Novel gene therapy

Abstract

There is provided a vector; a pharmaceutical composition comprising said vector, a method, and a use of the vector in the manufacture of a medicament, for preventing, ameliorating, and / or treating obesity or an obesity-associated condition or disease, said vector comprising a promoter operably linked to a melanocortin-4-receptor (MC4R) gene.

Description

[0001] Novel Gene Therapy

[0002] Field of the Invention

[0003] The present invention relates to gene therapy vectors for the prevention, amelioration, and / or treatment of neurometabolic disorders, in particular obesity and obesity- associated conditions and diseases.

[0004] Background to the Invention

[0005] Obesity is a medical condition in which excess body fat accumulates in adipose tissue due to an imbalance in energy intake and expenditure. Obesity is closely linked to a cluster of metabolic diseases, including diabetes, hyperlipidaemia, fatty liver disease, hypertension, and cardiovascular disease, in addition to a wide range of cancers, including liver cancer, prostate cancer and breast cancer. In the US, the age-adjusted prevalence of obesity in 2017 / 8 for adults was 42 % and the estimated annual medical cost of obesity in the US was $147 billion in 2008 (US Centers for Disease Control and Prevention, https: / / www.cdc.gov / obesity).

[0006] Diet and exercise changes have globally contributed to the increased prevalence of obesity. Nevertheless, there is evidence of a substantial hereditary contribution to body weight. The genetic components include polygenic susceptibility and inheritance of rare, large-effect gene mutations and monogenic obesity syndromes (Walley AJ et al., Nat. Rev. Genet., 2009, 10, 431-42).

[0007] Among the single-gene causes of severe childhood obesity, genetic mutations in the leptin-melanocortin system have been shown to cause severe obesity (Farooqi S et al., Endocr Rev., 2006, 27, 710-718). The melanocortin-4-receptor (MC4R) gene is located on chromosome 18q21.3a. It is an intronless gene with an open reading frame of 999 bp that encodes a 332 amino acid protein that is a key regulator of energy homeostasis, food intake and body weight regulation at the level of the hypothalamus. MC4R plays an integral role within the leptin-pro-opiomelanocortin pathway regulating food intake and energy expenditure. MC4R gene mutations are one of the most common forms of monogenic obesity and have been implicated in 1 % to 6 % of early-onset severe obesity (Oswal A et al., Obes. Rev., 2007, 8, 293-306). Evidence shows that MC4R loss of function (LoF) mutations in homozygous and carriers have a substantial impact in BMI, weight, fat mass and lean mass, detectable as early as 5 years after birth (Wade, Kaitlin H et al. Nature medicine vol. 27,6 (2021): 1088-1096).

[0008] MC4R activity is regulated by two endogenous ligands, an anorexigenic agonist, a melanocyte-stimulating hormone (a-MSH) and an orexigenic antagonist / inverse agonist, agouti-related peptide (AgRP). These neuropeptides are produced by neurons of the arcuate nucleus (ARC) of the hypothalamus under the control of the adipocyte-secreted hormone leptin. As leptin levels are proportional to fat mass, the observation that MC4R can localise to neuronal primary cilia suggests that signalling from the primary cilia of MC4R-expressing neurons is a rate-limiting step in the sensing of energy stores and conditional modulation of energy intake (Wang Y et al., J. Clin. Invest., 2021 , 131(9), 1- 12). The generation of cyclic adenosine monophosphate (cAMP) as a second messenger for the activity of MC4R have been widely investigated within the context of energy and obesity regulation and balance (Selkirk JV, et al. Neuropharmacology 52:459-466). Patients with heterozygous frameshift or nonsense mutations have a complete loss of function and no cAMP generation. Other missense mutations have reduced ability to generate cAMP (Wade et al. Nature Medicine. 2021 Jun;27(6):1088-109).

[0009] Adeno-associated virus-mediated knockdown of MC4R in the paraventricular nucleus of the hypothalamus using AAV vectors encoding short hairpin RNAs has been reported to promote high-fat diet-induced hyperphagia and obesity in rats (Garza JC et al., Journal of Endocrinology, 2008, 197, 471-482).

[0010] Several pharmacological agonists of MC4R are in clinical development for the treatment of obesity (Gongalves JPL et al., Trends Pharmacol. Sci., 2018, 39, 2018). Of these, IMICIVREE (setmelanotide) was the first treatment to receive FDA approval for chronic weight management in patients six years and older with obesity due to proopiomelanocortin (POMC), proprotein convertase subtilisin / kexin type 1 (PCSK1) or leptin receptor (LEPR) deficiency confirmed by genetic testing demonstrating variants in POMC, PCSK1 or LEPR genes that are interpreted as pathogenic, likely pathogenic or of uncertain significance.

[0011] There remains a significant unmet medical need for alternative treatments for neurometabolic driven obesity and obesity-associated disorders. Gene therapy offers an alternative approach to conventional treatments via the transfer of gene coding or noncoding sequences to produce proteins that restore and maintain metabolic homeostasis. In obesity cases where the genetic defect is clearly identified, transfer copies of a functional gene to diseased cells may provide a cure similar to other monogenic diseases applicable in gene therapy.

[0012] US 2021 / 0222196 discloses recombinant adeno-associated virus (rAAV) vectors and rAAVs (e. g. viral particles) engineered to express a transgene comprising an inhibitory nucleic acid (e. g. an artificial miRNA) having a pri-miRNA scaffold and a guide strand that targets a human target gene. Such vectors are indicated to be useful in the treatment of obesity.

[0013] US 2005 / 0002905 discloses compositions comprising an adeno-associated viral vector that comprises a nucleic acid segment encoding a pro-opiomelanocortin polypeptide operably linked to a promoter capable of expressing the nucleic acid segment in a host cell. Such vectors are indicated to be useful in the treatment or amelioration of a proopiomelanocortin polypeptide deficiency which may result in, inter alia, polyphagia, hyperinsulinemia, adiposity, and weight gain.

[0014] Summary of the Invention

[0015] In a first aspect of the invention, there is provided a vector for preventing, ameliorating, and / or treating obesity or an obesity-associated condition or disease, said vector comprising a promoter operably linked to a melanocortin-4-receptor (MC4R) gene.

[0016] In a second aspect of the invention, there is provided a pharmaceutical composition comprising a vector, which vector comprises a promoter operably linked to a melanocortin-4-receptor (MC4R) gene, and one or more pharmaceutically acceptable excipients. Said pharmaceutical composition may be used to prevent, ameliorate and / or treat obesity or an obesity-associated condition or disease.

[0017] In a third aspect of the invention, there is provided a method of preventing, ameliorating or treating obesity or an obesity-associated condition or disease, which method comprises administering to a patient in need thereof a vector comprising a promoter operably linked to a melanocortin-4-receptor (MC4R) gene. In a fourth aspect of the present invention, there is provided a vector comprising a promoter operably linked to a melanocortin-4-receptor (MC4R) gene for use in therapy, in particular for the prevention, amelioration and / or treatment of obesity or an obesity- associated condition or disease.

[0018] In a fifth aspect of the invention, there is provided a vector comprising a promoter operably linked to a melanocortin-4-receptor (MC4R) gene, for use in the manufacture of a medicament for preventing, ameliorating, or treating obesity or an obesity-associated condition or disease.

[0019] Further aspects of invention are described in more detail below.

[0020] Brief Description of the Drawings

[0021] The invention will now be described in detail by way of example only with reference to the figures, which are as follows:

[0022] Figure 1 shows Human MC4R transgene and protein expression in HEK293T cells following CAG-hCOMC4R or CAG-eGFP transfection.

[0023] Figure 2 shows hCOMC4R transgene and protein expression detected in HEK293T cells following AAV9-CAG-hCOMC4R or AAV9-CAG-eGFP transduction.

[0024] Figure 3 shows dose dependent biological activity levels following AAV2-CAG- hCOMC4R transduction.

[0025] Figure 4 shows a construct comprising a CAG promoter and hMC4R COGS nucleotide sequence.

[0026] Figure 5 shows a construct comprising a CAG promoter and hMC4R COGA nucleotide sequence.

[0027] Figure 6 shows a construct comprising a CAG promoter and hMC4R COIDT nucleotide sequence. Figure 7 shows the results of an in vivo study of the efficacy of a vector according to an embodiment of the invention.

[0028] Figure 8 shows dose dependent biological activity levels following AAV9-CAG- hCOMC4R transduction.

[0029] Figures 9 and 10 show the average body weight curve from week 1 to week 16 in wildtype (WT) and Mc4rLoxTB / LoxTB(Mut) mice, untreated or injected at postnatal day 7 (P7) with AAV9-CAG-hMC4R_GA (AXV-201).

[0030] Figures 11 and 12 show normalisation of Insulin, Leptin and Ghrelin serum levels in AXV- 201 treated Mc4rLoxTB / LoxTBmice.

[0031] Detailed Description of the Invention

[0032] The present invention provides a therapeutic MC4R gene-containing vector for the prevention, amelioration and / or treatment of obesity or an obesity-associated condition or disease. The invention uses adeno-associated virus vectors which mediate highly efficient MC4R gene delivery to target sites within the body.

[0033] Figure 1 shows human MC4R transgene and protein expression in HEK293T cells following CAG-hCOMC4R or CAG-eGFP transfection. (A) hCOBBSI transgene is detected in HEK293T cells transfected with CAG-hCOMC4R compared to controls. Plasmid CAG-hCOMC4RGA had a fold change of 139153.185, 93033.97 and 153733.87 for 0.1 , 0.05 and 0.025 ug DNA. Plasmid CAG-hCOMC4RGS had a fold change of 3x107, 3687516.22 and 1359586.845 for 0.1 , 0.05 and 0.025 ug DNA, respectively. Plasmid CAG-hCOMC4RSS had a fold change of 2670534.785, 4944795.475 and 182481.4 for 0.1 , 0.05 and 0.025 ug DNA, respectively. Plasmid CAG-hCOMC4RSC had a fold change of 1529716.74, 244680.6 and 70027.56 for 0.1 , 0.05 and 0.025 ug DNA, respectively. (B) MC4R protein is over expressed in cells transfected with all CAG- hCOMC4R plasmids relative to GAPDH and compared to CAG-eGFP transfected control cells. Plasmid CAG-hCOMC4RGS had a relative intensity of 1.7, 5.2 and 6.7 for 0.1 , 0.05 and 0.025 ug transfected DNA, respectively. Plasmid CAG-hCOMC4RGA had a relative intensity of 1.9, 2.6 and 3.4 for 0.1 , 0.05 and 0.025 ug transfected DNA, respectively. Plasmid CAG-hCOMC4RSS had a relative intensity of 0.5, 0.2 and 0.002 for 0.1 , 0.05 and 0.025 ug transfected DNA, respectively. Plasmid CAG-hCOMC4RSC had a relative intensity of 0.7, 2.9 and 3.2 for 0.1 , 0.05 and 0.025 ug transfected DNA respectively compared to a relative intensity of 0.7 in the control. (C) MC4R protein is over expressed in cells transfected with CAG-hCOMC4R_GS plasmid in all DNA concentrations tested relative to GAPDH loading control.

[0034] Figure 2 shows hCOMC4R transgene and protein expression detected in HEK293T cells following AAV9-CAG-hCOMC4R or AAV9-CAG-eGFP transduction. hCOMC4R transgene and MC4R protein identified in lysates extracted from human embryonic kidney 293T (HEK293T) cells that were transduced at an MOI of 2x105, 2x104and 2x103using AAV9-CAG-COMC4R compared to control cells. (A) hCOMC4R transgene is over expressed in HEK293T cells transduced with all of the AAV9-CAG-hCOMC4R vectors compared to controls. Vector AAV9-CAG-hCOMC4RGA had a fold change of 1.8x108, 29953 and 2134 for 0.1 , 0.05 and 0.025 ug DNA. Vector AAV9-CAG-hCOMC4RGS had a fold change of 2. 107, 1590334 and 145370 for 0.1 , 0.05 and 0.025 ug DNA, respectively. Vector AAV9-CAG-hCOMC4RSS had a fold change of 1644, 158 and 16.4 for 0.1 , 0.05 and 0.025 ug DNA, respectively. Vector AAV9-CAG-hCOMC4RSC had a fold change of 334413, 43846 and 4047 for 0.1 , 0.05 and 0.025 ug DNA, respectively. (B) MC4R protein is over expressed in cells transduced with all AAV9-CAG-hCOMC4R vectors relative to GAPDH and compared to AAV9-CAG-eGFP transduced control cells. Vector AAV9-CAG-hCOMC4RGA had a relative intensity of 1.5, 2.9 and 2.1 for an MOI of 2x105, 2x104and 2x103, respectively. Vector AAV9-CAG-hCOMC4RGS had a relative intensity of 1.8, 1.7 and 2.1 for an MOI of 2x105, 2x104and 2x103, respectively. Vector AAV9-CAG-hCOMC4RSS had a relative intensity of 2.3, 1 and 1.5 for an MOI of 2x105, 2x104and 2x103, respectively. Vector AAV9-CAG-hCOMC4RSC had a relative intensity of 1.5, 1 and 0.7 for an MOI of 2x105, 2x104 and 2x103 respectively compared to a relative intensity of 0.19 in the control. (C) MC4R protein is over expressed in cells transduced with AAV9-CAG-hCOMC4R_GS vector in all MOI tested relative to GAPDH loading control.

[0035] Figure 3 shows cAMP activation functional potency assay shows dose dependent biological activity levels of following AAV2-CAG-hCOMC4R transduction. After transduction of different MOI (“Multiplicity Of Infection” or viral genome particles / number of cells infected) of AAV2-CAG-hCOMC4R vectors cAMP levels were measured. We can see there is a dose response generation of cAMP of the AAV2 vectors expressing the human codon-optimised self-complementary sequences (MC4R-hCOGA and MC4R- hCOGS) and the MC4R hWTsc. All of the average production of 250 nM of cAMP with 2 x 105MOI compared with the human single strand sequence vector (MC4R-hWTss) that average less than 100 nM of cAMP. The dose response is also mimic by the cAMP production after the control mouse MC4R (Mc4r-mWT). In one aspect of the invention, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 70 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 72 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 74 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 76 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 78 % sequence identity thereto, and encodes a functional MC4R protein Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 80 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 82 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 84 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 85 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 86 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 88 % sequence identity thereto, and encodes a functional BBS1 protein. In other embodiments, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 90 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 92 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 94 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 95 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 96 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 97 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 98 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1 or has at least 99 % sequence identity thereto, and encodes a functional MC4R protein.

[0036] Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 1.

[0037] In one aspect of the invention, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 70 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 72 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 74 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 76 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 78 % sequence identity thereto, and encodes a functional MC4R protein Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 80 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 82 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 84 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 85 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 86 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 88 % sequence identity thereto, and encodes a functional BBS1 protein. In other embodiments, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 90 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 92 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 94 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 95 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 96 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 97 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 98 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2 or has at least 99 % sequence identity thereto, and encodes a functional MC4R protein. Alternatively, the MC4R gene has the nucleotide sequence of SEQ ID NO. 2.

[0038] In one aspect of the present invention, the nucleotide sequence of the MC4R gene may be codon optimised to maximise expression of the functional MC4R protein. In codon optimisation, the amino acid sequence of the encoded protein remains the same so it will still be functional; it is simply the nucleotide sequence that is modified. SEQ ID NOs. 3, 4 and 5 are codon optimised nucleotide sequences encoding MC4R. Accordingly, in one aspect of the present invention, the MC4R gene has the nucleotide sequence of any one of SEQ ID Nos. 3, 4 and 5 or has at least 70 % sequence identity thereto, for example, at least 80 %, at least 85 %, at least 90 %, at least 95 % or at least 99 % sequence identity thereto.

[0039] The term “identity” is used herein to refer to the similarity of two sequences. For the purpose of this invention, it is defined here that in order to determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (e. g., gaps can be introduced in the sequence of a first sequence for optimal alignment with a second amino or nucleic acid sequence). The nucleotide / amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i. e., % identity = number of identical positions / total number of positions (i. e. overlapping positions) x 100). Generally, the two sequences are the same length. A sequence comparison is typically carried out over the entire length of the two sequences being compared.

[0040] Computer programs that may be used to determine the identity between two sequences are publicly available and well known in the art. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two nucleic acid sequences is determined using the sequence alignment software Clone Manager 9 (Sci-Ed software - www.scied.com) using global DNA alignment; parameters: both strands; scoring matrix: linear (mismatch 2, OpenGap 4, ExtGap 1). Alternatively, the percent identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. A further method to assess the percent identity between two amino acid or nucleic acid sequences can be to use the BLAST sequence comparison tool available on the National Center for Biotechnology Information (NCBI) website (www.blast.ncbi.nlm.nih.gov), for example using BLASTn for nucleotide sequences or BLASTp for amino acid sequences using the default parameters.

[0041] The MC4R gene employed in the present invention encodes a “functional” MC4R protein. This means that the protein, when expressed, has the same function and activity as the wild type human protein. Protein expression and functionality may be readily determined by methods known in the art. The protein encoded by the MC4R gene may be the wild type human protein. The human sequence of MC4R is 332 amino acids in length and the sequence is given in SEQ ID NO. 10.

[0042] The promoter contained in the vector of the present invention is preferably ubiquitous promoter. The promoter is operably linked to the MC4R gene so that the promoter directs expression of the gene. A ubiquitous promoter is one which is strongly active in a wide range of cells and tissues and provides constitutive expression. Suitable ubiquitous promoters are well known to those skilled in the art. A ubiquitous promoter is not tissue specific. It provides expression in multiple tissues / organs. The ubiquitous promoter results in expression of the MC4R gene so that the expressed protein ameliorates the pathologies associated with obesity or an obesity-associated condition or disease.

[0043] Suitable ubiquitous promoters include short elongation factor promoter (EFS), CAG promoter, cytomegalovirus immediate-early promoter (CMV), Ubiquitin C promoter (UBC), phosphoglycerate kinase promoter (PGK) and beta-actin promoter, e. g. chicken beta-actin promoter (CBA). These promoters and the according sequences are known to the person skilled in the art. Preferably, the promoter is a CAG promoter and, more preferably, a CAG promoter having the nucleotide sequence of SEQ ID NO. 6. In one aspect, the present invention provides a vector comprising a CAG promoter operably linked to an MC4R gene, for the prevention, amelioration and / or treatment of obesity or an obesity-associated condition or disease.

[0044] In another aspect, the present invention provides a vector comprising a CAG promoter operably linked to an MC4R gene with a nucleotide sequence selected from any one of SEQ ID Nos. 1-5, or a nucleotide having at least 70 % sequence identity thereto, for example, at least 80 %, at least 85 %, at least 90 %, at least 95 % or at least 99 % sequence identity thereto, for the prevention, amelioration and / or treatment of obesity or an obesity-associated condition or disease.

[0045] In a further aspect, the present invention provides a vector comprising a CAG promoter with a nucleotide sequence of SEQ ID No. 6 operably linked to an MC4R gene with having a nucleotide selected from any one of SEQ ID Nos. 1-5, or a nucleotide having at least 70 % sequence identity thereto, for example, at least 80 %, at least 85 %, at least 90 %, at least 95 % or at least 99 % sequence identity thereto, for the prevention, amelioration and / or treatment of obesity or an obesity-associated condition or disease.

[0046] Suitable vectors for use in accordance with the present invention are commercially available and / or may be prepared in accordance with methods known in the art. In one aspect of the invention, the vector can cross the blood brain barrier. This allows transduction to occur in the brain and central nervous system. Therefore, a single vector can be used to provide gene expression in multiple sites to ameliorate the pathologies associated with obesity or an obesity-associated condition or disease. This gene expression may be systemic as it can occur in multiple sites throughout the body. Further, administration of the vector by a limited number of routes can be used to provide systemic gene expression to ameliorate the pathologies associated with obesity or an obesity-associated condition or disease throughout the body.

[0047] Suitable vectors for use in the present invention include adeno-associated virus (AAV) vectors, recombinant adeno-associated virus (rAAV) vectors, single strand recombinant adeno-associated virus (ssAAV) vector and self-complementary recombinant adeno- associated virus (scAAV) vectors. Examples of particularly suitable vectors include but are not limited to adeno-associated virus-9 (AAV9) and also other AAVs (e. g. AAV8 and / or AAV2) which have been pseudotyped with the capsid proteins from AAV8 or AAV9. Such vectors are described in WO 2005 / 033321. Other suitable vectors include AAV-PHP.A and AAVPHP.B (Nature Biotechnology 34, 204-209 (2016)), AAV9.47 (Hum Gene Ther. 2016 Jul;27(7):497-508), AAV-B1 (Mol. Ther. 24, 1247-1257), AAV8(Y733F) (Mol Ther 2009; 17: 463-471) and AAV2-TT (described in W02015 / 121501). Lentiviral vectors can also be used, for example, as described in Trends in Molecular Medicine, April 2016, Vol. 22, No. 4 and Ther Deliv. 2010 October; 1 (4): 517-534.

[0048] In some aspects, the vector is an AAV vector such as AAV8, AAV9, AAV vectors pseudotyped with the capsid proteins from AAV8 or AAV9, AAV-PHP.A, AAV-PHP.B, AAV9.47, AAV-B1 , AAV8(Y733F) or AAV2-TT. In other aspects, the vector is selected from AAV8, AAV9, AAV vectors pseudotyped with the capsid proteins from AAV8 or AAV9, AAV-PHP.A, AAV-PHP.B, AAV9.47 and AAV-B1. In various aspects, the vector is selected from AAV8, AAV9, AAV vectors pseudotyped with the capsid proteins from AAV8 or AAV9, AAV-PHP.A and AAV-PHP.B. In a number of aspects, the vector is selected from AAV8, AAV9, AAV vectors pseudotyped with the capsid proteins from AAV8 or AAV9, and AAV-PHP.B. In particular aspects, the vector is selected from AAV8, AAV9, and AAV vectors pseudotyped with the capsid proteins from AAV8 or AAV9. In some aspects, the vector is selected from AAV8 and AAV vectors pseudotyped with the capsid proteins from AAV8 (e. g. AAV2 pseudotyped with the capsid proteins from AAV8 (AAV2 / 8)). In other aspects, the vector is selected from AAV9 and AAV vectors pseudotyped with the capsid proteins from AAV9 (e. g. AAV2 pseudotyped with the capsid proteins from AAV9 (AAV2 / 9)). Preferably, the vector is a self-complimentary recombinant adeno-associated virus-9 (scAAV9) vector.

[0049] The adeno-associated viral vector may be a recombinant adeno-associated viral (rAAV) vector. AAV is a member of the family Parvoviridae which is described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996).

[0050] The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1 , -2 and -3) form the capsid. The terminal 145 nt are self- complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild type (wt) AAV infection in mammalian cells the Rep genes (i. e. encoding Rep78 and Rep52 proteins) are expressed from the P5 promoter and the P19 promoter, respectively, and both Rep proteins have a function in the replication of the viral genome. A splicing event in the Rep ORF results in the expression of actually four Rep proteins (i. e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also, in insect cells the Rep78 and Rep52 proteins suffice for AAV vector production.

[0051] In an AAV suitable for use as a gene therapy vector, the vector genome typically comprises a nucleic acid (e. g. an MC4R gene) to be packaged for delivery to a target cell. According to this particular aspect of the invention, the heterologous nucleotide sequence is located between the viral ITRs at either end of the vector genome. In further preferred aspects of the invention, the parvovirus (e. g. AAV) cap genes and parvovirus (e. g. AAV) rep genes are deleted from the template genome (and thus from the virion DNA produced therefrom). This configuration maximizes the size of the nucleic acid sequence(s) that can be carried by the parvovirus capsid.

[0052] According to this particular aspect, the nucleic acid is located between the viral ITRs at either end of the substrate. It is possible for a parvoviral genome to function with only one ITR. Thus, in a gene therapy vector based on a parvovirus, the vector genome is flanked by at least one ITR, but, more typically, by two AAV ITRs (generally with one either side of the vector genome, i. e. one at the 5’ end and one at the 3’ end). There may be intervening sequences between the nucleic acid in the vector genome and one or more of the ITRs. The MC4R gene may be incorporated into a parvoviral genome located between two regular ITRs or located on either side of an ITR engineered with two D regions. The specific sequences of the ITRs will be such that they could be used to package single strand AAV (ssAAV) or self-complementary AAV (scAAV).

[0053] In one aspect, the present invention provides a pharmaceutical composition comprising a vector as described herein and one or more pharmaceutically acceptable excipients. The one or more pharmaceutically acceptable excipients may include one or more pharmaceutically acceptable carriers, diluents, buffers, isotonic adjuvants, and / or other pharmaceutical agents. Suitable pharmaceutically acceptable excipients are commercially available and well known in the art. Examples of suitable carriers include, but are not limited to, water, ethanol, a polyol (such as glycerol, liquid polyethylene glycol and propylene glycol), vegetable oils and any combination thereof. Preferably, the pharmaceutical composition is in the form of a liquid. The liquid composition may be in the form of a solution, suspension, dispersion, or emulsion and may contain liposomes, nanocapsules, lipid particles or vesicles for the introduction of the pharmaceutical composition into suitable host cells.

[0054] The present invention also provides a method of preventing, ameliorating, or treating obesity or an obesity-associated condition or disease, which method comprises administering a therapeutically effective amount of a vector as described herein to a patient in need thereof. Preferably, the patient is human. In one aspect, the patient to be treated is an adult. In an alternative aspect, the patient to be treated is a child. In a further aspect of the invention, the patient to be treated is MC4R deficient, i. e. has one or more MC4R gene mutations, such as one or more heterozygous mutations (e. g. one or more heterozygous loss-of-function mutations) in the MC4R-coding sequence or one or more homozygous null mutations.

[0055] Therefore, the vectors and pharmaceutical compositions of this invention are for use in methods of treatment of a subject, in particular a human subject, and the treatment particularly relates to treatment as described herein, i. e. treatment of obesity or an obesity-associated condition or disease as described herein. Any diagnostic methods disclosed herein can be carried out in vitro, in vivo, or ex vivo. The methods of treatment as mentioned herein are in particular therapeutic methods, i. e. non-cosmetic methods of treatment.

[0056] The MC4R gene-containing vectors of the present invention are indicated to be useful for the prevention, amelioration or treatment of obesity or an obesity-associated condition or disease. In one aspect of the present invention, the obesity is monogenic obesity. In another aspect of the invention, the obesity is autosomal dominant obesity. In another aspect of the invention, the obesity is MC4R-I inked obesity. In a further aspect of the invention, the obesity is severe early-onset obesity. In an alternative aspect of the invention, the obesity is severe early-onset obesity with abnormal feeding behaviour and / or endocrine disorders.

[0057] Obesity is defined as an excess of body fat mass with an impact upon physical health. It may be assessed by standard methods, for example by calculating the body mass index (BMI) of an adult patient to be treated. BMI corresponds to the weight of an individual expressed in kilograms divided by the square of their height expressed in metres. BMI values of between 25 and 29.9 kg / m2are generally categorised “overweight”, values between 30 and 39.9 kg / m2are categorised as “obese” and values of 40 kg / m2or more are categorised as “severely obese”. In a further aspect of the invention, the patient to be treated has a B Ml of from about 25 to about 29.9 kg / m2, from about 30 to about 39.9 kg / m2, or about 40 kg / m2or more.

[0058] Obesity-associated conditions and diseases include, but are not limited to, adiposity, polyphagia, weight management, overeating, cardiovascular diseases, type 2 diabetes, hypertension, atherosclerosis, asthma, metabolic syndrome, osteoarthritis, and certain types of cancer, including without limitation, bowel cancer, breast cancer and womb cancer. Accordingly, in one aspect, the present invention provides a method of preventing, ameliorating, or treating an obesity-associated condition or disease, which method comprises administering a therapeutically effective amount of a vector as described herein to a patient in need thereof.

[0059] As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as raising the level of functional protein in a subject, so as to lead to a level sufficient to ameliorate the symptoms of the condition being treated. Typically, pharmaceutical compositions of the type disclosed herein may contain at least about 0.1 % of the active ingredient(s), such as from about 1 % to about 80 %, preferably from about 1 % to about 20 %, of the weight or volume of the total formulation.

[0060] The method of treatment causes an increase in the level of functional MC4R protein in the subject. In some aspects, the method of treatment causes an increase in the level of functional MC4R protein to about a normal level (i. e. the level found in a normal healthy subject). In one aspect, the method of treatment causes an increase in the level of functional MC4R protein to, at most, normal levels.

[0061] The vector may be administered in any suitable way so as to allow expression of the MC4R gene in one or more sites within the body. In particular embodiments, a single administration of the vector can be used to provide gene expression to prevent, ameliorate or treat the pathologies associated with obesity or an obesity-associated condition or disease. Administration of the vector may provide systemic gene expression to ameliorate the pathologies associated with obesity or an obesity-associated condition or disease throughout the body. The vector may be administered by a number of methods known in the art, for example by intravenous, intracerebroventricular, intrathecal and intracisternal administration. Intracerebroventricular administration is the direct delivery of the vector to specific areas of the brain by means of a stereotaxic injection. In one aspect, the vector is administered intravenously. In another aspect, the vector is administered intracranially. In some aspects of the invention, the vector is administered intravenously and intracranially. The vector may be also administered intrathecally. This can be alone or in addition to intravenous and / or intracranial administration.

[0062] If the vector is administered by multiple routes of administration, the vector is preferably administered at both sites on the same day. In one aspect of the invention, the multiple administrations are given sequentially, for example, within the space of about six hours, within the space of about four hours, or within the space of about two hours. In another aspect of the invention, multiple administrations of the vector are given simultaneously.

[0063] The vector may be administered at a single point in time. For example, a single injection may be given. If the vector is administered by multiple routes of administration, for example, intravenously and intracranially, the vector is administered at both sites only once (and at least on the same day as described above).

[0064] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0065] Experimental Studies pAAV-CAG-hCOMC4R plasmid transfection in HEK293T cells

[0066] HEK293T cells were seeded at a density of 105cells / cm2in a 24 well plate or 104cells / cm2in a 96 well plate the day prior to transfection in DMEM (Fisher Scientific, Cat No: 11594446) with 10 % FBS (Fisher Scientific, Cat No: 11563387) and 1 % Penicillin / Streptomycin (Fisher Scientific, Cat No: 12090216). Plasmid DNA was diluted in 150 pl of Opti-MEM I Reduced Serum Medium for 24 well plate and 25pl for 96 well plate (Fisher Scientific, Cat No: 12559099) and mixed gently. Lipofectamine 2000 (Fisher Scientific, Cat No: 10696153) was mixed gently before use. A master mix was made by diluting 164 pl of Lipofectamine 2000 in 10.2 ml Opti-MEM I Medium for 24 well plates, a master mix of 21 pl Lipofectamine 2000 in 1050 pl Opti-MEM I Medium for 96-well plates. Complexes were incubated for 5 minutes at room temperature. The diluted DNA was incubated with the lipofectamine, mixed gently, and incubated for 20 minutes at room temperature. 300pl or 50pl of the complexes were added to each well in the 24- well plate or 96 well plate respectively and mixed gently. Cells were incubated for 48 hours (96 well plates) or 72 hours (24 well plates). Material was collected for qRT-PCR and western blot 72 hours post transfection.

[0067] AAV9 vector in-vitro Transduction in HEK293T cells

[0068] HEK293T cells were seeded at a density of 5x104cells / cm2in a 24-well plate in DMEM with 10 % FBS and 1 % P / S the day prior to the transduction. Neuraminidase (Fisher Scientific, Cat No: 11486145) was added to certain wells at a dilution of 50 mll / ml for 2 hours at 37 °C. The thawed AAV9 vectors were added in 5 % FBS transduction medium to obtain the appropriate Multiplicity Of Infection (2x106MOI). 600 pl of the diluted vector was added to the corresponding wells in the plate. Material was collected for qRT-PCR and western blot 48 hours post transduction.

[0069] Western blot analysis

[0070] Cells from 24 well plates were washed with 2 ml cold PBS, aspirated and kept on ice. For protein expression by Western blot, ice cold RIPA lysis buffer (Fisher Scientific, Cat No: 10014424) was added with protease inhibitors, incubated for 20 minutes on ice and the cells were scraped off using a plastic spatula. The RIPA buffer with cells was transferred to a 2ml microcentrifuge tube and spun for 10 minutes at 10,000 RPM at

[0071] 4 °C. For qRT-PCR, 350 pl RLT buffer with 1 % p-mercaptoethanol was added on ice and the cells were scraped off using a plastic spatula. The lysate was transferred to a 2 ml microcentrifuge tube, vortexed for 5 minutes and then spun for 3 minutes at 10,000 RPM at 4 °C. Protein was quantified using the DC protein assay (Biorad, Cat No: 5000111) following the manufacturer’s instructions. 10 pg of protein sample was loaded onto Mini-PROTEAN Precast 4-15 % gels (BioRad, Cat No: 4561084) and gel was run at a constant voltage of 300 V for 20 minutes. Transfer was done using the BioRad TransBlot T urbo System with the 1 Mini TGX protocol (2.5 A constant; up to 25 V for 3 minutes). Membranes were blocked in EveryBlot Blocking buffer (BioRad, Cat No: 12010020) for

[0072] 5 min at room temperature. Membranes were incubated in 5 ml of blocking buffer with primary Rabbit anti-MC4R (ab24233 Lot: GR3295430-10) at a dilution of 1 :500 overnight. Membranes were incubated with secondary antibody goat anti-xxx HRP (Fisher Scientific, Cat No: 15306987) at a dilution of 1 :10,000 for 1 h at room temperature. HRP was visualised using the Clarity Western ECL Substrate (Biorad, Cat No: 1705061) and imaged on the i Bright digital imager. qRT-PCR analysis

[0073] For RNA extraction, cells from 24 well plates were washed 2x with ice cold PBS and processed using the Qiagen RNeasy mini-RNA extraction kit (Qiagen, Cat No: 80204) according to the manufacturer’s instructions. RNA was quantified using the Qiagen QIAxpert. 1 pg of RNA was used to retrotranscribe into cDNA using the Maxima First Strand cDNA Synthesis Kit for q RT-PCR (ThermoFisher Scientific, Cat No: K1641). Gene expression analysis performed by qRT-PCR and PowerllP SYBR Green Master Mix quantification with 20 ng of cDNA (ThermoFisher Scientific, Cat No: A25741). qRT- PCR was performed on the QuantStudio 6 Real-Time machine to quantify the expression of human BBS1 relative to Gapdh using the following primers: hC0MC4RGA F: TGCTGGTGTCTGTGTCTAATG hC0MC4RGA R: GTCGATCACGTTGTCGATGT hC0MC4RGS F: CCAGACTGCACATCAAGAGAA hC0MC4RGS R: CACACCACGAACACTCCTATC hWTMC4R F TTGCAGTGGACAGGTACTTTAC hWTMC4R R GCTGCCCAGATACAACTTATGA mWTmc4rF. CAGTACGGATACGGATGCCC mWTmc4rR GCGAGCAAGGAGCTACAGAT

[0074] Gapdh F: ATGACATCAAGAAGGTGGTG

[0075] Gapdh R: CATACCAGGAAATGAGCTTG

[0076] The following standard qRT-PCR program was used:

[0077] 1. 50 °C, 2 min (UDG activation)

[0078] 2. 95 °C, 2 min (Dual-Lock DNA polymerase)

[0079] 3. 95 °C, 15 sec (Denature)

[0080] 4. 60 °C,1 min (Anneal / extend)

[0081] 5. Repeat steps 2.-4. 39 times

[0082] Gene expression was analysed using Ct values as previously described (MW P. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic acids research. 2001 ;29(9):E45. doi:10.1093 / NAR / 29.9.E45). cAMP analysis cAMP levels were measured following transfection or transduction in 96 well plates using the in-vitro Promega cAMP-Glo™ Assay kit (#V1501). Prepare the kit reagents (Induction buffer, cAMP solution and Kinase-Glo® solution) following the user manuals guidelines. Prepare the standards on a separate 96-well plate before transferring to the assay plate. Add 100ul of Induction buffer to wells A2 to A12, then add 200ul of cAMP solution to well A1. Perform a serial twofold dilution by transferring 100 pl from well A1 to well A2 with a pipette, pipetting to mix. Transfer 100 pl to well A3. Repeat for wells A4 through A11. Discard the extra 100 pl from well A11 . Do not add cAMP solution to the no-cAMP control reactions in well A12. Transfer 20ul of each standard to the 96 well plate that contains your cells, transfer 20ul in triplicate into A1 , B1 , C1. Add 20ul / well of cAMP-Glo™ Lysis Buffer to all wells, including the cAMP standards. Incubate the plate whilst shaking at room temperature for 15 minutes. Add 7.5 pl of Protein Kinase A to 3.0ml of cAMP-Glo™ Reaction Buffer and mix by inversion, then add 40ul of cAMP-Glo™ Detection Solution to all wells. Mix the plate by shaking for 30-60 seconds and incubate the plate at room temperature for 20 minutes. Add 80ul of room-temperature Kinase-Glo® Reagent to all wells. Mix the plate by shaking for 30-60 seconds and incubate at room temperature for 10 minutes. Proceed to measure luminescence on a luminometer. To calculate cAMP levels in each target well, use the following calculations from the standard curve produced by the cAMP standard curve: The luminesence output of the assay is affected by the presence of cells, so it is not possible to directly compare the raw RLU values of the samples with those of the cAMP standards. Instead, calculate the change in RLU (ARLU) for the standards and the samples as follows: For each standard concentration: ARLU = RLU (0 nM) - RLU (X nM). Plot ARLU (Y-axis) versus cAMP concentration in nM (X-axis). Perform a regression analysis. Figure 4 shows an example of a plotted standard curve. For each sample: ARLU = RLU (untreated sample) - RLU (treated sample). Using this ARLU value and the linear equation generated from the standard curve, calculate the cAMP concentration.

[0083] In vivo study

[0084] Concept

[0085] An in vivo study has been conducted to show the efficacy of a vector according to an embodiment of the invention. In brief, a scAAV-CAG-hCOMC4R vector was administered intracerebroventricularly (ICV) at postnatal day 7 (P7) in Mc4rLoxTB mouse model (Mus musculus). In more detail, the drug substance (DS) comprises self- complementary adeno-associated virus (scAAV) vectors expressing a human-codon- optimized MC4R cDNA. The scAAV9-CAG-hCOMC4R variant of the vector was utilized, distinguished by its capsid, AAV9. The expression cassette includes a human-codon- optimized MC4R cDNA, the CAG promoter, which combines the cytomegalovirus (CMV) enhancer, chicken p-actin promoter, and rabbit p-globin splice acceptor site for robust gene expression, and flanking AAV9 inverted terminal repeats (ITRs) for packaging and replication. These vectors have been manufactured using the plasmid DNA (pDNA) transfection technology in the HEK293T cell line.

[0086] The DS was formulated as drug product (DP) AXV-201 (ICV). The buffer treatment (PBS) does not contain viral materials. The DP, AXV-201 , was diluted with PBS if required, which also served as the vehicle for control animals. Control animals were injected with PBS in the same volume (5 pl) as the treated animals.

[0087] The experiment began with neonatal mice (P7 and above), starting with genotyping, treatment, and weekly weighing, and concluded with sacrifice at 4 months. All animals received a single dose of 1 E13 vg (viral genomes) in 5 pL at P7 following genotyping and sexing.

[0088] The primary endpoint was the attenuation of body weight gain in treated mutant mice by week 16, achieving normalisation relative to wild-type control mice. The extent to which AXV-201 normalises weight in mutant mice was also determined.

[0089] Secondary aim of the study was the measurement of circulating serum biomarkers (Leptin, Adiponectin, Insulin, Cholesterol, HDL / LDL, and Ghrelin) following routine ELISA protocols.

[0090] The study consisted of three arms: untreated, vehicle-treated (PBS), and AXV-201 - treated (see Table 1).

[0091]

[0092] Animals

[0093] Wild type (WT) MC4R and mutant (Mut) B6.129S4-Mc4rtm1 Lowl / J Mc4r LoxTB were obtained from Jackson Biolabs, C57BL / 6J, Stock number 032518. WT mice had fully functional MC4R, whereas in Mut mice, the MC4R gene was knocked out.

[0094] Animals were genotyped and tattooed at birth (tattoos on paw not correlating to treatment groups). All animals were housed in individually ventilated cages (IVCs) with communal air filtering. Food and drink were consistent for all animals. Ear clipping was performed after weaning if the paw tattoo faded. Each animal had an individual ID in SoftMouse colony management software where all procedures were recorded. Animals were housed under the same conditions, avoiding specific housing rows or columns for treated or control groups. Litters were kept together, and single housing was avoided.

[0095] Blinding was achieved by assigning animals individually to different groups, creating randomisations for all groups. Group sizes were increased proportionally to avoid differences in group composition. Statistical analysis of weight data

[0096] The primary method was repeated measures analysis with weekly weight comparisons. Statistical analysis (ANOVA; p<0.0001) was performed between all treated and control groups. The primary comparison of interest was between the Mc4rLoxTB / LoxTBtreated and vehicle-treated groups.

[0097] AAV injection

[0098] All animals in the Mut-AAV9 and WT-AAV9 groups were treated with the same batch of AAV9 hMC4R. All animals received treatments at P7. All animals received a single dose of 1 E13 viral genomes (vg) in 5 pL at P7 following genotyping and sexing. One single unilateral intracerebroventricular (ICV) was performed. This procedure was performed under general anaesthesia delivered intraperitonially (IP), following a Royal Veterinary College protocol.

[0099] In-life measurements

[0100] Animals were weighed weekly using a scientific scale, with all measurements taken in the morning.

[0101] Results

[0102] The results of this study are shown in Figure 7. Mean body weight (g; n=8) of each group (Mut-AAV9, Mut-UnT, Mut-PBS, WT-AAV9, WT-PBS, WT-LInT) are plotted over time (weeks). Error bars represent the standard deviation. **** indicates significant differences with a p-value smaller than 0.0001 , while “ns” indicates no statistically significant difference. Group differences are shown for each sex separately. For male and female animals alike, the diagrams show that mutant untreated and vehicle animals, starting from around weeks 5-7, gained more weight than wild type animals and substantially became obese. In contrast, vector treated mutant animals express significantly lower weight gain as compared to mutant untreated and vehicle animals, while weight gain of the vector treated animals as compared to all wild type groups was non-significant. In conclusion, it can be taken from Figure 7 that treatment with a vector comprising a promoter operably linked to a melanocortin-4-receptor (MC4R) gene in accordance with an embodiment of this invention is efficacious in ameliorating and / or treating obesity. No adverse effects were reported for the administered doses. Further results are shown in Figures 9 and 10. Figures 9 (A-B) and 10 (C-D) show the average body weight curve from week 1 to week 16 in wild-type (WT) and Mc4rLoxTB / LoxTB(Mut) mice, untreated or injected at postnatal day 7 (P7) with AAV9-CAG-hMC4R_GA (AXV-201). Figures 9 (A-B) illustrate the treatment effect of AXV-201 : (A) Male MC4fj-oxTB LoxTBmjcetreated with AXV-201 showed significantly reduced body weight compared to untreated Mut controls, (B) Female MC4fLoxTB / LoxTBmice treated with AXV- 201 also exhibited significantly reduced body weight compared to untreated Mut controls. Figures 10 (C-D) show a weight normalization effect: (C) Male Mc4rLoxTB / LoxTBmice treated with AXV-201 exhibited no significant difference in body weight compared to untreated WT controls. (D) Female MC4IJ-OXTB / LOXTBmice treated with AXV-201 weighed less than untreated WT controls up to week 7; however, from week 8 onward, there was no significant difference in body weight compared to untreated WT controls, (n = 6 - 8 per group; mean ± SD; unpaired T-test).

[0103] Yet further results are shown in Figures 11 and 12. Figures 11 (A-B) and 12 (C-D) demonstrate that Insulin, Leptin and Ghrelin serum levels are completely normalised in AXV-201 treated Mc4rLoxTB / LoxTBmice: (A) shows significant differences between the 3 different study groups. Significant differences between mutants treated with AXV-201 and Mc4rLox TB / LoxTBtreated with vehicle (p=<0.0001). (B) shows significant differences between groups (p=0.0007) and when analysing by sex. (C) shows significant differences between groups (p=0.0001). (D) shows significant differences in LDL Cholesterol level between groups (p=0.0016). Mutant treated animals show lower level of LDL-Cholesterol than mutants treated with vehicle or mutants control. (E) shows a significant difference of HDL-Cholesterol between the 3 study groups (p=0.0023) and between sexes in both MC4R genotypes and across all study groups. Ordinary one-way ANOVA (normally distributed) or Kruskall-Wallis test (skewed data) with multiple comparisons was applied for statistical analysis.

[0104] In summary, these results show that, surprisingly, codon-optimized human MC4R constructs packaged into adeno-associated viral (AAV) vectors, demonstrate significantly enhanced transgene expression compared to wild-type sequences, with a higher ability to activate cAMP signalling in vitro.

[0105] Most importantly, in vivo administration of AXV-201 (AAV9-hCOMC4R) in Mc4r-nu\\ mice prevented the development of obesity in males and females, restoring a normal weight trajectory comparable to wild-type controls. The rescue included also practical normalisation of neurometabolic markers. No toxicity issues were observed when wildtype cohorts were dosed.

[0106] Aberrancies in Insulin, Leptin and / or Ghrelin serum levels can be indicative of obesity or an obesity-associated condition or disease. Thus, it has been demonstrated by the inventors that a vector according to embodiments of the invention is not only capable of ameliorating and / or treating obesity, but also of ameliorating and / or treating obesity- associated (metabolic) conditions or disease.

[0107] SEQUENCES

[0108] The sequences as used in accordance with the present invention are defined by the appended sequence listing.

[0109] SEQ ID NO. 1 : Human melanocortin receptor 4 (hMC4R) nucleotide sequence (WT)

[0110] ATGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCA GCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGA TGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGT GTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAA TCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGG TGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGAT ACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTGACTCGGTGATCTG TAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACT TTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTAAGCGGGTTGGG ATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCAT TTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGC TGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATT AAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATG AAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGCCC CATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGT GCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGTGTAATTCAATCATCG ATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATC ATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAA

[0111] SEQ ID NO. 2: Human melanocortin receptor 4 (hMC4R) mRNA codon sequence (CDS) TACCACTTGAGGTGGGTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCG

[0112] TCGTCAATGTCTGACGTGTCGTTACGGTCACTCAGGGAACCTTTTCCGATGAGAC

[0113] TACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCC

[0114] ACAGTAGTCGAACAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTTGTTCTT

[0115] AGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGAC

[0116] CACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAATTTGTCATGTCT

[0117] ATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAACTGAGCCACTAGA

[0118] CATCGAGGAACGAACGTAGGTAAACGTCGGACGAAAGTTAACGTCACCTGTCCAT

[0119] GAAATGATAGAAGATACGAGAGGTCATGGTATTGTAATACTGTCAATTCGCCCAAC

[0120] CCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTA

[0121] GTAAATGAGTCTATCATCACGACAGTAGTAGACGGAGTAGTGGTACAAGAAGTGG

[0122] TACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAA

[0123] GTGTAATTCTCCTAACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGG

[0124] TTATACTTCCCTCGCTAATGGAACTGGTAGGACTAACCGCAGAAACAACAGACGA

[0125] CCCGGGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTAT

[0126] AACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTACACATTAA

[0127] GTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTTTGGAAG

[0128] TTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAACACTGAACAGATCGTCTA TAATT

[0129] SEQ ID NO. 3: Codon optimised nucleotide sequence encoding human MC4R protein (referred to as hMC4R COGS)

[0130] ATGGTGAACTCTACACACAGAGGCATGCACACCAGCCTGCACCTGTGGAACCGG

[0131] AGCTCCTACCGGCTGCACAGCAATGCCAGCGAAAGCCTGGGAAAGGGCTATTCT

[0132] GATGGCGGCTGCTACGAGCAGCTGTTTGTCAGCCCTGAGGTGTTCGTGACCCTG

[0133] GGCGTGATCTCTCTGCTGGAAAACATCCTGGTGATCGTGGCCATCGCAAAGAACA

[0134] AGAACCTGCATTCTCCAATGTACTTTTTTATCTGCAGCCTAGCTGTTGCCGACATG

[0135] CTGGTCTCCGTGTCCAACGGCAGCGAGACAATCGTGATCACACTGCTGAACAGCA

[0136] CCGACACCGACGCCCAGAGCTTCACCGTGAATATCGACAACGTGATCGACAGCG

[0137] TGATCTGCAGCTCTCTGCTGGCTAGCATCTGTAGCCTGCTGAGCATCGCCGTGGA

[0138] CAGATACTTCACCATTTTCTACGCCCTGCAATACCACAACATCATGACCGTGAAAA

[0139] GAGTGGGCATCATCATCAGCTGCATCTGGGCCGCCTGCACAGTGTCCGGCATCC

[0140] TCTTCATCATCTACTCAGATTCTAGCGCCGTGATCATTTGTCTGATCACCATGTTCT

[0141] TTACAATGCTGGCCCTGATGGCCAGCCTGTACGTGCACATGTTCCTGATGGCCAG

[0142] ACTGCACATCAAGAGAATCGCCGTTCTGCCTGGCACAGGCGCCATCCGGCAGGG

[0143] AGCTAATATGAAAGGCGCTATCACCCTGACCATCCTGATAGGAGTGTTCGTGGTG TGTTGGGCCCCTTTCTTCCTGCACCTGATCTTCTACATCAGCTGTCCTCAGAACCC

[0144] CTACTGCGTGTGCTTCATGAGCCACTTCAACCTGTACCTGATCCTGATTATGTGCA

[0145] ACAGCATCATTGACCCCCTGATCTACGCCCTGAGGTCCCAGGAGCTGAGAAAGAC

[0146] CTTCAAGGAAATCATCTGCTGCTATCCTCTGGGCGGTCTCTGTGATCTGAGCAGT AGATACTGA

[0147] SEQ ID NO. 4: Codon optimised nucleotide sequence encoding human MC4R protein (referred to as hMC4R COGA)

[0148] ATGGTCAACAGCACCCACAGAGGCATGCACACCAGCCTGCACCTGTGGAACAGA

[0149] AGCAGCTACCGGCTGCACAGCAATGCCTCTGAGTCTCTCGGCAAGGGCTACTCC

[0150] GATGGCGGCTGTTATGAGCAGCTGTTCGTGTCCCCTGAGGTGTTCGTGACACTG

[0151] GGCGTGATCAGCCTGCTGGAAAACATCCTGGTCATCGTGGCCATTGCCAAGAACA

[0152] AGAACCTGCACAGCCCCATGTATTTCTTCATCTGCAGCCTGGCCGTGGCCGACAT

[0153] GCTGGTGTCTGTGTCTAATGGCAGCGAGACAATCGTGATCACACTGCTGAACAGC

[0154] ACCGACACAGACGCCCAGAGCTTCACCGTGAACATCGACAACGTGATCGACTCC

[0155] GTGATCTGCAGCTCCCTGCTGGCCTCTATCTGTAGCCTGCTGTCTATCGCCGTGG

[0156] ACCGGTACTTCACCATCTTCTACGCCCTGCAGTACCACAACATCATGACCGTGAA

[0157] GAGAGTGGGCATCATCATCAGCTGCATCTGGGCCGCCTGTACCGTGTCCGGCAT

[0158] CCTGTTCATCATCTACAGCGACAGCAGCGCCGTGATCATCTGCCTGATCACCATG

[0159] TTCTTCACCATGCTGGCCCTGATGGCCAGCCTGTACGTGCACATGTTTCTGATGG

[0160] CCCGGCTGCATATCAAGCGGATCGCTGTGCTTCCTGGCACAGGCGCTATTAGACA

[0161] GGGCGCCAATATGAAGGGCGCCATCACACTGACCATCCTGATCGGCGTGTTCGT

[0162] CGTGTGTTGGGCCCCTTTCTTTCTGCACCTGATCTTCTACATCAGCTGTCCTCAGA

[0163] ACCCCTACTGCGTGTGCTTCATGAGCCACTTCAACCTGTACCTGATCCTGATCATG

[0164] TGCAACAGCATCATCGACCCTCTGATCTATGCCCTGCGGAGCCAAGAGCTGAGAA

[0165] AGACCTTCAAAGAGATTATCTGCTGCTACCCTCTCGGCGGCCTGTGCGATCTGTC CAGCAGATATTGA

[0166] SEQ ID NO. 5: Codon optimised nucleotide sequence encoding human MC4R protein (referred to as hMC4R COIDT)

[0167] ATGGTCAATTCTACACATCGAGGGATGCATACGTCCCTTCACCTTTGGAACAGGT

[0168] CTTCTTATAGATTGCATTCAAACGCAAGTGAGAGCCTGGGCAAGGGGTATAGCGA

[0169] CGGGGGCTGCTACGAACAACTCTTCGTTTCACCTGAAGTATTTGTAACCCTTGGT

[0170] GTCATTTCCTTGCTCGAAAATATTCTCGTTATAGTGGCCATCGCAAAAAATAAGAA

[0171] CCTTCATTCTCCCATGTACTTCTTCATCTGCTCTCTCGCGGTCGCAGATATGCTGG TATCAGTCTCTAACGGCTCAGAGACTATTGTCATCACACTTTTGAATAGTACCGAT ACAGATGCACAAAGTTTTACCGTAAACATAGATAATGTCATCGACTCAGTCATATG

[0172] CTCATCCCTCCTTGCTAGTATATGCAGCTTGCTTAGTATTGCAGTAGACAGGTACT TTACTATATTCTACGCACTCCAATATCACAATATAATGACCGTCAAAAGGGTTGGTA

[0173] TAATCATAAGCTGCATCTGGGCTGCGTGCACGGTCAGTGGCATTTTGTTCATTATA TATAGTGACTCTAGTGCGGTCATCATTTGTTTGATAACAATGTTCTTCACGATGTTG GCACTTATGGCCTCTTTGTACGTTCATATGTTTTTGATGGCCCGGTTGCACATTAA

[0174] AAGAATCGCGGTACTTCCAGGCACAGGGGCGATACGCCAGGGGGCAAATATGAA AGGTGCGATAACGCTGACAATCTTGATCGGGGTATTTGTAGTGTGTTGGGCGCCT

[0175] TTTTTTCTGCACCTCATCTTCTATATCTCCTGCCCGCAAAACCCATATTGCGTTTGC TTCATGTCTCATTTCAATTTGTATTTGATCTTGATAATGTGTAACAGTATAATAGACC CATTGATCTATGCCCTTAGAAGCCAAGAGCTGAGGAAAACATTCAAGGAGATTATC TGTTGTTATCCACTTGGGGGGTTGTGCGACCTCTCCAGTCGATATTAA

[0176] SEQ ID NO. 6: CAG promoter sequence

[0177] CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA

[0178] TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAA GTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG

[0179] CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC TACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCAC TCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATT

[0180] ATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGG GCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCA

[0181] GCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGG CGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0182] SEQ ID NO. 7: Construct comprising CAG promoter and hMC4R COGS nucleotide sequence, as depicted in Figure 4 ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacg gtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg gtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattatt ttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcgggg cggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggc ggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccg ctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcc cttctcctccgggctgtaattagcgcttggaattcgccaccATGGTGAACTCTACACACAGAGGCATGC ACACCAGCCTGCACCTGTGGAACCGGAGCTCCTACCGGCTGCACAGCAATGCCA GCGAAAGCCTGGGAAAGGGCTATTCTGATGGCGGCTGCTACGAGCAGCTGTTTG TCAGCCCTGAGGTGTTCGTGACCCTGGGCGTGATCTCTCTGCTGGAAAACATCCT GGTGATCGTGGCCATCGCAAAGAACAAGAACCTGCATTCTCCAATGTACTTTTTTA TCTGCAGCCTAGCTGTTGCCGACATGCTGGTCTCCGTGTCCAACGGCAGCGAGA CAATCGTGATCACACTGCTGAACAGCACCGACACCGACGCCCAGAGCTTCACCGT GAATATCGACAACGTGATCGACAGCGTGATCTGCAGCTCTCTGCTGGCTAGCATC TGTAGCCTGCTGAGCATCGCCGTGGACAGATACTTCACCATTTTCTACGCCCTGC AATACCACAACATCATGACCGTGAAAAGAGTGGGCATCATCATCAGCTGCATCTG

[0183] GGCCGCCTGCACAGTGTCCGGCATCCTCTTCATCATCTACTCAGATTCTAGCGCC GTGATCATTTGTCTGATCACCATGTTCTTTACAATGCTGGCCCTGATGGCCAGCCT GTACGTGCACATGTTCCTGATGGCCAGACTGCACATCAAGAGAATCGCCGTTCTG CCTGGCACAGGCGCCATCCGGCAGGGAGCTAATATGAAAGGCGCTATCACCCTG ACCATCCTGATAGGAGTGTTCGTGGTGTGTTGGGCCCCTTTCTTCCTGCACCTGA TCTTCTACATCAGCTGTCCTCAGAACCCCTACTGCGTGTGCTTCATGAGCCACTTC AACCTGTACCTGATCCTGATTATGTGCAACAGCATCATTGACCCCCTGATCTACGC CCTGAGGTCCCAGGAGCTGAGAAAGACCTTCAAGGAAATCATCTGCTGCTATCCT CTGGGCGGTCTCTGTGATCTGAGCAGTAGATACTGAgcgccgtcgacctcgagggggggccg cgactctagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacct gaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaa tttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttaaggcctaggtgagctctg gtaccctctagtcaaggatc

[0184] SEQ ID NO. 8: Construct comprising CAG promoter and hMC4R COGA nucleotide sequence, as depicted in Figure 5 ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacg gtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg gtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattatt ttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcgggg cggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggc ggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccg ctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcc cttctcctccgggctgtaattagcgcttggaattcgccaccATGGTCAACAGCACCCACAGAGGCATGC ACACCAGCCTGCACCTGTGGAACAGAAGCAGCTACCGGCTGCACAGCAATGCCT CTGAGTCTCTCGGCAAGGGCTACTCCGATGGCGGCTGTTATGAGCAGCTGTTCGT GTCCCCTGAGGTGTTCGTGACACTGGGCGTGATCAGCCTGCTGGAAAACATCCT GGTCATCGTGGCCATTGCCAAGAACAAGAACCTGCACAGCCCCATGTATTTCTTC ATCTGCAGCCTGGCCGTGGCCGACATGCTGGTGTCTGTGTCTAATGGCAGCGAG ACAATCGTGATCACACTGCTGAACAGCACCGACACAGACGCCCAGAGCTTCACCG TGAACATCGACAACGTGATCGACTCCGTGATCTGCAGCTCCCTGCTGGCCTCTAT CTGTAGCCTGCTGTCTATCGCCGTGGACCGGTACTTCACCATCTTCTACGCCCTG CAGTACCACAACATCATGACCGTGAAGAGAGTGGGCATCATCATCAGCTGCATCT GGGCCGCCTGTACCGTGTCCGGCATCCTGTTCATCATCTACAGCGACAGCAGCG CCGTGATCATCTGCCTGATCACCATGTTCTTCACCATGCTGGCCCTGATGGCCAG CCTGTACGTGCACATGTTTCTGATGGCCCGGCTGCATATCAAGCGGATCGCTGTG CTTCCTGGCACAGGCGCTATTAGACAGGGCGCCAATATGAAGGGCGCCATCACA CTGACCATCCTGATCGGCGTGTTCGTCGTGTGTTGGGCCCCTTTCTTTCTGCACC TGATCTTCTACATCAGCTGTCCTCAGAACCCCTACTGCGTGTGCTTCATGAGCCAC TTCAACCTGTACCTGATCCTGATCATGTGCAACAGCATCATCGACCCTCTGATCTA TGCCCTGCGGAGCCAAGAGCTGAGAAAGACCTTCAAAGAGATTATCTGCTGCTAC CCTCTCGGCGGCCTGTGCGATCTGTCCAGCAGATATTGAgcgccgtcgacctcgagggggg gccgcgactctagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctga acctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatca caaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttaaggcctaggtgag ctctggtaccctctagtcaaggatc

[0185] SEQ ID NO. 9: Construct comprising CAG promoter and hMC4R COIDT nucleotide sequence, as depicted in Figure 6 ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacg gtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg gtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattatt ttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcgggg cggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggc ggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccg ctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcc cttctcctccgggctgtaattagcgcttggaattcgccaccATGGTCAATTCTACACATCGAGGGATGC ATACGTCCCTTCACCTTTGGAACAGGTCTTCTTATAGATTGCATTCAAACGCAAGT GAGAGCCTGGGCAAGGGGTATAGCGACGGGGGCTGCTACGAACAACTCTTCGTT TCACCTGAAGTATTTGTAACCCTTGGTGTCATTTCCTTGCTCGAAAATATTCTCGTT ATAGTGGCCATCGCAAAAAATAAGAACCTTCATTCTCCCATGTACTTCTTCATCTG CTCTCTCGCGGTCGCAGATATGCTGGTATCAGTCTCTAACGGCTCAGAGACTATT

[0186] GTCATCACACTTTTGAATAGTACCGATACAGATGCACAAAGTTTTACCGTAAACAT

[0187] AGATAATGTCATCGACTCAGTCATATGCTCATCCCTCCTTGCTAGTATATGCAGCT

[0188] TGCTTAGTATTGCAGTAGACAGGTACTTTACTATATTCTACGCACTCCAATATCACA

[0189] ATATAATGACCGTCAAAAGGGTTGGTATAATCATAAGCTGCATCTGGGCTGCGTG

[0190] CACGGTCAGTGGCATTTTGTTCATTATATATAGTGACTCTAGTGCGGTCATCATTT

[0191] GTTTGATAACAATGTTCTTCACGATGTTGGCACTTATGGCCTCTTTGTACGTTCATA

[0192] TGTTTTTGATGGCCCGGTTGCACATTAAAAGAATCGCGGTACTTCCAGGCACAGG

[0193] GGCGATACGCCAGGGGGCAAATATGAAAGGTGCGATAACGCTGACAATCTTGATC

[0194] GGGGTATTTGTAGTGTGTTGGGCGCCTTTTTTTCTGCACCTCATCTTCTATATCTC

[0195] CTGCCCGCAAAACCCATATTGCGTTTGCTTCATGTCTCATTTCAATTTGTATTTGAT

[0196] CTTGATAATGTGTAACAGTATAATAGACCCATTGATCTATGCCCTTAGAAGCCAAG

[0197] AGCTGAGGAAAACATTCAAGGAGATTATCTGTTGTTATCCACTTGGGGGGTTGTG

[0198] CGACCTCTCCAGTCGATATTAAgcgccgtcgacctcgagggggggccgcgactctagatcataatcagc cataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaa ttgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttc actgcattctagttgtggtttgtccaaactcatcaatgtatcttaaggcctaggtgagctctggtaccctctagtcaaggatc

[0199] SEQ ID NO. 10: Human melanocortin receptor 4 full protein sequence

[0200] MVNSTHRGMHTSLHLWNRSSYRLHSNASESLGKGYSDGGCYEQLFVSPEVFVTLGV

[0201] ISLLENILVIVAIAKNKNLHSPMYFFICSLAVADMLVSVSNGSETIVITLLNSTDTDAQSFT

[0202] VNIDNVIDSVICSSLLASICSLLSIAVDRYFTIFYALQYHNIMTVKRVGIIISCIWAACTVSG

[0203] I LFI IYSDSSAVI ICLITMFFTMLALMASLYVHMFLMARLHI KRIAVLPGTGAI RQGANM KG AITLTILIGVFVVCWAPFFLHLIFYISCPQNPYCVCFMSHFNLYLILIMCNSIIDPLIYALRS QELRKTFKEIICCYPLGGLCDLSSRY

[0204] SEQ ID NO. 11 : Mouse melanocortin receptor 4 (mMC4R) nucleotide sequence (WT)

[0205] ATGAACTCCACCCACCACCATGGCATGTATACTTCCCTCCACCTCTGGAACCGCA

[0206] GCAGCTACGGGCTGCACGGCAATGCCAGCGAGTCGCTGGGGAAGGGCCACCCG

[0207] GACGGAGGATGCTATGAGCAACTTTTTGTTTCCCCCGAGGTGTTTGTGACTCTGG

[0208] GTGTCATAAGCCTGTTGGAGAACATTCTAGTGATCGTGGCGATAGCCAAGAACAA

[0209] GAACCTGCACTCACCCATGTACTTTTTCATCTGTAGCCTGGCTGTGGCAGATATGC

[0210] TGGTGAGCGTTTCGAATGGGTCGGAAACCATCGTCATTACCCTGTTAAACAGTAC

[0211] GGATACGGATGCCCAGAGCTTCACCGTGAACATTGATAATGTCATTGACTCTGTG

[0212] ATCTGTAGCTCCTTGCTCGCATCCATTTGCAGCCTGCTTTCCATTGCGGTGGACA GGTATTTCACTATCTTTTACGCGCTCCAGTACCATAACATCATGACGGTTAGGCGG

[0213] GTCGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACTGTGTCAGGCGTCCTCT

[0214] TCATCATTTACTCGGACAGCAGCGCTGTCATCATCTGCCTCATTTCCATGTTCTTC

[0215] ACTATGCTAGTTCTCATGGCCTCTCTCTATGTCCACATGTTCCTGATGGCGAGGCT TCACATTAAGAGGATTGCTGTCCTCCCAGGCACAGGGACCATCCGCCAGGGTAC

[0216] CAACATGAAGGGGGCGATTACCTTGACCATCCTGATTGGAGTCTTTGTTGTCTGC

[0217] TGGGCCCCGTTCTTTCTCCATTTACTGTTCTACATCTCTTGCCCTCAGAATCCATA

[0218] CTGCGTGTGCTTCATGTCTCATTTTAATTTGTATCTCATACTGATCATGTGTAACGC

[0219] CGTCATCGACCCTCTCATTTATGCCCTCCGGAGTCAAGAACTGAGGAAAACTTTCA AAGAGATCATCTGTTTCTATCCTCTGGGAGGCATCTGTGAGTTGTCTAGCAGGTAT

[0220] TAA

Claims

Claims1. A vector for preventing, ameliorating, and / or treating obesity or an obesity-associated condition or disease, said vector comprising a promoter operably linked to a melanocortin-4-receptor (MC4R) gene.

2. A vector according to claim 1 , wherein the vector is an adeno-associated viral (AAV) vector.

3. A vector according to claim 2, wherein the AAV vector is a recombinant adeno-associated virus (rAAV) vector, single strand recombinant adeno-associated virus (ssAAV) vector or a self-complementary recombinant adeno-associated virus (scAAV) vector.

4. A vector according to any preceding claim, wherein the vector is AAV9.

5. A vector according to any preceding claim, wherein the promoter is selected from the group consisting of short elongation factor promoter (EFS), CAG promoter, cytomegalovirus immediate-early promoter (CMV), Ubiquitin C promoter (UBC), phosphoglycerate kinase promoter (PGK), beta-actin promoter, and chicken beta-actin promoter (CBA).

6. A vector according to any preceding claim, wherein the promoter is a CAG promoter.

7. A vector according to any preceding claim, wherein the promoter is a CAG promoter having the nucleotide sequence of SEQ ID NO.6.

8. A vector according to any preceding claim, wherein the MC4R gene has the nucleotide sequence of any one of SEQ ID Nos. 1-5 or has at least 70 % sequence identity thereto.

9. A vector according to claim 8 wherein the MC4R gene has the nucleotide sequence of any one of SEQ ID Nos. 1-5 or has at least 80 % sequence identity thereto.

10. A vector according to claim 8, wherein the MC4R gene has the nucleotide sequence of any one of SEQ ID Nos. 1-5 or has at least 90 % sequence identity thereto.

11. A vector according to any preceding claim, wherein the vector is an AAV9 vector, and wherein the promoter is a CAG promoter.

12. A vector according to any preceding claim, wherein the vector is an AAV9 vector, the promoter is a CAG promoter and wherein the MC4R gene has the nucleotide sequence of any one of SEQ ID Nos. 1-5 or has at least 70 % sequence identity thereto.

13. A vector according to any preceding claim, wherein the vector is an AAV9 vector, the promoter is a CAG promoter having the nucleotide sequence of SEQ ID NO. 6 and wherein the MC4R gene has the nucleotide sequence of any one of SEQ ID Nos. 1-5 or has at least 70 % sequence identity thereto.

14. A pharmaceutical composition comprising the vector according to any one of claims 1 to 13 and one or more pharmaceutically acceptable excipients.

15. A pharmaceutical composition according to claim 14, for intravenous or intracranial administration.

16. A method of preventing, ameliorating and / or treating obesity or an obesity-associated condition or disease, which method comprises administering a therapeutically effective amount of a vector according to any one of claims 1 to 13, or a pharmaceutical composition according to claim 14 or 15, to a patient in need thereof.

17. The method of claim 16, wherein the vector is given by intravenous, intracerebroventricular, intrathecal and / or intracisternal administration18. The vector according to any one of claims 1 to 13, or the pharmaceutical composition according to claim 14 or 15, for use in preventing, ameliorating, and / or treating obesity or an obesity-associated condition or disease.

19. The use of the vector according to any one of claims 1 to 13, or the pharmaceutical composition according to claim 14 or 15, in the manufacture of a medicament for preventing, ameliorating, and / or treating obesity or an obesity-associated condition or disease.

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