treatment, wherein said medical treatment is intended for the treatment and / or prevention of a metabolic disorder and comprises the expression of the gene construct according to the present disclosure in the CNS, preferably in the brain, more preferably in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb, most preferably in the hypothalamus.

[0127] In the context of gene constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses according to the present invention, “include expression of the gene construct” (“provide for expression of the gene construct”) can be replaced by “cause expression of the gene construct” or “induce expression of the gene construct”.

[0128] In the context of gene constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses in accordance with the said invention, the therapy and / or treatment and / or medicament may involve the expression of the gene construct in the CNS, preferably in the brain, more preferably in the hypothalamus. According to some embodiments, the expression of the gene construct in the brain may mean the expression of the gene construct in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb, preferably in the hypothalamus.Thus, expression of the gene construct in the brain may mean expression of the gene construct in at least one, or at least two, or at least three, or all brain regions selected from the group consisting of the hypothalamus, cerebral cortex, hippocampus, cerebellum, and olfactory bulb. According to a preferred embodiment, the therapy involves expression of the gene construct in the hypothalamus. According to some embodiments, expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb may mean specific expression in the CNS and / or in the brain and / or in the hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb.In an embodiment, the expression does not include expression in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. In some embodiments, the expression does not include expression in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, and heart. A description of CNS-specific expression and / or brain-specific expression and / or hypothalamus-specific expression and / or cerebral cortex-specific expression and / or hippocampus-specific expression and / or cerebellum-specific expression and / or olfactory bulb-specific expression is provided in the section entitled "General Information."

[0129] In the context of gene constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses according to the said invention, the gene construct and / or expression vector and / or pharmaceutical composition can be administered by administration into the CSF (cerebrospinal fluid) (by delivery into the cisterna magna, intrathecal or intraventricular delivery).

[0130] In the context of gene constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses in accordance with the said invention, the gene construct and / or expression vector and / or pharmaceutical composition can be administered by intraparenchymal administration.

[0131] In the context of gene constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses in accordance with the said invention, the gene construct and / or expression vector and / or pharmaceutical composition can be administered by intranasal administration.

[0132] The terms "administration into CSF", "intranasal administration", "intraparenchymal administration", "administration into the cisterna magna", "intrathecal administration" and "intraventricular administration" as used herein are described in the part of this application under the heading "general information".

[0133] In the context of gene constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses in accordance with the said invention, the therapy and / or treatment and / or medicinal product may be intended for use in the treatment and / or prevention of a metabolic disorder, preferably obesity and / or diabetes. Complications of a metabolic disorder may also be envisaged.

[0134] Metabolic disorders may include metabolic syndrome, diabetes, obesity, obesity-related comorbidities, diabetes-related comorbidities, hyperglycemia, insulin resistance, glucose intolerance, hepatic steatosis, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), coronary artery disease (CAD), hyperlipidemia, atherosclerosis, endocrinopathies, osteosarcopenic obesity (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, carcinoma.

[0135] Diabetes may include prediabetes, hyperglycemia, type 1 diabetes, type 2 diabetes, adult-onset diabetes in the young (MODY), monogenic diabetes, neonatal diabetes, gestational diabetes, brittle diabetes, idiopathic diabetes, drug- or chemical-induced diabetes, stiff-person syndrome, lipoatrophic diabetes, latent autoimmune diabetes of adults (LADA).

[0136] Obesity may include overweight, upper / central obesity, peripheral / lower obesity, morbid obesity, osteosarcopenic obesity (OSO), childhood obesity, Mendelian (monogenic) syndromic obesity, Mendelian non-syndromic obesity, polygenic obesity.

[0137] The preferred metabolic disorders are obesity and / or diabetes.

[0138] According to a preferred embodiment, the treatment, therapy, use, or administration of the medicament according to the present disclosure need not be repeated. According to some embodiments, the treatment, therapy, use, or administration of the medicament according to the present disclosure can be repeated annually, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, including intervals between any two of the listed values.

[0139] The subject receiving the treatment may be a higher mammal such as a cat, a rodent (preferably mice, rats, gerbils and guinea pigs, and more preferably mice and rats), a dog or a human.

[0140] In the context of gene constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses in accordance with said invention, the gene construct and / or expression vector and / or pharmaceutical composition according to the present description preferably exhibits an anti-diabetic effect and / or an anti-obesity effect.

[0141] An antidiabetic effect can be achieved by increasing blood glucose utilization and / or improving glucose tolerance and / or increasing insulin sensitivity. This can be assessed using methods known to those skilled in the art, such as measuring glycemia, insulinemia, and / or performing an insulin tolerance test and / or a glucose tolerance test, as, for example, done in the experimental section. In this context, the term "increase" (and, accordingly, "improvement") means at least a detectable increase (and, accordingly, a detectable improvement) using an assay known to those skilled in the art.The 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%, at least 100% in the results of tests such as measuring glycemia, insulinemia, and / or performing an insulin tolerance test and / or a glucose tolerance test.

[0142] The anti-obesity effect can be achieved by reducing body weight, body weight gain, and / or body fat percentage. The anti-obesity effect can also be achieved by reducing body mass index (BMI), waist circumference, waist-to-hip ratio (WHR), and / or waist-to-height ratio (WHR). The anti-obesity effect can also be achieved by reducing tissue mass, such as the liver. This can be assessed using methods known to those skilled in the art, such as was done in the experimental section. In this context, the term "reduction" (and, respectively, "improvement") means at least a detectable reduction (respectively, a detectable improvement) using an assay known to those skilled in the art, such as the assays performed in the experimental section. Anti-obesity effects include both the prevention of obesity and the reversal of obesity.

[0143] An antidiabetic and / or antiobesity effect may also be observed when the progression of a typical symptom (e.g., insulitis, beta-cell loss, decreased beta-cell mass, weight gain) is slowed, as assessed by a physician. A reduction in a typical symptom may mean a slowing of the progression of the symptom or a complete resolution of symptoms. Symptoms, and therefore symptom reduction, can be assessed using a variety of methods, largely the same as those used in the diagnosis of diabetes and / or obesity, including clinical examination and routine laboratory testing. Such methods include both macroscopic and microscopic methods, as well as molecular methods, radiographic methods such as X-rays, biochemical methods, immunohistochemical methods, and others.Beta cell loss and / or reduction in beta cell mass can be assessed using immunohistochemical methods, preferably as performed in the experimental part.

[0144] An antidiabetic effect and / or an antiobesity effect can also be observed when assessing the reduction in systemic inflammation (reduction in the level of proinflammatory cytokines such as F4 / 80, IL6, and TNF-alpha). In this context, the term "reduction" means at least a detectable reduction using an assay known to those skilled in the art. The reduction may be a reduction 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%, at least 100% using studies such as measuring proinflammatory cytokines such as F4 / 80, IL6 and / or TNF-alpha using methods known to the person skilled in the art, preferably those used in the experimental part (e.g. RT-PCR).

[0145] In the context of gene constructs for use, expression vectors for use, pharmaceutical compositions for use, methods and uses in accordance with said invention, the gene construct and / or expression vector and / or pharmaceutical composition according to the present description preferably alleviates one or more symptoms of a metabolic disorder, such as diabetes and / or obesity, in an individual, in a cell, tissue or organ of said individual, or alleviates one or more characteristics or symptoms in a cell, tissue or organ of said individual.

[0146] The gene construct and / or expression vector and / or pharmaceutical composition according to the present description is preferably capable of alleviating a symptom or characteristic in a patient or in a cell, tissue or organ of said patient if, after at least one week, one month, six months, one year or more of treatment time using the gene construct and / or expression vector and / or pharmaceutical composition according to the present invention, said symptom or characteristic has decreased (e.g., is no longer detectable or has slowed down) according to the present description.

[0147] The gene construct and / or expression vector and / or pharmaceutical composition according to the present description may be suitable for administration to a cell, tissue and / or organ in vivo in individuals suffering from a metabolic disorder such as diabetes and / or obesity, or at risk of developing it, and can also be administered in vivo, ex vivo or in vitro. The said gene construct and / or expression vector and / or pharmaceutical composition can be directly or indirectly administered to a cell, tissue and / or organ in vivo in an individual suffering from a metabolic disorder such as diabetes and / or obesity, or at risk of developing it, and can also be directly or indirectly administered in vivo, ex vivo or in vitro.

[0148] The route of administration can be intravenous, intramuscular, intratracheal, intraventricular, intraperitoneal, by inhalation, intranasal, intraocular and / or intraparenchymal administration. Preferred routes of administration are intranasal, intraparenchymal and intracerebrospinal fluid (CISF) administration (via cisterna magna delivery, intrathecal or intraventricular delivery). CSF administration is most preferred.

[0149] The viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition of the present invention can be administered directly or indirectly using suitable methods known in the art. It is envisaged to improve the methods for providing an individual or a cell, tissue, or organ of said individual with the viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition of the present invention, taking into account the progress that has already been achieved to date. Such future improvements can, of course, be envisaged to achieve the aforementioned effect of the present invention. The viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition can be delivered to an individual or to a cell, tissue, or organ of said individual in its original form.Depending on the disease or condition, the cell, tissue, or organ of said individual may be as previously described herein. When administering a viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition according to the present invention, it is preferred that such viral expression construct and / or viral vector and / or nucleic acid molecule and / or composition be dissolved in a solution compatible with the delivery method.

[0150] As provided by the present description, a therapeutically effective dose of a viral expression construct and / or a viral vector and / or a nucleic acid molecule and / or a composition as mentioned above is preferably administered once in a single dose, thus avoiding multiple periodic administration.

[0151] General information

[0152] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and should be construed in light of this description.

[0153] Sequence identity / similarity

[0154] In the context of the present invention, a nucleic acid molecule, such as a nucleic acid molecule encoding FGF-21, is represented by a nucleic acid or nucleotide sequence that encodes a protein fragment, or a polypeptide, or a peptide, or a resulting peptide. In the context of the present invention, a protein fragment of FGF-21, or a polypeptide, or a peptide, or a resulting peptide as fibroblast growth factor 21 (FGF-21) is represented by an amino acid sequence.

[0155] It should be understood that each nucleic acid molecule, or protein fragment, or polypeptide, or peptide, or resulting peptide, or construct, as identified herein by a specified sequence identification number (SEQ ID NO) is not limited to that particular disclosed sequence. Each coding sequence identified herein encodes the specified protein fragment, or polypeptide, or peptide, or resulting peptide, or construct, or is itself a protein fragment, or a polypeptide, or a construct, or a peptide, or a resulting peptide. In the text of this document, whenever a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as an example) is referred to, which encodes the specified protein fragment, or polypeptide, or peptide, or resulting peptide, it can be replaced by:

[0156] i. a nucleotide sequence comprising a nucleotide sequence that has at least 60% identity to SEQ ID NO: X;

[0157] ii. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule by (i) the degeneracy of the genetic code; or

[0158] iii. a nucleotide sequence that encodes an amino acid sequence that has at least 60% identity or similarity to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: X.

[0159] In the text of this application, each time when a specific amino acid sequence SEQ ID NO is indicated (take SEQ ID NO: Y as an example), it can be replaced with: a polypeptide that contains an amino acid sequence that has at least 60% sequence identity or similarity with the amino acid sequence of SEQ ID NO: Y.

[0160] Each nucleotide sequence or amino acid sequence that is described in the present document by percentage identity or similarity (at least 60%) with said nucleotide sequence or amino acid sequence, respectively, has, according to another preferred embodiment, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity or similarity to the said nucleotide or amino acid sequence, respectively.

[0161] Each non-coding nucleotide sequence (e.g., a promoter or other regulatory region) can be replaced with a nucleotide sequence that comprises a nucleotide sequence having at least 60% sequence identity or similarity with a specific nucleotide sequence of SEQ ID NO (take SEQ ID NO: A as an example).A preferred nucleotide sequence has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: A.According to a preferred embodiment, said non-coding nucleotide sequence, such as a promoter, exhibits or displays at least an activity of said non-coding nucleotide sequence, such as a promoter activity known to a person skilled in the art.

[0162] The terms "homology," "sequence identity," "identity," and the like are used interchangeably herein. Sequence identity is described herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, which is determined by comparing the sequences. "Similarity" or "sequence similarity" between two amino acid sequences is determined by comparing the amino acid sequence and the corresponding amino acid sequence variants with conservative substitutions of one polypeptide with the sequence of the second polypeptide.“Identity” and “similarity” can be easily calculated using known methods, including, but not limited to, those described in the publication: Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and Transcriptomics, Xia X., Springer International Publishing, New York, 2018; and the publications Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004.

[0163] Sequence identity or similarity can be calculated based on the full length of two said SEQ ID NOs or on a portion thereof. According to some embodiments, a portion thereof means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NOs. According to a preferred embodiment, sequence identity or similarity is determined by comparing the full length of the sequences identified herein. Unless otherwise stated herein, identity or similarity with a said SEQ ID NO means identity or similarity based on the full length of the said sequence (e.g., over the entire length of the sequence, or the entire sequence). In the art, the term "identity" also refers to the degree of relatedness of amino acid or nucleic acid sequences, as the case may be, which is determined by comparing portions of such sequences.

[0164] Sequence identity or similarity can be determined by aligning two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch), which optimally aligns sequences over their entire length, while sequences of significantly different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman).Accordingly, sequences may be said to be "substantially identical" or "substantially similar" if they (after optimal alignment, such as with EMBOSS Needle or EMBOSS Water using default parameters) share at least a certain minimum percentage of sequence identity or similarity (as described below).

[0165] Global alignment is used when appropriate to determine sequence identity or similarity when two sequences are of similar length. When sequences have significantly different overall lengths, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS Needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their full length, maximizing the number of matches and minimizing the number of gaps. EMBOSS Water uses the Smith-Waterman local alignment algorithm. EMBOSS Needle and EMBOSS Water typically use default parameters, which are a gap opening penalty of 10 (for nucleotide sequences) / 10 (for proteins) and a gap extension penalty of 0.5 (for nucleotide sequences) / 0.5 (for proteins).For nucleotide sequences, the default substitution matrix is ​​DNAfull, and for proteins, the default substitution matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).

[0166] Alternatively, the percentage similarity or identity can be determined by searching publicly available databases using algorithms such as FASTA, BLAST, etc. Thus, nucleic acid and protein sequences according to some embodiments of the present invention can be further used as a "query sequence" to search publicly available databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) according to Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, alignment score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecules of the present invention.BLAST protein searches can be performed using the BLASTx program, alignment score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. For gapped alignments, the Gapped BLAST program can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389–3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the home page of the National Center for Biotechnology Information, accessible on the World Wide Web at www.ncbi.nlm.nih.gov / .

[0167] Optionally, when determining the degree of similarity of amino acids, a person skilled in the art may also take into account so-called conservative amino acid substitutions.

[0168] In this description, "conservative" amino acid substitutions refer to the interchangeability of residues with similar side chains. Examples of amino acid residue classes are provided in the tables below.

[0169] Acid residues Asp (D) and Glu (E) Main balances Lys (K), Arg (R), and His (H) Hydrophilic uncharged residues Ser (S), Thr (T), Asn (N) and Gln (Q) Aliphatic uncharged residues Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar uncharged residues Cys (C), Met (M), and Pro (P) Aromatic residues Phe (F), Tyr (Y), and Trp (W)

[0170] Alternative classes of conservative substitutions of amino acid residues:

[0171] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0172] Alternative physical and functional classifications of amino acid residues:

[0173] Residues containing an alcohol group S and T Aliphatic residues I, L, V and M Cycloalkenyl-associated residues F, H, W and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residues D and E Polar remnants C, D, E, H, K, N, Q, R, S and T Positively charged residues H, K and R Small size remains A, C, D, G, N, P, S, T and V Remains of very small size A, G and S Residues involved in the formation of bends A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible balances Q, T, K, S, G, P, D, E, and R

[0174] For example, the group of amino acids having aliphatic side chains includes glycine, alanine, valine, leucine and isoleucine; the group of amino acids having aliphatic-hydroxyl side chains includes serine and threonine; the group of amino acids having amide-containing side chains includes asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; the group of amino acids having basic side chains includes lysine, arginine and histidine; and the group of amino acids having sulfur-containing side chains includes cysteine ​​and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.Substitution variants of the amino acid sequence disclosed herein are those variants wherein at least one residue of the disclosed sequences is deleted and another residue is inserted in its place. Preferably, said amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are the following: substitution of Ala for Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser or Ala; Gln for Asn; Glu for Asp; Gly for Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Ile or Val; Lys for Arg; Gln or Glu; Met for Leu or Ile; Phe for Met, Leu, or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp or Phe; and Val for Ile or Leu.

[0175] Gene or coding sequence

[0176] The term "gene" means a fragment of DNA containing a region (the transcribed region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell, operably linked to a suitable regulatory region (e.g., a promoter). Typically, a gene contains several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) containing, for example, a polyadenylation site and / or a transcription termination site. A chimeric or recombinant gene (such as the FGF-21 gene) is a gene that does not normally occur in nature, such as a gene in which the promoter is not naturally linked to part or all of the transcribed region of DNA."Gene expression" refers to the process by which a region of DNA that is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into RNA that is biologically active, such as one that is capable of being translated into a biologically active protein or peptide.

[0177] "Transgene" as used herein is described as a gene, or a coding sequence, or a nucleic acid molecule (i.e., a molecule that encodes FGF-21), which has been reintroduced into a cell, i.e., a gene that may be present but may not normally be expressed or may be expressed in the cell at an insufficient level. In this context, "insufficient" means that although said FGF-21 is expressed in the cell, the condition and / or disease as described herein may still develop. In this case, said invention provides the possibility of overexpression of FGF-21. The transgene may contain sequences that are native to the cell, sequences that are not naturally found in the cell, and may also contain combinations of the first and second sequences.The transgene may contain sequences encoding FGF-21 and / or additional proteins previously identified herein, which may be operably linked to appropriate regulatory sequences for expression of the FGF-21 encoding sequences in the cell. Preferably, the transgene is not integrated into the host cell genome.

[0178] Promoter

[0179] As used herein, the term "promoter" or "transcription control sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences and is located in the 5' direction based on the direction of transcription relative to the transcription initiation site of the coding sequence and is structurally identifiable by the presence of a DNA-dependent RNA polymerase binding site, transcription initiation sites, and other DNA sequences, including, but not limited to, transcription factor binding sites, repressor protein binding sites, activator protein binding sites, and any other nucleotide sequences that are known to one skilled in the art to directly or indirectly act to regulate the level of transcription by a promoter.A "constitutive" promoter is one that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is one that is regulated by physiological and developmental conditions, such as by the use of a chemical inducer.

[0180] "Universal Promoter" is active in essentially all tissues, organs and cells of the body.

[0181] An "organ-specific" or "tissue-specific" promoter is a promoter that is active in a specific organ or tissue type, respectively. Organ-specific and tissue-specific promoters regulate the expression of one or more genes (or coding sequences) primarily in one organ or tissue, but may also provide detectable expression ("residual expression") in other organs or tissues. Expression at a residual level in other organs or tissues means at least one-fold, at least two-fold, at least three-fold, at least four-fold, at least five-fold lower, but still detectable, expression compared to organ-specific or tissue-specific expression, as assessed based on the mRNA or protein level using standard assays known to those skilled in the art (e.g., qPCR, Wernblot, ELISA).The maximum number of organs or tissues in which leaky expression can be detected is five, six, seven, or eight.

[0182] A "CNS-specific, brain-specific, or hypothalamic-specific promoter" is a promoter that is capable of initiating transcription in the CNS and / or brain and / or hypothalamus, while still allowing some residual expression in other (maximum five, six, seven, or eight) organs and parts of the body. Transcription in the CNS and / or brain and / or hypothalamus can be detected in relevant regions, such as the hypothalamus, cerebral cortex, hippocampus, cerebellum, and olfactory bulb, as well as in cells such as neurons and / or glial cells.

[0183] In the context of the present invention, promoters specific for the CNS and / or the brain and / or the hypothalamus and / or the cerebral cortex and / or the hippocampus and / or the cerebellum and / or the olfactory bulb may be promoters that are capable of driving 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 FGF-21 in the CNS and / or the brain brain, and / or in the hypothalamus, and / or in the cerebral cortex, and / or in the hippocampus, and / or in the cerebellum, and / or in the olfactory bulb compared to other organs or tissues.Other organs or tissues may include the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, etc. Preferably, the other organs are the liver and / or heart. Expression can be assessed as described in the text of this document in the section titled "General Information."

[0184] As used herein, the term "specific for the CNS and / or for the brain and / or for the hypothalamus and / or for the cerebral cortex and / or for the hippocampus and / or for the cerebellum and / or for the olfactory bulb" in the context of expression also includes cell type-specific expression in the cell type(s) that make up the CNS and / or the brain and / or the hypothalamus and / or for the cerebral cortex and / or the hippocampus and / or for the cerebellum and / or for the olfactory bulb, respectively.

[0185] Functionally related

[0186] As used herein, the term "operably linked" refers to the joining of polynucleotide elements by a functional relationship. A nucleic acid is "operably linked" if it is placed into a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. The term "operably linked" means that the linked DNA sequences are typically contiguous and, when necessary to connect two protein-coding regions, are contiguous and in-frame. Linking can be achieved by ligation at convenient restriction sites, or by using adapters or linkers inserted in their place, or by gene synthesis.

[0187] MicroRNA

[0188] As used herein, the term "microRNA," or "miRNA," or "miR" has the generally accepted, common meaning understood by those skilled in the art based on this disclosure. MicroRNA is a small non-coding RNA molecule found in plants, animals, and some viruses and can function in RNA silencing and post-transcriptional regulation of gene expression. The target sequence of a microRNA can be referred to as "miRT." For example, the target sequence of microRNA-1, or miRNA-1, or miR-1 can be referred to as miRT-1.

[0189] Proteins and amino acids

[0190] The terms "protein," "polypeptide," or "amino acid sequence" are used interchangeably and refer to molecules composed of a chain of amino acids, regardless of their specific mode of action, size, three-dimensional structure, or origin. Amino acids or "residues" in amino acid sequences are designated herein by three-letter symbols.These three-letter symbols, as well as the corresponding single-letter symbols, are well known to those skilled in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine.The residue may be any proteinogenic amino acid, as well as any non-proteinogenic amino acid, such as D-amino acids and modified amino acids obtained by post-translational modifications, as well as any non-naturally occurring amino acid.

[0191] CNS and brain

[0192] As used herein, the term "central nervous system" or "CNS" refers to the part of the nervous system that includes the head, brain, and spinal cord, to which sensory impulses are transmitted, from which motor impulses are propagated, and which coordinates the activity of the entire nervous system.

[0193] As used herein, the term "brain" refers to the central organ of the nervous system and consists of the cerebral hemispheres, brainstem, and cerebellum. It controls ****most *****of the body's activities, processing, integrating, and coordinating information it receives from the senses, as well as making decisions regarding instructions sent to the rest of the body.

[0194] In particular, in this description, the term "hypothalamus" refers to the region of the forebrain that is located below the thalamus and coordinates both the autonomic nervous system and the activity of the pituitary gland, which controls body temperature, thirst, hunger and other homeostasis systems, and is also involved in sleep processes and emotional activity.

[0195] Gene constructs

[0196] The gene constructs according to the present description can be obtained using any cloning and / or recombinant DNA techniques known to one of skill in the art, wherein the nucleotide sequence encoding said FGF-21 is expressed in a suitable cell, for example, in cultured cells or in the cells of a multicellular organism, such as those described in Ausubelet et al., "Current Protocols in Molecular Biology", Greene Publishing and Wiley Interscience, New York (1987) and in Sambrook and Russell (2001, supra); both of which are incorporated herein by reference in their entireties. See also Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describes site-directed mutagenesis) and Robertset al.(1987) Nature 328:731734 or Wells, JA, et al.(1985) Gene 34:315 (describes cassette mutagenesis).

[0197] Expression vectors

[0198] The term "expression vector" or "vector" generally refers to a nucleotide sequence capable of expressing a gene or coding sequence in a host compatible with such sequences. An expression vector carries a genome capable of being stabilized and maintained in an episomal state within a cell. In the context of the present invention, "cell" may include the cell used to produce the construct or the cell into which the construct is introduced. Alternatively, the vector is capable of integrating into the cell's genome, for example, through homologous recombination or other means.

[0199] These expression vectors typically comprise at least suitable promoter sequences and, optionally, transcription termination signals. An additional factor necessary or beneficial for expression may also be used according to the present disclosure. The nucleic acid, or DNA, or nucleotide sequence encoding FGF-21 is included in a DNA construct suitable for introduction into and expression of cells in vitro. In particular, the DNA construct is suitable for replication in a prokaryotic host, such as bacteria, for example, E. coli, or can be introduced into cultured mammalian, plant, insect (for example, Sf9), yeast, fungal, or other eukaryotic cell lines.

[0200] A DNA construct prepared for introduction into a specific host may comprise a replication system recognized by the host, a putative DNA segment encoding the desired polypeptide, and sequences operatively linked to the segment encoding the polypeptide that regulate the initiation and termination of translation and transcription. The term "operably linked" has already been described herein. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates transcription of said sequence. A signal sequence DNA is operably linked to DNA encoding a polypeptide if it is expressed as a precursor protein that participates in the secretion of the polypeptide. Typically, operably linked DNA sequences are contiguous and, in the case of a signal sequence, are simultaneously contiguous and in-frame.However, enhancers do not need to be adjacent to the coding sequence whose transcription they control. Linkage is achieved by ligation at convenient restriction sites, by adaptors or linkers inserted in their place, or by gene synthesis.

[0201] The selection of a suitable promoter sequence generally depends on the host cell chosen to express the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters, which are well known in the art (see, e.g., Sambrook and Russell, 2001, supra). The transcriptional regulatory sequence typically comprises a heterologous enhancer or promoter that is recognized by the host. The selection of a suitable promoter depends on the host, but promoters such as the trp, lac, and phage promoters, tRNA (transfer RNA) promoters, and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell, 2001, supra). The expression vector contains a replication system and transcriptional and translational regulatory sequences, together with an insertion site for the protein-coding segment.In most cases, the replication system is functional only in the cell used to produce the vector (a bacterial cell, such as E. coli). Most plasmids and vectors do not replicate in cells infected with the vector. Examples of viable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, above) and Metzger et al. (1988) Nature. 334 : 31-36. For example, suitable expression vectors can be expressed in yeast, such as S. cerevisiae, insect cells, such as Sf9 cells, mammalian cells, such as CHO cells, and bacterial cells, such as E. coli. The cell can thus be a prokaryotic or eukaryotic host cell. The cell can be a cell that is suitable for culture in a liquid or solid medium.

[0202] In an alternative embodiment, the host cell is a cell that is part of a multicellular organism, such as a transgenic plant or animal.

[0203] The selection of a suitable promoter sequence generally depends on the host cell chosen to express the DNA segment. Examples of suitable promoter sequences include prokaryotic and eukaryotic promoters, which are well known in the art (see, e.g., Sambrook and Russell, 2001, supra). The transcriptional regulatory sequence typically comprises a heterologous enhancer or promoter that is recognized by the host. The selection of a suitable promoter depends on the host, but promoters such as the trp, lac, and phage promoters, tRNA promoters, and glycolytic enzyme promoters are known and available (see, e.g., Sambrook and Russell, 2001, supra). The expression vector contains a replication system and transcriptional and translational regulatory sequences, together with an insertion site for the protein-coding segment.In most cases, the replication system is functional only in the cell used to produce the vector (a bacterial cell, such as E. coli). Most plasmids and vectors do not replicate in cells infected with the vector. Examples of viable cell line and expression vector combinations are described in Sambrook and Russell (2001, above) and Metzger et al. (1988) Nature. 334 : 31-36. For example, suitable expression vectors can be expressed in yeast, such as S. cerevisiae, insect cells, such as Sf9 cells, mammalian cells, such as CHO cells, and bacterial cells, such as E. coli. The cell can thus be a prokaryotic or eukaryotic host cell. The cell can be a cell that is suitable for culture in a liquid or solid medium.

[0204] In an alternative embodiment, the host cell is a cell that is part of a multicellular organism, such as a transgenic plant or animal.

[0205] Viral vector

[0206] A viral vector, or viral expression vector, or viral gene therapy vector is a vector that contains the gene construct according to the present description.

[0207] A viral vector or viral gene therapy vector is a vector suitable for gene therapy. Vectors suitable for gene therapy are described in Anderson 1998, Nature 392 : 25-30; Walther and Stein, 2000, Drugs 60 : 249-71; Kayet al., 2001, Nat. Med. 7 : 33-40; Russell, 2000, J. Gen. Virol. 81 : 2573-604; Amado and Chen, 1999, Science 285 : 674-6; Federico, 1999, Curr. Opin. Biotechnol. 10 : 448-53; Vigna and Naldini, 2000, J. Gene Med. 2: 308-16; Marinet al., 1997, Mol. Med. Today 3 : 396-403; Peng and Russell, 1999, Curr. Opin. Biotechnol. 10 : 454-7; Sommerfelt, 1999, J. Gen. Virol. 80 : 3049-64; Reiser, 2000, Gene Ther. 7 : 910-3; and materials cited in the indicated sources.

[0208] In particular, suitable gene therapy vectors include adenovirus and adeno-associated virus (AAV) vectors. These vectors infect a significant number of dividing and non-dividing cell types, including synovial cells and liver cells. The episomal nature of adenovirus and AAV vectors after cell entry makes these vectors suitable for therapeutic applications (Russell, 2000, J. Gen. Virol. 81: 2573-2604; Goncalves, 2005, Virol J. 2(1):43), as noted above. AAV vectors are even more preferred because they are known to result in very stable long-term transgene expression (up to 9 years in dogs (Niemeyer et al., Blood. 2009 Jan 22;113(4):797-806) and ~10 years in humans (Buchlis, G. et al., Blood. 2012 Mar 29;119(13):3038-41)). Preferred adenoviral vectors are modified to reduce the host response as described in Russell (2000, supra).Gene therapy approaches using AAV vectors are described by Wang et al., 2005, J Gene Med. March 9 (Epub ahead of print), Mandel et al., 2004, Curr Opin Mol Ther. 6(5):482–90 and Martin et al., 2004, Eye 18(11):1049–55, Nathwani et al., N Engl J Med. 2011 Dec 22;365(25):2357–65, Apparailly et al., Hum Gene Ther. 2005 Apr;16(4):426–34.

[0209] Another vector suitable for gene therapy includes a retroviral vector. A preferred retroviral vector for use in the present invention is a lentiviral expression construct. Lentiviral vectors have the ability to infect dividing and non-dividing cells and stably integrate into their genome (Amado and Chen, 1999 Science 285: 674-6). Methods for constructing and using lentivirus-based expression constructs are described in U.S. Patent Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633, and 6,323,031, as well as in Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med 2000; 2: 308-16).

[0210] Other vectors suitable for gene therapy include adenovirus vector, herpesvirus vector, polyoma virus vector, or vaccinia virus vector.

[0211] Adeno-associated virus vector (AAV vector)

[0212] The terms "adeno-associated virus," "AAV virus," "AAV virion," "AAV viral particle," and "AAV particle," used synonymously herein, refer to a viral particle consisting of at least one AAV capsid protein (preferably consisting of the entire capsid protein of a particular AAV serotype) and an encapsidated polynucleotide of the AAV genome. If the particle contains a heterologous polynucleotide (e.g., a polynucleotide different from the wild-type AAV genome, such as a transgene to be delivered into a mammalian cell) flanked by AAV inverted terminal repeats, it is commonly known as an "AAV vector particle" or "AAV viral vector" or "AAV vector." AAV refers to a virus that belongs to the genus Dependovirus of the family Parvoviridae. The AAV genome is approximately 4.7 kb in length. and consists of single-stranded deoxyribonucleic acid (ssDNA), which can be positively or negatively detected.The present invention also encompasses the use of a double-stranded AAV, also referred to as dsAAV or scAAV. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs), rep and cap. The rep frame consists of four overlapping genes that encode Rep proteins required for the AAV life cycle. The cap frame contains nucleotide sequences that overlap with the capsid proteins VP1, VP2, and VP3, which interact to form a capsid with icosahedral symmetry (see Carter and Samulski, 2000, and Gao et al., 2004).

[0213] A preferred viral vector or a preferred gene therapy vector is an AAV vector. According to the present invention, the AAV vector preferably comprises a recombinant AAV vector (rAAV vector). As used herein, the term "rAAV vector" refers to a recombinant vector that comprises a portion of an AAV genome encapsidated in a protein coat of a capsid protein derived from an AAV serotype, as explained herein. The portion of the AAV genome may comprise inverted terminal repeats (ITRs) derived from an adeno-associated virus serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, and others. Preferred ITRs are those AAV2 ITRs that are represented by sequences comprising, consisting essentially of, or consisting of SEQ ID NO: 30 (5'-ITR) and SEQ ID NO: 31 (3'-ITR). The present invention also preferably encompasses the use of a sequence that has at least 80% (or at least 81%,at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 30 as the 5'-ITR and a sequence that has at least 80% (or at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 31 as the 3'-ITR.

[0214] The protein coat, consisting of the capsid protein, may be from any AAV serotype. The protein coat may also be referred to as the capsid protein coat. One or preferably all of the wild-type AAV genes may be deleted from the rAAV vector and still contain functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue, and packaging of AAV virions. The ITR sequences may be wild-type sequences, or may have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to the wild-type sequences, or may be altered by, for example, insertion, mutation, deletion, or nucleotide substitution, provided that they remain functional.In this context, functionality refers to the ability to direct genome packaging within the capsid shell, subsequently enabling expression in the host cell to be infected or in the target cell. In the context of the present invention, the capsid protein shell may belong to a different serotype than that of the rAAV vector genome ITR.

[0215] A nucleic acid molecule represented by the selected nucleic acid sequence is preferably inserted between the rAAV genome or the ITR sequences identified above, for example, into an expression construct that contains an expression regulatory element operably linked to the coding sequence and a 3' termination sequence. Said nucleic acid molecule may also be referred to as a transgene.

[0216] The term "AAV accessory functions" generally refers to the relevant AAV functions that are required for rAAV replication and rAAVin trans vector packaging. AAV accessory functions are those AAV functions that are lacking in the rAAV vector but lacking the AAV ITRs (which the rAAV vector genome provides). AAV accessory functions include the two major AAV OFRs, namely the rep coding region and the cap coding region, or sequences that are substantially functionally identical to them. The Rep and Cap regions are well known in the art, see, for example, Chiorini et al. (1999, J. of Virology, Vol 73(2): 1309-1319) or U.S. Patent 5,139,941, incorporated herein by reference. AAV accessory functions can be provided by an AAV helper construct.The introduction of the helper construct into the host cell can be accomplished, for example, by transformation, transfection, or transduction, prior to or simultaneously with the introduction of the rAAV genome present in the rAAV vector identified herein. Therefore, the helper AAV constructs of the present invention can be selected such that they produce the desired combination of serotypes for the capsid protein coat of the rAAV vector, on the one hand, and for the replication and packaging of the rAAV genome present in said rAAV vector, on the other hand.

[0217] An "AAV helper virus" provides additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2), and vaccinia viruses. Additional functions provided by helper viruses can also be introduced into the host cell via plasmids, as described in U.S. Patent 6,531,456, incorporated herein by reference.

[0218] The term "transduction" refers to the delivery of FGF-21 into a recipient host cell via a viral vector. For example, transduction of a target cell with an rAAV vector according to the present invention results in the transfer of the rAAV genome contained in the vector to the transduced cell. The term "host cell" or "target cell" refers to the cell into which the DNA is delivered, such as the subject's muscle cells. AAV vectors are capable of transducing both dividing and non-dividing cells.

[0219] Getting AAV vector

[0220] The production of recombinant AAV (rAAV) for transgene vectorization has been described previously. See Ayuso E, et al., Curr. Gene Ther. 2010; 10:423-436, Okada T, et al., Hum. Gene Ther. 2009; 20:1013-1021, Zhang H, et al., Hum. Gene Ther. 2009; 20:922-929, and Virag T, et al., Hum. Gene Ther. 2009; 20:807-817. These protocols can be used or adapted to generate the AAV of the present invention. According to one embodiment, a producer cell line is transiently transfected with a polynucleotide of the present invention (comprising an expression cassette flanked by an ITR) and a construct(s) encoding the repeat proteins and providing auxiliary functions. According to another embodiment, the cell line stably performs auxiliary functions and is transiently transfected with a polynucleotide of the present invention (comprising an expression cassette flanked by an ITR) and a construct(s) encoding the repeat proteins.According to another embodiment, the cell line stably provides repeat proteins and auxiliary functions and is transiently transfected with a polynucleotide of the present invention. According to another embodiment, the cell line stably provides repeat proteins and is transiently transfected with a polynucleotide of the present invention and a polynucleotide encoding auxiliary functions. According to yet another embodiment, the cell line stably provides a polynucleotide of the present invention, repeat proteins, and auxiliary functions. Methods for producing and using these and other AAV production systems have been described in the art. See sources: Muzyczka N, et al., US Patent 5,139,941, Zhou X, et al., US Patent 5,741,683, Samulski R, et al., US Patent 6,057,152, Samulski R, et al., US Patent 6,204,059, Samulski R, et al., US Patent 6,268,213, Rabinowitz J, et al., US Patent 6,491,907, Zolotukhin S, et al., US Patent 6,660,514, Shenk T, et al., US Patent 6951753, Snyder R, et al., US Patent 7094604, Rabinowitz J, et al., US Patent 7172893, Monahan P, et al., US Patent 7201898, Samulski R, et al., US Patent 7229823 and Ferrari F, et al., US Patent 7439065.

[0221] The rAAV genome present in the rAAV vector comprises at least the nucleotide sequences of the inverted terminal repeat (ITR) regions of one of the AAV serotypes (preferably the AAV2 serotype, as previously specified herein), or nucleotide sequences substantially identical thereto, or nucleotide sequences that have at least 60% identity thereto, or a nucleotide sequence that encodes FGF-21 (under the control of a suitable regulatory element), inserted between two ITRs. The vector genome requires the use of sequences flanking the 5'- and 3'-ITRs to ensure the possibility of efficient packaging of said vector genome into the rAAV capsid.

[0222] The complete genome of several AAV serotypes and the corresponding ITRs have been sequenced (Chiorini et al. 1999, J. of Virology Vol. 73, No. 2, p1309-1319). They can either be cloned or produced by chemical synthesis as is known in the art, for example, using an oligonucleotide synthesizer such as that supplied by, for example, Applied Biosystems Inc. (Foster City, CA, USA), or using standard molecular biology techniques. ITRs can be cloned from the AAV viral genome or excised from a vector containing AAV ITRs. One can either ligate the ITR nucleotide sequences at either end with a nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or replace the AAV sequence between the ITRs with the desired nucleotide sequence.

[0223] Preferably, the rAAV genome present in the rAAV vector does not contain any nucleotide sequences encoding viral proteins, such as the AAVrep (replication) or capsid (capsid) genes. This 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 product that is chemically, enzymatically, or otherwise detectable and / or selectable (e.g., lacZ, aph, etc.), as known in the art.

[0224] The rAAV genome present in the said rAAV vector further comprises a promoter sequence operably linked to a nucleotide sequence that encodes FGF-21.

[0225] A suitable 3'-untranslated sequence may also be operably linked to a nucleotide sequence encoding FGF-21. Suitable 3'-untranslated regions may be regions naturally associated with the nucleotide sequence or may be derived from other genes, such as the SV40 polyadenylation signal (SEQ ID NO: 32) and the rabbit β-globin polyadenylation signal (SEQ ID NO: 33).

[0226] Expression

[0227] Expression can be assessed using any method known to those skilled in the art. For example, expression can be assessed by measuring transgene expression levels in the liver, either as mRNA or protein, using standard assays known to those skilled in the art, such as qPCR, Wernblot, or ELISA.

[0228] Expression can be assessed at any time after administration of the gene construct, expression vector, or composition according to the present description. According to some embodiments of the present invention, expression can be assessed after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, or more.

[0229] In the context of the present invention, CNS-specific and / or brain-specific and / or hypothalamus-specific and / or cerebral cortex-specific and / or hippocampus-specific and / or cerebellum-specific and / or olfactory bulb-specific expression refers to a predominant or pre-dominant (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 FGF-21 in the CNS and / or brain and / or hypothalamus and / or in the cerebral cortex and / or in the hippocampus and / or in the cerebellum and / or in the olfactory bulb compared to other organs or tissues.The other organs or tissues may be the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, colon, hematopoietic tissue, lung, ovary, spleen, stomach, testis, and others. Preferably, the other organs are the liver and / or heart. According to an embodiment, expression is not detected in the liver, pancreas, adipose tissue, skeletal muscle, and / or heart. According to some embodiments, expression is not detected in at least one, at least two, at least three, at least four, or all organs selected from the group consisting of the liver, pancreas, adipose tissue, skeletal muscle, heart, kidney, intestine, hematopoietic tissue, lung, ovary, spleen, stomach, and testis. Expression can be assessed as described above.

[0230] As used herein, the term "specific for the CNS and / or for the brain and / or for the hypothalamus and / or for the cerebral cortex and / or for the hippocampus and / or for the cerebellum and / or for the olfactory bulb" in the context of expression also includes cell type-specific expression in the cell type(s) that make up the CNS and / or the brain and / or the hypothalamus and / or for the cerebral cortex and / or the hippocampus and / or for the cerebellum and / or for the olfactory bulb, respectively.

[0231] Introduction

[0232] "Administration into the CSF" as used herein means direct administration into the CSF, which is located in the subarachnoid space between the arachnoid and pia mater layers surrounding the brain. Administration into the CSF can be accomplished by administration into the cisterna magna, intraventricular administration, or intrathecal administration. "Administration into the cisterna magna" as used herein means administration into the cisterna magna, an opening of the subarachnoid space located between the cerebellum and the dorsal surface of the medulla oblongata. The term "intraventricular administration" as used herein means administration into either of the two lateral ventricles of the brain. The term "intrathecal administration" as used herein includes direct administration into the CSF within the intrathecal space of the spinal column. The term "intraparenchymal administration" as used in this document means local administration directly into any area of ​​the brain parenchyma.The term "intranasal administration" in this application means administration through the nasal structures.

[0233] Codon optimization

[0234] "Codon optimization" as used herein refers to processes used to modify an existing coding sequence or to design a coding sequence, for example, to improve the translation of an RNA transcript molecule transcribed from the coding sequence when expressed in a host cell or organism, or to improve the transcription of the coding sequence. Codon optimization includes, but is not limited to, processes that involve selecting codons for a coding sequence to match the codon preference inherent in expression in the host organism; for example, to match the codon preference inherent in the codon preference inherent in expression in mammalian hosts, preferably mice, dogs, or humans.Codon optimization also eliminates elements that potentially have a negative impact on RNA stability and / or translation (e.g., termination sequences, TATA boxes, splice sites, ribosome entry sites, repetitive and / or GC-rich sequences, and secondary structures or RNA instability motifs). According to some embodiments, codon-optimized sequences exhibit at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater increase in transcription, RNA stability, and / or translation.

[0235] In the description and claims of the present invention, the verb "comprise" and its conjugations are used in an open-ended sense, implying that the following items are included, and items not specifically mentioned are not excluded. In addition, the verb "consist" can be replaced by the phrase "consist essentially of," which means that the peptide or peptide mimetic, culture medium, or composition according to the present description may contain an additional component (or components) in addition to those specifically identified, and said additional component (or components) does not change the unique characteristics of the present invention.Furthermore, the verb "consist" may be replaced by the phrase "consist essentially of," which means that the method according to the present description may include an additional step (or steps) in addition to those specifically identified, and said additional step (or steps) do not change the unique characteristics of the said invention.

[0236] A reference to an element in the singular does not exclude the possibility of more than one element being present, unless the context clearly specifies the presence of one and only one of the elements. Thus, the use of the singular generally implies "at least one."

[0237] In this specification, the wording "at least" for a specific value means that specific value or more. For example, the wording "at least 2" means the same as "2 or more", such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... etc.

[0238] Individual numerical values ​​are claimed as approximate values ​​as if the stated values ​​were preceded by the word "about" or "approximately." Similarly, numerical values ​​in various ranges set forth in this application, unless expressly stated otherwise, are claimed as approximate values ​​as if both the minimum and maximum values ​​within the stated range were preceded by the word "about" or "approximately." In this specification, the terms "about" and "approximately," when applied to numerical values, have the plain, ordinary meanings to one skilled in the art closest to the disclosed subject matter or relevant to the range or element in question. The extent to which the strict numerical boundaries are extended depends on many factors.For example, some factors that may be considered include the criticality of an element and / or the effect of a given amount of variability on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. In the absence of any contrary considerations, the term "about" or "approximately" when used in connection with a numerical value (e.g., approximately 10) preferably means that the stated value may be 1% greater or less than the given value (equal to 10).

[0239] In the present description, the term "and / or" indicates that one or more of the claimed cases may occur, alone or in combination with at least one of the claimed cases, up to all of the claimed cases.

[0240] Each of the embodiments identified herein may be combined with others unless otherwise indicated.

[0241] All patent applications, patents, and printed publications referenced herein are herein incorporated by reference in their entirety, except for any definitions, disclaimers, or disclaimers with respect to the subject matter of the inventions, and except to the extent the incorporated material is inconsistent with the express disclosure herein, in which case the wording herein shall control.

[0242] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in practicing the present invention. Therefore, the present invention is in no way limited to the methods and materials described.

[0243] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0244] Description of drawings

[0245] Figure 1. Expression of moFGF21 in the brain of db / db mice. Expression levels of the codon-optimized coding sequence of mouse FGF-21 (moFgf21) were measured by RT-qPCR (quantitative reverse transcription PCR, RTqPCR) in the hypothalamus, cerebral cortex, hippocampus, and cerebellum of db / db mice and normalized to Rplp0 values. Analyses were performed 12 weeks after CSF administration of AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5 × 10 10 vg per mouse. Results are presented as mean ± SEM, n = 9 animals per group. N / A: not detected.

[0246] Figure 2. Reduced body and tissue weight in db / db mice after treatment with AAV9-FGF21 vectors. (A) Process of body weight change. Body weight was measured weekly after AAV administration. (B) Body weight gain. Body weight gain was calculated as the percentage of increased weight divided by body weight at the time of AAV administration. (C) Weights of iWAT, eWAT, mWAT, BAT, and liver in untreated and AAV9-FGF21-treated db / db mice. Analyses were performed 12 weeks after CSF administration of AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5x10 10 vg / mouse. Results are presented as mean ± SEM, n = 9 animals per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 for comparison with untreated mice. iWAT: inguinal white adipose tissue; eWAT: epididymal white adipose tissue; mWAT: mesenteric white adipose tissue; BAT: interscapular brown adipose tissue; L: liver.

[0247] Figure 3. Reversal of diabetes in db / db mice by intracerebrospinal fluid administration of AAV9-FGF21 vectors. Changes in postprandial blood glucose levels in untreated and AAV9-CAG-moFGF21-dmiRT-treated db / db mice after intracerebrospinal fluid administration of vectors. Results are presented as mean ± SEM, n = 9 animals per group. ***p < 0.001 for comparison with untreated mice.

[0248] Figure 4. Reduced brain inflammation in db / db mice treated with AAV9-FGF21 vectors. The expression levels of astrocyte markers (Gfap and S100b), microglia marker (Aif1), and inflammatory molecules (Nfkb, Il1b-, and IL6) were measured by RT-qPCR in the hypothalamus of db / db mice and normalized to Rplp0 levels. Analyses were performed 12 weeks after CSF administration of AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5×10 10vg / mouse. Results are presented as mean ± SEM, n = 9 animals per group. * p < 0.05 compared with untreated mice. Gfap, glial fibrillary acidic protein; S100b, calcium-binding protein B; Aif1, allograft inflammatory factor 1; Nfkb, nuclear factor kappa B; Il1b-, interleukin 1 beta; IL6, interleukin 6.

[0249] Figure 5. Brain expression of moFGF21 in SAMP8 mice. Expression levels of the codon-optimized coding sequence of mouse FGF-21 (moFGF21) were measured by RT-qPCR in the hypothalamus, cerebral cortex, hippocampus, and cerebellum of SAMP8 mice and normalized to Rplp0 levels. Analyses were performed 14 weeks after CSF administration of AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5×10 10 vg / mouse. Results are presented as mean ± SEM, n = 9 animals per group. N / A: not detected.

[0250] Figure 6. Reduced body and tissue weight in SAMP8 mice after treatment with AAV9-FGF21 vectors. (A) Process of body weight change. Body weight was measured weekly after AAV administration. (B) Body weight gain. Body weight gain was calculated as the percentage of increased weight divided by body weight at the time of AAV administration. (C) Weights of iWAT, eWAT, mWAT, BAT, and liver in untreated and AAV9-FGF21-treated SAMP8 mice. Analyses were performed 14 weeks after CSF administration of AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5 × 10 10 vg / mouse. Results are presented as mean ± SEM, n = 9 animals per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 for comparison with untreated mice. iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue; mWAT, mesenteric white adipose tissue; BAT, interscapular brown adipose tissue; L, liver.

[0251] Figure 7. Reduction of brain inflammation in SAMP8 mice treated with AAV9-FGF21 vectors. The expression levels of astrocyte markers (Gfap and S100b), microglia marker (Aif1), and inflammatory molecules (Nfkb, Il1b-, and IL6) were measured by RT-qPCR in the hypothalamus of SAMP8 mice and normalized to Rplp0 levels. Analyses were performed 14 weeks after the administration of AAV9-CAG-moFGF21-dmiRT vectors into the CSF at a dose of 5×10 10 vg / mouse. Results are presented as mean ± SEM, n = 9 animals per group. ** p < 0.01 for comparison with untreated mice. Gfap, glial fibrillary acidic protein; S100b, calcium-binding protein B; Aif1, allograft inflammatory factor 1; Nfkb, nuclear factor kappa B; Il1b-, interleukin 1 beta; IL6, interleukin 6.

[0252] Figure 8. Expression of moFGF21 in the brain after CSF administration of AAV1-FGF21, AAV2-FGF21, and AAV9-FGF21 vectors. Expression levels of the sequence encoding the codon-optimized mouse FGF21 (moFGF21) were measured by RT-qPCR in the hypothalamus, cerebral cortex, hippocampus, and cerebellum of wild-type mice 3 weeks after CSF administration of AAV1-CAG-moFGF21-dmiRT, AAV2-CAG-moFGF21-dmiRT, or AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5×10 10 vg / mouse. Results were normalized by Rplp0 levels and presented as mean ± SEM, n = 5 animals per group. N / A: not detected.

[0253] Figure 9. FGF-21 protein levels in the brain. FGF-21 protein content was determined by ELISA in brain homogenates of wild-type mice 3 weeks after administration of AAV1-CAG-moFGF21-dmiRT, AAV2-CAG-moFGF21-dmiRT, or AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5×10 10vg / mouse. Results were normalized to total protein levels and presented as mean ± SEM, n = 5 animals per group. N / A: not detected.

[0254] Figure 10. Reduced adiposity and increased thermogenesis after treatment with AAV9-FGF21 vectors. Representative images of hematoxylin and eosin-stained sections of (A) eWAT and (B) BAT of untreated and AAV9-FGF21-treated db / db mice. Baseline magnification x200. (C) Expression levels of thermogenesis markers (Ucp1 and Cidea) were measured by RT-qPCR in BAT of db / db mice and normalized to Rplp0 levels. Analyses were performed 12 weeks after CSF administration of AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5 × 10 10vg / mouse. Results are presented as mean ± SEM, n = 9 animals per group. *** p < 0.001 for comparison with untreated mice. Ucp1, uncoupling protein 1; Cidea, cell death-inducing factor alpha-like effector A; eWAT, epididymal white adipose tissue; BAT, brown adipose tissue.

[0255] Figure 11. Reduced liver triglyceride content in AAV9-FGF21-treated mice. (A) Liver triglyceride content. (B) Serum triglyceride and (C) FFA levels. Analyses were performed 12 weeks after CSF vector administration. Results are presented as mean ± SEM, n = 9 animals per group. *p < 0.05 for comparison with untreated mice. FFA, free fatty acids.

[0256] Figure 12. Improvement of β-cell mass in FGF21-treated db / db mice. (A) Islet numbers and (B) β-cell mass were calculated in untreated and AAV9-FGF21-treated db / db mice after immunohistochemistry of pancreatic sections stained with insulin antibody. Results are shown as mean ± SEM, n = 3 animals per group. *p < 0.05 for comparison with untreated mice.

[0257] Figure 13. Reduced inflammation in adipose tissue and liver of db / db mice after treatment with AAV9-FGF21 vectors. (A) Representative images of MAC-2 immunohistochemistry of eWAT from untreated and AAV9-FGF21-treated db / db mice (n = 6 animals per group) (B) Expression levels of the inflammatory marker F4 / 80 were measured by RT-qPCR in eWAT of db / db mice and normalized to Rplp0 levels. Analyses were performed 12 weeks after CSF administration of AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5 × 10 10 vg / mouse. (CD) Expression levels of inflammatory markers F4 / 80, IL6, and TNF-α were measured by RT-qPCR in BAT (C) and liver (D) of db / db mice and normalized to Rplp0 levels. Analyses were performed 12 weeks after CSF administration of AAV9-CAG-moFGF21-dmiRT vectors at a dose of 5×10 10vg / mouse. Results are presented as mean ± SEM, n = 9 animals per group. * p < 0.05, ** p < 0.01 and *** p < 0.001 for comparison with untreated mice. F4 / 80, G protein-coupled adhesion receptor E1; IL6, interleukin 6; TNF-α, tumor necrosis factor alpha; eWAT, epididymal white adipose tissue; BAT, brown adipose tissue. MAC-2, soluble galactose-binding lectin 3; Arrows indicate MAC-2 signal.

[0258] Figure 14. Brain expression of FGF21 in db / db mice treated with AAV1-FGF21. Expression levels of the sequence encoding the codon-optimized mouse FGF21 (moFgf21) were measured by RT-qPCR in the hypothalamus, cerebral cortex, hippocampus, and cerebellum of db / db mice and normalized to Rplp0 levels. Analyses were performed 16 weeks after CSF administration of AAV1-CAG-moFGF21 vectors at a dose of 5×10 10vg / mouse. Results are presented as mean ± SEM, n = 7 animals per group. N / A: not detected.

[0259] Figure 15. Reduced body weight in db / db mice after treatment with AAV1-CAG-FGF21 vectors. Body weight was measured weekly after AAV administration in untreated db / + (lean) mice, untreated db / db mice, and AAV1-CAG-FGF21-treated db / db mice. Results are presented as mean ± SEM, n = 7 animals per group. * p < 0.05, ** p < 0.01, and *** p < 0.001 for comparison with db / + mice. $$$ p < 0.001 for comparison with untreated db / db mice.

[0260] Figure 16. Reversal of diabetes in db / db mice treated with AAV1-FGF21. (A) Progression of post-prandial blood glucose levels in lean (db / +) mice, untreated db / db mice, and AAV9-CAG-moFGF21-dmiRT-treated db / db mice after CSF vector administration. (B) Fasting blood glucose levels were measured 11 weeks after AAV1-CAG-FGF21 vector administration. Results are shown as mean ± SEM, n = 7 animals per group. ** p < 0.01 and *** p < 0.001 for comparison with db / + mice. $$$ p < 0.001 for comparison with untreated db / db mice.

[0261] Figure 17. Enhanced insulin sensitivity in AAV1-FGF21-treated db / db mice. Intraperitoneal insulin tolerance test. Lean (db / +), untreated db / db mice, and AAV9-CAG-moFGF21-dmiRT-treated db / db mice were given an intraperitoneal injection of 0.75 U / kg insulin, and blood glucose levels were measured at the indicated time points. The test was performed 14 weeks after AAV administration. Results are presented as mean ± SEM, n = 7 animals per group. * p < 0.05, ** p < 0.01, and *** p < 0.001 for comparison with db / + mice. $ p < 0.05 and $$ p < 0.01 for comparison with untreated db / db mice.

[0262] Figure 18. AAV1-CAG-FGF21 Treatment Improves Glucose Tolerance. Glucose tolerance was examined 11 weeks after AAV administration in untreated db / + (lean) mice, untreated db / db mice, and AAV1-CAG-FGF21-treated db / db mice following intraperitoneal injection of glucose (1 g / kg body weight). Results are presented as mean ± SEM, n = 7 animals per group. * p < 0.05 and ** p < 0.01 for comparison with db / + mice. $$$ p < 0.001 for comparison with untreated db / db mice.

[0263] Figure 19. Reduced gluconeogenesis in db / db mice after AAV1-FGF21 administration. Pyruvate tolerance test was performed in lean (db / +) mice, untreated db / db mice, and db / db mice treated with AAV9-CAG-moFGF21-dmiRT. All groups received an intraperitoneal injection of pyruvate (1 g / kg body weight), and blood glucose levels were measured at the indicated time points. The test was performed 12 weeks after AAV administration. Results are presented as mean ± SEM, n = 7 animals per group. ***p < 0.001 for comparison with untreated mice. $$$ p < 0.001 for comparison with untreated db / db mice.

[0264] Examples

[0265] To investigate the effects of FGF-21 in the brain when overexpressed in the brain using AAV vectors, three experiments were performed:

[0266] • Treatment of db / db mice with AAV9-CAG-moFGF21-dmiRT. Application dose: 5×10 10 vg / mouse (example 1).

[0267] • Treatment of SAMP8 mice with AAV9-CAG-moFGF21-dmiRT. Applied dose: 5×10 10 vg / mouse (example 2).

[0268] • Treatment of db / db mice with AAV1-CAG-moFGF21. Applied dose: 5×10 10 vg / mouse (example 4).

[0269] Furthermore, the present inventors also examined the transduction efficiency in the brain of AAV1-FGF21, AAV2-FGF21, and AAV9-FGF21 vectors after administration into the CSF of wild-type mice (Example 3).

[0270] dmiRT refers to 4 copies of the miRT-122a sequence and 4 copies of the miRT-1 sequence.

[0271] The sequence of the CAG-moFGF21-dmiRT gene construct is contained in the sequence SEQ ID NO: 35. The sequence of the CAG-moFGF21 gene construct is contained in the sequence SEQ ID NO: 46.

[0272] General Procedures for Examples

[0273] Characteristics of subjects

[0274] Male BKS.Cg-+Lepr mice were used db / +Lepr dbOlaHsd (db / db), BKS.Cg-m+ / +Lepr db / OlaHsd (db / +, lean) SAMP8 / TaHsd (SAMP8) and C57Bl / 6J (wild-type). Mice were given free access to a standard diet (2018S Teklad Global Diets®, Harlan Labs, Inc., Madison, WI, USA) and maintained under 12-h light / dark cycles (lights on at 8:00 AM) and a constant temperature (22°C ± 2°C). For tissue sampling, mice were anesthetized with isoflurane inhalation anesthetic (IsoFlo®, Abbott Laboratories, Abbott Park, IL, USA) and decapitated. Tissues of interest were removed and stored at -80°C until analysis. All experimental procedures were approved by the Ethics Committee for Animal and Human Experimentation of the Autonomous University of Barcelona (Universitat Autònoma de Barcelona).

[0275] Recombinant AAV vectors

[0276] Single-stranded AAV vectors of serotypes 1, 2, and 9 were generated by triple transfection of HEK293 cells according to standard methods (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6) :423-36). Cells were cultured in 10 roller flasks (850 cm 2, flat; Corning™, Sigma-Aldrich Co., St. Louis, MO, USA) in DMEM with 10% FBS to 80% confluency and co-transfected using the calcium phosphate method with a plasmid carrying an expression cassette flanked by the AAV2 ITR (SEQ ID NO: 35), a helper plasmid carrying the repAAV2 gene and the capAAV gene of serotypes 1, 2, or 9, respectively, and a plasmid carrying adenovirus helper functions. The transgene used was a codon-optimized mouse FGF-21 coding sequence (SEQ ID NO: 9) under the control of the chicken early enhancer / beta-actin (CAG) promoter (SEQ ID NO: 27). In Examples 1, 2 and 3, the transgene also contained the addition of four tandem repeats of the miRT-122a sequence (5'CAAACACCATTGTCACACTCCA3', SEQ ID NO: 12) and four tandem repeats of the miRT-1 sequence (5'TTACATACTTCTTTACATTCCA3', SEQ ID NO: 13) cloned into the 3' untranslated region of the expression cassette.In Example 4, the cassette lacked miRT-122a and miRT-1; AAV was purified using an optimized method based on a polyethylene glycol precipitation step and two consecutive passes through a cesium chloride (CsCl) density gradient. This second-generation CsCl-based protocol dramatically reduced the amount of blank AAV capsids, as well as DNA and protein contaminants (Ayuso, E. et al., 2010. Curr Gene Ther. 10(6) :423-36). Purified AAV vectors were dialyzed against PBS, filtered, and stored at -80°C. Viral genome titers were determined by quantitative PCR according to the protocol described for the AAV2 reference standard material, using linearized plasmid DNA as a standard curve (Lock M, et al., Hum. Gene Ther. 2010; 21:1273-1285). Vectors were constructed according to molecular biology techniques well known in the art.

[0277] In vivo administration of AAV vectors into CSF

[0278] Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg), the back of the head was shaved, from behind the ears to approximately the shoulder blades, and rinsed with ethanol. Mice were held in a prone position with a slight head-down tilt. A 2 mm incision was made in the rostrocaudal direction to insert a Hamilton syringe at an angle of 45-55° into the cisterna magna, between the occipital bone and the C1 vertebra, and 5 μL (microliters) of the vector solution was injected. Considering that the CNS is the main target compartment for vector delivery, mice received a dose of the same number of vector genomes per mouse regardless of body weight (5 × 10 10 vg / mouse).

[0279] Immunohistochemical and morphometric analysis

[0280] Tissues were fixed for 24 hours in formalin (Panreac Química), embedded in paraffin, and sectioned. Tissue samples were stained with hematoxylin and eosin, and images were acquired using a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) coupled to a video camera equipped with a monitor and image analysis software (analySIS 3.0; Soft Imaging System, Center Valley, PA, EEUU).

[0281] Immunohistochemistry

[0282] Tissues were fixed for 12-24 h in 10% formalin, embedded in paraffin, and sectioned. For immunohistochemical detection, sections were deparaffinized and incubated overnight at 4°C with rat anti-MAC2 antibody (1:50; CL8942AP; Cedarlane) and guinea pig anti-insulin antibody (1:100; I-8510; Sigma-Aldrich). Biotinylated rabbit anti-rat antibody (1:300; E0467; Dako) and peroxidase-linked rabbit anti-guinea pig antibody (1:300; P0141; Dako) were used as secondary antibodies. An ABC peroxidase kit (Pierce) was used for immunodetection, and sections were counterstained with Mayer's hematoxylin. The percentage of β-cell area in the pancreas was analyzed on two insulin-stained sections separated by 200 μm by dividing the area of ​​all insulin-positive cells in one section by the total pancreatic area in that section.β-cell mass was calculated by multiplying pancreatic mass by the percentage of β-cell area as described previously (Casellas et al., 2006).

[0283] RNA analysis

[0284] Total RNA was prepared from the hypothalamus, cerebral cortex, hippocampus, cerebellum, and olfactory bulb using Tripure extraction reagent (Roche Diagnostics Corp., Indianapolis, IN, USA), and from white adipose tissue, brown adipose tissue, and liver using Qiazol lysis reagent (Qiagen NV, Venlo, the Netherlands) and RNeasy Mini Kit or RNeasy Micro Kit for hippocampal samples (Qiagen NV, Venlo, the Netherlands). For RT-PCR analysis, 1 μg of RNA was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (04379012001, Roche, CA, USA). Quantitative real-time PCR was performed in a SmartCyclerII® (Cepheid, Sunnyvale, USA) using TB Green Premix Ex TaqII (Takara Bio Europe, France). Data were normalized to Rplp0 levels and analyzed as described previously (Pfaffl, M., Nucleic Acids Res. 2001; 29(9):e45).

[0285] General information about the primers used is given below:

[0286] moFgf21-Fw:5'-CCTAACCAGGACGCCACAAG-3’ (SEQ ID NO: 47)

[0287] moFgf21-Rv:5'-GTTCCACCATGCTCAGAGGG -3’ (SEQ ID NO: 48)

[0288] Gfap-Fw:5'-ACAGACTTTCTCCAACCTCCAG-3’ (SEQ ID NO: 49)

[0289] Gfap-Rv: 5'-CCTTCTGACACGGATTTGGT-3’ (SEQ ID NO: 50)

[0290] S100b-Fw: 5'-AACAACGAGCTCTCTCACTTCC-3’ (SEQ ID NO: 51)

[0291] S100b-Rv: 5'-CGTCTCCATCACTTTGTCCA-3’ (SEQ ID NO: 52)

[0292] Aif1-Fw: 5'-TGAGCCAAAGCAGGGATTTG-3’ (SEQ ID NO: 53)

[0293] Aif1-Rv: 5'-TCAAGTTTGGACGGCAGATC-3’ (SEQ ID NO: 54)

[0294] Nfkb-Fw: 5'-GACCACTGCTCAGGTCCACT-3’ (SEQ ID NO: 55)

[0295] Nfkb-Rv: 5'-TGTCACTATCCCGGAGTTCA-3’ (SEQ ID NO: 56)

[0296] Il1b-Fw: 5'-ATGAAGGGCTGCTTCCAAAC-3’ (SEQ ID NO: 57)

[0297] Il1b-Rv: 5'-ATGTGCTGCTGCGAGATTTG-3’ (SEQ ID NO: 58)

[0298] Il6-Fw: 5'-TCGCTCAGGGTCACAAGAAA-3’ (SEQ ID NO: 59)

[0299] Il6-Rv: 5'-CATCAGAGGCAAGGAGGAAAAC-3’ (SEQ ID NO: 60)

[0300] Ucp1-Fw:5'-GGCCTCTACGACTCAGTCCA-3' (SEQ ID NO: 61)

[0301] Ucp1-Rv:5'-TAAGCCGGCTGAGATCTTGT-3' (SEQ ID NO: 62)

[0302] Cidea-Fw:5'-AAACCATGACCGAAGTAGCC-3' (SEQ ID NO: 63)

[0303] Cidea-Rv: 5'-AGGCCAGTTTGTGATGACTAAGAC-3' (SEQ ID NO: 64)

[0304] Tnfa-Fw:5'-CGGCATGGATCTCAAAGACAAC-3' (SEQ ID NO: 65)

[0305] Tnfa-Rv:5'-AGATAGCAAATCGGCTGACG-3' (SEQ ID NO: 66)

[0306] F4 / 80-Fw:5'-CTTTGGCTATGGGCTTCCAGTC-3' (SEQ ID NO: 67)

[0307] F4 / 80-Rv:5'-GCAAGGAGGACAGAGTTTATC-3' (SEQ ID NO: 68)

[0308] Rplp0-Fw: 5'-ACTGGTCTAGGACCCGAGAA-3' (SEQ ID NO: 69)

[0309] Rplp0-Fw:5'-TCCCACCTTGTCTCCAGTCT-3' (SEQ ID NO: 70)

[0310] Money and music

[0311] Blood glucose levels were measured using a Glucometer Elite™ analyzer (Bayer, Leverkusen, Germany). Brain FGF-21 protein levels were determined using a mouse / rat sandwich FGF-21 quantitative enzyme-linked immunosorbent assay (ELISA) kit (MF2100, R&D Systems, Abingdon, UK) and normalized to total protein content measured using the Bradford Protein Assay reagent (Bio-Rad, Germany) in whole brain homogenates. For liver lipid extraction, frozen samples of approximately 100 mg were weighed and homogenized in a chloroform:methanol (2:1) mixture as described by Carr et al. Liver triglycerides and serum triglycerides were quantified spectrophotometrically using an enzymatic assay kit (Horiba-ABX, Montpellier, France).Serum free fatty acids were measured using the acyl-CoA synthase and acyl-CoA oxidase assays (Wako Chemicals GmbH, Neuss, Germany). All biochemical parameters were determined using a Pentra 400 Analyzer (Horiba-ABX).

[0312] Insulin Tolerance Test

[0313] To conduct the insulin tolerance test, conscious, fed mice were injected intraperitoneally with insulin (0.75 IU / kg body weight; Humulin Regular; Eli Lilly, Indianapolis, IN). Glucose concentrations were determined in blood samples obtained from the tail vein at specified time points after insulin injection.

[0314] Glucose tolerance test

[0315] Conscious mice were fasted overnight (16 h) and given an intraperitoneal injection of glucose (1 g / kg body weight). Blood samples from the tail vein were collected for glycemia measurements at designated time points.

[0316] Pyruvate tolerance test

[0317] Conscious mice were fasted overnight (16 h) and given an intraperitoneal injection of pyruvate (1 g / kg body weight). Blood samples from the tail vein were collected for glycemia measurements at designated time points.

[0318] Example 1. Reversal of obesity and diabetes in db / db mice by CSF administration of AAV9-CAG-moFGF21-dmirT vectors

[0319] The present inventors determined the therapeutic potential of AAV-mediated brain genetic engineering using FGF-21 to prevent the development of diabetes and obesity in 7-week-old male db / db mice, which have a defect in leptin signaling and are a widely used genetic model of obesity and diabetes. For this purpose, db / db mice were locally injected into the cerebrospinal fluid (CSF) via the cisterna magna with 5×10 10AAV9 vectors encoding a codon-optimized murine FGF-21 coding sequence under the control of the universal CAG promoter containing target sites for liver-specific miR-122a and heart-specific miR-1 (AAV9-CAG-moFGF21-dmiRT) were used in mice per mouse. Untreated db / db animals served as controls.

[0320] Administration of AAV9-CAG-moFGF21-dmiRT vectors into CSF ​​mediated widespread overexpression of FGF-21 in the brain, as evidenced by increased expression levels of this factor in various brain regions such as the hypothalamus, cerebral cortex, hippocampus, and cerebellum 12 weeks after AAV administration (Fig. 1).

[0321] While untreated db / db mice continued to gain weight throughout the 12-week follow-up period (~50% weight gain), there was a clear reduction in weight gain in the cohort treated with FGF21-encoding vectors (~20% weight gain) (Figs. 2A and 2B). Consistent with this, animals treated with AAV9-CAG-moFGF21-dmiRT vectors exhibited reduced fat deposition and a 60% reduction in liver weight (Fig. 2C). Notably, db / db mice in which FGF21 gene transfer was targeted to the brain also exhibited complete normalization of postprandial glycemia, indicating that diabetes is counteracted in these animals (Fig. 3).

[0322] Obesity is associated with inflammation in the brain (O. Guillemot-Legris, GG Muccioli, Trends Neurosci. 40, 237-253 (2017). Inflammation in the brain was analyzed by the expression of astrocyte markers Gfap and S100b, microglia marker Aif1, and inflammatory molecules such as Nfkb, Il1b-, and IL6. db / db mice treated with AAV9-CAG-moFGF21-dmiRT vectors showed reduced expression of GFAP, S100b, Aif1, Nfkb, Il1b-, and IL6 in the hypothalamus (Fig. 4).

[0323] Example 1.1

[0324] Histological analysis of white adipose tissue by hematoxylin and eosin staining revealed a reduced size of white adipocytes in eWAT (Fig. 10A). Histological examination of BAT showed reduced lipid accumulation and more multilocular brown adipocytes (Fig. 10B). Consistent with these results, the expression levels of Ucp1 and Cidea were significantly increased in BAT of FGF21-treated mice (Fig. 10C), suggesting enhanced thermogenesis after AAV-FGF21 administration into the CNS. Hepatic triglyceride content was decreased in AAV-FGF21-treated db / db mice (Fig. 11A). Concomitantly, the levels of circulating triglycerides and free fatty acids in serum were also decreased in these mice (Figs. 11B and 11C). Immunohistochemistry of the pancreas revealed increased islet numbers (Fig. 12A) and improved β-cell mass (Fig. 12B) in db / db mice after treatment with AAV9-FGF21 vectors.

[0325] Obesity and diabetes are associated with systemic inflammation. Immunohistochemistry based on the proinflammatory marker MAC-2 in white adipose tissue indicated a decrease in macrophage infiltration in mice treated with AAV9-FGF21 (Fig. 13A), and this decrease was associated with a decrease in the mRNA expression levels of F4 / 80 (Fig. 13B). The expression levels of proinflammatory cytokines F4 / 80, IL6, and TNF-alpha in brown adipose tissue and liver of animals treated with FGF-21 were also decreased (Figs. 13C and 13D, respectively), indicating a reduction in systemic inflammation after FGF-21 gene therapy.

[0326] Example 2. Reduction of weight gain in SAMP8 mice by administration of AAV9-CAG-moFGF21-dmirT vectors into the CSF

[0327] Seven-week-old male rapidly aging prone 8 (SAMP8) mice, a widely used aging model with age-related brain pathologies, were injected locally into the CSF, via the cisterna magna, with 5×1010 vg / mouse of AAV9-CAG-moFGF21-dmiRT vectors. Untreated SAMP8 animals were used as controls.

[0328] Similar to the observations made in db / db mice, CSF administration of AAV9-CAG-moFGF21-dmiRT vectors mediated robust FGF21 overexpression in the hypothalamus, cerebral cortex, hippocampus, and cerebellum of SAMP8 mice (Fig. 5) 14 weeks after AAV administration. FGF21-treated mice exhibited lower body weight gain than the untreated cohort (Figs. 6A and 6B), along with decreased liver weight (Fig. 6C). Furthermore, SAMP8 mice overexpressing FGF21 in the brain had reduced hypothalamic expression of the proinflammatory cytokines Il1b- and IL6 (Fig. 7).

[0329] Example 3. Transduction in the brain after administration of AAV1-CAG-moFGF21-dmirT, AAV2-CAG-moFGF21-dmirT, and AAV9-CAG-moFGF21-dmirT vectors into the CSF.

[0330] To investigate the ability of several AAV serotypes to efficiently transduce in the brain after direct administration into the CSF via the cisterna magna, wild-type mice were treated with 5×10 10 vg / mouse of AAV1, AAV2, and AAV9 vectors encoding a codon-optimized murine FGF-21 coding sequence under the control of the universal CAG promoter, which contained target sites for liver-specific miR-122a and heart-specific miR-1 (AAV1-CAG-moFGF21-dmiRT, AAV2-CAG-moFGF21-dmiRT, and AAV9-CAG-moFGF21-dmiRT, respectively). Untreated wild-type mice served as controls.

[0331] Three weeks after the CSF administration of AAV vectors, brain samples were obtained and RT-PCR analysis revealed increased moFGF21 expression in various brain regions, such as the hypothalamus, cerebral cortex, hippocampus, and cerebellum (Fig. 8). Moreover, moFGF21 overexpression resulted in increased FGF-21 protein content throughout the brain (Fig. 9).

[0332] Example 4. Reversal of obesity and diabetes in db / db mice by CSF administration of AAV1-CAG-moFGF21 vectors.

[0333] The therapeutic potential of AAV-mediated brain genetic engineering using FGF-21 gene therapy in preventing the development of diabetes and obesity was also determined in 7-week-old male db / db mice, which were injected locally into the cerebrospinal fluid (CSF), via the cisterna magna, with 5×10 10vg / mouse of AAV1 vectors encoding a codon-optimized murine FGF-21 coding sequence under the control of the universal CAG promoter (AAV1-CAG-moFGF21). Untreated db / db mice and untreated db / + (lean) mice were used as controls.

[0334] Administration of AAV1-CAG-moFGF21 vectors into CSF ​​mediated widespread overexpression of FGF-21 in the brain, as evidenced by increased expression levels of this factor in various brain regions such as the hypothalamus, cerebral cortex, hippocampus, cerebellum, and olfactory bulb at 16 weeks after AAV administration (Fig. 14).

[0335] While untreated db / db mice continued to gain weight throughout the 14-week follow-up period, body weight did not increase in the cohort treated with AAV1-FGF21-encoding vectors (Fig. 15). Notably, db / db mice in which FGF21 gene transfer was targeted to the brain also showed complete normalization of fasting and postprandial glycemia (Figs. 16A and 16B), indicating that diabetes is counteracted in these animals.

[0336] The insulin tolerance test showed that insulin resistance in db / db mice was improved after treatment with AAV1-FGF21 viral vectors (Fig. 17), and the intraperitoneal glucose tolerance test in overnight-fasted mice showed that db / db mice treated with AAV1-CAG-moFGF21 were more glucose tolerant than untreated db / db mice (Fig. 18). The intraperitoneal pyruvate tolerance test was performed as an indicator of hepatic gluconeogenesis. Following the pyruvate challenge, blood glucose levels increased to 600 mg / dL (deciliter) in untreated db / db mice and remained elevated throughout the test, while glucose levels in FGF-21-treated db / db mice and in treated lean mice increased to a maximum of 150 mg / dL, indicating decreased gluconeogenesis following AAV1-CAG-FGF21 treatment (Fig. 19).

[0337] Sequences

[0338] SEQ ID NO: Description of the sequence 1 Amino acid sequence of FGF-21 from Homo sapiens 2 Amino acid sequence of FGF-21 from Mus musculus 3 Amino acid sequence of FGF-21 from Canis lupus familiaris 4 Nucleotide sequence of FGF-21 from Homo sapiens 5 Codon-optimized nucleotide sequence of Homo sapiens FGF-21 - variant 1 6 Codon-optimized nucleotide sequence of Homo sapiens FGF-21 - variant 2 7 Codon-optimized nucleotide sequence of Homo sapiens FGF-21 - variant 3 8 Nucleotide sequence of FGF-21 from Mus musculus 9 Codon-optimized nucleotide sequence of FGF-21 from Mus musculus 10 Nucleotide sequence of FGF-21 from Canis lupus familiaris 11 Codon-optimized nucleotide sequence of FGF-21 from Canis lupus familiaris 12 Nucleotide sequence encoding miRT-122a 13 Nucleotide sequence encoding miRT-1 14 Nucleotide sequence encoding miRT-152 15 Nucleotide sequence encoding miRT-199a-5p 16 Nucleotide sequence encoding miRT-199a-3p 17 Nucleotide sequence encoding miRT-215 18 Nucleotide sequence encoding miRT-192 19 Nucleotide sequence encoding miRT-148a 20 Nucleotide sequence encoding miRT-194 21 Nucleotide sequence encoding miRT-133a 22 Nucleotide sequence encoding miRT-206 23 Nucleotide sequence encoding miRT-208-5p 24 Nucleotide sequence encoding miRT-208a-3p 25 Nucleotide sequence encoding miRT-499-5p 26 Nucleotide sequence of a chimeric intron consisting of the introns of the human β-globin and immunoglobulin heavy chain genes 27 Nucleotide sequence of the CAG promoter 28 Nucleotide sequence of the CMV promoter 29 Nucleotide sequence of the CMV enhancer 30 Truncated 5'-ITR of AAV2 31 Truncated 3'-ITR of AAV2 32 SV40 polyadenylation signal 33 Rabbit β-globin polyadenylation signal 34 Sequence of the CMV promoter and CMV enhancer 35 pAAV-CAG-moFGF21-dmiRT 36 Minimal CMV promoter 37 EF1α promoter 38 RSV promoter 39 Synapsin-1 promoter 40 Calcium / calmodulin-dependent protein kinase II (CaMKII) promoter 41 Glial fibrillary acidic protein (GFAP) promoter 42 Nestin promoter 43 Homeobox protein 9 (HB9) promoter 44 Tyrosine hydroxylase (TH) promoter 45 Myelin basic protein (MBP) promoter 46 pAAV-CAG-moFGF21 47-70 RT-qPCR primers

[0339] Amino acid sequence of FGF-21Homo sapiens (SEQ ID NO: 1)

[0340] MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRF LCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS

[0341] Nucleotide sequence of FRF-21Homo sapiens(SEQ ID NO: 4)

[0342] ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACACA GATGATGCCCAGCAGACAGAAGCCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAG CGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACCGGGACCCTGCA CCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCCTGGCCCCCCAGCCCCCCGATGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTTCCCAGGGCCGAAGCCCCAGCTACGCTTCCTGA

[0343] Codon-optimized nucleotide sequence of FRF-21Homo sapiens -variant 1 (SEQ ID NO: 5)

[0344] ATGGATTCTGATGAGACAGGCTTCGAGCACAGGCCTGTGGGTTTCAGTTCTGGCTGGACTGCTGGGAGCCTGTCAGGCACACCCTATTCCAGATAGCAGCCCTCTGCTGCAGTTCGGCGGACAAGTGCGGCAGAGATACCTGTACACC GACGACGCCCAGCAGACAGAAGCCCACCTGGAAATCAGAGAGGATGGCACAGTTGGCGGAGCCGCCAGTCAGTCCTGAATCTCTGCTCCAGCTGAAGGCCCTGAAGCCCTGGCGTGATCCAGATCCTGGGCGTGAAAACCAGCCGGTTCCTGTGCCAA AGACCTGACGGCGCCCTGTATGGCAGCCTGCACTTTGATCCTGAGGCCTGCAGCTTCAGAGAGCTGCTGCTTGAGGACGGCTACAACGTGTACCAGTCTGAGGCCCATGGCCTGCCTCTGCATCTGCCTGGAAACAAGCCCTCAGAGATCCCGCT CCTAGAGGCCCTGCCAGATTTCTGCCTCTTCCTGGATTGCCTCCTGCTCTGCCAGAGCCTCCTGGAATTCTGGCTCCTCAGCCTCCTGATGTGGGCAGCTCTGATCCTCTGAGCATGGTCGGACCTAGCCAGGGCAGATCCTAGCTACGCCTCTTGA

[0345] Codon-optimized nucleotide sequence of FRF-21Homo sapiens -variant 2 (SEQ ID NO: 6)

[0346] ATGGACAGCGATGAAACCGGGTTCGAGCAGCGGTGTGGGTGTCCGTGCTGGCCGGACTGCTCCTGGGAGCCTGTCAGGCGCACCCCATCCCTGACTCCTCGCCGCTGCTGCAATTCGGCGACAAGTCCGCCAGAGATACCTGTACACC GACGACGCCCAGCAGACCGAAGCCCACCTGGAAATTCGGGAGGACGGGACTGTGGGAGGCGCTGCAGATCAGTCACCCGAGTCCCTCCTCCAACTGAAGGCCTTGAAGCCCGGCGTGATTCAGATCCTGGGCGTGAAAACTTCCCGCTTCCTTTGCCAA CGGCCGGATGGAGCTCTGTACGGATCCCTGCACTTCGACCCCGAGCTGCTCATTCCGCGCTGCTCCTTGAGGACGGCTATAACGTGTACCAGTCTGAGGCCCATGGACTCCCCCTGCATCTGCCCGGCAACAAGTCCCCTCACCGGGATCCTGCC CCAAGAGGCCCAGCTCGGTTTCTGCCTCTGCCGGGACTGCCTCCAGCGTTGCCCGAACCCCCTGGTATCCTGGCCCCGCAACCACCTGACGTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAGCCAGGAAGGTCCCCGTCCTACGCATCCTGA

[0347] Codon-optimized nucleotide sequence of FRF-21Homo sapiens -variant 3 (SEQ ID NO: 7)

[0348] ATGGATTCCGACGAAACTGGATTTGAACATTCAGGCTGTGGGTCTCTGTGCTGGCTGGACTGCTGGGGGCTTGTCAGGCTCACCCCATCCCTGACAGCTCCCCTCTGCTGCAGTTCGGAGGACAGGTGCGAGAGAGATACCTGTATACC GACGATGCCCAGCAGACAGAGGCACACCTGGAGATCAGGGAGGACGGAACCGTGGGAGGAGCAGCCAGTCTCCCGAGCCTGCTGCAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAGACATCTCGGTTTCTGTGCCAG CGGCCCGACGGCGCCCTGTACGGCTCCCTGCACTTCGATCCCGAGGCCTGTTCTTTAGGGAGCTGCTGGAGGACGGCTACAACGTGTATCAGAGCGAGGCACAGGCCTGCCACTGCACCTGCCTGGCAATAAGTCCCCTCACCGCGATCCAGCA CCCAGGGGCCCAGCACGCTTCCTGCCTCTGCCAGGCCTGCCCCCTGCCCTGCCAGAGCCACCCGGCATCCTGGCCCCCCAGCCTCCAGATGTGGGCTCCAGCGATCCTCTGTCAATGGTGGGGCCAAGTCAGGGGCGGAGTCCTTCATACGCATCATAA

[0349] Nucleotide sequence of codon-optimized mouse FRF-21 (SEQ ID NO: 9)

[0350] ATGGAATGGATGAGAAGCAGAGTGGGCACCCTGGGCCTGTGGGTGCGACTGCTGGCTGTGTTTCTGCTGGGCGTTACCAGGCCTACCCCATCCCTGACTCTAGCCCCCTGCTGCAGTTTGGCGGACAAGTGCGGAGAGAGTCCTGCT ACCGACGACGACCAGGACACCGAGGCCCACCTGGAAATCCGCGAGGATGGCACAGTCGTGGGCGCTGCTCACAGAAGCCCTGAGAGCCTGCTGGAACTGAAGGCCCTGAAGCCCGGCGTGATCCAGATCCTGGGCGTGAAGGCCAGCAGATTCCTGTGCC AGCAGCCTGACGGCGCCCTGTACGGCTCTCCTCACTTCGATCCTGAGGCCTGCAGCTTCAGAGAGCTGCTGCTGGAGGACGGCTACAACGTGTACCAGTCTGAGGCCCACGGCCTGCCCCTGAGACTGCCTCAGAAGGACAGCCCTAAACCAGGACGCCAC AAGCTGGGGACCTGTGCGGTTCCTGCCTATGCCTGGACTGCTGCACGAGCCCCAGGATCAGGCTGGCTTTCTGCCTCCTGAGCCTCCAGACGTGGGCAGCAGCGACCCTCTGAGCATGGTGGAACCTCTGCAGGGCAGAAGCCCCAGCTACGCCTCTTGA

[0351] CAG promoter nucleotide sequence (SEQ ID NO: 27)

[0353] CMV promoter nucleotide sequence (SEQ ID NO: 28)

[0354] GTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCATTGACGTCAATGGGAGTTTGTTTTGGC ACCAAAATCAACGGGACTTTCCAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAATGGGCGGTAGGCGTGTAGGTGGGAGGTCTATATAAGCAGAGCT

[0355] CMV enhancer nucleotide sequence (SEQ ID NO: 29)

[0356] GGCATTGATTATTGACTAGTTTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCCCATTGACGTCATAATGACGTATTAACCCCCATAGGACCATAGGACCATAGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCCCATTGACGTC TTGACGTCCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACTTACGTATCATTCATTCGCCATTCATGCCAGTATCATGACCTTACGGGACTTTCCTACTGGCAGTACATCTATCATGTATCATTCATGCCATTCATTTGACGTCATGATCATGGGACTCCTACTGGCAGTACATCTATTTACGTATCATTCATGCCATTC

[0357] CMV promoter and CMV enhancer sequence (SEQ ID NO: 34)

[0358] GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT

[0359] 5’-ITR AAV2 (SEQ ID NO: 30)

[0360] GCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC

[0361] CCGGGCGTCG GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG

[0362] CGCAGAGAGG GAGTGGCCAA CTCCATCACT AGGGGTTCCT

[0363] 3’-ITR AAV2 (SEQ ID NO: 31)

[0364] AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTCTGCG

[0365] CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGC

[0366] Rabbit β-globin polyadenylation signal (3'-UTR and flanking region of rabbit beta-globin, including polyA (polyadenylation) signal) (SEQ ID NO: 33)

[0367] GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAA TTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAA CAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC

[0368] miRT sequences

[0369] miRT-122a (SEQ ID NO: 12): 5' CAAACACCATTGTCACACTCCA 3', a target of microRNA-122a (miRBase accession number MI0000442), which is expressed in the liver.

[0370] miRT-152 (SEQ ID NO: 14): 5' CCAAGTTCTGTCATGCACTGA 3', a target of microRNA-152 (MI0000462) expressed in the liver.

[0371] miRT-199a-5p (SEQ ID NO: 15): 5' GAACAGGTAGTCTGAACACTGGG 3', target of microRNA 199a (MI0000242), which is expressed in the liver.

[0372] miRT-199a-3p (SEQ ID NO: 16): 5' TAACCAATGTGCAGACTACTGT 3', a target of microRNA-199a (MI0000242) expressed in the liver.

[0373] miRT-215 (SEQ ID NO: 17): 5' GTCTGTCAATTCATAGGTCAT 3', a target of microRNA-215 (MI0000291) expressed in the liver.

[0374] miRT-192 (SEQ ID NO: 18): 5' GGCTGTCAATTCATAGGTCAG 3', a target of microRNA-192 (MI0000234) expressed in the liver.

[0375] miRT-148a (SEQ ID NO: 19): 5' ACAAAGTTCTGTAGTGCACTGA 3', target of microRNA-148a (MI0000253) expressed in the liver.

[0376] miRT-194 (SEQ ID NO: 20): 5' TCCACATGGAGTTGCTGTTACA 3', a target of microRNA-194 (MI0000488) expressed in the liver.

[0377] miRT-133a (SEQ ID NO: 21): 5' CAGCTGGTTGAAGGGGACCAAA 3', target of microRNA-133a (MI0000450) expressed in the heart.

[0378] miRT-206 (SEQ ID NO: 22): 5' CCACACACTTCCTTACATTCCA 3', target of microRNA-206 (MI0000490) expressed in the heart.

[0379] miRT-1 (SEQ ID NO: 13): 5' TTACATACTTCTTTACATTCCA 3', target of microRNA-1 (MI0000651) expressed in the heart.

[0380] miRT-208a-5p (SEQ ID NO: 23): 5' GTATAACCCGGGCCAAAAGCTC 3', a target of microRNA-208a (MI0000251) expressed in the heart.

[0381] miRT-208a-3p (SEQ ID NO: 24): 5' ACAAGCTTTTTGCTCGTCTTAT 3', a target of microRNA-208a (MI0000251) expressed in the heart.

[0382] miRT-499-5p (SEQ ID NO: 25): 5' AAACATCACTGCAAGTCTTAA 3', target for microRNA-499 (MI0003183) which is expressed in the heart.

[0383] pAAV-CAG-moFGF21-dmiRT (SEQ ID NO: 35)

[0384] 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAATCGGCC AACGCGCGGG

[0385] 51 GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT

[0386] 101 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG

[0387] 151 GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT

[0388] 201 GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC

[0389] 251 TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA

[0390] 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC

[0391] 351 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC

[0392] 401 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT

[0393] 451 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA

[0394] 501 GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT

[0395] 551 CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA

[0396] 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG

[0397] 651 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA

[0398] 701 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA

[0399] 751 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT

[0400] 801 TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAGGA TCTCAAGAAG

[0401] 851 ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA

[0402] 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT

[0403] 951 CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT

[0404] 1001 AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA

[0405] 1051 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA

[0406] 1101 GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA

[0407] 1151 TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG

[0408] 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC

[0409] 1251 CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG

[0410] 1301 TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA

[0411] 1351 CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGTCAAG

[0412] 1401 GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG

[0413] 1451 GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG

[0414] 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG

[0415] 1551 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA

[0416] 1601 TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG

[0417] 1651 CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG

[0418] 1701 GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC

[0419] 1751 CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT

[0420] 1801 TCTGGGTGAG CAAAACAGG AAGGCAAAAAT GCCGCAAAAA AGGGATAAG

[0421] 1851 GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT

[0422] 1901 GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT

[0423] 1951 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT

[0424] 2001 GCCACCTGAC GTCTAAGAAA CCTATTAT ASSISTANT ACCTAAAAA

[0425] 2051 ATAGGCGTAT CACGAGGCCC TTTCGTCTCG CGCGTTTCGG TGATGACGGT

[0426] 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA

[0427] 2151 AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG

[0428] 2201 GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG

[0429] 2251 AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA

[0430] 2301 ATACCGCATC AGGCGATTCC AACATCCAAT AAATCATACA GGCAAGGCAA

[0431] 2351 CONNECTION AAATAGCCTC CONNECTIONAA GCTAAATCGG

[0432] 2401 TTGTACCAAA AACATTATGA CCCTGTAATA CTTTTGCGGG AGAAGCCTTT

[0433] 2451 ATTTCAACGC AAGGATAAAA ATTTTTAGAA CCCTCATATA TTTTAAATGC

[0434] 2501 AATGCCTGAG TAATGTGTAG GTAAAGATTC AAACGGGTGA GAAAGGCCGG

[0435] 2551 AGACAGTCAA ATCACCATCA ATATGATATT CAACCGTTCT AGCTGATAAA

[0436] 2601 TTCATGCCGG AGAGGGTAGC TATTTTTGAG AGGTCTCTAC AAAGGCTATC

[0437] 2651 AGGTCATTGC CTGAGAGTCT GGAGCAAACA AGAGAATCGA TGAACGGTAA

[0438] 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC CCCGGTTGAT AATCAGAAAAA

[0439] 2751 GCCCCAAAA CAGGAAGATT GTATAAGCAA ATATTTAAAT TGTAAGCGTT

[0440] 2801 AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATC GCTCATTTTT

[0441] 2851 TAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA

[0442] 2901 CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA

[0443] 2951 AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA

[0444] 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT

[0445] 3051 GCCGTAAAGC ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT

[0446] 3101 TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA

[0447] 3151 AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCCGGTAA

[0448] 3201 CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTACTATGGT

[0449] 3251 TGCTTTGACG AGCACGTATA ACGTGCTTTC CTCGTTAGAA TCAGAGCGGG

[0450] 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT TAGACAGGAA CGGTACGCCA

[0451] 3351 GAATCCTGAG AAGTGTTTTT ATAATCAGTG AGGCCACCGA GTAAAAGAGT

[0452] 3401 CTGTCCATCA CGCAAATTAA CCGTTGTCGC AATACTTCTT TGATTAGTAA

[0453] 3451 TAACATCACT TGCCTGAGTA GAAGAACTCA AACTATCGGC CTTGCTGGTA

[0454] 3501 ATATCCAGAA CAATATTACC GCCAGCCATT GCAACGGAAT CGCCATTCGC

[0455] 3551 CATTCAGGCT GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCC

[0456] 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC ACTGAGGCCG CCCGGGCAAA

[0457] 3651 GCCCGGGCGT CGGGCGACCT TTGGTCGCCC GGCCTCAGTG AGCGAGCGAG

[0458] 3701 CGCGCAGAGA GGGAGTGGCC AACTCCATCA CTAGGGGTTC CTTGTAGTTA

[0459] 3751 ATGATTAACC CGCCATGCTA CTTATCTACT CGACATTGAT TATTGACTAG

[0460] 3801 GROW GROW CGGGGTCATT AGTTCATAGC CCATATATGG

[0461] 3851 AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC

[0462] 3901 AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC

[0463] 3951 GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA

[0464] 4001 CTGCCCACTT GGCAGTACAT CAAGTGTATC ATATGCCAAG TACGCCCCCT

[0465] 4051 ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG CCCAGTACAT

[0466] 4101 GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG

[0467] 4151 CTATTACCAT GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC

[0468] 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT TTATTTATTT TTTAATTT

[0469] 4251 TTGTGCAGCG ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG CCAGGCGGGG

[0470] 4301 CGGGGCGGGG CGAGGGGCGG GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA

[0471] 4351 GCCAATCAGA GCGGCGCGCT CCGAAAGTTT CCTTTTATGG CGAGGCGGCG

[0472] 4401 GCGGCGGCGG CCCTATAAAA AGCGAAGCGC GCGGCGGGCG GGAGTCGCTG

[0473] 4451 CGTTGCCTTC GCCCCGTGCC CCGCTCCGCG CCGCCTCGCG CCGCCCGCCC

[0474] 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG GTGAGCGGGC GGGACGGCCC

[0475] 4551 TTCTCCTCCG GGCTGTAATT AGCGCTTGGT TTAATGACGG CTTGTTTCTT

[0476] 4601 TTCTGTGGCT GCGTGAAAGC CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC

[0477] 4651 GGGGGGAGCG GCTCGGGGGG TGCGTGCGTG TGTGTGTGCG TGGGGAGCGC

[0478] 4701 CGCGTGCGGC TCCGCGCTGC CCGGCGGCTG TGAGCGCTGC GGGCGCGGCG

[0479] 4751 CGGGGCTTTG TGCGCTCCGC AGTGTGCGCG AGGGGAGCGC GGCCGGGGGC

[0480] 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG GAACAAAGGC TGCGTGCGGG

[0481] 4851 GTGTGTGCGT GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC GGTCGGGCTG

[0482] 4901 CAACCCCCCC TGCACCCCCC TCCCCGAGTT GCTGAGCACG GCCCGGCTTC

[0483] 4951 GGGTGCGGGG CTCCGTACGG GGCGTGGCGC GGGGCTCGCC GTGCCGGGCG

[0484] 5001 GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG GGGCGGGGCC GCCTCGGGCC

[0485] 5051 GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC CCCCGGAGCG CCGGCGGCTG

[0486] 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT TTTATGGTAA TCGTGCGAGA

[0487] 5151 GGGCGCAGGG ACTTCCTTTG TCCCAAATCT GTGCGGAGCC GAAATCTGGG

[0488] 5201 AGGCGCCGCC GCACCCCCTC TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC

[0489] 5251 CGGCAGGAAG GAAATGGGCG GGGAGGGCCT TCGTGCGTCG CCGCGCCGCC

[0490] 5301 GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT GTCCGCGGGG GGACGGCTGC

[0491] 5351 CTTCGGGGGG GACGGGGCAG GGCGGGGTTC GGCTTCTGGC GTGTGACCGG

[0492] 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA TGCCTTCTTC TTTTTCCTAC

[0493] 5451 AGCTCCTGGG CAACGTGCTG GTTATTGTGC TGTCTCATCA TTTTGGCAAA

[0494] 5501 GAATTGATTA ATTCGAGCGA ACGCGTCGAG TCGCTCGGTA CGATTTAAAT

[0495] 5551 TGAATTGGCC TCGAGCGCAA GCTTGAGCTA GCGCCACCAT GGAATGGATG

[0496] 5601 AGAAGCAGAG TGGGCACCCT GGGCCTGTGG GTGCGACTGC TGCTGGCTGT

[0497] 5651 GTTTCTGCTG GGCGTGTACC AGGCCTACCC CATCCCTGAC TCTAGCCCCC

[0498] 5701 TGCTGCAGTT TGGCGGACAA GTGCGGCAGA GATACCTGTA CACCGACGAC

[0499] 5751 GACCAGGACA CCGAGGCCCA CCTGGAAATC CGCGAGGATG GCACAGTCGT

[0500] 5801 GGGCGCTGCT CACAGAAGCC CTGAGAGCCT GCTGGAACTG AAGGCCCTGA

[0501] 5851 AGCCCGGCGT GATCCAGATC CTGGGCGTGA AGGCCAGCAG ATTCCTGTGC

[0502] 5901 CAGCAGCCTG ACGGCGCCCT GTACGGCTCT CCTCACTTCG ATCCTGAGGC

[0503] 5951 CTGCAGCTTC AGAGAGCTGC TGCTGGAGGA CGGCTACAAC GTGTACCAGT

[0504] 6001 CTGAGGCCCA CGGCCTGCCC CTGAGACTGC CTCAGAAGGA CAGCCCTAAC

[0505] 6051 CAGGACGCCA CAAGCTGGGG ACCTGTGCGG TTCCTGCCTA TGCCTGGACT

[0506] 6101 GCTGCACGAG CCCCAGGATC AGGCTGGCTT TCTGCCTCCT GAGCCTCCAG

[0507] 6151 ACGTGGGCAG CAGCGACCCT CTGAGCATGG TGGAACCTCT GCAGGGCAG

[0508] 6201 AGCCCCAGCT ACGCCTCTTG AGAATGCGGG CCCGGTACCC CCGACGCGGC

[0509] 6251 CGCTAATTCT AGATCGCGAA CAAACACCAT TGTCACACTC CAGTATACAC

[0510] 6301 AAACACCATT GTCACACTCC AGATATCACA AACACCATTG TCACACTCCA

[0511] 6351 ACACCATTGT CACACTCCA GGCTATTCTA GATCGCGAAT

[0512] 6401 TACATACTTC TTTACATTCC ACATACTTCT TTACATTCCA

[0513] 6451 GTATCATTA CATACTTCTT GGCGAATTAC ATACTTCTTT

[0514] 6501 ACATTCCAAG GCTACCTGAG GCCCGGGGGT ACCTCTTAAT TAACTGGCCT

[0515] 6551 CATGGGCCTT CCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC

[0516] 6601 CAGTCAGGTG CAGGCTGCCT ATCAGAAGGT GGTGGCTGGT GTGGCCAATG

[0517] 6651 CCCTGGCTCA CAAATACCAC TGAGATCTTT TTCCCTCTGC CAAAAATTAT

[0518] 6701 GGGGACATCA TGAAGCCCCT TGAGCATCTG ACTTCTGGCT AATAAAGGA

[0519] 6751 ATTTATTTTC ATTGCAATAG TGTGTTGGAA TTTTTTGTGT CTCTCACTCG

[0520] 6801 GAAGGACATA TGGGAGGGCA AATCATTTAA AACATCAGAA TGAGTATTTG

[0521] 6851 GTTTAGAGTT TGGCAACATA TGCCCATATG CTGGCTGCCA TGAACAAAGG

[0522] 6901 TTGGCTATAA AGAGGTCATC AGTATATGAA ACAGCCCCCT GCTGTCCATT

[0523] 6951 CCTTATTCCA TAGAAAAGCC TTGACTTGAG GTTAGATTTT TTTTATATTT

[0524] 7001 TGTTTTGTGT TATTTTTTTC TTTAACATCC CTAAAATTTT CCTTACATGT

[0525] 7051 TTTACTAGCC AGATTTTTCC TCCTCTCCTG ACTACTCCCA GTCATAGCTG

[0526] 7101 TCCCTCTTCT CTTATGGAGA TCCCTCGACC TGCAGCCCAA GCTGTAGATA

[0527] 7151 AGTAGCATGG CGGGTTAATC ATTAACTACA AGGAACCCCT AGTGATGGAG

[0528] 7201 TTGGCACACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC

[0529] 7251 AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC

[0530] 7301 GAGCGCGCAG CTGGCGTAA

[0531] 5'-ITR AAV2: 3615-3742 p.o.

[0532] CAG Promoter: 3782-5452 p.o.

[0533] Codon-optimized FGF-21Mus musculus(moFGF21): 5589-6221 bp

[0534] dmiRT (4 copies of miRT-122a and 4 copies of miRT-1): 6254-6514 bp

[0535] Rabbit β-globin polyA signal (3'-UTR and 3'-flanking region of rabbit β-globin, including polyA signal): 6674-6764 bp

[0536] 3'-ITR AAV2: 7181-7308 bp

[0537] pAAV-CAG-moFGF21 (SEQ ID NO: 46)

[0538] 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAATCGGCC AACGCGCGGGG GAGAGGCGGT

[0539] 61 TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG

[0540] 121 CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG

[0541] 181 GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG

[0542] 241 GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA

[0543] 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT

[0544] 361 GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC

[0545] 421 TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG

[0546] 481 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC

[0547] 541 TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA

[0548] 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG

[0549] 661 TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT

[0550] 721 CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC

[0551] 781 ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAAGGA

[0552] 841 TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA

[0553] 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT

[0554] 961 TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC

[0555] 1021 CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT

[0556] 1081 GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT

[0557] 1141 GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG

[0558] 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT

[0559] 1261 ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT

[0560] 1321 GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC

[0561] 1381 TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT

[0562] 1441 AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG

[0563] 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG

[0564] 1561 ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT

[0565] 1621 TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC

[0566] 1681 ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT

[0567] 1741 TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT

[0568] 1801 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG

[0569] 1861 AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT

[0570] 1921 TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAAA ATAAACAAAT AGGGGTTCCG

[0571] 1981 CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA CCATTATTAT CATGACATTA

[0572] 2041 ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTCTCG CGCGTTTCGG TGATGACGGT

[0573] 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC

[0574] 2161 GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT

[0575] 2221 AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC ATATGCGGTG TGAAATACCG

[0576] 2281 CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGATTCC AACATCCAAT AAATCATACA

[0577] 2341 GGCAAGGCAA AGAATTAGCA AAATTAAGCA ATAAAGCCTC AGAGCATAAA GCTAAATCGG

[0578] 2401 TTGTACCAAA AACATTATGA CCCTGTAATA CTTTTGCGGG AGAAGCCTTT ATTTCAACGC

[0579] 2461 AAGGATAAAA ATTTTTAGAA CCCTCATATA TTTTAAATGC AATGCCTGAG TAATGTGTAG

[0580] 2521 GTAAAGATTC AAACGGGTGA GAAAGGCCGG AGACAGTCAA ATCACCATCA ATATGATATT

[0581] 2581 CAACCGTTCT AGCTGATAAA TTCATGCCGG AGAGGGTAGC TATTTTTGAG AGGTCTCTAC

[0582] 2641 AAAGGCTATC AGGTCATTGC CTGAGAGTCT GGAGCAAACA AGAGAATCGA TGAACGGTAA

[0583] 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC CCCGGTTGAT AATCAGAAAA GCCCCAAAAAA

[0584] 2761 CAGGAAGATT GTATAAGCAA ATATTTAAAT TGTAAGCGTT AATATTTTGT TAAAATTCGC

[0585] 2821 GTTAAATTTT TGTTAAATC GCTCATTTTT TAACCAATAG GCCGAAATCG GCAAAATCCC

[0586] 2881 TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG

[0587] 2941 TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA

[0588] 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC

[0589] 3061 ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA AGCCGGCGGA

[0590] 3121 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT

[0591] 3181 AGCGGTCACG CTGCCGGTAA CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC

[0592] 3241 GTACTATGGT TGCTTTGACG AGCACGTATA ACGTGCTTTC CTCGTTAGAA TCAGAGCGGG

[0593] 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT TAGACAGGAA CGGTACGCCA GAATCCTGAG

[0594] 3361 AAGTGTTTTT ATAATCAGTG AGGCCACCGA GTAAAAGGT CTGTCCATCA CGCAAATTAA

[0595] 3421 CCGTTGTCGC AATACTTCTT TGATTAGTAA TAACATCACT TGCCTGAGTA GAAGAACTCA

[0596] 3481 AACTATCGGC CTTGCTGGTA ATATCCAGAA CAATATTACC GCCAGCCATT GCAACGGAAT

[0597] 3541 CGCCATTCGC CATTCAGGCT GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCC

[0598] 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC ACTGAGGCCG CCCGGGCAAA GCCCGGGCGT

[0599] 3661 CGGGCGACCT TTGGTCGCCC GGCCTCAGTG AGCGAGCGAG CGCGCAGAGA GGGAGTGGCC

[0600] 3721 AACTCCATCA CTAGGGGTTC CTTGTAGTTA ATGATTAACC CGCCATGCTA CTTATCTACT

[0601] 3781 CGACATTGAT TATTGACTAG TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC

[0602] 3841 CCATATATGG AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC

[0603] 3901 AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG

[0604] 3961 ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT

[0605] 4021 CAAGTGTATC ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC

[0606] 4081 TGGCATTATG CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA

[0607] 4141 TTAGTCATCG CTATTACCAT GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC

[0608] 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT TTATTTATTT TTGTGTGCAGCG

[0609] 4261 ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG CCAGGCGGGG CGGGGCGGGG CGAGGGGCGG

[0610] 4321 GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA GCCAATCAGA GCGGCGCGCT CCGAAAGTTT

[0611] 4381 CCTTTTATGG CGAGGCGGCG GCGGCGGCGG CCCTATAAAA AGCGAAGCGC GCGGCGGGCG

[0612] 4441 GGAGTCGCTG CGTTGCCTTC GCCCCGTGCC CCGCTCCGCG CCGCCTCGCG CCGCCCGCCC

[0613] 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG GTGAGCGGGC GGGACGGCCC TTCTCCTCCG

[0614] 4561 GGCTGTAATT AGCGCTTGGT TTAATGACGG CTTGTTTCTT TTCTGTGGCT GCGTGAAAGC

[0615] 4621 CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC GGGGGGAGCG GCTCGGGGGG TGCGTGCGTG

[0616] 4681 TGTGTGTGCG TGGGGAGCGC CGCGTGCGGC TCCGCGCTGC CCGGCGGCTG TGAGCGCTGC

[0617] 4741 GGGCGCGGCG CGGGGCTTTG TGCGCTCCGC AGTGTGCGCG AGGGGAGCGC GGCCGGGGGC

[0618] 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG GAACAAAGGC TGCGTGCGGG GTGTGTGCGT

[0619] 4861 GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC GGTCGGGCTG CAACCCCCCC TGCACCCCCC

[0620] 4921 TCCCCGAGTT GCTGAGCACG GCCCGGCTTC GGGTGCGGGG CTCCGTACGG GGCGTGGCGC

[0621] 4981 GGGGCTCGCC GTGCCGGGCG GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG GGGCGGGGCC

[0622] 5041 GCCTCGGGCC GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC CCCCGGAGCG CCGGCGGCTG

[0623] 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT TTTATGGTAA TCGTGCGAGA GGGCGCAGGG

[0624] 5161 ACTTCCTTTG TCCCAAATCT GTGCGGAGCC GAAATCTGGG AGGCGCCGCC GCACCCCCTC

[0625] 5221 TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC CGGCAGGAAG GAAATGGGCG GGGAGGGCCT

[0626] 5281 TCGTGCGTCG CCGCGCCGCC GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT GTCCGCGGGG

[0627] 5341 GGACGGCTGC CTTCGGGGGG GACGGGGCAG GGCGGGGTTC GGCTTCTGGC GTGTGACCGG

[0628] 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA TGCCTTCTTC TTTTTCCTAC AGCTCCTGGG

[0629] 5461 CAACGTGCTG GTTATTGTGC TGTCTCATCA TTTTGGCAAA GAATTGATTA ATTCGAGCGA

[0630] 5521 ACGCGTCGAG TCGCTCGGTA CGATTTAAAT TGAATTGGCC TCGAGCGCAA GCTTGAGCTA

[0631] 5581 GCGCCACCAT GGAATGGATG AGAAGCAGAG TGGGCACCCT GGGCCTGTGG GTGCGACTGC

[0632] 5641 TGCTGGCTGT GTTTCTGCTG GGCGTGTACC AGGCCTACCC CATCCCTGAC TCTAGCCCCC

[0633] 5701 TGCTGCAGTT TGGCGGACAA GTGCGGCAGA GATACCTGTA CACCGACGAC GACCAGGACA

[0634] 5761 CCGAGGCCCA CCTGGAAATC CGCGAGGATG GCACAGTCGT GGGCGCTGCT CACAGAAGCC

[0635] 5821 CTGAGAGCCT GCTGGAACTG AAGGCCCTGA AGCCCGGCGT GATCCAGATC CTGGGCGTGA

[0636] 5881 AGGCCAGCAG ATTCCTGTGC CAGCAGCCTG ACGGCGCCCT GTACGGCTCT CCTCACTTCG

[0637] 5941 ATCCTGAGGC CTGCAGCTTC AGAGAGCTGC TGCTGGAGGA CGGCTACAAC GTGTACCAGT

[0638] 6001 CTGAGGCCCA CGGCCTGCCC CTGAGACTGC CTCAGAAGGA CAGCCCTAAC CAGGACGCCA

[0639] 6061 CAAGCTGGGG ACCTGTGCGG TTCCTGCCTA TGCCTGGACT GCTGCACGAG CCCCAGGATC

[0640] 6121 AGGCTGGCTT TCTGCCTCCT GAGCCTCCAG ACGTGGGCAG CAGCGACCCT CTGAGCATGG

[0641] 6181 TGGAACCTCT GCAGGGCAGA AGCCCCAGCT ACGCCTCTTG AGAATGCGGG CCCGGTACCC

[0642] 6241 CCGACGCGGC CTAACTGGCC TCATGGGCCT TCCGCTCACT GCCCGCTTTC CAGTCGGGAA

[0643] 6301 ACCTGTCGTG CCAGTCAGGT GCAGGCTGCC TATCAGAAGG TGGTGGCTGG TGTGGCCAAT

[0644] 6361 GCCCTGGCTC ACAAATACCA CTGAGATCTT TTTCCCTCTG CCAAAAATTA TGGGGACATC

[0645] 6421 ATGAAGCCCC TTGAGCATCT GACTTCTGGC TAATAAAGGA AATTATTTT CATTGCAATA

[0646] 6481 GTGTGTTGGA ATTTTTTGTG TCTCTCACTC GGAAGGACAT ATGGGAGGGC AAATCATTTA

[0647] 6541 AAACATCAGA ATGAGTATTT GGTTTAGAGT TTGGCAACAT ATGCCCATAT GCTGGCTGCC

[0648] 6601 ATGAACAAAG GTTGGCTATA AAGAGGTCAT CAGTATATGA AACAGCCCCCC TGCTGTCCAT

[0649] 6661 TCCTTATTCC ATAGAAAAGC CTTGACTTGA GGTTAGATTT TTTTTATATT TTGTTTTGTG

[0650] 6721 TTATTTTTTT CTTTAACATC CCTAAAATTT TCCTTACATG TTTTACTAGC CAGATTTTTC

[0651] 6781 CTCCTCTCCT GACTACTCCC AGTCATAGCT GTCCCTCTTC TCTTATGGAG ATCCCTCGAC

[0652] 6841 CTGCAGCCCA AGCTGTAGAT AAGTAGCATG GCGGGTTAAT CATTAACTAC AAGGAACCCC

[0653] 6901 TAGTGATGGA GTTGGCCACT CCCTCTCTGC GCGCTCGCTC GCTCACTGAG GCCGGGCGAC

[0654] 6961 CAAAGGTCGC CCGACGCCCG GGCTTTGCCC GGGCGGCCTC AGTGAGCGAG CGAGCGCGCA

[0655] 7021 GCTGGCGTAA

[0656] 5'-ITR AAV2: 3601-3742 bp

[0657] CAG promoter: bp 3779-5423

[0658] Codon-optimized FGF-21Mus musculus(moFGF21): 5588-6221 bp

[0659] Rabbit β-globin polyA signal (3'-UTR and 3'-flanking region of rabbit β-globin, including polyA signal): 6315-6833 bp

[0660] 3'-ITR AAV2: 6892-7024 bp

[0661] CMV minimal promoter: CMV intermediate early promoter (SEQ ID NO: 36)

[0662] TATGCCAAGTACGCCCCCTATTTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCAC CCCATTGACGTCCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGT ACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTAACTGGCTTATCGAAATTAATTACGACTCACTATAGGGAGACCCAAGCTT

[0663] Nucleotide sequence of EF1α promoter (SEQ ID NO: 37)

[0665] Нуклеотидная последовательность промотора RSV (SEQ ID NO: 38)

[0666] CATGTTTGACAGCTTATCATCGCAGATCCGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATTCGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTAAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTGGGTACCAGCT

[0667] Промотор синапсина-1 (SEQ ID NO: 39)

[0668] ctgcgctctcaggcacgacacgactccgctgccaccgcagactgaggcagcgctcgcggcgcgcgcgcgcgccgtgccccctct cgcgcctcgcgtggtgcggcggctgggcggcggcggcggcgcgaccaaggtggccgggaaggggagtttgcgggcggcggcgcgtcgcgcttcgtgc tgaggcggcggtggcgcgcgccaggcgggggcgaaggcactgtcgcggtgctgaagctggcagtgcgcgccgcgcatcctgtttcccccccctctgat aggggatgcgcaatttggggaatgggggttggtgctgtccagtgggtcggggtcggtcggtaggcacccccacccgcctcatcctggctctaaaacccacttgcact

[0669] Calcium / calmodulin-dependent protein kinase II (CaMKII) promoter (SEQ ID NO: 40)

[0671] Glial Fibrillary Acidic Protein (GFAP) Promoter (SEQ ID NO: 41)

[0672] cgcgtgatctaacatatcctgtgtggagggcgctgctctgacagaggctcgggggcctgctggctctgct gaatgactcaccttggcacagacaatgttcggggtgggcacagtgcctgctccgcgcaccccagccccctcaaatgccttccgagaagcccattgagcggggcttgcattgcaccccagcctgacagcctggcatcttgggataaaagcagcagcccc cccttgctgtgtggcgccaccggcggtggaaaggcttattcagcctgcccaggaaaggggatcaggggatgcccaggcatggacagtgggcggggggaggggctctgcttcccagaagtccaaggacaaatgggtgagggagctctcccatagc tgggctgcggcccaaccccaccccctgccagggggtgttgccaggggccccgcatcgccagtctagcccactctcataaagccctcgcatcccaggagcgagcagccagagcaggttggagagacgcatcaccgctgctcgcggcgcgcga

[0673] Nestin Promoter (SEQ ID NO: 42)

[0675] Промотор гомеобоксного белка 9 (HB9) (SEQ ID NO: 43)

[0676] tgaataaatttaagcaggctaattaatatataaactagctcaatttgtcaagttgatttgtattttagttaattgtgaaagtaattaccacatggtcaaattaacagctttctggaaatgaccaagcctgaggttttatttccttcctgggtgaagaaaattcatttttccaagctcttgatgtgatgaataaaagtcataaatctgggtgattggtgcaggcagagtctaaatggcttcatatttcattttaggtttaatagaaatattcatgctctgttttaatgaaattaaattgaagggggatggggctagagtggttagctgatgaattgacaaaaactaatcagctttattgggaaacaggtttaagggcacggacgtgtcaataacgctcagcctgaccccctcttccattagctaggcaggctgattaga

[0677] Промотор тирозингидроксилазы (TH) (SEQ ID NO: 44)

[0679] Myelin basic protein (MBP) promoter (SEQ ID NO: 45)

[0681] --->

[0682] SEQUENCE LISTING

[0683] <110> Universitat Autònoma de Barcelona

[0684] <120> Gene therapy using fibroblast growth factor 21 (FGF21)

[0685] <130> P6075726PCT

[0686] <150> EP18382857.3

[0687] <151> 2018-11-26

[0688] <160> 70

[0689] <170> PatentIn version 3.5

[0690] <210> 1

[0691] <211> 209

[0692] <212> protein (PRT)

[0693] <213> Homo sapiens

[0694] <400> 1

[0695] Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser

[0696] 1 5 10 15

[0697] Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro

[0698] 20 25 30

[0699] Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr

[0700] 35 40 45

[0701] Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg

[0702] 50 55 60

[0703] Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu

[0704] 65 70 75 80

[0705] Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val

[0706] 85 90 95

[0707] Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly

[0708] 100 105 110

[0709] Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu

[0710] 115 120 125

[0711] Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu

[0712] 130 135 140

[0713] His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly

[0714] 145 150 155 160

[0715] Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu

[0716] 165 170 175

[0717] Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp

[0718] 180 185 190

[0719] Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala

[0720] 195 200 205

[0721] Ser

[0722] <210> 2

[0723] <211> 210

[0724] <212> белок

[0725] <213> Mus musculus

[0726] <400> 2

[0727] Met Glu Trp Met Arg Ser Arg Val Gly Thr Leu Gly Leu Trp Val Arg

[0728] 1 5 10 15

[0729] Leu Leu Leu Ala Val Phe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile

[0730] 20 25 30

[0731] Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg

[0732] 35 40 45

[0733] Tyr Leu Tyr Thr Asp Asp Asp Gln Asp Thr Glu Ala His Leu Glu Ile

[0734] 50 55 60

[0735] Arg Glu Asp Gly Thr Val Val Gly Ala Ala His Arg Ser Pro Glu Ser

[0736] 65 70 75 80

[0737] Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly

[0738] 85 90 95

[0739] Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr

[0740] 100 105 110

[0741] Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu

[0742] 115 120 125

[0743] Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro

[0744] 130 135 140

[0745] Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp

[0746] 145 150 155 160

[0747] Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln

[0748] 165 170 175

[0749] Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser

[0750] 180 185 190

[0751] Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro Ser Tyr

[0752] 195 200 205

[0753] Ala Ser

[0754] 210

[0755] <210> 3

[0756] <211> 209

[0757] <212> белок

[0758] <213> Canis lupus familiaris

[0759] <400> 3

[0760] Met Gly Trp Ala Glu Ala Gly Phe Glu His Leu Gly Leu Trp Val Pro

[0761] 1 5 10 15

[0762] Val Leu Ala Val Leu Leu Leu Glu Ala Cys Arg Ala His Pro Ile Pro

[0763] 20 25 30

[0764] Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr

[0765] 35 40 45

[0766] Leu Tyr Thr Asp Asp Ala Gln Glu Thr Glu Ala His Leu Glu Ile Arg

[0767] 50 55 60

[0768] Ala Asp Gly Thr Val Val Gly Ala Ala Arg Gln Ser Pro Glu Ser Leu

[0769] 65 70 75 80

[0770] Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val

[0771] 85 90 95

[0772] Lys Thr Ser Arg Phe Leu Cys Gln Gly Pro Asp Gly Thr Leu Tyr Gly

[0773] 100 105 110

[0774] Ser Leu His Phe Asp Pro Val Ala Cys Ser Phe Arg Glu Leu Leu Leu

[0775] 115 120 125

[0776] Glu Asp Gly Tyr Asn Ile Tyr His Ser Glu Thr Leu Gly Leu Pro Leu

[0777] 130 135 140

[0778] Arg Leu Arg Pro His Asn Ser Ala Tyr Arg Asp Leu Ala Pro Arg Gly

[0779] 145 150 155 160

[0780] Pro Wing Arg Phe Leu Pro Leu Pro Gly Leu Leu Pro Wing Pro Pro Glu

[0781] 165 170 175

[0782] Pro Pro Gly Ile Leu Ala Pro Glu Pro Pro Asp Val Gly Ser Ser Asp

[0783] 180 185 190

[0784] Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala

[0785] 195 200 205

[0786] Ser

[0787] <210> 4

[0788] <211> 630

[0789] <212> DNA

[0790] <213> Homo sapiens

[0791] <400> 4

[0792] atggactcgg acgagaccgg gttcgagcac tcaggactgt gggttctgt gctggctggt 60

[0793] cttctgctgg gagcctgcca ggcacacccc atccctgact ccagtcctct cctgcaattc 120

[0794] gggggccaag tccggcagcg gtacctctac acagatgatg cccagcagac agaagcccac 180

[0795] ctggagatca gggaggatgg gacggtgggg ggcgctgctg accagagccc cgaaagtctc 240

[0796] ctgcagctga aagccttgaa gccgggagtt attcaaatct tgggagtcaa gacatccagg 300

[0797] ttcctgtgcc agcggccaga tggggccctg tatggatcgc tccactttga ccctgaggcc 360

[0798] tgcagcttc gggagctgct tcttgaggac ggatacaatg tttaccagtc cgaagcccac 420

[0799] ggcctcccgc tgcacctgcc agggaacaag tccccacacc gggaccctgc accccgagga 480

[0800] ccagctcgct tcctgccact accaggcctg cccccgcac tcccggagcc acccggaatc 540

[0801] ctggccccc agcccccga tgtgggctc tcggaccctc tgagcatggt gggaccttcc 600

[0802] cagggccgaa gccccagcta cgcttcctga 630

[0803] <210> 5

[0804] <211> 630

[0805] <212> DNA

[0806] <213> Artificial sequence

[0807] <220>

[0808] <223> Codon-optimized FRF21 of Homo sapiens – variant

[0809] <400> 5

[0810] atggattctg atgagacagg cttcgagcac agcggcctgt gggtttcagt tctggctgga 60

[0811] ctgctgctgg gagcctgtca ggcacaccct attccagata gcagccctct gctgcagttc 120

[0812] ggcggacaag tgcggcagag atacctgtac accgacgacg ccagcagac agaagcccac 180

[0813] ctggaaatca gagaggatgg cacagttggc ggagccgccg atcagtctcc tgaatctctg 240

[0814] ctccagctga aggccctgaa gcctggcgtg atccagatcc tgggcgtgaa aaccagccgg 300

[0815] ttcctgtgcc aaagacctga cggcgccctg tatggcagcc tgcactttga tcctgaggcc 360

[0816] tgcagcttca gagagctgct gcttgaggac ggctacaacg tgtaccagtc gaggcccat 420

[0817] ggcctgcctc tgcatctgcc tggaaacaag agccctcaca gagatcccgc tcctagaggc 480

[0818] cctgccagat tctgcctct tcctggattg cctcctgctc tgccagagcc tcctggaatt 540

[0819] ctggctcctc agcctcctga tgtgggcagc tctgatcctc tgagcatggt cggacctagc 600

[0820] cagggcagat ctcctagcta cgcctcttga 630

[0821] <210> 6

[0822] <211> 630

[0823] <212> DNA

[0824] <213> Artificial sequence

[0825] <220>

[0826] <223> Codon-optimized FRF21 of Homo sapiens – variant 2

[0827] <400> 6

[0828] atggacagcg atgaaaccgg gttcgagcac agcggtctgt gggtgtccgt gctggccgga 60

[0829] ctgctcctgg gagcctgtca ggcgcacccc atccctgact cctcgccgct gctgcaattc 120

[0830] ggcggacaag tccgccagag atacctgtac accgacgacg cccagcagac cgaagcccac 180

[0831] ctggaaattc gggaggacgg gactgtggga ggcgctgcag atcagtcacc cgagtccctc 240

[0832] ctccaactga aggccttgaa gcccggcgtg attcagatcc tgggcgtgaa aacttcccgc 300

[0833] ttcctttgcc aacggccgga tggagctctg tacggatccc tgcacttcga ccccgaagcc 360

[0834] tgctcattcc gcgagctgct ccttgaggac ggctataacg tgtaccagtc tgaggcccat 420

[0835] ggactccccc tgcatctgcc cggcaacaag tcccctcacc gggatcctgc cccaagaggc 480

[0836] ccagctcggt ttctgcctct gccgggactg cctccagcgt tgcccgaacc ccctggtatc 540

[0837] ctggccccgc aaccacctga cgtcggttcg tcggacccgc tgagcatggt cggtccgagc 600

[0838] cagggaaggt ccccgtccta cgcatcctga 630

[0839] <210> 7

[0840] <211> 630

[0841] <212> DNA

[0842] <213> Artificial sequence

[0843] <220>

[0844] <223> Codon-optimized FRF21 of Homo sapiens – variant 3

[0845] <400> 7

[0846] atggattccg acgaaactgg atttgaacat tcagggctgt gggtctctgt gctggctgga 60

[0847] ctgctgctgg gggcttgtca ggctcacccc atccctgaca gctcccctct gctgcagttc 120

[0848] ggaggacagg tgcggcagag atacctgtat accgacgatg cccagcagac agaggcacac 180

[0849] ctggagatca gggaggacgg aaccgtggga ggagcagccg atcagtctcc cgagagcctg 240

[0850] ctgcagctga aggccctgaa gcctggcgtg atccagatcc tgggcgtgaa gacatctcgg 300

[0851] tttctgcc agcggcccga cggcgccctg tacggctccc tgcacttcga tcccgaggcc 360

[0852] tgttctttta gggagctgct gctggaggac ggctacaacg tgtatcagag cgaggcacac 420

[0853] ggcctgccac tgcacctgcc tggcaataag tcccctcacc gcgatccagc acccaggggc 480

[0854] ccagcacgct tctgcctct gccaggcctg cccctgccc tgccagagcc acccggcatc 540

[0855] ctggccccc agcctccaga tgtgggctcc agcgatcctc tgtcaatggt ggggccaagt 600

[0856] caggggcgga gtccttcata cgcatcataa 630

[0857] <210> 8

[0858] <211> 633

[0859] <212> DNA

[0860] <213> Mus musculus

[0861] <400> 8

[0862] atggaatgga tgagatctag agttgggacc ctgggactgt gggtccgact gctgctggct 60

[0863] gtcttcctgc tgggggcta ccaagcatac cccatccctg actccagccc cctcctccag 120

[0864] tttgggggtc aagtccggca gaggtacctc tacacagatg acgaccaaga cactgaagcc 180

[0865] cacctggaga tcagggagga tggaacagtg gtaggcgcag cacaccgcag tccagaaagt 240

[0866] ctcctggagc tcaaagcctt gaagccaggg gtcattcaaa tcctgggtgt caaagcctct 300

[0867] aggtttcttt gccaacagcc agatggagct ctctatggat cgcctcactt tgatcctgag 360

[0868] gcctgcagct tcagagaact gctgctggag gacggttaca atgtgtacca gtctgaagcc 420

[0869] catggcctgc cctgcgtct gcctcagaag gactccccaa accaggatgc aacatcctgg 480

[0870] ggacctgtgc gcttcctgcc catgccaggc ctgctccacg agccccaaga ccaagcagga 540

[0871] ttcctgcccc cagagccccc agatgtgggc tcctctgacc ccctgagcat ggtagagcct 600

[0872] ttacagggcc gaagccccag ctatgcgtcc tga 633

[0873] <210> 9

[0874] <211> 633

[0875] <212> DNA

[0876] <213> Artificial sequence

[0877] <220>

[0878] <223> Codon-optimized FRF21 of Mus musculus

[0879] <400> 9

[0880] atggaatgga tgagaagcag agtgggcacc ctgggcctgt gggtgcgact gctgctggct 60

[0881] gtgtttctgc tgggcgtgta ccaggcctac cccatccctg actctagccc cctgctgcag 120

[0882] tttggcggac aagtgcggca gagatacctg tacaccgacg acgaccagga caccgaggcc 180

[0883] cacctggaaa tccgcgagga tggcacagtc gtgggcgctg ctcacagaag ccctgagagc 240

[0884] ctgctggaac tgaaggccct gaagcccggc gtgatccaga tcctgggcgt gaaggccagc 300

[0885] agattcctgt gccagcagcc tgacggcgcc ctgtacggct ctcctcactt cgatcctgag 360

[0886] gcctgcagct tcagagagct gctgctggag gacggctaca acgtgtacca gtctgaggcc 420

[0887] cacggcctgc ccctgagact gcctcagaag gacagcccta accaggacgc cacaagctgg 480

[0888] ggacctgtgc ggttcctgcc tatgcctgga ctgctgcacg agccccagga tcaggctggc 540

[0889] tttctgcctc ctgagcctcc agacgtgggc agcagcgacc ctctgagcat ggtggaacct 600

[0890] ctgcagggca gaagccccag ctacgcctct tga 633

[0891] <210> 10

[0892] <211> 629

[0893] <212> ДНК

[0894] <213> Canis lupus familiaris

[0895] <400> 10

[0896] atgggctggg ccgaggccgg gttcgagcac ctgggactgt gggtccctgt gctggctgtg 60

[0897] cttttgctgg aagcctgccg ggcacatccg atccctgact ccagccccct cctacaattt 120

[0898] ggaggtcaag ttcgacagcg gtacctctac accgacgatg cccaggagac agaggcccac 180

[0899] ctagagatca gggccgatgg cacagggtgg gggctgcccg ccagagccct gaaagtctcc 240

[0900] tggagctgaa agccctaaag ccaggggtca ttcaaatctt gggagtcaaa acatccaggt 300

[0901] tcctgtgcca gggcccagat gggacactat atggctcgct ccatttcgac cctgtggcct 360

[0902] gcagtttccg agaactgctt cttgaggatg ggtacaacat ctaccactcc gagacccttg 420

[0903] gtctcccgct tcgcctgcgc ccccacaact ccgcataccg ggacttggca ccccgcggc 480

[0904] ctgcccgct cctgccactg ccaggcctgc ttccagcacc cccagagcct ccagggatcc 540

[0905] tggccccgga gcctcctgac gtgggctcct cggaccctct gagcatggtg gggccttcac 600

[0906] agggccggag tcccagctat gcttcctaa 629

[0907] <210> 11

[0908] <211> 630

[0909] <212> DNA

[0910] <213> Artificial sequence

[0911] <220>

[0912] <223> Codon-optimized FRF21 of Canis lupus familiaris

[0913] <400> 11

[0914] atgggatggg ctgaggctgg attcgaacac ctgggactct gggtgcccgt cctggccgtg 60

[0915] ctgctcctgg aggcttgcag ggctcatccc atccctgaca gctccccact cctgcagttt 120

[0916] ggaggacagg tgaggcagcg gtacctgtat accgacgatg cccaggagac agaagctcac 180

[0917] ctggaaattc gggctgatgg aacagtggtc ggagctgccc gacagtcccc agagtctctc 240

[0918] ctggaactga aggccctcaa acccggagtg atccagattc tgggcgtcaa gacttctaga 300

[0919] ttcctgtgcc agggaccaga cggcaccctg tacggcagcc tgcatttcga tcctgtggcc 360

[0920] tgttcctttc gagagctcct gctcgaagac ggctacaaca tctatcactc tgagaccctg 420

[0921] ggactcccac tgcgactcag acctcataat agtgcctatc gagatctggc tcccaggggc 480

[0922] ccagctaggt ttctgccact ccccggactg ctccctgctc cacctgagcc acccggcatt 540

[0923] ctggctccag aacctccaga cgtgggctct agtgatccac tgagtatggt cggcccctca 600

[0924] caggggaggt cacctagcta cgccagctga 630

[0925] <210> 12

[0926] <211> 22

[0927] <212> DNA

[0928] <213> Artificial sequence

[0929] <220>

[0930] <223> miRT-122a

[0931] <400> 12

[0932] caaacaccat tgtcacactc ca 22

[0933] <210> 13

[0934] <211> 22

[0935] <212> DNA

[0936] <213> Artificial sequence

[0937] <220>

[0938] <223> miRT-1

[0939] <400> 13

[0940] ttacatactt ctttacattc ca 22

[0941] <210> 14

[0942] <211> 21

[0943] <212> DNA

[0944] <213> Artificial sequence

[0945] <220>

[0946] <223> miRT-152

[0947] <400> 14

[0948] ccaagttctg tcatgcactg a 21

[0949] <210> 15

[0950] <211> 23

[0951] <212> DNA

[0952] <213> Artificial sequence

[0953] <220>

[0954] <223> miRT-199a-5p

[0955] <400> 15

[0956] gaacaggtag tctgaacact ggg 23

[0957] <210> 16

[0958] <211> 22

[0959] <212> DNA

[0960] <213> Artificial sequence

[0961] <220>

[0962] <223> miRT-199a-3p

[0963] <400> 16

[0964] taaccaatgt gcagactact gt 22

[0965] <210> 17

[0966] <211> 21

[0967] <212> DNA

[0968] <213> Artificial sequence

[0969] <220>

[0970] <223> miRT-215

[0971] <400> 17

[0972] gtctgtcaat tcataggtca t 21

[0973] <210> 18

[0974] <211> 21

[0975] <212> DNA

[0976] <213> Artificial sequence

[0977] <220>

[0978] <223> miRT-192

[0979] <400> 18

[0980] ggctgtcaat tcataggtca g 21

[0981] <210> 19

[0982] <211> 22

[0983] <212> DNA

[0984] <213> Artificial sequence

[0985] <220>

[0986] <223> miRT-148a

[0987] <400> 19

[0988] acaaagttct gtagtgcact ga 22

[0989] <210> 20

[0990] <211> 22

[0991] <212> DNA

[0992] <213> Artificial sequence

[0993] <220>

[0994] <223> miRT-194

[0995] <400> 20

[0996] tccacatgga gttgctgtta ca 22

[0997] <210> 21

[0998] <211> 22

[0999] <212> DNA

[1000] <213> Artificial sequence

[1001] <220>

[1002] <223> miRT-133a

[1003] <400> 21

[1004] cagctggttg aaggggacca aa 22

[1005] <210> 22

[1006] <211> 22

[1007] <212> DNA

[1008] <213> Artificial sequence

[1009] <220>

[1010] <223> miRT-206

[1011] <400> 22

[1012] ccacacactt ccttacattc ca 22

[1013] <210> 23

[1014] <211> 22

[1015] <212> DNA

[1016] <213> Artificial sequence

[1017] <220>

[1018] <223> miRT-208a-5p

[1019] <400> 23

[1020] gtataacccg ggccaaaagc tc 22

[1021] <210> 24

[1022] <211> 22

[1023] <212> DNA

[1024] <213> Artificial sequence

[1025] <220>

[1026] <223> miRT-208a-3p

[1027] <400> 24

[1028] acaagctttt tgctcgtctt at 22

[1029] <210> 25

[1030] <211> 21

[1031] <212> DNA

[1032] <213> Artificial sequence

[1033] <220>

[1034] <223> miRT-499-5p

[1035] <400> 25

[1036] aaacatcact gcaagtctta a 21

[1037] <210> 26

[1038] <211> 133

[1039] <212> DNA

[1040] <213> Artificial sequence

[1041] <220>

[1042] <223> a chimeric intron consisting of introns from the human β-globin and immunoglobulin heavy chain genes

[1043] <400> 26

[1044] gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60

[1045] cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120

[1046] ttttctctcca cag 133

[1047] <210> 27

[1048] <211> 1671

[1049] <212> DNA

[1050] <213> Artificial sequence

[1051] <220>

[1052] <223> CAG promoter

[1053] <400> 27

[1054] gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

[1055] catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

[1056] acgacccccg cccatgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

[1057] ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

[1058] aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

[1059] ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

[1060] tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 420

[1061] cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 480

[1062] tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 540

[1063] gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 600

[1064] cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 660

[1065] gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc 720

[1066] ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 780

[1067] gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 840

[1068] ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt 900

[1069] gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg 960

[1070] ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg 1020

[1071] gtgccccgcg gtgcgggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg 1080

[1072] ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct 1140

[1073] ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg 1200

[1074] gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg 1260

[1075] cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt 1320

[1076] cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga 1380

[1077] cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct 1440

[1078] agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500

[1079] cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg 1560

[1080] gacggctgcc ttcgggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 1620

[1081] ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca g 1671

[1082] <210> 28

[1083] <211> 212

[1084] <212> DNA

[1085] <213> Artificial sequence

[1086] <220>

[1087] <223> CMV promoter

[1088] <400> 28

[1089] gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt 60

[1090] ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120

[1091] tttccaaaat gtcgtaacaa ctgcgatcgc ccgccccgtt gacgcaaatg ggcggtaggc 180

[1092] gtgtacggtg ggaggtctat ataagcagag ct 212

[1093] <210> 29

[1094] <211> 380

[1095] <212> DNA

[1096] <213> Artificial sequence

[1097] <220>

[1098] <223> CMV enhancer

[1099] <400> 29

[1100] ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

[1101] catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

[1102] acgaccccg cccattgacg tcaataatga cgtatgttcc catagtaacg caataggga 180

[1103] ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

[1104] aagtgtatca tatgccaagt ccgccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

[1105] ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 360

[1106] tagtcatcgc tattaccatg 380

[1107] <210> 30

[1108] <211> 128

[1109] <212> DNA

[1110] <213> Artificial sequence

[1111] <220>

[1112] <223> Truncated 5'-ICP of AAV2

[1113] <400> 30

[1114] gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgaccttttgg 60

[1115] tcgcccggcc tcagtgagcg agcgagcgcg cagagagggga gtggccaact ccatcactag 120

[1116] gggttcct 128

[1117] <210> 31

[1118] <211> 128

[1119] <212> DNA

[1120] <213> Artificial sequence

[1121] <220>

[1122] <223> AAV2 3'-truncated IPC

[1123] <400> 31

[1124] aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

[1125] ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

[1126] gagcgcgc 128

[1127] <210> 32

[1128] <211> 122

[1129] <212> DNA

[1130] <213> Artificial sequence

[1131] <220>

[1132] <223> SV40 polyadenylation signal

[1133] <400> 32

[1134] taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 60

[1135] tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 120

[1136] tt 122

[1137] <210> 33

[1138] <211> 449

[1139] <212> DNA

[1140] <213> Artificial sequence

[1141] <220>

[1142] <223> Rabbit beta-globin polyadenylation signal

[1143] <400> 33

[1144] gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 60

[1145] tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 120

[1146] tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt 180

[1147] tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga 240

[1148] ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg 300

[1149] acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta 360

[1150] aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc 420

[1151] atagctgtcc ctcttctctt atggagatc 449

[1152] <210> 34

[1153] <211> 592

[1154] <212> DNA

[1155] <213> Artificial sequence

[1156] <220>

[1157] <223> CMV promoter and CMV enhancer sequence

[1158] <400> 34

[1159] ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

[1160] catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

[1161] acgaccccg cccattgacg tcaataatga cgtatgttcc catagtaacg caataggga 180

[1162] ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

[1163] aagtgtatca tatgccaagt ccgccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

[1164] ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat 360

[1165] tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc 420

[1166] ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

[1167] ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctgcgatcgc ccgccccgtt 540

[1168] gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ct 592

[1169] <210> 35

[1170] <211> 7319

[1171] <212> DNA

[1172] <213> Artificial sequence

[1173] <220>

[1174] <223> pAAV-CAG-moFGF21-dmiRT

[1175] <400> 35

[1176] agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 60

[1177] ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 120

[1178] ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 180

[1179] gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 240

[1180] gccgcgttgc tggcgtttt ccataggctc cgcccccctg acgagcatca caaaaatcga 300

[1181] cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 360

[1182] ggaagctccc tcgtgcgctc tcctgttccg acctgccgc ttaccggata cctgtcgcc 420

[1183] tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 480

[1184] gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gccgaccgc 540

[1185] tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 600

[1186] ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 660

[1187] ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 720

[1188] ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 780

[1189] accgctggta gcggtggtttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 840

[1190] tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 900

[1191] cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 960

[1192] taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1020

[1193] caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 1080

[1194] gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 1140

[1195] gctgcaatga taccgcgaga cccacgctca ccggctccag attatcagc aataaaccag 1200

[1196] ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgccctc catccagtct 1260

[1197] atttattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 1320

[1198] gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 1380

[1199] tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaagcggtt 1440

[1200] agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 1500

[1201] gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 1560

[1202] actggtgagt actcaaccaa gtcattctga gatagtgta tgcggcgacc gagttgctct 1620

[1203] tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 1680

[1204] attggaaac gttcttcgggg gcgaaaactc tcaggatct taccgctgtt gagatccagt 1740

[1205] tcgatgtaac ccactcgtgc acccaactga tcttcagcat ctttacttt caccagcgtt 1800

[1206] tctggggtgag aaaaacagg aaggcaaat gccgcaaaaa agggaatag ggcgacacgg 1860

[1207] aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 1920

[1208] tgtctcatga gcggatacat atttgaatgt atttagaaaa aaacaat aggggttccg 1980

[1209] cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 2040

[1210] acctataaaa ataggcgtat cacgaggcccc tttcgtctcg cgcgtttcgg tgatgacggt 2100

[1211] gaaaacctct gandacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 2160

[1212] gggagcagac aagcccgtca gggcgcgtca gcggggttgttg gcggggtgtcg gggctggctt 2220

[1213] aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 2280

[1214] cacagatgcg taaggaaa ataccgcatc aggcgattcc aacatccaat aaatcataca 2340

[1215] ggcaaggcaa agattagca aaattaagca attaagccctc agagcataa gctaaatcgg 2400

[1216] ttgtaccaaaacattatga ccctgtataa cttttgcgggg agaagcctttt atttcaacgc 2460

[1217] aaggataaaa atttttagaa ccctcatata ttttaaatgc atgcctgag taatgtgtag 2520 1218

[1219] caaccgttct agctgataaa ttcatgccgg agaggggtagc tatttttgag aggtctctac 2640

[1220] aaaggctatc aggtcattgc ctgagagtct ggagcaaaca agagaatcga tgaacggtaa 2700

[1221] tcgtaaaact agcatgtcaa tcatatgtac cccggttgat aatcagaaaa gccccaaaaa 2760

[1222] caggaagaatt gtataagcaa atatttaaat tgtaagcgtt aatattttgt taaaattcgc 2820

[1223] gttaaatttt tgttaaatca gctcatttt taaccaatag gccgaaatcg gcaaaatccc 2880

[1224] ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag 2940

[1225] tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 3000

[1226] tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc 3060

[1227] actaaatcgg aaccctaaag ggagcccccg atttagagct tgacgggaa agccggcgaa 3120

[1228] cgtggcgaga agaaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 3180

[1229] agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 3240

[1230] gtactatggt tgctttgacg agcacgtata acgtgctttc ctcgttagaa tcagagcggg 3300

[1231] agctaaacag gaggccgatt aaagggattt tagacaggaa cggtacgcca gaatcctgag 3360

[1232] aagtgtttt ataatcagtg aggccaccga gtaaaagagt ctgtccatca cgcaaattaa 3420

[1233] ccgttgtcgc aatacttctt tgattagtaa taacatcact tgcctgagta gaagaactca 3480

[1234] aactatcggc cttgctggta atatccagaa caatattacc gccagccatt gcaacggaat 3540

[1235] cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcc 3600

[1236] actgaggccc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt 3660

[1237] cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcaga gggagtggcc 3720

[1238] aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctact 3780

[1239] cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 3840

[1240] catatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc

[1241] aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg

[1242] actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat

[1243] caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc

[1244] tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta

[1245] ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc 4200

[1246] tcccccccct ccccaccccc aatttttgtat ttttttttttttttttttgtgcagcg 4260.

[1247] atggggggcgg gggggggggg ggggcggcggc ccaggcgggg cggggcgggg cgaggggcgg 4320

[1248] ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt

[1249] ccttttatgg cgaggcggcg gcggcggcgg ccctaaaa agcgaagcgc gcggcggggcg 4440

[1250] ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcccgccc 4500

[1251] cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 4560

[1252] ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc 4620

[1253] cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg 4680

[1254] tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc 4740

[1255] gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc 4800

[1256] ggtgccccgc ggtgcggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt 4860

[1257] gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc 4920

[1258] tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc 4980

[1259] ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc 5040

[1260] gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg 5100

[1261] tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg 5160

[1262] acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc 5220

[1263] tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 5280

[1264] tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg 5340

[1265] ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg 5400

[1266] cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg 5460

[1267] caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattgatta attcgagcga 5520

[1268] acgcgtcgag tcgctcggta cgatttaaat tgaattggcc tcgagcgcaa gcttgagcta 5580

[1269] gcgccaccat ggaatggatg agaagcagag tgggcaccct gggcctgtgg gtgcgactgc 5640

[1270] tgctggctgt gtttctgctg ggcgtgtacc aggcctaccc catccctgac tctagccccc 5700

[1271] tgctgcagtt tggcggacaa gtgcggcaga gatacctgta caccgacgac gaccaggaca 5760

[1272] ccgaggccca cctggaaatc cgcgaggatg gcacagtcgt gggcgctgct cacagaagcc 5820

[1273] ctgagagcct gctggaactg aaggccctga agcccggcgt gatccagatc ctgggcgtga 5880

[1274] aggccagcag attcctgtgc cagcagcctg acggcgccct gtacggctct cctcacttcg 5940

[1275] atcctgaggc ctgcagcttc agagagctgc tgctggagga cggctacaac gtgtaccagt

[1276] ctgaggccca cggcctgccc ctgagactgc ctcagaagga cagccctaac caggacgcca

[1277] caagctgggg acctgtgcgg ttcctgccta tgcctggact gctgcacgag ccccaggatc 6120

[1278] aggctggctt tctgcctcct gagcctccag acgtgggcag cagcgaccct ctgagcatgg

[1279] tggaacctct gcagggcaga agccccagct acgcctcttg agaatgcggg cccggtaccc 6240

[1280] ccgacgcggc cgctaattct agatcgcgaa caaacaccat tgtcacactc cagtatacac

[1281] aaacaccatt gtcacactcc agatatcaca aacaccattg tcacactcca aggcgaacaa

[1282] acaccattgt cacactccaa ggctattcta gatcgcgaat tacatacttc tttacattcc

[1283] agtatacatt ttacattcca 6480.

[1283] agtatacatt ttacattcca

[1284] ggcgaattac atacttcttt acattccaag gctacctgag gcccgggggt acctcttaat 6540

[1285] taactggcct catgggcctt ccgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 6600

[1286] cagtcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca 6660

[1287] caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct 6720

[1288] tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa 6780

[1289] ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa 6840

[1290] tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg 6900

[1291] ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca 6960

[1292] tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc 7020

[1293] tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg 7080

[1294] actactccca gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa 7140

[1295] gctgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag 7200

[1296] ttggccactc cctctctgcg cgctcgctcg ctcactgagg cggggcgacc aaaggtcgcc 7260

[1297] cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctggcgtaa 7319

[1298] <210> 36

[1299] <211> 411

[1300] <212> DNA

[1301] <213> Artificial sequence

[1302] <220>

[1303] <223> The minimal promoter of CMV

[1304] <400> 36

[1305] tatgccaagt acgccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 60

[1306] ccagtacatg acctatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 120

[1307] tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 180

[1308] acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 240

[1309] tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 300

[1310] gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc 360

[1311] ttaactggct tatcgaaatt aatacgactc actataggga gacccaagct t 411

[1312] <210> 37

[1313] <211> 1178

[1314] <212> DNA

[1315] <213> Artificial sequence

[1316] <220>

[1317] <223> The promoter of EF1-alpha

[1318] <400> 37

[1319] ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60

[1320] ggaggggtg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120

[1321] gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180

[1322] gtagtcgccg tgaacgttct tttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240

[1323] gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300

[1324] acttccactg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360

[1325] agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420

[1326] ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480

[1327] tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540

[1328] aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600

[1329] cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660

[1330] cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720

[1331] gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780

[1332] ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840

[1333] cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900

[1334] cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 960

[1335] agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020

[1336] agttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080

[1337] tctccttgga atttgcccttt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140

[1338] tggttcaaag ttttttctt ccatttcagg tgtcgtga 1178

[1339] <210> 38

[1340] <211> 623

[1341] <212> DNA

[1342] <213> Artificial sequence

[1343] <220>

[1344] <223> The promoter of RSV

[1345] <400> 38

[1346] catgtttgac agcttatcat cgcagatccg tatggtgcac tctcagtaca atctgctctg 60

[1347] atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120

[1348] gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180

[1349] tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat tcgcgtatct 240

[1350] gaggggacta gggtgtgttt aggcgaaaag cggggcttcg gttgtacgcg gttaggagtc 300

[1351] ccctcaggat atagtagttt cgcttttgca tagggagggg gaaatgtagt cttatgcaat 360

[1352] actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa 420

[1353] aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag 480

[1354] gcaacagacg ggtctgacat ggattggacg aaccactaaa ttccgcattg cagagatatt 540

[1355] gtatttaagt gcctagctcg atacaataaa cgccatttga ccattcacca cattggtgtg 600

[1356] cacctccaag ctgggtacca gct 623

[1357] <210> 39

[1358] <211> 448

[1359] <212> DNA

[1360] <213> Artificial sequence

[1361] <220>

[1362] <223> Promoter of synapsin-1

[1363] <400> 39

[1364] ctgcgctctc aggcacgaca cgactcctc gctgcccacc gcagactgag gcagcgctga 60

[1365] gtcgcggcg ccgcagcgca gatggtcgcg cccgtgcccc cctatctcgc gcctcgcgtg 120

[1366] gtgcggtccg gctgggccgg cggcggcgcg gacgcgacca aggtggccgg gaaggggagt 180

[1367] ttgcggggga ccggcgagtg acgtcagcgc gccttcagtg ctgaggcggc ggtggcgcgc 240

[1368] gccgccaggc gggggcgaag gcactgtccg cggtgctgaa gctggcagtg cgcacgcgcc 300

[1369] tcgccgcatc ctgtttcccc tccccctctc tgatagggga tgcgcaattt ggggaatggg 360

[1370] ggttgggtgc ttgtccagtg ggtcggggtc ggtcgtcagg taggcacccc caccccgcct 420

[1371] catcctggtc ctaaaaccca cttgcact 448

[1372] <210> 40

[1373] <211> 1296

[1374] <212> DNA

[1375] <213> Artificial sequence

[1376] <220>

[1377] <223> The promoter of calcium / calmodulin-dependent protein kinase II (CaMKII)

[1378] <400> 40

[1379] taacattatg gccttaggtc acttcatctc catggggttc ttcttctgat tttctagaaa 60

[1380] atgagatggg ggtgcagaga gcttcctcag tgacctgcc agggtcacat cagaaatgtc 120

[1381] agagctagaa cttgaactca gattactaat cttaaattcc atgccttggg ggcatgcaag 180

[1382] tacgatatac agaaggagtg aactcattag ggcagatgac caatgagttt aggaaagaag 240

[1383] agtccagggc agggtacatc tacaccaccc gcccagccct gggtgagtcc agccacgttc 300

[1384] acctcattat agttgcctct ctccagtcct accttgacgg gaagcacaag cagaaactgg 360

[1385] gacaggagcc ccaggagacc aaatcttcat ggtccctctg ggaggatggg tggggagagc 420

[1386] tgtggcagag gcctcaggag gggccctgct gctcagtggt gacagatagg ggtgagaaag 480

[1387] cagacagagt cattccgtca gcattctggg tctgtttggt acttcttctc acgctaaggt 540

[1388] ggcggtgtga tatgcacaat ggctaaaaag cagggagagc tggaaagaaa caaggacaga 600

[1389] gacagaggcc aagtcaacca gaccaattcc cagaggaagc aaagaaacca ttacagagac 660

[1390] tacaaggggg aagggaagga gagatgaatt agcttcccct gtaaacctta gaacccagct 720

[1391] gttgccaggg caacggggca atacctgtct cttcagagga gatgaagttg ccagggtaac 780

[1392] tacatcctgt cttctcaag gaccatccca gaatgtggca cccactagcc gttaccatag 840

[1393] caactgcctc ttgccccac ttaatcccat cccgtctgtt aaaagggccc tatagttgga 900

[1394] ggtgggggag gtaggaagag cgatgatcac ttgtggacta agtttgttcg catccccttc 960

[1395] tccaaccccc tcagtacatc accctggggg aacagggtcc acttgctcct gggcccacac 1020

[1396] agtcctgcag tattgtgtat ataaggccag ggcaaagagg agcaggtttt aaagtgaaag 1080

[1397] gcaggcaggt gttggggagg cagttaccgg ggcaacgggga acagggcgtt tcggaggtgg 1140

[1398] ttgccatggg gacctggatg ctgacgaagg ctcgcgaggc tgtgagcagc cacagtgccc 1200

[1399] tgctcagaag ccccaagctc gtcagtcaag ccggttctcc gtttgcactc aggagcacgg 1260

[1400] gcaggcgagt ggcccctagt tctgggggca gcgggg 1296

[1401] <210> 41

[1402] <211> 706

[1403] <212> DNA

[1404] <213> Artificial sequence

[1405] <220>

[1406] <223> Glial fibrillary acidic protein (GFAP) promoter

[1407] <400> 41

[1408] cgcgtgatct aacatatcct ggtgtggagt aggggacgct gctctgacag aggctcgggg 60

[1409] gcctgagctg gctctgtgag ctggggagga ggcagacagc caggccttgt ctgcaagcag 120

[1410] acctggcagc attgggctgg ccgcccccca gggcctcctc ttcatgccca gtgaatgact 180

[1411] caccttggca cagacacaat gttcggggtg ggcacagtgc ctgcttcccg ccgcacccca 240

[1412] gcccccctca aatgccttcc gagaagccca ttgagcaggg ggcttgcatt gcaccccagc 300

[1413] ctgacagcct ggcatcttgg gataaaagca gcacagcccc ctaggggctg cccttgctgt 360

[1414] gtggcgccac cggcggtgga gaacaaggct ctattcagcc tgtgcccagg aaaggggatc 420

[1415] aggggatgcc caggcatgga cagtgggtgg caggggggga gaggagggct gtctgcttcc 480

[1416] cagaagtcca aggacacaaa tgggtgaggg gagagctctc cccatagctg ggctgcggcc 540

[1417] caaccccacc ccctcaggct atgccagggg gtgttgccag gggcacccgg gcatcgccag 600

[1418] tctagcccac tccttcataa agccctcgca tcccaggagc gagcagagcc agagcaggtt 660

[1419] ggagaggaga cgcatcacct ccgctgctcg cggggtctag agtcga 706

[1420] <210> 42

[1421] <211> 1153

[1422] <212> DNA

[1423] <213> Artificial sequence

[1424] <220>

[1425] <223> Nestin promoter

[1426] <400> 42

[1427] gaaggcagcc cccggaggtc aaaggctggg cacgcggggag gagaggccag agtcagaggc 60

[1428] tgcgggtatc tcagatatga aggaaagatg agagaggctc aggaagaggt aagaaaagac 120

[1429] acaagagacc agagaaggga gaagaattag agagggaggc agaggaccgc tgtctctaca 180

[1430] gacatagctg gtagagactg ggaggaaggg atgaaccctg agcgcatgaa gggaaggagg 240

[1431] tggctggtgg tatatggagg atgtagctgg gccagggaaa agatcctgca ctaaaaatct 300

[1432] gaagctaaaa ataacaggac acggggtgga gaggcgaaag gagggcagat tgaggcagag 360

[1433] agactgagag gcctggggat gtgggcattc cggtagggca cacagttcac ttgtcttctc 420

[1434] tttttccagg aggccaaaga tgctgacctc aagaactcat aataccccag tggggaccac 480

[1435] cgcattcata gccctgttac aagaagtggg agatgttcct ttttgtccca gactggaaat 540

[1436] ccattacatc ccgaggctca ggttctgtgg tggtcatctc tgtgtggctt gttctgtggg 600

[1437] cctacctaaa gtcctaagca cagctctcaa gcagatccga ggcgactaag atgctagtag 660

[1438] gggttgtctg gagaagag ccgaggaggt gggctgtgat ggatcagttc agctttcaaa 720

[1439] taaaaaggcg ttttatatt ctgtgtcgag ttcgtgaacc cctgtggtgg gcttctccat 780

[1440] ctgtctgggt tagtacctgc cactatactg gaataaggag acgcctgctt ccctcgagtt 840

[1441] ggctggacaa ggtatgagc atccgtgtac ttatggggtt gccagcttgg tcctggatcg 900

[1442] cccgggccct tccccaccc gttcggttcc ccaccaccac ccgcgctcgt acgtgcgtct 960

[1443] ccgcctgcag ctcttgactc atcggggccc cgggtcaca tgcgctcgct cggctctata 1020

[1444] ggcgccgcc cctgcccacc ccccgcccgc gctgggagcc gcagccgccg ccactcctgc 1080

[1445] tctctctgcg ccgccgccgt caccaccgcc accgccaccg gctgagtctg cagtcctccg 1140

[1446] aaacgggcc tct 1153

[1447] <210> 43

[1448] <211> 438

[1449] <212> DNA

[1450] <213> Artificial sequence

[1451] <220>

[1452] <223> Homeobox protein 9 (HB9) promoter

[1453] <400> 43

[1454] tgaataaatt tagcaggct attaatata taaactagct caatttgtca agttgatttg 60

[1455] tattttagtt aattgtgaaa gtaattacca catggtcaaa ttaacagctt tctggaaatg 120

[1456] accaagcctg aggttttatt tccttcctgg gtgaagaaaa ttcatttttc caagctcttg 180

[1457] atgtgatgaa taaaagtcat aaatctgggt gattggtgca ggcagagtct aaatggcttc 240

[1458] atatttcatt ttaggtttaa tagaaatatt catgctctgt tttaatgaaa ttaaattgaa 300

[1459] gggggatggg gctagagtgg ttagctgatg aattgacaaa aactaatcag cttattggg 360

[1460] aaacaggttt aagggcacgg acgtgtcaat aacgctcagc ctgaccccct cttccattag 420

[1461] ctaggcaggc tgattaga 438

[1462] <210> 44

[1463] <211> 3015

[1464] <212> DNA

[1465] <213> Artificial sequence

[1466] <220>

[1467] <223> Promoter tyrosine hydroxylase (TH)

[1468] <400> 44

[1469] ctgctagggg ctgcttccca gctacctc ttggctccgt ggcttgcctt ccagcctgtg 60

[1470] tgctgtctgg agagccttta aagcctcact tccaccaact agaagtctct ccccaaccct 120

[1471] gcctgacct caagtgcacc tcttcaaagt caggtttagc agctgcagct gggggccctg 180

[1472] aatcccacc ctgctgtctt ccttgaagac agaagtgttg ggagctgagg atctgggcta 240

[1473] gagactggct gtatgatcca gagaagtagt gtgcttctgg gcctcagatt tcccttctgt 300

[1474] agaacaggtt tgtctgaaat ggagaggttg gtgctcctct gcagggccta gtgggagtca 360

[1475] ccatgagtgg ttaaaagatc cagcttgtct tttggtgagc tttgagagga ggtaacaggg 420

[1476] ctgagttctg gaagcctgac caagggcaga cttaaggggc ctcttggagt tgttctcatc 480

[1477] aaatggggat gggacacagc taaagtgccc agggcttctc tgtgcccaca gatgctttag 540

[1478] atcttggcac agtgtggtct accagctgtc tctctctgtg tatatatatg tatttcatag 600

[1479] acagtgtaca gtggctggt ttgtgctatc aggctggata tggacagagg caagagtttg 660

[1480] tggcagcagt tatctcccaa gagagtccaa agacatcatg tttcaagtt taggccaggt 720

[1481] gctacttgag agagctcaga cacagacaaa ggtctggaga gcacatgtcc tccaccccca 780

[1482] cctagcttct gttgcaagca cctccagccg agacaagaga acgaattaa aagcaatatt 840

[1483] tgtgtcagtg taagacattt gccgaaaggt taaatccaca ttcgtgttgc tgcagagcag 900

[1484] cccctatgc aggatttgtt agatacagct ccgtcctacc ctgtgccagc tgagcaaacg 960

[1485] ccaggctggg tggggtggaa cccagcctgg gttgcctca ccctgcaatc cccccagcac 1020

[1486] cctactaaagg aggaccctgt ggtggggcatg cagacctagg gactggggcat agataacctt 1080

[1487] tgggttttgggg cacagcccc cactccctcag gattgaggc taaggtgcag ccagctctgc 1140

[1488] cttcatggtg ggaatgtctc cacgtgaccc ctttctgggc tgtggagaac actcagagaa 1200

[1489] gagtcctggg atgccaggca ggccagggat gtgctgggca tgttgagaca ggagtgggct 1260

[1490] aagccagcag agttgctgac ccaggaagag ttcagaaagg ggcatggaac atggggaggg

[1491] gtccatagtg agagagagca ggcagtgcag agtaaatagt ccctgagctg ggggttatgg

[1492] gatttgcagg agcttgctca gagaaggcag aggagagatg ctgcgccaag ctgggtatca

[1493] cagagcctca gactcctgga acaggaactg tgggggtcag gtcagcaggg gaggttaggg

[1494] agtgttccct ttgtactgac ttagcattta tcctgcttct aggggggag gggggccagt

[1495] gggggatgca cagcaaggca gtgatgtggc aggcagcctg cgggagctcc tggttcctgg

[1496] tgtgaaaaag ctgggaagga agagggctgg gtctggtaag tacagcaggc agttggctcc

[1497] tgagagtcca agccctgtct agagggtgga gtgagatttc agagggag ctaaacgggg

[1498] tgggggctgg ggagtccagg cttctggctc ctgctaatac tcagtgtgct gggtcctcag

[1499] aacctcaggg tggccatttt cagggtgaga gctctgtcct ttggcacttc tgcagactcc

[1500] 1500 tggtactctt cctcagttcc cttagtgaga ggacacctttt

[1501] ctctgaaggg cttgggcagt tgtcctgaac cattgcctga aggaaggact tgactccagg

[1502] they are the gggctcagca they are the ccccc gthey are the aaggtcccct ctctggtctc

[1503] cttagatc ctgtttcctt ggctgagga gctagggtgg atctttgtgt aagtgggtgt

[1504] ggatgctcac tggaatcaa aaggcccctt ggtgttagac cttggggtgc catgggagag

[1505] ttgatcactg agtgcgccct tacatggggg ccagctgaga atggggctgc ctctagctcg

[1506] agaccatgat gcagggagtg agtggggag ttcaggatac tcttaactaa agcagaggtc

[1507] tgtcccccca gggaggggag gtcagaagac cctagggaga tgccaaaggc tagggttggc

[1508] accatgttgc aggctgtgtc ttcaaggaga tgataatcag aggatcgaa cctgcaaaag

[1509] tgggccagtc ttagatacac tataggga taatcttctg aaacattctg tgtctcatag

[1510] gacctgcctg cccagtgcca gcacatacac tggggcagtg

[1511] atactttgtt acatgggctg gggggacatg gcctgtgccc tggaggggac ttgaagacat

[1512] ccaaaaagct agtgagaggg ctcctagatt tatttgtctc caagggctat atatagcctt 2640

[1513] cctaacatga acccttgggt aatccagcat gggcgctccc atatgccctg gtttgattag 2700

[1514] agagctctag atgtctcctg tcccagaaca ccagccagcc cctgtcttca tgtcgtgtct 2760

[1515] agggcggagg gtgattcaga ggcaggtgcc tgcgacagtg gatgcaatta gatctaatgg 2820

[1516] gacggaggcc tctctcgtcc gtcgccctcg ctctgtgccc acccccgcct ccctcaggca 2880

[1517] cagcaggcgt ggagaggatg cgcaggaggt aggaggtggg ggacccagag gggctttgac 2940

[1518] gtcagcctgg cctttaagag gccgcctgcc tggcaagggc cgtggagaca gaactcggga 3000

[1519] ccaccagctt gcact 3015

[1520] <210> 45

[1521] <211> 1353

[1522] <212> DNA

[1523] <213> Artificial sequence

[1524] <220>

[1525] <223> Myelin basic protein (MPP) promoter

[1526] <400> 45

[1527] caccgtggct ttaacactta gagaaaatgc atcccctcta atcaataagt catcgacagt 60

[1528] gggtagatgg aggaacggca gtgcgtagta ggatgcgtgc aagcatagtc tcgtgcatgg 120 1529

[1530] tgatgttagg taaatatcac tgggtggaca gatgggtggt aggtggatgg atggttagaa 240

[1531] tagtcagaag agggatggat tgataaggtg aacagatgat aaatgggtga tagactgggaa 300

[1532] gggttgtcaa aagagattaa gggaagtgtg agctagccgt atttctaagg tcagtaatag 360

[1533] agttgggaga agaggttaag ttacatccat ttaaaacctca cacgaagctg agagggaatg 420

[1534] gacttgctgc cgttggtgag gaaagcgttg catttcccgt gtgcttggtt gtgaagtgct 480

[1535] caggtcccac atgaagcagt caggttactg cggctttacag aggagccaga tccaaatgcc 540

[1536] ccgagtaagc acgtccccga gccagaggcc tccagcggaa tccgggagag ggattgctca 600

[1537] gtgccctgct tccctggact gtaagctgca gaagatgtg ggaagtcctg ttctccactg 660

[1538] agaacactaa aagcaccttt tgtcaaacga ccgcttcaca tctggggctt gtgcactggt 720

[1539] ggccttttaa accagagaca acccacaaga tacctaacct gcggggctct ctggtacagt 780

[1540] gagcaactca ggaaatgctt tggcttgatt gctgtgggct ctcaggccat cgccctctgg 840

[1541] agtggttctt ttaatgagaa cctgaagatt ggcccctgag ccatgtatac caagcaagct 900

[1542] caatccaggt tagctccctc tggttggggc aagctaacgt gctccttggg ccccgcgcgt 960

[1543] aactgtgcgt tttataggag acagctagtt caagacccca ggaagaaagc ggctttgtcc 1020

[1544] ccctctaggc ctcgtacagg cccacattca tatctcattg ttgttgcagg ggaggcagat 1080

[1545] gcgatccaga acaatgggac ctcggctgag gacacggcgg tgacagactc caagcacaca 1140

[1546] gcagacccaa agaataactg gcaaggcgcc cacccagctg acccagggaa ccgcccccac 1200

[1547] ttgatccgcc tcttttcccg agatgccccg ggaagggagg acaacacctt caaagacagg 1260

[1548] ccctcagagt ccgacgagct tcagaccatc caagaagatc ccacagcagc ttccgaagaa 1320

[1549] ttctgcagtc gacggtaccg cgggcccggg atc 1353

[1550] <210> 46

[1551] <211> 7030

[1552] <212> DNA

[1553] <213> Artificial sequence

[1554] <220>

[1555] <223> pAAV-CAG-moFGF21

[1556] <400> 46

[1557] agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 60

[1558] ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 120

[1559] ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 180

[1560] gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 240

[1561] gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 300

[1562] cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 360

[1563] ggaagctccc tcgtgcgctc tctgttccg acctgccgc ttaccggata cctgtcgcc 420

[1564] tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 480

[1565] gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gccgaccgc 540

[1566] tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 600

[1567] ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 660

[1568] ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 720

[1569] ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 780

[1570] accgctggta gcggtggtttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 840

[1571] tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 900

[1572] cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 960

[1573] taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1020

[1574] caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 1080

[1575] gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 1140

[1576] gctgcaatga taccgcgaga cccacgctca ccggctccag attatcagc aataaaccag 1200

[1577] ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgccctc catccagtct 1260

[1578] atttaatgtt gccgggaagc tagagtagt agttcgccag ttaatagttt gcgcaacgtt 1320

[1579] gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 1380

[1580] tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaagcggtt 1440

[1581] agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 1500

[1582] gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 1560

[1583] actggtgagt actcaaccaa gtcattctga gatagtgta tgcggcgacc gagttgctct 1620

[1584] tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 1680

[1585] attggaaac gttcttcgggg gcgaaaactc tcaggatct taccgctgtt gagatccagt 1740

[1586] tcgatgtaac ccactcgtgc acccaactga tcttcagcat ctttacttt caccagcgtt 1800

[1587] tctggggtgag aaaaacagg aaggcaaat gccgcaaaaa agggaataag ggcgacacgg 1860

[1588] aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 1920

[1589] tgtctcatga gcggatacat atttgaatgt atttagaaaa aaacaat aggggttccg 1980

[1590] cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 2040

[1591] acctataaaa ataggcgtat cacgaggcccc tttcgtctcg cgcgtttcgg tgatgacggt 2100

[1592] gaaaacctct gandacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 2160

[1593] gggagcagac aagcccgtca gggcgcgtca gcggggttgttg gcggggtgtcg gggctggctt 2220

[1594] aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 2280

[1595] cacagatgcg taaggaaa ataccgcatc aggcgattcc aacatccaat aaatcataca 2340

[1596] ggcaaggcaa agattagca aaattaagca attaagccctc agagcataa gctaaatcgg 2400

[1597] ttgtaccaaa aacattatga ccctgtataa cttttgcgggg agaagcctttt atttcaacgc 2460

[1598] aaggataaaa atttttagaa ccctcatata ttttaaatgc atgcctgag taatgtgtag 2520 1599

[1600] caaccgttct agctgataaa ttcatgccgg agaggggtagc tatttttgag aggtctctac 2640

[1601] aaaggctatc aggtcattgc ctgagagtct ggagcaaaca agagaatcga tgaacggtaa 2700

[1602] tcgtaaaact agcatgtcaa tcatatgtac cccggttgat aatcagaaaa gccccaaaaa 2760

[1603] caggaagaatt gtataagcaa atatttaaat tgtaagcgtt aatattttgt taaaattcgc 2820

[1604] gttaaatttt tgttaaatca gctcatttt taaccaatag gccgaaatcg gcaaaatccc 2880

[1605] ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag 2940

[1606] tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 3000

[1607] tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc 3060

[1608] actaaatcgg aaccctaaag ggagcccccg atttagagct tgacgggaa agccggcgaa 3120

[1609] cgtggcgaga agaaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 3180

[1610] agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 3240

[1611] gtactatggt tgctttgacg agcacgtata acgtgctttc ctcgttagaa tcagagcggg 3300

[1612] agctaaacag gaggccgatt aaagggattt tagacaggaa cggtacgcca gaatcctgag 3360

[1613] aagtgtttt ataatcagtg aggccaccga gtaaaagagt ctgtccatca cgcaaattaa 3420

[1614] ccgttgtcgc aatacttctt tgattagtaa taacatcact tgcctgagta gaagaactca 3480

[1615] aactatcggc cttgctggta atatccagaa caatattacc gccagccatt gcaacggaat 3540

[1616] cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcc 3600

[1617] actgaggccc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt 3660

[1618] cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc 3720

[1619] aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta cttatctact 3780

[1620] cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 3840

[1621] catatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc

[1622] aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg

[1623] actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat

[1624] caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc

[1625] tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta

[1626] ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc

[1627] tcccccccct ccccaccccc aatttttgtat ttttttttttttttttttgtgcagcg 4260.

[1628] atggggggcgg gggggggggg ggggcggcggc ccaggcgggg cggggcgggg cgaggggcgg 4320

[1629] ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt

[1630] ccttttatgg cgaggcggcg gcggcggcgg ccctaaaa agcgaagcgc gcggcggggcg 4440

[1631] ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcccgccc 4500

[1632] cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 4560

[1633] ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc 4620

[1634] cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg 4680

[1635] tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc 4740

[1636] gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc 4800

[1637] ggtgccccgc ggtgcggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt 4860

[1638] gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc 4920

[1639] tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc 4980

[1640] ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc 5040

[1641] gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg 5100

[1642] tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg 5160

[1643] acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc 5220

[1644] tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 5280

[1645] tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg 5340

[1646] ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg 5400

[1647] cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg 5460

[1648] caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattgatta attcgagcga 5520

[1649] acgcgtcgag tcgctcggta cgatttaaat tgaattggcc tcgagcgcaa gcttgagcta 5580

[1650] gcgccaccat ggaatggatg agaagcagag tgggcaccct gggcctgtgg gtgcgactgc 5640

[1651] tgctggctgt gtttctgctg ggcgtgtacc aggcctaccc catccctgac tctagccccc 5700

[1652] tgctgcagtt tggcggacaa gtgcggcaga gatacctgta caccgacgac gaccaggaca 5760

[1653] ccgaggccca cctggaaatc cgcgaggatg gcacagtcgt gggcgctgct cacagaagcc 5820

[1654] ctgagagcct gctggaactg aaggccctga agcccggcgt gatccagatc ctgggcgtga 5880

[1655] aggccagcag attcctgtgc cagcagcctg acggcgccct gtacggctct cctcacttcg 5940

[1656] atcctgaggc ctgcagcttc agagagctgc tgctggagga cggctacaac gtgtaccagt 6000

[1657] ctgaggccca cggcctgccc ctgagactgc ctcagaagga cagccctaac caggacgcca 6060

[1658] caagctgggg acctgtgcgg ttcctgccta tgcctggact gctgcacgag ccccaggatc 6120

[1659] aggctggctt tctgcctcct gagcctccag acgtgggcag cagcgaccct ctgagcatgg 6180

[1660] tggaacctct gcagggcaga agccccagct acgcctcttg agaatgcggg cccggtaccc 6240

[1661] ccgacgcggc ctaactggcc tcatgggcct tccgctcact gcccgctttc cagtcgggaa 6300

[1662] acctgtcgtg ccagtcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat 6360

[1663] gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc 6420

[1664] atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata 6480

[1665] gtgtgttgga atttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta 6540

[1666] aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc 6600

[1667] atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat 6660

[1668] tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg 6720

[1669] ttatttttt cttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc 6780

[1670] ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag atccctcgac 6840

[1671] ctgcagccca agctgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc 6900

[1672] taggatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 6960

[1673] caaaggtcgc ccgacgcccg ggctttgcc gggcggcctc agtgagcgag cgagcgcgca 7020

[1674] gctggcgtaa 7030

[1675] <210> 47

[1676] <211> 20

[1677] <212> DNA

[1678] <213> Artificial sequence

[1679] <220>

[1680] <223> moFgf21-Fw

[1681] <400> 47

[1682] cctaaccagg acgccacaag 20

[1683] <210> 48

[1684] <211> 20

[1685] <212> DNA

[1686] <213> Artificial sequence

[1687] <220>

[1688] <223> moFgf21-Rv

[1689] <400> 48

[1690] gttccaccat gctcagagg 20

[1691] <210> 49

[1692] <211> 22

[1693] <212> DNA

[1694] <213> Artificial sequence

[1695] <220>

[1696] <223> GFCB-Fw

[1697] <400> 49

[1698] acagactttc tccaacctcc ag 22

[1699] <210> 50

[1700] <211> 20

[1701] <212> DNA

[1702] <213> Artificial sequence

[1703] <220>

[1704] <223> GFCB-Rv

[1705] <400> 50

[1706] ccttctgaca cggatttggt 20

[1707] <210> 51

[1708] <211> 22

[1709] <212> DNA

[1710] <213> Artificial sequence

[1711] <220>

[1712] <223> S100b-Fw

[1713] <400> 51

[1714] aacaacgagc tctctcactt cc 22

[1715] <210> 52

[1716] <211> 20

[1717] <212> DNA

[1718] <213> Artificial sequence

[1719] <220>

[1720] <223> S100b-Rv

[1721] <400> 52

[1722] cgtctccatc actttgtcca 20

[1723] <210> 53

[1724] <211> 20

[1725] <212> DNA

[1726] <213> Artificial sequence

[1727] <220>

[1728] <223> Aif1-Fw

[1729] <400> 53

[1730] tgagccaaag cagggatttg 20

[1731] <210> 54

[1732] <211> 20

[1733] <212> DNA

[1734] <213> Artificial sequence

[1735] <220>

[1736] <223> Aif1-Rv

[1737] <400> 54

[1738] tcaagtttgg acggcagatc 20

[1739] <210> 55

[1740] <211> 20

[1741] <212> DNA

[1742] <213> Artificial sequence

[1743] <220>

[1744] <223> Nfkb-Fw

[1745] <400> 55

[1746] gaccactgct caggtccact 20

[1747] <210> 56

[1748] <211> 20

[1749] <212> DNA

[1750] <213> Artificial sequence

[1751] <220>

[1752] <223> Nfkb-Rv

[1753] <400> 56

[1754] tgtcactatcccggagttca 20

[1755] <210> 57

[1756] <211> 20

[1757] <212> DNA

[1758] <213> Artificial sequence

[1759] <220>

[1760] <223> IL-1b-Fw

[1761] <400> 57

[1762] atgaagggct gcttccaaac 20

[1763] <210> 58

[1764] <211> 20

[1765] <212> DNA

[1766] <213> Artificial sequence

[1767] <220>

[1768] <223> IL-1b-Rv

[1769] <400> 58

[1770] atgtgctgct gcgagatttg 20

[1771] <210> 59

[1772] <211> 20

[1773] <212> DNA

[1774] <213> Artificial sequence

[1775] <220>

[1776] <223> IL-6-Fw

[1777] <400> 59

[1778] tcgctcaggg tcacaagaaa 20

[1779] <210> 60

[1780] <211> 22

[1781] <212> DNA

[1782] <213> Artificial sequence

[1783] <220>

[1784] <223> IL-6-Rv

[1785] <400> 60

[1786] catcagaggc aaggaggaaa ac 22

[1787] <210> 61

[1788] <211> 20

[1789] <212> DNA

[1790] <213> Artificial sequence

[1791] <220>

[1792] <223> Ucp1-Fw

[1793] <400> 61

[1794] ggcctctacg actcagtcca 20

[1795] <210> 62

[1796] <211> 20

[1797] <212> DNA

[1798] <213> Artificial sequence

[1799] <220>

[1800] <223> Ucp1-Rv

[1801] <400> 62

[1802] taagccggct gagatcttgt 20

[1803] <210> 63

[1804] <211> 20

[1805] <212> DNA

[1806] <213> Artificial sequence

[1807] <220>

[1808] <223> Cidea-Fw

[1809] <400> 63

[1810] aaaccatgac cgaagtagcc 20

[1811] <210> 64

[1812] <211> 23

[1813] <212> DNA

[1814] <213> Artificial sequence

[1815] <220>

[1816] <223> Cidea-Rv

[1817] <400> 64

[1818] aggccagttg tgatgactaa gac 23

[1819] <210> 65

[1820] <211> 22

[1821] <212> DNA

[1822] <213> Artificial sequence

[1823] <220>

[1824] <223> TNF-a-Fw

[1825] <400> 65

[1826] cggcatggat ctcaaagaca ac 22

[1827] <210> 66

[1828] <211> 20

[1829] <212> DNA

[1830] <213> Artificial sequence

[1831] <220>

[1832] <223> TNF-a-Rv

[1833] <400> 66

[1834] agatagcaaa tcggctgacg 20

[1835] <210> 67

[1836] <211> 22

[1837] <212> DNA

[1838] <213> Artificial sequence

[1839] <220>

[1840] <223> F4 / 80-Fw

[1841] <400> 67

[1842] ctttggctat gggcttccag tc 22

[1843] <210> 68

[1844] <211> 21

[1845] <212> DNA

[1846] <213> Artificial sequence

[1847] <220>

[1848] <223> F4 / 80-Rv

[1849] <400> 68

[1850] gcaagggagga cagagtttat from 21

[1851] <210> 69

[1852] <211> 20

[1853] <212> DNA

[1854] <213> Artificial sequence

[1855] <220>

[1856] <223> Rplp0-Fw

[1857] <400> 69

[1858] actggtctag gacccgagaa 20

[1859] <210> 70

[1860] <211> 20

[1861] <212> DNA

[1862] <213> Artificial sequence

[1863] <220>

[1864] <223> Rplp0-Fw

[1865] <400> 70

[1866] tccaccttg tctccagtct 20

[1867] <---

Claims

1. A recombinant adeno-associated virus (rAAV) vector for use in the treatment or prevention of diabetes, wherein said rAAV vector comprises (i) a gene construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF-21) comprising the amino acid sequence of SEQ ID NO: 1, wherein said nucleotide sequence is operably linked to a universal promoter, and (ii) an AAV capsid protein, wherein said rAAV vector is administered by administration into the cerebrospinal fluid (CSF), and FGF-21 is expressed in the central nervous system (CNS).

2. A recombinant adeno-associated virus (rAAV) vector for use in treating or preventing obesity, wherein said rAAV vector comprises (i) a gene construct comprising a nucleotide sequence encoding fibroblast growth factor 21 (FGF-21) comprising the amino acid sequence of SEQ ID NO: 1, wherein said nucleotide sequence is operably linked to a universal promoter, and (ii) an AAV capsid protein, wherein said rAAV vector is administered by injection into the cerebrospinal fluid (CSF), and FGF-21 is expressed in the central nervous system (CNS).

3. An rAAV vector for use according to claim 1 or 2, wherein the universal promoter is selected from the group consisting of a CAG promoter and a CMV promoter.

4. An rAAV vector for use according to any one of claims 1 to 3, wherein the universal promoter is a CAG promoter.

5. An rAAV vector for use according to any one of claims 1 to 4, wherein said gene construct comprises at least one target sequence of a microRNA expressed in the tissue in which FGF-21 expression is prevented.

6. An rAAV vector for use according to any one of claims 1 to 5, wherein at least one target microRNA sequence is selected from those target sequences that bind to microRNAs expressed in the heart and / or liver of a mammal.

7. An rAAV vector for use according to any one of claims 1 to 6, wherein the nucleotide sequence encoding FGF-21 is also operably linked to at least one microRNA target sequence expressed in the liver and at least one microRNA target sequence expressed in the heart.

8. An rAAV vector for use according to claim 6 or 7, wherein the target sequence of the microRNA expressed in the heart is selected from SEQ ID NOs: 13 and 21-25, and the target sequence of the microRNA expressed in the liver is selected from SEQ ID NOs: 12 and 14-20.

9. An rAAV vector for use according to any one of claims 6-8, wherein said gene construct comprises a microRNA-122a target sequence and a microRNA-1 target sequence.

10. An rAAV vector for use according to any one of claims 1 to 9, wherein the nucleotide sequence encoding FGF-21 comprises: (a) a nucleotide sequence that has at least 95% identity to the nucleotide sequence of SEQ ID NO: 5, 6 or 7; or (b) a nucleotide sequence the sequence of which differs from the sequence of the nucleotide sequence in (a) due to the degeneracy of the genetic code.

11. An rAAV vector for use according to any one of claims 1 to 10, wherein the AAV capsid protein is serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype rh10 (AAVrh10), serotype rh8 (AAVrh8), serotype Cb4 (AAVCb4), serotype rh74 (AAVrh74), serotype DJ (AAV-DJ), serotype 2 / 5 (AAV2 / 5), serotype 2 / 1 (AAV2 / 1), serotype 1 / 2 (AAV1 / 2) or serotype Anc80 (AAV-Anc80).

12. An rAAV vector for use according to any one of claims 1 to 11, wherein the capsid protein is AAV1, AAV2 or AAV9.

13. An rAAV vector for use according to any one of claims 1-12, wherein the FGF-21 is expressed in the brain.

14. An rAAV vector for use according to any one of claims 1-12, wherein FGF-21 is expressed in the hypothalamus.

15. An rAAV vector for use according to any one of claims 1-12, wherein FGF-21 is expressed in the cerebral cortex.

16. An rAAV vector for use according to any one of claims 1-12, wherein the FGF-21 is expressed in the hippocampus or cerebellum.

17. An rAAV vector for use according to any one of claims 1-12, wherein FGF-21 is expressed in the olfactory bulb.

18. A pharmaceutical composition for use in the treatment or prevention of diabetes, which comprises an rAAV vector for use according to any one of claims 1 and 3-17 and a pharmaceutically acceptable ingredient selected from a carrier, filler, preservative, solvent, vehicle, diluent and / or excipient.

19. A pharmaceutical composition for use in the treatment or prevention of obesity, which comprises an rAAV vector for use according to any one of claims 2-17 and a pharmaceutically acceptable ingredient selected from a carrier, a filler, a preservative, a solvent, a vehicle, a diluent and / or an excipient.

Images