VIRAL EXPRESSION CONSTRUCT COMPRISING A FIBROBLAST GROWTH FACTOR 21 (FGF21) CODING SEQUENCE

Abstract

The invention relates to a viral expression construct and related viral vector and nucleic acid molecule and composition and to their use wherein said construct and vector are suitable for expression in a mammal and comprise a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) to be expressed in liver, adipose tissue and/or skeletal muscle.

Claims

1-28. (canceled)

29. A viral expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) operably linked to at least one heterologous regulatory element selected from: (a) a liver-specific promoter, (b) an adipose tissue-specific promoter, (c) a combination of a ubiquitous promoter and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the liver and at least one nucleotide sequence encoding a target sequence of a microRNA expressed in the heart, (d) a skeletal muscle promoter, and (e) a ubiquitous promoter.

30. The viral expression construct of claim 29, wherein the regulatory element comprises a ubiquitous promoter.

31. The viral expression construct of claim 30, wherein the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter or a CAG promoter.

32. The viral expression construct of claim 29, wherein the regulatory element comprises a liver-specific promoter.

33. The viral expression construct of claim 29, wherein the regulatory element comprises an adipose tissue-specific promoter.

34. The viral expression construct of claim 29, wherein the regulatory element comprises skeletal muscle promoter.

35. The viral expression construct of claim 29, wherein the nucleotide sequence encoding a target sequence of a microRNA expressed in the liver and the nucleotide sequence encoding a target sequence of a microRNA expressed in the heart is selected from a group consisting of sequences SEQ ID NO: 12 to 30 and any combinations thereof.

36. The viral expression construct of claim 29, wherein the nucleotide sequence encoding the FGF21 is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that has at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 1, 2 or 3, (b) a nucleotide sequence that has at least 60% sequence identity with the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11, and (c) a nucleotide sequence the sequence which differs from the sequence of a nucleotide sequence of (b) due to the degeneracy of the genetic code.

37. The viral expression construct of claim 29, wherein the nucleotide sequence encoding the FGF21 is selected from the group consisting of a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6, or 7.

38. The viral expression construct of claim 29, wherein the nucleotide sequence encoding the FGF21 is selected from the nucleotide sequence of SEQ ID NO: 5, 6, or 7.

39. The viral expression construct of claim 29, wherein the FGF21 is expressed in a mammalian liver, adipose tissue and/or skeletal muscle.

40. A recombinant adeno-associated virus (rAAV) vector comprising a vector genome and an AAV capsid, the vector genome comprising an inverted terminal repeat (ITR) and the viral expression construct of claim 29.

41. The rAAV vector of claim 40, wherein the AAV capsid has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, or AAV9.

42. The rAAV vector of claim 40, wherein the AAV capsid has a serotype selected from AAV1, AAV3, AAV8, and AAV9.

43. A recombinant adeno-associated virus (rAAV) vector comprising a vector genome and a capsid has an AAV1 serotype, the vector genome comprising an inverted terminal repeat (ITR) and a viral expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) operably linked to a ubiquitous promoter.

44. The rAAV vector of claim 43, wherein the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter or a CAG promoter.

45. The rAAV vector of claim 43, wherein the nucleotide sequence encoding the FGF21 is selected from the group consisting of a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6, or 7.

46. A composition comprising the rAAV vector of claim 40 and a pharmaceutically acceptable excipient or vehicle.

47. A method for treatment and/or prevention of a metabolic disorder, liver inflammation and/or fibrosis, a cancer, and/or extending healthy lifespan in a subject in need thereof comprising administering the viral expression construct of claim 29.

48. A method for treatment and/or prevention of a metabolic disorder, liver inflammation and/or fibrosis, a cancer, and/or extending healthy lifespan in a subject in need thereof comprising administering the rAAV vector of claim 40.

49. The method of claim 48, wherein the metabolic disorder is a metabolic disorder associated with aging.

50. The method of claim 48, wherein the metabolic disorder comprises a metabolic syndrome, diabetes, obesity, obesity-related comorbidities, diabetes-related comorbidities, hyperglycaemia, insulin resistance, glucose intolerance, hepatic steatosis, alcoholic liver diseases (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), coronary heart disease (CHD), hyperlipidemia, atherosclerosis, endocrinophaties, osteosarcopenic obesity syndrome (OSO), diabetic nephropaty, chronic kidney disease (CKD), cardiac hypertrophy, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, arthritis, sepsis, ocular neovascularization, neurodegeneration, and/or dementia.

51. The method of claim 48, wherein the metabolic disorder is non-alcoholic steatohepatitis (NASH), diabetes and/or obesity.

52. The method of claim 48, wherein the cancer is liver cancer.

53. A method for preventing, delaying, reverting, curing and/or treating a metabolic disorder comprising administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) vector comprising a vector genome and an AAV1 serotype capsid, the vector genome comprising an inverted terminal repeat (ITR) and a viral expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) operably linked to a ubiquitous promoter.

54. The method of claim 53, wherein the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter or a CAG promoter.

55. The method of claim 53, wherein the nucleotide sequence encoding the FGF21 is selected from the group consisting of a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6, or 7.

56. The method of claim 53, wherein the metabolic disorder is diabetes and/or obesity.

57. The method of claim 53, wherein the metabolic disorder is NASH.

58. The method of claim 53, wherein the FGF21 is expressed in skeletal muscle of the subject.

59. The method of claim 53, wherein the rAAV vector is administered intramuscularly.

60. A nucleic acid molecule comprising a nucleotide sequence encoding a FGF21, wherein the nucleotide sequence encoding the FGF21 is selected from the group consisting of a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 5, 6, or 7.

61. The nucleic acid molecule of claim 60, wherein the nucleotide sequence encoding the FGF21 is selected from the group consisting the nucleotide sequence of SEQ ID NO: 5, 6, or 7.

62. The nucleic acid molecule of claim 60, further comprising one or more of a promoter, a polyadenylation signal, and/or an enhancer sequence.

63. An expression cassette comprising the nucleic acid molecule of claim 62, wherein the promoter is operably linked to the nucleic acid sequence encoding FGF21.

64. An rAAV vector genome comprising the expression cassette of claim 63 flanked by a 5 ITR and a 3 ITR.

65. A recombinant adeno-associated virus (rAAV) vector comprising the AAV vector genome of claim 64 encapsidated in an AAV capsid.

66. A composition comprising the rAAV vector of claim 65 and a pharmaceutically acceptable excipient or vehicle.

67. A host cell comprising the rAAV vector genome of claim 64.

Description

FIGURE LEGENDS

[0221] FIGS. 1A-1H. Prevention of obesity by intra-eWAT administration of AAV9-CAG-moFGF21-dmiRT vectors in C57Bl6 mice. (FIG. 1A) Schematic representation of the AAV-CAG-moFGF21-doublemiRT vectors. The expression cassette contained the CAG promoter, a murine codon-optimized FGF21 coding sequence and four tandem repeats of the miRT122a sequence and four tandems repeats of the miRT1 sequence cloned in the 3 untranslated region of the expression cassette. ITRs from AAV2 flanked the expression cassette. The schematic representation is not to scale. CAG: chicken ?-actin promoter/CMV enhancer; pA: polyA. (FIG. 1B) Expression levels of FGF21 in metabolic tissues. The expression levels of the murine codon-optimized FGF21 coding sequence were measured by RTqPCR in eWAT, iWAT, iBAT and liver of C57Bl6 mice, and normalized with Rplp0 values (n=8-11 animals/group). (FIG. 1C) Circulating levels of FGF21 (n=8-11 animals/group). (FIGS. 1D-1E) Expression levels of FGF21R1 (FIG. 1D) and ?-Klotho (FIG. 1E) in metabolic tissues. The expression levels of the FGF21 receptor 1 (FGF21R1) and ?-Klotho were measured by RTqPCR in eWAT, iWAT, iBAT and liver of C57Bl6 mice, and normalized with Rplp0 values (n=7 animals/group). (FIG. 1F) Body weight evolution. Body weight was measured weekly (n=8-11 animals/group). (FIG. 1G) Representative image of animals. (FIG. 1H) Weight of tissues. Weight of eWAT, iWAT, rWAT, mWAT, iBAT and liver of chow- and HFD-fed C57Bl6 mice treated intra-eWAT with AAV vectors (n=8-11 animals/group). Analyses were performed 14 weeks after intra-eWAT administration of 10.sup.12 vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vectors. Results are expressed as the mean?SEM. ND, not detected. HFD, high fat diet. AU, arbitrary units. eWAT, epididymal white adipose tissue. iWAT, inguinal white adipose tissue. rWAT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue. iBAT interscapular brown adipose tissue. *p<0.05 vs AAV9-CAG-null chow, **p<0.01 vs AAV9-CAG-null chow, ***p<0.001 vs AAV9-CAG-null chow, $ p<0.05 vs AAV9-CAG-null HFD, $$ p<0.01 vs AAV9-CAG-null HFD, $$$ p<0.001 vs AAV9-CAG-null HFD.

[0222] FIGS. 2A-2C. Histological analysis of adipose tissue and liver of C57Bl6 mice treated intra-eWAT with AAV9-CAG-moFGF21-doublemiRT vectors. (FIG. 2A) Representative images of sections stained with hematoxylin and eosin of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (TWAT) interscapular brown adipose tissue (iBAT) and liver of chow- and HFD-fed C57Bl6 mice treated intra-eWAT with AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vectors. Original magnification ?100. (FIG. 2B) Mean area of white adipocytes of eWAT (n=4 animals/group). (FIG. 2C) Frequency distribution of area of white adipocytes of eWAT (n=4 animals/group). Analyses were performed 14 weeks after intra-eWAT administration of 10.sup.12 vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vectors. Results are expressed as the mean?SEM. HFD, high fat diet. **p<0.01 vs AAV9-CAG-null chow, ***p<0.001 vs AAV9-CAG-null chow, $$ p<0.01 vs AAV9-CAG-null HFD, $$$ p<0.001 vs AAV9-CAG-null HFD.

[0223] FIGS. 3A-3H. Increased energy expenditure and insulin sensitivity in C57Bl6 mice treated intra-eWAT with AAV9-CAG-moFGF21-doublemiRT vectors. (FIGS. 3A-3B) Expression levels of UCP1 (FIG. 3A) and Dio2 (FIG. 3B). The expression levels of UCP1 and Dio2 were measured by RTqPCR in iWAT and normalized with Rplp0 values (n=7 animals/group). (FIG. 3C) Energy metabolism. The energy expenditure (EE) was measured with indirect open circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were taken 9 weeks post-AAV administration during the light cycle (basal state) and dark cycle (activity phase) and adjusted for body weight (n=8-11 animals/group). (FIG. 3D) Liver triglyceride content (n=8-10 animals/group). (FIGS. 3E-3F) Serum triglyceride (FIG. 3E) and cholesterol (FIG. 3F) levels (n=8-11 animals/group). (FIG. 3G) Intraperitoneal insulin tolerance test. Mice were given an intraperitoneal injection of 0.75 U insulin/kg body weight and blood glucose levels were measured at the indicated time points (n=6-11 animals/group). The test was performed 11 weeks post-AAV administration. (FIG. 3H) Fasted insulin circulating levels. Unless otherwise indicated, analyses were performed 14 weeks after intra-eWAT administration of 10.sup.12 vg of AAV9-CAG-moFGF21-doublemiRT or AAV9-CAG-null vectors. Results are expressed as the mean?SEM. HFD, high fat diet. TG, triglycerides. Chol, cholesterol. *p<0.05 vs AAV9-CAG-null chow, **p<0.01 vs AAV9-CAG-null chow, ***p<0.001 vs AAV9-CAG-null chow, $ p<0.05 vs AAV9-CAG-null HFD, $$ p<0.01 vs AAV9-CAG-null HFD, $$$ p<0.001 vs AAV9-CAG-null HFD.

[0224] FIGS. 4A-4E. Reversion of obesity by intra-eWAT administration of AAV8-CAG-moFGF21-dmiRT vectors in ob/ob mice. (FIG. 4A) Expression levels of FGF21 in metabolic tissues. The expression levels of the murine codon-optimized FGF21 coding sequence were measured by RTqPCR in eWAT, iWAT, iBAT and liver of ob/ob mice, and normalized with Rplp0 values (FIG. 4B) Circulating levels of FGF21. (FIGS. 4C-4D) Body weight (FIG. 4C) and body weight gain (FIG. 4D) evolution. Body weight was measured weekly. (FIG. 4E) Weight of tissues. Weight of eWAT, iWAT, rWAT, mWAT, iBAT and liver of ob/ob mice treated intra-eWAT with AAV vectors. Analyses were performed 16 weeks after intra-eWAT administration of 10.sup.10 vg, 5?10.sup.10 vg, 2?10.sup.11 vg or 10.sup.12 vg of AAV8-CAG-moFGF21-doublemiRT or 10.sup.12 vg of AAV8-CAG-null vectors. Results are expressed as the mean?SEM. n=7-8 animals/group. ND, not detected. AU, arbitrary units. eWAT, epididymal white adipose tissue. iWAT, inguinal white adipose tissue. rWAT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue. iBAT interscapular brown adipose tissue. *p<0.05 vs AAV8-CAG-null, **p<0.01 vs AAV8-CAG-null, ***p<0.001 vs AAV8-CAG-null.

[0225] FIGS. 5A-5B. Improved insulin sensitivity in ob/ob mice treated intra-eWAT with AAV8-CAG-moFGF21-doublemiRT vectors. (FIG. 5A) Intraperitoneal insulin tolerance test. Ob/ob mice were given an intraperitoneal injection of 0.75 U insulin/kg body weight and blood glucose levels were measured at the indicated time points. The test was performed 9 weeks post-AAV administration. (FIG. 5B) Fasted insulin circulating levels 2 months post-AAV. Results are expressed as the mean?SEM, n=7-8 animals/group. *p<0.05 vs AAV8-CAG-null, **p<0.01 vs AAV8-CAG-null, ***p<0.001 vs AAV8-CAG-null.

[0226] FIGS. 6A-6I. Reversion of obesity and amelioration of glucose metabolism by intravenous administration of AAV8-hAAT-moFGF21-vectors in ob/ob mice. (FIG. 6A) Schematic representation of AAV-hAAT-moFGF21 vectors. The expression cassette contained the human al-antitrypsin (hAAT) promoter and a murine codon-optimized FGF21 coding sequence. ITRs from AAV2 flanked the expression cassette. The schematic representation is not to scale. pA: polyA. (FIG. 6B) Expression levels of FGF21. The expression levels of the murine codon-optimized FGF21 coding sequence were measured by RTqPCR in the liver of ob/ob mice, and normalized with Rplp0 values. (FIG. 6C) Circulating levels of FGF21. (FIGS. 6D-6E) Body weight (FIG. 6C) and body weight gain (FIG. 6D) evolution. Body weight was measured weekly. (FIG. 6F) Representative image of animals. (FIG. 6G) Weight of tissues. Weight of eWAT, iWAT, rWAT, mWAT, iBAT and liver of ob/ob mice treated intravenously with AAV vectors. (FIG. 6H) Intraperitoneal insulin tolerance test. Ob/ob mice were given an intraperitoneal injection of 0.75 U insulin/kg body weight and blood glucose levels were measured at the indicated time points. The test was performed 9 weeks post-AAV administration. (FIG. 6I) Fasted insulin circulating levels 3 months post-AAV. Unless otherwise indicated, analyses were performed 20 weeks after intravenous administration of 10.sup.11 vg or 5?10.sup.11 vg of AAV8-hAAT-moFGF21 or 5?10.sup.11 vg of AAV8-hAAT-null vectors. Results are expressed as the mean?SEM. n=9-10 animals/group. ND, not detected. AU, arbitrary units. eWAT, epididymal white adipose tissue. iWAT, inguinal white adipose tissue. rWAT, retroperitoneal white adipose tissue. mWAT, mesenteric white adipose tissue. iBAT interscapular brown adipose tissue. *p<0.05 vs AAV8-hAAT-null, **p<0.01 vs AAV8-hAAT-null, ***p<0.001 vs AAV8-hAAT-null.

[0227] FIGS. 7A-7C. Long-term reversion of obesity by intravenous administration of AAV-hAAT-moFGF21 vectors in HFD-fed C57b16 mice. (FIG. 7A) Circulating levels of FGF21. (FIGS. 7B-7C) Body weight (FIG. 7B) and body weight gain (FIG. 7C) evolution. Body weight was measured weekly. Analyses were performed 52 weeks after intravenous administration of 10.sup.10 vg or 5?10.sup.10 vg of AAV8-hAAT-moFGF21 or 5?10.sup.10 vg of AAV8-hAAT-null vectors. Results are expressed as the mean?SEM, n=9-12 animals/group. ***p<0.001 vs AAV8-hAAT-null chow, $$ p<0.01 vs AAV8-hAAT-null HFD, $$$ p<0.001 vs AAV8-hAAT-null HFD.

[0228] FIGS. 8A-8C. Long-term increased energy expenditure and insulin sensitivity by intravenous administration of AAV-hAAT-moFGF21 vectors in HFD-fed C57Bl6 mice. (FIG. 8A) Energy metabolism. The energy expenditure (EE) was measured with indirect open circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were taken 4 weeks post-AAV administration during the light cycle (basal state) and dark cycle (activity phase) and adjusted for body weight. (FIG. 8B) Intraperitoneal insulin tolerance test. C57Bl6 mice were given an intraperitoneal injection of 0.75 U insulin/kg body weight and blood glucose levels were measured at the indicated time points. The test was performed 7 weeks post-AAV administration. (FIG. 8C) Fasted and fed insulin circulating levels. Results are expressed as the mean?SEM, n=9-12 animals/group. HFD, high fat diet. *p<0.05 vs AAV8-hAAT-null chow, **p<0.01 vs AAV8-hAAT-null chow, ***p<0.001 vs AAV8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD, $$$ p<0.001 vs AAV8-hAAT-null HFD.

[0229] FIGS. 9A-9C. Reversion of obesity by intravenous administration of AAV-hAAT-moFGF21 vectors in old HFD-fed mice. (FIG. 9A) Circulating levels of FGF21. (FIGS. 9B-9C) Body weight (FIG. 9B) and body weight gain (FIG. 9C) evolution. Body weight was measured weekly. Analysis were performed 21 weeks after intravenous administration of 10.sup.10 vg, 2?10.sup.10 vg or 5?10.sup.10 vg of AAV8-hAAT-moFGF21 or 5?10.sup.10 vg of AAV8-hAAT-null vectors. Results are expressed as the mean?SEM, n=7-8 animals/group. HFD, high fat diet. ***p<0.05 vs AAV8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD. $$$ p<0.001 vs AAV8-hAAT-null HFD.

[0230] FIGS. 10A-10C. Increased energy expenditure and insulin sensitivity by intravenous administration of AAV-hAAT-moFGF21 vectors in old HFD-fed mice. (FIG. 10A) Energy metabolism. The energy expenditure (EE) was measured with indirect open circuit calorimeter. Oxygen consumption and carbon dioxide production were monitored simultaneously. Data were taken 6 weeks post-AAV administration during the light cycle (basal state) and dark cycle (activity phase) and adjusted for body weight. (FIG. 10B) Intraperitoneal insulin tolerance test. Old C57Bl6 mice were given an intraperitoneal injection of 0.75 U insulin/kg body weight and blood glucose levels were measured at the indicated time points. The test was performed 9 weeks post-AAV administration. (FIG. 10C) Fasted and fed insulin circulating levels. Results are expressed as the mean?SEM, n=7-8 animals/group. HFD, high fat diet. **p<0.01 vs AAV8-hAAT-null chow, ***p<0.001 vs AAV8-hAAT-null chow, $ p<0.05 vs AAV8-hAAT-null HFD, $$ p<0.01 vs AAV8-hAAT-null HFD, $$$ p<0.001 vs AAV8-hAAT-null HFD.

[0231] FIGS. 11A-11D. Body weight loss by intramuscular administration of AAV-CMV-moFGF21 vectors in C57Bl6 mice. (FIG. 11A) Schematic representation of the AAV-CMV-moFGF21 vectors. The expression cassette contained the cytomegalovirus (CMV) promoter and a murine codon-optimized FGF21 coding sequence. ITRs from AAV2 flanked the expression cassette. The schematic representation is not to scale. pA: polyA. (FIG. 11B) Circulating FGF21 levels. (FIGS. 11C-11D) Body weight (FIG. 11C) and body weight gain (FIG. 11D) evolution. Body weight was measured weekly. Results are expressed as the mean?SEM. n=6-7 animals/group. *p<0.05 vs AAV1-CMV-null, **p<0.01 vs AAV1-CMV-null. FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0232] FIG. 12. Increased FGF21 protein production by codon-optimization of nucleotide sequences encoding human FGF21. (A) Levels of hFGF21 protein in the culture media of BEK293 cells transfected with wild-type hFGF21 or three different versions of codon-optimized human FGF21 sequences. Results are expressed as the mean?SEM. n=3 wells/group. ND, non detected. *p<0.05 vs Non-transfected cells.

[0233] FIGS. 13A-13D. Intra-eWAT administration of AAV8-CAG-moFGF21-dmiRT vectors in ob/ob mice.

[0234] FIGS. 13A, 13B Representative images of the hematoxylin-eosin staining of (FIG. 13A) eWAT and (FIG. 13B) liver tissue sections obtained from ob/ob animals injected intra-eWAT either null or FGF21-encoding AAV8 vectors at all doses tested. Scale bars: 100 ?m for eWAT and 200 ?m for liver.

[0235] FIG. 13C Glycemia in the fed state.

[0236] FIG. 13D Insulinemia in the fed state 3 months post-AAV.

[0237] FGF21 labels in the figure refer to moFGF21.

[0238] Data information: All values are expressed as mean?SEM. In (FIGS. 13A, 13B) n=6-9 animals/group. In (FIGS. 13C-13D) n=4-8 animals/group. In (I) n=6-8 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the Null-injected group.

[0239] FIGS. 14A-14F. Impact of FGF21 gene transfer to the eWAT of ob/ob mice.

[0240] FIG. 14A Serum adiponectin levels in 25-week-old ob/ob animals injected intra-eWAT at 11 weeks of age with either AAV8-CAG-null vectors or AAV8-CAG-moFGF21-dmiRT vectors at 4 different doses (1?10.sup.10, 5?10.sup.10, 2?10.sup.11, 1?10.sup.12 vg/mouse).

[0241] FIG. 14B Quantification by qRT-PCR of the expression of the macrophage marker F4/80 the same groups of animals as in (FIG. 14A).

[0242] FIG. 14C Representative images of the immunostaining of eWAT sections from ob/ob mice that received AAV8-CAG-moFGF21-dmiRT vectors for the macrophage-specific marker Mac2. n=4-8/group. Scale bars: 200 ?m.

[0243] FIG. 14D Weight of the liver in all intra-eWAT treatment groups.

[0244] FIGS. 14E, 14F Hepatic triglyceride and cholesterol content in the fed stated in the same cohorts as in (FIG. 14A)

[0245] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend. Data information: All values are expressed as mean?SEM. In (FIGS. 14A, 14B, 14D) n=4-8 animals/group. *P<0.05, **<001 and ***P<0.001 versus null-injected ob/ob group.

[0246] FIGS. 15A-15E. Reduced obesity and improved insulin sensitivity in ob/ob mice treated with AAV8-hAAT-moFGF21 vectors.

[0247] FIG. 15A Representative images of the hematoxylin-eosin staining of eWAT tissue sections obtained from ob/ob animals injected with either null or FGF21-encoding AAV vectors at 1?10.sup.11 or 5?10.sup.11 vg/mouse.

[0248] FIG. 15B Serum adiponectin levels in all groups.

[0249] FIG. 15C Representative images of the hematoxylin-eosin staining of liver tissue sections obtained from ob/ob animals injected with either null or FGF21-encoding AAV vectors at 1?10.sup.11 or 5?10.sup.11 vg/mouse.

[0250] FIG. 15D Fed blood glucose levels.

[0251] FIG. 15E Fed serum Insulin levels 5 months post-AAV.

[0252] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend. Data information: All data represent the mean?SEM. In (FIGS. 15A-15C, 15E) n=9-10 animals/group. *P<0.05, **P<001 and ***P<0.001 versus null-injected ob/ob group.

[0253] FIGS. 16A-16G. Effects of FGF21 liver gene transfer on ob/ob mice.

[0254] FIG. 16A Immunohistochemistry for the macrophage-specific marker Mac2 in eWAT sections from ob/ob mice that received AAV8-hAAT-moFGF21 vectors. Scale bars: 500 ?m.

[0255] FIGS. 16B, 16C Quantification by qRT-PCR of the expression of the markers of inflammation F4/80 (FIG. 16B) and TNF-? (FIG. 16C) in the same cohorts of mice.

[0256] FIGS. 16D, 16E Weight (FIG. 16D) and representative images of the liver (FIG. 16E) obtained from animals belonging to the same experimental groups as in (FIG. 16A).

[0257] FIGS. 16F, 16G Hepatic triglyceride and cholesterol content in the fed state in the same cohorts as in (FIG. 16A)

[0258] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0259] Data information: All values are expressed as mean?SEM. In (FIGS. 16B, 16D-16F) n=9-10 animals/group. *P<0.05, **<001 and ***P<0.001 versus null-injected ob/ob group.

[0260] FIGS. 17A-17G. AAV8-hAAT-moFGF21 treatment increases the expression of genes involved in glucose uptake and thermogenesis in adipose tissue of ob/ob mice.

[0261] FIGS. 17A, 17B Quantification by qRT-PCR of liver PEPCK and G6Pase expression in ob/ob mice injected at 2 months of age with either AAV8-hAAT-null vectors or AAV8-hAAT-moFGF21 vectors.

[0262] FIGS. 17C-17F Quantification by qRT-PCR of GLUT1 (FIG. 17C), GLUT4 (FIG. 17D), HKI (FIG. 17E) and HKII (FIG. 17F) expression in eWAT, TWAT and iBAT in the same animals as in (FIG. 17A)

[0263] FIG. 17G Relative expression of UCP1 in iBAT in the same cohorts as in (FIG. 17A).

[0264] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0265] Data information: All values are expressed as mean?SEM. In (FIGS. 17A-17G) n=9-10 animals/group. *P<0.05, **P<001 and ***P<0.001 versus null-injected ob/ob group.

[0266] FIGS. 18A-18B. AAV8-mediated liver gene transfer of FGF21 counteracts HFD-induced obesity.

[0267] FIG. 18A Weight of the epididymal (eWAT), inguinal (iWAT) and retroperitoneal (rWAT) white adipose tissue depots, the liver and the quadriceps obtained from mice treated with AAV8-hAAT-moFGF21 vectors as young adults (top panel) or as adults (bottom panel).

[0268] FIG. 18B Circulating levels of FGF21 at different time-points after vector administration.

[0269] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0270] Data information: All values are expressed as mean?SEM. In (FIGS. 18A-18B) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the chow-fed Null-injected group. .sup.#P<0.05, .sup.##P<0.01 and .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0271] FIGS. 19A-19E. FGF21 gene transfer to the liver counteracts HFD-induced obesity.

[0272] FIGS. 19A, 19B Representative images of animals belonging to all experimental groups of the studies performed in young adults (FIG. 19A) or in adults (FIG. 19B).

[0273] FIG. 19C Representative images of the epididymal white adipose (eWAT) pad obtained at sacrifice from animals treated with several doses of AAV8-hAAT-moFGF21 as young adults (left) or adults (right).

[0274] FIG. 19D Representative images of the liver obtained from animals treated as young adults (left) or adults (right).

[0275] FIG. 19E AAV-derived FGF21 expression in the liver of animals treated as young adults or adults. The qPCR was performed with primers that specifically detected the codon-optimized murine FGF21 (coFGF21) coding sequence.

[0276] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0277] Data information: All values are expressed as mean?SEM. In (FIG. 19E) n=7-10 animals/group. HFD, High-fat diet. ND, non-detected.

[0278] FIGS. 20A-20E. AAV8-hAAT-moFGF21-mediated increased energy expenditure and decreased fat accumulation in iBAT and TWAT.

[0279] FIG. 20A Assessment of the locomotor activity through the Open field test in animals that had been subjected to HFD-feeding since ?2 months of age and were treated with either null or FGF21-encoding vectors 2 months later (young adults).

[0280] FIG. 20B Hematoxylin-eosin staining of iBAT tissue sections obtained from animals treated as young adults (left) or adults (right).

[0281] FIG. 20C Western-blot analysis of UCP1 content in iBAT from the same cohort of animals as in (FIG. 20A). A representative immunoblot is shown (left). The histogram depicts the densitometric analysis of two different immunoblots (right).

[0282] FIG. 20D Hematoxylin-eosin staining of TWAT tissue sections obtained from animals treated as young adults (left) or adults (right).

[0283] FIG. 20E Quantification by qRT-PCR of the expression of Phosphol in TWAT in the groups of animals that initiated the HFD feeding and received FGF21 vectors as young adults or adults.

[0284] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0285] Data information: All values are expressed as mean?SEM. In (FIGS. 20A-20C) n=7-10 animals/group. In (FIG. 20E) n=4 animals/group. In (G) n=7-10 animals/group. *P<0.05, **P<001 and ***P<0.001 versus the chow-fed Null-injected group. .sup.#P<0.05 and .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0286] FIGS. 21A-21C. Energy expenditure 10 months after gene transfer to the liver.

[0287] FIG. 21A Energy expenditure was measured 10 months after AAV8-hAAT-null or AAV8-hAAT-moFGF21 vector delivery in the cohort of animals that initiated HFD-feeding at 2 months of age. Data were taken during the light and dark cycles.

[0288] FIG. 21B Western-blot analysis of UCP1 content in TWAT from the same cohort of animals. A representative immunoblot is shown (left). The graph shows the densitometric analysis of two different immunoblots (right).

[0289] FIG. 21C Relative expression of Serca2b and RyR2 in the TWAT of the groups of animals that initiated the HFD feeding and received FGF21 vectors as young adults or adults

[0290] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0291] Data information: All values are expressed as mean?SEM. In (FIG. 21A) n=7-10 animals/group. In (FIG. 21B) n=4 animals/group. In (FIG. 21C) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the chow-fed Null-injected group. .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0292] FIGS. 22A-22D. AAV8-hAAT-moFGF21-mediated reversal of islet hyperplasia.

[0293] FIG. 22A Fasted glucagon levels in the group of animals that initiated the HFD feeding and received FGF21 vectors as young adults.

[0294] FIG. 22B ?-cell mass in the group of animals that initiated the HFD feeding and received FGF21 vectors as adults.

[0295] FIG. 22C Representative images of the immunostaining against insulin in pancreas sections from animals that received 5?10.sup.10 vg/mouse of AAV8-hAAT-moFGF21 as adults. Scale bars: 400 ?m. Inset scale bars: 100 ?m.

[0296] FIG. 22D Representative images of the double immunostaining against insulin (dark grey) and glucagon (light grey) in pancreas sections from animals that received 5?10.sup.10 vg/mouse of AAV8-hAAT-moFGF21 as young adults (upper panel) or adults (lower panel). Scale bars: 100 ?m.

[0297] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0298] Data information: All values are expressed as mean?SEM. In (FIGS. 22A-22C) n=7-10 animals/group. In (FIG. 22D) n=4-5 animals/group. *P<0.05, **P<001 and ***P<0.001 versus the chow-fed Null-injected group. .sup.#P<0.05, .sup.##P<0.01 and .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0299] FIGS. 23A-23B. Treatment with AAV8-hAAT-moFGF21 improves glucose tolerance.

[0300] FIG. 23A Glucose tolerance was studied in the group of mice that initiated the HFD feeding and received FGF21 vectors as young adults after an intraperitoneal injection of glucose (2 g/kg body weight).

[0301] FIG. 23B Serum insulin levels during the glucose tolerance test shown in (FIG. 23A).

[0302] Data information: All data represent the mean mean?SEM. In (FIGS. 23A-23B) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the chow-fed Null-injected group. .sup.#P<0.05, .sup.##P<0.01 and .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0303] FIGS. 24A-24H. Reversal of WAT hypertrophy and inflammation by AAV8-hAAT-moFGF21 treatment.

[0304] FIG. 24A Representative images of the hematoxylin-eosin staining of the eWAT from animals fed a chow or a HFD and administered with either AAV8-hAAT-null or 5?10.sup.10 vg/mouse AAV8-hAAT-moFGF21 vectors as young adults (left panels) or adults (right panels). While HFD-fed, null-injected mice had larger adipocytes, HFD-fed, FGF21-treated animals had adipocytes of reduced size. Scale bars: 100 ?m.

[0305] FIG. 24B Morphometric analysis of the area of WAT adipocytes in animals treated as young adults or as adults.

[0306] FIGS. 24C, 24D Circulating levels of adiponectin (FIG. 24C) and leptin (FIG. 24D).

[0307] FIG. 24E Immunohistochemistry for the macrophage-specific marker Mac2 in eWAT sections from animals that received 5?10.sup.10 vg/mouse AAV8-hAAT-moFGF21 as adults. The micrographs illustrate the presence of crown-like structures (arrows and inset) in the eWAT of HFD-fed, null-injected animals but no in the eWAT of HFD-fed, FGF21-treated mice. Scale bars: 200 ?m and 50 ?m (inset).

[0308] FIGS. 24F-24H Quantification by qRT-PCR of the expression of the markers of inflammation F4/80 (FIG. 24F), IL 1-? (FIG. 24G) and TNF-? (FIG. 24H) in the group of animals that initiated the HFD feeding and received FGF21 vectors as adults.

[0309] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0310] Data information: All values are expressed as mean?SEM. In (FIG. 24B) n=4 animals/group. In (FIGS. 24F-24H) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the chow-fed Null-injected group. .sup.#P<0.05, .sup.##P<0.01 and .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0311] FIGS. 25A-25E. Adipocyte size and inflammation in AAV8-hAAT-moFGF21-treated animals.

[0312] FIG. 25A Frequency distribution of adipocyte area in the groups of animals that initiated the chow or HFD feeding and received either AAV8-hAAT-null or 5?10.sup.10 vg/mouse AAV8-hAAT-moFGF21 vectors as young adults (top graph) or adults (bottom graph).

[0313] FIG. 25B Mac2 immunohistochemistry in eWAT of animals in which the study was initiated as young adults. The crown-like structures formed by infiltrating macrophages in the eWAT of HFD-fed, null-injected mice are indicated by arrows. Scale bars: 200 ?m and 50 ?m (inset).

[0314] FIGS. 25C-25E Relative expression by qRT-PCR of the markers of inflammation F4/80, CD68 and TNF-? in the same cohort of animals as in (FIG. 25B).

[0315] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0316] Data information: All values are expressed as mean?SEM. In (FIG. 25A) n=4 animals/group. In (FIGS. 25C-25E) n=7-10 animals/group. ***P<0.001 versus the chow-fed Null-injected group. .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0317] FIGS. 26A-26D. Treatment with FGF21-encoding vectors reverses hepatic steatosis and inflammation.

[0318] FIG. 26A Representative images of the hematoxylin-eosin staining of liver sections obtained from animals fed a chow or a HFD and administered with either AAV8-hAAT-null or 5?10.sup.10 vg/mouse AAV8-hAAT-moFGF21 vectors. HFD clearly induced the deposition of lipid droplets in the liver, and this was reverted by AAV8-hAAT-moFGF21 treatment both in young adults and in adults. Scale bars: 100 ?m.

[0319] FIGS. 26B, 26C Fed hepatic triglyceride and cholesterol content in the same cohorts of animals.

[0320] FIG. 26D Immunostaining for the macrophage-specific marker Mac-2 of liver sections from animals fed a HFD that received either AAV8-hAAT-null or 5?10.sup.10 vg/mouse AAV8-hAAT-moFGF21 vectors. Arrows indicate the presence of crown-like structures. Scale bars: 200 ?m and 50 ?m (inset).

[0321] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0322] Data information: All values are expressed as mean?SEM. In (FIGS. 26B-26C) n=7-10 animals/group. **P<001 and ***P<0.001 versus the chow-fed Null-injected group. .sup.##P<0.01 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0323] FIG. 27. AAV8-hAAT-moFGF21-mediated amelioration of liver fibrosis.

[0324] Analysis of hepatic fibrosis through Masson's trichrome staining in animals fed a HFD that received 5?10.sup.10 vg/mouse of either AAV8-hAAT-null or AAV8-hAAT-moFGF21 vectors. AAV8-hAAT-moFGF21 treatment (right panels) markedly decreased the detection of collagen fibers that were readily detectable (in blue) in animals treated with the null vector (left panels). Scale bars: 50 ?m. FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0325] FIGS. 28A-28C. AAV8-hAAT-moFGF21 treatment improves liver fibrosis.

[0326] FIG. 28A Analysis of hepatic fibrosis through PicroSirius staining in animals fed a HFD that received 5?10.sup.10 vg/mouse of either AAV8-hAAT-null or AAV8-hAAT-moFGF21 vectors. AAV8-hAAT-moFGF21 treatment (right panels) markedly decreased the detection of collagen fibers that were readily detectable (in black) in animals treated with the null vector (left panels). Scale bars: 50 ?m.

[0327] FIGS. 28B, 28C Quantification by qRT-PCR of the expression of collagen 1 in the liver in the group of animals that initiated the HFD feeding and received FGF21 vectors as young adults (FIG. 28B) or adults (FIG. 28C).

[0328] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0329] Data information: All values are expressed as mean?SEM. In (FIGS. 28B-28C) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the chow-fed Null-injected group. .sup.#P<0.05 and .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.

[0330] FIGS. 29A-29Q. No bone abnormalities were observed in AAV8-hAAT-moFGF21-treated animals. The long-term effects of FGF21 gene transfer on bones were studied by comparison of HFD-fed mice treated with the highest dose (5?10.sup.10 vg/mouse) of AAV8-hAAT-moFGF21 vectors as young adults or adults with null-injected, chow or HFD-fed animals.

[0331] FIG. 29A Total naso-anal length.

[0332] FIG. 29B Tibial length.

[0333] FIGS. 29C-29O Micro-computed tomography (?CT) analysis of the epiphysis (FIGS. 29C-29J) and the diaphysis (FIGS. 29K-29O) of tibiae obtained at the time of sacrifice, i.e. when animals were 18 months of age, from HFD-fed mice administered with either null or FGF21-encoding AAV vectors.

[0334] FIGS. 29P, 29Q Circulating IGFBP1 (FIG. 29P) and IGF1 (FIG. 29Q) levels measured by ELISA.

[0335] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0336] Data information: All data represent the mean?SEM. In (FIGS. 29A, 29P-29Q) n=7-10 animals/group. In (FIGS. 29B-29O) n=4 animals/group. **P<0.01 and ***P<0.001 versus the chow-fed Null-injected group. HFD, High-fat diet; BMD, bone mineral density; BMC, bone mineral content; BV, bone volume; BV/TV, bone volume/tissue volume ratio; BS/BV, bone surface/bone volume ratio; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation.

[0337] FIG. 30. Analysis of glycaemic profiles in C57Bl6 mice treated with AAV8-hAAT-moFGF21 vectors. Blood glucose levels were evaluated under fed conditions. AAV, IV administration of 5?10.sup.10 vg or 2?10.sup.11 vg of AAV8-hAAT-moFGF21 (n=13 and 15, respectively) or 2?10.sup.11 vg of AAV8-null vectors (n=15). STZ, treatment with streptozotocin (5?50 mg/kg). Results shown are means+SEM. *p<0.05; ***p<0.001 vs AAV8-hAAT-Null. FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0338] FIGS. 31A-31H. Gene transfer of FGF21 to the skeletal muscle of healthy animals.

[0339] FIG. 31A Circulating levels of FGF21 measured 40 weeks after injection of 3?10.sup.11 vg/mouse of either AAV1-CMV-Null or AAV1-CMV-moFGF21 vectors to the skeletal muscle of healthy animals fed a chow diet.

[0340] FIG. 31B AAV-derived FGF21 expression in the muscles and liver of healthy animals injected intramuscularly with AAV1-CMV-Null or AAV1-CMV-moFGF21 vectors.

[0341] FIG. 31C Evolution of the body weight in the 40-week follow-up period.

[0342] FIG. 31D Wet tissue weight of different muscles, adipose pads and liver.

[0343] FIGS. 31E, 31F Hepatic triglyceride and cholesterol content in the fed state.

[0344] FIG. 31G Fed serum insulin levels.

[0345] FIG. 31H Insulin sensitivity assessed through intraperitoneal injection of insulin (0.75 units/kg body weight) and represented as percentage of initial blood glucose.

[0346] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0347] Data information: All values are expressed as mean?SEM. In (FIGS. 31A-31H) n=5-7 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the Null-injected group.

[0348] FIGS. 32A-32F. AAV1-mediated skeletal muscle gene transfer of FGF21 counteracts HFD-induced obesity and insulin resistance.

[0349] FIGS. 32A, 32B Evolution of body weight (FIG. 32A) and body weight gain (FIG. 32B) in animals treated with AAV1-CMV-moFGF21. C57Bl6 mice were fed a HFD for ?12 weeks and then administered with 3?10.sup.11 vg/mouse of AAV1-CMV-moFGF21 vectors. Control obese mice and control chow-fed mice received 3?10.sup.11 vg of AAV1-CMV-null.

[0350] FIG. 32C Circulating levels of FGF21 at different time-points after vector administration.

[0351] FIGS. 32D, 32E Fasted blood glucose (FIG. 32D) and fed serum insulin (FIG. 32E) levels in the same groups of animals as in (FIGS. 32A, 32B).

[0352] FIG. 32F Insulin sensitivity was determined in all experimental groups after an intraperitoneal injection of insulin (0.75 units/kg body weight). Results were calculated as the percentage of initial blood glucose levels.

[0353] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.

[0354] Data information: All values are expressed as mean?SEM. In (FIGS. 32A-32F) HFD-fed mice n=10 animals/group; chow-fed mice n=5 animals/group. ***P<0.001 versus the HFD-fed null-injected group.

[0355] FIG. 33. In vivo increased FGF21 circulating levels by codon-optimization of nucleotide sequences encoding human FGF21. Circulating levels of hFGF21 in C57Bl6 mice administered hydrodynamically with plasmids encoding wild-type hFGF21 or three different variants of codon-optimized human FGF21 sequences. Results are expressed as the mean?SEM. n=9-10 mice/group. ND, non detected. Negative control, untreated mice. *p<0.05 vs Non-treated mice.

[0356] FIGS. 34A-34E. In vitro increased FGF21 expression levels by hAAT-moFGF21, CAG-moFGF21-doublemiRT and CMV-moFGF21 expression cassettes. (FIG. 34A) Expression levels of FGF21 in HEK293 cells transfected with plasmids encoding the WT murine FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21) or of the CAG promoter in conjunction with four tandem repeats of the miRT122a sequence and four tandems repeats of the miRT1 sequence (CAG-moFGF21-doublemiRT). (FIGS. 34B and 34C) Intracellular FGF21 protein content (FIG. 34B) and FGF21 protein levels in the culture medium (FIG. 34C) in the same cells as in (FIG. 34A). (FIG. 34D) Expression levels of FGF21 in C2C12 cells transfected with plasmids encoding the WT murine FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21). (FIG. 34E) Expression levels of FGF21 in HepG2 cells transfected with plasmids encoding the WT murine FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) or a codon-optimized murine FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF21). The qPCR was performed with primers that detected both the wt and the codon-optimized FGF21 coding sequences. Results are expressed as the mean?SEM. n=3 wells/group. ND, non detected. *p<0.05 vs control. ###p<0.001 vs EF1a-mFGF21.

[0357] FIGS. 35A-35B. In vivo increased hepatic FGF21 expression and FGF21 circulating levels by hAAT-moFGF21 and CMV-moFGF21 expression cassettes. (FIG. 35A) Expression levels of FGF21 in the liver of C57Bl6 mice hydrodynamically administered with plasmids encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (EF1a-mFGF21) or a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21) or the hAAT promoter (hAAT-moFGF21). The qPCR was performed with primers that detected both the wt and the codon-optimized FGF21 coding sequences. (FIG. 35B) FGF21 circulating levels in the same cohorts as in (FIG. 35A). Results are expressed as the mean?SEM. n=5 mice/group. **p<0.01 vs. EF1a-mFGF21

[0358] FIGS. 36A-36B. In vivo increased hepatic FGF21 expression and FGF21 circulating levels by AAV8-hAAT-moFGF21. (FIG. 36A) Expression levels of FGF21 in the liver of C57Bl6 mice intravenously administered with 1?10.sup.10 vg, 2?10.sup.10 vg or 5?10.sup.10 vg of AAV8 vectors encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or a codon-optimized murine FGF21 coding sequence under the control of the hAAT promoter (AAV8-hAAT-moFGF21). The qPCR was performed with primers that detected both the wt and the codon-optimized FGF21 coding sequences. (FIG. 36B) FGF21 circulating levels in the same cohorts as in (FIG. 36A). Analyses were performed two weeks post-AAV. Results are expressed as the mean?SEM. n=4-5 mice/group. Control, untreated mice. **p<0.01 and ***p<0.001 vs. control. ##p<0.01 and ###p<0.001 vs. AAV8-EF1a-mFGF21

[0359] FIGS. 37A-37B. In vivo increased adipose FGF21 expression by AAV8-CAG-moFGF21-dmiRT. (FIGS. 37A-37B) Expression levels of FGF21 in the eWAT (FIG. 37A) or the liver (FIG. 37B) of C57Bl6 mice administered intra-eWAT with 2?10.sup.10 vg, 5?10.sup.0 vg or 1?10.sup.11 vg of either AAV8 vectors encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or AAV8 vectors encoding a codon-optimized murine FGF21 coding sequence under the control of the CAG promoter in conjunction with four tandem repeats of the miRT122a sequence and four tandems repeats of the miRT1 sequence (AAV8-CAG-moFGF21-doublemiRT). The qPCR was performed with primers that detected both the wt and the codon-optimized FGF21 coding sequences. Analyses were performed two weeks post-AAV. Results are expressed as the mean?SEM. n=4-5 mice/group. Control, untreated mice. eWAT, epidydimal white adipose tissue. *p<0.05, **p<0.01 and

[0360] FIGS. 38A-38B. In vivo increased FGF21 expression in the skeletal muscle by AAV1-CMV-moFGF21. (FIGS. 38A-38B) Expression levels of FGF21 in the quadriceps (FIG. 38A) or the liver (FIG. 38B) of C57Bl6 mice administered intramuscularly with 5?10.sup.10 vg, 1?10.sup.11 vg or 3?10.sup.11 vg of either AAV8 vectors encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or AAV1 vectors encoding a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (AAV1-CMV-FGF21). The qPCR was performed with primers that detected both the wt and the codon-optimized FGF21 coding sequences. Analyses were performed two weeks post-AAV. Results are expressed as the mean?SEM. n=4-5 mice/group. Control, untreated mice. *p<0.05, **p<0.01 and ***p<0.001 vs. control. #p<0.05, ##p<0.01 and ###p<0.001 vs. AAV8-EF1a-mFGF21.

EXAMPLES

General Procedures to the Examples

Subject Characteristics

[0361] Male C57Bl/6J mice and B6.V-Lep.sup.ob/OlaHsd (ob/ob) mice were used. Mice were fed ad libitum with a standard diet (2018S Teklad Global Diets?, Harlan Labs., Inc., Madison, WI, US) or a high fat diet (TD.88137 Harlan Teklad Madison, WI, US) and kept under a light-dark cycle of 12 h (lights on at 8:00 a.m.) and stable temperature (22? C.?2). For tissue sampling, mice were anesthetized by means of inhalational anesthetic isoflurane (IsoFlo?, Abbott Laboratories, Abbott Park, IL, US) and decapitated. Tissues of interest were excised and kept at ?80? C. or with formalin until analysis. All experimental procedures were approved by the Ethics Committee for Animal and Human Experimentation of the Universitat Aut?noma de Barcelona.

Recombinant AAV Vectors

[0362] Single-stranded AAV vectors of serotype 1, 8 or 9 were produced by triple transfection of HEK293 cells according to standard methods (Ayuso, E. et al, 2010. Curr Gene Ther. 10(6):423-36). Cells were cultured in 10 roller bottles (850 cm.sup.2, flat; Corning?, Sigma-Aldrich Co., Saint Louis, MO, US) in DMEM 10% FBS to 80% confluence and co-transfected by calcium phosphate method with a plasmid carrying the expression cassette flanked by the AAV2 ITRs, a helper plasmid carrying the AAV2 rep gene and the AAV of serotypes 1, 8 or 9 cap gene, and a plasmid carrying the adenovirus helper functions. Transgenes used were: murine, canine or human codon-optimized or wt FGF21 coding-sequence driven by 1) the cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter with the addition of four tandem repeats of the miRT122a sequence (5CAAACACCATTGTCACACTCCA3) (SEQ ID NO:12) and four tandems repeats of the miRT1 sequence (5TTACATACTTCTTTACATTCCA3) (SEQ ID NO:13) cloned in the 3 untranslated region of the expression cassette; 2) the CMV promoter; or 3) the human al-antitrypsin promoter (hAAT). Noncoding plasmids carrying the CAG, hAAT or CMV promoters were used to produce null vectors. AAV were purified with an optimized method based on a polyethylene glycol precipitation step and two consecutive cesium chloride (CsCl) gradients. This second-generation CsCl-based protocol reduced empty AAV capsids and DNA and protein impurities dramatically (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. Titers of viral genomes were determined by quantitative PCR following the protocol described for the AAV2 reference standard material using linearized plasmid DNA as standard curve (Lock M, et al., Hum. Gene Ther. 2010; 21:1273-1285). The vectors were constructed according to molecular biology techniques well known in the art.

In Vivo Intra-eWAT Administration of AAV Vectors

[0363] Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). A laparotomy was performed in order to expose the epididymal white adipose tissue. AAV vectors were resuspended in PBS with 0.001% Pluronic? F68 (Gibco) and injected directly into the epididymal fat pad. Each epididymal fat pad was injected twice with 50 ?L of the AAV solution (one injection close to the testicle and the other one in the middle of the fat pad). The abdomen was rinsed with sterile saline solution and closed with a two-layer approach.

Systemic Administration of AAV Vectors

[0364] The appropriate amount of the AAV solution was diluted in 200 ?L of PBS with 0.001% Pluronic? and was manually injected into the lateral tail vein without exerting pressure at the moment of delivery. Before the injection, the animals were put under a 250 W infrared heat lamp (Philips NV, Amsterdam, NL) for a few minutes to dilate the blood vessels and facilitate viewing and easier access to the tail vein. A plastic restrainer (Harvard Apparatus, Holliston, MA, US) was used to secure the animal for injection. No anesthesia was used since an appropriate restraining device was employed. A 30-gauge needle was utilized to inject the animals.

Intramuscular Administration of AAV Vectors

[0365] Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Hind limbs were shaved and vectors were administered by intramuscular injection in a total volume of 180 ?l divided into six injection sites distributed in the quadriceps, gastrocnemius, and tibialis cranealis of each hind limb.

Immunohistochemical and Morphometric Analysis

[0366] Tissues were fixed for 24 h in formalin (Panreac Quimica), embedded in paraffin, and sectioned. Tissue samples were stained with hematoxylin-eosin. Adipocyte area was determined in 12 hematoxylin/eosin WAT images per animal taken at 10? with the Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) connected to a videocamera with a monitor with an image analysis software (analySlS 3.0; Soft Imaging System, Center Valley, PA, EEUU) and each adipocyte area was quantified in ?m.sup.2. Mean adipocyte area was calculated for each experimental group and distribution of adipocytes according to size categories was represented in a histogram. Four animals per group were used and at least 250 adipocytes per animal were analyzed.

Immunohistochemistry

[0367] Tissues were fixed for 12-24 h in 10% formalin, embedded in paraffin and sectioned. Sections were incubated overnight at 4? C. with rat anti-Mac2 (1:50; CL8942AP; Cedarlane), guinea pig anti-insulin (1:100; I-8510; Sigma-Aldrich) or rabbit anti-glucagon (1:100; 219-01; Signet Labs). Biotinylated rabbit anti-rat (1:300; E0467; Dako), goat anti-rabbit IgG (Alexa Fluor 568-conjugated) (1:200; A11011; ThermoFisher), goat anti-guinea pig IgG (Alexa Fluor 488-conjugated) (1:300; A11073; ThermoFisher) or rabbit anti-guinea pig coupled to peroxidase (1:300; P0141; Dako) were used as secondary antibodies. The ABC peroxidase kit (Pierce) was used for immunodetection, and sections were counterstained in Mayer's hematoxylin. Hoechst (B2261; Sigma-Aldrich) was used for nuclear counterstaining of fluorescent specimens. PicroSirius Red staining and Masson's trichrome staining were used to evaluate fibrosis. The percentage of ?-cell area in the pancreas was analyzed in two insulin-stained sections 200 ?m apart, by dividing the area of all insulin+cells in one section by the total pancreas area of that section. ?-cell mass was calculated by multiplying pancreas weight by percentage of ?-cell area, as previously described (Jimenez et al, 2011).

RNA Analysis

[0368] Total RNA was obtained from adipose depots or liver by using QIAzol Lysis Reagent (Qiagen NV, Venlo, NL) or Tripure isolation reagent (Roche Diagnostics Corp., Indianapolis, IN, US), respectively, and RNeasy Lipid Tissue Minikit (Qiagen NV, Venlo, NL). In order to eliminate the residual viral genomes, total RNA was treated with DNAseI (Qiagen NV, Venlo, NL). For RT-PCR, 1 ?g of RNA samples was reverse-transcribed using Transcriptor First Strand cDNA Synthesis Kit (04379012001, Roche, California, USA). Real-time quantitative PCR was performed in a SmartCyclerII? (Cepheid, Sunnyvale, USA) using EXPRESS SYBRGreen qPCR supermix (Invitrogen?, Life Technologies Corp., Carslbad, CA, US). Data was normalized with Rplp0 values and analyzed as previously described (Pfaffl, M., Nucleic Acids Res. 2001; 29(9):e45).

Hormone and Metabolite Assays

[0369] Blood glucose levels were measured with a Glucometer Elite? analyzer (Bayer, Leverkusen, Germany). Circulating levels of FGF21 were determined by quantitative sandwich enzyme immunoassay Mouse/Rat FGF-21 ELISA kit (MF2100, R&Dsystems, Abingdon, UK). Serum insulin concentrations were determined by Rat Insulin ELISA sandwich assay (90010, Crystal Chem INC. Downers Grove, IL 60515, USA). To extract lipids from tissue, frozen samples of approximately 100 mg were weighted and homogenized in 15 ml chloroform:methanol (2:1). Lipid and aqeuous phases were then separated by adding 3 ml of H.sub.2SO.sub.4 0.05% and keeping them overnight at 4? C. Once the phases were separated, the aqueous superior phase was eliminated using a Pasteur pipet and 1 ml of the inferior lipid phase was recuperated in a glass tube. 1 ml of a chloroform and Triton X-100 at 1% solution was added to the glass tube and it was incubated at 90? C. in a bath, to evaporate the chloroform. By the use of the chloroform and Triton X-100 mix, any remaining aqeuous particle was eliminated from the lipid phase. After the evaporation, chloroform was rinsed to the walls of the tube to concentrate the sample and, it was warmed again at 90? C. to evaporate the chloroform. Once the sediment was completely dry and concentrated, it was resuspended by the addition of 500 ?l of H20 miliQ at 37? C. The amount of triglycerides was finally determined using the commercial product GPO-PAP (Roche Diagnostics, Basel, Switzerland). Serum triglycerides and cholesterol were quantified spectrophotometrically using an enzymatic assay kit (Horiba-ABX, Montpellier, France). All biochemical parameters were determined using Pentra 400 Analyzer (Horiba-ABX).

[0370] Glycemia was determined using a Glucometer Elite? (Bayer). Glucagon levels were measured using a glucagon Radioimmunoassay (#GL-32K, EMD Millipore). Adiponectin, leptin, IGFBP1 and IGF1 were determined using the Mouse Adiponectin ELISA kit (80569, Crystal Chem), the Mouse Leptin ELISA kit (90030, Crystal Chem), the IGFBP1 (Mouse) ELISA kit (KA3054, Abnova) and the m/r IGF-I-ELISA kit (E25, Mediagnost), respectively.

Insulin Tolerance Tests

[0371] For insulin tolerance tests, insulin (0.75 IU/kg body wt; Humulin Regular; Eli Lilly, Indianapolis, IN) was injected intraperitoneally into awake fed mice. Glucose concentration was determined in blood samples obtained from the tail vein at the indicated time points after the insulin injection.

Glucose Tolerance Test

[0372] Awake mice were fasted overnight (16 h) and administered with an intraperitoneal injection of glucose (2 g/kg body weight). Glycemia was measured in tail vein blood samples at the indicated time points. Venous blood was collected from tail vein in tubes (Microvette? CB 300, SARSTEDT) at the same time points and immediately centrifuged to separate serum, which was used to measure insulin levels.

Oximetry

[0373] An indirect open circuit calorimeter (Oxylet, Panlab, Cornelia, Spain) was used to monitor oxygen consumption, carbon dioxide production in eight metabolic chambers simultaneously. Mice were individualized and acclimated to the metabolic chambers for 24 h, and data were collected every 15 min for 3 min in each cage for other 24 h. Data were taken from the light and dark cycle and adjusted for body weight. To calculate energy expenditure the Metabolism software provided by the manufacturer was used.

Transfection of HEK293, C2C12 and HepG2 Cells

[0374] Cells were cultured in a 24-well plate and transfected with 0.8 ?g of DNA per well using Lipofectamine 2000 following the manufacturer's instructions (Thermo Fisher Scientific).

Bone Analysis

[0375] Bone volume and architecture were evaluated by ?CT. Mouse tibiae were fixed in neutral buffered formalin (10%) and scanned using the eXplore Locus CT scanner (General Electric) at 27-micron resolution. Trabeculae were analyzed in 1 mm3 of proximal tibial epiphysis and 1.8 mm3 of cortical tibial diaphysis in 4 mice/group. Bone parameters were calculated with the MicroView 3D Image Viewer & Analysis Tool. The length of the tibia was measured from the intercondilar eminence to the medial malleolus.

Western Blot Analysis

[0376] iWAT and iBAT were homogenized in QIAzol Lysis Reagent (Qiagen) and the protein fraction was isolated from the organic phase following the manufacturer's instructions. Proteins were separated by 12% SDS-PAGE, and analyzed by immunoblotting with rabbit polyclonal anti-UCP1 (ab10983; Abcam) and rabbit polyclonal anti-?-tubulin (ab4074; Abcam) antibodies. Detection was performed using ECL Plus detection reagent (Amersham Biosciences).

Open Field Test

[0377] The open field test was performed between 9:00 am and 1:00 pm as previously reported (Haurigot et al, 2013). Briefly, animals were placed in the center of a brightly lit chamber (41?41?30 cm) crossed by 2 bundles of photobeams (LE 8811; Panlab) that detect horizontal and vertical movements. Motor and exploratory activities were evaluated during the first 6 minutes. The total distance covered was evaluated using a video tracking system (SMART Junior; Panlab).

Statistical Analysis

[0378] All values are expressed as mean?SEM. Differences between groups were compared by Student's t-test. Differences were considered significant at p<0.05.

EXAMPLES

Example 1. Prevention of Obesity and Diabetes by Intra-eWAT Administration of AAV-CAG-moFGF21-dmiRT Vectors in C57Bl6 Mice

[0379] We evaluated the therapeutic potential of the AAV-mediated genetic engineering of adipose tissue with FGF21 to prevent obesity and diabetes in 8-week-old male C57Bl6. Intra-eWAT (eWAT: epididymal white adipose tissue) administration of 10.sup.12 viral genomes (vg) of AAV9 vectors encoding a murine codon-optimized FGF21 coding sequence under the control of the CAG ubiquitous promoter which included target sites of miR122 and miR1 (AAV9-CAG-moFGF21-doublemiRT) (FIG. 1A) mediated adipose-specific overexpression of FGF21 (FIG. 1B) as well as high secretion of the protein into the bloodstream (FIG. 1C). AAV9-CAG-moFGF21-doublemiRT-treated mice also showed overexpression of the FGF21 receptor1 (FGF21R1) in eWAT (FIG. 1D) and ?-Klotho (a FGF21 co-receptor) in adipose tissue and liver (FIG. 1E) in comparison with AAV9-CAG-null vectors (vectors that retain equal infectivity but do not encode any transgene). The CAG-moFGF21-doublemiRT construct is comprised in SEQ ID NO: 32 and the CAG-null construct is comprised in SEQ ID NO:31.

[0380] Following AAV-mediated gene transfer of FGF21 to eWAT, mice fed a chow diet showed loss of body weight (FIGS. 1F and 1G). When challenged with a high fat diet (HFD), animals overexpressing FGF21 in adipose tissue remained lean for the duration of the experiment whereas AAV9-CAG-null treated mice became progressively obese (FIGS. 1F and 1G). According to their lower body weight, both chow- and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice showed decreased weight of adipose depots and liver (FIG. 1H).

[0381] Histological analysis of white adipose tissue by hematoxylin-eosin staining revealed decreased white adipocyte size in eWAT and iWAT (iWAT: inguinal white adipose tissue) and multiple multilocular adipocytes in iWAT, suggesting that browning of this depot had occurred (FIG. 2A). Morphometric analysis further confirmed decreased mean area of white adipocytes in AAV9-CAG-moFGF21-doublemiRT-treated mice (FIG. 2B). The frequency distribution of the area of white adipocytes was also different between groups. Both chow- and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice presented increased number of small adipocytes and fewer big adipocytes (FIG. 2C). Noticeably, the frequency distribution of the area of white adipocytes in HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice was almost identical to that of chow-fed AAV9-CAG-null-treated animals (FIG. 2C). Therefore, the HFD-induced hypertrophy of adipocytes observed in AAV9-CAG-null-treated mice was blocked in mice overexpressing FGF21. Overexpression of UCP1 and Dio2 in iWAT (FIGS. 3A and 3B) further confirmed browning of iWAT in chow- and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice.

[0382] Histologic analisis of iBAT (iBAT: interscapular brown adipose tissue) showed lower lipid accumulation in this depot in chow- and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice in comparison with AAV9-CAG-null mice (FIG. 2A). According to this result and to browning of iWAT, energy expenditure (FIG. 3C) of HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice during the light and dark cycles was higher than that of HFD-fed AAV9-CAG-null mice. Altogether, these data suggest that AAV9-CAG-moFGF21-doublemiRT-treated mice have increased thermogenic activity.

[0383] Liver histologic sections showed decreased lipid accumulation in hepatocytes of mice overexpressing FGF21 compared with AAV9-CAG-null-treated mice both under chow or HFD (FIG. 2A). Accordingly, HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice normalized their hepatic content of tryglycerides (TG) (FIG. 3D). In parallel, circulating levels of TG, total cholesterol, HDL-cholesterol and LDL-cholesterol were normalized in HFD-fed mice overexpressing FGF21 (FIGS. 3E and 3F).

[0384] HFD-fed mice overexpressing FGF21 were more insulin sensitive than HFD-fed AAV9-null treated mice (FIG. 3G) and both chow- and HFD-fed AAV9-CAG-moFGF21-doublemiRT-treated mice showed decreased insulin circulating levels in comparison with their AAV9-CAG-null treated counterparts (FIG. 3H).

Example 2. Reversion of Obesity and Improvement of Glucose Metabolism by Intra-eWAT Administration of AAV-CAG-moFGF21-dmiRT Vectors in Ob/Ob Mice

[0385] We evaluated the anti-diabetic and anti-obesogenic therapeutic potential of the AAV-mediated genetic engineering of adipose tissue with FGF21 in 11-week-old male ob/ob mice, which have defective leptin signalling and are a widely used genetic model of obesity and diabetes. To this end, a dose-response study was performed. Ob/ob mice were administered locally into the eWAT with four different doses (10.sup.10 vg, 5?10.sup.10 vg, 2?10.sup.11 vg or 10.sup.12 vg) of AAV8-CAG-moFGF21-doublemiRT vectors (FIG. 1A). As control, ob/ob animals were administered intra-eWAT with 10.sup.12 vg of AAV8-CAG-null vectors.

[0386] Intra-eWAT administration of AAV8-CAG-moFGF21-doublemiRT vectors mediated specific overexpression of FGF21 in white adipose tissue as well as high secretion of the protein into the bloodstream in a dose-dependent manner (FIGS. 4A and 4B). Specifically, the dose of 10.sup.12 vg of AAV8-CAG-moFGF21-doublemiRT vectors mediated a very robust overexpression of FGF21 in eWAT and iWAT (FIG. 4A) and achieved the highest circulating FGF21 levels (FIG. 4B). In contrast, the lowest dose administered, 10.sup.10 vg of AAV8-CAG-moFGF21-doublemiRT vectors, only produced a very modest overexpression of FGF21 in eWAT (FIG. 4A) and animals treated with this dose showed no differences in the serum FGF21 levels in comparison with AAV8-CAG-null treated animals (FIG. 4B), probably because FGF21 acted in a paracrine-autocrine manner. Accordingly, whereas AAV8-CAG-null-treated animals progressively increased their body weight, animals treated with AAV8-CAG-moFGF21-doublemiRT vectors showed decreased body weight gain proportional to the dose of vectors administered (FIGS. 4C and 4D). Noticeably, animals treated with 10.sup.12 vg of AAV8-CAG-moFGF21-doublemiRT vectors lost approximately 15% of weight during the first two weeks after AAV administration and afterwards increased their body weight until they reached the initial body weight (FIGS. 4C and 4D). Thus, animals administered intra-eWAT with 10.sup.12 vg of AAV8-CAG-moFGF21-doublemiRT vectors showed a 40% difference in total body weight in comparison with AAV8-CAG-null-treated animals at the end of the experiment (FIG. 4D). In agreement, animals treated with 10.sup.12 vg of AAV8-CAG-moFGF21-doublemiRT vectors showed marked decreased adiposity and a 60% reduction of the weight of the liver (FIG. 4E). iBAT weight was increased in this cohort of mice (FIG. 4E), probably due to increased thermogenic activity.

[0387] Animals treated with 5?10.sup.10 vg, 2?10.sup.11 vg or 10.sup.12 vg of AAV8-CAG-moFGF21-doublemiRT vectors presented improved insulin sensitivity in comparison with AAV8-CAG-null-treated mice (FIG. 5A). Animals treated with 2?10.sup.11 vg or 10.sup.12 vg of AAV8-CAG-moFGF21-doublemiRT vectors also showed lower insulin circulating levels than ob/ob mice treated with AAV8-CAG-null vectors (FIG. 5B).

Example 3. Reversion of Obesity and Improvement of Glucose Metabolism by Intravenous Administration of AAV-hAAT-moFGF21 Vectors in Ob/Ob Mice

[0388] We also evaluated the anti-diabetic and anti-obesogenic effects mediated by the increased circulating levels of FGF21 by means of AAV-mediated genetic engineering of the liver in 8-week-old male ob/ob mice. Ob/ob mice were administered intravenously (IV) with 10.sup.11 vg or 5?10.sup.11 vg of AAV8 vectors encoding a murine codon-optimized FGF21 coding sequence under the control of the liver-specific human al-antitrypsin (hAAT) promoter (AAV8-hAAT-moFGF21) (FIG. 6A). As control, ob/ob animals were administered IV with 5?10.sup.11 vg of AAV8-hAAT-null vectors. The hAAT-moFGF21 construct is comprised in SEQ ID NO: 34 and the hAAT-null construct is comprised in SEQ ID NO:33.

[0389] Intravenous administration of AAV8-hAAT-moFGF21 vectors mediated specific overexpression of FGF21 in the liver as well as high secretion of the protein into the bloodstream in a dose-dependent manner (FIGS. 6B and 6C). Specifically, the dose of 5?10.sup.11 vg of AAV8-hAAT-moFGF21 vectors mediated a very robust overexpression of FGF21 in the liver (FIG. 6B) and achieved the highest circulating FGF21 levels (FIG. 6C). The body weight of animals treated with this dose of AAV8-hAAT-moFGF21 vectors decreased approximately 7% during the two first weeks after AAV administration and afterwards slightly increased whereas AAV8-hAAT-null-treated mice progressively put on weight (FIGS. 6D, 6E and 6F). Mice administered with 10.sup.11 vg of AAV8-hAAT-moFGF21 vectors gained markedly much less weight than AAV8-hAAT-null-treated animals (FIGS. 6D, 6E and 6F). Specifically, AAV8-hAAT-null animals showed a 50% increase in their body weight at the end of the experiment in comparison with the 10% weight gain of animals treated with 10.sup.11 vg of AAV8-hAAT-moFGF21 vectors (FIG. 6E). According to their lower body weight, animals overexpressing FGF21 in the liver showed significant decreased adiposity, particularly in those animals treated with the highest dose of vectors, and approximately a 60% reduction of the liver weight (FIG. 6G). iBAT weight was similarly increased in both groups of AAV8-hAAT-moFGF21-treated mice (FIG. 6G), probably due to higher thermogenic activity in these animals in comparison with mice administered with AAV8-hAAT-null vectors.

[0390] Animals treated with AAV8-hAAT-moFGF21 vectors showed improved insulin sensitivity and decreased insulin circulating levels in comparison with AAV8-hAAT-null-treated mice (FIGS. 6H and 6I)

Example 4. Long-Term Reversion of Obesity and Diabetes by Intravenous Administration of AAV-hAAT-moFGF21 Vectors in HFD-Fed Mice

[0391] We also evaluated the anti-diabetic and anti-obesogenic effects mediated by the increased circulating levels of FGF21 by means of AAV-mediated genetic engineering of the liver in obese C57Bl6 mice. Nine-week-old male C57Bl6 mice (young adults) were fed a HFD for 9 weeks and then administered IV with 10.sup.10 vg or 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors (FIG. 6A). After AAV administration, AAV8-hAAT-moFGF21-treated mice were maintained on HFD for 52 weeks. As controls, 5?10.sup.10 vg of AAV8-hAAT-null were administered IV to chow- and HFD-fed C57Bl6 mice. These two latter cohorts of mice were maintained either on chow diet or HFD thereafter.

[0392] Intravenous administration of AAV8-hAAT-moFGF21 vectors in HFD-fed mice mediated high secretion of FGF21 into the bloodstream in a dose-dependent manner (FIG. 7A).

[0393] No differences in body weight were observed between HFD-fed AAV8-null-treated mice and HFD-fed animals administered with 10.sup.10 vg of AAV8-hAAT-moFGF21 vectors (FIGS. 7B and 7C). However, HFD-fed animals treated with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors initially lost 20% of body weight after AAV administration and then progressively gained weight similarly to chow-fed AAV8-hAAT-null-treated mice (FIGS. 7B and 7C). Noticeably, from week 9 after AAV administration onwards no statistical significant differences were observed in the total body weight and body weight gain between HFD-fed animals administered with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors and chow-fed AAV8-hAAT-null-treated mice (FIGS. 7B and 7C).

[0394] The energy expenditure of HFD-fed mice treated with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors during the light and dark cycles was higher than that of chow- and HFD-fed AAV8-hAAT-null mice (FIG. 8A). No differences in energy expenditure were observed among chow- and HFD-fed AAV8-hAAT-null-treated animals and mice administered with 10.sup.10 vg of AAV8-hAAT-moFGF21 vectors (FIG. 8A). Altogether, these data suggest that mice treated with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 have increased thermogenic activity.

[0395] Animals treated with 10.sup.10 vg of AAV8-hAAT-moFGF21 vectors presented improved insulin sensitivity in comparison with HFD-fed mice administered with AAV8-hAAT-null vectors and their insulin sensitivity was similar to that of chow-fed mice treated with AAV8-hAAT-null vectors (FIG. 8B). Noticeably, animals administered with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors presented improved insulin sensitivity in comparison with chow-fed mice administered with AAV8-hAAT-null vectors and showed normalized insulin circulating levels (FIGS. 8B and 8C).

Example 5. Reversion of Obesity and Diabetes by Intravenous Administration of AAV-hAAT-moFGF21 Vectors in Old HFD-Fed Mice

[0396] We also evaluated the anti-diabetic and anti-obesogenic effects of FGF21 in obese old (adults) C57Bl6 mice. Seven and a half-month-old male C57Bl6 mice were fed a HFD for 8 weeks and then administered IV with 10.sup.10 vg, 2?10.sup.10 vg or 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors (FIG. 6A). After AAV administration, AAV8-hAAT-moFGF21-treated mice were maintained on HFD for 22 weeks. As controls, 5?10.sup.10 vg of AAV8-hAAT-null were administered IV to chow- and HFD-fed old C57Bl6 mice. These two latter cohorts of mice were maintained either on chow diet or HFD thereafter. Intravenous administration of AAV8-hAAT-moFGF21 vectors in old HFD-fed mice mediated high secretion of FGF21 into the bloodstream in a dose-dependent manner (FIG. 9A).

[0397] No differences in body weight were observed between HFD-fed AAV8-null-treated mice and HFD-fed animals administered with 10.sup.10 vg of AAV8-hAAT-moFGF21 vectors (FIGS. 9B and 9C). However, HFD-fed animals treated with either 2?10.sup.10 vg or 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors initially lost 15 and 20%, respectively, of body weight after AAV administration (FIGS. 9B and 9C). Thereafter, animals treated with 2?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors progressively gained weight similarly to chow-fed AAV8-hAAT-null-treated mice whereas no significant changes in body weight of animals treated with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors were observed (FIGS. 9B and 9C). Noticeably from week 3 after AAV administration onwards no statistical significant differences were observed in the total body weight and body weight gain between HFD-fed animals administered with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors and chow-fed AAV8-hAAT-null-treated mice (FIGS. 9B and 9C).

[0398] The energy expenditure of HFD-fed mice treated with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors during the light and dark cycles was higher than that of chow- and HFD-fed AAV8-hAAT-null mice (FIG. 10A). Animals treated with 2?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors showed increased energy expenditure during the light cycle and a tendency to increase energy expenditure during the dark cycle (FIG. 10A). Animals treated with 10.sup.10 vg of AAV8-hAAT-moFGF21 vectors showed increased energy expenditure during the dark cycle (FIG. 10A). No differences were observed among chow- and HFD-fed AAV8-hAAT-null-treated animals and mice administered with 10.sup.10 vg of AAV8-hAAT-moFGF21 vectors (FIG. 10A). Altogether, these data suggest that old mice treated with AAV8-hAAT-moFGF21 have increased thermogenic activity.

[0399] Animals treated with 10.sup.10 vg or 2?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors presented improved insulin sensitivity in comparison with HFD-fed mice administered with AAV8-hAAT-null vectors and their insulin sensitivity was similar to that of chow-fed mice treated with AAV8-hAAT-null vectors (FIG. 10B). Noticeably, animals administered with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors presented improved insulin sensitivity in comparison with chow-fed mice administered with AAV8-hAAT-null vectors (FIG. 10A). Animals treated with 10.sup.10 vg, 2?10.sup.10 vg or 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors showed lower fasted and fed insulin circulating levels than HFD-fed AAV8-hAAT-null-treated mice (FIG. 10C). Noticeably, no differences in fed insulin circulating levels were observed between old animals administered IV with 2?10.sup.10 vg or 5?10.sup.10 vg of AAV8-hAAT-moFGF21 vectors and chow-fed AAV8-hAAT-null-treated mice (FIG. 1C).

Example 6. Evaluation of Weight Loss by Intramuscular Administration of AAV-CMV-moFGF21 Vectors in C57Bl6 Mice

[0400] We also evaluated the therapeutic potential of increasing FGF21 circulating levels by the AAV-mediated genetic engineering of skeletal muscle in C57Bl6 mice. In order to target the skeletal muscle, the CMV promoter and the AAV1 serotype were selected. Although the CMV promoter is an ubiquitous promoter, its concomitant use together with the AAV1 capsids enables to very efficiently target the skeletal muscle without transducing the liver, as previously published (Mas et al., Diabetes 2006; Callejas et al., Diabetes 2013).

[0401] A dose of 3?10.sup.11 vg of AAV1 vectors encoding a murine codon-optimized FGF21 coding sequence under the control of the ubiquitous CMV promoter (AAV1-CMV-moFGF21) (FIG. 11A) were administered by intramuscular injection in the quadriceps, gastrocnemius, and tibialis cranealis of each hind limb (5?10.sup.10 vg/muscle) of 6 to 12-week-old male C57BL6 mice. As control, age-matched C57Bl6 animals were administered intramuscularly in the same muscles with 3?10.sup.11 vg of AAV1-CMV-null vectors (5?10.sup.10 vg/muscle). The CMV-moFGF21 construct is comprised in SEQ ID NO: 36 and the CMV-null construct is comprised in SEQ ID NO:35.

[0402] Intramuscular administration of AAV1-CMV-moFGF21 vectors mediated high secretion of FGF21 into the bloodstream (FIG. 11B). Animals treated with AAV1-CMV-moFGF21 vectors showed decreased body weight and body weight gain in comparison with AAV1-CMV-null-treated mice (FIGS. 11C and 11D).

Example 7. Increased Protein Production by Codon-Optimized Human FGF21 Nucleotide Sequences

[0403] To evaluate if codon-optimization was able to mediate increased FGF21 protein production, HEK293 cells were transfected with plasmids encoding three different codon-optimized human FGF21 nucleotide sequences (SEQ ID NO's: 40-42). As control, non-transfected cells and cells transduced with wild-type hFGF21 coding sequence were used. Expression of the three codon-optimized human FGF21 sequences and the WT human FGF21 sequence was under the control of the hAAT promoter (SEQ ID NO:47). Cells transduced with either codon-optimized human FGF21 version 1 or 3 were able to secrete higher human FGF21 levels into the culture media in comparison with wild-type or codon-optimized FGF21 variant 2 (FIG. 12), thus demonstrating increased FGF21 protein production by codon-optimization of variants 1 and 3.

Example 8. Reversion of Obesity and Diabetes in Mice by Administration of AAV Vectors Encoding Human FGF21 (In Vivo Experiment Proving the Activity of FGF21)

[0404] HFD-fed mice are treated with AAV vectors encoding human FGF21. As controls, the same dose of AAV-null vectors is administered to chow- and HFD-fed mice.

[0405] To evaluate the capacity of human FGF21 to induce browning of WAT and thermogenic activity of BAT, to increase energy expenditure and to improve glucose and energy metabolism, the following tests are performed: [0406] measurement of body weight and food and liquid intake weekly [0407] measurement of body temperature [0408] measurement of energy expenditure and respiratory quotient by indirect calorimetry [0409] measurement of glycemia [0410] evaluation of whole-body glucose disposal by intraperitoneal glucose tolerance test [0411] evaluation of insulin sensitivity by intraperitoneal insulin tolerance test [0412] analyses in tissue and serum samples, including [0413] examination of the level of overexpression of human FGF21 in the targeted tissue and into the bloodstream [0414] morphological and histological analysis. [0415] determination of circulating levels of hormones and cytokines [0416] determination of serum metabolic parameters, such as free fatty acids, glycerol, triglycerides, cholesterol and ketone bodies [0417] evaluation of browning capacity by examination of the presence of beige adipocytes in the inguinal fat pad by immunohistochemistry, and gene expression of classic white, brown and beige adipocyte markers

Example 9: In Vitro Assay for Assessing FGF21 Activity

[0418] FGF21 is expected to increase glucose uptake and GLUT1 expression in 3T3-L1 cells (Kharitonenkov, A. et al., 2005. J Clin. Invest 115:1627-1635).

Example 10. Reversion of Obesity and Improvement of Glucose Metabolism by Intra-eWAT Administration of AAV8-CAG-moFGF21-dmiRT Vectors in Ob/Ob Mice: Further Observations

[0419] We further evaluated the anti-diabetic and anti-obesogenic therapeutic potential of the AAV-mediated genetic engineering of adipose tissue with FGF21 in ob/ob mice (see Example 2).

[0420] Ob/ob mice that received intra-eWAT injections of AAV8-CAG-moFGF21-dmirT vectors showed a reduction in the size of white adipocytes of the epididymal pad (FIG. 13A). Circulating adiponectin levels also increased with dose (FIG. 14A). eWAT inflammation, evaluated through Mac2 staining, was also reduced as a function of the dose of vector, as did the expression of the macrophage marker F4/80 (FIGS. 14B and C). The liver of ob/ob mice injected with null vectors or the lowest dose of AAV8-CAG-moFGF21-dmirT showed accumulation of lipid droplets in hepatocytes (FIG. 13B). The administration of doses of 5?10.sup.10 vg/mouse or higher of FGF21-encoding vectors completely prevented the development of hepatic steatosis (FIG. 13B), which correlated with the weight of the organ (FIG. 14D) and its total triglyceride and cholesterol content (FIGS. 14E and F). Further evidence that the dose of 5?10.sup.10 vg/mouse represented a threshold for therapeutic efficacy came from the analysis of glycemia and insulinemia. While the dose of 1?10.sup.10 vg/mouse did not modify the levels of blood glucose in the fed state and only partially reduced insulin levels, doses of 5?10.sup.10 vg/mouse and higher completely normalized glycemia and insulinemia (FIGS. 13C and D). Altogether, this example confirms the therapeutic potential of overexpressing FGF21 in adipose tissue.

Example 11. Reversion of Obesity and Improvement of Glucose Metabolism by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors in Ob/Ob Mice: Further Observations

[0421] We further evaluated the anti-diabetic and anti-obesogenic therapeutic potential of intravenous administration of AAV8-hAAT-moFGF21 vectors in ob/ob mice (see Example 3).

[0422] In agreement with their lower body weight, ob/ob animals overexpressing FGF21 in the liver showed significantly decreased size of white adipocytes, particularly those animals treated with 5?10.sup.11 vg (FIG. 15A). This was parallel with a dose-dependent increase in circulating adiponectin levels (FIG. 15B) and decreased WAT inflammation, as evidenced by decreased staining for Mac2 and expression of F4/80 and TNF-? in eWAT (FIG. 16A-C). Noticeably, ob/ob mice treated with 5?10.sup.11 vg showed a remarkable reduction in crown-like structures in eWAT (FIG. 16A).

[0423] While 7-month-old ob/ob mice showed marked hepatic steatosis, the liver of FGF21-treated ob/ob mice did not show accumulation of lipids in hepatocytes (FIG. 15C). This agreed with a 60% reduction in the weight of this organ (FIGS. 16D and E) as well as with a marked reduction in the total liver triglyceride and cholesterol content (FIGS. 16F and G) in ob/ob mice receiving therapeutic vectors. Ob/ob animals treated with both doses of AAV8-hAAT-moFGF21 also showed decreased fed glycemia, and their insulinemia in the fed state was reduced by ?70% (FIGS. 15D and E).

[0424] We evaluated whether the decrease in circulating glucose levels observed in ob/ob mice after AAV8-hAAT-moFGF21 treatment resulted from suppression of hepatic gluconeogenesis by measuring the expression by qPCR of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). No changes in the expression of these enzymes were observed in the liver of AAV8-hAAT-moFGF21-treated ob/ob mice, except for the animals treated with 1?10.sup.11 vg of AAV8-hAAT-moFGF21 that showed increased PEPCK expression (FIGS. 17A and B). These results suggested that AAV-mediated long-term expression of FGF21 in the liver, and the subsequent increase of circulating FGF21, did not lower glucose by inhibiting hepatic glucose production.

[0425] The glucose-lowering effects of FGF21 have also been attributed to increased glucose uptake by adipocytes and enhanced energy expenditure (Xu J. et al., 2009. AJP Endocrinol. Metab. 297:E1105-E1114; Ding X. et al., 2012. Cell Metab. 16:387-393; Camacho R. C. et al., 2013. Eur. J. Pharmacol. 715:41-45; Emanuelli B. et al., 2014. Clin. Invest. 124:515-527; Kharitonenkov A. et al., 2005. Endocrinology 148: 774-781; Hondares E. et al., 2010. Cell Metab. 11:206-212; Samms R. J. et al., 2015. Cell Rep. 11:991-999). Thus, we assessed in different pads of adipose tissue (iWAT, eWAT and iBAT) the expression of key components of the glucose uptake machinery by qPCR, such as the glucose transporters Glut1 and Glut4, the glucose phosphorylating enzymes hexokinase I and II (HKI and HKI), and UCP1 in the case of iBAT. In AAV8-FGF21 treated ob/ob mice, the expression of Glut1 was increased in iWAT and iBAT (FIG. 17C), and that of Glut4 was increased in eWAT, iWAT and iBAT (FIG. 17D). HKI and HKII were upregulated only in iBAT (FIGS. 17E and F). Moreover, UCP1 expression was increased in the iBAT of ob/ob mice treated with the high dose of AAV8-hAAT-moFGF21 vectors (FIG. 17G). Altogether, these results suggest that the long-term amelioration of glycemia observed in ob/ob mice following treatment with AAV8-hAAT-moFGF21 vectors probably results from increased glucose uptake by white and brown adipocytes and enhanced thermogenesis in iBAT.

Example 12. Long-Term Reversion of Obesity and Diabetes by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors in HFD-Fed Mice and HFD-Fed Old Mice: Decreased Tissue Weight and Stable Expression Up to 1 Year

[0426] Representative images of animals belonging to all experimental groups of the studies performed in young adults or in adults (see Examples 4 and 5) are shown in FIG. 19A-B. The reversion of obesity by AAV8-hAAT-moFGF2l treatment was parallel to a dose-dependent decrease in the weight of the main white adipose tissue (WAT) depots, such as the epididymal (eWAT), inguinal (iWAT) and retroperitoneal (rWAT) fat pads, both in animals treated as young adults or as adults (FIG. 18A and FIG. 19C). The HFD-induced increase in the weight of the liver was completely normalized by FGF21 gene transfer at the highest doses of vector used, whereas the weight of the quadriceps was unchanged by the diet or AAV delivery (FIG. 18A and FIG. 19D).

[0427] AAV8-hAAT-moFGF21-treated mice of both ages showed specific overexpression of codon-optimized FGF21 in the liver (FIG. 19E), which resulted in secretion of FGF21 into the bloodstream in a dose-dependent manner in both groups of mice, with levels remaining stable for up to 1 year after a single administration of the vector (FIG. 18B).

Example 13. Long-Term Reversion of Obesity and Diabetes by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors in HFD-Fed Mice and HFD-Fed Old Mice: Increased Locomotor Activity and Investigating the Thermogenic Mechanism

[0428] Increased energy expenditure (see Examples 4 and 5) was also seen in animals treated as young adults 10 months after AAV8-hAAT-moFGF21 delivery (FIG. 21A).

[0429] This observation was in agreement with AAV8-hAAT-moFGF21-mediated effects on locomotor activity. In contrast to the hypoactivity observed in the open field test in the animals fed a HFD that received AAV8-null vectors, mice treated with 5?10.sup.10 vg AAV8-hAAT-moFGF21 as young adults showed the same degree of spontaneous locomotor activity than chow-fed, null-injected animals. As shown in FIG. 20A, 1 year after AAV8-hAAT-moFGF21 delivery, treated animals travelled more distance, rested less time, and spent more time doing slow and fast movements than untreated HFD-fed controls.

[0430] Given that changes in energy expenditure may reflect changes in thermogenesis, we evaluated the degree of activation of the brown adipose tissue (BAT). Both mice treated as young adults or adults with 5?10.sup.10 vg AAV8-hAAT-moFGF21 showed decreased lipid deposition in iBAT (FIG. 20B). The content of UCP1 protein in BAT was increased in a dose-dependent manner in mice treated with AAV8-hAAT-moFGF21 vectors as young adults (FIG. 20C), consistent with an increase in non-shivering thermogenesis induced by FGF21 gene transfer to the liver.

[0431] The browning of the subcutaneous WAT, characterized by the appearance of beige adipocytes, is also associated with increases in energy expenditure (Harms & Seale, 2013). To evaluate if browning was accountable for the enhancement of energy expenditure observed following AAV8-hAAT-moFGF21 treatment, histological evaluation of iWAT was performed. In agreement with the decreased weight of this pad (FIG. 18A), the adipocytes of HFD-fed AAV8-hAAT-moFGF21-treated animals were smaller than those of HFD-fed null-injected animals (FIG. 20D). Treatment with AAV8-hAAT-moFGF21 vectors, nevertheless, did not result in increased detection of multilocular beige adipocytes in iWAT at any of the doses tested, either in animals treated as young adults or adults (FIG. 20D). Accordingly, there were no statistically significant differences in the levels of UCP1 protein in iWAT between the HFD-fed groups (FIG. 21B).

[0432] The creatine-driven substrate cycle and sarco/endoplasmic reticulum Ca2+-ATPase 2b (Serca2b)-mediated calcium cycling can increase thermogenesis in iWAT independently of UCP1 (Kazak L. et al., 2015. Cell 163:643-655; Ikeda K. et al., 2017. Nat. Med. 23:1454-1465). Higher levels of expression of phosphatase orphan 1 (Phosphol), an enzyme involved in the creatine-driven substrate cycle, were observed in iWAT of HFD-fed mice treated with 5?10.sup.10 vg of AAV8-hAAT-moFGF21 when compared with age-matched, chow- and HFD-fed control groups (FIG. 20E), suggesting that the activity of the creatine-driven cycle was probably increased as a result of FGF21 gene transfer. Regarding the calcium cycling-dependent thermogenic mechanism, no differences in the expression levels of Serca2b were detected in the iWAT of animals treated with AAV8-hAAT-moFGF21 vectors when compared with chow- or HFD-fed null-treated animals (FIG. 21C). On the other hand, the iWAT expression of ryanodine receptor 2 (RyR2), another enzyme involved in the same cycle, was increased by HFD-feeding in both null- and AAV8-hAAT-moFGF21-treated mice (FIG. 21C). Altogether, these results suggest that the calcium cycling-dependent thermogenic mechanism is not involved in the improvement of whole-body energy homeostasis observed after AAV-FGF21 treatment.

Example 14. Long-Term Reversion of Obesity and Diabetes by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors in HFD-Fed Mice and HFD-Fed Old Mice: Glucagon Levels, Islet Hyperplasia and Glucose Tolerance

[0433] Moreover, HFD-fed animals treated as young adults with AAV8-hAAT-moFGF21 vectors showed decreased circulating levels of glucagon compared with HFD-fed null-treated mice (FIG. 22A).

[0434] While AAV8-null-treated mice developed islet hyperplasia as a consequence of HFD feeding, the ?-cell mass of animals treated with AAV8-hAAT-FGF21 vectors (at the doses of 2?10.sup.10 or 5?10.sup.10 vg/mouse) was similar to that of control mice fed a chow diet (FIGS. 22B and C). Double immunostaining for insulin and glucagon of pancreatic sections from HFD-fed AAV8-hAAT-moFGF21-treated mice showed normal distribution of a and ? cells in the islets of these animals, with localization of glucagon-expressing cells in the periphery of the islet and of insulin-expressing cells in the core (FIG. 22D).

[0435] To evaluate glucose tolerance in FGF21-treated mice, an intraperitoneal glucose tolerance test (GTT) (2 g glucose/kg bw) was performed 10 weeks after AAV administration. HFD-fed animals injected with either null or FGF21-encoding vectors at a dose of 1?10.sup.10 vg/mouse were glucose intolerant and showed markedly increased circulating levels of insulin during the GTT (FIGS. 23A and B). In contrast, animals treated with 5?10.sup.10 vg/mouse of AAV8-hAAT-moFGF21 showed improved glucose clearance when compared to chow-fed control mice (FIG. 23A). Insulin levels were indistinguishable between these two experimental groups (FIG. 23B). These results further confirmed improved insulin sensitivity in HFD-fed mice treated with 5?10.sup.10 vg/mouse of AAV8-hAAT-moFGF21.

Example 15. Reversion of HFD-Associated WAT Hypertrophy and Inflammation by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors

[0436] HFD-feeding induces an increase in the size of WAT adipocytes (Sattar N. & Gill J. M. R., 2014. BMC Med. 12:123). Administration of FGF21-encoding vectors counteracted this increase (FIG. 24A). Morphometric analysis of WAT revealed that the area of white adipocytes of animals treated as young adults with 1?10.sup.10 or 5?10.sup.10 vg of vector, and of mice treated as adults with 2?10.sup.10 or 5?10.sup.10 vg of vector was similar to that of animals fed a chow diet (FIG. 24B). In both groups of FGF21-treated animals, there was a redistribution of the size of adipocytes, with a greater proportion of smaller adipocytes (FIG. 25A). In agreement with the decrease in adiposity and reversal of WAT hypertrophy, adiponectin and leptin levels were also normalized in animals treated with highest doses of AAV8-hAAT-moFGF21 vectors, irrespective of the age of initiation of the treatment (FIGS. 24C and D).

[0437] Obesity also causes the inflammation of WAT (Hafer G. R. et al., 2008. Eur. Heart J. 29:2959-2571). Thus, we analyzed inflammation in this tissue through immunostaining for the macrophage-specific marker Mac2 and the expression of pro-inflammatory molecules. While HFD-fed mice showed increased presence of macrophages, revealed as crown-like structures, in the eWAT, animals treated as young adults or adults with 5?10.sup.10 vg AAV8-hAAT-moFGF2l had no sign of macrophage infiltration (FIG. 24E and FIG. 25B). This was parallel to the normalization in the expression of the macrophage markers F480 and CD68 and of the pro-inflammatory cytokines TNF? and IL-1? (FIG. 24F-H and FIG. 25C-E), indicating that FGF21 expression counteracted the inflammation of WAT associated to obesity.

Example 16. Reversal of Hepatic Steatosis, Inflammation and Fibrosis by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors

[0438] Histological analysis of the liver showed that all null-treated animals fed a HFD had marked hepatic steatosis at the time of sacrifice (FIG. 26A-D). In contrast, HFD-fed mice receiving 5?10.sup.10 vg AAV8-hAAT-moFGF21 as young adults or as adults evidenced reversal of this pathological deposition of lipids (FIG. 26A). These histological findings were parallel to a marked reduction in the total liver triglyceride and cholesterol content of 5?10.sup.10 vg AAV8-hAAT-moFGF21-treated animals (FIGS. 26B and C). In addition, animals fed a HFD and treated with 5?10.sup.10 vg AAV8-hAAT-moFGF21 vectors when young adults or adults showed no sign of hepatic inflammation, as evidenced by the lack of staining for Mac2, which revealed increased presence of macrophages in the livers of null-treated HFD-fed mice (FIG. 26D). Finally, FGF21 gene transfer to the liver reversed hepatic fibrosis. While collagen fibers were readily detectable following PicroSirius Red staining or Masson's trichrome staining of liver sections from animals fed a HFD and injected with control null vectors, they were undetectable in the livers of AAV8-hAAT-moFGF21 treated mice (FIG. 28A and FIG. 27). These mice also showed markedly reduced hepatic expression of collagen 1 (FIGS. 28B and C). Altogether these findings indicated that AAV8-hAAT-moFGF21 treatment protected from the development of HFD-induced non-alcoholic steatohepatitis (NASH).

Example 17. Long-Term Safety of Liver-Directed AAV-FGF21 Treatment

[0439] Pharmacological treatment with FGF21 or transgenic overexpression have been associated with perturbation of bone homeostasis through increased bone resorption, which could cause bone loss (Wei W. et al., 2012. Proc. Natl. Acad. Sci. 109:3143-3148; Wang X. et al., 2015. Cell Metab. 22:811-824; Charoenphandhu N. et al., 2017. J. Bone Miner. Metab. 35:142-149; Talukdar S. et al., 2016. Cell Metab. 23:427-440; Kim A. M. et al., 2017. Diabetes, Obes. Metab). Given the therapeutic potential of AAV8-hAAT-moFGF21 for the treatment of obesity and diabetes, we evaluated the long-term effects of gene transfer on the bones of the animals treated with the highest dose of vector. At the time of sacrifice (?16.5 months of age), the naso-anal length and the tibial length were normal in the animals that were administered with AAV8-hAAT-moFGF21 vectors at 9 or 29 weeks of age (FIGS. 29A and B). We then examined bone structure by micro-computed tomography (?CT). Analysis of the proximal epiphysis of the tibia revealed no significant differences in the trabecular and cortical bone of mice fed a HFD and administered with 5?10.sup.10 vg AAV8-hAAT-moFGF21 in comparison with age-matched mice treated with null vectors. Specifically, no differences were documented in the bone mineral density (BMD) (FIG. 29C), bone mineral content (BMC) (FIG. 29D), bone volume (BV) (FIG. 29E), bone volume/tissue volume ratio (BV/TV) (FIG. 29F), bone surface/bone volume ratio (BS/BV) (FIG. 29G), trabecular number (Tb.N) (FIG. 29H), trabecular thickness (Tb.Th) (FIG. 29I) or trabecular separation (Tb.Sp) (FIG. 29J). Similarly, the analysis of the compact bone at the tibial diaphysis showed no differences in the BMC, BMD, BV, BV/TV or BS/BV between the HFD-fed null-injected or FGF21-treated groups (FIG. 29K-O).

[0440] The pathological effects of FGF21 have been reported to be mediated, at least in part, by increased production of Insulin-like Growth Factor Binding Protein 1 (IGFPB1) by the liver (Wang X. et al., 2015. Cell Metab. 22:811-824). In agreement with the lack of bone alterations, high-dose AAV8-hAAT-moFGF21 treatment did not lead to an increase in the levels of circulating IGFBP1 protein in animals treated 12 (young adults) or 6 (adults) months earlier when compared to null-injected HFD-fed mice (FIG. 29P). Circulating IGF1 levels were also normal in all experimental groups (FIG. 29Q). Altogether these results support the safety for bone tissue of AAV-mediated FGF21 gene transfer to the liver.

Example 18. Prevention of HFD-Induced Liver Tumours by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors

[0441] Long-term feeding (>60 weeks) with a HFD has been associated with increased incidence of liver neoplasms in C57BL/6J mice (Hill-Baskin A. E. et al., 2009. Hum. Mol. Genet. 18:2975-2988; Nakagawa H., 2015. World J. Hepatol. 7:2110). In our study in animals that initiated the HFD as young adults and maintained it for 60 weeks we found liver tumours in 66.7% (6/9) of animals injected with null-vectors. Animals treated with AAV8-hAAT-moFGF21 vectors were protected from HFD-induced development of liver neoplasms: 0% (0/8) of animals treated with the 5?10.sup.10 vg of FGF21-encoding vectors showed tumours, and the incidence was 40% (4/10) in the cohort treated with the lowest dose (1?10.sup.10 vg). None (0/11) of the chow-fed mice developed tumours in the same period of time (Table 1).

TABLE-US-00001 TABLE 1 Liver tumour incidence in young adults. Hepatocarcinoma Group Hepatocarcinoma (%) Chow AAV8-null 0/11 0% HFD AAV8-null 6/9 66.7% HFD AAV8-FGF21 4/10 40% (1 ? 10.sup.10 vg/mouse) HFD AAV8-FGF21 0/8 0% (5 ? 10.sup.10 vg/mouse)

Example 19. Amelioration of STZ-Induced Hyperglycemia by Liver-Specific AAV8-Mediated FGF21 Overexpression

Material and Methods

Animals

[0442] We used 9-week-old male C57b16 mice. Mice had free access to a standard diet and were kept under a 12 h light-dark cycle (lights on at 08:00 hours). For diabetes induction, mice received five intraperitoneal injections, on consecutive days, of streptozotocin (50 mg/kg) dissolved in 0.1 mol/l citrate buffer (pH 4.5). Blood glucose levels were assessed using an analyser (Glucometer Elite; Bayer, Leverkusen, Germany). Animal care and experimental procedures were approved by the Ethics Committee in Animal and Human Experimentation of the Universitat Autonoma de Barcelona.

In Vivo Administration of AAV Vectors

[0443] For systemic administration, AAV vectors were diluted in 200 ?l of 0.001% F68 Pluronic? (Gibco) in PBS and injected via the tail vein.

Results

[0444] In order to test the protective potential against type 1 diabetes of AAV-derived FGF21, 5?10.sup.10 vg or 2?10.sup.11 vg of AAV8 vectors encoding a codon-optimized murine FGF21 coding sequence under the control of the hAAT promoter (AAV8-hAAT-moFGF21) were administered IV to male 9-week-old C57Bl6 mice. Control mice received 2?10.sup.11 vg of AAV8-hAAT-Null vectors. Two weeks post-AAV administration, all animals were treated with streptozotocin (STZ) (5 doses of 50 mg/kg; 1 dose per day) to trigger the diabetic process.

[0445] Analysis of the blood glucose levels revealed that animals treated with AAV8 vectors encoding moFGF21 displayed lower circulating glucose levels than C57Bl6 mice treated with AAV8-hAAT-Null vectors (FIG. 30).

Example 20. Extension of Healthy Lifespan by Intramuscular Administration of AAV-CMV-moFGF21 Vectors in C57Bl6 Mice Due to the Prevention of Weight Gain and Insulin Resistance Associated with Aging

[0446] Skeletal muscle (Skm) is a readily accessible tissue and has been used to produce secretable therapeutic proteins (Haurigot V. et al., 2010. J. Clin. Invest. 123:3254-3271; Callejas D. et al., 2013. Diabetes 62:1718-1729; Jaen M. L. et al., 2017. Mol. Ther. Methods Clin. Dev. 6:1-7). To explore if the Skm could represent a viable source of circulating FGF21, AAV vectors of serotype 1, which show a high tropism for Skm (Chao L. et al., 2000. J. Clin. Invest. 106: 1221-1228; Wu Z. et al., 2006. J. Virol. 80:9093-9103; Lisowski L. et al., 2015. Curr. Opin. Pharmacol. 24:59-67), carrying murine optimized FGF21 under the control of the CMV promoter were used (AAV1-CMV-moFGF21). Vectors were injected at a dose of 5?10.sup.10 vg/muscle to the quadriceps, gastrocnemius and tibialis cranialis of both legs (total dose, 3?10.sup.11 vg/mouse) of 8-week-old C57Bl6 mice. Control animals were injected with AAV1-CM V-Null vectors at the same dose. The use of healthy mice fed a standard diet further allowed us to evaluate the long-term safety of FGF21 gene therapy.

[0447] Eleven-month-old animals injected with FGF21-encoding vectors at 8 weeks of age showed a marked increase in circulating FGF21 (FIG. 31A), which was parallel to high levels of expression of vector-derived FGF21 in the 3 injected muscles (FIG. 31B). In agreement with previous reports, this combination of vector serotype, promoter and route of administration did not lead to expression of the transgene in the liver (FIG. 31B).

[0448] At the end of the ?10-month follow-up period, mice injected intramuscularly with AAV1-CMV-moFGF21 maintained the body weight they had at the initiation of the study and were ?38% slimmer than controls, which steady increased their weight as animals aged (FIG. 31C). While the weight of the muscles was barely affected by FGF21 gene transfer, the weight of the white and brown depots as well as the liver were considerably reduced (FIG. 31D). Indeed, the weight of the WAT pads analysed was reduced by >50% (FIG. 31D). Moreover, mice treated with AAV1-CMV-moFGF21 showed a marked reduction in the hepatic total triglyceride content (FIG. 31E). No changes in hepatic cholesterol levels were observed (FIG. 31F). As opposed to null-injected animals, animals treated with AAV1-CMV-moFGF21 showed normoglycemia (data not shown) and reduced insulinemia when they were approximately 1-year-old (FIG. 31G). Accordingly, FGF21-treated mice showed markedly improved insulin sensitivity at the end of the study (FIG. 31H). Altogether, this study demonstrates that administration of AAV vectors that leads to therapeutically-relevant levels of circulating FGF21 is safe in the long-term in healthy, and may be used to reverse the increase in body weight and insulin resistance associated to aging.

Example 21. Reversion of Obesity and Diabetes by Intramuscular Administration of AAV1-CMV-moFGF21 Vectors in HFD-Fed C57Bl6 Mice

[0449] We next evaluated whether im administration of AAV1-CMV-moFGF21 vectors was also able to reverse obesity and insulin resistance. To this end, two-month-old C57Bl6 mice were fed either a chow or a HFD for 12 weeks. During these first 3 months of follow-up, while the weight of chow-fed animals increased by 20%, animals fed a HFD became obese (95% body weight gain) (FIGS. 32A and B). Vectors were then injected at a dose of 5?10.sup.10 vg/muscle to the quadriceps, gastrocnemius and tibialis cranialis of both legs (total dose, 3?10.sup.11 vg/mouse) of obese C57Bl6 mice. As controls, another cohort of obese mice and the cohort of chow-fed mice received 3?10.sup.11 vg of non-coding null vectors (AAV1-CMV-null). Following AAV delivery, mice were maintained on chow or HFD feeding. Animals treated with AAV1-CMV-moFGF21 experienced progressive loss of body weight (FIGS. 32A and B). The reversion of obesity by AAV1-CMV-FGF21 treatment was parallel to an increase in the circulating levels of FGF21 (FIG. 32C).

[0450] Null-treated mice fed a HFD showed normal fed glycemia (FIG. 32D), but were hyperinsulinemic (FIG. 32E), suggesting that these mice had developed insulin resistance. In contrast, HFD-fed mice treated with AAV1-CMV-moFGF21 were, by the end of the study, normoglycemic and normoinsulinemic (FIGS. 32D and E). Moreover, animals administered with AAV1-CMV-moFGF21 showed greater insulin sensitivity than their HFD-fed controls (FIG. 32F).

Example 22. Increased FGF21 Circulating Levels by Codon-Optimized Human FGF21 Nucleotide Sequences

[0451] To evaluate if codon-optimization was able to mediate increased FGF21 circulating levels, 8-week-old male C57Bl6 mice were hydrodynamically injected with plasmids encoding three different codon-optimized human FGF21 nucleotide sequences (SEQ ID NO's: 40-42) under the control of the hAAT promoter. As control, non-treated mice and mice hydrodynamically injected with a plasmid encoding wild-type hFGF21 coding sequence under the control of the hAAT promoter were used.

Material and Methods

[0452] In vivo delivery of plasmids into mice by hydrodynamic tail vein injection Plasmid DNA was diluted in saline in a volume (ml) equal to ?10% of the animals' average body weight (grams) and was manually injected into the lateral tail vein in less tan 5 seconds. Before the injection, the animals were put under a 250 W infrared heat lamp (Philips) for a few minutes to dilate the blood vessels and facilitate viewing and easier access to the tail vein. A plastic restrainer (Harvard Apparatus) was used to secure the animal for injection. No anaesthesia was used as it is not necessary so long as an appropriate restraining device is employed. We used 26G 3/8 in. gauge hypodermic needles (BD), the largest feasible needle gauge that fit snugly into the access vein, to inject the animals.

Results

[0453] Mice treated with either codon-optimized human FGF21 version 2 or 3 were able to secrete higher human FGF21 levels into the circulation in comparison with wild-type or codon-optimized FGF21 variant 1 (FIG. 33, thus demonstrating increased FGF21 protein production by codon-optimization of variants 2 and 3.

Example 23. In Vitro and In Vivo Increased FGF21 Expression and Protein Production Levels by hAAT-moFGF21, CAG-moFGF21-doublemiRT and CMV-moFGF21 Expression Cassettes

Material and Methods

[0454] In vivo delivery of plasmids into mice by hydrodynamic tail vein injection Plasmid DNA was diluted in saline in a volume (ml) equal to ?10% of the animals' average body weight (grams) and was manually injected into the lateral tail vein in less tan 5 seconds. Before the injection, the animals were put under a 250 W infrared heat lamp (Philips) for a few minutes to dilate the blood vessels and facilitate viewing and easier access to the tail vein. A plastic restrainer (Harvard Apparatus) was used to secure the animal for injection. No anaesthesia was used as it is not necessary so long as an appropriate restraining device is employed. We used 26 G ? in. gauge hypodermic needles (BD), the largest feasible needle gauge that fit snugly into the access vein, to inject the animals.

Results

In Vitro

[0455] HEK293 cells were transfected with plasmids encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (EF1a-mFGF21) (Zhang et al., EBioMedicine 15 (2017) 173-183) (SEQ ID NO:57) or a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21) or the CAG promoter in conjunction with four tandem repeats of the miRT122a sequence and four tandems repeats of the miRT1 sequence (CAG-moFGF21-doublemiRT). As control, non-transfected cells were used. HEK293 cells transduced with CAG-moFGF21-doublemiRT expressed higher levels of FGF21 in comparison with cells transduced with EF1a-mFGF21 or non-transduced cells (FIG. 34A). Moreover, HEK293 cells transduced with CAG-moFGF21-doublemiRT also showed higher intracellular FGF21 protein content and higher FGF21 protein levels in the culture medium (FIGS. 34B and C). Although HEK293 cells transduced with EF1a-mFGF21 or CMV-moFGF21 expressed similar levels of FGF21 (FIG. 34A), HEK293 cells transduced with CMV-moFGF21 showed higher intracellular FGF21 protein content and higher FGF21 protein levels in the culture medium (FIGS. 34B and C).

[0456] C2C12 cells were transfected with plasmids encoding the WT murine FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) (Zhang et al., EBioMedicine 15 (2017) 173-183) or a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21). As control, non-transfected cells were used. C2C12 cells transduced with CMV-moFGF21 expressed higher levels of FGF21 in comparison with cells transduced with EF1a-mFGF21 or non-transduced cells (FIG. 34D).

[0457] HepG2 cells were transfected with plasmids encoding the WT murine FGF21 coding sequence under the control of the EF1a promoter (EF1a-mFGF21) (Zhang et al., EBioMedicine 15 (2017) 173-183) or a codon-optimized murine FGF21 coding sequence under the control of the hAAT promoter (hAAT-moFGF21). As control, non-transfected cells were used. HepG2 cells transduced with hAAT-moFGF21 expressed higher levels of FGF21 in comparison with cells transduced with EF1a-mFGF21 or non-transduced cells (FIG. 34E).

In Vivo

[0458] 8-week-old male C57Bl6 mice were hydrodynamically administered with 5 ?g of plasmids encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (EF1a-mFGF21) (Zhang et al., EBioMedicine 15 (2017) 173-183) or a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (CMV-moFGF21) or the hAAT promoter (hAAT-moFGF21). Analysis of FGF21 expression levels in the liver 24 h post-administration of plasmids revealed that animals treated with hAAT-moFGF21 or CMV-moFGF21 expressed much higher levels of FGF21 than animals receiving EF1a-mFGF21 (FIG. 35A). In addition, animals treated with hAAT-moFGF21 or CMV-moFGF21 showed higher FGF21 circulating levels than animals receiving EF1a-mFGF21 (FIG. 35B).

Example 24. In Vivo Increased FGF21 Expression in Target Tissues and FGF21 Circulating Levels by AAV8-hAAT-moFGF21, AAV8-CAG-moFGF21-doublemiRT and AAV1-CMC-moFGF21 in Comparison with AAV8-Ef1a-mFGF21

Hepatic Expression

[0459] Male C57Bl6 mice were intravenously administered with 1?10.sup.10 vg, 2?10.sup.10 vg or 5?10.sup.10 vg of AAV8 vectors encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or a codon-optimized murine FGF21 coding sequence under the control of the liver-specific hAAT promoter (AAV8-hAAT-moFGF21). Two weeks post-AAV administration, animals treated with AAV8-hAAT-moFGF21 showed both higher expression levels of FGF21 in the liver and higher FGF21 circulating levels than animals treated with AAV8-EF1a-mFGF21, irrespective of the dose of vector (FIGS. 36A and B).

Adipose Expression

[0460] Male C57Bl6 mice were administered intra-eWAT with 2?10.sup.10 vg, 5?10.sup.10 vg or 1?10.sup.11 vg of either AAV8 vectors encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or AAV8 vectors encoding a codon-optimized murine FGF21 coding sequence under the control of the CAG promoter in conjunction with four tandem repeats of the miRT122a sequence and four tandems repeats of the miRT1 sequence (AAV8-CAG-moFGF21-doublemiRT). Two weeks post-AAV administration, animals treated with AAV8-CAG-moFGF21-doublemiRT showed higher expression levels of FGF21 in WAT than animals administered with AAV8-EF1a-mFGF21 (FIG. 37A). Moreover, animals treated with AAV8-CAG-moFGF21-doublemiRT showed much lower expression of FGF21 in the liver than animals administered with AAV8-EF1a-mFGF21 (FIG. 37B), demonstrating that intra-eWAT administration of AAV8-CAG-moFGF21-doublemiRT vectors efficiently precluded transgene expression in off-target tissues.

Skeletal Muscle Expression

[0461] Male C57Bl6 mice were administered intramuscularly with 5?10.sup.10 vg, 1?10.sup.11 vg or 3?10.sup.11 vg of either AAV8 vectors encoding the WT murine FGF21 coding sequence under the control of the elongation factor 1a (EF1a) promoter (AAV8-EF1a-mFGF21) or AAV1 vectors encoding a codon-optimized murine FGF21 coding sequence under the control of the CMV promoter (AAV1-CMV-FGF21). Two weeks post-AAV administration, animals treated with AAV1-CMV-FGF21 showed much higher expression levels of FGF21 in skeletal muscle than animals administered with AAV8-EF1a-mFGF21 (FIG. 38A). Moreover, animals treated with AAV8-EF1a-mFGF21 showed high expression of FGF21 in the liver whereas intramuscular administration of AAV1-CMV-FGF21 vectors efficiently precluded hepatic transgene expression (FIG. 38B).

SEQUENCES

[0462]

TABLE-US-00002 SEQUENCES SEQ IDNO: Typeofsequence 1 AminoacidsequenceofhomosapiensFGF21 2 AminoacidsequenceofmusmusculusFGF21 3 AminoacidsequenceofcanislupusfamiliarisFGF21 4 NucleotidesequenceofhomosapiensFGF21 5 CodonoptimizednucleotidesequenceofhomosapiensFGF21- variant1 6 CodonoptimizednucleotidesequenceofhomosapiensFGF21- variant2 7 CodonoptimizednucleotidesequenceofhomosapiensFGF21- variant3 8 NucleotidesequenceofmusmusculusFGF21 9 CodonoptimizednucleotidesequenceofmusmusculusFGF21 10 NucleotidesequenceofcanislupusfamiliarisFGF21 11 Codonoptimizednucleotidesequenceofcanislupusfamiliaris FGF21 12 NucleotidesequenceencodingmiRT122a 13 NucleotidesequenceencodingmiRT1 14 NucleotidesequenceencodingmiRT152 15 NucleotidesequenceencodingmiRT199a-5p 16 NucleotidesequenceencodingmiRT199a-3p 17 NucleotidesequenceencodingmiRT215 18 NucleotidesequenceencodingmiRT192 19 NucleotidesequenceencodingmiRT148a 20 NucleotidesequenceencodingmiRT194 21 NucleotidesequenceencodingmiRT124 22 NucleotidesequenceencodingmiRT216 23 NucleotidesequenceencodingmiRT125 24 NucleotidesequenceencodingmiRT133a 25 NucleotidesequenceencodingmiRT206 26 NucleotidesequenceencodingmiRT130 27 NucleotidesequenceencodingmiRT99 28 NucleotidesequenceencodingmiRT208-5p 29 NucleotidesequenceencodingmiRT208a-3p 30 NucleotidesequenceencodingmiRT499-5p 31 ConstructA 32 ConstructB 33 ConstructC 34 ConstructD 35 ConstructE 36 ConstructF 37 ConstructG 38 ConstructH 39 ConstructI 40 ConstructJ 41 ConstructK 42 ConstructL 43 Nucleotidesequenceofchimericintroncomposedofintronsfrom human?-globinandimmunoglobulinheavychaingenes 44 NucleotidesequenceofCAGpromoter 45 NucleotidesequenceofCMVpromoter 46 NucleotidesequenceofCMVenhancer 47 NucleotidesequenceofhAATpromoter 48 TruncatedAAV25ITR 49 TruncatedAAV23ITR 50 SV40polyadenylationsignal 51 Rabbit?-Globinpolyadenylationsignal 52 CMVpromoterandCMVenhancersequence 53 Hepatocytecontrolregion(HCR)enhancerfromapolipoproteinE 54 mini/aP2promoter 55 mini/UCP1promoter 56 C5-12promoter 57 pAAV-EF1a-mmFGF21-pA AminoacidsequenceofhomosapiensFGF21 (SEQIDNO:1) MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDD AQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPD GALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRG PARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS NuleotidesequenceofhomosapiensFGF21 (SEQIDNO:4) ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTG CTGGCTGGTCTTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCA GTCCTCTCCTGCAATTCGGGGGCCAAGTCCGGCAGCGGTACCTCTACACAG ATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACG GTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCC TTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTG TGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAG GCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAG TCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACAC CGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTG CCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGAT GTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGC CCCAGCTACGCTTCCTGA CodonoptimizednucleotidesequenceofhomosapiensFGF21-variant1 (SEQIDNO:5) ATGGATTCTGATGAGACAGGCTTCGAGCACAGCGGCCTGTGGGTTTCAGTT CTGGCTGGACTGCTGCTGGGAGCCTGTCAGGCACACCCTATTCCAGATAGC AGCCCTCTGCTGCAGTTCGGCGGACAAGTGCGGCAGAGATACCTGTACACC GACGACGCCCAGCAGACAGAAGCCCACCTGGAAATCAGAGAGGATGGCAC AGTTGGCGGAGCCGCCGATCAGTCTCCTGAATCTCTGCTCCAGCTGAAGGC CCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAAACCAGCCGGTTCCT GTGCCAAAGACCTGACGGCGCCCTGTATGGCAGCCTGCACTTTGATCCTGA GGCCTGCAGCTTCAGAGAGCTGCTGCTTGAGGACGGCTACAACGTGTACCA GTCTGAGGCCCATGGCCTGCCTCTGCATCTGCCTGGAAACAAGAGCCCTCA CAGAGATCCCGCTCCTAGAGGCCCTGCCAGATTTCTGCCTCTTCCTGGATTG CCTCCTGCTCTGCCAGAGCCTCCTGGAATTCTGGCTCCTCAGCCTCCTGATG TGGGCAGCTCTGATCCTCTGAGCATGGTCGGACCTAGCCAGGGCAGATCTC CTAGCTACGCCTCTTGA CodonoptimizednucleotidesequenceofhomosapiensFGF21-variant2 (SEQIDNO:6) ATGGACAGCGATGAAACCGGGTTCGAGCACAGCGGTCTGTGGGTGTCCGTG CTGGCCGGACTGCTCCTGGGAGCCTGTCAGGCGCACCCCATCCCTGACTCC TCGCCGCTGCTGCAATTCGGCGGACAAGTCCGCCAGAGATACCTGTACACC GACGACGCCCAGCAGACCGAAGCCCACCTGGAAATTCGGGAGGACGGGAC TGTGGGAGGCGCTGCAGATCAGTCACCCGAGTCCCTCCTCCAACTGAAGGC CTTGAAGCCCGGCGTGATTCAGATCCTGGGCGTGAAAACTTCCCGCTTCCTT TGCCAACGGCCGGATGGAGCTCTGTACGGATCCCTGCACTTCGACCCCGAA GCCTGCTCATTCCGCGAGCTGCTCCTTGAGGACGGCTATAACGTGTACCAG TCTGAGGCCCATGGACTCCCCCTGCATCTGCCCGGCAACAAGTCCCCTCAC CGGGATCCTGCCCCAAGAGGCCCAGCTCGGTTTCTGCCTCTGCCGGGACTG CCTCCAGCGTTGCCCGAACCCCCTGGTATCCTGGCCCCGCAACCACCTGAC GTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAGCCAGGGAAGGTCC CCGTCCTACGCATCCTGA CodonoptimizednucleotidesequenceofhomosapiensFGF21-variant3 (SEQIDNO:7) ATGGATTCCGACGAAACTGGATTTGAACATTCAGGGCTGTGGGTCTCTGTG CTGGCTGGACTGCTGCTGGGGGCTTGTCAGGCTCACCCCATCCCTGACAGC TCCCCTCTGCTGCAGTTCGGAGGACAGGTGCGGCAGAGATACCTGTATACC GACGATGCCCAGCAGACAGAGGCACACCTGGAGATCAGGGAGGACGGAAC CGTGGGAGGAGCAGCCGATCAGTCTCCCGAGAGCCTGCTGCAGCTGAAGG CCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAGACATCTCGGTTTC TGTGCCAGCGGCCCGACGGCGCCCTGTACGGCTCCCTGCACTTCGATCCCG AGGCCTGTTCTTTTAGGGAGCTGCTGCTGGAGGACGGCTACAACGTGTATC AGAGCGAGGCACACGGCCTGCCACTGCACCTGCCTGGCAATAAGTCCCCTC ACCGCGATCCAGCACCCAGGGGCCCAGCACGCTTCCTGCCTCTGCCAGGCC TGCCCCCTGCCCTGCCAGAGCCACCCGGCATCCTGGCCCCCCAGCCTCCAG ATGTGGGCTCCAGCGATCCTCTGTCAATGGTGGGGCCAAGTCAGGGGCGGA GTCCTTCATACGCATCATAA NucleotidesequenceencodingmiRT122a(targetsequenceofmicroRNA122a) (SEQIDNO:12) 5CAAACACCATTGTCACACTCCA3 NucleotidesequenceencodingmiRT1(targetsequenceofmicroRNA1) (SEQIDNO:13) 5TTACATACTTCTTTACATTCCA3 NucleotidesequenceencodingmiRT152(targetsequenceofmicroRNA152) (SEQIDNO:14) 5CCAAGTTCTGTCATGCACTGA3 NucleotidesequenceencodingmiRT199a-5p(targetsequenceofmicroRNA199a) (SEQIDNO:15) 5GAACAGGTAGTCTGAACACTGGG3 NucleotidesequenceencodingmiRT199a-3p(targetsequenceofmicroRNA199a) (SEQIDNO:16) 5TAACCAATGTGCAGACTACTGT3 NucleotidesequenceencodingmiRT215(targetsequenceofmicroRNA215) (SEQIDNO:17) 5GTCTGTCAATTCATAGGTCAT3 NucleotidesequenceencodingmiRT192(targetsequenceofmicroRNA192) (SEQIDNO:18) 5GGCTGTCAATTCATAGGTCAG3 NucleotidesequenceencodingmiRT148a(targetsequenceofmicroRNA148a) (SEQIDNO:19) 5ACAAAGTTCTGTAGTGCACTGA3 NucleotidesequenceencodingmiRT194(targetsequenceofmicroRNA194) (SEQIDNO:20) 5TCCACATGGAGTTGCTGTTACA3 NucleotidesequenceencodingmiRT124(targetsequenceofmicroRNA124) (SEQIDNO:21) 5GGCATTCACCGCGTGCCTTA3 NucleotidesequenceencodingmiRT216(targetsequenceofmicroRNA216) (SEQIDNO:22) 5TCACAGTTGCCAGCTGAGATTA3 NucleotidesequenceencodingmiRT125(targetsequenceofmicroRNA125) (SEQIDNO:23) 5TCACAGGTTAAAGGGTCTCAGGGA3 NucleotidesequenceencodingmiRT133a(targetsequenceofmicroRNA133a) (SEQIDNO:24) 5CAGCTGGTTGAAGGGGACCAAA3 NucleotidesequenceencodingmiRT206(targetsequenceofmicroRNA206) (SEQIDNO:25) 5CCACACACTTCCTTACATTCCA3 NucleotidesequenceencodingmiRT130(targetsequenceofmicroRNA130) (SEQIDNO:26) 5ATGCCCTTTTAACATTGCACTG3 NucleotidesequenceencodingmiRT99(targetsequenceofmicroRNA99) (SEQIDNO:27) 5CACAAGATCGGATCTACGGGTT3 NucleotidesequenceencodingmiRT208-5p(targetsequenceofmicroRNA208a) (SEQIDNO:28) 5GTATAACCCGGGCCAAAAGCTC3 NucleotidesequenceencodingmiRT208a-3p(targetsequenceofmicroRNA208a) (SEQIDNO:29) 5ACAAGCTTTTTGCTCGTCTTAT3 NucleotidesequenceencodingmiRT499-5p(targetsequenceofheart-specific microRNA499) (SEQIDNO:30) 5AAACATCACTGCAAGTCTTAA3 NucleotidesequenceofCAGpromoter (SEQIDNO:44) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGT TCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCC GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTA TGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTC TCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAA TTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGG CGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCG GCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGG CGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGT CGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCG CCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGC CCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTT TCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGG GGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCG CGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGG GGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGT GCCCCGCGGTGCGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTG TGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAA CCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGT GCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGG GTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGG AGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGA GGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCG CAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGC CGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAG GAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCT TCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGG GGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAG AGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAG NucleotidesequenceofCMVpromoter (SEQIDNO:45) GTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCA CGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGC ACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGC CCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAG CAGAGCT NucleotidesequenceofCMVenhancer (SEQIDNO:46) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGT TCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCC GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTA TGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC AAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA TGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATG NucleotidesequenceofhAATpromoter (SEQIDNO:47) GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAG GGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCT CCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCT TTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCA GCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCT CCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGT TGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTC CTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT TruncatedAAV25ITR (SEQIDNO:48) GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGC CCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAG CGAGCGAGCG CGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT TruncatedAAV23ITR (SEQIDNO:49) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCG CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC SV40polyadenylationsignal (SEQIDNO:50) TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAA AAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTAT AAGCTGCAATAAACAAGTT Rabbit?-Globinpolyadenylationsignal (SEQIDNO:51) GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGC ATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTG GAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTT AAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATAT GCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAA ACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGT TAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAA TTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCC AGTCATAGCTGTCCCTCTTCTCTTATGGAGATC CMVpromoterandCMVenhancersequence (SEQIDNO:52) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGT TCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCC GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTA TGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC AAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA TGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTA TTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGC GTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACG TCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTC GTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGT ACGGTGGGAGGTCTATATAAGCAGAGCT Hepatocytecontrolregion(HCR)enhancerfromapolipoproteinE (SEQIDNO:53) CAGAGAGGTCTCTGACCTCTGCCCCAGCTCCAAGGTCAGCAGGCAGGGAGG GCTGTGTGTTTGCTGTTTGCTGCTTGCAATGTTTGCCCATTTTAGGGACATG AGTAGGCTGAAGTTTGTTCAGTGTGGACTTCAGAGGCAGCACACAAACAGC miniaP2promoter (SEQIDNO:54) GATTA ACCCGCCATGCTACTTATCTACTCGACATTGATTATTGACTAGGGGAATT CCAGCAGGAATCAGGTAGCTGGAGAATCGCACAGAGCCAT GCGATTCTTG GCAAGCCATGCGACAAAGGCAGAAATGCACATTTCACCCA GAGAGAAGGG ATTGATGTCAGCAGGAAGTCACCACCCAGAGAGCAAATGG AGTTCCCAGA TGCCTGACATTTGCCTTCTTACTGGATCAGAGTTCACTAGTGGAAGTGTC ACAGCCCAAACACTCCCCCAAAGCTCAGCCCTTCCTTGCCTTGTAACAAT CAAGCCGCTCCTGGATGAACTGCTCCGCCCTCTGTCTCTTTGGCAGGGTT GGAGCCCACTGTGGCCTGAGCGACTTCTATGGCTCCCTTTTCTGTGATTT TCATGGTTTCTGAGCTCTTTTCCCCCGCTTTATGATTTTCTCTTTTTGTC TCTCTCTTGCTAAACCTCCTTCGTATATATGCCCTCTCAGGTTTCATTTC TGAATCATCTACTGTGAACTATTCCCATTGTTTGCCAGAAGCCCCCTGGT TCTTCCTTCTAGACACCAGGCAAGGGGCAGGAGGTAAGAG GCAGGAGTCC ATAAAACAGCCCTGAGAGCCTGCTGGGTCAGTGCCTGCTGTCAGAA miniUCP1promoter (SEQIDNO:55) GACGTCACAGTGGGTCAGTCACCCTTGATCACACTGCACCAGTCTTCACC TTTCCACGCTTCCTGCCAGAGCATGAATCAGGCTCTCTGGGGATACCGGC CTCACCCCTACTGAGGCAAACTTTCTCCCACTTCTCAGAGGCTCTGAGGG CAGCAAGGTCAGCCCTTTCTTTGGAATCTAGAACCACTCCCTGTCTTGAG CTGACATCACAGGGCAGGCAGATGCAGCAGGGAAGGGCCT GGGACTGGGA CGTTCATCCTACAAGAAAGCTGTGGAACTTTTCAGCAACATCTCAGAAAT CAGATCGCACTTATTCAAAGGAGCCAGGCCCTGCTCTGCGCCCTGGTGGA GGCTCCTCATGTGAAGAGTGACAAAAGGCACCATGTTGTG GATACGGGGC GAAGCCCCTCCGGTGTGTCCTCCAGGCATCATCAGGAACT AGTGCCAAAG CAGAGGTGCTGGCCAGGGCTTTGGGAGTGACGCGCGTCTG GGAGGCTTGT GCGCCCAGGGCACGCCCCTGCCGATTCCCACTAGCAGGTC TTGGGGGACC TGGGCCGGCTCTGCCCCTCCTCCAGCAATCGGGCTATAAAGCTCTTCCAA GTCAGGGCGCAGAAGTGCCGGGCGATCCGGGCTTAAAGAG CGAGAGGAAG GGACGCTCACCTTTGAGCTCCTCCACAAATAGCCCTGGTGGCTGCCACAG AAGTTCGAAGTTGAGAGTTCGG C5-12promoter (SEQIDNO:56) CGGCCGTCCGCCTTCGGCACCATCCTCACGACACCCAAATATGGCGACGG GTGAGGAATG GTGGGGAGTTATTTTTAGAGCGGTGAGGAAGGTGGGCAGG CAGCAGGTGTTGGCGCTCTA AAAATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGG TGGACACCCAAATATGGCGA CGGTTCCTCACCCGTCGCCATATTTGGGTGTCCGCCCTCGGCCGGGGCCG CATTCCTGGG GGCCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCG GGGCCGGCGGCGGCCCACGA GCTACCCGGAGGAGCGGGAGGCGCCA pAAV-EF1a-mmFGF21-pA (SEQIDNO:57) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGC CCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGC GCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCGGCT CCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGT TGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGG GTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGT GGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGC AACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGG CCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTG GCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGA GAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGA GGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCG CGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGA CCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAG ATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCC GTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCAC CGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTG GCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGT CGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAG GGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA CCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGAC TCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTT GGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTC CCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGT AATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAG CCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAA TTTCGACTGCTAGCACGCGTGATATCAATGGAATGGATGAGATCTAGAGTT GGGACCCTGGGACTGTGGGTCCGACTGCTGCTGGCTGTCTTCCTGCTGGGG GTCTACCAAGCATACCCCATCCCTGACTCCAGCCCCCTCCTCCAGTTTGGGG GTCAAGTCCGGCAGAGGTACCTCTACACAGATGACGACCAAGACACTGAA GCCCACCTGGAGATCAGGGAGGATGGAACAGTGGTAGGCGCAGCACACCG CAGTCCAGAAAGTCTCCTGGAGCTCAAAGCCTTGAAGCCAGGGGTCATTCA AATCCTGGGTGTCAAAGCCTCTAGGTTTCTTTGCCAACAGCCAGATGGAGC TCTCTATGGATCGCCTCACTTTGATCCTGAGGCCTGCAGCTTCAGAGAACTG CTGCTGGAGGACGGTTACAATGTGTACCAGTCTGAAGCCCATGGCCTGCCC CTGCGTCTGCCTCAGAAGGACTCCCCAAACCAGGATGCAACATCCTGGGGA CCTGTGCGCTTCCTGCCCATGCCAGGCCTGCTCCACGAGCCCCAAGACCAA GCAGGATTCCTGCCCCCAGAGCCCCCAGATGTGGGCTCCTCTGACCCCCTG AGCATGGTAGAGCCTTTACAGGGCCGAAGCCCCAGCTATGCGTCCTGAGAT ATCAAAGAATTCTAAGCTTGTCGACGAATGCAATTGTTGTTAATTAATTGTT AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACA AATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCA AACTCATCAATGTATCTTAGTCGAGTTAATTAACGGCGGCCGCAGGAACCC CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGG CCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG TGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTC TCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATA GTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCG CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTC TTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCG GGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAA AAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACG GTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTT CCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAA GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAA AAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCA CTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACC CGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCG CTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTT CACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTAT TTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACAT TCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAA TATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTC CCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTG AAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGA ACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACG TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAG AATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGC ATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACT GCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCT TTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCG GAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGT AGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCT AGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAG GACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATC TGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGA TGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAAC TATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATC TGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG CAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACG ATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCA CACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCC AGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA CTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG CTCACATGT Elongationfactor1alphapromoter:from150to1327(1178bp) MusmusculusFGF21:from1359to1991(633bp) SEQIDNO:57alsocontainsthetruncatedAAV25and3ITRandtheSV40polyA (alreadyincludedinsequencelisting,SEQIDNO:48,49and50)