EP4615967A1 - Compositions and methods for treating obesity - Google Patents

Compositions and methods for treating obesity

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Publication number
EP4615967A1
EP4615967A1 EP23889277.2A EP23889277A EP4615967A1 EP 4615967 A1 EP4615967 A1 EP 4615967A1 EP 23889277 A EP23889277 A EP 23889277A EP 4615967 A1 EP4615967 A1 EP 4615967A1
Authority
EP
European Patent Office
Prior art keywords
rabla
gdp
seq
subject
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23889277.2A
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German (de)
French (fr)
Inventor
Mohsen Amir Alipour
Zemin Yao
Kuo-Tang Tseng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Reliable Holdings Co Ltd
Motigenix Singapore Pte Ltd
Original Assignee
Reliable Holdings Co Ltd
Motigenix Singapore Pte Ltd
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Application filed by Reliable Holdings Co Ltd, Motigenix Singapore Pte Ltd filed Critical Reliable Holdings Co Ltd
Publication of EP4615967A1 publication Critical patent/EP4615967A1/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the present invention relates generally to treatment of obesity. More specifically, the present invention relates to compositions and methods for enhancing microautophagy to treat obesity and related diseases or disorders.
  • cytoplasmic target or cargo e.g. protein, lipid, glycogen or pathogen
  • Microautophagy may contribute to cytosolic protein degradation via multivesicular bodies (MVBs) of the late endosomes.
  • microautophagy may also support direct glycogen delivery to lysosomes and its degradation.
  • malfunctioning or insufficient microautophagy may be associated with development of a variety of metabolic and/or neurological diseases, for example.
  • proteins, lipids, and/or glycogens may be associated with a variety of important diseases, disorders, and conditions.
  • Methods for targeting such proteins, lipids, and/or glycogens are desirable.
  • Obesity is a leading preventable cause of death worldwide, with increasing rates in adults and children. Indeed, the prevalence of obesity has increased markedly over the past decades and has reached epidemic proportions; over one- third of adults are overweight (BMI 25-29.9 kg/m2) or obese (BMI > 30 kg/m2) worldwide (WHO).
  • Obesity is a complex disease involving an excessive amount of body fat that increases the risk of other diseases and health problems, such as heart disease, diabetes, high blood pressure and certain cancers (Zhang et al., 2018). Obesity is widely considered as one of the most serious public health problems of the 21st century as the efficacy of current therapies remains poor and focus mainly on suppressing caloric intake.
  • a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabi a, or one or more expressible nucleic acids encoding such a Rabla GDP , may be used to reduce obesity in an obese subject.
  • a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabi a, or one or more expressible nucleic acids encoding such a Rabla GDP , may be used to reduce obesity in an obese subject.
  • a method for reducing obesity, or for preventing or treating obesity, in a subject in need thereof comprising: treating the subject with a GDP-bound form of Rabla (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof; thereby increasing cellular levels of Rabla GDP in the subject, resulting in decreased obesity in the subject.
  • Rabla GDP a GDP-bound form of Rabla
  • the obesity is accompanied by at least one of the following conditions: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • inflammatory response increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • a method for reducing bodyweight in an overweight subject comprising: treating the overweight subject with a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof; thereby increasing cellular levels of Rabla GDP in the overweight subject, resulting in decreased bodyweight in the subject.
  • Rabla GDP a GDP-bound form of Rabi a
  • the Rabla GDP may be or may comprise Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP- bound form of Rabi a.
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may consist of the amino acid sequence:
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may comprise or consist of the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, wherein the Rabla GDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabla GDP .
  • the fusion protein may comprise the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
  • the one or more expressible nucleic acids may encode any one or more of the Rabla GDP s as defined herein.
  • the one or more expressible nucleic acids may be DNA-based, or RNA-based.
  • the one or more expressible nucleic acids may transiently express the Rabla GDP in one or more cells of the subject, or wherein the one or more expressible nucleic acids may integrate in the one or more cells genome and express the Rabla GDP in the one or more cells of the subject.
  • the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the Rabla GDP inside the one or more cells of the subject.
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • Luciferase Tag (SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a Rabla GDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
  • a use of a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, for reducing obesity in a subject, or for preventing or treating obesity, in a subject in need thereof.
  • a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • a use of a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing bodyweight in an overweight subject.
  • a use of a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, in the manufacture of a medicament for reducing obesity, or for preventing or treating obesity, in a subject in need thereof.
  • a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • Rabla GDP a GDP-bound form of Rabi a
  • one or more expressible nucleic acids encoding Rabla GDP or a combination thereof, in the manufacture of a medicament for reducing body weight in an overweight subject.
  • the Rabla GDP may be or may comprise Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabi a.
  • the Rabla GDP may comprise the amino acid sequence:
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may consist of the amino acid sequence:
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may comprise or consist of the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, wherein the Rabla GDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabla GDP .
  • the fusion protein may comprise the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
  • the one or more expressible nucleic acids may encode one or more Rabla GDP s as defined herein.
  • the one or more expressible nucleic acids may be DNA-based, or RNA-based.
  • the one or more expressible nucleic acids may transiently express the Rabla GDP in one or more cells of the subject, or wherein the one or more expressible nucleic acids may integrate in the one or more cells genome of the subject and express the Rabla GDP in the one or more cells of the subject.
  • the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the Rabla GDP inside one or more cells of the subject.
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • Luciferase Tag (SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a Rabla GDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
  • polypeptide comprising the amino acid sequence:
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP; for use in reducing obesity, or for preventing or treating obesity, in a subject in need thereof; or for use in reducing body weight in an overweight subject.
  • the above polypeptide is for use in reducing obesity, or for preventing or treating obesity, in a subject in need thereof, wherein the obesity is accompanied by at least one of the following conditions: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • inflammatory response increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • kits comprising any one or more of: a GDP-bound form of Rabi a (Rabla GDP ); one or more expressible nucleic acids encoding Rabla GDP ; an anti-obesity agent; instructions for performing any of the method or methods as described herein; or any combinations thereof.
  • FIGURE 1 shows that MG008-treated obese mice lost bodyweight as determined, 12 days postinjection, as described in Example 1 ;
  • FIGURE 2 shows certain amino acid sequences and nucleic acid sequences as described herein.
  • FIGURE 3 shows the timeline for mice treated with Fl l and the corresponding points at which data were collected.
  • FIGURE 4 shows the baseline of fat and lean mass in diet-induced-obese (DIO) mice before Fl l treatment, as assessed by an NMR analyzer.
  • FIG. 4A shows the correlation between total fat and body weight.
  • FIG. 4B shows the correlation between total lean mass and body weight.
  • FIG. 4C shows the correlation between total fat and total lean mass.
  • FIG. 4D shows the correlation between the percentage of total fat and body weight.
  • FIG. 4E shows the correlation between the percentage of total lean mass and body weight.
  • FIG. 4F shows the correlation between the percentage of total fat and the percentage of total lean mass.
  • FIGURE 5 shows the fat and lean mass in diet-induced-obese (DIO) mice after 3 days of Fl l treatment, as determined by an NMR analyzer.
  • FIG. 5A shows the correlation between total fat and body weight.
  • FIG. 5B shows the correlation between total lean mass and body weight.
  • FIG. 5C shows the correlation between total fat and total lean mass.
  • FIG. 5D shows the correlation between the percentage of total fat and body weight.
  • FIG. 5E shows the correlation between the percentage of total lean mass and body weight.
  • FIG. 5F shows the correlation between the percentage of total fat and the percentage of total lean mass.
  • FIGURE 6 shows the fat and lean mass in diet-induced-obese (DIO) mice after 17 days of Fl l treatment, as determined by an NMR analyzer.
  • FIG. 6A shows the correlation between total fat and body weight.
  • FIG. 6B shows the correlation between total lean mass and body weight.
  • FIG. 6C shows the correlation between total fat and total lean mass.
  • FIG. 6D shows the correlation between the percentage of total fat and body weight.
  • FIG. 6E shows the correlation between the percentage of total lean mass and body weight.
  • FIG. 6F shows the correlation between the percentage of total fat and the percentage of total lean mass.
  • FIGURE 7 shows the change in values for body weight, fat mass, and lean mass in diet-induced- obese (DIO) mice after 3 days of Fl 1 treatment, as assessed by an NMR analyzer.
  • FIG. 7 A shows the body weight change in DIO mice after 3 days of Fl l treatment.
  • FIG. 7B shows the total fat change in DIO mice after 3 days of Fl l treatment.
  • FIG. 7C shows the total lean mass change in DIO mice after 3 days of Fl 1 treatment.
  • FIGURE 8 shows the change in values for body weight, fat mass, and lean mass in diet-induced- obese (DIO) mice after 17 days of Fl 1 treatment, as assessed by an NMR analyzer.
  • FIG. 8 A shows the body weight change in DIO mice after 17 days of Fl 1 treatment.
  • FIG. 8B shows the total fat change in DIO mice after 17 days of Fl l treatment.
  • FIG. 8C shows the total lean mass change in DIO mice after 17 days of Fl 1 treatment.
  • FIGURE 9 shows the multiple logistic regression analysis of Fl l efficacy in diet-induced-obese (DIO) mice after 3 days of Fl l treatment.
  • FIG. 9 A shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 3 days of Fl 1 treatment with respect to body weight change.
  • FIG. 9B shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 3 days of Fl 1 treatment with respect to fat mass change.
  • FIG. 9C shows the multiple logistic regression analysis of Fl l efficacy in DIO mice after 3 days of Fl l treatment with respect to lean mass change.
  • FIGURE 10 shows the multiple logistic regression analysis of Fl l efficacy in diet-induced-obese (DIO) mice after 17 days of Fl l treatment.
  • FIG. 10A shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 17 days of Fl 1 treatment with respect to body weight change.
  • FIG. 10B shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 17 days of Fl l treatment with respect to fat mass change.
  • FIG. 10C shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 17 days of Fl 1 treatment with respect to lean mass change.
  • FIGURE 11 shows the body weight (A) and percentage body weight (B) change of diet-induced- obese (DOI) mice before and after Fl 1 treatment.
  • Described herein are compounds, compositions, uses, and methods for reducing obesity in a subject, and/or for preventing or treating obesity in a subject in need thereof. It will be appreciated that embodiments and examples are provided for illustrative purposes intended for those skilled in the art and are not meant to be limiting in any way.
  • Obesity is a complex disease as the cause is often multifactorial. Indeed, several factors such as diet, physical activity, automation, urbanization, genetic susceptibility, medications, mental disorders, economic policies, endocrine disorders, and exposure to endocrine-disrupting chemicals may contribute to obesity in individuals.
  • One of the hallmarks of obesity is the accumulation of dysfunctional adipose tissue when energy intake exceeds energy expenditure, which triggers metabolic stress by increasing inflammatory responses and levels of fatty acids, triglycerides and LDL cholesterol, resulting in a cluster of interrelated complications including insulin resistance, glucose intolerance, diabetes mellitus, hyper- tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • NAFLD nonalcoholic fatty liver disease
  • Autophagy is a major intracellular degradation system that derives its degradative abilities from the lysosome. It is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles to adapt to adverse microenvironmental conditions, including limited nutrient supplies (Allyson et al. Cleaning House: Selective Autophagy of Organelles. (2017) Developmental Cell 41, 10-22). Several sensors interacting with the autophagic machinery have evolved to detect fluctuations in key metabolic parameters.
  • autophagy fulfills tissue-inherent metabolic tasks within the major organs involved in the maintenance of organismal energetic balance, including adipose tissue, liver, and exocrine pancreas.
  • insufficient autophagy is associated with metabolic syndrome which is characterized by at least three of the following five medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum high-density lipoprotein (HDL).
  • metabolic syndrome which is characterized by at least three of the following five medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum high-density lipoprotein (HDL).
  • HDL low serum high-density lipoprotein
  • Autophagy is a highly regulated process by which misfolded proteins and organelles reach lysosomes for their degradation (Kim & Lee, 2014).
  • autophagy There are three different types of autophagy, namely macroautophagy (commonly known as autophagy), chaperon mediated autophagy, and microautophagy. Lysosomal degradation of substrates in chaperon-mediated autophagy and microautophagy occurs directly within the lysosomes (Parzych & Klionsky, 2014).
  • Autophagy is regulated by a number of signalling molecules, particularly mechanistic target of rapamycin (mTOR) kinase and autophagy-related protein (ATG) family.
  • Food intake transiently increases plasma levels of branched- chain amino acids (BCAAs), including leucine, which subsequently activate mTOR signalling, thereby inhibiting autophagy (Nicklin et al., 2009; Broer & Broer, 2017).
  • BCAAs branched- chain amino acids
  • Activation of mTOR as a result of increased growth factor and insulin signalling and/or increased consumption of BCAAs is common in human obesity and in experimental models and is considered to be the main driving force for nutrition- triggered suppression of autophagy (Shimobayashi & Hall, 2016; Meijer et al., 2015).
  • autophagy is able to maintain normal levels of amino acids during short- term fasting.
  • elevated levels of plasma glucose activate insulin-like growth factor 1 (IGF1) signalling, leading to AKT1 dependent activation of mTOR through inhibition of TSC2-RHEB signalling.
  • IGF1 insulin-like growth factor 1
  • both glucose and amino acids share similar means of activating mTORCl in a RAG- dependent manner.
  • autophagy also controls glucose and energy metabolism through the regulation of gluconeogenesis (Kim & Lee, 2014).
  • Elevated lipid levels are common in obesity and might suppress autophagy by interrupting the fusion of the autophagolysosome, lysosomal acidification and hydrolase activity (Wang, 2016). Indeed, elevated lipid levels suppresses autophagosome-lysosome fusion (Koga et al., 2010). Autophagy may regulate lipid homeostasis through the selective degradation of lipid droplets via lipophagy and the utilization of lipids (Nguyen & Olzmann, 2017) as an energy source. Conversely, evidence point to a role for adipose autophagy in the regulation of adipose tissue development, adipogenesis and lipid metabolism (Tao et al., 2016).
  • adipose lysosomal dysfunction was shown to contribute to autophagosome accumulation and early adipose pathologies in obesity (Mizunoe et al., 2017). Furthermore, autophagy insufficiency may facilitate the transition from obesity to diabetes mellitus, highlighting the therapeutic potential of autophagy regulators for prevention and treatment of diabetes mellitus and obesity (Lim et al., 2014).
  • the specialized functions of autophagy in metabolic regulation include adipocyte differentiation, accumulation of fat deposits in the liver, maintenance of pancreatic P-cell fitness, central nervous system (CNS)-mediated regulation of food intake and inflammatory reactions, among other processes (Klionsky et al. Autophagy in major human diseases. (2021) EMBO 40: el08863- el08863).
  • CNS central nervous system
  • regulation of autophagy in the central nervous system was shown to contribute to bodyweight regulation. More specifically, inhibition of autophagy in a subtype of neurons producing proopiomelanocortin (POMC) was shown to promote the development of obesity by stimulating hyperphagia (Quan et al., 2012).
  • POMC proopiomelanocortin
  • ROS Reactive oxygen species
  • autophagic lysosome-mediated catabolic processes particularly the process of microautophagy, may be used for developing an effective treatment strategy for obesity.
  • Stimulation of microautophagy (which in certain embodiments may involve piecemeal engulfment of target membrane(s)) by promoting lysosome movement toward the target membranes) may be particularly desirable for treatment of a variety of diseases and/or disorders and/or conditions.
  • Specific lysosome positioning inside the cells may be associated with different types of lysosome activity.
  • lysosome positioning may correlate with the activity of mTOR and may regulate autophagic flux.
  • mTORCl may be inactive and lysosomes may be accumulated in the perinuclear region of the cells, which may perform macroautophagy by stimulation of fusion of encased target membrane with lysosomes.
  • lysosomes may move in a bidirectional manner along the microtubules, and such a lysosomal motility is governed by different sets of motor proteins that are recruited through different mechanisms.
  • microautophagy may involve direct engulfment of cytoplasmic cargo at a boundary membrane by autophagic tubes, which may mediate both invagination and vesicle scission into the lumen of lysosomes (see Li, W.-W., Li, J. & Bao, J.-K. Microautophagy: lesser- known self-eating. Cell. Mol. Life Sci. 69, 1125-1136 (2011)).
  • Direct lysosomal degradation of target substrates may occur for DNA, for example (referred to as a piecemeal autophagy - see Fujiwara, Y. et al. Direct uptake and degradation of DNA by lysosomes. - PubMed - NCBI.
  • lysosomes maybe able to move toward the different organelles and/or membranes substrates (e.g. plasma membrane, mitochondria) and may interact with them directly by recruitment of motor proteins and SNAREs proteins (see Andrews, N. W. Lysosomes and the plasma membrane. J. Cell Biol. 158, 389-394 (2002); Hofmann, I. & Munro,
  • Movement of lysosomes toward the cell periphery and their positioning within the cells may be associated with signaling (see R. & Bonifacino, J. S. Lysosome Positioning Influences mT0RC2 and AKT Signaling. Molecular Cell 75, 26-38.e3 (2019)).
  • mTORCl, mT0RC2 and AKT activation may be important for lysosomal peripheral distribution (see Poiis, C. & Codogno, P. Lysosome positioning coordinates mTORCl activity and autophagy. Nature Cell Biology 13, 342-344 (2011); and Cabukusta, B. & Neefjes, J. Mechanisms of lysosomal positioning and movement. Traffic 19, 761-769 (2016)).
  • microautophagy and/or piecemeal degradation of target substrates and/or membranes may involve movement of lysosomes toward the peripheral and target membrane in cytosol (e.g. direct interaction with target membrane) (see Pu, J., Guardia, C. M., Keren-Kaplan,
  • movement of lysosomes toward the cell periphery may be associated with the activation of mT0RCl/mT0RC2 (see Rabanal-Ruiz, Y. & Korolchuk, V. I.
  • Rabi a DN (dominant negative form of Rabi a) may stimulate lysosome peripheral distribution (from perinuclear region) through activating mT0RCl/mT0RC2 proteins inside the cells without the need of external (or extracellular) signal for their activation (to support lysosome peripheral distribution).
  • Rabla GDP or Rabla GTP in reducing obesity:
  • a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as Rabla S25N , Rabla N124I (a mouse Rabi sequence), Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabi a, or one or more expressible nucleic acids encoding such a Rabla GDP , may be used to reduce obesity in animal models.
  • a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as Rabla S25N , Rabla N124I (a mouse Rabi sequence), Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabi a, or one or more expressible nucleic acids encoding such a Rabla GDP , may be used to reduce obesity in animal models.
  • methods as described herein may be in vitro methods, in vivo methods, or both.
  • a method for reducing obesity comprising: treating the subject with a GDP-bound form of Rabla (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof; thereby increasing cellular levels of Rabla GDP in the subject, resulting in reduced obesity in the subject.
  • Rabla GDP a GDP-bound form of Rabla
  • the obesity is accompanied by at least one of the following conditions: increased inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • increased inflammatory response increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • a method for reducing bodyweight in an overweight subject comprising: treating the overweight subject with a GDP-bound form of Rabla (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof; thereby increasing cellular levels of Rabla GDP in the overweight subject, resulting in decreased bodyweight.
  • the Rabla GDP may be or may comprise Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP- bound form of Rabi a.
  • the Rabla GDP may comprise the amino acid sequence:
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may consist of the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may comprise or consist of the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, wherein the Rabla GDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabla GDP .
  • the fusion protein can comprise any protein or tag, for example, GFP, YFP, mCherry, luciferase specific antibody, aptamer for identification or delivery to a specific organ or the like.
  • the fusion protein may comprise the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
  • the one or more expressible nucleic acids may encode any one or more of the Rabla GDP s as defined herein.
  • the one or more expressible nucleic acids may be DNA-based, or RNA-based.
  • the one or more expressible nucleic acids may transiently express the Rabla GDP in one or more cells of the subject, or wherein the one or more expressible nucleic acids may integrate in the one or more cells genome and express the Rabla GDP in the one or more cells of the subject.
  • the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the Rabla GDP inside the one or more cells of the subject.
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • Luciferase Tag (SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a Rabla GDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
  • a use of a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, for reducing obesity in a subject, or for preventing or treating obesity, in a subject in need thereof.
  • a use of a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • inflammatory response increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep
  • RablaGDP GDP-bound form of Rabi a
  • one or more expressible nucleic acids encoding RablaGDP or a combination thereof, for reducing body weight in an overweight subject.
  • a use of a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, in the manufacture of a medicament for reducing obesity, or for preventing or treating obesity, in a subject in need thereof.
  • a use of a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof, in the manufacture of a medicament for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • inflammatory response increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation,
  • Rabla GDP a GDP-bound form of Rabi a
  • one or more expressible nucleic acids encoding Rabla GDP or a combination thereof, in the manufacture of a medicament for reducing body weight in an overweight subject, or for preventing or treating excess weight.
  • the Rabla GDP may be or may comprise Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabi a.
  • the Rabla GDP may comprise the amino acid sequence:
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may consist of the amino acid sequence:
  • TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC SEQ ID NO: 9; Mouse Rabl N1241 ); the amino acid sequence of human Rabla D41N ; or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
  • the Rabla GDP may comprise or consist of the amino acid sequence:
  • the Rabla GDP may be in the form of a fusion protein, wherein the Rabla GDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabla GDP .
  • the fusion protein may comprise the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
  • the Rabla GDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
  • the one or more expressible nucleic acids may encode one or more Rabla GDP s as defined herein.
  • the one or more expressible nucleic acids may be DNA-based, or RNA-based.
  • the one or more expressible nucleic acids may transiently express the Rabla GDP in one or more cells, or wherein the one or more expressible nucleic acids may integrate in the one or more cells genome and express the Rabla GDP in the one or more cells.
  • the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the Rabla GDP inside the one or more cells.
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
  • Luciferase Tag (SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a Rabla GDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
  • polypeptide comprising the amino acid sequence:
  • SEQ ID NO: 18 Human Rabla D47N ; or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP; for use in reducing obesity, or for preventing or treating obesity, in a subject in need thereof; or for use in reducing body weight in an overweight subject.
  • the obesity is accompanied by at least one of the following conditions: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • inflammatory response increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • a pharmaceutical composition comprising a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof; and another anti-obesity agent.
  • kits comprising any one or more of: a GDP-bound form of Rabi a (Rabla GDP ); one or more expressible nucleic acids encoding Rabla GDP ; an anti-obesity agent; instructions for performing any of the method or methods as described herein; or any combinations thereof.
  • the obesity may be accompanied by an increased inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • increased abdominal obesity high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
  • HDL low serum high-density lipoprotein
  • NAFLD nonalcoholic fatty liver disease
  • lysosome-mediated microautophagy may refer to a cellular process in which a cellular lipid or protein or glycogen substrate, or a portion thereof, is engulfed and degraded by a lysosome.
  • lysosome-mediated microautophagy is an integral component of cell’s bioenergetic process (i.e. ATP generation) as well as biosynthesis process (i.e. synthesizing new biomass through recycling existing “old” materials).
  • references to increasing lysosome-mediated microautophagy may refer to an increase in the rate, extent, capacity, or efficacy of the lysosome-mediated microautophagy process in a cell as compared to baseline levels of the cell, or as compared with a corresponding treated or untreated control cell, or as compared to levels in a reference diseased cell or reference cell having accumulation of a protein, lipid, or glycogen substrate.
  • increasing lysosome-mediated microautophagy may include restoring or increasing lysosomal motility or lysosomal bidirectional motility in a cell, and/or may include enhancing, increasing, activating, or otherwise restoring or rescuing lysosomal motility activity in a cell.
  • Such restoration or rescue may result in an increase in autophagy (such as, for example, micro- and/or macro- autophagy) and/or lysosomal degradation capacity.
  • autophagy such as, for example, micro- and/or macro- autophagy
  • lysosomal degradation capacity there are several diseases, conditions, and cellular states in which cellular lysosomal motility may be impaired, reduced, blocked, or suppressed.
  • microautophagyenhancing agents may restore or increase lysosomal motility in a cell.
  • Lysosomal motility may play an important role in several cellular functions, including lysosome-mediated microautophagy, lysosome-mediated macroautophagy, lysosomal regeneration, and lysosomal maturation processes.
  • references to restoring lysosomal motility and/or lysosomal bidirectional motility may refer to modulating cellular lysosomal motility/bidirectional motility levels back to those of corresponding normal or healthy control cells having baseline levels of lysosomal motility/bidirectional motility. Such modulation may, in certain embodiments, also modulate degradation capacity (i.e. phagy) levels back to those of normal or healthy cells.
  • degradation capacity i.e. phagy
  • lysosomal association-dissociation events between lysosomes and the lipid or protein or glycogen substrate may refer to events where a lysosome associates with a lipid or protein or glycogen substrate (i.e. a lipid droplet or a protein aggregate, for example), acquires at least a portion of the lipid or protein or glycogen substrate, and then dissociates from the lipid or protein or glycogen substrate.
  • Lysosomal association-dissociation i.e. the "on” and "off
  • events between lysosomes and the lipid or protein or glycogen substrate may be considered as "kiss-and- run"-type events.
  • a small piece of substrate i.e. lipid
  • the substrate i.e. cytosolic lipid droplets, or CLD, for example.
  • this may be achieved through the formation of a fusion pore between lysosome and the CLD.
  • dissociation or "run" event, dissociation of the lysosome from the substrate (i.e. CLD) may occur.
  • An increase in lysosomal association-dissociation events may refer to an increase in the rate, extent, or efficacy of lysosomal association-dissociation events in a cell as compared to baseline levels of a corresponding treated or untreated control cell, for example an identical cell treated under identical conditions but without a microautophagy-modulating agent or with a compound or composition that is known not to affect the process.
  • microautophagy-enhancing agents may be used to correct a microautophagy deficiency in a cell, or a cellular condition in which microautophagy is decreased.
  • a microautophagy-enhancing agent may be any suitable agent which increases or facilitates the rate, activity, extent, or efficacy of lysosome-mediated microautophagy in a cell, or that increases lysosomal motility or bidirectional motility.
  • a suitable microautophagyenhancing agent may be or comprise a GDP-bound form of Rabi a (Rabla GDP ), one or more expressible nucleic acids encoding Rabla GDP , or a combination thereof.
  • Ras-related protein Rab-IA is a protein that in humans is encoded by the RABI A gene. It may control vesicle transport from the endoplasmic reticulum (ER) to the Golgi compartment and on to the cell surface, and may play an important role in IL-8 and growth hormone secretion. In addition, it may play a role in autophagosome assembly in macroautophagy and cellular defense reactions against pathogenic bacteria when it is in its GTP-bound form. It also may regulate motility of endocytic compartment(s).
  • lysosome-mediated microautophagy of a target protein, lipid, or glycogen substrate in a cell may be increased by treatment with a microautophagy-enhancing agent comprising a GDP-bound form of Rabi a, such as Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabla.
  • a microautophagy-enhancing agent comprising a GDP-bound form of Rabi a, such as Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or another dominant negative (DN) GDP-bound form of Rabla.
  • the specific amino acid or nucleic acid sequence of a particular gene may vary from species to species.
  • the human Rabla amino acid sequence may have homologs in other species having sequence variation from the human sequence.
  • the general effect for example, the phenotypic effect
  • the general effect of a homolog sequence may be substantially similar to the effect of the wild- type sequence in a given cell or subject.
  • a microautophagy-enhancing agent may be, or comprise, a GDP-bound form of Rabla (Rabla GDP ) such as, for example, Rabla S25N , Rabla N1241 , Rabla D41N , Rabla D47N , or a functional equivalent thereof, or another dominant negative (DN) GDP-bound form of Rabla.
  • Rabla GDP a GDP-bound form of Rabla
  • DN dominant negative
  • Suitable GDP-bound forms of Rabla may include any suitable Rabla variant which is "dominant negative", or which preferentially binds GDP over GTP.
  • Such Rabla GDP variants may be identified using techniques known in the art (see, for example, Chan, C.-C. et al.
  • Figure 2 shows certain sequences of nucleic acids and amino acids/proteins as described herein.
  • SEQ ID NOs: 1-3 provide human Rabla WT DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively;
  • SEQ ID NOs:4-6 provide human Rabla S25N DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively;
  • SEQ ID NOs:7-9 provide mouse Rabl N1241 DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively;
  • SEQ ID NOs: 10-12 provide human Rabla Q70L DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively;
  • SEQ ID NOs: 13-15 provide human Rabla Q63L DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively;
  • SEQ ID NOs: 16-18 provide human Rabla D47N DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; and
  • SEQ ID NOs: 19-21 provide MG-008 ORF DNA sequence with 5’ luciferase tag
  • nucleic acid or amino acid comprising any of these sequences.
  • nucleic acid or amino acid comprising a nucleic acid sequence or amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences (i.e. with any of SEQ ID NOs: 1-21 or the sequence of Rabla D41N ) or an active fragment thereof.
  • Suitable functional equivalents of Rabla S25N , Rabla D41N , Rabla D47N , and Rabla N1241 may include, for example, suitable Rabla variants or mutants having at least 80% (or >85%, or >90%, or >95%, or >99%) sequence identity to Rabla WT or Rabla S25N or Rabla N1241 or Rabla D41N or Rabla D47N and which preferentially bind GDP over GTP while also retaining the relevant cellular/biochemical functions of Rabla S25N or Rabla N1241 or Rabla D41N or Rabla D47N as described in detail herein.
  • a microautophagy-enhancing agent may be, or comprise, one or more expressible nucleic acids encoding Rabla GDP , such as any suitable nucleic acid/expression vector (i.e. vector, cassette, mRNA, modified mRNA, plasmid, for example) which encodes for/expresses a GDP-bound form of Rabla (Rabla GDP ) such as, for example, Rabla S25N or Rabla N1241 or Rabla D41N or Rabla D47N , or a functional equivalent thereof.
  • any suitable nucleic acid/expression vector i.e. vector, cassette, mRNA, modified mRNA, plasmid, for example
  • Rabla GDP a GDP-bound form of Rabla
  • sequences are mainly described herein with reference to human and/or mouse homologs. It will be understood that functional equivalents and/or variants may be found in a variety of different species, such as among different mammals. References herein to particular sequence modifications and/or mutants providing for Rabla GDP (DN) or Rabla GTP (DA) forms often provide position and modification/mutation information (e.g.
  • Rabl Q67L is a modification/mutation of a mouse sequence, and there is no Q at the 67 th position of human Rabi a; rather, there is a Q in the human sequence at the 63 rd position, and so the modification/mutation with reference to the human sequence is Rabi a ()63L .
  • a microautophagy-reducing agent may be or comprise any one or more of a GTP-bound form of Rabi a (Rabla GTP ), one or more expressible nucleic acids encoding Rabla GTP , Rabi a wild-type (Rabla WT ), or one or more expressible nucleic acids encoding Rabla WT .
  • a Rabla GTP may be or comprise Rabla Q70L , Rabla Q67L (in mouse sequence), Rabla Q63L (in human Rabi a sequence), or a functional equivalent thereof, or another dominant active (DA) GTP-bound form of Rabi a.
  • GTP-bound forms of Rabi a may include any Rabi a variant which is "dominant active", or which preferentially binds GTP over GDP.
  • Such Rabla GTP variants may be identified using techniques known in the art (see, for example, Chan, C.-C. et al. Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila. Current Biology 21, 1704-1715 (2011); Tabancay, A. P. et al. Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K. J. Biol. Chem. 278, 39921-39930 (2003); and Dumas, J.
  • treatment with a microautophagy-enhancing agent such as Rabla GDP
  • treatment with a microautophagy-enhancing agent may comprise introducing the Rabla GDP protein to a cell, expressing Rabla GDP within the cell, or both, for example.
  • expression of a particular protein within a cell may refer to the production of a polypeptide from a nucleic acid sequence encoding the polypeptide.
  • Gene expression may include both transcription and translation processes, and so gene expression may refer to production of a nucleic acid sequence such as an mRNA (i.e. transcription), production of a protein (i.e. translation), or both.
  • overexpression of a particular gene in a cell may refer to increasing the expression of a particular gene within a cell as compared to wildtype, baseline, or untreated levels. Overexpression, or introduction of a mutant gene, into cells may be accomplished using any of several methods known in the art.
  • a vector (either viral, plasmid, or other) comprising one or more copies of the particular gene each driven by a suitable promoter sequence (for example, a constitutive or inducible promoter), or an mRNA or chemically modified version thereof may be introduced into cells via transfection, electroporation, or viral infection, or another suitable method know in the art.
  • suitable expression vector techniques for overexpressing or introducing a particular gene into a cell are known in the art (see, for example, Molecular Cloning: A Laboratory Manual (4th Ed.), 2012, Cold Spring Harbor Laboratory Press).
  • compositions comprising or consisting of one or more of the nucleic acids and/or proteins as described herein may be used.
  • Compositions may additionally comprise one or more pharmaceutically acceptable diluents, carriers, excipients, or buffers.
  • Compositions may be used for administering one or more nucleic acids and/or proteins to a cell in vitro or in vivo.
  • Introduction of a gene in the context of inserting a nucleic acid sequence into a cell, may refer to "transfection", “transformation”, or “transduction”, and may include the incorporation or introduction of a nucleic acid sequence into a eukaryotic cell where the nucleic acid sequence may optionally be incorporated into the genome of the cell, or transiently expressed (for example, transfected mRNA).
  • a protein or enzyme may be introduced into a cell by delivering the protein or enzyme itself into the cell, or by expressing an mRNA encoding the protein or enzyme within the cell, leading to its translation.
  • nucleic acids for expressing a particular gene may encode or include features as described in "Genes VII", Lewin, B. Oxford University Press (2000) or “Molecular Cloning: A Laboratory Manual”, Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition (2001).
  • a nucleotide sequence encoding a polypeptide or protein may be incorporated into a suitable vector, such as a commercially available vector.
  • Vectors may also be individually constructed or modified using standard molecular biology techniques, as outlined, for example, in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)).
  • a vector may include nucleotide sequences encoding desired elements that may be operably linked to a nucleotide sequence encoding a polypeptide or protein.
  • nucleotide sequences encoding desired elements may include transcriptional promoters, transcriptional enhancers, transcriptional terminators, translational initiators, translational terminators, ribosome binding sites, 5'- untranslated region, 3'- untranslated regions, cap structure, poly A tail, and/or an origin of replication.
  • Selection of a suitable vector may depend upon several factors, including, without limitation, the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational control elements desired, the level of expression desired, copy number desired, whether chromosomal integration is desired, the type of selection process that is desired, or the host cell or the host range that is intended to be transformed.
  • biomolecules and/or compounds described herein may be provided in pharmaceutical compositions together with a pharmaceutically acceptable diluent, carrier, or excipient, and/or together with one or more separate active agents or drugs as part of a pharmaceutical combination or pharmaceutical composition.
  • the biomolecules, compounds, and/or pharmaceutical compositions may be administered in a treatment regimen simultaneously, sequentially, or in combination with other drugs or pharmaceutical compositions, either separately or as a combined formulation or combination.
  • Biomolecules, compounds, and/or compositions as described herein may include one or more pharmaceutically acceptable excipients, diluents, and/or carriers.
  • a pharmaceutically acceptable carrier, diluent, or excipient may include any suitable carrier, diluent, or excipient known to the person of skill in the art.
  • Examples of pharmaceutically acceptable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch.
  • pharmaceutically acceptable excipients may include suitable fillers, binders, lubricants, buffers, glidants, and disentegrants known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2006)).
  • Examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000 — 20th edition) and in the United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • a conservative amino acid substitution may include one in which an amino acid is substituted for another amino acid having similar properties such that the folding, activity, or other functionality of the protein is not significantly affected.
  • aromatic amino acids which may be substitutable, may include phenylalanine, tryptophan, and tyrosine.
  • interchangeable hydrophobic amino acids which may be substitutable, may include leucine, isoleucine, methionine, and valine.
  • interchangeable polar amino acids which may be substitutable, may include glutamine and asparagine.
  • interchangeable basic amino acids which may be substitutable, may include arginine, lysine, and histidine.
  • interchangeable acidic amino acids may include aspartic acid and glutamic acid.
  • interchangeable small amino acids which may be substitutable, may include alanine, serine, threonine, cysteine, and glycine.
  • dominant-negative (DN) Rabi a e.g. GDP-bound Rabi a, Rabla GDP
  • DN dominant-negative
  • any suitable Rabi a DN or Rabla GDP may be used.
  • a variety of Rabi a DN proteins will be known to the person of skill in the art having regard to the teachings herein.
  • generally any suitable dominant negative form of Rabi a i.e.
  • a constant/locked Rabi a in its GDP form may be used to promote lysosomal movement toward the cytosol and peripheral of the cells (from perinuclear region of the cells), and stimulate direct lysosome interaction with target substrates, for example.
  • Rabi a DN may stimulate activation of mT0RCl/mT0RC2 and AKT based on their effect on lysosomal positioning and promoting lysosome movement toward the peripheral and toward the target substrates (see also Jia, R. & Bonifacino, J. S. Lysosome Positioning Influences mT0RC2 and AKT Signaling. Molecular Cell 75, 26-38.e3 (2019)).
  • Rabi a constant GDP-bound form is generally not available in normal physiological conditions, in which the native protein Rabi a is constantly shifting between its GTP and GDP forms. It is contemplated that in certain embodiments, genetic mutation and/or amino acid substitution/modification may be used to lock this GTPase in its GDP-bound form (or constant GTP form), and prevent it from going to its GTP state (or GDP state).
  • any suitable modification(s) that can substantially keep the integrity of the GDP form/state of GTPases may promote microautophagy and degradation of target substrate(s) through lysosomal piecemeal engulfment.
  • Rabla GDP may be administered to a particular cell type or subject in need thereof in generally any suitable manner which may be selected to suit the particular cell type, subject, and/or indication.
  • a nucleic acid sequences encoding and capable of expressing the Rabla GDP may be administered to the subject or introduced to the cell type via any suitable transfection or nucleic acid delivery approach as will be known to the person of skill in the art having regard to the teachings herein.
  • delivery may be based on DNA or RNA transfection (for example, using common transfection regent(s) such as lipofectamine (Invitrogen), FuGENE (Rosche), using DNA adenovirus (gene therapy), or using Modified RNA (i.e.
  • protein may be administered or delivered to cells in need thereof, optionally assisted with any suitable technique or delivery vehicle for facilitating protein delivery to a cell or cells.
  • Fl l can be administered to an obese or overweight subject to reduce body weight in said subject.
  • Fl l is lipid nanoparticles that encapsulate Rabi, which is commercially available from Precision NanoSystems (#50 655 W Kent Ave N, Vancouver, BC V6P 6T7).
  • Fl l may be administered intravenously, orally, subcutaneously, intramuscularly, sublingually, or any other route that would be known to a person of skill in the art.
  • Fl l may be dosed at 0.001-1 mg/kg (mpk), but other doses may be beneficial depending on the subject.
  • Fl l treatment may be done in a single dose or multiple doses.
  • Fl l may be administered repeatedly, the individual treatments may be separated by hours, days, weeks, months, or years, as would be necessary to establish, maintain, or re-establish body weight reduction.
  • Administration of Fl 1 may decrease subject body weight and/or fat content, and/or may positively affect HbAlc, alanine amino transferase (ALT), asparatate amino transferase (AST), blood urea nitrogen (BUN), creatinine (CRE) or other blood chemistry measurements.
  • ALT alanine amino transferase
  • AST asparatate amino transferase
  • BUN blood urea nitrogen
  • CRE creatinine
  • Fl 1 treatment may also improve other body health indicators, such as high blood pressure, high blood sugar, elevated cholesterol levels, inflammation, high serum triglycerides, high resting heart rate, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders, sleep apnea, and/or elevated cancer progression markers, without being limiting.
  • body health indicators such as high blood pressure, high blood sugar, elevated cholesterol levels, inflammation, high serum triglycerides, high resting heart rate, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders, sleep apnea, and/or elevated cancer progression markers, without being limiting.
  • HDL low serum high-
  • Rabi encapsulated in a lipid nanoparticle may be in the form of a fusion protein, wherein the Rabi is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabi.
  • the Rabla DN is also referred to as MG-008 which is encoded by mRNA and encapsulated with an ANM formulation.
  • Figure 1 Shown on the left (red bars) of Figure 1 is the body weight loss of mice at day 12 post-MG008 injection (a single dose), and on the right (black bars) is the bodyweight of control mice at day 12.
  • the bodyweight loss in MG008-treated mice was apparent throughout the entire 18-day post-MG008 treatment, and the earliest significant body weight loss was registered as short as day 4 post- MG008 injection (data not shown).
  • EXAMPLE 2 Encapsulated Rabi lipid nanoparticles (Fll) reduces body weight in obese mice
  • mice at approximately 6 weeks of age were fed a high-fat diet (HFD) until they weighed at least 40 g (40-50 g). Mice were weighed at the start of the HFD and their weight was monitored weekly. The appropriate weight gain was usually achieved at around 18-26 weeks of age, at which point NMR was used to determine body composition and Vevo LAZR-X was used to determine steatosis. Mice were then treated with Fl 1 via intravenous administration. Fl 1 was administered at 0.00625, 0.0125, 0.025, 0.05, or 0.5 mg/kg (mpk). NMR body composition measurements were then made at 1, 3, 5 and 7 weeks post Fl l treatment.
  • HFD high-fat diet
  • LAZR-X steatosis measurements were taken at weeks 4 and 7 post Fl l treatment. Additionally, at week 7 post Fl l treatment, blood was taken from the submandibular region (200 pl) to determine HbAlc levels. Eight weeks after Fl l administration the mice were sacrificed and the following measurements were taken: (1) measurement of body weight, liver weight and epididymal fat weight; (2) blood was collected for measurement of alanine amino transferase (ALT), aspartate amino transferase (AST), blood urea nitrogen (BUN), creatinine (CRE), and cytokines; (3) pictures were taken of the liver and epididymal fat; (4) optical coherence tomography (OCT) was conducted on the liver; and (5) mice were perfused and tissues were processed for immunohistochemical and H&E staining.
  • ALT alanine amino transferase
  • AST aspartate amino transferase
  • BUN blood urea nitrogen
  • CRE creatinine
  • cytokines
  • Fl l administered at 0.05-0.5 mpk reduced body weight by reducing either fat or lean mass or both after 3 days of Fl 1 treatment (FIG. 7).
  • the most dramatic effect on body weight was observed when Fl l was administered at 0.5 mpk as it lead to a substantial drop in both fat and lean mass (FIG. 7).
  • the lower doses of Fl 1 (0.025-0.05 mpk) did not change the fat mass significantly while even lower levels of Fl 1 ( ⁇ 0.0125) appeared to induce fat accumulation (FIG. 7B).
  • total body weight changes by all Fl l doses were largely determined by changing fat content because the mice continuously fed on 60% HFD (FIGs 8A and 8B).
  • the multiple regression analysis suggests that 0.32 mpk and 0.4 mpk may be the best Fl l doses for reducing fat and body weight, respectively, after 3 days of treatment.
  • Nrf2 promotes autophagy- dependent osteoblastic differentiation of adipose- derived mesenchymal stem cells. Exp. Cell Res. 349, 221-229 (2016). 87.

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Abstract

Compounds, compositions, uses, and methods for reducing obesity in a subject, or for preventing or treating obesity, are provided herein. In certain examples, methods for reducing obesity in a subject and/or for preventing or treating obesity in a subject in need thereof are provided which may include a step of treatment with a GDP-bound form of Rab1a (Rab1aGDP), one or more expressible nucleic acids encoding Rab1aGDP, or a combination thereof.

Description

COMPOSITIONS AND METHODS FOR TREATING OBESITY
FIELD OF INVENTION
The present invention relates generally to treatment of obesity. More specifically, the present invention relates to compositions and methods for enhancing microautophagy to treat obesity and related diseases or disorders.
BACKGROUND
There are three known cellular processes, in eukaryotes, through which the cytosolic content and/or subcellular organelles can be delivered to or acquired by lysosomes for degradation: namely macroautophagy (commonly known as autophagy), microautophagy, and chaperone-mediated autophagy. Microautophagy, unlike macroautophagy or chaperone-mediated autophagy, is a type of autophagic process that is mediated by direct lysosomal (in mammals) or vacuolar (in plants and fungi) piecemeal engulfment of a cytoplasmic target or cargo (e.g. protein, lipid, glycogen or pathogen).
Microautophagy may contribute to cytosolic protein degradation via multivesicular bodies (MVBs) of the late endosomes. In addition, microautophagy may also support direct glycogen delivery to lysosomes and its degradation. In this regard, it is contemplated that malfunctioning or insufficient microautophagy may be associated with development of a variety of metabolic and/or neurological diseases, for example.
Clearly, accumulation of particular proteins, lipids, and/or glycogens may be associated with a variety of important diseases, disorders, and conditions. Methods for targeting such proteins, lipids, and/or glycogens are desirable.
Obesity is a leading preventable cause of death worldwide, with increasing rates in adults and children. Indeed, the prevalence of obesity has increased markedly over the past decades and has reached epidemic proportions; over one- third of adults are overweight (BMI 25-29.9 kg/m2) or obese (BMI > 30 kg/m2) worldwide (WHO). Obesity is a complex disease involving an excessive amount of body fat that increases the risk of other diseases and health problems, such as heart disease, diabetes, high blood pressure and certain cancers (Zhang et al., 2018). Obesity is widely considered as one of the most serious public health problems of the 21st century as the efficacy of current therapies remains poor and focus mainly on suppressing caloric intake. Moreover, one of the most effective agents for weight loss, Sibutramine, was withdrawn from the US market owing to cardiovascular safety concerns. Thus, the clinical management of obesity represents a major challenge in the absence of effective medications. Anti-obesity medications targeting the molecular and cellular mechanisms implicated in obesity are thus highly desirable.
Alternative, additional, and/or improved anti-obesity agents, compositions, and/or methods for treating obesity and related diseases or disorders are desirable.
SUMMARY OF INVENTION
As described in detail herein, it has now been identified that treatment with a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabi a, or one or more expressible nucleic acids encoding such a RablaGDP, may be used to reduce obesity in an obese subject.
In an embodiment, there is provided herein a method for reducing obesity, or for preventing or treating obesity, in a subject in need thereof, said method comprising: treating the subject with a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing cellular levels of RablaGDP in the subject, resulting in decreased obesity in the subject.
In an embodiment, the obesity is accompanied by at least one of the following conditions: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
In another embodiment, there is provided herein a method for reducing bodyweight in an overweight subject, said method comprising: treating the overweight subject with a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing cellular levels of RablaGDP in the overweight subject, resulting in decreased bodyweight in the subject.
In another embodiment of any of the above method or methods, the RablaGDP may be or may comprise RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP- bound form of Rabi a.
In still another embodiment of any of the above method or methods, the RablaGDP may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI
KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In yet another embodiment of any of the above method or methods, the RablaGDP may consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In another embodiment of any of the above method or methods, the RablaGDP may comprise or consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above method or methods, the RablaGDP may be in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP.
In yet another embodiment of any of the above method or methods, the fusion protein may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. In another embodiment of any of the above method or methods, the RablaGDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above method or methods, the one or more expressible nucleic acids may encode any one or more of the RablaGDPs as defined herein.
In yet another embodiment of any of the above method or methods, the one or more expressible nucleic acids may be DNA-based, or RNA-based.
In another embodiment of any of the above method or methods, the one or more expressible nucleic acids may transiently express the RablaGDP in one or more cells of the subject, or wherein the one or more expressible nucleic acids may integrate in the one or more cells genome and express the RablaGDP in the one or more cells of the subject. In still another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the one or more cells of the subject.
In yet another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG
ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC
AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT
AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT
TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA
TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT
AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG
CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG
GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT
GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA
ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA
TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA
CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT
CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT
CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT
TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT
GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA
CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT
CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA
AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA
ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC
AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC
CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA
AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing obesity in a subject, or for preventing or treating obesity, in a subject in need thereof. In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing bodyweight in an overweight subject.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing obesity, or for preventing or treating obesity, in a subject in need thereof.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing body weight in an overweight subject.
In another embodiment of any of the above use or uses, the RablaGDP may be or may comprise RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabi a. In still another embodiment of any of the above use or uses, the RablaGDP may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In yet another embodiment of any of the above use or uses, the RablaGDP may consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIEEDGKTIKEQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In another embodiment of any of the above use or uses, the RablaGDP may comprise or consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above use or uses, the RablaGDP may be in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP.
In yet another embodiment of any of the above use or uses, the fusion protein may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In another embodiment of any of the above use or uses, the RablaGDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT
AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST
PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above use or uses, the one or more expressible nucleic acids may encode one or more RablaGDPs as defined herein.
In yet another embodiment of any of the above use or uses, the one or more expressible nucleic acids may be DNA-based, or RNA-based.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may transiently express the RablaGDP in one or more cells of the subject, or wherein the one or more expressible nucleic acids may integrate in the one or more cells genome of the subject and express the RablaGDP in the one or more cells of the subject.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside one or more cells of the subject.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG
ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC
CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA
GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC
AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT
GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA
GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG
AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT
CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG
TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG
TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG
AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA
TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In still another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG
GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC
CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA
AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA
UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA
AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA
AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment, there is provided herein a polypeptide comprising the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTES
YISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQ ESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFL ETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGC C
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELD GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP; for use in reducing obesity, or for preventing or treating obesity, in a subject in need thereof; or for use in reducing body weight in an overweight subject.
In an embodiment, the above polypeptide is for use in reducing obesity, or for preventing or treating obesity, in a subject in need thereof, wherein the obesity is accompanied by at least one of the following conditions: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
In another embodiment, there is provided herein a pharmaceutical composition comprising a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; and another anti-obesity agent.
In yet another embodiment, there is provided herein a kit comprising any one or more of: a GDP-bound form of Rabi a (RablaGDP); one or more expressible nucleic acids encoding RablaGDP; an anti-obesity agent; instructions for performing any of the method or methods as described herein; or any combinations thereof.
BRIEF DESCRIPTION OF DRAWINGS
These and other features will become further understood having regard to the following drawing in which obese mice were treated with mRNA-encoded RablaGDP:
FIGURE 1 shows that MG008-treated obese mice lost bodyweight as determined, 12 days postinjection, as described in Example 1 ;
FIGURE 2 shows certain amino acid sequences and nucleic acid sequences as described herein.
FIGURE 3 shows the timeline for mice treated with Fl l and the corresponding points at which data were collected.
FIGURE 4 shows the baseline of fat and lean mass in diet-induced-obese (DIO) mice before Fl l treatment, as assessed by an NMR analyzer. FIG. 4A shows the correlation between total fat and body weight. FIG. 4B shows the correlation between total lean mass and body weight. FIG. 4C shows the correlation between total fat and total lean mass. FIG. 4D shows the correlation between the percentage of total fat and body weight. FIG. 4E shows the correlation between the percentage of total lean mass and body weight. FIG. 4F shows the correlation between the percentage of total fat and the percentage of total lean mass.
FIGURE 5 shows the fat and lean mass in diet-induced-obese (DIO) mice after 3 days of Fl l treatment, as determined by an NMR analyzer. FIG. 5A shows the correlation between total fat and body weight. FIG. 5B shows the correlation between total lean mass and body weight. FIG. 5C shows the correlation between total fat and total lean mass. FIG. 5D shows the correlation between the percentage of total fat and body weight. FIG. 5E shows the correlation between the percentage of total lean mass and body weight. FIG. 5F shows the correlation between the percentage of total fat and the percentage of total lean mass.
FIGURE 6 shows the fat and lean mass in diet-induced-obese (DIO) mice after 17 days of Fl l treatment, as determined by an NMR analyzer. FIG. 6A shows the correlation between total fat and body weight. FIG. 6B shows the correlation between total lean mass and body weight. FIG. 6C shows the correlation between total fat and total lean mass. FIG. 6D shows the correlation between the percentage of total fat and body weight. FIG. 6E shows the correlation between the percentage of total lean mass and body weight. FIG. 6F shows the correlation between the percentage of total fat and the percentage of total lean mass.
FIGURE 7 shows the change in values for body weight, fat mass, and lean mass in diet-induced- obese (DIO) mice after 3 days of Fl 1 treatment, as assessed by an NMR analyzer. FIG. 7 A shows the body weight change in DIO mice after 3 days of Fl l treatment. FIG. 7B shows the total fat change in DIO mice after 3 days of Fl l treatment. FIG. 7C shows the total lean mass change in DIO mice after 3 days of Fl 1 treatment. The changed value is calculated by the formula: Changed value (gram) = value (gram) at 3 day post i.v. - value (gram) at baseline (before i.v.).
FIGURE 8 shows the change in values for body weight, fat mass, and lean mass in diet-induced- obese (DIO) mice after 17 days of Fl 1 treatment, as assessed by an NMR analyzer. FIG. 8 A shows the body weight change in DIO mice after 17 days of Fl 1 treatment. FIG. 8B shows the total fat change in DIO mice after 17 days of Fl l treatment. FIG. 8C shows the total lean mass change in DIO mice after 17 days of Fl 1 treatment. The changed value is calculated by the formula: Changed value (gram) = value (gram) at 17 day post i.v. - value (gram) at baseline (before i.v.).
FIGURE 9 shows the multiple logistic regression analysis of Fl l efficacy in diet-induced-obese (DIO) mice after 3 days of Fl l treatment. FIG. 9 A shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 3 days of Fl 1 treatment with respect to body weight change. FIG. 9B shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 3 days of Fl 1 treatment with respect to fat mass change. FIG. 9C shows the multiple logistic regression analysis of Fl l efficacy in DIO mice after 3 days of Fl l treatment with respect to lean mass change.
FIGURE 10 shows the multiple logistic regression analysis of Fl l efficacy in diet-induced-obese (DIO) mice after 17 days of Fl l treatment. FIG. 10A shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 17 days of Fl 1 treatment with respect to body weight change. FIG. 10B shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 17 days of Fl l treatment with respect to fat mass change. FIG. 10C shows the multiple logistic regression analysis of Fl 1 efficacy in DIO mice after 17 days of Fl 1 treatment with respect to lean mass change.
FIGURE 11 shows the body weight (A) and percentage body weight (B) change of diet-induced- obese (DOI) mice before and after Fl 1 treatment.
DETAILED DESCRIPTION
Described herein are compounds, compositions, uses, and methods for reducing obesity in a subject, and/or for preventing or treating obesity in a subject in need thereof. It will be appreciated that embodiments and examples are provided for illustrative purposes intended for those skilled in the art and are not meant to be limiting in any way.
Obesity is a complex disease as the cause is often multifactorial. Indeed, several factors such as diet, physical activity, automation, urbanization, genetic susceptibility, medications, mental disorders, economic policies, endocrine disorders, and exposure to endocrine-disrupting chemicals may contribute to obesity in individuals. One of the hallmarks of obesity is the accumulation of dysfunctional adipose tissue when energy intake exceeds energy expenditure, which triggers metabolic stress by increasing inflammatory responses and levels of fatty acids, triglycerides and LDL cholesterol, resulting in a cluster of interrelated complications including insulin resistance, glucose intolerance, diabetes mellitus, hyper- tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
Autophagy is a major intracellular degradation system that derives its degradative abilities from the lysosome. It is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles to adapt to adverse microenvironmental conditions, including limited nutrient supplies (Allyson et al. Cleaning House: Selective Autophagy of Organelles. (2017) Developmental Cell 41, 10-22). Several sensors interacting with the autophagic machinery have evolved to detect fluctuations in key metabolic parameters. Indeed, because the lysosomal disposal of intracellular macromolecules invariably leads to their breakdown into essential metabolic intermediates, including amino acids, glucose, nucleotides, and free fatty acids (FAs), autophagy stands out as a key coordinator of the response to energetic stresses, at both the tissue-specific and systemic levels (Rabinowitz JD, White E (2010) Autophagy and metabolism. Science 330:1344 - 1348; Galluzzi L, Pietrocola F, Levine B, Kroemer G (2014) Metabolic control of autophagy. Cell 159: 1263 - 1276). Thus, autophagy fulfills tissue-inherent metabolic tasks within the major organs involved in the maintenance of organismal energetic balance, including adipose tissue, liver, and exocrine pancreas. As such, insufficient autophagy is associated with metabolic syndrome which is characterized by at least three of the following five medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum high-density lipoprotein (HDL).
Autophagy is a highly regulated process by which misfolded proteins and organelles reach lysosomes for their degradation (Kim & Lee, 2014). There are three different types of autophagy, namely macroautophagy (commonly known as autophagy), chaperon mediated autophagy, and microautophagy. Lysosomal degradation of substrates in chaperon-mediated autophagy and microautophagy occurs directly within the lysosomes (Parzych & Klionsky, 2014).
Global and tissue-specific deletion and/or mutation of certain autophagy genes or autophagy regulatory molecules result in altered lipid metabolism, hepatic steatosis and an obese or diabetic phenotype in animal models (Kim & Lee, 2014; Lee et al. 2016). More specifically, genetic ablation or loss-of-function of several autophagy-associated genes, including Atg7 (Lim et al, 2014), Atg4b (Fernandez et al, 2017), Becn2 (He et al, 2013), and Tfeb (Settembre et al, 2013), at the whole-body level or in a tissue restricted manner predisposes these animals to metabolic disorders, both under a normal diet and high fat diets. Moreover, obesity is associated with increased plasma levels of autophagy inhibitory factors including DB1/ACBP, both in humans and in mice (Bravo-San Pedro et al., 2019; Joseph et al., 2020). These findings support a role for autophagy in the development of obesity (obesogenesis) and obesity-related complications (Zhang et al., 2018).
Autophagy is regulated by a number of signalling molecules, particularly mechanistic target of rapamycin (mTOR) kinase and autophagy-related protein (ATG) family. Food intake transiently increases plasma levels of branched- chain amino acids (BCAAs), including leucine, which subsequently activate mTOR signalling, thereby inhibiting autophagy (Nicklin et al., 2009; Broer & Broer, 2017). Activation of mTOR as a result of increased growth factor and insulin signalling and/or increased consumption of BCAAs is common in human obesity and in experimental models and is considered to be the main driving force for nutrition- triggered suppression of autophagy (Shimobayashi & Hall, 2016; Meijer et al., 2015). In contrast, autophagy is able to maintain normal levels of amino acids during short- term fasting. Similarly, elevated levels of plasma glucose activate insulin-like growth factor 1 (IGF1) signalling, leading to AKT1 dependent activation of mTOR through inhibition of TSC2-RHEB signalling. Interestingly, both glucose and amino acids share similar means of activating mTORCl in a RAG- dependent manner. Moreover, autophagy also controls glucose and energy metabolism through the regulation of gluconeogenesis (Kim & Lee, 2014).
Elevated lipid levels are common in obesity and might suppress autophagy by interrupting the fusion of the autophagolysosome, lysosomal acidification and hydrolase activity (Wang, 2016). Indeed, elevated lipid levels suppresses autophagosome-lysosome fusion (Koga et al., 2010). Autophagy may regulate lipid homeostasis through the selective degradation of lipid droplets via lipophagy and the utilization of lipids (Nguyen & Olzmann, 2017) as an energy source. Conversely, evidence point to a role for adipose autophagy in the regulation of adipose tissue development, adipogenesis and lipid metabolism (Tao et al., 2016). Moreover, adipose lysosomal dysfunction was shown to contribute to autophagosome accumulation and early adipose pathologies in obesity (Mizunoe et al., 2017). Furthermore, autophagy insufficiency may facilitate the transition from obesity to diabetes mellitus, highlighting the therapeutic potential of autophagy regulators for prevention and treatment of diabetes mellitus and obesity (Lim et al., 2014).
The specialized functions of autophagy in metabolic regulation include adipocyte differentiation, accumulation of fat deposits in the liver, maintenance of pancreatic P-cell fitness, central nervous system (CNS)-mediated regulation of food intake and inflammatory reactions, among other processes (Klionsky et al. Autophagy in major human diseases. (2021) EMBO 40: el08863- el08863). Moreover, regulation of autophagy in the central nervous system was shown to contribute to bodyweight regulation. More specifically, inhibition of autophagy in a subtype of neurons producing proopiomelanocortin (POMC) was shown to promote the development of obesity by stimulating hyperphagia (Quan et al., 2012).
Reactive oxygen species (ROS) were shown to play an important role in obesity (Lavallard et al., 2012). Excessive ROS production promotes cytochrome c to be released from mitochondria into the cytoplasm and contributes to the onset and development of insulin resistance and diabetes mellitus in obesity (Houstis et al., 2006; Petersen et al., 2004). Unfortunately, ROS is an inevitable byproduct of the bioenergetic process, which is most produced in the mitochondria during the ATP synthesis process known as oxidative phosphorylation.
In this regard, the present inventors recognized that in addition to suppressing caloric intake, autophagic lysosome-mediated catabolic processes, particularly the process of microautophagy, may be used for developing an effective treatment strategy for obesity.
Stimulation of microautophagy (which in certain embodiments may involve piecemeal engulfment of target membrane(s)) by promoting lysosome movement toward the target membranes) may be particularly desirable for treatment of a variety of diseases and/or disorders and/or conditions. Specific lysosome positioning inside the cells may be associated with different types of lysosome activity. In addition, lysosome positioning may correlate with the activity of mTOR and may regulate autophagic flux. During macroautophagy, mTORCl may be inactive and lysosomes may be accumulated in the perinuclear region of the cells, which may perform macroautophagy by stimulation of fusion of encased target membrane with lysosomes. In contrast, in the process of microautophagy, piecemeal engulfment of target membrane(s) by lysosomes may be happening through lysosome movement and direct interaction with target membrane(s) without need of formation of autophagosome(s). Lysosomes may move in a bidirectional manner along the microtubules, and such a lysosomal motility is governed by different sets of motor proteins that are recruited through different mechanisms.
In certain embodiments, microautophagy may involve direct engulfment of cytoplasmic cargo at a boundary membrane by autophagic tubes, which may mediate both invagination and vesicle scission into the lumen of lysosomes (see Li, W.-W., Li, J. & Bao, J.-K. Microautophagy: lesser- known self-eating. Cell. Mol. Life Sci. 69, 1125-1136 (2011)). Direct lysosomal degradation of target substrates may occur for DNA, for example (referred to as a piecemeal autophagy - see Fujiwara, Y. et al. Direct uptake and degradation of DNA by lysosomes. - PubMed - NCBI. Autophagy 9, 1167-1171 (2014)). In addition, lysosomes maybe able to move toward the different organelles and/or membranes substrates (e.g. plasma membrane, mitochondria) and may interact with them directly by recruitment of motor proteins and SNAREs proteins (see Andrews, N. W. Lysosomes and the plasma membrane. J. Cell Biol. 158, 389-394 (2002); Hofmann, I. & Munro,
S. An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility. J. Cell. Sci. 119, 1494-1503 (2006); Fraldi, A. et al. Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders. The EMBO Journal 29, 3607-3620 (2010); and Pankiv, S. et al. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. J. Cell Biol. 188, 253-269 (2010)). Movement of lysosomes toward the cell periphery and their positioning within the cells may be associated with signaling (see R. & Bonifacino, J. S. Lysosome Positioning Influences mT0RC2 and AKT Signaling. Molecular Cell 75, 26-38.e3 (2019)). In this regard, it is contemplated that mTORCl, mT0RC2 and AKT activation may be important for lysosomal peripheral distribution (see Poiis, C. & Codogno, P. Lysosome positioning coordinates mTORCl activity and autophagy. Nature Cell Biology 13, 342-344 (2011); and Cabukusta, B. & Neefjes, J. Mechanisms of lysosomal positioning and movement. Traffic 19, 761-769 (2018)).
In certain embodiments, microautophagy and/or piecemeal degradation of target substrates and/or membranes may involve movement of lysosomes toward the peripheral and target membrane in cytosol (e.g. direct interaction with target membrane) (see Pu, J., Guardia, C. M., Keren-Kaplan,
T. & Bonifacino, J. S. Mechanisms and functions of lysosome positioning. J. Cell. Sci. 129, 4329- 4339 (2016); and Katherine R Parzych, D. J. K. An Overview of Autophagy: Morphology, Mechanism, and Regulation. Antioxid. Redox Signal. 20, 460-473 (2014)). In certain embodiments, movement of lysosomes toward the cell periphery (from perinuclear region of the cell) and interaction with target membranes/substrates (e.g. glycogen, lipid, protein) in cell periphery may be associated with the activation of mT0RCl/mT0RC2 (see Rabanal-Ruiz, Y. & Korolchuk, V. I. mTORCl and Nutrient Homeostasis: The Central Role of the Lysosome. Int J Mol Sci 19, 818 (2018); Jia, R. & Bonifacino, J. S. Lysosome Positioning Influences mT0RC2 and AKT Signaling. Molecular Cell 75, 26-38.e3 (2019)).
Without wishing to be bound by theory, it is contemplated that Rabi a DN (dominant negative form of Rabi a) may stimulate lysosome peripheral distribution (from perinuclear region) through activating mT0RCl/mT0RC2 proteins inside the cells without the need of external (or extracellular) signal for their activation (to support lysosome peripheral distribution). The use of RablaGDP or RablaGTP in reducing obesity:
As described in detail herein, it has now been identified that treatment with a microautophagyenhancing agent comprising a GDP-bound form of Rabi a, such as RablaS25N, RablaN124I(a mouse Rabi sequence), RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabi a, or one or more expressible nucleic acids encoding such a RablaGDP, may be used to reduce obesity in animal models.
As will be understood, in certain embodiments, methods as described herein may be in vitro methods, in vivo methods, or both.
In an embodiment, there is provided herein a method for reducing obesity, in a subject in need thereof, said method comprising: treating the subject with a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing cellular levels of RablaGDP in the subject, resulting in reduced obesity in the subject.
In an embodiment of the above method, the obesity is accompanied by at least one of the following conditions: increased inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
In another embodiment, there is provided herein a method for reducing bodyweight in an overweight subject, said method comprising: treating the overweight subject with a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing cellular levels of RablaGDP in the overweight subject, resulting in decreased bodyweight. In another embodiment of any of the above method or methods, the RablaGDP may be or may comprise RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP- bound form of Rabi a.
In still another embodiment of any of the above method or methods, the RablaGDP may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In yet another embodiment of any of the above method or methods, the RablaGDP may consist of the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In another embodiment of any of the above method or methods, the RablaGDP may comprise or consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above method or methods, the RablaGDP may be in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP. In an embodiment that is not meant to be limiting in any manner, the fusion protein can comprise any protein or tag, for example, GFP, YFP, mCherry, luciferase specific antibody, aptamer for identification or delivery to a specific organ or the like.
In yet another embodiment of any of the above method or methods, the fusion protein may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In another embodiment of any of the above method or methods, the RablaGDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above method or methods, the one or more expressible nucleic acids may encode any one or more of the RablaGDPs as defined herein.
In yet another embodiment of any of the above method or methods, the one or more expressible nucleic acids may be DNA-based, or RNA-based.
In another embodiment of any of the above method or methods, the one or more expressible nucleic acids may transiently express the RablaGDP in one or more cells of the subject, or wherein the one or more expressible nucleic acids may integrate in the one or more cells genome and express the RablaGDP in the one or more cells of the subject.
In still another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the one or more cells of the subject.
In yet another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG
ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT
AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT
TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA
TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT
AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA
TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC
CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA
GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC
AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT
GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA
GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment of any of the above method or methods, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG
GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA
CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT
CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA
AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA
ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC
AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing obesity in a subject, or for preventing or treating obesity, in a subject in need thereof.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing body weight in an overweight subject.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing obesity, or for preventing or treating obesity, in a subject in need thereof.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
In another embodiment, there is provided herein a use of a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing body weight in an overweight subject, or for preventing or treating excess weight.
In another embodiment of any of the above use or uses, the RablaGDP may be or may comprise RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabi a.
In still another embodiment of any of the above use or uses, the RablaGDP may comprise the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N); MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In yet another embodiment of any of the above use or uses, the RablaGDP may consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA
TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP.
In another embodiment of any of the above use or uses, the RablaGDP may comprise or consist of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In still another embodiment of any of the above use or uses, the RablaGDP may be in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP.
In yet another embodiment of any of the above use or uses, the fusion protein may comprise the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP.
In another embodiment of any of the above use or uses, the RablaGDP may be in the form of a fusion protein, and may comprise the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. In still another embodiment of any of the above use or uses, the one or more expressible nucleic acids may encode one or more RablaGDPs as defined herein.
In yet another embodiment of any of the above use or uses, the one or more expressible nucleic acids may be DNA-based, or RNA-based.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may transiently express the RablaGDP in one or more cells, or wherein the one or more expressible nucleic acids may integrate in the one or more cells genome and express the RablaGDP in the one or more cells.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the one or more cells.
In another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG
ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In still another embodiment of any of the above use or uses, the one or more expressible nucleic acids may comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG
GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT
CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG
GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG
AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC
CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA
TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT
GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA
ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA
TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA
CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT
CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT
CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT
TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT
GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA
CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT
CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA
AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA
ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC
AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC
CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA
AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA
UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA
AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA
AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG
UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA
UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU
CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU
CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA
ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA
GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG
UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
In another embodiment, there is provided herein a polypeptide comprising the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTES
YISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQ ESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFL ETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGC C
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP; for use in reducing obesity, or for preventing or treating obesity, in a subject in need thereof; or for use in reducing body weight in an overweight subject.
In another embodiment of the above polypeptide for use, the obesity is accompanied by at least one of the following conditions: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
In another embodiment, there is provided herein a pharmaceutical composition comprising a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; and another anti-obesity agent.
In yet another embodiment, there is provided herein a kit comprising any one or more of: a GDP-bound form of Rabi a (RablaGDP); one or more expressible nucleic acids encoding RablaGDP; an anti-obesity agent; instructions for performing any of the method or methods as described herein; or any combinations thereof.
As will be understood, in certain embodiments of any of the methods, uses, or polypeptides for use as described herein, the obesity may be accompanied by an increased inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea. As will be understood, lysosome-mediated microautophagy may refer to a cellular process in which a cellular lipid or protein or glycogen substrate, or a portion thereof, is engulfed and degraded by a lysosome. In this particular regard, lysosome-mediated microautophagy is an integral component of cell’s bioenergetic process (i.e. ATP generation) as well as biosynthesis process (i.e. synthesizing new biomass through recycling existing “old” materials).
It will also be understood that references to increasing lysosome-mediated microautophagy may refer to an increase in the rate, extent, capacity, or efficacy of the lysosome-mediated microautophagy process in a cell as compared to baseline levels of the cell, or as compared with a corresponding treated or untreated control cell, or as compared to levels in a reference diseased cell or reference cell having accumulation of a protein, lipid, or glycogen substrate.
It will further be understood that increasing lysosome-mediated microautophagy may include restoring or increasing lysosomal motility or lysosomal bidirectional motility in a cell, and/or may include enhancing, increasing, activating, or otherwise restoring or rescuing lysosomal motility activity in a cell. Such restoration or rescue may result in an increase in autophagy (such as, for example, micro- and/or macro- autophagy) and/or lysosomal degradation capacity. As detailed herein, there are several diseases, conditions, and cellular states in which cellular lysosomal motility may be impaired, reduced, blocked, or suppressed. As lysosome-mediated microautophagy may involve lysosomal motility and kiss-and-run events, microautophagyenhancing agents may restore or increase lysosomal motility in a cell. Lysosomal motility may play an important role in several cellular functions, including lysosome-mediated microautophagy, lysosome-mediated macroautophagy, lysosomal regeneration, and lysosomal maturation processes. References to restoring lysosomal motility and/or lysosomal bidirectional motility may refer to modulating cellular lysosomal motility/bidirectional motility levels back to those of corresponding normal or healthy control cells having baseline levels of lysosomal motility/bidirectional motility. Such modulation may, in certain embodiments, also modulate degradation capacity (i.e. phagy) levels back to those of normal or healthy cells.
It will be understood that lysosomal association-dissociation events between lysosomes and the lipid or protein or glycogen substrate may refer to events where a lysosome associates with a lipid or protein or glycogen substrate (i.e. a lipid droplet or a protein aggregate, for example), acquires at least a portion of the lipid or protein or glycogen substrate, and then dissociates from the lipid or protein or glycogen substrate. Lysosomal association-dissociation (i.e. the "on" and "off) events between lysosomes and the lipid or protein or glycogen substrate may be considered as "kiss-and- run"-type events. As part of the association (or "kiss") event, at least a small piece of substrate (i.e. lipid) may be "grabbed" or engulfed by the lysosome from the substrate (i.e. cytosolic lipid droplets, or CLD, for example). In the case of lipid droplet substrates, this may be achieved through the formation of a fusion pore between lysosome and the CLD. As part of the dissociation (or "run") event, dissociation of the lysosome from the substrate (i.e. CLD) may occur. An increase in lysosomal association-dissociation events may refer to an increase in the rate, extent, or efficacy of lysosomal association-dissociation events in a cell as compared to baseline levels of a corresponding treated or untreated control cell, for example an identical cell treated under identical conditions but without a microautophagy-modulating agent or with a compound or composition that is known not to affect the process.
It will be understood that in certain embodiments, microautophagy-enhancing agents may be used to correct a microautophagy deficiency in a cell, or a cellular condition in which microautophagy is decreased.
The person of skill in the art having regard to the teachings herein will understand that a microautophagy-enhancing agent may be any suitable agent which increases or facilitates the rate, activity, extent, or efficacy of lysosome-mediated microautophagy in a cell, or that increases lysosomal motility or bidirectional motility. In an embodiment, a suitable microautophagyenhancing agent may be or comprise a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof.
Ras-related protein Rab-IA (i.e. Rabla) is a protein that in humans is encoded by the RABI A gene. It may control vesicle transport from the endoplasmic reticulum (ER) to the Golgi compartment and on to the cell surface, and may play an important role in IL-8 and growth hormone secretion. In addition, it may play a role in autophagosome assembly in macroautophagy and cellular defense reactions against pathogenic bacteria when it is in its GTP-bound form. It also may regulate motility of endocytic compartment(s).
As described in detail herein, it has now been identified that lysosome-mediated microautophagy of a target protein, lipid, or glycogen substrate in a cell may be increased by treatment with a microautophagy-enhancing agent comprising a GDP-bound form of Rabi a, such as RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabla.
It will be understood that the specific amino acid or nucleic acid sequence of a particular gene may vary from species to species. By way of example, the human Rabla amino acid sequence may have homologs in other species having sequence variation from the human sequence. In some embodiments, although homologous sequences may vary between species, the general effect (for example, the phenotypic effect) of a homolog sequence may be substantially similar to the effect of the wild- type sequence in a given cell or subject.
In particular embodiments, a microautophagy-enhancing agent may be, or comprise, a GDP-bound form of Rabla (RablaGDP) such as, for example, RablaS25N, RablaN1241, RablaD41N, RablaD47N, or a functional equivalent thereof, or another dominant negative (DN) GDP-bound form of Rabla. Sequences for human and/or mouse RablaWT and certain GDP-bound forms thereof are shown in Figure 2. Suitable GDP-bound forms of Rabla may include any suitable Rabla variant which is "dominant negative", or which preferentially binds GDP over GTP. Such RablaGDP variants may be identified using techniques known in the art (see, for example, Chan, C.-C. et al. Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila. Current Biology 21, 1704-1715 (2011); Tabancay, A. P. et al. Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K. J. Biol. Chem. 278, 39921-39930 (2003); and Dumas, J. J., Zhu, Z., Connolly, J. L. & Lambright, D. G. Structural basis of activation and GTP hydrolysis in Rab proteins. Structure 7, 413 — s2 (1999), each of which is herein incorporated by reference in its entirety).
Figure 2 shows certain sequences of nucleic acids and amino acids/proteins as described herein. In Figure 2, SEQ ID NOs: 1-3 provide human RablaWT DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs:4-6 provide human RablaS25N DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs:7-9 provide mouse RablN1241 DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs: 10-12 provide human RablaQ70L DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs: 13-15 provide human RablaQ63L DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; SEQ ID NOs: 16-18 provide human RablaD47N DNA gene sequence, ORF codon sequence, and amino acid sequence, respectively; and SEQ ID NOs: 19-21 provide MG-008 ORF DNA sequence with 5’ luciferase tag, MG-008 ORF mRNA sequence with 5’ Eucif erase tag, and MG-008 Protein sequence with N-terminal luciferase tag (see Example 1 for further discussion of SEQ ID NOs: 19-21), respectively. Sequence of human RablaD41N may be found at https://www.addgene.org/49581/ (which is herein incorporated by reference in its entirety), and is commercially available. In certain embodiments, there is provided herein a nucleic acid or amino acid comprising any of these sequences. In certain embodiments, there is provided herein a nucleic acid or amino acid comprising a nucleic acid sequence or amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences (i.e. with any of SEQ ID NOs: 1-21 or the sequence of RablaD41N) or an active fragment thereof.
Suitable functional equivalents of RablaS25N, RablaD41N, RablaD47N, and RablaN1241 may include, for example, suitable Rabla variants or mutants having at least 80% (or >85%, or >90%, or >95%, or >99%) sequence identity to RablaWT or RablaS25N or RablaN1241 or RablaD41N or RablaD47N and which preferentially bind GDP over GTP while also retaining the relevant cellular/biochemical functions of RablaS25N or RablaN1241 or RablaD41N or RablaD47N as described in detail herein. In further embodiments, it will be understood that a microautophagy-enhancing agent may be, or comprise, one or more expressible nucleic acids encoding RablaGDP, such as any suitable nucleic acid/expression vector (i.e. vector, cassette, mRNA, modified mRNA, plasmid, for example) which encodes for/expresses a GDP-bound form of Rabla (RablaGDP) such as, for example, RablaS25N or RablaN1241 or RablaD41N or RablaD47N, or a functional equivalent thereof.
As will be understood, sequences are mainly described herein with reference to human and/or mouse homologs. It will be understood that functional equivalents and/or variants may be found in a variety of different species, such as among different mammals. References herein to particular sequence modifications and/or mutants providing for RablaGDP (DN) or RablaGTP (DA) forms often provide position and modification/mutation information (e.g. S25N, D41N, D47N, N124I; Q70L, Q67L, Q63L) with reference to human and/or mouse homolog/sequence for convenience; however, it will be understood that equivalent DN and/or DA forms may be implemented in homologous sequences from other species and/or other sequences related to the human and/or mouse sequences provided herein, although the positioning and/or nature of the modification/mutation may vary somewhat depending on the particular sequence of interest. By way of example, RablN1241 is medication/mutation of a mouse sequence. By way of another example, RablQ67L is a modification/mutation of a mouse sequence, and there is no Q at the 67th position of human Rabi a; rather, there is a Q in the human sequence at the 63rd position, and so the modification/mutation with reference to the human sequence is Rabi a()63L.
A person of skill in the art will understand that a microautophagy-reducing agent may be or comprise any one or more of a GTP-bound form of Rabi a (RablaGTP), one or more expressible nucleic acids encoding RablaGTP, Rabi a wild-type (RablaWT), or one or more expressible nucleic acids encoding RablaWT. In certain embodiments, a RablaGTP may be or comprise RablaQ70L, RablaQ67L(in mouse sequence), RablaQ63L (in human Rabi a sequence), or a functional equivalent thereof, or another dominant active (DA) GTP-bound form of Rabi a. GTP-bound forms of Rabi a may include any Rabi a variant which is "dominant active", or which preferentially binds GTP over GDP. Such RablaGTP variants may be identified using techniques known in the art (see, for example, Chan, C.-C. et al. Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila. Current Biology 21, 1704-1715 (2011); Tabancay, A. P. et al. Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K. J. Biol. Chem. 278, 39921-39930 (2003); and Dumas, J. J., Zhu, Z., Connolly, J. L. & Lambright, D. G. Structural basis of activation and GTP hydrolysis in Rab proteins. Structure 7, 413 — s2 (1999), each of which is herein incorporated by reference in its entirety), such as those used to identify Rab 1 a<)70L and/or Rabi a()67L and/or Rab 1 aQ63L.
It will be understood that in certain embodiments, treatment with a microautophagy-enhancing agent, such as RablaGDP, may comprise introducing the RablaGDP protein to a cell, expressing RablaGDP within the cell, or both, for example.
It will be understood that expression of a particular protein within a cell may refer to the production of a polypeptide from a nucleic acid sequence encoding the polypeptide. Gene expression may include both transcription and translation processes, and so gene expression may refer to production of a nucleic acid sequence such as an mRNA (i.e. transcription), production of a protein (i.e. translation), or both. It will further be understood that overexpression of a particular gene in a cell may refer to increasing the expression of a particular gene within a cell as compared to wildtype, baseline, or untreated levels. Overexpression, or introduction of a mutant gene, into cells may be accomplished using any of several methods known in the art. By way of example, a vector (either viral, plasmid, or other) comprising one or more copies of the particular gene each driven by a suitable promoter sequence (for example, a constitutive or inducible promoter), or an mRNA or chemically modified version thereof may be introduced into cells via transfection, electroporation, or viral infection, or another suitable method know in the art. Suitable expression vector techniques for overexpressing or introducing a particular gene into a cell are known in the art (see, for example, Molecular Cloning: A Laboratory Manual (4th Ed.), 2012, Cold Spring Harbor Laboratory Press). The skilled person having regard to the teachings herein will be aware of a wide variety of expressible nucleic acids encoding a particular protein, such as RablaGDP, that may be prepared for introduction to a cell (either transiently or long-term via integration into the genome, for example) to provide expression of the protein of interest.
It will be understood that compounds and/or compositions comprising or consisting of one or more of the nucleic acids and/or proteins as described herein may be used. Compositions may additionally comprise one or more pharmaceutically acceptable diluents, carriers, excipients, or buffers. Compositions may be used for administering one or more nucleic acids and/or proteins to a cell in vitro or in vivo.
Introduction of a gene, in the context of inserting a nucleic acid sequence into a cell, may refer to "transfection", "transformation", or "transduction", and may include the incorporation or introduction of a nucleic acid sequence into a eukaryotic cell where the nucleic acid sequence may optionally be incorporated into the genome of the cell, or transiently expressed (for example, transfected mRNA). A protein or enzyme may be introduced into a cell by delivering the protein or enzyme itself into the cell, or by expressing an mRNA encoding the protein or enzyme within the cell, leading to its translation.
As will be known to one of skill in the art, expressible nucleic acids for expressing a particular gene may encode or include features as described in "Genes VII", Lewin, B. Oxford University Press (2000) or "Molecular Cloning: A Laboratory Manual", Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition (2001). A nucleotide sequence encoding a polypeptide or protein may be incorporated into a suitable vector, such as a commercially available vector. Vectors may also be individually constructed or modified using standard molecular biology techniques, as outlined, for example, in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd edition (2001)). The person of skill in the art will recognize that a vector may include nucleotide sequences encoding desired elements that may be operably linked to a nucleotide sequence encoding a polypeptide or protein. Such nucleotide sequences encoding desired elements may include transcriptional promoters, transcriptional enhancers, transcriptional terminators, translational initiators, translational terminators, ribosome binding sites, 5'- untranslated region, 3'- untranslated regions, cap structure, poly A tail, and/or an origin of replication. Selection of a suitable vector may depend upon several factors, including, without limitation, the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational control elements desired, the level of expression desired, copy number desired, whether chromosomal integration is desired, the type of selection process that is desired, or the host cell or the host range that is intended to be transformed.
The person of skill in the art will understand that biomolecules and/or compounds described herein may be provided in pharmaceutical compositions together with a pharmaceutically acceptable diluent, carrier, or excipient, and/or together with one or more separate active agents or drugs as part of a pharmaceutical combination or pharmaceutical composition. In certain embodiments, the biomolecules, compounds, and/or pharmaceutical compositions may be administered in a treatment regimen simultaneously, sequentially, or in combination with other drugs or pharmaceutical compositions, either separately or as a combined formulation or combination.
Biomolecules, compounds, and/or compositions as described herein may include one or more pharmaceutically acceptable excipients, diluents, and/or carriers. A pharmaceutically acceptable carrier, diluent, or excipient may include any suitable carrier, diluent, or excipient known to the person of skill in the art. Examples of pharmaceutically acceptable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch. The person of skill in the art will recognize that pharmaceutically acceptable excipients may include suitable fillers, binders, lubricants, buffers, glidants, and disentegrants known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2006)). Examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000 — 20th edition) and in the United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
It will also be understood that one or more conservative amino acid substitutions may be possible. As will be recognized, a conservative amino acid substitution may include one in which an amino acid is substituted for another amino acid having similar properties such that the folding, activity, or other functionality of the protein is not significantly affected. Examples of aromatic amino acids, which may be substitutable, may include phenylalanine, tryptophan, and tyrosine. Examples of interchangeable hydrophobic amino acids, which may be substitutable, may include leucine, isoleucine, methionine, and valine. Examples of interchangeable polar amino acids, which may be substitutable, may include glutamine and asparagine. Examples of interchangeable basic amino acids, which may be substitutable, may include arginine, lysine, and histidine. Examples of interchangeable acidic amino acids, which may be substitutable, may include aspartic acid and glutamic acid. Finally, examples of interchangeable small amino acids, which may be substitutable, may include alanine, serine, threonine, cysteine, and glycine.
As described in detail herein, dominant-negative (DN) Rabi a (e.g. GDP-bound Rabi a, RablaGDP) may be used for increasing microautophagy, which may provide for degradation of a cellular target of interest such as a disease-related protein, glycogen, or lipid. As will be understood, any suitable Rabi a DN or RablaGDP may be used. A variety of Rabi a DN proteins will be known to the person of skill in the art having regard to the teachings herein. In certain embodiments, generally any suitable dominant negative form of Rabi a (i.e. a constant/locked Rabi a in its GDP form) may be used to promote lysosomal movement toward the cytosol and peripheral of the cells (from perinuclear region of the cells), and stimulate direct lysosome interaction with target substrates, for example.
In certain embodiments, it is contemplated that expression of Rabi a DN (GDP form) may stimulate activation of mT0RCl/mT0RC2 and AKT based on their effect on lysosomal positioning and promoting lysosome movement toward the peripheral and toward the target substrates (see also Jia, R. & Bonifacino, J. S. Lysosome Positioning Influences mT0RC2 and AKT Signaling. Molecular Cell 75, 26-38.e3 (2019)).
It is contemplated herein that different genetic modifications of Rabi a which lock this protein in its constant GDP-bound state may be used to promote direct lysosome interaction and piecemeal engulfment of different target substrate(s) (such as one or more protein, lipid, and/or glycogen target(s)). Rabi a constant GDP-bound form (DN) is generally not available in normal physiological conditions, in which the native protein Rabi a is constantly shifting between its GTP and GDP forms. It is contemplated that in certain embodiments, genetic mutation and/or amino acid substitution/modification may be used to lock this GTPase in its GDP-bound form (or constant GTP form), and prevent it from going to its GTP state (or GDP state). It is contemplated that in certain embodiments, any suitable modification(s) that can substantially keep the integrity of the GDP form/state of GTPases (which may exert an impact on signaling as well as an effect on lysosome movement to peripheral/cytosol) may promote microautophagy and degradation of target substrate(s) through lysosomal piecemeal engulfment.
In certain embodiments, RablaGDP may be administered to a particular cell type or subject in need thereof in generally any suitable manner which may be selected to suit the particular cell type, subject, and/or indication. In certain embodiments, a nucleic acid sequences encoding and capable of expressing the RablaGDP may be administered to the subject or introduced to the cell type via any suitable transfection or nucleic acid delivery approach as will be known to the person of skill in the art having regard to the teachings herein. In certain embodiments, delivery may be based on DNA or RNA transfection (for example, using common transfection regent(s) such as lipofectamine (Invitrogen), FuGENE (Rosche), using DNA adenovirus (gene therapy), or using Modified RNA (i.e. stabilized RNA) delivery to body (using, for example, virus or microvesicle, exosome, or ectosome). In certain embodiments, protein may be administered or delivered to cells in need thereof, optionally assisted with any suitable technique or delivery vehicle for facilitating protein delivery to a cell or cells.
In an embodiment of the present invention, Fl l can be administered to an obese or overweight subject to reduce body weight in said subject. Fl l is lipid nanoparticles that encapsulate Rabi, which is commercially available from Precision NanoSystems (#50 655 W Kent Ave N, Vancouver, BC V6P 6T7). Fl l may be administered intravenously, orally, subcutaneously, intramuscularly, sublingually, or any other route that would be known to a person of skill in the art. Fl l may be dosed at 0.001-1 mg/kg (mpk), but other doses may be beneficial depending on the subject. Fl l treatment may be done in a single dose or multiple doses. If Fl l is given repeatedly, the individual treatments may be separated by hours, days, weeks, months, or years, as would be necessary to establish, maintain, or re-establish body weight reduction. Administration of Fl 1 may decrease subject body weight and/or fat content, and/or may positively affect HbAlc, alanine amino transferase (ALT), asparatate amino transferase (AST), blood urea nitrogen (BUN), creatinine (CRE) or other blood chemistry measurements. Fl 1 treatment may also improve other body health indicators, such as high blood pressure, high blood sugar, elevated cholesterol levels, inflammation, high serum triglycerides, high resting heart rate, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders, sleep apnea, and/or elevated cancer progression markers, without being limiting.
In an embodiment of the present invention, Rabi encapsulated in a lipid nanoparticle may be in the form of a fusion protein, wherein the Rabi is fused or otherwise directly or indirectly linked, optionally via a linker, with a signaling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the Rabi.
EXAMPLE 1: RablaDN (GDP form) Reduces body weight in Obese Mice
Female C57BE/6 mice (n = 10), age of 6 weeks old, were fed a high-fat diet for 10 months to generate diet-induced-obesity (DIO) mouse model. The mice were divided into two groups, one group (n = 5) was administered, intravenously, with RablaDN. In this Example, the RablaDN is also referred to as MG-008 which is encoded by mRNA and encapsulated with an ANM formulation. The control group (n = 5) was injected with PBS. The mice were continued on HFD for another 18 days, and the diet intake and the bodyweight were recorded every 2-3 days. Shown on the left (red bars) of Figure 1 is the body weight loss of mice at day 12 post-MG008 injection (a single dose), and on the right (black bars) is the bodyweight of control mice at day 12. The bodyweight loss in MG008-treated mice was apparent throughout the entire 18-day post-MG008 treatment, and the earliest significant body weight loss was registered as short as day 4 post- MG008 injection (data not shown).
EXAMPLE 2: Encapsulated Rabi lipid nanoparticles (Fll) reduces body weight in obese mice
Male C57BL/6 mice at approximately 6 weeks of age were fed a high-fat diet (HFD) until they weighed at least 40 g (40-50 g). Mice were weighed at the start of the HFD and their weight was monitored weekly. The appropriate weight gain was usually achieved at around 18-26 weeks of age, at which point NMR was used to determine body composition and Vevo LAZR-X was used to determine steatosis. Mice were then treated with Fl 1 via intravenous administration. Fl 1 was administered at 0.00625, 0.0125, 0.025, 0.05, or 0.5 mg/kg (mpk). NMR body composition measurements were then made at 1, 3, 5 and 7 weeks post Fl l treatment. LAZR-X steatosis measurements were taken at weeks 4 and 7 post Fl l treatment. Additionally, at week 7 post Fl l treatment, blood was taken from the submandibular region (200 pl) to determine HbAlc levels. Eight weeks after Fl l administration the mice were sacrificed and the following measurements were taken: (1) measurement of body weight, liver weight and epididymal fat weight; (2) blood was collected for measurement of alanine amino transferase (ALT), aspartate amino transferase (AST), blood urea nitrogen (BUN), creatinine (CRE), and cytokines; (3) pictures were taken of the liver and epididymal fat; (4) optical coherence tomography (OCT) was conducted on the liver; and (5) mice were perfused and tissues were processed for immunohistochemical and H&E staining.
The results from these experiments indicated that body weight is positively correlated with total fat and not lean mass at baseline, 3 days post Fl l treatment, and 7 days post Fl l treatment (FIGs 4-6, panels A and B). Moreover, the percentage of total fat mass and the percentage of total lean mass are negatively correlated at the same three time points (FIGs 4-6, panel F). The consistent results from baseline, day 3 post-i.v. administration of Fl 1, and day 17 post-i.v. administration of Fl l indicate that Fl l proportionally changes fat and lean content within the same mouse (FIGs 4- 6, panel F).The total body weight changes at all Fl 1 doses were largely determined by lean mass change after 3 days of Fl 1 treatment (FIGs 7A and 7C). Fl l administered at 0.05-0.5 mpk reduced body weight by reducing either fat or lean mass or both after 3 days of Fl 1 treatment (FIG. 7). The most dramatic effect on body weight was observed when Fl l was administered at 0.5 mpk as it lead to a substantial drop in both fat and lean mass (FIG. 7). The lower doses of Fl 1 (0.025-0.05 mpk) did not change the fat mass significantly while even lower levels of Fl 1 (<0.0125) appeared to induce fat accumulation (FIG. 7B). After 17 days of Fl l treatment, total body weight changes by all Fl l doses were largely determined by changing fat content because the mice continuously fed on 60% HFD (FIGs 8A and 8B). Consistent with the results of the 3 day treatment with Fl l, only the 0.5 mpk Fl l treatment was effective at reducing body weight, fat content, and lean mass content (FIG. 8). The Fl l doses below 0.5 mpk were ineffective at reducing fat content compared to baseline (before i.v. administration of Fl l) (FIG. 8).
FIGs. 9 and 10 show significant standard curves for Fl 1 efficacy on body weight change (R2 = 0.9821) and fat change (R2= 0.9639) after 3 days of Fl l treatment using multiple logistic regression analysis (Figures 9A and 9B), compared to the standard curve for Fl 1 efficacy on body weight change (R2 = 0.7278) and fat change (R2= 0.7023) 17 days after Fl 1 treatment. The multiple regression analysis suggests that 0.32 mpk and 0.4 mpk may be the best Fl l doses for reducing fat and body weight, respectively, after 3 days of treatment.
One or more illustrative embodiments have been described by way of example. It will be understood to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
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W02017/008141 - Lysosomal Degradation of Lipids and Proteins and Method of Use Thereof
All references cited herein and throughout this specification are herein incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:
1. A method for reducing obesity, or for preventing or treating obesity, in a subject in need thereof, said method comprising: treating the subject with a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing a cellular levels of RablaGDP in the subject, resulting in decreased obesity in the subject.
2. The method of claim 1 , wherein the obesity is accompanied by at least one of the following conditions: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea.
3. A method for reducing bodyweight in an overweight subject, said method comprising: treating the overweight subject with a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; thereby increasing cellular levels of RablaGDP in the overweight subject, resulting in decreased bodyweight in the subject.
4. The method of any one of claims 1-3, wherein the RablaGDP is or comprises RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabla.
5. The method of any one of claims 1-4, wherein the RablaGDP comprises the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP. The method of any one of claims 1-5, wherein the RablaGDP consists of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP. The method of any one of claims 1-5, wherein the RablaGDP comprises or consists of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The method of any one of claims 1-5, wherein the RablaGDP is in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signalling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP. The method of claim 8, wherein the fusion protein comprises the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The method of claim 8 or 9, wherein the RablaGDP is in the form of a fusion protein, and comprises the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The method of any one of claims 1-3, wherein the one or more expressible nucleic acids encode one or more RablaGDP as defined in any one of claims 4-10. The method of claim 11, wherein the one or more expressible nucleic acids are DNA-based, or RNA-based. The method of claim 11 or 12, wherein the one or more expressible nucleic acids transiently express the RablaGDP in one or more cells of the subject, or wherein the one or more expressible nucleic acids integrate in the one or more cells genome and express the RablaGDP in the one or more cells of the subject. The method of any one of claims 1-3 or 10, wherein the one or more expressible nucleic acids comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the one or more cells of the subject. The method of claim 14, wherein the one or more expressible nucleic acids comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5); ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT
GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy. The method of claim 14, wherein the one or more expressible nucleic acids comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU
AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC
CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA
AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA
UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA
AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA
AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG
UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA
UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU
CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU
CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA
ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA
GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG
UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy. Use of a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing obesity, or for preventing or treating obesity, in a subject in need thereof. Use of a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea. Use of a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, for reducing body weight in a subject. Use of a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing obesity, or for preventing or treating obesity, in a subject in need thereof. Use of a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea. Use of a GDP-bound form of Rabla (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof, in the manufacture of a medicament for reducing bodyweight in an overweight subject. The use of any one of claims 17-22, wherein the RablaGDP is or comprises RablaS25N, RablaN1241, RablaD41N, RablaD47N, or another dominant negative (DN) GDP-bound form of Rabi a. The use of any one of claims 17-23, wherein the RablaGDP comprises the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP. The use of any one of claims 17-24, wherein the RablaGDP consists of the amino acid sequence: MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYIS
TIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNN
VKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNA TNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or
MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELDGKT
IKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASEN VNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAEI KKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP. The use of any one of claims 17-24, wherein the RablaGDP comprises or consists of the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK
TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE
NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE
IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The use of any one of claims 17-24, wherein the RablaGDP is in the form of a fusion protein, wherein the RablaGDP is fused or otherwise directly or indirectly linked, optionally via a linker, with a signalling or targeting peptide, a fluorescent peptide or other marker or tracer, or another peptide or non-peptide moiety for targeted delivery, facilitating cell uptake, increasing stability or in vivo half-life, or improving another therapeutic, diagnostic, or in vivo property of the RablaGDP. The use of claim 27, wherein the fusion protein comprises the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELDGK TIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDRYASE NVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSFMTMAAE IKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 6; Human RablaS25N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The use of claim 27 or 28, wherein the RablaGDP is in the form of a fusion protein, and comprises the amino acid sequence:
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERE LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHL PPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQI IPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPT LFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAI LITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP NIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKSKLMSSMNPEYDYLFKLLLIGDSGVGK NCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRG AHGIIVVYDVTDQESFNNVKQWLQEIDRYASENVNKLLVGNKCDLTTKKVVDYTT AKEFADSLGIPFLETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQST PVKQSGGGCC
(SEQ ID NO: 21); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and preferentially binding GDP. The use of any one of claims 17-22, wherein the one or more expressible nucleic acids encode one or more RablaGDP as defined in any one of claims 23-29. The use of claim 30, wherein the one or more expressible nucleic acids are DNA-based, or RNA-based. The use of claim 30 or 31 , wherein the one or more expressible nucleic acids transiently express the RablaGDP in one or more cells of the subject, or wherein the one or more expressible nucleic acids integrate in the one or more cells genome and express the RablaGDP in the one or more cells of the subject. The use of any one of claims 17-22 or 30, wherein the one or more expressible nucleic acids comprise one or more expression vectors, plasmids, or mRNAs encoding and capable of expressing the RablaGDP inside the one or more cells of the subject. The use of claim 33, wherein the one or more expressible nucleic acids comprise a nucleic acid sequence of:
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCG ACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAGATGATACATATAC AGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAAGAACTATAGAGTT AGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGAT TTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTA TGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAAT
AGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGA
TCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGGAATTTGCTGATTC
CCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAACGAATGTAGAACA
GTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGGTCCCGGAGCAAC
AGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCACTCCAGTCAAGCA GTCAGGTGGAGGTTGCTGCTAA
(Human RablaS25N ORF Codon Sequence, SEQ ID NO: 5);
ATGGGGGACTACAAGGACGACGATGACAAGGGGGGTAGCGGTGGATCCAGCAT
GAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGATTCTGGGGTT
GGAAAGTCCTGCCTTCTCCTTAGGTTTGCAGATGATACGTATACGGAAAGCTACA
TCAGCACAATTGGTGTGGATTTCAAGATACGAACTATAGAGTTAGATGGGAAAA
CAATCAAGCTACAGATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAATCA
CTTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAACGTTAAACAGTGGCTGCAGGAGATAGATCGCTACGC
CAGTGAAAATGTCAACAAGTTGTTGGTAGGGATCAAATGTGACCTGACCACAAA
GAAAGTAGTAGACTACACAACAGCAAAGGAATTTGCAGATTCCCTTGGAATTCC
ATTTTTGGAAACCAGTGCTAAGAACGCAACGAATGTAGAACAGTCTTTCATGAC
GATGGCAGCTGAGATTAAAAAGCGAATGGGTCCTGGAGCTACAGCTGGTGGTGC
CGAGAAGTCCAATGTTAAAATCCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGG CTGCTGCTAA
(Mouse RablaN1241 ORF Codon Seqeunce, SEQ ID NO: 8); or
ATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTACTTCTGATTGGCGACT
CAGGGGTTGGAAAGTCTTGCCTTCTTCTTAGGTTTGCAGATGATACATATACAGAAA
GCTACATCAGCACAATTGGTGTGAACTTCAAAATAAGAACTATAGAGTTAGACGGG
AAAACAATCAAGCTTCAAATATGGGACACAGCAGGCCAGGAAAGATTTCGAACAAT
CACCTCCAGTTATTACAGAGGAGCCCATGGCATCATAGTTGTGTATGATGTGACAGA
TCAGGAGTCCTTCAATAATGTTAAACAGTGGCTGCAGGAAATAGATCGTTATGCCAG TGAAAATGTCAACAAATTGTTGGTAGGGAACAAATGTGATCTGACCACAAAGAAAG TAGTAGACTACACAACAGCGAAGGAATTTGCTGATTCCCTTGGAATTCCGTTTTTGG AAACCAGTGCTAAGAATGCAACGAATGTAGAACAGTCTTTCATGACGATGGCAGCT GAGATTAAAAAGCGAATGGGTCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAA TGTTAAAATTCAGAGCACTCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
(Human RablaD47N ORF Codon Sequence, SEQ ID NO: 17); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy.
35. The use of claim 33, wherein the one or more expressible nucleic acids comprise a nucleic acid sequence of:
ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTAGAG GATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCG GAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTG AATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGC CGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTA TAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTT GTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATA ATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGA TGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTA CCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAACTCCTCTGGAT CTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATT CTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATT TTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTT GATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTA CGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTT CATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGA AATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAA
ACGCTTCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATC
AGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGT
TGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGC
GTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGTTATG
TAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATT
CTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT
GAAGTCTTTAATTAAATACAAAGGATACCAGGTGGCCCCCGCTGAATTGGAGTC
GATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGAC
GATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACG
ATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCCAAA
AAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGA
AAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGG
AAAGTCCAAATTGATGTCCAGCATGAATCCCGAATATGATTATTTATTCAAGTTA
CTTCTGATTGGCGACTCAGGGGTTGGAAAGAATTGCCTTCTTCTTAGGTTTGCAG
ATGATACATATACAGAAAGCTACATCAGCACAATTGGTGTGGATTTCAAAATAA
GAACTATAGAGTTAGACGGGAAAACAATCAAGCTTCAAATATGGGACACAGCAG
GCCAGGAAAGATTTCGAACAATCACCTCCAGTTATTACAGAGGAGCCCATGGCA
TCATAGTTGTGTATGATGTGACAGATCAGGAGTCCTTCAATAATGTTAAACAGTG
GCTGCAGGAAATAGATCGTTATGCCAGTGAAAATGTCAACAAATTGTTGGTAGG
GAACAAATGTGATCTGACCACAAAGAAAGTAGTAGACTACACAACAGCGAAGG
AATTTGCTGATTCCCTTGGAATTCCGTTTTTGGAAACCAGTGCTAAGAATGCAAC
GAATGTAGAACAGTCTTTCATGACGATGGCAGCTGAGATTAAAAAGCGAATGGG
TCCCGGAGCAACAGCTGGTGGTGCTGAGAAGTCCAATGTTAAAATTCAGAGCAC
TCCAGTCAAGCAGTCAGGTGGAGGTTGCTGCTAA
£SEQ ID NO: 19, MG-008 ORF DNA Sequence with 5’ Luciferase Tag); or
AUGGAAGACGCCAAAAACAUAAAGAAAGGCCCGGCGCCAUUCUAUCCGCUAG
AGGAUGGAACCGCUGGAGAGCAACUGCAUAAGGCUAUGAAGAGAUACGCCCU
GGUUCCUGGAACAAUUGCUUUUACAGAUGCACAUAUCGAGGUGAACAUCACG
UACGCGGAAUACUUCGAAAUGUCCGUUCGGUUGGCAGAAGCUAUGAAACGAU AUGGGCUGAAUACAAAUCACAGAAUCGUCGUAUGCAGUGAAAACUCUCUUCA
AUUCUUUAUGCCGGUGUUGGGCGCGUUAUUUAUCGGAGUUGCAGUUGCGCCC
GCGAACGACAUUUAUAAUGAACGUGAAUUGCUCAACAGUAUGAACAUUUCGC
AGCCUACCGUAGUGUUUGUUUCCAAAAAGGGGUUGCAAAAAAUUUUGAACGU
GCAAAAAAAAUUACCAAUAAUCCAGAAAAUUAUUAUCAUGGAUUCUAAAACG
GAUUACCAGGGAUUUCAGUCGAUGUACACGUUCGUCACAUCUCAUCUACCUCC
CGGUUUUAAUGAAUACGAUUUUGUACCAGAGUCCUUUGAUCGUGACAAAACA
AUUGCACUGAUAAUGAACUCCUCUGGAUCUACUGGGUUACCUAAGGGUGUGG
CCCUUCCGCAUAGAACUGCCUGCGUCAGAUUCUCGCAUGCCAGAGAUCCUAUU
UUUGGCAAUCAAAUCAUUCCGGAUACUGCGAUUUUAAGUGUUGUUCCAUUCC
AUCACGGUUUUGGAAUGUUUACUACACUCGGAUAUUUGAUAUGUGGAUUUCG
AGUCGUCUUAAUGUAUAGAUUUGAAGAAGAGCUGUUUUUACGAUCCCUUCAG
GAUUACAAAAUUCAAAGUGCGUUGCUAGUACCAACCCUAUUUUCAUUCUUCG
CCAAAAGCACUCUGAUUGACAAAUACGAUUUAUCUAAUUUACACGAAAUUGC
UUCUGGGGGCGCACCUCUUUCGAAAGAAGUCGGGGAAGCGGUUGCAAAACGC
UUCCAUCUUCCAGGGAUACGACAAGGAUAUGGGCUCACUGAGACUACAUCAG
CUAUUCUGAUUACACCCGAGGGGGAUGAUAAACCGGGCGCGGUCGGUAAAGU
UGUUCCAUUUUUUGAAGCGAAGGUUGUGGAUCUGGAUACCGGGAAAACGCUG
GGCGUUAAUCAGAGAGGCGAAUUAUGUGUCAGAGGACCUAUGAUUAUGUCCG
GUUAUGUAAACAAUCCGGAAGCGACCAACGCCUUGAUUGACAAGGAUGGAUG
GCUACAUUCUGGAGACAUAGCUUACUGGGACGAAGACGAACACUUCUUCAUA
GUUGACCGCUUGAAGUCUUUAAUUAAAUACAAAGGAUACCAGGUGGCCCCCG
CUGAAUUGGAGUCGAUAUUGUUACAACACCCCAACAUCUUCGACGCGGGCGU
GGCAGGUCUUCCCGACGAUGACGCCGGUGAACUUCCCGCCGCCGUUGUUGUUU
UGGAGCACGGAAAGACGAUGACGGAAAAAGAGAUCGUGGAUUACGUCGCCAG
UCAAGUAACAACCGCCAAAAAGUUGCGCGGAGGAGUUGUGUUUGUGGACGAA
GUACCGAAAGGUCUUACCGGAAAACUCGACGCAAGAAAAAUCAGAGAGAUCC
UCAUAAAGGCCAAGAAGGGCGGAAAGUCCAAAUUGAUGUCCAGCAUGAAUCC
CGAAUAUGAUUAUUUAUUCAAGUUACUUCUGAUUGGCGACUCAGGGGUUGGA
AAGAAUUGCCUUCUUCUUAGGUUUGCAGAUGAUACAUAUACAGAAAGCUACA
UCAGCACAAUUGGUGUGGAUUUCAAAAUAAGAACUAUAGAGUUAGACGGGAA AACAAUCAAGCUUCAAAUAUGGGACACAGCAGGCCAGGAAAGAUUUCGAACA AUCACCUCCAGUUAUUACAGAGGAGCCCAUGGCAUCAUAGUUGUGUAUGAUG UGACAGAUCAGGAGUCCUUCAAUAAUGUUAAACAGUGGCUGCAGGAAAUAGA UCGUUAUGCCAGUGAAAAUGUCAACAAAUUGUUGGUAGGGAACAAAUGUGAU CUGACCACAAAGAAAGUAGUAGACUACACAACAGCGAAGGAAUUUGCUGAUU CCCUUGGAAUUCCGUUUUUGGAAACCAGUGCUAAGAAUGCAACGAAUGUAGA ACAGUCUUUCAUGACGAUGGCAGCUGAGAUUAAAAAGCGAAUGGGUCCCGGA GCAACAGCUGGUGGUGCUGAGAAGUCCAAUGUUAAAAUUCAGAGCACUCCAG UCAAGCAGUCAGGUGGAGGUUGCUGCUAA
(SEQ ID NO: 20, MG-008 ORF mRNA Sequence with 5’ Luciferase Tag); or a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith and encoding a RablaGDP preferentially binding GDP; or a nucleic acid sequence equivalent to any of the above sequences due to codon redundancy. A polypeptide comprising the amino acid sequence:
MSSMNPEYDYLFKLLLIGDSGVGKNCLLLRFADDTYTESYISTIGVDFKIRTIELD
GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC (SEQ ID NO: 6; Human RablaS25N);
MGDYKDDDDKGGSGGSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTES
YISTIGVDFKIRTIELDGKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQ ESFNNVKQWLQEIDRYASENVNKLLVGIKCDLTTKKVVDYTTAKEFADSLGIPFL ETSAKNATNVEQSFMTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGC C
(SEQ ID NO: 9; Mouse RablN1241); the amino acid sequence of human RablaD41N; or MSSMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVNFKIRTIELD GKTIKLQIWDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQWLQEIDR YASENVNKLLVGNKCDLTTKKVVDYTTAKEFADSLGIPFLETSAKNATNVEQSF MTMAAEIKKRMGPGATAGGAEKSNVKIQSTPVKQSGGGCC
(SEQ ID NO: 18; Human RablaD47N); or a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of these sequences and preferentially binding GDP; for use in reducing obesity, or for preventing or treating obesity, in a subject in need thereof; or for use in reducing body weight in an overweight subject. The polypeptide of claim 36, wherein the obesity is accompanied by at least one of the following conditions: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders and sleep apnea. A pharmaceutical composition comprising a GDP-bound form of Rabi a (RablaGDP), one or more expressible nucleic acids encoding RablaGDP, or a combination thereof; and another anti-obesity agent. A kit comprising any one or more of: a GDP-bound form of Rabi a (RablaGDP); one or more expressible nucleic acids encoding RablaGDP; an anti-obesity agent; instructions for performing a method as defined in any one of claims 1-16; or any combinations thereof.
40. Use of Rabi encapsulated lipid nanoparticles for reducing, treating or preventing obesity in a subject in need thereof.
41. Use of Rabi encapsulated lipid nanoparticles for reducing body weight in an overweight subject.
42. Use of Rabi encapsulated lipid nanoparticles in the manufacture of a medicament for reducing, treating or preventing obesity in a subject in need thereof.
43. Use of Rabi encapsulated lipid nanoparticles in the manufacture of a medicament for reducing body weight in an overweight subject.
44. The use of any one of claims 40-43, wherein the Rabi encapsulated lipid nanoparticles are for administration at 0.005-0.5 mg/kg.
45. The use of claim 44, wherein the Rabi encapsulated lipid nanoparticles are for administration at 0.3, 0.4, or 0.5 mg/kg.
46. The use of claim 44 or 45, wherein the Rabi encapsulated lipid nanoparticles are for administration via intravenous injection.
47. Use of Rabi encapsulated lipid nanoparticles for reducing, preventing or treating at least one of the following conditions in a subject in need thereof: inflammatory response, increased abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, low serum high-density lipoprotein (HDL), insulin resistance, glucose intolerance, diabetes mellitus, hyper-tension, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), heart failure, atrial fibrillation, musculoskeletal disorders, and sleep apnea.
48. A method of reducing obesity in an obese subject or reducing body weight in an overweight subject, said method comprising: administering to the subject 0.005-0.5 mg/kg Rabi encapsulated lipid nanoparticles. A pharmaceutical composition comprising:
Rabi encapsulated lipid nanoparticles and another anti-obesity agent. A kit comprising:
Rabi encapsulated lipid nanoparticles;
Optionally another anti-obesity agent; and instructions for performing the method defined in claim 48.
EP23889277.2A 2022-11-09 2023-11-08 Compositions and methods for treating obesity Pending EP4615967A1 (en)

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