Fibroblast growth factor 21 (FGF21) gene therapy for central nervous system disorders
20230201306 · 2023-06-29
Assignee
Inventors
- Maria Fatima Bosch Tubert (Cerdanyola del Valles, ES)
- Veronica Jiménez Cenzano (Sabadell, ES)
- Ivet Elias Puigdomenech (Cerdanyola del Vallès, ES)
- Ignasi Grass Costa (Sabadell, ES)
- Claudia Jambrina Pallarés (Tarragona, ES)
- Victor Sacristan Fraile (Montcada i Reixac Barcelona, ES)
Cpc classification
C12N2750/14143
CHEMISTRY; METALLURGY
International classification
Abstract
Described herein is a gene construct comprising a nucleotide sequence encoding a fibroblast growth factor 21 (FGF21), for use in the treatment and/or prevention of a central nervous system (CNS) disorder or disease, or a condition associated therewith.
Claims
1. A method for the treatment and/or prevention of a central nervous system (CNS) disorder or disease, the method comprising administering a gene construct comprising a nucleotide sequence encoding a fibroblast growth factor 21 (FGF21).
2. The method according to claim 1, wherein the nucleotide sequence encoding FGF21 is operably linked to a ubiquitous promoter, preferably wherein the ubiquitous promoter is selected from the group consisting of a CAG promoter and a CMV promoter.
3. The method according to claim 1, wherein the gene construct comprises at least one target sequence of a microRNA expressed in a tissue where the expression of FGF21 is wanted to be prevented, preferably wherein the at least one target sequence of a microRNA is selected from those target sequences that bind to microRNAs expressed in heart and/or liver of a mammal.
4. The method according to claim 3, wherein the gene construct comprises at least one target sequence of a microRNA expressed in the liver and at least one target sequence of a microRNA expressed in the heart, preferably wherein a target sequence of a microRNA expressed in the heart is selected from SEQ ID NO's: 13 and 21-25 and a target sequence of a microRNA expressed in the liver is selected from SEQ ID NO's: 12 and 14-20, more preferably wherein the gene construct comprises a target sequence of microRNA-122a (SEQ ID NO: 12) and a target sequence of microRNA-1 (SEQ ID NO: 13).
5. The method according to claim 1, wherein the nucleotide sequence encoding FGF21 is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity 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 of which differs from the sequence of a nucleotide sequence of (b) due to the degeneracy of the genetic code.
6. The method according to claim 16, wherein the expression vector is a viral vector, more preferably wherein the expression vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors.
7. The method according to claim 6, wherein the expression vector is an adeno-associated viral vector, preferably an adeno-associated viral vector of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh10, rh8, Cb4, rh74, DJ, 2/5, 2/1, 1/2 or Anc80, more preferably an adeno-associated viral vector of serotype 1, 8 or 9.
8. The method according to claim 1, wherein the gene construct is comprised in a pharmaceutical composition.
9. The method according to claim 1, wherein the central nervous system (CNS) disorder or disease is associated with and/or caused by aging and/or a metabolic disorder or disease, preferably obesity and/or diabetes.
10. The method according to claim 1, wherein the central nervous system (CNS) disorder or disease is neuroinflammation, neurodegeneration, cognitive decline and/or a disease or condition associated therewith.
11. The method according to claim 10, wherein the disease or condition associated with and/or caused by neuroinflammation, neurodegeneration and/or cognitive decline is selected from the group consisting of: a cognitive disorder, dementia, Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia (FTD), Parkinson's disease, Parkinson-like disease, Parkinsonism, Huntington's disease, traumatic brain injury, prion disease, dementia/neurocognitive issues due to HIV infection, dementia/neurocognitive issues due to aging, tauopathy, multiple sclerosis and other neuroinflammatory/neurodegenerative diseases, preferably selected from the group consisting of Alzheimer's disease, Parkinson's disease, Parkinson-like disease and Huntington's disease, more preferably selected from the group consisting of Alzheimer's disease and Parkinson's disease, most preferably Alzheimer's disease.
12. The method according to claim 1, wherein the central nervous system (CNS) disorder or disease is a behavioral disorder, preferably an anxiety disorder or a depressive disorder.
13. The method according to claim 1, wherein the central nervous system (CNS) disorder or disease is a neuromuscular disorder, preferably wherein the neuromuscular disorder is, or is associated with, declined muscle function, declined muscle strength, declined coordination, declined balance and/or hypoactivity.
14. A method for improving memory and/or learning in a subject, the method comprising administering to the subject a gene construct comprising a nucleotide sequence encoding a fibroblast growth factor 21 (FG21).
15. A method for improving muscle function, muscle strength, coordination, balance and/or hypoactivity in a subject, the method comprising administering to the subject a gene construct comprising a nucleotide sequence encoding a fibroblast growth factor 21 (FG21).
16. The method of claim 1, wherein the gene construct is comprised in an expression vector.
17. The method of claim 16, wherein the expression vector is comprised in a pharmaceutical composition.
18. The method of claim 14, wherein the subject is an elderly subject and/or a subject diagnosed with a metabolic disorder or disease, preferably diabetes and/or obesity.
19. The method of claim 15, wherein the subject is an elderly subject and/or a subject diagnosed with a metabolic disorder or disease, preferably diabetes and/or obesity.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
[0319] In example 1, intramuscular administration of AAV1-CMV-moFGF21 mediates robust overexpression and increases circulating levels of FGF21 and has the following benefits: [0320] improved coordination, balance, neuromuscular performance, strength and locomotor activity [0321] enhanced memory and learning [0322] decreased neurodegeneration by improving mitochondrial function and diminution of oxidative stress
[0323] In Example 2, intramuscular administration of AAV1-CMV-moFGF21 and intra-eWAT administration of AAV8-CAG-moFGF21-dmiRT mediates robust overexpression and increases circulating levels of FGF21 and has the following benefits: [0324] improved locomotor activity and neuromuscular performance [0325] reduced anxiety-like behavior
[0326] In Example 3, intramuscular administration of AAV1-CMV-moFGF21 mediates robust overexpression and increases circulating levels of FGF21 and has the following benefits: [0327] improved coordination, balance, neuromuscular performance, strength and locomotor activity [0328] reduced anxiety-like behavior [0329] improved cognitive performance, memory, learning and exploratory capacity
[0330] In Example 4, intra-CSF administration of AAV1-CAG-moFGF21 mediates robust overexpression and has the following benefits: [0331] improved locomotor activity [0332] reduced anxiety-like behavior [0333] improved cognitive performance, memory and exploratory capacity
[0334] In Example 5, intra-CSF administration of AAV9-CAG-moFGF21-dmiRT mediates robust overexpression and has the following benefits: [0335] decreased neuroinflammation indicating improvement of depression
[0336] In Examples 8 and 9, intramuscular administration of AAV1-CMV-moFGF21 is shown to mediate a positive therapeutic effect in SAMP8 mice (widely used mouse model of senescence with age-related brain pathologies such as neuroinflammation) and in 3×Tg-AD mice (Alzheimer disease model).
[0337] In Example 10, intramuscular administration of AAV1-CMV-moFGF21 is shown to lead to improved coordination, balance and motor learning as well as short- and long-term memory.
[0338] In Example 11, it was shown that intramuscular administration of AAV1-CMV-moFGF21 inhibited neurodegeneration and cognitive decline by improvement of mitochondrial function, increase of glucose metabolism and autophagia, diminution of oxidative and ER stress, and amelioration of cholesterol homeostasis and synaptic function in cortex and hippocampus of old mice.
[0339] In Examples 12 and 13, intra-CSF administration of AAV1-CAG-moFGF21 improved the neuromuscular and cognitive decline associated with diabetes and obesity and improved neuromuscular performance and enhanced learning and short and long-term memory in old mice.
General Procedures to the Examples
[0340] Subject Characteristics
[0341] Male SAMP8/TaHsd (SAMP8), male and female C57Bl/6J mice, male BKS.Cg-+Lepr.sup.db/+Lepr.sup.db/OlaHsd (db/db) and male BKS.Cg-m+/+Lepr.sup.db/OlaHsd (db/+, lean) mice, male SAMR1/TaHsd (SAMR1) mice, male 3×Tg-AD (B6; 129Tg(APPSwe,tauP301L)1Lfa Psen1.sup.tm1Mpm) and male B6129SF2/J 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). When stated, mice were fasted for 16 h. 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 Autònoma de Barcelona.
[0342] Recombinant AAV Vectors
[0343] Single-stranded AAV vectors of serotype 1 or 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 or 8 cap gene, and a plasmid carrying the adenovirus helper functions. Transgenes used were: the murine codon-optimized FGF21 coding-sequence driven by 1) the cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter; 2) the cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter with the addition of four tandem repeats of the miRT122a sequence (5′CAAACACCATTGTCACACTCCA3′) (SEQ ID NO:12) and four tandems repeats of the miRT1 sequence (5′TTACATACTTCTTTACATTCCA3′) (SEQ ID NO:13) cloned in the 3′ untranslated region of the expression cassette; or 3) the CMV promoter. A Noncoding plasmid carrying the CMV promoter was 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.
[0344] In Vivo Intra-eWAT Administration of AAV Vectors
[0345] 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 μL 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.
[0346] Intramuscular Administration of AAV Vectors
[0347] 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 μl divided into six injection sites distributed in the quadriceps, gastrocnemius, and tibialis cranealis of each hind limb.
[0348] In Vivo Intra-CSF Administration of AAV Vectors
[0349] Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and the skin of the posterior part of the head, from behind the ears to approximately between the scapulas, was shaved and rinsed with ethanol. Mice were held in prone position, with the head at a slightly downward inclination. A 2-mm rostro-caudal incision was made to introduce a Hamilton syringe at an angle of 45-55° into the cisterna magna, between the occiput and the Cl-vertebra and 5 μl of vector dilution was administered. Given that the CNS is the main target compartment for vector delivery, mice were dosed with the same number of vector genomes/mouse irrespective of body weight (5×10.sup.9, 1×10.sup.10 and 5×10.sup.19 vg/mice).
[0350] RNA Analysis
[0351] Total RNA was obtained from adipose depots or skeletal muscle 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 μg 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., Carlsbad, Calif., US). Data was normalized with Rplp0 values and analyzed as previously described (Pfaffl, M., Nucleic Acids Res. 2001; 29(9):e45). Total RNA was obtained from hypothalamus, cortex, hippocampus, cerebellum and olfactory bulb using Tripure isolation reagent (Roche Diagnostics Corp., Indianapolis, Ind., US), and RNeasy Mini Kit or RNeasy Micro Kit for hippocampus samples (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 analysis, 1 μg 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 TB Green Premix Ex TaqII (Takara Bio Europe, France). Data was normalized with Rplp0 values and analyzed as previously described (Pfaffl, M., Nucleic Acids Res. 2001; 29(9):e45).
[0352] An overview of the primers used is shown below:
TABLE-US-00004 moFgf21-Fw: (SEQ ID NO: 47) 5′-CCTTTCCTGGTCGCCTCTTAG-3′ moFgf21-Rv: (SEQ ID NO: 48) 5′-GTTCCACCATGCTCAGAGGG-3′ Gfap-Fw: (SEQ ID NO: 49) 5′-ACAGACTTTCTCCAACCTCCAG-3′ Gfap-Rv: (SEQ ID NO: 50) 5′-CCTTCTGACACGGATTTGGT-3′ S100b-Fw: (SEQ ID NO: 51) 5′-AACAACGAGCTCTCTCACTTCC-3′ S100b-Rv: (SEQ ID NO: 52) 5′-CGTCTCCATCACTTTGTCCA-3′ Aif1-Fw: (SEQ ID NO: 53) 5′-TGAGCCAAAGCAGGGATTTG-3′ Aif1-Rv: (SEQ ID NO: 54) 5′-TCAAGTTTGGACGGCAGATC-3′ Nfkb-Fw: (SEQ ID NO: 55) 5′-GACCACTGCTCAGGTCCACT-3′ Nfkb-Rv: (SEQ ID NO: 56) 5′-TGTCACTATCCCGGAGTTCA-3′ Il1b-Fw: (SEQ ID NO: 57) 5′-ATGAAGGGCTGCTTCCAAAC-3′ Il1b-Rv: (SEQ ID NO: 58) 5′-ATGTGCTGCTGCGAGATTTG-3′ Il6-Fw: (SEQ ID NO: 59) 5′-TCGCTCAGGGTCACAAGAAA-3′ Il6-Rv: (SEQ ID NO: 60) 5′-CATCAGAGGCAAGGAGGAAAAC-3′ Rplp0-Fw: (SEQ ID NO: 61) 5′-ACTGGTCTAGGACCCGAGAA-3′ Rplp0-Fw: (SEQ ID NO: 62) 5′-TCCCACCTTGTCTCCAGTCT-3′ Ccl19-Fw: (SEQ ID NO: 69) 5′-GCGGGCTCACTGGGGCACAC-3′ Ccl19-Rv: (SEQ ID NO: 70) 5′-TGGGAAGGTCCAGAGAACCAG-3′ Ppargc1a-Fw: (SEQ ID NO: 71) 5′-TTTGGCCGACGACACGACTTTTC-3′ Ppargc1a-Rv: (SEQ ID NO: 72) 5′-TTGTGTTGGGCGAGAGAAAG-3′ Ppargc1b-Fw: (SEQ ID NO: 73) 5′-AGAAGCGCTTTGAGGTGTTC-3′ Ppargc1b-Rv: (SEQ ID NO: 74) 5′-GGTGATAAAACCGTGCTTCTGG-3′ Atp5f1a-Fw: (SEQ ID NO: 75) 5′-TCTCGGCCAGAGACTAGGAC-3′ Atp5f1a-Rv. (SEQ ID NO: 76) 5′-GCACTTGCACCAATGAATTT-3′ Mt-co1-Fw: (SEQ ID NO: 77) 5′-ATGAGCAAAAGCCCACTTCG-3′ Mt-co1-Rv: (SEQ ID NO: 78) 5′-ACCGTGGAGATTTGGTCCAG-3′ Cox6-Fw: (SEQ ID NO: 79) 5′-AGTCCCTCTGTCCCGTGTC-3′ Cox6-Rv: (SEQ ID NO: 80) 5′-ATATGCTGAGGTCCCCCTTT-3′ Cox5a-Fw: (SEQ ID NO: 81) 5′-CTCGTCAGCCTCAGCCAGT-3′ Cox5a-Rv: (SEQ ID NO: 82) 5′-TAGCAGCGAATGGAACAGAC-3′ Sod1-Fw: (SEQ ID NO: 83) 5′-TACACAAGGCTGTACCAGTGC-3′ Sod1-Rv: (SEQ ID NO: 84) 5′-TTTCCAGCAGTCACATTGCC-3′ Nrf2-Fw: (SEQ ID NO: 85) 5′-AGTCGCTTGCCCTGGATATC-3′ Nrf2-Rv: (SEQ ID NO: 86) 5′-TGCCAAACTTGCTCCATGTC-3′ Cat-Fw: (SEQ ID NO: 87) 5′-TGTGCATGCATGACAACCAG-3′ Cat-Rv: (SEQ ID NO: 88) 5′-GCACTGTTGAAGCGTTTCAC-3′ Gapdh-Fw: (SEQ ID NO: 89) 5′-CCTTCCGTGTTCCTACCC-3′ Gapdh-Rv: (SEQ ID NO: 90) 5′-CAACCTGGTCCTCACTGTAG-3′ Hkl-Fw: (SEQ ID NO: 91) 5′-ACGGTCAAAATGCTGCCTTC-3′ Hkl-Rv: (SEQ ID NO: 92) 5′-AATCGTTCCTCCGAGATCCA-3′ Pfkp-Fw: (SEQ ID NO: 93) 5′-TGTGTCTGAAGGAGCAATCG-3′ Pfkp-Rv: (SEQ ID NO: 94) 5′-GGCCAAAATCCTGTCAAATG-3′ Gpd1-Fw: (SEQ ID NO: 95) 5′-AGACACCCAACTTTCGCATC-3′ Gpd1-Rv: (SEQ ID NO: 96) 5′-TATTCTTCAAGGCCCCACAG-3′ Gpd2-Fw: (SEQ ID NO: 97) 5′-TTGCCTTGGGAGAAGATGAC-3′ Gpd2-Rv: (SEQ ID NO: 98) 5′-AGTTCCGCACTTCATTCAGG-3′ Pkm-Fw: (SEQ ID NO: 99) 5′-GCTTTGCATCTGATCCCATT-3′ Pkm-Rv: (SEQ ID NO: 100) 5′-AGTCCAGCCACAGGATGTTC-3′ Syp-Fw: (SEQ ID NO: 101) 5′-ACATGGACGTGGTGAATCAG-3′ Syp-Rv: (SEQ ID NO: 102) 5′-AAGATGGCAAAGACCCACTG-3′ Gria1-Fw: (SEQ ID NO: 103) 5′-CCATGCTGGTTGCCTTAATC-3′ Gria1-Rv: (SEQ ID NO: 104) 5′-CCGTATGGCTTCATTGATGG-3′ Gria2-Fw: (SEQ ID NO: 105) 5′-AAGGGCGTGTAATCCTTGAC-3′ Gria2-Rv: (SEQ ID NO: 106) 5′-TTTCAGCAGGTCTCCATCAG-3′ Grin1-Fw: (SEQ ID NO: 107) 5′-TGACTACCCGAATGTCCATC-3′ Grin1-Rv: (SEQ ID NO: 108) 5′-TTGTAGACGCGCATCATCTC-3′ Grin2a-Fw: (SEQ ID NO: 109) 5′-TGTGAAGAAGTGCTGCAAGG-3′ Grin2a-Rv: (SEQ ID NO: 110) 5′-CGCCTATCATTCCATTCCAC-3′ Grin2b-Fw: (SEQ ID NO: 111) 5′-TTGGTGAGGTGGTCATGAAG-3′ Grin2b-Rv: (SEQ ID NO: 112) 5′-TGCGTGATACCATGACACTG-3′ Sqstm1-Fw: (SEQ ID NO: 113) 5′-TGCTGGCGGCTTTACATTTG-3′ Sqstm1-Rv: (SEQ ID NO: 114) 5′-CAGAAGCAGAGAAGGAAAAGCC-3′ Atg5-Fw: (SEQ ID NO: 115) 5′-AGATGGACAGCTGCACACAC-3′ Atg5-Rv: (SEQ ID NO: 116) 5′-TTGGCTCTATCCCGTGAATC-3′ Atf4-Fw: (SEQ ID NO: 117) 5′-ATGATGGCTTGGCCAGTG-3′ Atf4-Rv: (SEQ ID NO: 118) 5′-CCATTTTCTCCAACATCCAATC-3′ Bip-Fw: (SEQ ID NO: 119) 5′-CTGAGGCGTATTGGGAAG-3′ Bip-Rv: (SEQ ID NO: 120) 5′-TCATGACATTCAGTCCAGCAA-3′ Cyp46a1-Fw: (SEQ ID NO: 121) 5′-TCGTTGAACGTCTCCATCAG-3′ Cyp46a1-Rv: (SEQ ID NO: 122) 5′-TTTGGGGAGAGACTGTTTGG-3′
[0353] Analysis of mRNA Expression with Microarrays
[0354] cDNA synthesis and array hybridization. For the analysis of the mRNA expression Affymetrix Clariom S Mouse microarray (Affymetrix, Thermo Fisher Scientific, Waltham, Mass., USA) was used. Approximately 300 ng of total RNA were processed using the GeneChip WT Plus Reagent kit (Affymetrix, Thermo Fisher Scientific, Waltham, Mass., USA) following the manufacturer instructions and hybridized to Affymetrix Clariom S Mouse microarray plates. The Affymetrix GeneChip Hybridization, Wash, and Stain kit were used for array processing. The chips were subsequently scanned with an Affymetrix GeneChip Scanner 3000.
[0355] Array quality control and normalization. The Expression Console™ Software (Affymetrix, Thermo Fisher Scientific, Waltham, Mass., USA) was also used to perform quality control of microarrays and to normalize the data of all the microarrays. RMA algorithm was used to perform background correction, log 2 transformation, and quantile normalization to allow the comparison of values across microarrays. Afterwards, Affymetrix Transcriptome Analysis Console Software (Affymetrix, Thermo Fisher Scientific, Waltham, Mass., USA) was used to annotate and compare FGF21 treated brain samples vs Null treated brain samples to generate a list of genes with computed fold change and p-value.
[0356] Measurement of FGF21 Circulating Levels
[0357] Circulating levels of FGF21 were determined by quantitative sandwich enzyme immunoassay Mouse/Rat FGF-21 ELISA kit (MF2100, R&Dsystems, Abingdon, UK).
[0358] Amyloid Beta Extraction and Quantification
[0359] Dissected cortex was homogenized using a sonicator (Sonics, Vibra-Cell, Newtown, USA) in cold T-PER buffer (ThermoScientific, Rockford, Ill., USA) supplemented with a protease inhibitor cocktail (Complete EDTA-free, Roche, Mannheim, Germany). After a brief sonication the samples were centrifuged at 100,000×g at 4° C. for 1 h in an Ultracentrifugue (Optima XPN-100, Beckman Coulter, Brea, Calif., USA) using a SW-55Ti rotor. The supernatant was labelled as the soluble fraction. The pellet was re-suspended in 70% formic acid solution. Sonication and centrifugation steps were repeated and the supernatant was recovered and dried for 4 hours in a vacuum concentrator (Savant SpeedVac DNA130 Concentrator, ThermoFischer Scientific). The dried formic extract was re-suspended in DMSO and labeled as insoluble fraction. All fractions were immediately stored at −80° C. until further use.
[0360] Aβ40 levels were quantified in the insoluble fraction by ELISA following the protocol recommended by the manufacturer (Human Aβ40 ELISA kit, Invitrogen, ref. KHB3481). Data were normalized to the total amount of protein in each sample (Pierce BCA Protein Assay Kit, Thermo Scientific, ref. 23225).
[0361] Open Field Test
[0362] 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 a corner of a white plastic walls and floor box (45×45×40 cm). For C57Bl/6J mice motor and exploratory activities were evaluated during the first 6 minutes using a video tracking system (SMART Junior; Panlab). For db/db mice and their control groups, mice were first habituated for 5 minutes in the open field arena. Then, they were placed for 5 minutes in the home cage and afterwards, they were placed again in the open field arena and motor and exploratory activities were evaluated during the first 12 minutes.
[0363] Novel Object Recognition Test
[0364] The novel object recognition tests were conducted in the open field box. Open-field test was used to acclimatize the mice to the box. The next day, to conduct the first trial, two identical objects (A and B) were placed in the upper right and upper left quadrants of the box, and then mice were placed backwards to both objects. After 10 min of exploration, mice were removed from the box, and allowed for 10 min break. In the second trial, one of the identical objects (A and B) was replaced with object C (new object). Mice were then put back into the box for a further 10 minutes of exploration for the short-term memory trial. For the long-term memory trial, the day after, the object C was replaced by a new object (D), allowing the mice to explore objects A and D for a further 10 minutes. The amount of time animals spent exploring the novel object was recorded and evaluated using a video tracking system (SMART Junior; Panlab). The evaluation of novel object recognition test memory was expressed as a percentage of the discrimination ratio calculated according to the following formula: Discrimination ratio (%)=(N−F)/(N+F)×100%, where N represents the time spent in exploring the new object and F represents the time spent in exploring the same object.
[0365] Rotarod Test
[0366] Mice were placed on a rotating rod (Panlab, Barcelona, Spain), spinning at 4 RPM. Lane width, 50 mm; rod diameter, 30 mm. Once stabilized, mice were subjected to an incrementally increasing speed of x RPM per x s. The first day of the experiment was used to train the animals in the use of the device. Each animal underwent 3 trials. The length of time that the mice managed to remain on the rod was recorded. Then, animals underwent 1 day resting and the third day, mice took 3 more trials on the rod. The average of 3 trials was analyzed. For evaluation of motor learning, performance in each individual trial was analyzed.
[0367] Grip Strength Test
[0368] A grip strength test meter (Panlab, Barcelona, Spain) was used to assess forelimb grip strength. The grip strength meter was positioned horizontally and mice were held by the tail and lowered towards the apparatus. Animals were allowed to grasp the metal bar with their front paws and were then pulled backwards in the horizontal plane. The force applied to the bar just before it lost grip was recorded as the peak tension. The average of 3 trials was analyzed.
[0369] Hang Wire Test
[0370] The wire hang test was conducted using a 55 cm wide 2-mm thick metallic wire which was secured to two vertical stands. The wire was maintained 35 cm above a layer of bedding material to prevent injury to the animal when it falls down. Mice, handled by the tail, were allowed to grasp the middle of the wire with its fore limbs. The time until mice fell down was measured. Mice that reached the limit suspension time of 180 seconds, independent on the trial number, were allowed to stop the experiment, while the others were directly retested for a maximum of three trials (a 30 seconds recovery period was used between trials).
[0371] Barnes Maze Test
[0372] The Barnes maze test consisted of an elevated circular platform with a 20 evenly-spaced holes around the perimeter. An escape box is mounted under one hole while the remaining 19 holes are left covered. During training and test, aversive stimulus such as bright light (more than 1000 lumens), open space and noise (more than 90 db) served as a motivation factor to induce escape behavior. Barnes maze was conducted in an empty room and visual cues in the walls were used as a reference. During the first day animals were acclimated during 1 minute in the scape box followed by 140 seconds in the open platform. Once all animals were acclimated, escape box was moved to another hole in the Barnes maze where it was maintained for the duration of the trainings. In the first training, mice were placed inside a PVC tube during 15 seconds in the middle of the Barnes maze and then PVC was released and animals were free to explore the platform and find the escape box for 140 seconds. If they found the correct hole and entered the escape box, animals remained inside for 30 seconds, if not the animals were guided to the scape box. In the following days (2, 3 and 4), two trainings per day were assessed as the first training. The last day (day 5), the scape box was removed, and a probe trial was conducted to assess memory for 180 seconds. The amount of time that animals spent exploring the Barnes maze was recorded and evaluated using a video tracking system (SMART Junior; Panlab). The time that animals spend until they found the scape box was calculated as a measure of memory.
[0373] Elevated Plus Maze
[0374] The elevated plus maze test was conducted in an apparatus which consists of open and closed arms, crossed in the middle, and a center area. The structure was elevated 90-100 cm from the floor. During the test, mice were placed in the center area and were allowed to move freely between arms for 5 minutes. The amount of time that animals spent exploring the open and closed arms was recorded and evaluated using a video tracking system (SMART Junior; Panlab). The number of entries into the open arms and the time spent in the open arms are used as index of open space-induced anxiety in mice.
[0375] Statistical Analysis
[0376] All values are expressed as mean±SEM. Data were analyzed by one-way ANOVA with Tukey's post hoc correction, except for those parameters involving comparison of only two experimental groups, in which case an unpaired Student's t-test was used. Differences were considered significant when P<0.05.
Example 1. Improved Neuromuscular Performance and Cognition and Decreased Neurodegeneration in Old Mice Treated with AAV Vectors Encoding FGF21
[0377] To evaluate whether genetic engineering of the skeletal muscle with FGF21 may exert therapeutic benefit in old animals, 13.5-month-old male C57Bl6 mice were administered intramuscularly with 3×10.sup.11 viral genomes (vg) of AAV vectors of serotype 1 encoding a murine codon-optimized FGF21 coding sequence (moFGF21) under the control of the CMV promoter (AAV1-CMV-moFGF21). Age-matched control animals were treated with the same dose of AAV1-CMV-Null vectors. Untreated cohorts of younger mice served as additional control groups. All experimental groups were fed with a chow diet.
[0378] AAV1-CMV-moFGF21 treated mice showed overexpression of codon-optimized FGF21 in the three injected muscles but not in off-target tissues such as the liver and heart (
[0379] Treatment of old mice with AAV1-CMV-moFGF21 vectors improved coordination and balance. Noticeably, no differences between 22-month-old AAV1-CMV-moFGF21-treated mice and 3-month-old untreated mice were observed (
[0380] To gain insight into the molecular mechanisms underlying the AAV-FGF21 mediated improvement of cognition, RNA from brain of old mice treated with AAV1-CMV-moFGF21 or AAV1-CMV-Null vectors was obtained, and transcriptomic analysis was performed using the Affymetrix Clariom S Mouse microarray technology. Pre-processing of the data was done using the Affymetrix Expression Console. Afterwards, the Affymetrix Transcriptome Analysis Console was used to compare brain samples from old mice treated with AAV1-CMV-moFGF21 or AAV1-CMV-Null vectors to generate a list of genes with computed fold change and p-value. Gene Set Enrichment Analysis (GSEA) was performed for interpreting transcriptomic data obtained from microarray analysis. This method relies on gene sets, that is, groups of genes that share common features based on prior biological knowledge, e.g., biological function, biological pathway, or cellular compartment (Subramanian, A. et al., 2005). These sets contain a variable number of genes (Size of gene set) and were retrieved from several databases such as Hallmark, KEGG, Reactome, or Gene Ontology (GO) and then overrepresentation analysis was computed. The goal of GSEA is to determine whether members of a gene set tend to correlate with treated vs non-treated samples. The degree to which a set is overrepresented was calculated and normalized to account for the size of the set, yielding a normalized enrichment score (NES), and the associated p-value to account for statistical significance.
[0381] In agreement with previous reports describing improvement of neurodegeneration and cognitive decline in animals treated with recombinant FGF21 protein mainly due to enhanced mitochondrial function and diminution of oxidative stress (Yu, Y. et al., 2015; Wang, X-M. et al., 2016; Sa-nguanmoo P. et al 2016; Sa-nguanmoo P. et al 2018; Chen S. et al., 2019; Amiri M. et al., 2018), the GSEA revealed that pathways related to oxidative phosphorylation, respiratory electron transport, uncoupling protein-mediated thermogenesis, reactive oxygen species, mitochondrial complexes and components, cristae formation and mitochondrial transmembrane transport were enriched in old-animals treated with AAV1-CMV-moFGF21 vectors in comparison with mice receiving AAV1-CMV-Null vectors (Table 1). The data thus indicates that FGF21 gene therapy inhibits neurodegeneration by improvement of mitochondrial function and diminution of oxidative stress.
TABLE-US-00005 TABLE 1 Enriched Gene Sets relevant to oxidative and mitochondrial metabolism obtained from GSEA analysis NES Size (# (normalized genes in enrichment Enriched set Database the set) score) p-value Oxidative hallmark 191 2.28 <0.001 phosphorylation Reactive oxygen hallmark 45 1.62 0.015 species pathway Oxidative KEGG 110 2.19 <0.001 phosphorylation Respiratory electron REACTOME 103 2.32 <0.001 transport atp synthesis by chemiosmotic coupling and heat production by uncoupling proteins Respiratory electron REACTOME 83 2.18 <0.001 transport Mitochondrial REACTOME 92 2.1 <0.001 translation Complex i REACTOME 48 2.06 <0.001 biogenesis The citric acid REACTOME 152 2.02 <0.001 tca cycle and respiratory electron transport Intrinsic component GO cellular 38 1.78 <0.001 of mitochondrial component inner membrane Intrinsic component GO cellular 65 1.75 0.004 of mitochondrial component membrane Mitochondrial GO cellular 237 1.69 0.027 protein complex component Inner mitochondrial GO cellular 115 1.68 0.018 membrane protein component complex Proton transporting GO cellular 20 1.65 <0.001 two sector atpase component complex proton transporting domain Organelle inner GO cellular 486 1.65 0.019 membrane component Proton transporting GO cellular 48 1.64 <0.001 two sector atpase component complex Inner mitochondrial GO biological 43 1.77 <0.001 membrane process organization Mitochondrial GO biological 90 1.73 <0.001 transmembrane process transport Cristae formation GO biological 30 1.69 <0.001 process Establishment GO biological 17 1.67 0.022 of protein process localization to mitochondrial membrane
Example 2. Reversal of Hypoactivity and Anxiety- and Depression-Like Symptoms in HFD-Fed Male Mice Treated with AAV Vectors Encoding FGF21
[0382] We evaluated the therapeutic potential of the AAV-mediated genetic engineering of adipose tissue or skeletal muscle with FGF21 to revert obesity- and diabetes-associated anxiety and decreased neuromuscular performance. To this end, 10-week-old male C57Bl6 mice were fed a HFD for 18 weeks. During these first 4 months of follow-up, while the weight of chow-fed animals increased by 25%, animals fed a HFD became obese (91% body weight gain) (
[0383] Animals treated with 5×10.sup.10 vg or 1×10.sup.11 vg of AAV8-CAG-moFGF21-dmiRT vectors initially lost 14% and 25% of body weight, respectively, and continued to progressively lose weight (
[0384] A clear dose-dependent loss of body weight was observed in the groups treated with AAV1-CMV-moFGF21. The lowest dose of vector did not counteract the weight gain associated to HFD-feeding, although the mean weight of these animals was always lower than that of control HFD-fed mice (
[0385] Animals treated intra-eWAT with 5×10.sup.10 or 1×10.sup.11 vg of AAV8-CAG-moFGF21-dmiRT vectors showed high levels of FGF21 in the bloodstream (
[0386] Treatment with AAV8-CAG-moFGF21-dmiRT or AAV1-CMV-moFG F21 vectors mediated effects on locomotor activity. In contrast to the hypoactivity observed in the open field test in the untreated animals fed a HFD, mice treated with 5×10.sup.10 vg or 1×10.sup.11 vg of AAV8-CAG-moFGF21-dmiRT showed the same degree of spontaneous locomotor activity than chow-fed animals (
[0387] Mice displaying diet-induced obesity have been reported to mimic the anxiety-like behaviour observed in obese and diabetes patients (Asato et al, Nihon Shinkei Seishin Yakurigaku Zasshi, 32 (5-6), 251-5 (2012)). We examined the anxiety-like behaviour by means of the open field test, which is widely used to assess this parameter in mice (Zhang, L-L. et al., 2011, Neuroscience, 196, 203-14). Mice prefer to move around the periphery of an apparatus when they are placed in an open field of a novel environment. Therefore, the time spent in the central area of the open field is considered to be inversely correlated to their level of anxiety-related proneness. 16.5-month-old untreated HFD-fed mice spent less time in the central zone as compared to age-matched chow-fed controls, suggesting an enhanced level of anxiety (
[0388] All these results suggest that treatment with FGF21-encoding AAV vectors improved the behavioural deficits associated with diabetes and obesity.
Example 3. Counteraction of Anxiety and Improvement of Neuromuscular Performance and Cognition in HFD-Fed Female Mice Treated with AAV Vectors Encoding FGF21
[0389] Next, we evaluated whether im administration of AAV1-CMV-moFGF21 vectors may mediate therapeutic benefit in obese and insulin resistance female mice. To this end, 11-week-old female C57Bl6 mice were fed a HFD for 8 weeks and subsequently treated in the quadriceps, gastrocnemius and tibialis cranialis skeletal muscle with AAV1-CMV-moFGF21 vectors at doses of 1×10.sup.11 or 3×10.sup.11 vg/mouse. Untreated chow and HFD-fed cohorts served as controls.
[0390] Female mice treated with 1×10.sup.11 vg of AAV1-CMV-moFGF21 vectors initially lost 5% body weight and showed always a mean weight lower than that of control HFD-fed mice (
[0391] Similar to the observations made in HFD-fed male mice treated im with AAV1-CMV-moFGF21 vectors, genetic engineering of the skeletal muscle of female mice with the same vectors also mediated a marked increase in circulating FGF21 levels (
[0392] To assess neuromuscular performance, the open field, the rotarod, and the grip strength tests were performed. During the open field test, female mice fed a HFD and overexpressing FGF21 in the skeletal muscle showed increased locomotor activity (
[0393] To test the effect of the treatment with AAV1-CMV-FGF21 vectors on cognitive performance, the novel object recognition and the Y-maze tests were performed. HFD-fed female mice treated with FGF21-encoding vectors performed markedly better than the untreated HFD-fed cohort in both tests. In the novel object recognition test, mice receiving 3×10.sup.11 vg/mouse of AAV1-CMV-FGF21 vectors had a recognition index equivalent to that of the chow-fed control cohort whereas mice treated with the dose of 1×10.sup.11 vg displayed better learning and memory than control lean animals (
[0394] All these results suggest that treatment with FGF21-encoding AAV vectors improved the neuromuscular and cognitive decline associated with diabetes and obesity.
Example 4. Increased Locomotor Activity and Amelioration of Anxiety-Like Behaviour, Exploratory Capacity and Cognition in db/db Mice Treated with AAV Vectors Encoding FGF21
[0395] The therapeutic potential for cognitive decline of the AAV-mediated genetic engineering of the brain with FGF21 gene therapy was evaluated in db/db mice. db/db mice are a widely used genetic mouse model of obesity and diabetes, characterized by a deficit in leptin signalling. Moreover, db/db mice have also been used as a mice model of neuroinflammation and cognitive decline (Dey et al, J. Neuroimmmunol. 2014; Dinel et al Plos one 2011; Stranahan et al Nat Neurosci 2008; Zheng, Biochimica and Biophysica Acta 2017).
[0396] Two-month-old db/db male mice were administered locally intra-cerebrospinal fluid (CSF), through the cisterna magna, with 5×10.sup.10 vg/mouse of AAV1 vectors encoding a murine codon-optimized FGF21 coding sequence under the control of the CAG ubiquitous promoter (AAV1-CAG-moFGF21). As controls, non-treated db/db and non-treated db/+(lean) mice were used.
[0397] Intra-CSF administration of AAV1-CAG-moFGF21 vectors mediated widespread overexpression of FGF21 in the brain, as evidenced by the increased expression levels of the factor in different areas of the brain such as hypothalamus, cortex, hippocampus, cerebellum and olfactory bulb, 16 weeks after AAV administration (
[0398] An open field test was performed at 9 weeks of age to all groups of mice. Non-treated db/db mice showed a reduction in the distance travelled, the maximum velocity and in the fast time (
[0399] The anxiety-like behaviour was also studied in the open field, and the impairment observed in db/db non-treated mice (increased distance in the border and reduced distance in the center) (
[0400] An Y-maze test was performed to all groups of mice at 10 weeks of age and showed that non-treated db/db mice had less exploratory capacity than db/+ lean mice (
[0401] To test the effect of the intra-CSF treatment with AAV1-CAG-moFGF21 vectors on memory, the novel object recognition test was performed at 11 weeks of age. db/db mice treated with AAV1-CAG-moFGF21-encoding vectors performed markedly better than the untreated db/db cohort (
Example 5 Decreased Neuroinflammation Indicating Reduction of Depression in db/db and SAMP8 Mice Treated with AAV Vectors Encoding FGF21
[0402] We also evaluated the potential of the AAV-mediated FGF21 gene therapy for decreasing neuroinflammation.
[0403] First, we used a senescence-accelerated mouse-prone 8 (SAMP8) mice, which is a widely used mouse model of senescence with age-related brain pathologies such as neuroinflammation (Takeda T., Neurochem. Res. 2009, 34(4):639-659; Griñan-Ferré C. et al. Mol. Neurobiol. 2016, 53(4):2435-2450). Inflammation in the brain was analyzed through the expression of astrocyte markers Gfap and S100b, the microglia marker Aif1 and pro-inflammatory molecules, such as Nfkb, II1b and II6. Expression of the pro-inflammatory cytokines 111b and 116 was decreased in the hypothalamus of SAMP8 mice overexpressing FGF21 in the brain (
[0404] Second, we used db/db mice which are a widely used genetic mouse model of obesity and diabetes, characterized by a deficit in leptin signalling. Moreover, these mice present not only inflammation in peripheral tissues such as adipose tissue and liver but also in the brain (Dey et al, J. Neuroimmmunol. 2014). db/db mice treated intra-CSF with AAV9-CAG-moFGF21-dmiRT vectors showed decreased expression of Gfap, S100b, Aif1, Nfkb, 111b and 116 in the hypothalamus (
[0405] The decrease in astrocyte markers accompanied with a decrease in the expression levels of the inflammatory cytokines indicates that after FGF21 gene therapy treatment there is a decrease in the population of deleterious astrocytes (A1 astrocytes) and also a decrease in microglia.
[0406] Many studies have supported that inflammatory processes play a central role in the aetiology of depression (Wang et al. 2020 Front. Pharmacol., 28 Feb. 2020). Together with the reduced depression-like behaviour observed in examples 2, 3 and 4, this indicates that the FGF21 gene therapy has an anti-depressant effect.
Example 6. Intramuscular Administration of AAV1-CMV-moFGF21 Vectors in SAMP8 Mice
[0407] To further evaluate the therapeutic potential of the AAV-mediated genetic engineering of the skeletal muscle with FGF21 on cognitive decline, SAMP8 mice are used. The SAMP8 mouse model presents cognitive decline by the age of 8-12 months (Miyamoto, M., Physiol Behay. 1986; 38(3):399-406; Markowska, A L., Physiol Behay. 1998; 64(1):15-26).
[0408] SAMP8 mice are administered im with 3×10.sup.11 vg/mouse of AAV1-CMV-moFGF21 vectors. As control, non-treated SAMP8 and SAMR1 animals are used. Several behavioural and neuromuscular tests such as Y-Maze, Open-Field, novel object recognition test, rotarod, hang wire test, grip strength test and Morris Water Maze are performed in these mice. At sacrifice, serum and tissue samples are taken for analysis. Analysis of these samples include studies on neurogenesis (expression of neuronal markers such as Sox2, NeuN, and Dcx), neuroinflammation (expression of GFAP, Iba1 and several cytokine levels), studies on synaptic degeneration (protein levels of synaptophysin and spine density).
Example 7. Intramuscular Administration of AAV1-CMV-moFGF21 Vectors in an Alzheimer's Disease Mouse Model
[0409] To evaluate the therapeutic potential of the AAV-mediated genetic engineering of the skeletal muscle with FGF21 on Alzheimer's disease, the 3×Tg-AD (B6; 129Tg(APPSwe,tauP301L)1Lfa Psen1.sup.tm1Mpm) mouse model is used. The 3×Tg-AD is a widely used mouse model of Alzheimer's disease, homozygous for all three mutant alleles, homozygous for the Psen1 mutation and homozygous for the co-injected APPSwe and tauP301 L transgenes (Belfiore, R., Aging Cell. 2019, 18(1):e12873)
[0410] 3×Tg-AD mice are administered im with 3×10.sup.11 vg/mouse of AAV1-CMV-moFGF21 vectors. As control, non-treated 3×Tg-AD animals are used. Several behavioural and neuromuscular tests such as Y-Maze, Open-Field, novel object recognition test, rotarod, hang wire test, grip strength test and Morris Water Maze are performed in these mice. At sacrifice, serum and tissue samples are taken for analysis. Analysis of these samples include studies on neurogenesis (expression of neuronal markers such as Sox2, NeuN, and Dcx), neuroinflammation (expression of GFAP, Iba1 and several cytokine levels), levels of amyloid-beta (soluble amyloid and plaques), studies on synaptic degeneration (protein levels of synaptophysin and spine density), levels of tau phosphorylation.
Example 8. Improved Neuromuscular Performance and Cognition in SAMP8 Mice Treated Intramuscularly with AAV1-CMV-moFGF21 Vectors
[0411] Eight-week-old male SAMP8 mice were administered im with 3×10.sup.11 vg/mouse of AAV1-CMV-moFGF21 vectors. As control, non-treated SAMP8 and SAMR1 animals were used.
[0412] AAV1-CMV-moFGF21 treated SAMP8 mice showed specific overexpression of codon-optimized FGF21 in the three injected muscles and increased FGF21 circulating levels (
[0413] To test the effect of the treatment with AAV1-CMV-moFGF21 vectors on neuromuscular performance, the rotarod test was performed. SAMP8 mice administered im with 3×10.sup.11 vg of AAV1-CMV-moFGF21 vectors were able to stay longer on the accelerating rotarod than untreated SAMP8 and SAMR1, demonstrating improvement of coordination and balance (
[0414] Inflammation in the brain was analyzed through the expression of chemokine (C—C motif) ligand 19 (Ccl19) and II6. Cccl19 has been postulated to play a primary role on the neuropathological phenotype of SAMP8 (Carter T A. Genome Biol. 2005; 6(6):R48). SAMP8 showed markedly increased Ccl19 expression levels in cortex and hippocampus in comparison with SAMR1 mice (
[0415] All these results suggest that treatment with AAV1-FGF21 enhanced neuromuscular performance, motor learning and memory, and decreased brain inflammation in SAMP8 mice.
Example 9. Improved Memory in an Alzheimer's Disease Mouse Model Treated Intramuscularly with AAV1-CMV-moFGF21 Vectors
[0416] Eight-week-old male 3×Tg-AD mice were administered im with 3×10.sup.11 vg/mouse of AAV1-CMV-moFGF21 vectors. As control, non-treated 3×TG-AD and B6129SF2/J animals were used.
[0417] Similar to the observations made in SAMP8 mice treated im with AAV1-CMV-moFGF21 vectors, genetic engineering of the skeletal muscle of 3×Tg-AD mice with the same vectors also mediated a marked increase in circulating FGF21 levels (
[0418] Accumulation of amyloid plaques (primary made of amyloid-β (Aβ)) in brain and memory loss are key hallmarks of Alzheimer's disease (Belfiore R. et al. Aging Cell. 2019; 18(1):e12873). Treatment of 3×Tg-AD mice with AAV1-CMV-moFGF21 vectors precluded cognitive decline as demonstrated by the markedly improved short- and long-term memory in treated 3×Tg-AD mice in comparison with untreated 3×Tg-AD mice (
Example 10. Improved Neuromuscular Performance and Cognition in Old Mice Treated im with Different Doses of AAV1-CMV-moFGF21 Vectors
[0419] Thirteen-month-old male C57B16 mice were administered intramuscularly with 1×10.sup.11 or 3×10.sup.11 vg of AAV1-CMV-moFGF21 vectors. Untreated age-matched control animals served as controls.
[0420] AAV1-CMV-moFGF21-treated mice showed secretion of FGF21 into the bloodstream in a dose-dependent manner (
Example 11. Molecular Mechanisms and Brain Areas Involved in Preclusion of Neurodegeneration and Cognitive Decline in Old Mice Treated im with AAV1-CMV-moFGF21
[0421] As previously mentioned, whole brain transcriptomic analysis suggested that improvement of mitochondrial function and diminution of oxidative stress mediated inhibition of neurodegeneration and cognitive decline in old mice treated im AAV1-CMV-moFGF21 vectors (Table 1). Next, we characterized the specifically affected brain areas as well as decipher additional molecular mechanisms involved in the improvement of cognitive performance in AAV1-FGF21 treated mice.
[0422] Measurement of several oxidative phosphorylation (OXPHOS) and antioxidant markers by qPCR further corroborated GSEA findings (
[0423] The brain is an energy-demanding organ and relies heavily on efficient ATP production via glycolysis, the TCA cycle and oxidative phosphorylation (Butterfield D A. Nat Rev Neurosci 2019 March; 20(3):148-160). Given that glycolysis is in charge of metabolization of glucose for OXPHOS, expression levels of key glycolysis-related genes were determined. Old mice treated with AAV1-CMV-moFGF21 vectors showed increased expression of glycerldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase 1 (Hk1), platelet isoform of phosphofructokinase (Pfkp) and glycerol-3-Phosphate Dehydrogenase 1 and 2 (Gpd1 and Gpd2, respectively) in cortex and of pyruvate kinase M (Pkm) and Gpd2 in hippocampus, suggesting enhanced glycolysis in these brain areas (
[0424] All these results suggest that treatment of old mice with AAV1-CMV-moFGF21 precluded age-associated decreased glucose metabolism and mitochondrial dysfunction, which would ensure efficient ATP production for neuronal function.
[0425] It is worth to mention that mitochondrial perturbations and reduced ATP production have been reported to contribute to synaptic dysfunction and degeneration, which correlate strongly with cognitive deficits and memory loss (Butterfield D A. Nat Rev Neurosci 2019 March; 20(3):148-160; Cai Q. J Alzheimers Dis. 2017; 57(4):1087-1103). Noticeably, by 25 months of age, old animals treated with AAV1-CMV-moFGF21 showed robust increased expression of key synaptic proteins. (
[0426] Brain autophagic capacity has been reported to decrease with age and to cause neurodegeneration (Lipinski M M. Proc Natl Acad Sci USA 2010; 107:14164-9; Hara T. Nature 2006; 441:885-9; Komatsu M. Nature 2006; 441:880-4). Old mice treated im with AAV1-CMV-moFGF21 vectors showed increased expression of the autophagy markers p62 (encoded by the Sqstm1 gene) and autophagy related 5 (Atg5) in cortex (
[0427] Finally, a strong association between abnormalities in brain cholesterol homeostasis (especially high concentrations in neurons) and several neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and Huntington's disease, has been observed (Vance J E. Dis Model Mech 2012; 5:746-55). Cholesterol 24-hydroxylase, encoded by Cyp46a1, controls cholesterol efflux from the brain and thereby plays a major role in regulating brain cholesterol homeostasis. Moreover, increasing evidence suggest that Cyp46a1 has a role in the pathogenesis and progression of neurodegenerative disorders, and that increasing its levels in the brain is neuroprotective (Kacher R. Brain. 2019; 142(8):2432-2450; Djelti F. Brain 2015; 138(Pt 8):2383-98). In agreement, treatment with AAV1-CMV-moFGF21 vectors increased the expression of Cyp46a1 in cortex of old mice (
[0428] Altogether, these results indicate that FGF21 gene therapy inhibited neurodegeneration and cognitive decline by improvement of mitochondrial function, increase of glucose metabolism and autophagia, diminution of oxidative and ER stress, and amelioration of cholesterol homeostasis and synaptic function in cortex and hippocampus of old mice.
Example 12. Counteraction of Anxiety and Improvement of Neuromuscular Performance and Cognition in HFD-Fed Male Mice Treated Intra-CSF with AAV Vectors Encoding FGF21
[0429] We next evaluated whether intra-CSF administration of AAV1-CAG-moFGF21 vectors may mediate therapeutic benefit in obese and insulin resistance male mice. To this end, 8-week-old male C57Bl6 mice were fed a HFD for 3 months. During these first 3 months of follow-up the body weight of chow-fed animals increased by 32% while animals fed a HFD became obese (84% body weight gain). Obese animals were then administered intra-CSF with 5×10.sup.9 or 1×10.sup.10 vg/mouse of AAV1-CAG-moFGF21 vectors. Untreated chow and HFD-fed cohorts served as controls. Initially, HFD-fed mice treated with AAV1 vectors lost body weight, reaching similar levels than those of age-matched chow-diet fed mice (
[0430] Similar to the observations made in db/db male mice treated intra-CSF with AAV1-CAG-moFGF21 vectors, genetic engineering of the brain of HFD-fed mice with the same vectors also mediated a specific overexpression of the factor in different brain areas (
[0431] To assess neuromuscular performance, at the end of the follow-up period, the open field test was performed. During the open field test, HFD-fed mice administered intra-CSF with the two doses of the AAV1 vectors showed increased locomotor activity (
[0432] To test the effect of the intra-CSF treatment with AAV1-CAG-FGF21 vectors on cognitive performance, the novel object recognition and the Barnes maze tests were performed. In the novel object recognition test, mice receiving both doses of AAV1-CAG-FGF21 vectors had a recognition index equivalent to that of the chow-fed control cohort, both at the short and long-term memory trial (
[0433] All these results suggest that intra-CSF treatment with FGF21-encoding AAV vectors improved the neuromuscular and cognitive decline associated with diabetes and obesity.
Example 13. Improved Neuromuscular Performance and Cognition in Old Mice Treated with AAV Vectors Encoding FGF21
[0434] To evaluate whether intra-CSF gene therapy with FGF21 may exert therapeutic benefit in old animals, 13-month-old male C57Bl6 mice were administered intra-CSF with 5×10.sup.9 and 1×10.sup.10 vg/mouse of AAV1-CAG-moFGF21 vectors. Untreated cohorts served as controls. All experimental groups were fed with a chow diet during all the experiment.
[0435] To assess neuromuscular performance, the rotarod test was performed to all groups at 23 months of age. Old mice treated intra-CSF with all doses of AAV1-CAG-moFGF21 were able to stay longer on the accelerating rotarod than untreated old counterparts (
[0436] Moreover, by 24-25 months of age, mice treated with 5×10.sup.9 vg/mouse of FGF21-encoding vectors performed markedly better in the novel object recognition test, both at the short- and long-term trials (
TABLE-US-00006 Sequences SEQ ID NO: Description of the sequence 1 Amino acid sequence of homo sapiens FGF21 2 Amino acid sequence of mus musculus FGF21 3 Amino acid sequence of canis lupus familiaris FGF21 4 Nucleotide sequence of homo sapiens FGF21 5 Codon optimized nucleotide sequence of homo sapiens FGF21-variant 1 6 Codon optimized nucleotide sequence of homo sapiens FGF21-variant 2 7 Codon optimized nucleotide sequence of homo sapiens FGF21-variant 3 8 Nucleotide sequence of mus musculus FGF21 9 Codon optimized nucleotide sequence of mus musculus FGF21 10 Nucleotide sequence of canis lupus familiaris FGF21 11 Codon optimized nucleotide sequence of canis lupus familiaris FGF21 12 Nucleotide sequence encoding miRT-122a 13 Nucleotide sequence encoding miRT-1 14 Nucleotide sequence encoding miRT-152 15 Nucleotide sequence encoding miRT-199a-5p 16 Nucleotide sequence encoding miRT-199a-3p 17 Nucleotide sequence encoding miRT-215 18 Nucleotide sequence encoding miRT-192 19 Nucleotide sequence encoding miRT-148a 20 Nucleotide sequence encoding miRT-194 21 Nucleotide sequence encoding miRT-133a 22 Nucleotide sequence encoding miRT-206 23 Nucleotide sequence encoding miRT-208-5p 24 Nucleotide sequence encoding miRT-208a-3p 25 Nucleotide sequence encoding miRT-499-5p 26 Nucleotide sequence of chimeric intron composed of introns from human β-globin and immunoglobulin heavy chain genes 27 Nucleotide sequence of CAG promoter 28 Nucleotide sequence of CMV promoter 29 Nucleotide sequence of CMV enhancer 30 Truncated AAV2 5′ ITR 31 Truncated AAV2 3′ ITR 32 SV40 polyadenylation signal 33 Rabbit β-globin polyadenylation signal 34 CMV promoter and CMV enhancer sequence 35 pAAV-CAG-moFGF21-dmiRT 36 mini-CMV promoter 37 EF1α promoter 38 RSV promoter 39 Synapsin 1 promoter 40 Calcium/calmodulin-dependent protein kinase II (CaMKII) promoter 41 Glial fibrillary acidic protein (GFAP) promoter 42 Nestin promoter 43 Homeobox Protein 9 (HB9) promoter 44 Tyrosine hydroxylase (TH) promoter 45 Myelin basic protein (MBP) promoter 46 pAAV-CAG-moFGF21 47-62 and RT-qPCR primers 69-122 63 pAAV-CMV-moFGF21 64 Nucleotide sequence of hAAT promoter 65 Hepatocyte control region (HCR) enhancer from apolipoprotein E 66 mini/aP2 promoter 67 mini/UCP1 promoter 68 C5-12 promoter Amino acid sequence of homo sapiens FGF21 (SEQ ID NO: 1) MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIR EDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLE DGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDP LSMVGPSQGRSPSYAS Nucleotide sequence of homo sapiens FGF21 (SEQ ID NO: 4) ATGGACTCGGACGAGACCGGGTTCGAGCACTCAGGACTGTGGGTTTCTGTGCTGGCTGGTC TTCTGCTGGGAGCCTGCCAGGCACACCCCATCCCTGACTCCAGTCCTCTCCTGCAATTCGG GGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTG GAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTG CAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCT GTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGC TTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCC CGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTC GCTTCCTGCCACTACCAGGCCTGCCCCCCGCACTCCCGGAGCCACCCGGAATCCTGGCCC CCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCC GAAGCCCCAGCTACGCTTCCTGA Codon optimized nucleotide sequence of homo sapiens FGF21-variant 1 (SEQ ID NO: 5) ATGGATTCTGATGAGACAGGCTTCGAGCACAGCGGCCTGTGGGTTTCAGTTCTGGCTGGAC TGCTGCTGGGAGCCTGTCAGGCACACCCTATTCCAGATAGCAGCCCTCTGCTGCAGTTCGG CGGACAAGTGCGGCAGAGATACCTGTACACCGACGACGCCCAGCAGACAGAAGCCCACCT GGAAATCAGAGAGGATGGCACAGTTGGCGGAGCCGCCGATCAGTCTCCTGAATCTCTGCTC CAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAAACCAGCCGGTTCC TGTGCCAAAGACCTGACGGCGCCCTGTATGGCAGCCTGCACTTTGATCCTGAGGCCTGCAG CTTCAGAGAGCTGCTGCTTGAGGACGGCTACAACGTGTACCAGTCTGAGGCCCATGGCCTG CCTCTGCATCTGCCTGGAAACAAGAGCCCTCACAGAGATCCCGCTCCTAGAGGCCCTGCCA GATTTCTGCCTCTTCCTGGATTGCCTCCTGCTCTGCCAGAGCCTCCTGGAATTCTGGCTCCT CAGCCTCCTGATGTGGGCAGCTCTGATCCTCTGAGCATGGTCGGACCTAGCCAGGGCAGAT CTCCTAGCTACGCCTCTTGA Codon optimized nucleotide sequence of homo sapiens FGF21-variant 2 (SEQ ID NO: 6) ATGGACAGCGATGAAACCGGGTTCGAGCACAGCGGTCTGTGGGTGTCCGTGCTGGCCGGA CTGCTCCTGGGAGCCTGTCAGGCGCACCCCATCCCTGACTCCTCGCCGCTGCTGCAATTCG GCGGACAAGTCCGCCAGAGATACCTGTACACCGACGACGCCCAGCAGACCGAAGCCCACC TGGAAATTCGGGAGGACGGGACTGTGGGAGGCGCTGCAGATCAGTCACCCGAGTCCCTCC TCCAACTGAAGGCCTTGAAGCCCGGCGTGATTCAGATCCTGGGCGTGAAAACTTCCCGCTT CCTTTGCCAACGGCCGGATGGAGCTCTGTACGGATCCCTGCACTTCGACCCCGAAGCCTGC TCATTCCGCGAGCTGCTCCTTGAGGACGGCTATAACGTGTACCAGTCTGAGGCCCATGGAC TCCCCCTGCATCTGCCCGGCAACAAGTCCCCTCACCGGGATCCTGCCCCAAGAGGCCCAGC TCGGTTTCTGCCTCTGCCGGGACTGCCTCCAGCGTTGCCCGAACCCCCTGGTATCCTGGCC CCGCAACCACCTGACGTCGGTTCGTCGGACCCGCTGAGCATGGTCGGTCCGAGCCAGGGA AGGTCCCCGTCCTACGCATCCTGA Codon optimized nucleotide sequence of homo sapiens FGF21-variant 3 (SEQ ID NO: 7) ATGGATTCCGACGAAACTGGATTTGAACATTCAGGGCTGTGGGTCTCTGTGCTGGCTGGACT GCTGCTGGGGGCTTGTCAGGCTCACCCCATCCCTGACAGCTCCCCTCTGCTGCAGTTCGGA GGACAGGTGCGGCAGAGATACCTGTATACCGACGATGCCCAGCAGACAGAGGCACACCTG GAGATCAGGGAGGACGGAACCGTGGGAGGAGCAGCCGATCAGTCTCCCGAGAGCCTGCTG CAGCTGAAGGCCCTGAAGCCTGGCGTGATCCAGATCCTGGGCGTGAAGACATCTCGGTTTC TGTGCCAGCGGCCCGACGGCGCCCTGTACGGCTCCCTGCACTTCGATCCCGAGGCCTGTT CTTTTAGGGAGCTGCTGCTGGAGGACGGCTACAACGTGTATCAGAGCGAGGCACACGGCCT GCCACTGCACCTGCCTGGCAATAAGTCCCCTCACCGCGATCCAGCACCCAGGGGCCCAGCA CGCTTCCTGCCTCTGCCAGGCCTGCCCCCTGCCCTGCCAGAGCCACCCGGCATCCTGGCC CCCCAGCCTCCAGATGTGGGCTCCAGCGATCCTCTGTCAATGGTGGGGCCAAGTCAGGGG CGGAGTCCTTCATACGCATCATAA Nucleotide sequence of murine codon-optimized FGF21 (SEQ ID NO: 9) ATGGAATGGATGAGAAGCAGAGTGGGCACCCTGGGCCTGTGGGTGCGACTGCTGCTGGCT GTGTTTCTGCTGGGCGTGTACCAGGCCTACCCCATCCCTGACTCTAGCCCCCTGCTGCAGTT TGGCGGACAAGTGCGGCAGAGATACCTGTACACCGACGACGACCAGGACACCGAGGCCCA CCTGGAAATCCGCGAGGATGGCACAGTCGTGGGCGCTGCTCACAGAAGCCCTGAGAGCCT GCTGGAACTGAAGGCCCTGAAGCCCGGCGTGATCCAGATCCTGGGCGTGAAGGCCAGCAG ATTCCTGTGCCAGCAGCCTGACGGCGCCCTGTACGGCTCTCCTCACTTCGATCCTGAGGCC TGCAGCTTCAGAGAGCTGCTGCTGGAGGACGGCTACAACGTGTACCAGTCTGAGGCCCACG GCCTGCCCCTGAGACTGCCTCAGAAGGACAGCCCTAACCAGGACGCCACAAGCTGGGGAC CTGTGCGGTTCCTGCCTATGCCTGGACTGCTGCACGAGCCCCAGGATCAGGCTGGCTTTCT GCCTCCTGAGCCTCCAGACGTGGGCAGCAGCGACCCTCTGAGCATGGTGGAACCTCTGCA GGGCAGAAGCCCCAGCTACGCCTCTTGA Nucleotide sequence of CAG promoter (SEQ ID NO: 27) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATT ACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCAC CCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGG GGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGC GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTG CCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACC GCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCT TGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAG GGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAG CGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGG CTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTG CGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCA GGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCT GAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGT GCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGC CGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGC GCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTT TGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGC GCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCG TCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGC TGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTC TAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAG Nucleotide sequence of CMV promoter (SEQ ID NO: 28) GTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGT ACGGTGGGAGGTCTATATAAGCAGAGCT Nucleotide sequence of CMV enhancer (SEQ ID NO: 29) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC CAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATT ACCATG CMV promoter and CMV enhancer sequence (SEQ ID NO: 34) GGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC CAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATT ACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGG GATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGG GACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAG GCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT AAV2 5′ ITR (SEQ ID NO: 30) GCGCGCTC GCTCGCTCAC TGAGGCCGCC CGGGCAAAGC CCGGGCGTCG GGCGACCTTT GGTCGCCCGG CCTCAGTGAG CGAGCGAGCG CGCAGAGAGG GAGTGGCCAA CTCCATCACT AGGGGTTCCT AAV2 3′ ITR (SEQ ID NO: 31) AGGAACCCCT AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGC Rabbit β-globin polyadenylation signal (3′ UTR and flanking region of rabbit beta-globin, including polyA signal) (SEQ ID NO: 33) GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCT GGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG GAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTAT ATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATT TTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTAC TAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGA GATC miRT seguences miRT-122a (SEQ ID NO: 12): 5′ CAAACACCATTGTCACACTCCA 3′, target for the microRNA-122a (Accession Number to the miRBase database MI0000442), which is expressed in the liver. miRT-152 (SEQ ID NO: 14): 5′ CCAAGTTCTGTCATGCACTGA 3′, target for the microRNA-152 (MI0000462), which is expressed in the liver. miRT-199a-5p (SEQ ID NO: 15): 5′ GAACAGGTAGTCTGAACACTGGG 3′, target for the microRNA 199a (MI0000242), which is expressed in the liver. miRT-199a-3p (SEQ ID NO: 16): 5′ TAACCAATGTGCAGACTACTGT 3′, target for the microRNA-199a (MI0000242), which is expressed in the liver. miRT-215 (SEQ ID NO: 17): 5′ GTCTGTCAATTCATAGGTCAT 3′, target for the microRNA-215 (MI0000291), which is expressed in the liver. miRT-192 (SEQ ID NO: 18): 5′ GGCTGTCAATTCATAGGTCAG 3′, target for the microRNA-192 (MI0000234), which is expressed in the liver. miRT-148a (SEQ ID NO: 19): 5′ ACAAAGTTCTGTAGTGCACTGA 3′, target for the microRNA-148a (MI0000253), which is expressed in the liver. miRT-194 (SEQ ID NO: 20): 5′ TCCACATGGAGTTGCTGTTACA 3′, target for the microRNA-194 (MI0000488), which is expressed in the liver. miRT-133a (SEQ ID NO: 21): 5′ CAGCTGGTTGAAGGGGACCAAA 3′, target for the microRNA-133a (MI0000450), which is expressed in the heart. miRT-206 (SEQ ID NO: 22): 5′ CCACACACTTCCTTACATTCCA 3′, target for the microRNA-206 (MI0000490), which is expressed in the heart. miRT-1 (SEQ ID NO: 13): 5′ TTACATACTTCTTTACATTCCA 3′, target for the microRNA-1 (MI0000651), which is expressed in the heart. miRT-208a-5p (SEQ ID NO: 23): 5′ GTATAACCCGGGCCAAAAGCTC 3′, target for the microRNA-208a (MI0000251), which is expressed in the heart. miR1-208a-3p (SEQ ID NO: 24): 5′ ACAAGCTTTTTGCTCGTCTTAT 3′, target for the microRNA-208a (MI0000251), which is expressed in the heart. miRT-499-5p (SEQ ID NO: 25): 5′ AAACATCACTGCAAGTCTTAA 3′, target for the microRNA-499 (MI0003183), which is expressed in the heart. pAAV-CAG-moFGF21-dmiRT (SEQ ID NO: 35) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAATCGGCC AACGCGCGGG 51 GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT 101 CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG 151 GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT 201 GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC 251 TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC 351 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC 401 TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT 451 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA 501 GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT 551 CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG 651 TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA 701 CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA 751 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT 801 TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG 851 ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT 951 CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT 1001 AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA 1051 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA 1101 GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA 1151 TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC 1251 CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG 1301 TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA 1351 CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG 1401 GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG 1451 GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG 1551 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA 1601 TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG 1651 CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG 1701 GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC 1751 CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG 1851 GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT 1901 GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT 1951 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT 2001 GCCACCTGAC GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA 2051 ATAGGCGTAT CACGAGGCCC TTTCGTCTCG CGCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA 2151 AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG 2201 GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG 2251 AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA 2301 ATACCGCATC AGGCGATTCC AACATCCAAT AAATCATACA GGCAAGGCAA 2351 AGAATTAGCA AAATTAAGCA ATAAAGCCTC AGAGCATAAA GCTAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTAATA CTTTTGCGGG AGAAGCCTTT 2451 ATTTCAACGC AAGGATAAAA ATTTTTAGAA CCCTCATATA TTTTAAATGC 2501 AATGCCTGAG TAATGTGTAG GTAAAGATTC AAACGGGTGA GAAAGGCCGG 2551 AGACAGTCAA ATCACCATCA ATATGATATT CAACCGTTCT AGCTGATAAA 2601 TTCATGCCGG AGAGGGTAGC TATTTTTGAG AGGTCTCTAC AAAGGCTATC 2651 AGGTCATTGC CTGAGAGTCT GGAGCAAACA AGAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC CCCGGTTGAT AATCAGAAAA 2751 GCCCCAAAAA CAGGAAGATT GTATAAGCAA ATATTTAAAT TGTAAGCGTT 2801 AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT 2851 TAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA 2901 CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA 2951 AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT 3051 GCCGTAAAGC ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT 3101 TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA 3151 AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA 3201 CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTACTATGGT 3251 TGCTTTGACG AGCACGTATA ACGTGCTTTC CTCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT TAGACAGGAA CGGTACGCCA 3351 GAATCCTGAG AAGTGTTTTT ATAATCAGTG AGGCCACCGA GTAAAAGAGT 3401 CTGTCCATCA CGCAAATTAA CCGTTGTCGC AATACTTCTT TGATTAGTAA 3451 TAACATCACT TGCCTGAGTA GAAGAACTCA AACTATCGGC CTTGCTGGTA 3501 ATATCCAGAA CAATATTACC GCCAGCCATT GCAACGGAAT CGCCATTCGC 3551 CATTCAGGCT GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC ACTGAGGCCG CCCGGGCAAA 3651 GCCCGGGCGT CGGGCGACCT TTGGTCGCCC GGCCTCAGTG AGCGAGCGAG 3701 CGCGCAGAGA GGGAGTGGCC AACTCCATCA CTAGGGGTTC CTTGTAGTTA 3751 ATGATTAACC CGCCATGCTA CTTATCTACT CGACATTGAT TATTGACTAG 3801 TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC CCATATATGG 3851 AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC 3951 GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA 4001 CTGCCCACTT GGCAGTACAT CAAGTGTATC ATATGCCAAG TACGCCCCCT 4051 ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG CCCAGTACAT 4101 GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG 4151 CTATTACCAT GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT TTATTTATTT TTTAATTATT 4251 TTGTGCAGCG ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG CCAGGCGGGG 4301 CGGGGCGGGG CGAGGGGCGG GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA 4351 GCCAATCAGA GCGGCGCGCT CCGAAAGTTT CCTTTTATGG CGAGGCGGCG 4401 GCGGCGGCGG CCCTATAAAA AGCGAAGCGC GCGGCGGGCG GGAGTCGCTG 4451 CGTTGCCTTC GCCCCGTGCC CCGCTCCGCG CCGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG GTGAGCGGGC GGGACGGCCC 4551 TTCTCCTCCG GGCTGTAATT AGCGCTTGGT TTAATGACGG CTTGTTTCTT 4601 TTCTGTGGCT GCGTGAAAGC CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC 4651 GGGGGGAGCG GCTCGGGGGG TGCGTGCGTG TGTGTGTGCG TGGGGAGCGC 4701 CGCGTGCGGC TCCGCGCTGC CCGGCGGCTG TGAGCGCTGC GGGCGCGGCG 4751 CGGGGCTTTG TGCGCTCCGC AGTGTGCGCG AGGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG GAACAAAGGC TGCGTGCGGG 4851 GTGTGTGCGT GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC GGTCGGGCTG 4901 CAACCCCCCC TGCACCCCCC TCCCCGAGTT GCTGAGCACG GCCCGGCTTC 4951 GGGTGCGGGG CTCCGTACGG GGCGTGGCGC GGGGCTCGCC GTGCCGGGCG 5001 GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG GGGCGGGGCC GCCTCGGGCC 5051 GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC CCCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT TTTATGGTAA TCGTGCGAGA 5151 GGGCGCAGGG ACTTCCTTTG TCCCAAATCT GTGCGGAGCC GAAATCTGGG 5201 AGGCGCCGCC GCACCCCCTC TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC 5251 CGGCAGGAAG GAAATGGGCG GGGAGGGCCT TCGTGCGTCG CCGCGCCGCC 5301 GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT GTCCGCGGGG GGACGGCTGC 5351 CTTCGGGGGG GACGGGGCAG GGCGGGGTTC GGCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA TGCCTTCTTC TTTTTCCTAC 5451 AGCTCCTGGG CAACGTGCTG GTTATTGTGC TGTCTCATCA TTTTGGCAAA 5501 GAATTGATTA ATTCGAGCGA ACGCGTCGAG TCGCTCGGTA CGATTTAAAT 5551 TGAATTGGCC TCGAGCGCAA GCTTGAGCTA GCGCCACCAT GGAATGGATG 5601 AGAAGCAGAG TGGGCACCCT GGGCCTGTGG GTGCGACTGC TGCTGGCTGT 5651 GTTTCTGCTG GGCGTGTACC AGGCCTACCC CATCCCTGAC TCTAGCCCCC 5701 TGCTGCAGTT TGGCGGACAA GTGCGGCAGA GATACCTGTA CACCGACGAC 5751 GACCAGGACA CCGAGGCCCA CCTGGAAATC CGCGAGGATG GCACAGTCGT 5801 GGGCGCTGCT CACAGAAGCC CTGAGAGCCT GCTGGAACTG AAGGCCCTGA 5851 AGCCCGGCGT GATCCAGATC CTGGGCGTGA AGGCCAGCAG ATTCCTGTGC 5901 CAGCAGCCTG ACGGCGCCCT GTACGGCTCT CCTCACTTCG ATCCTGAGGC 5951 CTGCAGCTTC AGAGAGCTGC TGCTGGAGGA CGGCTACAAC GTGTACCAGT 6001 CTGAGGCCCA CGGCCTGCCC CTGAGACTGC CTCAGAAGGA CAGCCCTAAC 6051 CAGGACGCCA CAAGCTGGGG ACCTGTGCGG TTCCTGCCTA TGCCTGGACT 6101 GCTGCACGAG CCCCAGGATC AGGCTGGCTT TCTGCCTCCT GAGCCTCCAG 6151 ACGTGGGCAG CAGCGACCCT CTGAGCATGG TGGAACCTCT GCAGGGCAGA 6201 AGCCCCAGCT ACGCCTCTTG AGAATGCGGG CCCGGTACCC CCGACGCGGC 6251 CGCTAATTCT AGATCGCGAA CAAACACCAT TGTCACACTC CAGTATACAC 6301 AAACACCATT GTCACACTCC AGATATCACA AACACCATTG TCACACTCCA 6351 AGGCGAACAA ACACCATTGT CACACTCCAA GGCTATTCTA GATCGCGAAT 6401 TACATACTTC TTTACATTCC AGTATACATT ACATACTTCT TTACATTCCA 6451 GATATCATTA CATACTTCTT TACATTCCAA GGCGAATTAC ATACTTCTTT 6501 ACATTCCAAG GCTACCTGAG GCCCGGGGGT ACCTCTTAAT TAACTGGCCT 6551 CATGGGCCTT CCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC 6601 CAGTCAGGTG CAGGCTGCCT ATCAGAAGGT GGTGGCTGGT GTGGCCAATG 6651 CCCTGGCTCA CAAATACCAC TGAGATCTTT TTCCCTCTGC CAAAAATTAT 6701 GGGGACATCA TGAAGCCCCT TGAGCATCTG ACTTCTGGCT AATAAAGGAA 6751 ATTTATTTTC ATTGCAATAG TGTGTTGGAA TTTTTTGTGT CTCTCACTCG 6801 GAAGGACATA TGGGAGGGCA AATCATTTAA AACATCAGAA TGAGTATTTG 6851 GTTTAGAGTT TGGCAACATA TGCCCATATG CTGGCTGCCA TGAACAAAGG 6901 TTGGCTATAA AGAGGTCATC AGTATATGAA ACAGCCCCCT GCTGTCCATT 6951 CCTTATTCCA TAGAAAAGCC TTGACTTGAG GTTAGATTTT TTTTATATTT 7001 TGTTTTGTGT TATTTTTTTC TTTAACATCC CTAAAATTTT CCTTACATGT 7051 TTTACTAGCC AGATTTTTCC TCCTCTCCTG ACTACTCCCA GTCATAGCTG 7101 TCCCTCTTCT CTTATGGAGA TCCCTCGACC TGCAGCCCAA GCTGTAGATA 7151 AGTAGCATGG CGGGTTAATC ATTAACTACA AGGAACCCCT AGTGATGGAG 7201 TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC 7251 AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC 7301 GAGCGCGCAG CTGGCGTAA AAV2 5′ ITR: 3615-3742 bp CAG promoter: 3782-5452 bp Mus musculus codon-optimized FGF21 (moFGF21): 5589-6221 bp dmiRT (4 copies of the miRT-122a and 4 copies of the miRT-1): 6254-6514 bp Rabbit β-globin polyA signal (3′ UTR and 3′ flanking region of rabbit beta-globin, including polyA signal): 6674-6764 bp AAV2 3′ ITR: 7181-7308 bp pAAV-CAG-moFGF21 (SEQ ID NO: 46) 1 AGTGAGCGAG CGAGCGCGCA GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT 61 TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC GGTCGTTCGG 121 CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG 181 GATAACGCAG GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG 241 GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA 301 CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT 361 GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC 421 TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG 481 GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC 541 TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA 601 CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG 661 TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT 721 CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC 781 ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA 841 TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA 901 CGTTAAGGGA TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT 961 TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC 1021 CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT 1081 GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT 1141 GCTGCAATGA TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG 1201 CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT 1261 ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT 1321 GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC 1381 TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT 1441 AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG 1501 GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG 1561 ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT 1621 TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC 1681 ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT 1741 TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT 1801 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG 1861 AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT 1921 TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG 1981 CGCACATTTC CCCGAAAAGT GCCACCTGAC GTCTAAGAAA CCATTATTAT CATGACATTA 2041 ACCTATAAAA ATAGGCGTAT CACGAGGCCC TTTCGTCTCG CGCGTTTCGG TGATGACGGT 2101 GAAAACCTCT GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC 2161 GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT 2221 AACTATGCGG CATCAGAGCA GATTGTACTG AGAGTGCACC ATATGCGGTG TGAAATACCG 2281 CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGATTCC AACATCCAAT AAATCATACA 2341 GGCAAGGCAA AGAATTAGCA AAATTAAGCA ATAAAGCCTC AGAGCATAAA GCTAAATCGG 2401 TTGTACCAAA AACATTATGA CCCTGTAATA CTTTTGCGGG AGAAGCCTTT ATTTCAACGC 2461 AAGGATAAAA ATTTTTAGAA CCCTCATATA TTTTAAATGC AATGCCTGAG TAATGTGTAG 2521 GTAAAGATTC AAACGGGTGA GAAAGGCCGG AGACAGTCAA ATCACCATCA ATATGATATT 2581 CAACCGTTCT AGCTGATAAA TTCATGCCGG AGAGGGTAGC TATTTTTGAG AGGTCTCTAC 2641 AAAGGCTATC AGGTCATTGC CTGAGAGTCT GGAGCAAACA AGAGAATCGA TGAACGGTAA 2701 TCGTAAAACT AGCATGTCAA TCATATGTAC CCCGGTTGAT AATCAGAAAA GCCCCAAAAA 2761 CAGGAAGATT GTATAAGCAA ATATTTAAAT TGTAAGCGTT AATATTTTGT TAAAATTCGC 2821 GTTAAATTTT TGTTAAATCA GCTCATTTTT TAACCAATAG GCCGAAATCG GCAAAATCCC 2881 TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG 2941 TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA 3001 TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC 3061 ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA AGCCGGCGAA 3121 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT 3181 AGCGGTCACG CTGCGCGTAA CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC 3241 GTACTATGGT TGCTTTGACG AGCACGTATA ACGTGCTTTC CTCGTTAGAA TCAGAGCGGG 3301 AGCTAAACAG GAGGCCGATT AAAGGGATTT TAGACAGGAA CGGTACGCCA GAATCCTGAG 3361 AAGTGTTTTT ATAATCAGTG AGGCCACCGA GTAAAAGAGT CTGTCCATCA CGCAAATTAA 3421 CCGTTGTCGC AATACTTCTT TGATTAGTAA TAACATCACT TGCCTGAGTA GAAGAACTCA 3481 AACTATCGGC CTTGCTGGTA ATATCCAGAA CAATATTACC GCCAGCCATT GCAACGGAAT 3541 CGCCATTCGC CATTCAGGCT GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCC 3601 ACTGAGGCCC AGCTGCGCGC TCGCTCGCTC ACTGAGGCCG CCCGGGCAAA GCCCGGGCGT 3661 CGGGCGACCT TTGGTCGCCC GGCCTCAGTG AGCGAGCGAG CGCGCAGAGA GGGAGTGGCC 3721 AACTCCATCA CTAGGGGTTC CTTGTAGTTA ATGATTAACC CGCCATGCTA CTTATCTACT 3781 CGACATTGAT TATTGACTAG TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC 3841 CCATATATGG AGTTCCGCGT TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC 3901 AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG 3961 ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT 4021 CAAGTGTATC ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC 4081 TGGCATTATG CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA 4141 TTAGTCATCG CTATTACCAT GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC 4201 TCCCCCCCCT CCCCACCCCC AATTTTGTAT TTATTTATTT TTTAATTATT TTGTGCAGCG 4261 ATGGGGGCGG GGGGGGGGGG GGGGCGCGCG CCAGGCGGGG CGGGGCGGGG CGAGGGGCGG 4321 GGCGGGGCGA GGCGGAGAGG TGCGGCGGCA GCCAATCAGA GCGGCGCGCT CCGAAAGTTT 4381 CCTTTTATGG CGAGGCGGCG GCGGCGGCGG CCCTATAAAA AGCGAAGCGC GCGGCGGGCG 4441 GGAGTCGCTG CGTTGCCTTC GCCCCGTGCC CCGCTCCGCG CCGCCTCGCG CCGCCCGCCC 4501 CGGCTCTGAC TGACCGCGTT ACTCCCACAG GTGAGCGGGC GGGACGGCCC TTCTCCTCCG 4561 GGCTGTAATT AGCGCTTGGT TTAATGACGG CTTGTTTCTT TTCTGTGGCT GCGTGAAAGC 4621 CTTGAGGGGC TCCGGGAGGG CCCTTTGTGC GGGGGGAGCG GCTCGGGGGG TGCGTGCGTG 4681 TGTGTGTGCG TGGGGAGCGC CGCGTGCGGC TCCGCGCTGC CCGGCGGCTG TGAGCGCTGC 4741 GGGCGCGGCG CGGGGCTTTG TGCGCTCCGC AGTGTGCGCG AGGGGAGCGC GGCCGGGGGC 4801 GGTGCCCCGC GGTGCGGGGG GCTGCGAGGG GAACAAAGGC TGCGTGCGGG GTGTGTGCGT 4861 GGGGGGGTGA GCAGGGGGTG TGGGCGCGTC GGTCGGGCTG CAACCCCCCC TGCACCCCCC 4921 TCCCCGAGTT GCTGAGCACG GCCCGGCTTC GGGTGCGGGG CTCCGTACGG GGCGTGGCGC 4981 GGGGCTCGCC GTGCCGGGCG GGGGGTGGCG GCAGGTGGGG GTGCCGGGCG GGGCGGGGCC 5041 GCCTCGGGCC GGGGAGGGCT CGGGGGAGGG GCGCGGCGGC CCCCGGAGCG CCGGCGGCTG 5101 TCGAGGCGCG GCGAGCCGCA GCCATTGCCT TTTATGGTAA TCGTGCGAGA GGGCGCAGGG 5161 ACTTCCTTTG TCCCAAATCT GTGCGGAGCC GAAATCTGGG AGGCGCCGCC GCACCCCCTC 5221 TAGCGGGCGC GGGGCGAAGC GGTGCGGCGC CGGCAGGAAG GAAATGGGCG GGGAGGGCCT 5281 TCGTGCGTCG CCGCGCCGCC GTCCCCTTCT CCCTCTCCAG CCTCGGGGCT GTCCGCGGGG 5341 GGACGGCTGC CTTCGGGGGG GACGGGGCAG GGCGGGGTTC GGCTTCTGGC GTGTGACCGG 5401 CGGCTCTAGA GCCTCTGCTA ACCATGTTCA TGCCTTCTTC TTTTTCCTAC AGCTCCTGGG 5461 CAACGTGCTG GTTATTGTGC TGTCTCATCA TTTTGGCAAA GAATTGATTA ATTCGAGCGA 5521 ACGCGTCGAG TCGCTCGGTA CGATTTAAAT TGAATTGGCC TCGAGCGCAA GCTTGAGCTA 5581 GCGCCACCAT GGAATGGATG AGAAGCAGAG TGGGCACCCT GGGCCTGTGG GTGCGACTGC 5641 TGCTGGCTGT GTTTCTGCTG GGCGTGTACC AGGCCTACCC CATCCCTGAC TCTAGCCCCC 5701 TGCTGCAGTT TGGCGGACAA GTGCGGCAGA GATACCTGTA CACCGACGAC GACCAGGACA 5761 CCGAGGCCCA CCTGGAAATC CGCGAGGATG GCACAGTCGT GGGCGCTGCT CACAGAAGCC 5821 CTGAGAGCCT GCTGGAACTG AAGGCCCTGA AGCCCGGCGT GATCCAGATC CTGGGCGTGA 5881 AGGCCAGCAG ATTCCTGTGC CAGCAGCCTG ACGGCGCCCT GTACGGCTCT CCTCACTTCG 5941 ATCCTGAGGC CTGCAGCTTC AGAGAGCTGC TGCTGGAGGA CGGCTACAAC GTGTACCAGT 6001 CTGAGGCCCA CGGCCTGCCC CTGAGACTGC CTCAGAAGGA CAGCCCTAAC CAGGACGCCA 6061 CAAGCTGGGG ACCTGTGCGG TTCCTGCCTA TGCCTGGACT GCTGCACGAG CCCCAGGATC 6121 AGGCTGGCTT TCTGCCTCCT GAGCCTCCAG ACGTGGGCAG CAGCGACCCT CTGAGCATGG 6181 TGGAACCTCT GCAGGGCAGA AGCCCCAGCT ACGCCTCTTG AGAATGCGGG CCCGGTACCC 6241 CCGACGCGGC CTAACTGGCC TCATGGGCCT TCCGCTCACT GCCCGCTTTC CAGTCGGGAA 6301 ACCTGTCGTG CCAGTCAGGT GCAGGCTGCC TATCAGAAGG TGGTGGCTGG TGTGGCCAAT 6361 GCCCTGGCTC ACAAATACCA CTGAGATCTT TTTCCCTCTG CCAAAAATTA TGGGGACATC 6421 ATGAAGCCCC TTGAGCATCT GACTTCTGGC TAATAAAGGA AATTTATTTT CATTGCAATA 6481 GTGTGTTGGA ATTTTTTGTG TCTCTCACTC GGAAGGACAT ATGGGAGGGC AAATCATTTA 6541 AAACATCAGA ATGAGTATTT GGTTTAGAGT TTGGCAACAT ATGCCCATAT GCTGGCTGCC 6601 ATGAACAAAG GTTGGCTATA AAGAGGTCAT CAGTATATGA AACAGCCCCC TGCTGTCCAT 6661 TCCTTATTCC ATAGAAAAGC CTTGACTTGA GGTTAGATTT TTTTTATATT TTGTTTTGTG 6721 TTATTTTTTT CTTTAACATC CCTAAAATTT TCCTTACATG TTTTACTAGC CAGATTTTTC 6781 CTCCTCTCCT GACTACTCCC AGTCATAGCT GTCCCTCTTC TCTTATGGAG ATCCCTCGAC 6841 CTGCAGCCCA AGCTGTAGAT AAGTAGCATG GCGGGTTAAT CATTAACTAC AAGGAACCCC 6901 TAGTGATGGA GTTGGCCACT CCCTCTCTGC GCGCTCGCTC GCTCACTGAG GCCGGGCGAC 6961 CAAAGGTCGC CCGACGCCCG GGCTTTGCCC GGGCGGCCTC AGTGAGCGAG CGAGCGCGCA 7021 GCTGGCGTAA AAV2 5′ ITR: 3601-3742 bp CAG promoter: 3779-5423 bp Mus musculus codon-optimized FGF21 (moFGF21): 5588-6221 bp Rabbit β-globin polyA signal (3′ UTR and 3′ flanking region of rabbit beta-globin, including polyA signal): 6315-6833 bp AAV2 3′ ITR: 6892-7024 bp pAAV-CMV-moFGF21 (SEQ ID NO: 63) 1 GGGGCTAGCG CCACCATGGA ATGGATGAGA AGCAGAGTGG GCACCCTGGG 51 CCTGTGGGTG CGACTGCTGC TGGCTGTGTT TCTGCTGGGC GTGTACCAGG 101 CCTACCCCAT CCCTGACTCT AGCCCCCTGC TGCAGTTTGG CGGACAAGTG 151 CGGCAGAGAT ACCTGTACAC CGACGACGAC CAGGACACCG AGGCCCACCT 201 GGAAATCCGC GAGGATGGCA CAGTCGTGGG CGCTGCTCAC AGAAGCCCTG 251 AGAGCCTGCT GGAACTGAAG GCCCTGAAGC CCGGCGTGAT CCAGATCCTG 301 GGCGTGAAGG CCAGCAGATT CCTGTGCCAG CAGCCTGACG GCGCCCTGTA 351 CGGCTCTCCT CACTTCGATC CTGAGGCCTG CAGCTTCAGA GAGCTGCTGC 401 TGGAGGACGG CTACAACGTG TACCAGTCTG AGGCCCACGG CCTGCCCCTG 451 AGACTGCCTC AGAAGGACAG CCCTAACCAG GACGCCACAA GCTGGGGACC 501 TGTGCGGTTC CTGCCTATGC CTGGACTGCT GCACGAGCCC CAGGATCAGG 551 CTGGCTTTCT GCCTCCTGAG CCTCCAGACG TGGGCAGCAG CGACCCTCTG 601 AGCATGGTGG AACCTCTGCA GGGCAGAAGC CCCAGCTACG CCTCTTGAGA 651 ATGCGGGCCC GGTACCCCCT CGACGGTACC AGCGCTGTCG AGGCCGCTTC 701 GAGCAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC CACAACTAGA 751 ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT 801 ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA 851 TTCATTTTAT GTTTCAGGTT CAGGGGGAGA TGTGGGAGGT TTTTTAAAGC 901 AAGTAAAACC TCTACAAATG TGGTAAAATC GATTAGGATC TTCCTAGAGC 951 ATGGCTACCT AGACATGGCT CGACAGATCA GCGCTCATGC TCTGGAAGAT 1001 CTCGATTTAA ATGCGGCCGC AGGAACCCCT AGTGATGGAG TTGGCCACTC 1051 CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC AAAGGTCGCC 1101 CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGCAG 1151 CTGCCTGCAG GGGCGCCTGA TGCGGTATTT TCTCCTTACG CATCTGTGCG 1201 GTATTTCACA CCGCATACGT CAAAGCAACC ATAGTACGCG CCCTGTAGCG 1251 GCGCATTAAG CGCGGCGGGT GTGGTGGTTA CGCGCAGCGT GACCGCTACA 1301 CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC CTTCCTTTCT 1351 CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG GGGCTCCCTT 1401 TAGGGTTCCG ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT 1451 TTGGGTGATG GTTCACGTAG TGGGCCATCG CCCTGATAGA CGGTTTTTCG 1501 CCCTTTGACG TTGGAGTCCA CGTTCTTTAA TAGTGGACTC TTGTTCCAAA 1551 CTGGAACAAC ACTCAACCCT ATCTCGGGCT ATTCTTTTGA TTTATAAGGG 1601 ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA TTTAACAAAA 1651 ATTTAACGCG AATTTTAACA AAATATTAAC GTTTACAATT TTATGGTGCA 1701 CTCTCAGTAC AATCTGCTCT GATGCCGCAT AGTTAAGCCA GCCCCGACAC 1751 CCGCCAACAC CCGCTGACGC GCCCTGACGG GCTTGTCTGC TCCCGGCATC 1801 CGCTTACAGA CAAGCTGTGA CCGTCTCCGG GAGCTGCATG TGTCAGAGGT 1851 TTTCACCGTC ATCACCGAAA CGCGCGAGAC GAAAGGGCCT CGTGATACGC 1901 CTATTTTTAT AGGTTAATGT CATGATAATA ATGGTTTCTT AGACGTCAGG 1951 TGGCACTTTT CGGGGAAATG TGCGCGGAAC CCCTATTTGT TTATTTTTCT 2001 AAATACATTC AAATATGTAT CCGCTCATGA GACAATAACC CTGATAAATG 2051 CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC ATTTCCGTGT 2101 CGCCCTTATT CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACC 2151 CAGAAACGCT GGTGAAAGTA AAAGATGCTG AAGATCAGTT GGGTGCACGA 2201 GTGGGTTACA TCGAACTGGA TCTCAACAGC GGTAAGATCC TTGAGAGTTT 2251 TCGCCCCGAA GAACGTTTTC CAATGATGAG CACTTTTAAA GTTCTGCTAT 2301 GTGGCGCGGT ATTATCCCGT ATTGACGCCG GGCAAGAGCA ACTCGGTCGC 2351 CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCAC CAGTCACAGA 2401 AAAGCATCTT ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA 2451 TAACCATGAG TGATAACACT GCGGCCAACT TACTTCTGAC AACGATCGGA 2501 GGACCGAAGG AGCTAACCGC TTTTTTGCAC AACATGGGGG ATCATGTAAC 2551 TCGCCTTGAT CGTTGGGAAC CGGAGCTGAA TGAAGCCATA CCAAACGACG 2601 AGCGTGACAC CACGATGCCT GTAGCAATGG CAACAACGTT GCGCAAACTA 2651 TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT TAATAGACTG 2701 GATGGAGGCG GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG 2751 CTGGCTGGTT TATTGCTGAT AAATCTGGAG CCGGTGAGCG TGGGTCTCGC 2801 GGTATCATTG CAGCACTGGG GCCAGATGGT AAGCCCTCCC GTATCGTAGT 2851 TATCTACACG ACGGGGAGTC AGGCAACTAT GGATGAACGA AATAGACAGA 2901 TCGCTGAGAT AGGTGCCTCA CTGATTAAGC ATTGGTAACT GTCAGACCAA 2951 GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT TTTAATTTAA 3001 AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT 3051 AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA 3101 GGATCTTCTT GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC 3151 AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC 3201 CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT 3251 ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT 3301 AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG 3351 CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA 3401 CCGGATAAGG CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC 3451 CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC 3501 TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG 3551 GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG 3601 AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG 3651 AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC 3701 GCCAGCAACG CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC 3751 TCACATGTCC TGCAGGCAGC TGCGCGCTCG CTCGCTCACT GAGGCCGCCC 3801 GGGCAAAGCC CGGGCGTCGG GCGACCTTTG GTCGCCCGGC CTCAGTGAGC 3851 GAGCGAGCGC GCAGAGAGGG AGTGGCCAAC TCCATCACTA GGGGTTCCTG 3901 CGGCCGCGAT ATCTGTAGTT AATGATTAAC CCGCCATGCT ACTTATCTAC 3951 AGATCTCAAT ATTGGCCATT AGCCATATTA TTCATTGGTT ATATAGCATA 4001 AATCAATATT GGCTATTGGC CATTGCATAC GTTGTATCTA TATCATAATA 4051 TGTACATTTA TATTGGCTCA TGTCCAATAT GACCGCCATG TTGGCATTGA 4101 TTATTGACTA GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG 4151 CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT GGCCCGCCTG 4201 GCTGACCGCC CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT 4251 CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAAT GGGTGGAGTA 4301 TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAA 4351 GTCCGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT 4401 GCCCAGTACA TGACCTTACG GGACTTTCCT ACTTGGCAGT ACATCTACGT 4451 ATTAGTCATC GCTATTACCA TGGTGATGCG GTTTTGGCAG TACACCAATG 4501 GGCGTGGATA GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT 4551 GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGG ACTTTCCAAA 4601 ATGTCGTAAC AACTGCGATC GCCCGCCCCG TTGACGCAAA TGGGCGGTAG 4651 GCGTGTACGG TGGGAGGTCT ATATAAGCAG AGCTCGTTTA GTGAACCGTC 4701 AGATCACTAG GCTAGCTATT GCGGTAGTTT ATCACAGTTA AATTGCTAAC 4751 GCAGTCAGTG CTTCTGACAC AACAGTCTCG AACTTAAGCT GCAGTGACTC 4801 TCTTAAGGTA GCCTTGCAGA AGTTGGTCGT GAGGCACTGG GCAGGTAAGT 4851 ATCAAGGTTA CAAGACAGGT TTAAGGAGAC CAATAGAAAC TGGGCTTGTC 4901 GAGACAGAGA AGACTCTTGC GTTTCTGATA GGCACCTATT GGTCTTACTG 4951 ACATCCACTT TGCCTTTCTC TCCACAGGTG TCCACTCCCA GTTCAATTAC 5001 AGCTCTTAAG GCTAGAGTAC TTAATACGAC TCACTATAGA ATACGACTCA 5051 CTATAGGGAG ACGCTAGCGT CGA AAV2 5′ ITR: 3772-3899 bp CMV enhancer: 4093-4472 bp CMV promoter: 4473-4684 bp β-globin intron (chimeric intron composed of introns from human β-globin and immunoglobulin heavy chain genes): 4845-4977 bp Mus musculus codon-optimized FGF21 (moFGF21): 16-648 bp SV40 polyA signal: 713-834 bp AAV2 3′ ITR: 1021-1148 bp