VIRAL EXPRESSION CONSTRUCT COMPRISING A FIBROBLAST GROWTH FACTOR 21 (FGF21) CODING SEQUENCE
20200102361 · 2020-04-02
Assignee
Inventors
- Fàtima BOSCH TUBERT (Cerdanyola del Valles, ES)
- Verónica JIMÉNEZ CENZANO (Cerdanyola del Valles, ES)
- Claudia Jambrina Pallares (Cerdanyola del Valles, ES)
Cpc classification
A01K67/0275
HUMAN NECESSITIES
C12N2750/14143
CHEMISTRY; METALLURGY
A01K2267/0362
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
C12N2015/8518
CHEMISTRY; METALLURGY
International classification
C12N15/86
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
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. A viral expression construct suitable for expression in a mammal and comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) to be expressed in liver, adipose tissue and/or skeletal muscle.
2. The viral expression construct according to claim 1 comprising a nucleotide sequence encoding a FGF21 suitable for expression in a mammal and at least one of elements a), b), c), d) and e): (a) a liver-specific promoter (b) an adipose tissue-specific promoter (c) a combination of an 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, wherein said combination enables specific expression in adipose tissue (d) a skeletal muscle promoter and (e) a combination of an ubiquitous promoter and an adeno-associated virus (AAV) vector sequence, wherein said combination enables specific expression in skeletal muscle.
3. The viral expression construct according to claim 1, wherein the nucleotide sequence encoding a FGF21 suitable for expression in a mammal 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 (c) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of (b) due to the degeneracy of the genetic code.
4. The viral expression construct according to claim 2, 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/or combinations thereof.
5. The viral expression construct according to claim 1, wherein the liver-specific promoter is the human 1-antitrypsin (hAAT) promoter and/or the adipose tissue-specific promoter is the mini/aP2 and/or the mini/UCP1 promoter and/or the skeletal muscle promoter is the C5-12 promoter and/or the ubiquitous promoter is the cytomegalovirus (CMV) promoter and/or the CAG promoter.
6. The viral vector comprising a viral expression construct according to claim 1, wherein said viral vector is an adenovirus vector, an adeno-associated virus vector, a retrovirus vector or a lentivirus vector, preferably an adeno-associated virus vector selected from the group consisting of an adeno-associated virus 1 (AAV1) vector, an adeno-associated virus 8 (AAV8) vector, and an adeno-associated virus 9 (AAV9) vector.
7. A nucleic acid molecule suitable for expression in a mammal and represented by a mammalian codon optimized nucleotide sequence encoding a FGF21 to be expressed in liver, adipose tissue and/or skeletal muscle.
8. The nucleic acid molecule according to claim 7, wherein the nucleotide sequence has at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10 or 11.
9. A composition comprising a viral expression construct as defined in claim 1, together with one or more pharmaceutically acceptable excipients or vehicles.
10.-16. (canceled)
17. A method for preventing, delaying, reverting, curing and/or treating a metabolic disorder comprising the use of a viral expression construct as defined in claim 1.
18. The method according to claim 17, wherein the metabolic disorder is diabetes and/or obesity.
19. The method according to claim 17, wherein the metabolic disorder is NASH.
20. A method for preventing, delaying, reverting, curing and/or treating liver inflammation and/or fibrosis, comprising the use of a viral expression construct as defined in claim 1.
21. A method for extending healthy lifespan, comprising the use of a viral expression construct as defined in claim 1.
22. A method for preventing, delaying, reverting, curing and/or treating cancer, preferably liver cancer, comprising the use of a viral expression construct as defined in claim 1.
23.-28. (canceled)
Description
FIGURE LEGENDS
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[0266] FGF21 labels in the figure refer to moFGF21.
[0267] Data information: All values are expressed as meanSEM. In (A, B) n=6-9 animals/group. In (C-H) 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.
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[0274] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0275] Data information: All values are expressed as meanSEM. In (A, B, D) n=4-8 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus null-injected ob/ob group.
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[0282] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0283] Data information: All data represent the meanSEM. In (A-C, E, G-H) n=9-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus null-injected ob/ob group.
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[0289] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0290] Data information: All values are expressed as meanSEM. In (B, D-F, HI) n=9-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus null-injected ob/ob group.
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[0295] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0296] Data information: All values are expressed as meanSEM. In (A-G) n=9-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus null-injected ob/ob group.
[0297]
[0300] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0301] Data information: All values are expressed as meanSEM. In (A-D) 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.
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[0307] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0308] Data information: All values are expressed as meanSEM. In (E) n=7-10 animals/group. HFD, High-fat diet. ND, non-detected.
[0309]
[0315] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0316] Data information: All values are expressed as meanSEM. In (A-C) n=7-10 animals/group. In (E) n=4 animals/group. In (G) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus 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.
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[0321] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0322] Data information: All values are expressed as meanSEM. In (A) n=7-10 animals/group. In (B) n=4 animals/group. In (C) n=7-10 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the chow-fed Null-injected group. .sup.###P<0.001 versus the HFD-fed Null-injected group. HFD, High-fat diet.
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[0328] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0329] Data information: All values are expressed as meanSEM. In (A-C) n=7-10 animals/group. In (D) n=4-5 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.
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[0333] Data information: All data represent the mean meanSEM. In (A-D) 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.
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[0340] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0341] Data information: All values are expressed as meanSEM. In (B) n=4 animals/group. In (F-H) 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.
[0342]
[0346] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0347] Data information: All values are expressed as meanSEM. In (A) n=4 animals/group. In (C-E) 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.
[0348]
[0352] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0353] Data information: All values are expressed as meanSEM. In (B-C) n=7-10 animals/group. **P<0.01 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.
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[0355] Analysis of hepatic fibrosis through Masson's trichrome staining in animals fed a HFD that received 510.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.
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[0359] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0360] Data information: All values are expressed as meanSEM. In (B-C) 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.
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[0366] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0367] Data information: All data represent the meanSEM. In (A, P-Q) n=7-10 animals/group. In (B-O) 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.
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[0377] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0378] Data information: All values are expressed as meanSEM. In (A-H) n=5-7 animals/group. *P<0.05, **P<0.01 and ***P<0.001 versus the Null-injected group.
[0379]
[0384] FGF21 labels in the figure refer to moFGF21 in accordance with this Figure legend.
[0385] Data information: All values are expressed as meanSEM. In (A-F) HFD-fed mice n=10 animals/group; chow-fed mice n=5 animals/group. ***P<0.001 versus the HFD-fed null-injected group.
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EXAMPLES
General Procedures to the Examples
[0392] Subject Characteristics
[0393] 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, Wis., US) or a high fat diet (TD.88137 Harlan Teklad Madison, Wis., 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, Ill., 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 Autnoma de Barcelona.
[0394] Recombinant AAV Vectors
[0395] 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 1-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.
[0396] In Vivo Intra-eWAT Administration of AAV Vectors
[0397] 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 504, 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.
[0398] Systemic Administration of AAV Vectors
[0399] 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 Nev., 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, Mass., 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.
[0400] Intramuscular Administration of AAV Vectors
[0401] 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.
[0402] Immunohistochemical and Morphometric Analysis
[0403] 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 (analySIS 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.
[0404] Immunohistochemistry
[0405] 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; 1-8510; 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).
[0406] RNA Analysis
[0407] 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, Ind., 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, Calif., USA). Real-time quantitative PCR was performed in a SmartCyclerII (Cepheid, Sunnyvale, USA) using EXPRESS SYBRGreen qPCR supermix (Invitrogen, Life Technologies Corp., Carslbad, Calif., US). Data was normalized with Rplp0 values and analyzed as previously described (Pfaffl, M., Nucleic Acids Res. 2001; 29(9):e45).
[0408] Hormone and Metabolite Assays
[0409] 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, Ill. 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 aqueous 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 aqueous 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 ressuspended by the addition of 500 l of H.sub.2O 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).
[0410] 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.
[0411] Insulin Tolerance Tests
[0412] For insulin tolerance tests, insulin (0.75 IU/kg body wt; Humulin Regular; Eli Lilly, Indianapolis, Ind.) 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.
[0413] Glucose Tolerance Test
[0414] 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.
[0415] Oximetry
[0416] 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.
[0417] Transfection of HEK293, C2C12 and HepG2 Cells
[0418] 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).
[0419] Bone Analysis
[0420] 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.
[0421] Western Blot Analysis
[0422] 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).
[0423] Open Field Test
[0424] The open field test was performed between 9:00 am and 1:00 m as previously reported (Haurigot et al, 2013). Briefly, animals were placed in the center of a brightly lit chamber (414130 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).
[0425] Statistical Analysis
[0426] All values are expressed as meanSEM. 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 C57B16 Mice
[0427] 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 C57B16. 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) (
[0428] Following AAV-mediated gene transfer of FGF21 to eWAT, mice fed a chow diet showed loss of body weight (
[0429] 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 (
[0430] 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 (
[0431] Histologic analysis 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 (
[0432] 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 (
[0433] HFD-fed mice overexpressing FGF21 were more insulin sensitive than HFD-fed AAV9-null treated mice (
Example 2. Reversion of Obesity and Improvement of Glucose Metabolism by Intra-eWAT Administration of AAV-CAG-moFGF21-dmiRT Vectors in Ob/Ob Mice
[0434] 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, 510.sup.10 vg, 210.sup.11 vg or 10.sup.12 vg) of AAV8-CAG-moFGF21-doublemiRT vectors (
[0435] 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 (
[0436] Animals treated with 510.sup.10 vg, 210.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 (
Example 3. Reversion of Obesity and Improvement of Glucose Metabolism by Intravenous Administration of AAV-hAAT-moFGF21 Vectors in Ob/Ob Mice
[0437] 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 510.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) (
[0438] 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 (
[0439] Animals treated with AAV8-hAAT-moFGF21 vectors showed improved insulin sensitivity and decreased insulin circulating levels in comparison with AAV8-hAAT-null-treated mice (
Example 4. Long-Term Reversion of Obesity and Diabetes by Intravenous Administration of AAV-hAAT-moFGF21 Vectors in HFD-Fed Mice
[0440] 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 C57B16 mice. Nine-week-old male C57B16 mice (young adults) were fed a HFD for 9 weeks and then administered IV with 10.sup.10 vg or 510.sup.10 vg of AAV8-hAAT-moFGF21 vectors (
[0441] Intravenous administration of AAV8-hAAT-moFGF21 vectors in HFD-fed mice mediated high secretion of FGF21 into the bloodstream in a dose-dependent manner (
[0442] 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 (
[0443] The energy expenditure of HFD-fed mice treated with 510.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 (
[0444] 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 (
Example 5. Reversion of Obesity and Diabetes by Intravenous Administration of AAV-hAAT-moFGF21 Vectors in Old HFD-Fed Mice
[0445] We also evaluated the anti-diabetic and anti-obesogenic effects of FGF21 in obese old (adults) C57B16 mice. Seven and a half-month-old male C57B16 mice were fed a HFD for 8 weeks and then administered IV with 10.sup.10 vg, 210.sup.10 vg or 510 vg of AAV8-hAAT-moFGF21 vectors (
[0446] 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 (
[0447] The energy expenditure of HFD-fed mice treated with 510.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 (
[0448] Animals treated with 10.sup.10 vg or 210.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 (
Example 6. Evaluation of Weight Loss by Intramuscular Administration of AAV-CMV-moFGF21 Vectors in C57B16 Mice
[0449] We also evaluated the therapeutic potential of increasing FGF21 circulating levels by the AAV-mediated genetic engineering of skeletal muscle in C57B16 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).
[0450] A dose of 310.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) (
[0451] Intramuscular administration of AAV1-CMV-moFGF21 vectors mediated high secretion of FGF21 into the bloodstream (
Example 7. Increased Protein Production by Codon-Optimized Human FGF21 Nucleotide Sequences
[0452] 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 (
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)
[0453] 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.
[0454] 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: [0455] measurement of body weight and food and liquid intake weekly [0456] measurement of body temperature [0457] measurement of energy expenditure and respiratory quotient by indirect calorimetry [0458] measurement of glycemia [0459] evaluation of whole-body glucose disposal by intraperitoneal glucose tolerance test [0460] evaluation of insulin sensitivity by intraperitoneal insulin tolerance test [0461] analyses in tissue and serum samples, including [0462] examination of the level of overexpression of human FGF21 in the targeted tissue and into the bloodstream [0463] morphological and histological analysis. [0464] determination of circulating levels of hormones and cytokines [0465] determination of serum metabolic parameters, such as free fatty acids, glycerol, triglycerides, cholesterol and ketone bodies [0466] 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
[0467] 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
[0468] 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).
[0469] 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 (
Example 11. Reversion of Obesity and Improvement of Glucose Metabolism by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors in Ob/Ob Mice: Further Observations
[0470] 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).
[0471] 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 510.sup.11 vg (
[0472] 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 (
[0473] 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 110.sup.11 vg of AAV8-hAAT-moFGF21 that showed increased PEPCK expression (
[0474] 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 (
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
[0475] 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
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
[0476] Increased energy expenditure (see Examples 4 and 5) was also seen in animals treated as young adults 10 months after AAV8-hAAT-moFGF21 delivery (
[0477] 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 510.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
[0478] 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 510.sup.10 vg AAV8-hAAT-moFGF21 showed decreased lipid deposition in iBAT (
[0479] 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 (
[0480] 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 510.sup.10 vg of AAV8-hAAT-moFGF21 when compared with age-matched, chow- and HFD-fed control groups (
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
[0481] 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 (
[0482] 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 210.sup.10 or 510.sup.10 vg/mouse) was similar to that of control mice fed a chow diet (
[0483] 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 110.sup.10 vg/mouse were glucose intolerant and showed markedly increased circulating levels of insulin during the GTT (
Example 15. Reversion of HFD-Associated WAT Hypertrophy and Inflammation by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors
[0484] 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 (
[0485] 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 510.sup.10 vg AAV8-hAAT-moFGF21 had no sign of macrophage infiltration (
Example 16. Reversal of Hepatic Steatosis, Inflammation and Fibrosis by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors
[0486] Histological analysis of the liver showed that all null-treated animals fed a HFD had marked hepatic steatosis at the time of sacrifice (
Example 17. Long-Term Safety of Liver-Directed AAV-FGF21 Treatment
[0487] 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 (
[0488] 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 (
Example 18. Prevention of HFD-Induced Liver Tumours by Intravenous Administration of AAV8-hAAT-moFGF21 Vectors
[0489] 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 510.sup.10 vg of FGF21-encoding vectors showed tumours, and the incidence was 40% (4/10) in the cohort treated with the lowest dose (110.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
[0490] Material and Methods
[0491] Animals
[0492] 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 Autnoma de Barcelona.
[0493] In Vivo Administration of AAV Vectors
[0494] For systemic administration, AAV vectors were diluted in 200 l of 0.001% F68 Pluronic (Gibco) in PBS and injected via the tail vein.
[0495] Results
[0496] In order to test the protective potential against type 1 diabetes of AAV-derived FGF21, 510.sup.10 vg or 210.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 C57B16 mice. Control mice received 210.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.
[0497] Analysis of the blood glucose levels revealed that animals treated with AAV8 vectors encoding moFGF21 displayed lower circulating glucose levels than C57B16 mice treated with AAV8-hAAT-Null vectors (
Example 20. Extension of Healthy Lifespan by Intramuscular Administration of AAV-CMV-moFGF21 Vectors in C57B16 Mice Due to the Prevention of Weight Gain and Insulin Resistance Associated with Aging
[0498] 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 510.sup.10 vg/muscle to the quadriceps, gastrocnemius and tibialis cranialis of both legs (total dose, 310.sup.11 vg/mouse) of 8-week-old C57B16 mice. Control animals were injected with AAV1-CMV-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.
[0499] Eleven-month-old animals injected with FGF21-encoding vectors at 8 weeks of age showed a marked increase in circulating FGF21 (
[0500] 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 (
Example 21. Reversion of Obesity and Diabetes by Intramuscular Administration of AAV1-CMV-moFGF21 Vectors in HFD-Fed C57B16 Mice
[0501] 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 C57B16 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) (
[0502] Null-treated mice fed a HFD showed normal fed glycemia (
Example 22. Increased FGF21 Circulating Levels by Codon-Optimized Human FGF21 Nucleotide Sequences
[0503] To evaluate if codon-optimization was able to mediate increased FGF21 circulating levels, 8-week-old male C57B16 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.
[0504] Material and Methods
[0505] In Vivo Delivery of Plasmids into Mice by Hydrodynamic Tail Vein Injection
[0506] 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 3/8 in. gauge hypodermic needles (BD), the largest feasible needle gauge that fit snugly into the access vein, to inject the animals.
[0507] Results
[0508] 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 (
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
[0509] Material and Methods
[0510] 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 3/8 in. gauge hypodermic needles (BD), the largest feasible needle gauge that fit snugly into the access vein, to inject the animals.
[0511] Results
[0512] In Vitro
[0513] 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 (
[0514] 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 (
[0515] 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 (
[0516] In Vivo
[0517] 8-week-old male C57B16 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 (
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
[0518] Hepatic Expression
[0519] Male C57B16 mice were intravenously administered with 110.sup.10 vg, 210.sup.10 vg or 510.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 (
[0520] Adipose Expression
[0521] Male C57B16 mice were administered intra-eWAT with 210.sup.10 vg, 510.sup.10 vg or 110.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 (
[0522] Skeletal Muscle Expression
[0523] Male C57B16 mice were administered intramuscularly with 510.sup.10 vg, 110.sup.11 vg or 310.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 (
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) 5 CAAACACCATTGTCACACTCCA3 NucleotidesequenceencodingmiRT1(targetsequenceofmicroRNA1) (SEQIDNO:13) 5 TTACATACTTCTTTACATTCCA3 NucleotidesequenceencodingmiRT152(targetsequenceofmicroRNA152) (SEQIDNO:14) 5 CCAAGTTCTGTCATGCACTGA3 NucleotidesequenceencodingmiRT199a-5p(targetsequenceofmicroRNA199a) (SEQIDNO:15) 5 GAACAGGTAGTCTGAACACTGGG3 NucleotidesequenceencodingmiRT199a-3p(targetsequenceofmicroRNA199a) (SEQIDNO:16) 5 TAACCAATGTGCAGACTACTGT3 NucleotidesequenceencodingmiRT215(targetsequenceofmicroRNA215) (SEQIDNO:17) 5 GTCTGTCAATTCATAGGTCAT3 NucleotidesequenceencodingmiRT192(targetsequenceofmicroRNA192) (SEQIDNO:18) 5 GGCTGTCAATTCATAGGTCAG3 NucleotidesequenceencodingmiRT148a(targetsequenceofmicroRNA148a) (SEQIDNO:19) 5 ACAAAGTTCTGTAGTGCACTGA3 NucleotidesequenceencodingmiRT194(targetsequenceofmicroRNA194) (SEQIDNO:20) 5 TCCACATGGAGTTGCTGTTACA3 NucleotidesequenceencodingmiRT124(targetsequenceofmicroRNA124) (SEQIDNO:21) 5 GGCATTCACCGCGTGCCTTA3 NucleotidesequenceencodingmiRT216(targetsequenceofmicroRNA216) (SEQIDNO:22) 5 TCACAGTTGCCAGCTGAGATTA3 NucleotidesequenceencodingmiRT125(targetsequenceofmicroRNA125) (SEQIDNO:23) 5 TCACAGGTTAAAGGGTCTCAGGGA3 NucleotidesequenceencodingmiRT133a(targetsequenceofmicroRNA133a) (SEQIDNO:24) 5 CAGCTGGTTGAAGGGGACCAAA3 NucleotidesequenceencodingmiRT206(targetsequenceofmicroRNA206) (SEQIDNO:25) 5 CCACACACTTCCTTACATTCCA3 NucleotidesequenceencodingmiRT130(targetsequenceofmicroRNA130) (SEQIDNO:26) 5 ATGCCCTTTTAACATTGCACTG3 NucleotidesequenceencodingmiRT99(targetsequenceofmicroRNA99) (SEQIDNO:27) 5 CACAAGATCGGATCTACGGGTT3 NucleotidesequenceencodingmiRT208-5p(targetsequenceofmicroRNA208a) (SEQIDNO:28) 5 GTATAACCCGGGCCAAAAGCTC3 NucleotidesequenceencodingmiRT208a-3p(targetsequenceofmicroRNA208a) (SEQIDNO:29) 5 ACAAGCTTTTTGCTCGTCTTAT3 NucleotidesequenceencodingmiRT499-5p(targetsequenceofheart-specific microRNA499) (SEQIDNO:30) 5 AAACATCACTGCAAGTCTTAA3 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:57alsocontainsthetruncatedAAV25 and3 ITRandtheSV40polyA (alreadyincludedinsequencelisting,SEQIDNO:48,49and50)