Fusion protein therapies for targeting nutrient sensors upstream of mtorc1 for treatment of cancer and metabolic disease

Abstract

The present disclosure provides methods for treating cancer or metabolic disease (e.g., diabetes, obesity, and cardiovascular disease) using one or more FABP5 fusion proteins that target nutrient sensors upstream of mTORCl.

Description

Atty. Dkt. No.: 093873-1504FUSION PROTEIN THERAPIES FOR TARGETING NUTRIENT SENSORS UPSTREAM OF MTORC1 FOR TREATMENT OF CANCER AND METABOLIC DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 733,948, filed December 13, 2024, and PCT / US2025 / 050732, filed October 13, 2025, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates to methods for treating cancer or metabolic disease (e.g., diabetes, obesity, and cardiovascular disease) using one or more FABP5 fusion proteins that target nutrient sensors upstream of mTORCl. In some embodiments, the FABP5 fusion proteins of the present technology are useful for abrogating the onset of high- fat diet (HFD) induced obesity and inflammageing, and / or mitigating the onset of unhealthy aging and associated co-morbidities.GOVERNMENT SUPPORT CLAUSE

[0003] This invention was made with government support under GM051405, and CA046595 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Cancer cells must co-ordinate their growth and proliferation with nutrient availability and metabolic demand. The mammalian target of rapamycin (mTOR) pathway represents the major integrator of cellular nutrient status, and is dysregulated in up to 50% of breast cancers. Understanding how various nutrient inputs influence the signaling dynamics of this pathway could therefore be a powerful strategy for developing therapies that uncouple metabolic dependencies from disease pathogenesis.-1-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0006] There is an urgent need for therapeutic agents that circumvent many of the clinical limitations of current mTOR inhibitors which are often hindered by small therapeutic windows.SUMMARY OF THE PRESENT TECHNOLOGY

[0007] Disclosed herein is the lipid transporter fatty acid binding protein 5 (FABP5), a biomarker that dictates cellular responses to the essential dietary fatty acid co-6 linoleic acid (LA) by activating mTORCl in cancer (e.g., triple negative breast cancer).

[0008] In one aspect, the present disclosure provides a FABP5 fusion protein comprising a FABP5 TOR signaling (TOS) motif operably linked to a cell-penetrating peptide, wherein the FABP5 TOS motif comprises the amino acid sequence of FDEYM (SEQ ID NO: 4). In certain embodiments, the cell-penetrating peptide is located at the N- terminus or C-terminus of the FABP5 TOS motif. Additionally or alternatively, in some embodiments, the cell-penetrating peptide is selected from among Tat (YGRKKRRQRRR) (SEQ ID NO: 11) or a Tat peptide (RKKRRQRRR) (SEQ ID NO: 12), poly-arginine (RRRRRR (SEQ ID NO: 13); RRRRRRRR (SEQ ID NO: 14); RRRRRRRRR (SEQ ID NO: 15)), antennapedia (Antp) or Antp peptide (RQIKIWFQNRRMKW) (SEQ ID NO: 16), penetratin (RQIKIWFQNRRMKWKK) (SEQ ID NO: 17), SAP (VRLPPPVRLPPPVRLPPP) (SEQ ID NO: 18), PTD-5 (RRQRRTSKLMKR) (SEQ ID NO: 19), K-FGF (SN50 peptide) (AAVALLPAVLLALLAP) (SEQ ID NO: 20), HIV-1 Rev (TRQARRNRRRRWRERQR) (SEQ ID NO: 21), FHV (RRRRNRTRRNRRRVR) (SEQ ID NO: 22), HTLV-II (TRRQRTRRATTNR) (SEQ ID NO: 23), NLS (KRPAAIKKAGQAKKKK) (SEQ ID NO: 24), transportan (GWTLNSAGYLLGKINLKALAALAKKIL) (SEQ ID NO: 25), and pVEC (LLIILRRRIRKQAHAHSK) (SEQ ID NO: 26).

[0009] Additionally or alternatively, in certain embodiments, the FABP5 TOS motif is linked to the cell-penetrating peptide either directly or indirectly via a linker. The linker may be a flexible peptide linker or a rigid peptide linker. Examples of peptide linker sequences include, but are not limited to, (GGGGS)n (SEQ ID NO: 27), (G)n (SEQ ID NO: 28), (GSSSS)n (SEQ ID NO: 29), (GSSS)n (SEQ ID NO: 30), (AAY)n (SEQ ID NO: 31), (EAAAK)n (SEQ ID NO: 32), GPGPG (SEQ ID NO: 33), HEYGAEALERAG (SEQ ID -2-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504NO: 34), (KK)n (SEQ ID NO: 35), (RVRR)n (SEQ ID NO: 36), and (XP)n (SEQ ID NO: 37), where X denotes any amino acid, wherein n ranges from 1-15.

[0010] In any of the preceding embodiments, the fusion protein comprises the amino acid sequence of RQIKIWFQNRRMKWKK-FDEYMKE (SEQ ID NO:1), RQIKIWFQNRRMKWKK-QQLEGRWRLVDSKGFDEYMKELGVGIA (SEQ ID NO: 2), or RQIKIWFQNRRMKWKK-DSKGFDEYM (SEQ ID NO: 3).

[0011] Additionally or alternatively, in some embodiments, the fusion protein further comprises a tag selected from among a histidine tag, a glutathione-S-transferase tag, a maltose binding protein tag, a Streptavidin tag, a hemagglutinin tag, a FLAG tag, a MYC tag, or a biotin tag. In some embodiments, the tag is located at the N- or C-terminus of the cell-penetrating peptide. Additionally or alternatively, in certain embodiments, the tag is located at the N- or C-terminus of the FABP5 TOS motif. In any of the preceding embodiments, the tag is located at the N- or C-terminus of the fusion protein.

[0012] In any of the preceding embodiments, the fusion protein further comprises one or more covalent modifications selected from among capping, or PEGylation. In some embodiments, the fusion protein is formulated with micelles, liposomes or fatty acid derivates. Additionally or alternatively, in some embodiments, the FABP5 fusion protein may be stabilized through well established processes such as cyclization or stapling, and / or the use of D-amino acids.

[0013] In one aspect, the present disclosure provides a nucleic acid molecule encoding any and all embodiments of the fusion protein described herein. In another aspect, the present disclosure provides a vector comprising any and all embodiments of the nucleic acid molecule described herein. Vectors for delivery of nucleic acids encoding the fusion proteins of the present disclosure can be any that are known in the art. Adenoviral vectors and adeno-associated vectors are well known and widely used. Non-viral vectors can also be used, including nanoparticles, liposomes, and micelles. Retroviral vectors can also be used.

[0014] In one aspect, the present disclosure provides a host cell comprising any and all embodiments of the nucleic acid molecule disclosed herein or the vector described herein.-3-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0015] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein. In some embodiments, the cancer is prostate cancer or breast cancer. In certain embodiments, the breast cancer is triple negative breast cancer (TNBC).

[0016] In another aspect, the present disclosure provides a method for treating metabolic disease in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein. Examples of metabolic disease include but are not limited to diabetes, obesity, or cardiovascular disease.

[0017] In yet another aspect, the present disclosure provides a method for preventing the onset of high-fat diet (HFD) induced obesity or inflammageing in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein.

[0018] In one aspect, the present disclosure provides a method for mitigating the onset of premature aging in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein. In another aspect, the present disclosure provides a method for mitigating the onset of premature aging in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein, wherein mRNA or polypeptide expression and / or activity levels of one or more of FABP5, phosphorylated p70S6K (Ribosomal Protein S6 Kinase), and phosphorylated pS6 in a biological sample obtained from the subject are elevated compared to that observed in a control sample obtained from a healthy subject or a predetermined threshold.-4-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0019] Additionally or alternatively, in some embodiments, mRNA expression levels are detected via real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase- PCR (RT-PCR), Northern blotting, microarray, dot or slot blots, in situ hybridization, or fluorescent in situ hybridization (FISH). Additionally or alternatively, in certain embodiments, polypeptide expression levels are detected via Western blotting, enzyme- linked immunosorbent assays (ELISA), dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry. In any and all embodiments of the methods disclosed herein, the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.

[0020] In any of the preceding embodiments, the methods of the present technology further comprise sequentially, separately, or simultaneously administering to the subject an additional therapeutic agent. Examples of additional therapeutic agents comprises one or more of chemotherapeutic drugs, anti-tumor antibodies, anti-receptor antibodies, radiation therapy, radiolabeled drugs, and surgery. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is human. In any of the preceding embodiments, the fusion protein is administered orally, intranasally, intrathecally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, or topically.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1A shows genes overexpressed in triple negative (TN) breast cancer samples from the METABRIC study. FIG. IB shows elevation of FABP5 protein levels in TNBC cell lines. FIG. 1C demonstrates that siRNA-mediated knockdown of FABP5 in HCC1806 cells abrogates the activation of mTORCl signaling following stimulation with co-6 LA. FIG. ID demonstrates that FABP5 overexpression in MCF7 cells promotes the activation of mTORCl signaling following stimulation with co-6 LA. FIG. IE demonstrates that knockdown of FABP5 suppresses the proliferative effects of co-6 LA on HCC1806 cells. FIG. IF demonstrates that FABP5 overexpression induces proliferation in MCF7 cells.

[0022] FIGs. 2A-2D demonstrate that FABP5 directly binds Raptor through an N- terminal ‘FDEYM’ (SEQ ID NO: 4) TOS-motif to enhance mTORCl activation specifically-5-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 in response to co-6 PUFAs. FIG. 2E demonstrates that mutating the highly conserved phenylalanine residue (‘F19A’) in the N-terminal ‘FDEYM’ (SEQ ID NO: 4) TOS-motif prevents FABP5 interaction with mTORCl. FIG. 2F demonstrates that mutating the highly conserved phenylalanine residue (‘F19A’) in the N-terminal ‘FDEYM’ (SEQ ID NO: 4) TOS-motif prevents mTORCl signaling activation. FIG. 2G demonstrates that FABP5- F19A modestly but significantly reduces proliferation under full serum growth conditions. FIG. 2H demonstrates that FABP5-F19A uncouples the pro-proliferative effects of co-6 LA.

[0023] FIG. 3A demonstrates that co-6 LA promoted the interaction between Raptor and FABP5, but not FABP4. FIG. 3B demonstrates that siRNA-mediated knockdown of FABP5 reduced the proportion of Raptor interacting with mTOR in response to co-6 LA. FIG. 3C demonstrates that siRNA-mediated knockdown of FABP5 completely and partially prevented the co-6 LA and amino acid induced relocalization of mTOR to lysosomes, respectively. FIG. 3D demonstrates that treatment with co-3 ALA significantly reduced FABP5 protein levels over a cycloheximide time-course.

[0024] FIGs. 4A-4B demonstrate that FABP5 levels are elevated in TNBC derived xenograft (PDX) tumors and in the serum of newly diagnosed TNBC patients compared to receptor positive patients.

[0025] FIGs. 5A-5B show the identification of four specific epitopes on FABP5 that encompass all or a portion of the amino acids found in the ‘FDEYM’ (SEQ ID NO: 4) TOS motif. These epitopes are (with amino acids forming part of the FDEYM (SEQ ID NO: 4) TOS motif annotated with a black box): “DEYMKE” (SEQ ID NO: 5; amino acid residues 20-25 on FABP5); “QQLEGRWRLVDSKGFDEYMKELGVGIA” (SEQ ID NO: 6; amino acid residues 5-31 on FABP5); “DSKGFDE” (SEQ ID NO: 7; amino acid residues 15-21 on FABP5); and “VDSKGFD” (SEQ ID NO: 8; amino acid residues 14-20 on FABP5).

[0026] FIG. 6 shows High-performance liquid chromatography (HPLC) results validating purity of the FABP5 fusion proteins of the present technology (SEQ ID NOs: 1- 3).

[0027] FIGs. 7A-7D shows the effects of the FABP5 fusion proteins of the present technology (SEQ ID NOs: 1-3) on the proliferation of TNBC cancer cells in the presence of-6-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 increasing peptide concentrations (from 50nM to 5000nM) under fatty acid free conditions (FAF) or supplemented with 30pM co-6 linoleic acid (FAF + LA) for 72 hours.

[0028] FIGs. 8A-8B shows the effects of the FABP5 fusion proteins of the present technology (SEQ ID NOs: 1-3) on the 3D proliferation and viability in HCC1806 (triple negative, high FABP5 protein levels) and MCF7 (estrogen receptor positive, undetectable FABP5 protein) cell lines under fatty acid free conditions supplemented with co-6 linoleic acid (FAF + LA).

[0029] FIG. 9 shows a proposed model of how a peptide-mimetic composed of N- terminal residues of FABP5 (including the FDEYM TOS motif) would compete for binding to Raptor and abrogate formation of functional of mTORCl.

[0030] FIGs. 10A-10B: Treatment of male and female NSG mice (11-15 week old) with 5mg / kg control peptide, Peptide 2 or Peptide 3 for two weeks (6 injections total, including vehicle / water control) in conjunction with an co-6 linoleic acid enriched diet. Peptides 2 and 3 significantly reduce mTORCl signaling activity in the pancreas (FIG. 10A) and liver (FIG. 10B) of male and female mice as indicated by lower pS6 S240 / 244 phosphorylation levels compared to control peptide and vehicle treatments. N=3 male and n = 3 female mice per group.

[0031] FIGs. 11A-11D: Animals in FIG. 10 are weight stable throughout the study (FIG. 11A) and show no differences in serum C-reactive protein levels (FIG. 11B). Peptides 2 and 3 significantly reduce serum concentrations of pro-inflammatory TNF-a (FIG. 11C) and IL-6 (FIG. 11D). N=3 male and n = 3 female mice per group (6 animals total)

[0032] FIG. 12A shows tumor growth rate of HCC1806 TNBC xenografts in female NSG mice fed an co-6 LA enriched diet and treated for 3 weeks with 5mg / kg control peptide, Peptide 2 or Peptide 3 (9 injections total). FIG. 12B shows that animals are weight stable throughout the study, n = 8 mice per group.

[0033] FIG. 13 shows rotarod and grip-strength test in tumor free, 21 month-old C57B1 / 6 mice, treated with 5mg / kg of control, peptide2 or peptide 3, or vehicle control-7-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504(water) for a total of 10 injections over 3 weeks. n=2, 3, 4, 5 mice for water, peptide control, peptide 2 and peptide 3 groups, respectively.DETAILED DESCRIPTION

[0034] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0035] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N. Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. ( \ 999 Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0036] The present disclosure provides methods for preventing fatty acid sensors (e.g., FABP5) from directly activating mTORCl signaling in response to co-6 PUFA availability. As described in the Examples herein, introducing specific mutations in the FDEYM (SEQ-8-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504ID NO: 4) TOS motif of FABP5 abrogate its interaction and activation of mTORCl. The FABP5 fusion peptides disclosed herein encompass all or part of the amino acid sequence corresponding to the region of FABP5 that facilitates its interaction with mTOR. Together, these strategies provide a therapeutic approach to inhibit chronic mTOR activation in the context of high-fat diet and excessive co-6 PUFA intake and in doing so, minimize the health burden associated with selected lifestyles. Moreover, this approach can be used as method to treat specific cancers (i.e. breast cancer and triple negative in particular) that are dependent on this FABP5-mTORCl signaling axis. The FABP5 fusion peptides disclosed herein inhibit mTOR by targeting its upstream nutrient sensors, rather than the kinase itself. Without wishing to be bound by theory, it is expected that the FABP5 fusion peptides disclosed herein will circumvent many of the clinical limitations of current mTOR inhibitors which are often hindered by small therapeutic windows.

[0037] The FABP5 fusion proteins of the present technology are structurally and functionally distinct for the following reasons:

[0038] 1) current mTOR targeted therapies are divided into three main ‘generations’ - first (i.e. rapamycin and rapalog derivates), second (i.e. ATP competitive inhibitors such as Torin) and third (Rapalink which combines first and second inhibitors). The common feature of these inhibitors is that they directly bind and inhibit mTORCl / 2 kinase activity. However, translating these three main ‘generations’ inhibitors to patients however has been hindered by small therapeutic windows and side-effects associated with chronic mTOR inhibition including hyperglycemia and dyslipidemia. The FABP5 fusion proteins of the present technology are ‘fourth generation’ inhibitors that exploit nutrient sensors that converge on mTORCl activation. Accordingly, the FABP5 fusion proteins inhibit mTOR in defined contexts which mitigates many of the side effects associated with sustained mTOR suppression.

[0039] 2) While there are existing inhibitors for FABP5, their mechanism of action involves directly competing with fatty acids for binding to FABP5. The FABP5 fusion proteins of the present technology target the interaction between FABP5 and mTORCl, not fatty acid binding.-9-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0040] 3) In contrast to estrogen receptor and HER2 positive breast cancers, there are currently no targeted or personalized therapies for triple negative tumors. The FABP5 fusion proteins of the present technology highlights a therapeutic strategy for leveraging the unique metabolic dependency of triple negative tumors for FABP5.Definitions

[0041] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0042] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0043] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, intrathecally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, or topically. Administration includes self-administration and the administration by another.

[0044] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a-10-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0045] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0046] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0047] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0048] As used herein, the term “inflammageing” refers to the persistent chronic inflammation that emerges during ageing and has been coupled with age-related conditions, such as cardiovascular diseases (CVDs), metabolic disorders, non-alcoholic fatty liver disease, chronic kidney disease, and cognitive decline.-11-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0049] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20thedition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0050] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, z.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.

[0051] As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample. As used herein, prevention includes preventing or delaying the initiation of symptoms of a disease or condition described herein and / or preventing a recurrence of one or more signs or symptoms of a disease or condition described herein.

[0052] As used herein, a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and / or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term "sample" may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF),-12-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art. A blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.

[0053] As used herein, the terms “subject”, “patient”, or “individual” are used interchangeably, and can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient or individual is a human.

[0054] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0055] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.Compositions Related to the FABP5 Fusion Proteins of the Present Technology

[0056] In one aspect, the present disclosure provides a FABP5 fusion protein comprising a FABP5 TOR signaling (TOS) motif operably linked to a cell-penetrating peptide, wherein the FABP5 TOS motif comprises the amino acid sequence of FDEYM (SEQ ID NO: 4). In certain embodiments, the cell-penetrating peptide is located at the N- terminus or C-terminus of the FABP5 TOS motif. Additionally or alternatively, in some embodiments, the cell-penetrating peptide is selected from among Tat (YGRKKRRQRRR) (SEQ ID NO: 11) or a Tat peptide (RKKRRQRRR) (SEQ ID NO: 12), poly-arginine (RRRRRR (SEQ ID NO: 13); RRRRRRRR (SEQ ID NO: 14); RRRRRRRRR (SEQ ID-13-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504NO: 15)), antennapedia (Antp) or Antp peptide (RQIKIWFQNRRMKW) (SEQ ID NO: 16), penetratin (RQIKIWFQNRRMKWKK) (SEQ ID NO: 17), SAP (VRLPPPVRLPPPVRLPPP) (SEQ ID NO: 18), PTD-5 (RRQRRTSKLMKR) (SEQ ID NO: 19), K-FGF (SN50 peptide) (AAVALLPAVLLALLAP) (SEQ ID NO: 20), HIV-1 Rev (TRQARRNRRRRWRERQR) (SEQ ID NO: 21), FHV (RRRRNRTRRNRRRVR) (SEQ ID NO: 22), HTLV-II (TRRQRTRRATTNR) (SEQ ID NO: 23), NLS (KRPAAIKKAGQAKKKK) (SEQ ID NO: 24), transportan (GWTLNSAGYLLGKINLKALAALAKKIL) (SEQ ID NO: 25), and pVEC (LLIILRRRIRKQAHAHSK) (SEQ ID NO: 26).

[0057] Additionally or alternatively, in certain embodiments, the FABP5 TOS motif is linked to the cell-penetrating peptide either directly or indirectly via a linker. The linker may be a flexible peptide linker or a rigid peptide linker. Examples of peptide linker sequences include, but are not limited to, (GGGGS)n (SEQ ID NO: 27), (G)n (SEQ ID NO: 28), (GSSSS)n (SEQ ID NO: 29), (GSSS)n (SEQ ID NO: 30), (AAY)n (SEQ ID NO: 31), (EAAAK)n (SEQ ID NO: 32), GPGPG (SEQ ID NO: 33), HEYGAEALERAG (SEQ ID NO: 34), (KK)n (SEQ ID NO: 35), (RVRR)n (SEQ ID NO: 36), and (XP)n (SEQ ID NO: 37), where X denotes any amino acid, wherein n ranges from 1-15.

[0058] In any of the preceding embodiments, the fusion protein comprises the amino acid sequence of RQIKIWFQNRRMKWKK-FDEYMKE (SEQ ID NO: 1), RQIKIWFQNRRMKWKK-QQLEGRWRLVDSKGFDEYMKELGVGIA (SEQ ID NO: 2), or RQIKIWFQNRRMKWKK-DSKGFDEYM (SEQ ID NO: 3).

[0059] Slight deviations from the precise sequence may be used to optimize activity, such as by substitution of one, two or three residues to make conservative changes or by substitution with alanine. Conservative changes substitute similar residues for each other, such as an uncharged polar for an uncharged polar, or a non-polar for a non-polar, or an acidic for an acidic residue. Thus, G or S residues can be substituted with G, S, T, C, Y, N, and Q. L residues can be substituted with A,V, I, P, F, W, while M, A, V, and P residues can be substituted with A, V, L, I, P, F, W, and M residues. Y or N residues can be substituted with G, S. T. C. Y., N, and Q residues. E residues can be substituted with a D-14-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 residue. K residues can be substituted with an R or H residue. Any residue can be substituted with an alanine residue.

[0060] Additionally or alternatively, in some embodiments, the fusion protein further comprises a tag selected from among a histidine tag, a glutathione-S-transferase tag, a maltose binding protein tag, a Streptavidin tag, a hemagglutinin tag, a FLAG tag, a MYC tag, or a biotin tag. In some embodiments, the tag is located at the N- or C-terminus of the cell-penetrating peptide. Additionally or alternatively, in certain embodiments, the tag is located at the N- or C-terminus of the FABP5 TOS motif. In any of the preceding embodiments, the tag is located at the N- or C-terminus of the fusion protein.

[0061] Fusion peptides can be formulated or modified as defined in the literature. This may involve covalent modifications, such as capping, or PEGylation, or combination with micelles, liposomes or fatty acid derivates. Such modifications and formulations may increase stability in the body, therefore permitting higher percentages of the input dosage to reach the target cancer. In some embodiments, the FABP5 fusion protein may be stabilized through well established processes such as cyclization or stapling, and / or the use of D- amino acids.

[0062] In one aspect, the present disclosure provides a nucleic acid molecule encoding any and all embodiments of the fusion protein described herein. In another aspect, the present disclosure provides a vector comprising any and all embodiments of the nucleic acid molecule described herein. Vectors for delivery of nucleic acids encoding the fusion proteins of the present disclosure can be any that are known in the art. Adenoviral vectors and adeno-associated vectors are well known and widely used. Non-viral vectors can also be used, including nanoparticles, liposomes, and micelles. Retroviral vectors can also be used. In one aspect, the present disclosure provides a host cell comprising any and all embodiments of the nucleic acid molecule disclosed herein or the vector described herein.Formulations Including FABP5 Fusion Proteins of the Present Technology

[0063] The pharmaceutical compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders,-15-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. In certain embodiments, the compositions disclosed herein are formulated for administration to a mammal, such as a human.

[0064] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0065] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions formulated for-16-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.

[0066] In order to prolong the effect of a compound of the present disclosure, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0067] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents such as phosphates or carbonates.-17-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0068] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0069] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.Nucleic Acid Amplification and / or Detection

[0070] Polynucleotides associated with responsiveness to intraoperative opioid analgesics can be detected by the use of nucleic acid amplification techniques that are well known in the art. The starting material may be genomic DNA, cDNA, RNA or mRNA. Nucleic acid amplification can be linear or exponential. Specific variants or mutations may be detected by the use of amplification methods with the aid of oligonucleotide primers or probes designed to interact with or hybridize to a particular target sequence in a specific manner, thus amplifying only the target variant.

[0071] Non-limiting examples of nucleic acid amplification techniques include polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), digital PCR (dPCR), reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, ligase chain reaction (see Abravaya, K. et al., Nucleic Acids Res. (1995), 23:675-682), branched DNA-18-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 signal amplification (see Urdea, M. S. et al., AIDS (1993), 7(suppl 2):S11- S14), amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA) (see Kievits, T. et al., J Virological Methods (1991), 35:273-286), Invader Technology, next-generation sequencing technology or other sequence replication assays or signal amplification assays.

[0072] Primers'. Oligonucleotide primers for use in amplification methods can be designed according to general guidance well known in the art as described herein, as well as with specific requirements as described herein for each step of the particular methods described. In some embodiments, oligonucleotide primers for cDNA synthesis and PCR are 10 to 100 nucleotides in length, preferably between about 15 and about 60 nucleotides in length, more preferably 25 and about 50 nucleotides in length, and most preferably between about 25 and about 40 nucleotides in length.

[0073] Tm of a polynucleotide affects its hybridization to another polynucleotide (e.g., the annealing of an oligonucleotide primer to a template polynucleotide). In certain embodiments of the disclosed methods, the oligonucleotide primer used in various steps selectively hybridizes to a target template or polynucleotides derived from the target template (i.e., first and second strand cDNAs and amplified products). Typically, selective hybridization occurs when two polynucleotide sequences are substantially complementary (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary). See Kanehisa, M., Polynucleotides Res. (1984), 12:203, incorporated herein by reference. As a result, it is expected that a certain degree of mismatch at the priming site is tolerated. Such mismatch may be small, such as a mono-, di- or tri -nucleotide. In certain embodiments, 100% complementarity exists.

[0074] Probes'. Probes are capable of hybridizing to at least a portion of the nucleic acid of interest or a reference nucleic acid (i.e., wild-type sequence). Probes may be an oligonucleotide, artificial chromosome, fragmented artificial chromosome, genomic nucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of-19-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 cyclic heterocycles, or conjugates of nucleic acid. Probes may be used for detecting and / or capturing / purifying a nucleic acid of interest.

[0075] Typically, probes can be about 10 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 75 nucleotides, or about 100 nucleotides long. However, longer probes are possible. Longer probes can be about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 750 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 5,000 nucleotides, about 7,500 nucleotides, or about 10,000 nucleotides long.

[0076] Probes may also include a detectable label or a plurality of detectable labels. The detectable label associated with the probe can generate a detectable signal directly. Additionally, the detectable label associated with the probe can be detected indirectly using a reagent, wherein the reagent includes a detectable label, and binds to the label associated with the probe.

[0077] In some embodiments, detectably labeled probes can be used in hybridization assays including, but not limited to Northern blots, Southern blots, microarray, dot or slot blots, and in situ hybridization assays such as fluorescent in situ hybridization (FISH) to detect a target nucleic acid sequence within a biological sample. Certain embodiments may employ hybridization methods for measuring expression of a polynucleotide gene product, such as mRNA. Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis el al. Molecular Cloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif, 1987); Young and Davis, PNAS. 80: 1194 (1983).

[0078] Detectably labeled probes can also be used to monitor the amplification of a target nucleic acid sequence. In some embodiments, detectably labeled probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a-20-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 function of time. Examples of such probes include, but are not limited to, the 5'- exonuclease assay (TAQMAN® probes described herein (see also U.S. Pat. No. 5,538,848) various stem-loop molecular beacons (see for example, U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303- 308), stemless or linear beacons (see, e.g., WO 99 / 21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, for example, Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (see, for example, U.S. Pat. No. 6,150,097), Sunrise® / Amplifluor™ probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion probes (Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, selfassembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901 ; Mhlanga et al., 2001, Methods 25:463-471 ; Whitcomb e et al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281 :26-35; Wolffs et al., 2001, Biotechniques 766:769-771 ; Tsourkas et al., 2002, Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem. Res. Toxicol. 15: 118- 126; and Yu et al., 2001, J. Am. Chem. Soc 14: 11155-11161.

[0079] In some embodiments, the detectable label is a fluorophore. Suitable fluorescent moieties include but are not limited to the following fluorophores working individually or in combination: 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; Alexa Fluors: Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes); 5-(2- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-l- naphthyl)mal eimide; anthranilamide; Black Hole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes: BODIPY® R-6G, BOPIPY®530 / 550, BODIPY® FL; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4--21-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5" -dibrom opyrogallol- sulfonephthalein (Bromopyrogallol Red); 7- diethylamino-3-(4'-isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansyl chloride); 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4'- isothiocyanate (DABITC); Eclipse™ (Epoch Biosciences Inc.); eosin and derivatives: eosin, eosin isothiocyanate; erythrosin and derivatives: erythrosin B, erythrosin isothiocyanate; ethidium; fluorescein and derivatives:5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)amino fluorescein (DTAF), 2', 7'- dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), tetrachlorofluorescem (TET); fiuorescamine; IR144; IR1446; lanthamide phosphors; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B -phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY® 35 (Molecular Probes); Reactive Red 4 (Cibacron®Brilliant Red 3B-A); rhodamine and derivatives: 6- carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, riboflavin, rosolic acid, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); terbium chelate derivatives; N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); and VIC®. Detector probes can also comprise sulfonate derivatives of fluorescenin dyes with S03 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available for example from Amersham).

[0080] Detectably labeled probes can also include quenchers, including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate / carboxylate Quenchers (Epoch).-22-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0081] Detectably labeled probes can also include two probes, wherein for example a fluorophore is on one probe, and a quencher is on the other probe, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on the target alters the signal signature via a change in fluorescence.

[0082] In some embodiments, interchelating labels such as ethidium bromide, SYBR® Green I (Molecular Probes), and PicoGreen® (Molecular Probes) are used, thereby allowing visualization in real-time, or at the end point, of an amplification product in the absence of a detector probe. In some embodiments, real-time visualization may involve the use of both an intercalating detector probe and a sequence-based detector probe. In some embodiments, the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction, and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction.

[0083] In some embodiments, the amount of probe that gives a fluorescent signal in response to an excited light typically relates to the amount of nucleic acid produced in the amplification reaction. Thus, in some embodiments, the amount of fluorescent signal is related to the amount of product created in the amplification reaction. In such embodiments, one can therefore measure the amount of amplification product by measuring the intensity of the fluorescent signal from the fluorescent indicator.

[0084] Primers or probes may be designed to selectively hybridize to any portion of a nucleic acid sequence encoding a FABP5 polypeptide.

[0085] In some embodiments, detection can occur through any of a variety of mobility dependent analytical techniques based on the differential rates of migration between different nucleic acid sequences. Exemplary mobility-dependent analysis techniques include electrophoresis, chromatography, mass spectroscopy, sedimentation, gradient centrifugation, field-flow fractionation, multi-stage extraction techniques, and the like. In some embodiments, mobility probes can be hybridized to amplification products, and the identity of the target nucleic acid sequence determined via a mobility dependent analysis technique of the eluted mobility probes, as described in Published PCT Applications WO04 / 46344 and WOOl / 92579. In some embodiments, detection can be achieved by various microarrays and related software such as the Applied Biosystems Array System-23-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, Illumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al, Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9: 14045, including supplements, 2003).

[0086] It is also understood that detection can comprise reporter groups that are incorporated into the reaction products, either as part of labeled primers or due to the incorporation of labeled dNTPs during an amplification, or attached to reaction products, for example but not limited to, via hybridization tag complements comprising reporter groups or via linker arms that are integral or attached to reaction products. In some embodiments, unlabeled reaction products may be detected using mass spectrometry.Methods of Treatment of the Present Technology

[0087] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein. In some embodiments, the cancer is prostate cancer or breast cancer. In certain embodiments, the breast cancer is triple negative breast cancer (TNBC).

[0088] In another aspect, the present disclosure provides a method for treating metabolic disease in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein. Examples of metabolic disease include but are not limited to diabetes, obesity, or cardiovascular disease.

[0089] In yet another aspect, the present disclosure provides a method for preventing the onset of high-fat diet (HFD) induced obesity or inflammageing in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein.

[0090] In one aspect, the present disclosure provides a method for mitigating the onset of premature aging in a subject in need thereof comprising administering to the subject an-24-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein. In another aspect, the present disclosure provides a method for mitigating the onset of premature aging in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the fusion protein described herein, any and all embodiments of the nucleic acid molecule described herein, or any and all embodiments of the vector described herein, wherein mRNA or polypeptide expression and / or activity levels of one or more of FABP5, phosphorylated p70S6K (Ribosomal Protein S6 Kinase), and phosphorylated pS6 in a biological sample obtained from the subject are elevated compared to that observed in a control sample obtained from a healthy subject or a predetermined threshold.

[0091] Additionally or alternatively, in some embodiments, mRNA expression levels are detected via real-time quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase- PCR (RT-PCR), Northern blotting, microarray, dot or slot blots, in situ hybridization, or fluorescent in situ hybridization (FISH). Additionally or alternatively, in certain embodiments, polypeptide expression levels are detected via Western blotting, enzyme- linked immunosorbent assays (ELISA), dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry. In any and all embodiments of the methods disclosed herein, the biological sample obtained from the cancer patient comprises biopsied tumor tissue, whole blood, plasma, or serum.

[0092] In any of the preceding embodiments, the methods of the present technology further comprise sequentially, separately, or simultaneously administering to the subject an additional therapeutic agent. Examples of additional therapeutic agents comprises one or more of chemotherapeutic drugs, anti-tumor antibodies, anti-receptor antibodies, radiation therapy, radiolabeled drugs, and surgery. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is human. In any of the preceding embodiments, the fusion protein is administered orally, intranasally, intrathecally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, or topically.

[0093] For therapeutic applications, a composition comprising a FABP5 fusion protein disclosed herein, is administered to the subject. In some embodiments, the FABP5 fusion-25-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 protein is administered one, two, three, four, or five times per day. In some embodiments, the FABP5 fusion protein is administered more than five times per day. Additionally or alternatively, in some embodiments, the FABP5 fusion protein is administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the FABP5 fusion protein is administered weekly, bi-weekly, triweekly, or monthly. In some embodiments, the FABP5 fusion protein is administered for a period of one, two, three, four, or five weeks. In some embodiments, the FABP5 fusion protein is administered for six weeks or more. In some embodiments, the FABP5 fusion protein is administered for twelve weeks or more. In some embodiments, the FABP5 fusion protein is administered for a period of less than one year. In some embodiments, the FABP5 fusion protein is administered for a period of more than one year. In some embodiments, the FABP5 fusion protein is administered throughout the subject’s life.

[0094] In some embodiments of the methods of the present technology, the FABP5 fusion protein is administered daily for 1 week or more. In some embodiments of the methods of the present technology, the FABP5 fusion protein is administered daily for 2 weeks or more. In some embodiments of the methods of the present technology, the FABP5 fusion protein is administered daily for 3 weeks or more. In some embodiments of the methods of the present technology, the FABP5 fusion protein is administered daily for 4 weeks or more. In some embodiments of the methods of the present technology, the FABP5 fusion protein is administered daily for 6 weeks or more. In some embodiments of the methods of the present technology, the FABP5 fusion protein is administered daily for 12 weeks or more. In some embodiments, the FABP5 fusion protein is administered daily throughout the subject’s life.Determination of the Biological Effect of FABP5 Fusion Proteins of the Present Technology

[0095] In various embodiments, suitable in vitro or in vivo assays are performed to determine the effect of a specific FABP5 fusion protein and whether its administration is indicated for treatment. In various embodiments, in vitro assays can be performed with representative animal models, to determine if a given FABP5 fusion protein exerts the desired effect on reducing or eliminating signs and / or symptoms of cancer (e.g., TNBC) or metabolic disease (e.g., diabetes, obesity, and cardiovascular disease).-26-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0096] Compounds for use in therapy can be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art can be used prior to administration to human subjects. In some embodiments, in vitro or in vivo testing is directed to the biological function of one or more FABP5 fusion proteins.

[0097] Animal models of cancer (e.g., TNBC) or metabolic disease (e.g., diabetes, obesity, and cardiovascular disease) may be generated using techniques known in the art (see, e.g, Examples described herein). Such models may be used to demonstrate the biological effect of FABP5 fusion proteins in the prevention and treatment of conditions disclosed herein, and for determining what comprises a therapeutically effective amount of the one or more FABP5 fusion proteins disclosed herein in a given context.Modes of Administration and Effective Dosages

[0098] Any method known to those in the art for contacting a cell, organ or tissue with one or more FABP5 fusion proteins disclosed herein may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more FABP5 fusion proteins to a mammal, suitably a human. When used in vivo for therapy, the one or more FABP5 fusion proteins described herein are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the particular FABP5 fusion protein used, e.g., its therapeutic index, and the subject’s history.

[0099] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of one or more FABP5 fusion proteins useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The FABP5 fusion protein may be administered systemically or locally.

[0100] The one or more FABP5 fusion proteins described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment of cancer (e.g., TNBC) or metabolic disease (e.g, diabetes, obesity, and cardiovascular disease), preventing the onset of high-fat diet (HFD) induced obesity or-27-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 inflammageing, or mitigating the onset of premature aging. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0101] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).

[0102] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.-28-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0103] The pharmaceutical compositions having one or more FABP5 fusion proteins disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0104] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0105] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g, gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as-29-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0106] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0107] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

[0108] A therapeutic agent can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity. One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al. , Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann.Pharmacother., 34(7-8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.-30-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0109] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic / glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother ., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[0110] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99 / 15154 (Tracy, et al. U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO 96 / 40073 (Zale, et aL), and PCT publication WO 00 / 38651 (Shah, et aL). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96 / 40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[0111] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0112] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g, Chonn-31-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends BiotechnoL, 13(12):527-37 (1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.

[0113] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0114] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0115] Typically, an effective amount of the one or more FABP5 fusion proteins disclosed herein sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram-32-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 body weight per day to about 100 mg per kilogram body weight per day. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight every day, every two days or every three days or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, one or more FABP5 fusion protein concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0116] In some embodiments, a therapeutically effective amount of one or more FABP5 fusion proteins may be defined as a concentration of inhibitor at the target tissue of 10'32to 10'6molar, e.g., approximately 10'7molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg / kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).

[0117] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[0118] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.Kits

[0119] The present disclosure also provides kits for the treatment of cancer (e.g., TNBC) or metabolic disease (e.g., diabetes, obesity, and cardiovascular disease), preventing-33-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 the onset of high-fat diet (HFD) induced obesity or inflammageing, or mitigating the onset of premature aging comprising one or more FABP5 fusion proteins disclosed herein that target nutrient sensors upstream of mTORCl. Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for the treatment of cancer or metabolic disease (e.g., diabetes, obesity, and cardiovascular disease), preventing the onset of high-fat diet (HFD) induced obesity or inflammageing, or mitigating the onset of premature aging.

[0120] The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts. The kits may optionally include instructions customarily included in commercial packages of therapeutic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and / or warnings concerning the use of such therapeutic products.

[0121] The kit can also comprise, e.g., a buffering agent, a preservative or a stabilizing agent. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single-34-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. In certain embodiments, the use of the reagents can be according to the methods of the present technology.EXAMPLES

[0122] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.Example 1: Functional Convergence of FABP5 on mTORCl Signaling

[0123] According to inventors’ prior unpublished findings on nutrient sensing, a nutrient signaling axis nucleated by the lipid transporter fatty acid binding protein 5 (FABP5) and mTORCl that is activated by the essential co-6 polyunsaturated fatty acid (PUFA) linoleic acid (LA) was identified (FIG. 9). FABP5 was identified as a candidate of interest by compiling a list of 84 genes with Kyoto Encyclopedia of Genes and Genomes (KEGG) ontologies relating to essential fatty acid (FA) metabolism and transport, and their expression was compared between triple negative (TN) and receptor positive (RP) primary breast cancer samples from the METABRIC study (FIG. 1A). Amongst the most significantly upregulated genes in TNBC were fatty acid binding proteins (FABPs), a family of 10 lipid-chaperone proteins that facilitate the intracellular trafficking, compartmentalization and signaling of various FAs (FIG. 1A). FABP5 was of particular interest because of its previously reported high binding affinity for co-6 LA, and capacity to activate various transcription factors such as peroxisome proliferator activated receptors (PPARs) and NF-KB to regulate pro-survival and proliferative transcriptional programs.

[0124] After confirming that FABP5 protein levels were elevated in TNBC cell line models (FIG. IB), the functional convergence of FABP5 on mTORCl signaling was characterized. siRNA-mediated knockdown of FABP5 in HCC1806 (FIG. 1C) abrogated the activation of mTORCl signaling following stimulation with co-6 LA. In contrast, overexpression in MCF7 cells (FIG. ID) promoted the activation of mTORCl signaling when stimulated with co-6 LA. These signaling effects were also corroborated phenotypically, with knockdown of FABP5 suppressing the proliferative effects of co-6 LA-35-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 on HCC1806 cells (FIG. IE), and overexpression conversely inducing proliferation in MCF7 cells (FIG. IF).

[0125] Given that FABPs exert their biological functions in part by interacting with protein complexes and modulating their formation and activation in response to specific lipids or physiological stimuli, the direct convergence of FABP5 on mTORCl was investigated. FABP5 directly binds Raptor through an N-terminal ‘FDEYM’ (SEQ ID NO: 4) TOS-motif to enhance mTORCl activation specifically in response to co-6 PUFAs (FIGs. 2A-2D). Importantly, mutating the highly conserved phenylalanine residue (‘F19A’) in the motif prevents FABP5 interaction with mTORCl (FIG. 2E) and signaling activation (FIG. 2F). This had notable consequences phenotypically, with FABP5-F19A significantly reducing proliferation even under full growth conditions (FIG. 2G) ,and importantly uncoupling the pro-proliferative effects of co-6 LA specifically (FIG. 2H).

[0126] co-6 LA promoted the interaction between Raptor and FABP5, but not FABP4 which has been implicated in the formation of nutrient-responsive protein complexes (FIG. 3A). To evaluate the functional relevance of these interactions, their impact on two obligatory steps required for complete mTORCl activation were evaluated: 1) mTOR / Raptor complex formation, and 2) lysosomal localization. siRNA-mediated knockdown of FABP5 not only reduced the proportion of Raptor interacting with mTOR in response to co-6 LA (FIG. 3B), it also completely and partially prevented the co-6 LA and amino acid induced relocalization of mTOR to lysosomes, respectively (FIG. 3C). Treatment with co-3 ALA also significantly reduced FABP5 protein levels over a cycloheximide time-course, thus providing at least one explanation for why this PUFA could not activate mTORCl signaling (FIG. 3D). Finally, FABP5 levels were elevated in TNBC derived xenograft (PDX) tumors (FIG. 4A) and in the serum (FIG. 4B) of newly diagnosed TNBC patients compared to receptor positive patients.

[0127] These results demonstrate an obligatory and previously undefined role for FABP5 in mediating the sensing of essential co-6 PUFA availability and linking these nutrients to proliferation through mTORCl signaling.-36-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504Example 2: FABP5 Fusion Proteins of the Present Technology Specifically Abrogate the Interaction Between FABP5 and mTORCl

[0128] Disclosed herein are strategies for specifically abrogating the interaction between FABP5 and mTORCl that can uncouple dietary co-6 PUFA availability from mTORCl hyperactivation. One strategy involves mutating the highly conserved phenylalanine residue at position 19 in the ‘FDEYM’ (SEQ ID NO: 4) motif on FABP5 to an alanine (F19A mutant).

[0129] This amino acid substitution corresponds to the following DNA alterations we introduced through site-directed mutagenesis:

[0130] Wild-type FABP5:ATGGCCACAGTTCAGCAGCTGGAAGGAAGATGGCGCCTGGTGGACAGCAAAGG CTTTGATGAATACATGAAGGAGCTAGGAGTGGGAATAGCTTTGCGAAAAATGG GCGCAATGGCCAAGCCAGATTGTATCATCACTTGTGATGGTAAAAACCTCACCA TAAAAACTGAGAGCACTTTGAAAACAACACAGTTTTCTTGTACCCTGGGAGAGA AGTTTGAAGAAACCACAGCTGATGGCAGAAAAACTCAGACTGTCTGCAACTTT ACAGATGGTGCATTGGTTCAGCATCAGGAGTGGGATGGGAAGGAAAGCACAAT AACAAGAAAATTGAAAGATGGGAAATTAGTGGTGGAGTGTGTCATGAACAATG TCACCTGTACTCGGATCTATGAAAAAGTAGAA (SEQ ID NO: 9)

[0131] F19A mutant FABP5:

[0132] ATGGCCACAGTTCAGCAGCTGGAAGGAAGATGGCGCCTGGTGGACA GCAAAGGCG i TGATGAATACATGAAGGAGCTAGGAGTGGGAATAGCTTTGCGA AAAATGGGCGCAATGGCCAAGCCAGATTGTATCATCACTTGTGATGGTAAAAA CCTCACCATAAAAACTGAGAGCACTTTGAAAACAACACAGTTTTCTTGTACCCT GGGAGAGAAGTTTGAAGAAACCACAGCTGATGGCAGAAAAACTCAGACTGTCT GCAACTTTACAGATGGTGCATTGGTTCAGCATCAGGAGTGGGATGGGAAGGAA AGCACAATAACAAGAAAATTGAAAGATGGGAAATTAGTGGTGGAGTGTGTCAT GAACAATGTCACCTGTACTCGGATCTATGAAAAAGTAGAA (SEQ ID NO: 10)

[0133] This mutation in FABP5 prevents its interaction with mTORCl in response to co- 6 polyunsaturated fatty acids and abrogates their proliferative effects in triple negative breast cancer.-37-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0134] Four specific epitopes on FABP5 were identified that encompass all or a portion of the amino acids found in the ‘FDEYM’ (SEQ ID NO: 4) TOS motif (FIGs. 5A-5B). These epitopes are (with amino acids forming part of the FDEYM TOS motif highlighted in italics): “DEEWKE” (SEQ ID NO: 5; amino acid residues 20-25 on FABP5);“QQLEGRWRLVDSKG DEEWKELGVGIA” (SEQ ID NO: 6; amino acid residues 5- 31 on FABP5); “DSKG DE” (SEQ ID NO: 7; amino acid residues 15-21 on FABP5); “VDSKG D” (SEQ ID NO: 8; amino acid residues 14-20 on FABP5).

[0135] Based on the mechanistic insights for how FABP5 interacts with and activates mTORCl signaling, the third approach involves designing various peptide fusion proteins in which N-terminal epitopes of FABP5 that encompass all or part of its ‘FDEYM’ (SEQ ID NO: 4) TOS motif are fused to cell penetrating peptides that allow for transport across biological membranes and internalization by cells. The peptides are designed to compete with endogenous FABP5 for binding to Raptor, therefore abrogating the interaction between Raptor and functional FABP5, and consequently inhibiting mTORCl activation. The peptide sequences are shown below and have the general structure of A-B, where A corresponds to the penetratin cell penetrating peptide (which can be substitute for other peptides known in the literature), and B corresponds to N terminal sequences of FABP5 encompassing all or part of the ‘FDEYM’ TOS motif.

[0136] Peptide 1 : RQIKIWFQNRRMKWKK-FDEYMKE (SEQ ID NO: 1),

[0137] Peptide 2: RQIKIWFQNRRMKWKK- QQLEGRWRLVDSKGFDEYMKELGVGIA (SEQ ID NO:2),

[0138] Peptide 3 : RQIKIWFQNRRMKWKK-DSKGFDEYM (SEQ ID NO:3)

[0139] These 3 candidate peptides were successfully synthesized to 95-98% purity. High-performance liquid chromatography was performed and chromatograms validating purity are shown in FIG. 6.

[0140] To explore the cellular effects of these peptides, the highly aggressive HCC1806 triple negative breast cancer cell line were cultured in the presence of increasing peptide concentrations (from 50nM to 5000nM) under fatty acid free conditions (FAF) or supplemented with 30pM co-6 linoleic acid (FAF + LA) for 72 hours to recapitulate-38-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504 maximal activation of FABP5-mTORCl signaling axis (FIGs. 7A-7D). Notably, while proliferation of HCC1806 cells in the presence of co-6 linoleic acid was not affected by the negative control peptide (FIG. 7A), Peptides 1-3 significantly reduced proliferation in a dose dependent manner, with the most robust suppression observed for Peptide 2 (SEQ ID NO: 2) (FIGs. 7B-7D)

[0141] To further demonstrate the therapeutic potential of the three fusion peptides, three dimensional spheroid models of the HCC1806 (triple negative, high FABP5 protein levels) and MCF7 (estrogen receptor positive, undetectable FABP5 protein) cell lines were utilized to recapitulate physiological tumor growth. Peptides 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3) significantly slowed 3D proliferation and viability in HCC1806 (FIG. 8A), but not MCF7 (FIG. 8B) spheroids, thus demonstrating the selectivity of the FABP5 fusion proteins in killing aggressive triple negative breast cancers that are dependent on FABP5 to drive proliferation and survival.Example 3: In vivo Therapeutic Effects of the FABP5 Fusion Proteins of the Present Technology

[0142] The physiological effects of FABP5 fusion proteins in inhibiting tissue specific mTORCl signaling, decreasing tumor growth of highly aggressive triple negative breast cancer models, and improvement in strength-related phenotypes linked to aging were evaluated in animals.

[0143] In an in vivo tolerability study, disease-free male and female NOD scid gamma (NSG) mice were fed an co-6 LA-enriched diet in which all PUFAs are derived from safflower oil (composition is >80% co-6 LA) and corresponds to intake levels mirroring the modern Western diet (MWD), and administered six 5mg / kg peptides doses over two weeks. Strikingly, Peptides 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3) significantly reduced mTORCl signaling in the pancreas (FIG. 10A) and liver (FIG. 10B).

[0144] Moreover, not only were animals weight stable over the course of the study (FIG. 11 A) and had consistent levels of C-reactive protein (FIG. 11B)- indications of good overall tolerability- they also had significantly lower serum concentrations of the pro- inflammatory markers TNFa (FIG. 11C) and IL-6 (FIG. 11D).-39-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0145] The therapeutic potential of the FABP5 fusion proteins was assessed in xenograft models of triple negative breast cancer (TNBC). The highly aggressive HCC1806 TNBC cell line was injected orthotopically in the mammary fat pad of female NSG mice fed an co-6 LA enriched diet, and once palpable tumors formed, animals were injected with vehicle control (water), or 5mg / kg control, Peptide 2 (SEQ ID NO: 2) or Peptide 3 (SEQ ID NO: 3) three times weekly. Tumor growth rate was significantly inhibited by -50% in mice treated with Peptide 2 (SEQ ID NO: 2) or Peptide 3 (SEQ ID NO: 3) (FIG. 12A), and tumor-bearing animals were all weight stable throughout the treatment timeline with no adverse tolerability issues observed (FIG. 12B).

[0146] In addition to the cancer studies, the potential role of the FABP5 fusion proteins in improving phenotypes associated with aging-related metabolic dysfunction was explored. In these experiments, 21 -month old male C57B1 / 6 mice (equivalent to human age of 60-70 years) were injected with 5mg / kg control, Peptide 2 (SEQ ID NO: 2) or Peptide 3 (SEQ ID NO: 3) three times per week, for three weeks (10 injections total). Administration of FABP5 fusion proteins (especially Peptide 2 (SEQ ID NO: 2)) significantly improved age- associated declines in muscle strength and balance (FIG. 13).EQUIVALENTS

[0147] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.-40-4907-021 1 -4175.1Atty. Dkt. No.: 093873-1504

[0148] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0149] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0150] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.-41-4907-021 1 -4175.1

Claims

Atty. Dkt. No.: 093873-1504WHAT IS CLAIMED IS1. A fusion protein comprising a FABP5 TOR signaling (TOS) motif operably linked to a cell-penetrating peptide, wherein the FABP5 TOS motif comprises the amino acid sequence of FDEYM (SEQ ID NO: 4).

2. The fusion protein of claim 1, wherein the cell-penetrating peptide is selected from among Tat (YGRKKRRQRRR) (SEQ ID NO: 11) or a Tat peptide (RKKRRQRRR) (SEQ ID NO: 12), poly-arginine (RRRRRR (SEQ ID NO: 13); RRRRRRRR (SEQ ID NO: 14); RRRRRRRRR (SEQ ID NO: 15)), antennapedia (Antp) or Antp peptide (RQIKIWFQNRRMKW) (SEQ ID NO: 16), penetratin (RQIKIWFQNRRMKWKK) (SEQ ID NO: 17), SAP (VRLPPPVRLPPPVRLPPP) (SEQ ID NO: 18), PTD-5 (RRQRRTSKLMKR) (SEQ ID NO: 19), K-FGF (SN50 peptide) (AAVALLPAVLLALLAP) (SEQ ID NO: 20), HIV-1 Rev (TRQARRNRRRRWRERQR) (SEQ ID NO: 21), FHV (RRRRNRTRRNRRRVR) (SEQ ID NO: 22), HTLV-II (TRRQRTRRATTNR) (SEQ ID NO: 23), NLS (KRPAAIKKAGQAKKKK) (SEQ ID NO: 24), transportan (GWTLNSAGYLLGKINLKALAALAKKIL) (SEQ ID NO: 25), and pVEC (LLIILRRRIRKQAHAHSK) (SEQ ID NO: 26).

3. The fusion protein of claim 1 or 2, wherein the cell-penetrating peptide is located at the N-terminus or C-terminus of the FABP5 TOS motif.

4. The fusion protein of any one of claims 1-3, wherein the FABP5 TOS motif is linked to the cell-penetrating peptide via a linker.

5. The fusion protein of claim 4, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of (GGGGS)n (SEQ ID NO: 27), (G)n (SEQ ID NO: 28), (GSSSS)n (SEQ ID NO: 29), (GSSS)n (SEQ ID NO: 30), (AAY)n (SEQ ID NO: 31), (EAAAK)n (SEQ ID NO: 32), GPGPG (SEQ ID NO: 33), HEYGAEALERAG (SEQ ID NO: 34), (KK)n (SEQ ID NO: 35), (RVRR)n (SEQ ID NO: 36), and (XP)n (SEQ ID NO: 37), where X denotes any amino acid, wherein n ranges from 1-15.

6. The fusion protein of any one of claims 1-5, further comprising a tag selected from among a histidine tag, a glutathione-S-transferase tag, a maltose binding protein tag, a Streptavidin tag, a hemagglutinin tag, a FLAG tag, a MYC tag, or a biotin tag.

7. The fusion protein of claim 6, wherein the tag is located at the N- or C-terminus of the cell-penetrating peptide.-42-4907-021 1 -4175.1Atty. Dkt. No.: 093873-15048. The fusion protein of claim 6 or 7, wherein the tag is located at the N- or C-terminus of the FABP5 TOS motif9. The fusion protein of any one of claims 1-8, wherein the tag is located at the N- or C-terminus of the fusion protein.

10. The fusion protein of any one of claims 1-9, wherein the fusion protein comprises the amino acid sequence of RQIKIWFQNRRMKWKK-FDEYMKE (SEQ ID NO:1), RQIKIWFQNRRMKWKK-QQLEGRWRLVDSKGFDEYMKELGVGIA (SEQ ID NO: 2), or RQIKIWFQNRRMKWKK-DSKGFDEYM (SEQ ID NO: 3).

11. The fusion protein of any one of claims 1-10, further comprising one or more covalent modifications selected from among capping, or PEGylation.

12. The fusion protein of any one of claims 1-11, wherein the fusion protein is formulated with micelles, liposomes or fatty acid derivates or wherein the FABP5 fusion protein is stabilized via cyclization, stapling, or D-amino acids.

13. A nucleic acid molecule encoding the fusion protein of any one of claims 1-10.

14. A vector comprising the nucleic acid molecule of claim 13.

15. The vector of claim 14, wherein the vector is an adenoviral vector, an adeno- associated vector, a retroviral vector, a nanoparticle, a liposome, or a micelle.

16. A host cell comprising the nucleic acid molecule of claim 13 or the vector of claim 14 or 15.

17. A method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the fusion protein of any one of claims 1-12, a nucleic acid molecule of claim 13, or the vector of claim 14 or 15.

18. The method of claim 17, wherein the cancer is prostate cancer or breast cancer, optionally wherein the breast cancer is triple negative breast cancer (TNBC).

19. A method for treating metabolic disease in a subject in need thereof comprising administering to the subject an effective amount of the fusion protein of any one of claims 1-12, a nucleic acid molecule of claim 13, or the vector of claim 14 or 15.

20. The method of claim 19, wherein the metabolic disease is diabetes, obesity, or cardiovascular disease.-43-4907-021 1 -4175.1Atty. Dkt. No.: 093873-150421. A method for preventing the onset of high-fat diet (HFD) induced obesity or inflammageing in a subject in need thereof comprising administering to the subject an effective amount of the fusion protein of any one of claims 1-12, a nucleic acid molecule of claim 13, or the vector of claim 14 or 15.

22. A method for mitigating the onset of premature aging in a subject in need thereof comprising administering to the subject an effective amount of the fusion protein of any one of claims 1-12, a nucleic acid molecule of claim 13, or the vector of claim 14 or 15.

23. A method for mitigating the onset of premature aging in a subject in need thereof comprising administering to the subject an effective amount of the fusion protein of any one of claims 1-12, a nucleic acid molecule of claim 13, or the vector of claim 14 or 15, wherein mRNA or polypeptide expression and / or activity levels of one or more of FABP5, phosphorylated p70S6K (Ribosomal Protein S6 Kinase), and phosphorylated pS6 in a biological sample obtained from the subject are elevated compared to that observed in a control sample obtained from a healthy subject or a predetermined threshold.

24. The method of claim 23, wherein mRNA expression levels are detected via realtime quantitative PCR (qPCR), digital PCR (dPCR), Reverse transcriptase-PCR (RT-PCR), Northern blotting, microarray, dot or slot blots, in situ hybridization, or fluorescent in situ hybridization (FISH).

25. The method of claim 23, wherein polypeptide expression levels are detected via Western blotting, enzyme-linked immunosorbent assays (ELISA), dot blotting, immunohistochemistry, immunofluorescence, immunoprecipitation, immunoelectrophoresis, or mass-spectrometry.

26. The method of any one of claims 23-25, wherein the biological sample comprises biopsied tumor tissue, whole blood, plasma, or serum.

27. The method of any one of claims 17-26, further comprising sequentially, separately, or simultaneously administering to the subject an additional therapeutic agent.

28. The method of claim 27, wherein the additional therapeutic agent comprises one or more of chemotherapeutic drugs, anti-tumor antibodies, anti-receptor antibodies, radiation therapy, radiolabeled drugs, and surgery.-44-4907-021 1 -4175.1

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