Engineered cells expressing glucose transporter 5 (GLUTS) and uses thereof

Abstract

Provided herein are compositions, kits, and methods for manufacturing cells for treating or regenerating damaged or diseased tissue comprising engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells that overexpress glucose transporter 5 (GLUTS).

Description

ENGINEERED CELLS EXPRESSING GLUCOSE TRANSPORTER 5 (GLUT5) AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 674,986 filed July 24, 2024, the contents of which are incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present technology relates to compositions, kits, and methods for manufacturing cells for treatment or regeneration of damaged or diseased tissue comprising engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells that overexpress glucose transporter 5 (GLUT5).BACKGROUND

[0003] 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.

[0004] Repair of damaged and diseased tissues is an energy intensive process that requires the production of certain biomolecules (e.g., collagen in the skin, bones and cartilage, proline in muscle, and hyaluronic acid in bone) and for specific cellular processes to occur (e.g., myoblast fusion in muscle). These requirements put an intense metabolic strain on cells participating in tissue repair, such as fibroblasts, myoblasts, osteocytes, and mesenchymal stem cells. Limits on nutrient availability and the capacity of these cells to take up and use different energy sources further exacerbates the metabolic challenges associated with tissue repair. Current regenerative therapies are highly limited, and most do not address the metabolic pressures faced by regenerating tissues.

[0005] Accordingly, there is an urgent need for methods and compositions for treating or regenerating damaged or diseased tissue.SUMMARY OF THE PRESENT TECHNOLOGY

[0006] In one aspect, the present disclosure provides an engineered cell comprising a non-endogenous expression vector that includes a nucleic acid sequence encoding a mammalian Glucose Transporter 5 (GLUT5) amino acid sequence, wherein the engineered cell is a fibroblast, myoblast, osteocyte, chondrocyte, adipocyte, epithelial cell, or4922-4164-0535.1 1mesenchymal stem cell. In some embodiments, the GLUT5 amino acid sequence comprises SEQ ID NO: 1 or SEQ ID NO: 2. Additionally or alternatively, in certain embodiments, the GLUT5 amino acid sequence comprises a variant GLUT5 amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or a biological equivalent thereof, wherein the variant GLUT5 amino acid sequence improves transport capacity. In some embodiments, the variant GLUT5 amino acid sequence comprises at least one or more of Y31, H386, H418, A395, S391, Q288, Q287, 1173, 1169, Q166, W419, N324 or A387 of SEQ ID NO: 1, SEQ ID NO: 2, or a biological equivalent thereof.

[0007] In certain embodiments, the nucleic acid sequence comprises any one of SEQ ID NOs: 3-5. Additionally or alternatively, in some embodiments, the non-endogenous expression vector including the GLUT5 nucleic acid sequence is a plasmid, a cosmid, a bacmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, or a retroviral vector. In any of the preceding embodiments, the GLUT5 nucleic acid sequence is operably linked to an expression control sequence. The expression control sequence can be an inducible promoter, a constitutive promoter, a native GLUT5 promoter, or a heterologous promoter. Additionally or alternatively, in some embodiments, the engineered cell is derived from an autologous donor or an allogenic donor.

[0008] In one aspect, the present disclosure provides a composition comprising an effective amount of any and all embodiments of the engineered cell disclosed herein and a pharmaceutically acceptable carrier.

[0009] In another aspect, the present disclosure provides a method of preparing cells for regenerative therapy comprising: isolating cells from a donor subject, wherein the cells are fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells; and transducing the isolated cells with a non-endogenous expression vector that includes a nucleic acid sequence encoding a mammalian Glucose Transporter 5 (GLUT5) amino acid sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). In certain embodiments, the nucleic acid sequence comprises any one of SEQ ID NOs: 3-5. In yet another aspect, the present disclosure provides a method of treatment comprising: isolating cells from a donor subject, wherein the cells are fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells; transducing the isolated cells with a non-endogenous expression vector that includes a nucleic acid sequence encoding a mammalian Glucose Transporter 5 (GLUT5) amino acid sequence(e.g., SEQ ID NO: 1 or SEQ ID NO: 2); and administering the transduced cells to a recipient subject. In certain embodiments, the nucleic acid sequence comprises any one of SEQ ID NOs: 3-5.

[0010] Additionally or alternatively, in some embodiments of the methods disclosed herein, the donor subject and the recipient subject are the same or different. In certain embodiments of the methods disclosed herein, the transduced mesenchymal stem cells differentiate into cartilage, bone and the fat.

[0011] Also disclosed herein are methods for treating damaged or diseased tissue in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the engineered cell disclosed herein or any and all embodiments of the compositions disclosed herein.

[0012] In some embodiments, the engineered mesenchymal stem cells provided herein can be used to treat pathologies such as liver disorders, cardiac ischemia, atherosclerosis, heart disease, diabetes, skin diseases, and bone and cartilage diseases.

[0013] In some embodiments, the damaged or diseased tissue comprises skin tissue, and / or the engineered cell or the composition comprises a fibroblast, an epithelial cell, or a mesenchymal stem cell. In certain embodiments, the damaged or diseased skin tissue is caused by wounds, ulcers, cuts, crush injuries, punctures, surgical intervention, burns, infections, or compromised skin grafts.

[0014] In other embodiments, the damaged or diseased tissue comprises bone tissue and / or the engineered cell or the composition comprises an osteocyte or a mesenchymal stem cell. In certain embodiments, the damaged or diseased bone tissue is caused by wounds, crush injuries, breaks, fractures, osteoporosis, osteopenia, Paget’s disease, osteogenesis imperfecta, osteonecrosis, osteoarthritis, osteomyelitis, fibrous dysplasia, bone cancers, osteomalacia, rickets, rheumatoid arthritis, achondroplasia, hypocalcemia, hypercalcemia, hypochondroplasia, various dysplasias other than fibrous, hypophosphatasia, bone spurs, brachydactyly, and other genetic and developmental bone and skeletal diseases. In certain embodiments, the damaged or diseased tissue comprises cartilage tissue and / or the engineered cell or the composition comprises a chondrocyte or a mesenchymal stem cell.

[0015] In certain embodiments, the damaged or diseased tissue comprises muscle tissue and / or the engineered cell or the composition is a myocyte or a mesenchymal stem cell. Incertain embodiments, the damaged or diseased muscle tissue is caused by diabetes, cardiac ischemia, atherosclerosis, heart disease, wounds, crush injuries, puncture wounds, bums, infections, sarcopenia, muscular dystrophy, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, malnutrition, aging, or coma.

[0016] In any and all embodiments of the methods disclosed herein, the engineered cell or composition is administered pleurally, intravenously, subcutaneously, intranodally, intramuscularly, topically, intradermally, intrathecally, intrapleurally, intraosseously, or intraperitoneally. Additionally or alternatively, in some embodiments, the methods further comprise sequentially, separately, or simultaneously administering to the subject at least one additional therapy. The at least one additional therapy may comprise one or more of verteporfin, growth factors (e.g., FGFs, BMPs, VEGF, PDGF), adhesamine, 8- Bromoadenosine 3', 5 '-cyclic monophosphate (8-Br-cAMP), N6-Benzoyladenosine-3', 5'- cyclic monophosphate (6-Bnz-cAMP), forskolin, SB216763, Valproic acid, CHIR99021, Repsox, Y-27632, peptidomimetics of the N-cadherin HAVD motif, dimethyloxalylglycine, trimebutine, phenamil, tesolvin El, kartogenin, duloxetine, phenelzine sulfate, tacrine, ethinyl estradiol, crotamiton, honokiol, trimebutine-maleate, piceid, bioceramics (e.g., hydroxyapatite, tricalcium phosphate, bisphosphonates, bioactive glass, akermanite), statins, strontium and vanadium compounds, flavonoids, Calcium silicate, curcumin, and pyrintegrin.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGs. 1A-1D show that GLUT5 overexpression in primary murine skin fibroblasts renders them no longer reliant on glucose as a nutrient. FIG. 1A shows that GLUT5 overexpressing fibroblasts (GT5-fibroblasts) are able to perform glycolysis using fructose as the only nutrient source, in contrast to fibroblasts lacking GLUT5. FIG. IB shows that GT5 -fibroblasts are also able to operate the TCA cycle, a requirement for energy production, using fructose. FIGs. 1C-1D shows that GT5-fibroblasts are able to produce proline (FIG. 1C) and glycine (FIG. ID) using fructose at comparable levels to control fibroblasts grown with glucose. All n=3 replicates and * p<0.05, ** p<0.01, and ***p<0.005.

[0018] FIGs. 2A-2D show that the GLUT5 overexpression in myoblasts (GT5- myoblasts) allows them to use fructose for energy metabolism. FIGs. 2A-2B show that glycolysis occurs in GT5 myoblasts in a glucose-independent manner using fructose as theonly fuel source. FIG. 2C shows that GT5-myoblasts are able to produce as much glutamate, an important TCA cycle intermediate, from fructose as control EV (empty vehicle)-myoblasts from glucose. The ability to produce TCA cycle intermediates is highly relevant as these muscles are the most energy requiring cells in the body. FIG. 2D shows that GT5 myoblasts are able to produce proline in the absence of glucose. In order for myoblasts to function, they require the ability to mobilize proline to form fusion events as part of their collagen metabolism. All n=3 replicates and * p<0.05, ** p<0.01, and ***p<0.005.

[0019] FIG. 3 shows representative images of fusion in EV-myocytes grown on glucose and GT5-myocytes grown on fructose (left) and the fusion rate (the percentage of cells with two or more nuclei over the total number of cells) for EV-myocytes grown on glucose, EV- myocytes grown on fructose, and GT5-myocytes grown on fructose, with GT5-myocytes grown on fructose having the highest percentage of fusion (right).

[0020] FIGs. 4A-4F show that GLUT5 expression in U2OS human osteocytes renders them metabolically independent of glucose. GLUT5 overexpressing osteocytes (GT5- osteocytes) performed glycolysis (FIG. 4A) and the TCA cycle (FIG. 4B), as indicated by the measurement of key intermediates in the respective pathways, using fructose as the sole energy source. GT5 -osteocytes grown on fructose produced the type 1 collagen components glycine (FIG. 4C) and proline (FIG. 4D), which are components of type 1 collagen. GT5 -osteocytes grown on fructose produced glucosamine-6-phosphate (FIG. 4E) and UDP N-acetyl-glucosamine (FIG. 4F), two metabolic intermediates in the hyaluronic acid synthesis pathway. All n=3 replicates and * p<0.05, ** p<0.01, and ***p<0.005.

[0021] FIGs. 5A-5D show GLUT5 expressing primary human mesenchymal stem cells (hMSCs) are not metabolically reliant on glucose. GT5-hMSCs grown on fructose performed glycolysis (FIG. 5A) and the TCA cycle (FIG. 5B), as indicated by the measurement of key intermediates in the respective pathways. GT5-hMSCs grown on fructose produced the type 1 collagen components glycine (FIG. 5C) and proline (FIG. 5D). All n=3 replicates and * p<0.05, ** p<0.01, and ***p<0.005.DETAILED DESCRIPTION

[0022] 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.

[0023] The present disclosure 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 disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and variations of the disclosure 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 disclosure, 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 appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0024] It is to be understood that the present disclosure is not limited to particular uses, 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.

[0025] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, 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. (1999) 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.

[0026] Current regenerative medicine therapies are highly limited, plagued by inefficiencies, and do not address the metabolic needs of cells to regenerate diseased or damaged tissue. Without wishing to be bound by the theory, while most cells can take up glucose, very few can utilize fructose efficiently. Thus, the present disclosure provides strategies to modulate fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells to be able to use fructose as a substrate for various metabolic pathways, thus enhancing their regenerative capacity.Definitions

[0027] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0029] As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0030] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) byincrements of 1.0 or 0.1, as appropriate or alternatively by a variation of + / - 20% or + / - 15%, or alternatively 10% or alternatively 5% or alternatively 2%. As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.

[0031] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0032] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5 -fold, or within 2-fold, of a value.

[0033] As used herein, the term “administration” of an agent to a subject includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be carried out by any suitable route, including, but not limited to, intravenously, intramuscularly, intraperitoneally, subcutaneously, and other suitable routes as described herein. Administration includes self-administration and the administration by another. “Administration” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated.Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose isrepeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.

[0034] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, z.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form.

[0035] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.

[0036] As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0037] As used herein, the term “cell population” refers to a group of at least two cells expressing similar or different phenotypes. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1 x 106cells, at least about 1 x 107cells, at least about 1 x 108cells, at least about 1 x 109cells, at least about 1 x 1010cells, at least about 1 x 1011cells, at least about 1 x 1012cells, or more cells expressing similar or different phenotypes.

[0038] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some embodiments, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.

[0039] A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a nanoparticle, detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include carriers, such as pharmaceutically acceptable carriers. In some embodiments, the carrier (such as the pharmaceutically acceptable carrier) comprises, or consists essentially of, or yet further consists of a nanoparticle, such as an polymeric nanoparticle carrier or an lipid nanoparticle that can be used alone or in combination with another carrier, such as an adjuvant or solvent. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. A composition as disclosed herein can be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0040] 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 composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0041] As used herein, the phrase “derived” means isolated, purified, mutated, or engineered, or any combination thereof. For example, a cell derived from a donor refers to the cell isolated from a biological sample of the donor and optionally engineered.

[0042] As used herein, the term “effective amount” or “therapeutically effective amount” refers to a quantity of an agent sufficient to achieve a beneficial or desired clinical result upon treatment. In the context of therapeutic applications, the amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease or injury. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art.

[0043] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of long-term stabilization, bulking up solid formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.

[0044] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample following administration of the compositions disclosed herein. The term“expression” also refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription) within a cell; (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation) within a cell; (3) translation of an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presentation of a polypeptide or protein on the cell surface; and (6) secretion or presentation or release of a polypeptide or protein from a cell.

[0045] As used herein, an "expression vector" includes vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0046] As used herein, the term “fibroblasts” refers to cells of mesenchymal origin and are the primary source of extracellular matrix, including type I Collagen, production both in hemostatic conditions and in response to injury.

[0047] As used herein, the term “heterologous nucleic acid molecule or polypeptide” refers to a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is either not normally expressed or is expressed at an aberrant level in a cell or sample obtained from a cell. This nucleic acid can be from another organism, or it can be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

[0048] As used herein, a "host cell" is a cell that is used to receive, maintain, reproduce and amplify an expression vector. A host cell also can be used to express the polypeptide encoded by the expression vector. The nucleic acid contained in the expression vector is replicated when the host cell divides, thereby amplifying the nucleic acids. In some embodiments, the host cell as disclosed herein is a eukaryotic cell or a prokaryotic cell. Insome embodiments, the host cell is a human cell. In some embodiments, the host cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), or a 293 T cell. These cells are commercially available, for example, from the American Type Culture Collection (ATCC).

[0049] As used herein, the term “increase” or “enhance” means to alter positively by at least about 5%, including, but not limited to, alter positively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

[0050] The terms “ketohexokinase,” and “KHK” are used interchangeably and refer to a ketohexokinase that catalyzes conversion of fructose to fructose- 1 -phosphate. The product of this gene is the first enzyme with a specialized pathway that catabolizes dietary fructose. In some embodiments, the KHK is a human KHK. Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC02P027086, HGNC: 6315, NCBI Entrez Gene: 3795, Ensembl: ENSG00000138030, OMIM®: 614058, or UniProtKB / Swiss-Prot: P50053, each of which is incorporated by reference herein in its entirety.

[0051] As used herein, the term “ligand” refers to a molecule that binds to a receptor. In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition and / or interaction.

[0052] As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide affects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linkedto nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0053] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0054] As used herein, the term “mesenchymal stem cell(s)” refers to multipotent stem cells that can differentiate into any mesenchymal lineage cell, including, but not limited to, osteoclasts, chondrocytes, myocytes, and adipocytes.

[0055] As used herein, the term “myoblast(s)” refers to postmitotic, mononucleated cells capable of fusion and contractile protein synthesis. Myoblast fusion is important to muscle repair and regeneration.

[0056] As used herein, the term “osteocyte(s)” refers to a bone cell capable of bone deposition and resorption, and is commonly found within the bone itself.

[0057] As used herein, the “percent homology” between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (z.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0058] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. AppL Biosci., 4: 1 1-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0059] Additionally or alternatively, the amino acids sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215 :403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the specified sequences disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al.. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0060] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. In some embodiments, a pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier or an lipid nanoparticle (LNP). Additionally or alternatively, pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They can be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0061] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0062] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are a non -naturally occurring amino acid, e.g., an amino acid analog. The terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0063] The terms “pyruvate kinase M2,” and “PKM2” are used interchangeably and refer to a protein involved in glycolysis. The encoded protein is a pyruvate kinase that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP and pyruvate. In some embodiments, the PKM2 is a human PKM2. Non-limiting exemplary sequences of this protein or the underlying gene can be found under Gene Cards ID: GC15M072199, HGNC: 9021, NCBI Entrez Gene: 5315, Ensembl: ENSG00000067225, OMIM®: 179050, or UniProtKB / Swiss-Prot: P14618, each of which is incorporated by reference herein in its entirety.

[0064] As used herein, the term “PKM2 activator” refers to an agent that increases the level of pyruvate kinase activity of PKM2, such as from the state of inactive monomeric or dimeric form or maintains or increases the activity of active tetrameric form of PKM2 (e.g., in the presence of an endogenous inhibitor.

[0065] As used herein, the term “reduce” means to alter negatively by at least about 5%, including, but not limited to, alter negatively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

[0066] As used herein, “regulatory sequence” of a nucleic acid molecule means a cisacting nucleotide sequence that influences expression, positively or negatively, of an operably linked gene. Regulatory regions include sequences of nucleotides that confer inducible (z.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (z.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration, gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and tissue regeneration. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0067] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Polymerase II and III are examples of promoters. A polymerase II or “pol II” promoter catalyzes the transcription of DNA to synthesize precursors of mRNA, and most shRNA and microRNA. Examples of pol II promoters are known in the art and include without limitation, the phosphoglycerate kinase (“PGK”) promoter; EFl -alpha; CMV (minimal cytomegalovirus promoter); and LTRs from retroviral and lentiviral vectors. In some embodiments, the promoter is a constitutive promoter. As used herein, the term “constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control in all or most tissues of a subject at all or most developing stages. Non-limiting examples of the constitutive promoters include a CMV promoter, a simian virus 40 (SV40) promoter, a polyubiquitin C (UBC) promoter, an EFl -alpha promoter, a PGK promoter and a CAG promoter. In some embodiments, the promoter is a conditional promoter, which allows for continual transcription of the coding sequence or gene under certain conditions. In further embodiments, the conditional promoter is a tissue-specific (e.g., fibroblast, myoblast, osteocyte, chondrocyte, adipocyte, epithelial cell and / or mesenchymal stem cell) promoter,which allows for continual transcription of the coding sequence or gene in a targeted cell type.

[0068] Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

[0069] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0070] As used herein, the term “sample” refers to clinical samples obtained from a subject. In certain embodiments, a sample is obtained from a biological source (z.e., a "biological sample"), such as tissue, bodily fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchial alveolar lavage (BAL), bronchial wash (BW), whole blood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

[0071] As used herein, the term “secreted” in reference to a polypeptide means a polypeptide that is released from a cell via the secretory pathway through the endoplasmic reticulum, Golgi apparatus, and as a vesicle that transiently fuses at the cell plasma membrane, releasing the proteins outside of the cell. Small molecules, such as drugs, can also be secreted by diffusion through the membrane to the outside of cell.

[0072] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0073] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or daysbefore administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0074] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0075] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to an individual organism, a vertebrate, or a mammal and may include humans, non-human primates, rodents, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested). In certain embodiments, the individual, patient or subject is a human.

[0076] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0077] As used herein, "synthetic," with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods. As used herein, production by recombinant means by using recombinant DNA methods means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.

[0078] “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. Therapeutic effects of treatment include, without limitation, increasing the rate and / or total amount of tissue regeneration, inhibiting recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0079] It is also to be appreciated that the various modes of treatment of diseases 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. Thetreatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0080] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, z.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.Overview of Fructose Metabolism

[0081] Fructose Metabolism: Dietary fructose consumption, which has increased > 100-fold over the past two centuries, now accounts for -10% of total caloric intake in the United States (Vos et al. Medscape J Med 10, 160 (2008); Bray et al. Am J Clin Nutr 19, 537-543 (2004); and Marriott et al., J Nutr 139, 1228S-1235S (2009)). As fructose is more palatable but less satiating than glucose, overconsumption often leads to the development of obesity and metabolic syndrome (Macdonald et al. Eur J Nutr 55, 17-23 (2016)). Fructose represents a possible energy source for cells with the metabolic flexibility to use it. For nearly 100 years it has been known that most cancer cells exhibit a high glycolytic rate, even in the presence of oxygen (Warburg Effect). Recently, multiple studies have demonstrated that cancer cells are able to utilize fructose as an additional fuel for their proliferation and metastasis. For example, pancreatic cancer cells have been shown to increase flux through the non-oxidative pentose phosphate pathway in fructose-rich conditions, which leads to the preferential use of fructose over glucose for nucleotide synthesis (Liu et al. Cancer research 70, 6368-6376 (2010)). Breast cancer and colon cancer cells with elevated levels of aldolase-B, a key enzyme in fructose metabolism, were shown to metastasize at high levels to liver under a high-fructose diet (Bu et al. Cell metabolism27, 1-41 (2018)). GLUT5, a potent transporter of fructose, has been shown to be upregulated in some patients with leukemia or lung adenocarcinoma, which facilitates the fructose use of cancer cells in glucose-limited conditions (Chen et al. Cancer cell 30, 779- 791 (2016); Weng et al. Cell Death Discov 4, 38 (2018)). Fructose metabolism by cancer cells represents a form of natural, opportunistic metabolic engineering to promote cell metabolic flexibility and productivity.

[0082] In normal physiology, fructose is predominantly metabolized in the liver and small intestine, utilizing rapid transport via the insulin-independent transporters GLUT2 and GLUT5 (Douard & Ferraris. American journal of physiology Endocrinology and metabolism 295, E227-237 (2008); Jang et al. Cell Metab 27, 351-361 e353 (2018);Goncalves et al. Science 363, 1345-1349 (2019)). It is subsequently converted to fructose- 1 -phosphate by the enzyme ketohexokinase (KHK) and can participate in further biochemical transformations in glycolysis. Fructose can also be converted directly to fructose-6-phosphate by hexokinase, though at a rate significantly slower than that of KHK. Ultimately, transport and these first enzymatic conversions drive the metabolism of fructose and, in many instances, gluconeogenesis.

[0083] Fructose is typically taken up by the liver, kidneys and small intestine via the insulin independent transporters GLUT2 and GLUT5. In these tissues, the enzyme ketohexokinase (KHK) is expressed at high levels, facilitating the formation of fructose- 1- phosphate and in most cases gluconeogenesis. A mechanism has been identified whereby fructose is metabolized through the serine synthesis pathway (SSP), generating insignificant lactate via glycolysis. Without wishing to be bound by the theory, this can be modulated by the fructose transporter, titrating carbons from the SSP to glycolysis and deriving lactate from fructose equivalent to glucose.GLUT5

[0084] As used herein, the terms “glucose transporter 5,” “GLUT5,” “Solute Carrier Family 2 Member 5” and “SLC2A5” refer to a fructose transporter responsible for fructose uptake by the small intestine, or a gene encoding the fructose transporter. The encoded protein also is necessary for the increase in blood pressure due to high dietary fructose consumption. Non-limiting exemplary sequences of this protein or the underlying gene may be found under Gene Cards ID: GC01M009036 (retrieved from www.genecards.org / cgi- bin / carddisp.pl?gene=SLC2A5), HGNC: 1010 (www.genenames.org / data / gene-symbol-report / # ! / hgnc_id / l 1010), NCBI Entrez Gene: 6518 (retrieved from www.ncbi.nlm.nih.gov / gene / 6518), Ensembl: ENSG00000142583 (retrieved from uswest.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSGOOOOO 142583 ;r=l:9035106- 9088478), OMIM®: 138230 (retrieved from omim.org / entry / 138230), or UniProtKB / Swiss- Prot: P22732 (retrieved from www.uniprot.org / uniprot / P22732), which are incorporated by reference herein.

[0085] Exemplary amino acid sequences of human GLUT5 are set forth below:

[0086] Solute carrier family 2, facilitated glucose transporter member 5 isoform 1MEQQDQSMKEGRLTLVLALATLIAAFGSSFQYGYNVAAVNSPALLMQQFYNETYY GRTGEFMEDFPLTLLWSVTVSMFPFGGFIGSLLVGPLVNKFGRKGALLFNNIFSIVP AILMGCSRVATSFELIIISRLLVGICAGVSSNVVPMYLGELAPKNLRGALGVVPQLFI TVGILVAQIFGLRNLLANVDGWPILLGLTGVPAALQLLLLPFFPESPRYLLIQKKDEA AAKKALQTLRGWDSVDREVAEIRQEDEAEKAAGFISVLKLFRMRSLRWQLLSIIVL MGGQQLSGVNAIYYYADQIYLSAGVPEEHVQYVTAGTGAVNVVMTFCAVFVVEL LGRRLLLLLGFSICLIACCVLTAALALQDTVSWMPYISIVCVISYVIGHALGPSPIPAL LITEIFLQSSRPSAFMVGGSVHWLSNFTVGLIFPFIQEGLGPYSFIVFAVICLLTTIYIFL IVPETKAKTFIEINQIFTKMNKVSEVYPEKEELKELPPVTSEQ (SEQ ID NO: 1); or

[0087] Solute carrier family 2, facilitated glucose transporter member 5 isoform 2MEQQDQSMKEGRLTLVLALATLIAAFGSSFQYGYNVAAVNSPALLMQQFYNETYY GRTGEFMEDFPLTLLWSVTVSMFPFGGFIGSLLVGPLVNKFGRKGALLFNNIFSIVP AILMGCSRVATSFELIIISRLLVGICAGVSSNVVPMYLGELAPKNLRGALGVVPQLFI TVGILVAQIFGLRNLLANVDGEFRTSREHPHPFTTTLGPLLVFQSHHHRTGLSADWS LLTGWMSLGGPSCPEPT (SEQ ID NO: 2)

[0088] In some embodiments, the engineered cells express a heterologous amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or a biological equivalent thereof, wherein the engineered cells are fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells. In further embodiments, the biological equivalent of SEQ ID NO: 1 or SEQ ID NO: 2 comprises one or more conservative amino acid substitutions relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively. Additionally or alternatively, in some embodiments, the biological equivalent transports fructose substantially similar to or significantly more efficiently compared to the protein of SEQ ID NO: 1 or 2. Additionallyor alternatively, in certain embodiments, the GLUT5 amino acid sequence comprises a variant GLUT5 amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, or a biological equivalent thereof, wherein the variant GLUT5 amino acid sequence improves transport capacity. See, e.g., Ebert et al., J Biol Chem. 2018 Feb 9; 293(6): 2115-2124; Nomura et al., Nature,' 526(7573): 397-401 (2015). In some embodiments, the variant GLUT5 amino acid sequence comprises at least one or more of Y31, H386, H418, A395, S391, Q288, Q287, 1173, 1169, Q166, W419, N324 or A387 of SEQ ID NO: 1, SEQ ID NO: 2, or a biological equivalent thereof.

[0089] Exemplary nucleic acid sequences of human GLUT5 are set forth below: atggagcaacaggatcagagcatgaaggaagggaggctgacgcttgtgcttgccctggcaaccctgatagctgcctttgggtcatc cttccagtatgggtacaacgtggctgctgtcaactccccagcactgctcatgcaacaattttacaatgagacttactatggtaggaccg gtgaattcatggaagacttccccttgacgttgctgtggtctgtaaccgtgtccatgtttccatttggagggtttatcggatccctcctggtc ggccccttggtgaataaatttggcagaaaaggggccttgctgttcaacaacatattttctatcgtgcctgcgatcttaatgggatgcagc agagtcgccacatcatttgagcttatcattatttccagacttttggtgggaatatgtgcaggtgtatcttccaacgtggtccccatgtactt aggggagctggcccctaaaaacctgcggggggctctcggggtggtgccccagctcttcatcactgttggcatccttgtggcccaga tctttggtcttcggaatctccttgcaaacgtagatggtgagttcaggacatctcgggagcacccccaccccttcaccactacccttggc cccctccttgtgttccaaagccaccaccacaggacaggactttctgcagactggtctcttctaacaggctggatgtccttggggggcc catcctgtcccgagccaacatag (SEQ ID NO: 3); atggagcaacaggatcagagcatgaaggaagggaggctgacgcttgtgcttgccctggcaaccctgatagctgcctttgggtcatc cttccagtatgggtacaacgtggctgctgtcaactccccagcactgctcatgcaacaattttacaatgagacttactatggtaggaccg gtgaattcatggaagacttccccttgacgttgctgtggtctgtaaccgtgtccatgtttccatttggagggtttatcggatccctcctggtc ggccccttggtgaataaatttggcagaaaaggggccttgctgttcaacaacatattttctatcgtgcctgcgatcttaatgggatgcagc agagtcgccacatcatttgagcttatcattatttccagacttttggtgggaatatgtgcaggtgtatcttccaacgtggtccccatgtactt aggggagctggcccctaaaaacctgcggggggctctcggggtggtgccccagctcttcatcactgttggcatccttgtggcccaga tctttggtcttcggaatctccttgcaaacgtagatggctggccgatcctgctggggctgaccggggtccccgcggcgctgcagctcct tctgctgcccttcttccccgagagccccaggtacctgctgattcagaagaaagacgaagcggccgccaagaaagccctacagacg ctgcgcggctgggactctgtggacagggaggtggccgagatccggcaggaggatgaggcagagaaggccgcgggcttcatctc cgtgctgaagctgttccggatgcgctcgctgcgctggcagctgctgtccatcatcgtcctcatgggcggccagcagctgtcgggcgt caacgctatctactactacgcggaccagatctacctgagcgccggcgtgccggaggagcacgtgcagtacgtgacggccggcac cggggccgtgaacgtggtcatgaccttctgcgccgtgttcgtggtggagctcctgggtcggaggctgctgctgctgctgggcttctc catctgcctcatagcctgctgcgtgctcactgcagctctggcactgcaggacacagtgtcctggatgccatacatcagcatcgtctgt gtcatctcctacgtcataggacatgccctcgggcccagtcccatacccgcgctgctcatcactgagatcttcctgcagtcctctcggccatctgccttcatggtggggggcagtgtgcactggctctccaacttcaccgtgggcttgatcttcccgttcatccaggagggcctcgg cccgtacagcttcattgtcttcgccgtgatctgcctcctcaccaccatctacatcttcttgattgtcccggagaccaaggccaagacgtt catagagatcaaccagattttcaccaagatgaataaggtgtctgaagtgtacccggaaaaggaggaactgaaagagcttccacctgt cacttcggaacagtga (SEQ ID NO: 4); or atggagcaacaggatcagagcatgaaggaagggaggctgacgcttgtgcttgccctggcaaccctgatagctgcctttgggtcatc cttccagtatgggtacaacgtggctgctgtcaactccccagcactgctcatgcaacaattttacaatgagacttactatggtaggaccg gtgaattcatggaagacttccccttgacgttgctgtggtctgtaaccgtgtccatgtttccatttggagggtttatcggatccctcctggtc ggccccttggtgaataaatttggcagaaaaggggccttgctgttcaacaacatattttctatcgtgcctgcgatcttaatgggatgcagc agagtcgccacatcatttgagcttatcattatttccagacttttggtgggaatatgtgcaggtgtatcttccaacgtggtccccatgtactt aggggagctggcccctaaaaacctgcggggggctctcggggtggtgccccagctcttcatcactgttggcatccttgtggcccaga tctttggtcttcggaatctccttgcaaacgtagatggctggccgatcctgctggggctgaccggggtccccgcggcgctgcagctcct tctgctgcccttcttccccgagagccccaggtacctgctgattcagaagaaagacgaagcggccgccaagaaagccctacagacg ctgcgcggctgggactctgtggacagggaggtggccgagatccggcaggaggatgaggcagagaaggccgcgggcttcatctc cgtgctgaagctgttccggatgcgctcgctgcgctggcagctgctgtccatcatcgtcctcatgggcggccagcagctgtcgggcgt caacgctatctactactacgcggaccagatctacctgagcgccggcgtgccggaggagcacgtgcagtacgtgacggccggcac cggggccgtgaacgtggtcatgaccttctgcgccgtgttcgtggtggagctcctgggtcggaggctgctgctgctgctgggcttctc catctgcctcatagcctgctgcgtgctcactgcagctctggcactgcaggacacagtgtcctggatgccatacatcagcatcgtctgt gtcatctcctacgtcataggacatgccctcgggcccagtcccatacccgcgctgctcatcactgagatcttcctgcagtcctctcggc catctgccttcatggtggggggcagtgtgcactggctctccaacttcaccgtgggcttgatcttcccgttcatccaggagggcctcgg cccgtacagcttcattgtcttcgccgtgatctgcctcctcaccaccatctacatcttcttgattgtcccggagaccaaggccaagacgtt catagagatcaaccagattttcaccaagatgaataaggtgtctgaagtgtacccggaaaaggaggaactgaaagagcttccacctgt cacttcggaacagtga (SEQ ID NO: 5).

[0090] In some embodiments, the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells comprise a heterologous nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. Additionally or alternatively, in some embodiments, the expression levels and / or activity of GLUT5 in the engineered cell is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 times higher compared to that observed in a native cell, wherein the engineered cell is of the same lineage as the native cell.

[0091] In some embodiments, the engineered fibroblast, myoblast, osteocyte, chondrocyte, adipocyte, epithelial cell, or mesenchymal stem cell further comprises a first regulatory sequence operatively linked to the nucleic acid encoding the GLUT5. In further embodiments, the first regulatory sequence directs the expression of the GLUT5. Additionally or alternatively, in some embodiments, the first regulatory sequence comprises, or consists essentially of, or yet further consists of a promoter, for example a constitutive promoter or a conditional promoter. In further embodiments, the conditional promoter is a fibroblast-specific, a myoblast-specific, an osteocyte-specific, a chondrocytespecific, adipocyte-specific, epithelial cell-specific, or a mesenchymal stem cell-specific promoter.

[0092] In one aspect, the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells provided herein overexpress GLUT5 and / or comprise a heterologous nucleic acid encoding the GLUT5 gene. In certain embodiments, the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells of the present disclosure regenerate damaged or diseased tissue more efficiently at a tissue site. The engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells disclosed herein can be generated by in vitro transduction of cells with a nucleic acid as disclosed herein.Polynucleotides., Polypeptides and Analogs

[0093] Also included in the presently disclosed subject matter are GLUT5 polynucleotides and their corresponding polypeptides or fragments that may be modified in ways that enhance their tissue regenerative activity when expressed in an engineered fibroblast, myoblast, osteocyte, chondrocyte, adipocyte, epithelial cell, or mesenchymal stem cell. The presently disclosed subject matter provides methods for optimizing an amino acid sequence or a nucleic acid sequence by producing an alteration in the sequence. Such alterations can comprise certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter further comprises analogs of any naturally-occurring polypeptide of the presently disclosed subject matter. Analogs can differ from a naturally- occurring polypeptide of the presently disclosed subject matter by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the presently disclosed subject matter can generally exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%), about 98%, about99% or more identity or homology with all or part of a naturally-occurring amino, acid sequence of the presently disclosed subject matter. The length of sequence comparison is at least about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100 or more amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program can be used, with a probability score between e'3and e'100indicating a closely related sequence. Modifications comprise in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications can occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the presently disclosed subject matter by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethyl sulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L- amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., beta (P) or gamma (y) amino acids.

[0094] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptides or peptide domains of the presently disclosed subject matter. A fragment can be at least about 5, about 10, about 13, or about 15 amino acids. In some embodiments, a fragment is at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, or at least about 50 contiguous amino acids. In some embodiments, a fragment is at least about 60 to about 80, about 100, about 200, about 300 or more contiguous amino acids. Fragments of the presently disclosed subject matter can be generated by methods known to those of ordinary skill in the art or can result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0095] Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein. Such analogs are administered according to methods of the presently disclosed subject matter. Such analogs can exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures suchthat the resultant analogs increase the antineoplastic activity of the original polypeptide when expressed in an engineered fibroblast, myoblast, osteocyte, chondrocyte, adipocyte, epithelial cell, or mesenchymal stem cell. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide. The protein analogs can be relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0096] In accordance with the presently disclosed subject matter, the polynucleotides encoding GLUT5 can be modified by codon optimization. Codon optimization can alter both naturally occurring and recombinant gene sequences to achieve the highest possible levels of productivity in any given expression system. Factors that are involved in different stages of protein expression include codon adaptability, mRNA structure, and various ciselements in transcription and translation. Any suitable codon optimization methods or technologies that are known to ones skilled in the art can be used to modify the polynucleotides of the presently disclosed subject matter, including, but not limited to, OptimumGene™, Encor optimization, and Blue Heron.

[0097] In some embodiments, a nucleic acid as disclosed herein further comprises a regulatory sequence directing the expression of the GLUT5 gene. In further embodiments, the nucleic acid comprises a single regulatory sequence directing the expression of the GLUT5 gene.Vectors

[0098] Many expression vectors are available and known to those of skill in the art and can be used for non-endogenous expression of GLUT5. The choice of expression vector will be influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cells.

[0099] Vectors also can contain additional nucleotide sequences operably linked to theligated nucleic acid molecule, such as, for example, an epitope tag such as for localization, e.g., a hexa-his tag or a myc tag, hemagglutinin tag or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and / or membrane association.

[0100] Expression of the heterologous GLUT5 gene can be controlled by any promoter / enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is within the level of skill of the skilled artisan. Promoters which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310(1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto etal., Cell 22:787- 797(1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA I '. 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the P-lactamase promoter (Jay et al., Proc. Natl. Acad. Sci. USA 75:5543 (1981)) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 50:21-25(1983)); see also "Useful Proteins from Recombinant Bacteria": in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrera- Estrella et al., Nature 505:209- 213(1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 9:2871(1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 510: 1 15-120(1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 55:639-646 (1984); Omitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409(1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan et al., Nature 515: 115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 55:647-658 (1984); Adams et al., Nature 515:533-538 (1985); Alexander et al., Mol. Cell Biol. 7: 1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 15:485-495 (1986)),albumin gene control region which is active in liver (Pinckert et al., Genes andDevel. 1 :268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5: 1639-403 (1985)); Hammer et al., Science 255:53-58 (1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel. 7: 161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 515:338-340 (1985)); Kollias et al., Cell 5:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 15:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 514:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 254: 1372- 1378 (1986)).

[0101] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of GLUT5 in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the polypeptide chains of interest and signals required for efficient polyadenylation of the transcript, ribosome binding sites and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.

[0102] Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.

[0103] Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding any of the polypeptides provided herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesizednucleic acids encoding restriction endonuclease recognition sequences.

[0104] Exemplary plasmid vectors useful to produce the polypeptides provided herein contain a strong promoter, such as the HCMV immediate early enhancer / promoter or the MHC class I promoter, an intron to enhance processing of the transcript, such as the HCMV immediate early gene intron A, and a polyadenylation (poly A) signal, such as the late SV40 poly A signal.

[0105] Genetic modification of engineered cells (e.g., fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA or RNA construct. The vector can be a retroviral vector (e.g., gamma retroviral), which is employed for the introduction of the DNA or RNA construct into the host cell genome. For example, a polynucleotide encoding GLUT5 can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from an alternative internal promoter.

[0106] Non-viral vectors or RNA may be used as well. Random chromosomal integration, or targeted integration (e.g., using a nuclease, transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs), or transgene expression (e.g., using a natural or chemically modified RNA) can be used.

[0107] For initial genetic modification of the cells to provide GLUT5 overexpressing cells, a retroviral vector can be employed for transduction. However, any other suitable viral vector or non-viral delivery system can be used for genetic modification of cells. Combinations of retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virusproducing cell lines are known, including, but not limited to, PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos et al. (1988) roc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.

[0108] Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g, by the method of Bregni, et al., Blood 80: 1418-1422(1992), or culturing with viral supernatant alone or concentrated vector stocks with or withoutappropriate growth factors and poly cations, e.g., by the method of Xu, et al. , Exp. Hemat. 22:223-230 (1994); and Hughes, et al., J. Clin. Invest. 89: 1817 (1992).

[0109] In some embodiments, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido c / a / ., Current Eye Research 15:833-844 (1996); Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263 267 (1996); and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, (1997)). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, (1990); Friedman, Science 244: 1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614, (1988); Tolstoshev et al., Current Opinion in Biotechnology 1 :55-61(1990); Sharp, The Lancet 337 : 1277-1278 (1991); Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995)). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson et al., U.S. Pat. No. 5,399,346).

[0110] In certain non-limiting embodiments, the vector expressing GLUT5 is a retroviral vector, e.g., an oncoretroviral vector. In some instances, the retroviral vector is a SFG retroviral vector or murine stem cell virus (MSCV) retroviral vector. In certain nonlimiting embodiments, the vector expressing a GLUT5 nucleic acid sequence is a lentiviral vector. In certain non-limiting embodiments, the vector expressing a GLUT5 nucleic acid sequence is a transposon vector.

[0111] Non-viral approaches can also be employed for the expression of a protein in a cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Nat'L Acad. Sci. U.S.A. 84:7413, (1987); Ono et al., Neuroscience Letters 17:259 (1990); Brigham et al., Am. J. Med. Sci. 298:278, (1989); Staubinger et al., Methods in Enzymology 101 :512 (1983)), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263 : 14621 (1988); Wu et al., Journal of Biological Chemistry 264: 16985 (1989)), or by microinjection under surgical conditions (Wolff et al., Science 247: 1465 (1990)). Other non- viral means for gene transfer include transfection in vitro using calcium phosphate, DEAEdextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically. Recombinant proteins can also be derived or obtained using transposases or targeted nucleases (e.g., Zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression may be obtained by RNA electroporation.

[0112] cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g., the elongation factor la enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0113] The resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.

[0114] In some embodiments, a vector as disclosed herein further comprises a regulatory sequence directing the expression of the GLUT5 gene. In further embodiments, the vector comprises a single regulatory sequence directing the expression of the GLUT5 gene.Engineered Cells of the Present Technology

[0115] The presently disclosed subject matter provides engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells that overexpress GLUT5. In certain embodiments, the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells can be transduced with a vector comprising nucleic acid sequences that encode GLUT5.

[0116] The presently disclosed subject matter also provides methods of using such cells in therapies to repair or regenerate damaged and / or diseased tissues. In some embodiments, a particular engineered cell type is useful in methods of repairing a specific type of damaged and / or diseased tissue. For example, in some embodiments, an engineered fibroblast is useful in methods of repairing damaged and / or diseased skin tissue. In other embodiments, an engineered myoblast is useful in methods of repairing damaged and / or diseased muscle tissue. In certain embodiments, an engineered osteocyte is useful in methods of repairing damaged and / or diseased bone tissue. In other embodiments, an engineered mesenchymal stem cell is useful in methods of repairing damaged or diseased tissue that is derived from mesenchymal cell lineages (e.g., skeletal tissues such as cartilage, bone, and fat).

[0117] The engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells of the presently disclosed subject matter can express non-endogenous levels of GLUT5 for the repair of damaged and / or diseased tissues. Such engineered cells can be administered to a subject (e.g., a human subject) in need thereof for the treatment of damaged and / or diseased tissues. In some embodiments, the engineered cell is derived from a primary cell.

[0118] The engineered cells can be generated from donor cells, or from cells from a subject to be treated. The engineered cells can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0119] In certain embodiments, the presently disclosed engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells express from about 1 to about 5, from about 1 to about 4, from about 2 to about 5, from about 2 to about 4, from about 3 to about 5, from about 3 to about 4, from about 4 to about 5, from about 1 to about 2, from about 2 to about 3, from about 3 to about 4, or from about 4 to about 5 vector copy numbers per cell of a GLUT5 heterologous nucleic acid.

[0120] For example, the higher the non-endogenous levels of GLUT5 in an engineered cell, the greater the tissue repair and regeneration capacity the engineered cell exhibits. Additionally, or alternatively, the regenerative and repair capacity of a presently disclosed engineered cell can be proportional to the expression level of GLUT5 in the cell.

[0121] The unpurified source of primary cells can be any known in the art, such as the bones, including bone marrow, muscles, and skin. Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-target cellsinitially. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and / or stages of differentiation for both positive and negative selections.

[0122] A large proportion of terminally differentiated cells can be initially removed by a relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. In some embodiments, at least about 80%, usually at least 70% of the total cells will be removed prior to cell isolation.

[0123] Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g., plate, chip, elutriation or any other convenient technique.

[0124] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels.

[0125] The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). In some embodiments, the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium.

[0126] In some embodiments, the engineered cells are further modified to suppress expression of one or more genes. In some embodiments, the engineered cells are further modified via genome editing. Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, for example, U.S. Patent Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960, the disclosures of which are incorporated by reference in their entireties. These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such thatrepair of the break by an error born process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or the insertion of a sequence of interest (targeted integration). Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), or using the CRISPR / Cas system with an engineered crRNA / tracr RNA ('single guide RNA') to guide specific cleavage.Administration

[0127] Engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells overexpressing GLUT5 of the presently disclosed subject matter can be provided systemically or directly to a subject for treating damaged and / or diseased tissues. In certain embodiments, engineered cells are directly injected into an organ or tissue of interest. Additionally or alternatively, the engineered cells are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the vasculature) or into the tissue of interest (e.g., skin, bone, or muscle tissue). Expansion and differentiation agents can be provided prior to, during or after administration of cells and compositions to increase production of the engineered cells either in vitro or in vivo.

[0128] Engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells of the presently disclosed subject matter can be administered in any physiologically acceptable vehicle, systemically or regionally, normally intravascularly, intraperitoneally, intrathecally, or intrapleurally, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). In certain embodiments, at least 1 x 105cells can be administered, eventually reaching 1 x IO10or more. In certain embodiments, at least 1 x 106cells can be administered. A cell population comprising engineered cells can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of engineered cells in a cell population using various well-known methods, such as fluorescence activated cell sorting (FACS). The ranges of purity in cell populations comprising engineered cells can be from about 50% to about 55%, from about 55% to about 60%, about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%; from about 85% to about 90%, from about 90% to about 95%, or from about 95 to about 100%. Dosages canbe readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). The engineered cells can be introduced by injection, catheter, or the like.

[0129] In certain embodiments, compositions of the presently disclosed subject matter comprise pharmaceutical compositions comprising engineered cells overexpressing GLUT5 with a pharmaceutically acceptable carrier. Administration can be autologous or non- autologous. For example, engineered cells overexpressing GLUT5 and compositions comprising the same can be obtained from one subject, and administered to the same subject or a different, compatible subject. Engineered cells can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. Engineered cells can be administered intramuscularly, intraosseously, topically, or transdermaly, for example, and can be formulated for said route of administration by one of ordinary skill in the art. When administering a pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising engineered cells overexpressing GLUT5), it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).Formulations

[0130] Engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells over-expressing GLUT5 and compositions comprising the same can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0131] Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g., a composition comprising engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, andmesenchymal stem cells, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON1S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0132] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells of the presently disclosed subject matter.

[0133] The compositions can be isotonic, z.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the presently disclosed subject matter may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is suitable particularly for buffers containing sodium ions.

[0134] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on theexact route of administration and the nature of the particular dosage form, e.g., liquid dosage form e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0135] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells as described in the presently disclosed subject matter. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0136] One consideration concerning the therapeutic use of the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells of the presently disclosed subject matter is the quantity of cells necessary to achieve an optimal effect. The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, from about 102to about 1012, from about 103to about 1011, from about 104to about IO10, from about 105to about 109, or from about 106to about 108engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and / or mesenchymal stem cells of the presently disclosed subject matter are administered to a subject. More effective cells may be administered in even smaller numbers. In some embodiments, at least about 1 x 108, about 2 x 108, about 3 x 108, about 4 x 108, about 5 x 108, about 1 x 109, about 5 x 109, about 1 x IO10, about 5 x IO10, about 1 x 1011, about 5 x 1011, about 1 x 1012or more engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and / or mesenchymal stem cells of the presently disclosed subject matter are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Generally, engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells are administered at doses that are nontoxic or tolerable to the patient.

[0137] The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and / or carrier in compositions to be administered in methods of thepresently disclosed subject matter. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of from about 0.001% to about 50% by weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 wt % to about 5 wt %, from about 0.0001 wt% to about 1 wt %, from about 0.0001 wt% to about 0.05 wt%, from about 0.001 wt% to about 20 wt %, from about 0.01 wt% to about 10 wt %, or from about 0.05 wt% to about 5 wt %. For any composition to be administered to an animal or human, and for any particular method of administration, toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.Therapeutic Uses of the Engineered Cells of the Present Technology

[0138] For treatment, the amount of the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells provided herein administered is an amount effective in producing the desired effect, for example, promoting or enhancing tissue repair or regeneration in a subject in need thereof. An effective amount can be provided in one or a series of administrations of the engineered cells provided herein. An effective amount can be provided in a bolus or by continuous perfusion. For regenerative cell therapies cell doses in the range of about 106to about 1010may be infused. Lower doses of the engineered cells may be administered, e.g., about 104to about 108.

[0139] The engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells of the presently disclosed subject matter can be administered by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intramuscular administration, intranodal administration, intraosseous administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and intradermal. In certain embodiments, the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, and mesenchymal stem cells and the compositions comprising thereof are administered at a site of tissue damage (e.g., a wound site, a bone deterioration site, a burn, etc.) or diseased tissue. Methods for administering cells for regenerative celltherapies are known in the art and can be employed for administration of the engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells provided herein. In some embodiments, the engineered mesenchymal stem cells provided herein can be used to treat pathologies such as liver disorders, cardiac ischemia, atherosclerosis, heart disease, diabetes, skin diseases, and bone and cartilage diseases.

[0140] For example, the presently disclosed subject matter provides methods of treating or regenerating damaged or diseased skin tissue. Skin can be damaged or diseased for any number of reasons including, but not limited to, wounds (e.g., ulcers, cuts, crush injuries, puncture wounds, etc.), surgeries, bums, tissue damage from infections, or compromised skin grafts. In one non-limiting example, the method of treating or regenerating damaged or diseased skin tissue in a subject comprises administering an effective amount of the presently disclosed engineered fibroblasts and / or mesenchymal stem cells to the subject. In one embodiment, the method of treating or regenerating damaged or diseased skin tissue in a subject comprises administering an effective amount of the presently disclosed engineered fibroblasts to the subject. In another embodiment, the method of treating or regenerating damaged or diseased skin tissue in a subject comprises administering an effective amount of the presently disclosed engineered mesenchymal stem cells to the subject. In some embodiments, methods of the present disclosure cause skin tissue in the treated subject to regenerate at a faster rate as compared to an untreated control subject. In some embodiments, methods of the present disclosure cause skin to regenerate with a lower risk of infection in the treated subject as compared to an untreated control.

[0141] In another example, the presently disclosed subject matter provides methods of treating or regenerating damaged or diseased bone tissue in a subject. Bones can be damaged or diseased for any number of reasons including, but not limited to, wounds (e.g., crush injuries, breaks, fractures etc.), osteoporosis, osteopenia, Paget’s disease, osteogenesis imperfecta, osteonecrosis, osteoarthritis, osteomyelitis, fibrous dysplasia, bone cancers, osteomalacia, rickets, rheumatoid arthritis, achondroplasia, hypocalcemia, hypercalcemia, hypochondroplasia, various dysplasias other than fibrous, hypophosphatasia, bone spurs, brachydactyly, and other genetic and developmental bone and skeletal diseases. In one nonlimiting example, the method of treating or regenerating damaged or diseased bone tissue in a subject comprises administering an effective amount of the presently disclosed engineered osteocytes and / or mesenchymal stem cells to the subject. In some embodiments, themethods comprise administering an effective amount of the presently disclosed engineered osteocytes to the subject. In some embodiments, the methods comprise administering an effective amount of the presently disclosed engineered mesenchymal stem cells to the subject. In some embodiments, methods of the present disclosure cause bone tissue in the treated subject to regenerate at a faster rate as compared to an untreated control subject. In some embodiments, methods of the present disclosure improve bone strength in the treated subject compared to an untreated control subject.

[0142] In certain embodiments, the damaged or diseased tissue comprises cartilage tissue and / or the engineered cell or the composition comprises a chondrocyte or a mesenchymal stem cell.

[0143] For example, the presently disclosed subject matter provides methods of treating or regenerating damaged or diseased muscle tissue in a subject. Muscle can be damaged or diseased for any number of reasons including, but not limited to, wounds (e.g., ulcers, cuts, crush injuries, puncture wounds, etc.), burns, tissue damage from infections, sarcopenia, muscular dystrophy (e.g., Becker, congenital, Duchenne, distal, etc.), amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, malnutrition, aging, or comas. In one non-limiting example, the method of treating or regenerating damaged or diseased muscle tissue in a subject comprises administering an effective amount of the presently disclosed engineered myoblast and / or mesenchymal stem cells to the subject. In some embodiments, the methods comprise administering an effective amount of the presently disclosed engineered myoblasts to the subject. In some embodiments, the methods comprise administering an effective amount of the presently disclosed engineered mesenchymal stem cells to the subject. In some embodiments, methods of the present disclosure cause muscle tissue in the treated subject to regenerate at a faster rate as compared to an untreated control subject. In some embodiments, methods of the present disclosure cause a greater proportion of the muscle tissue in the treated subject to regenerate compared to an untreated control subject.

[0144] Suitable human subjects for therapy typically comprise any subjects with damaged or diseased tissue, identified by clinical criteria, that can be treated with the engineered cells of the present disclosure. One of skill in the art will be readily able to identify, for example, a subject with degeneration of skin, muscle, or bone tissue, who would be indicated for treatment with the engineered cells of the present disclosure. A pharmaceutical composition embodied in the presently disclosed subject matter isadministered to these subjects to induce a regenerative response, with the objective of ameliorating their condition. Ideally, increased rate of tissue repair occurs as a result, but any clinical improvement constitutes a benefit.

[0145] The subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and / or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.Combination Therapy

[0146] The compositions of the present technology may be employed in conjunction with other therapeutic agents useful in the regeneration of skin, bone, muscle, or mesenchymal tissue. For example, the GLUT5 over-expressing engineered fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells of the present technology may be separately, sequentially or simultaneously administered with at least one additional therapy.

[0147] In some embodiments, the at least one additional therapy comprises onr or more of verteporfin, growth factors (e.g., FGFs, BMPs, VEGF, PDGF), adhesamine, 8- Bromoadenosine 3', 5 '-cyclic monophosphate (8-Br-cAMP), N6-Benzoyladenosine-3', 5'- cyclic monophosphate (6-Bnz-cAMP), forskolin, SB216763, Valproic acid, CHIR99021, Repsox, Y-27632, peptidomimetics of the N-cadherin HAVD motif, dimethyloxalylglycine, trimebutine, phenamil, tesolvin El, kartogenin, duloxetine, phenelzine sulfate, tacrine, ethinyl estradiol, crotamiton, honokiol, trimebutine-maleate, piceid, bioceramics (e.g., hydroxyapatite, tricalcium phosphate, bisphosphonates, bioactive glass, akermanite), statins, strontium and vanadium compounds, flavonoids, Calcium silicate, curcumin, pyrintegrin, and the like.Kits

[0148] The presently disclosed subject matter provides kits for the treatment or regeneration of tissue damage or diseased tissue (e.g., skin, bone, muscle, cardiac, cartilage, vascular tissue). In certain embodiments, the kit comprises a therapeutic or prophylactic composition containing an effective amount of an engineered fibroblast, myoblast, osteocyte, chondrocyte, adipocyte, epithelial cell, or mesenchymal stem cell comprising a vector that overexpresses GLUT5.

[0149] In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0150] If desired, the engineered cell can be provided together with instructions for administering the engineered cell to a subject having tissue damage or degradation. The instructions will generally include information about the use of the composition for the treatment of tissue damage and degradation. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment of tissue damage or diseased tissue; precautions; warnings; indications; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0151] The at least one engineered cell of the present technology may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation (e.g., Kivitz et al., Clin. Ther. 28: 1619-29 (2006)).

[0152] A device capable of delivering the kit components through an administrative route may be included. Examples of such devices include syringes (for parenteral administration) or inhalation devices.]0153] The kit components may be packaged together or separated into two or more containers. In some embodiments, the containers may be vials that contain sterile, lyophilized formulations of engineered cell composition that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.

[0154] Also provided herein are kits for use in the manufacture of an engineered fibroblast, myoblast, osteocyte, chondrocyte, adipocyte, epithelial cell, or mesenchymal stem cell that overexpresses GLUT5. In certain embodiments, the kit comprises a vector comprising a heterologous GLUT5 nucleic acid. Additionally or alternatively, in some embodiments, the kits further comprise fructose, a pyruvate kinase M2 (PKM2) activator or a ketohexokinase (KHK) inhibitor.EXAMPLES

[0155] The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.Example 1: GLUT5 Expression in Primary Skin Fibroblasts|0156] GT5 fibroblasts were generated by overexpressing the GLUT5 gene in primary murine fibroblasts. GT5-fibroblasts were then cultured with fructose and metabolically profiled as compared to empty vector control fibroblasts (EV) cultured with glucose or fructose. The results are shown in FIGs. 1A-1D and 2A-2D. As shown in FIG. 1A, GT5- fibroblasts are able to perform glycolysis using fructose as the only nutrient source, as indicated by the production of lactate. FIG. IB shows that GT5 -fibroblasts are also able to run the TCA cycle using fructose, a requirement for energy production. Type 1 collagen, the major component of skin, is composed of 1 / 3 glycine and 2 / 3 proline. A primary function of fibroblasts to generate glycine and proline, however without glucose production, is typically limited. GT5-fibroblasts are able to produce proline, as shown in FIG. 1C, and glycine, as shown in FIG. ID, using fructose at equivalent levels to glucose fed cells. Accordingly, GT5 -fibroblasts are capable to perform collagen production functions independent of glucose as a nutrient.

[0157] These results show that GT5 expression in fibroblasts allows cells to metabolically function using fructose and to no longer require glucose. Accordingly, these results show that the engineered cells of the present technology are useful in compositions and methods for treating damaged or diseased tissue in a subject in need thereof.Example 2: GL UT5 Expression in Primary Myoblasts

[0158] Primary C2C12 murine skeletal muscle myoblasts were transformed with either a vector encoding GLUT5 or an empty control vector control (EV). FIGs. 2A-2D show that GT5-myoblasts can use fructose to drive energy metabolism. GT5-myoblasts can run glycolysis independent of glucose using fructose as the only fuel source, as shown by F16P and lactate production in FIGs 2A-2B respectively. Even more relevant to myoblast function is the ability to produce TCA cycle intermediates, as muscle cells are some of the most energy intensive cells in the body. FIG. 2C shows that GT5-myoblasts can use fructose to run the TCA cycle and produce glutamate at equivalent levels to EV-myoblasts grown in glucose. Myoblasts require the ability to mobilize proline to form fusion events aspart of collagen metabolism. FIG. 2D shows that GT5-my oblasts are able to produce proline in the absence of glucose, making them independent of glucose as a nutrient source.

[0159] Additionally, regeneration and repair of damaged or diseased muscle, as indicated by myoblast fusion, is enhanced in GT5 -expressing myoblasts in the presence of fructose. Muscle repair and regeneration is entirely dependent on efficient fusion of myoblasts, which serve as muscle precursor cells (Hochreiter-Hufford, et al., Nature, 2013). To assess fusion capacity, GT5-myoblasts and EV-myoblasts were maintained at subconfluent densities in DMEM with 20% heat-inactivated FBS at 8.5% CO2 at 37°C. Myoblast fusion was induced by rinsing 70-80% confluent cultures with ion-containing PBS, then switching to DMEM with 2% heat-inactivated horse serum. Fusion medium was replaced every 24h over 3d. For fusion index determination, nuclei (Hoechst, cyan) were labelled and analyzed per sample for each experiment. The fusion rate (the percentage of cells with two or more nuclei over the total number of cells) was calculated for each field. As shown in FIG. 3, GT5-my oblasts were not only capable of performing fusion when grown on fructose, but also that the GT5-my oblast fusion rate with fructose was actually higher than the rate for EV-myoblasts grown on glucose.

[0160] These results show that GT5 expression in myoblasts allows cells to metabolically function using fructose and to no longer require glucose. Accordingly, these results show that the engineered cells of the present technology are useful in compositions and methods for treating damaged or diseased tissue in a subject in need thereof.Example 3: GLUT5 Expression in Osteocytes.

[0161] U2OS human Osteocytes were transformed with an expression vector encoding GLUT5 or an empty control vector. GT5 -osteocytes grown on fructose and EV-osteocytes grown on glucose or fructose were metabolically profiled. GT5-osteocyctes grown on fructose can run glycolysis, as shown by lactate production (FIG. 4A), and the TCA cycle, as indicated by glutamate production shown in (FIG. 4B). Critical to bone formation are the production of collagen and hyaluronic acid. GT5 -osteocytes can generate collagen in the absence of glucose using fructose as the only carbon source as demonstrated by the production of type 1 collagen components glycine (FIG. 4C) and proline (FIG. 4D). Hyaluronic acid is produced by condensing 2 parallel metabolic pathways. GT5 -osteocytes can produce both sides of the HA pathway as indicated by the production of glucosamine-6- phosphate (FIG. 4E) and UDP N-acetyl-glucosamine (FIG. 4F). Accordingly, GT5- osteocytes are capable of important metabolic functions using only fructose.

[0162] These results show that GT5 expression in osteocytes allows cells to metabolically function using fructose and to no longer require glucose. Accordingly, these results show that the engineered cells of the present technology are useful in compositions and methods for treating damaged or diseased tissue in a subject in need thereof.Example 4. GLUT5 Expression in Primary Human Mesenchymal Stem Cells

[0163] Primary human mesenchymal stem cells (hMSCs) were transformed with an expression vector encoding GLUT5 or an empty control vector. GT5-hMSCs grown on fructose and EV-hMSCs grown on glucose or fructose were metabolically profiled. GT5- hMSCs were able to perform glycolysis (FIG. 5A) and the TCA cycle (FIG. 5B) as indicated by the measurement of key intermediates, lactate and glutamate, in the respective pathways. Critical to cartilage and intervertebral disc formation is the production of collagen. GT5-hMSCs can generate collagen in the absence of glucose using fructose as the only carbon source demonstrated by the production of type 1 collagen components glycine (FIG. 5C) and proline (FIG. 5D).

[0164] These results show that GT5 expression in hMSCs allows cells to metabolically function using fructose and to no longer require glucose. Accordingly, these results show that the engineered cells of the present technology are useful in compositions and methods for treating damaged or diseased tissue in a subject in need thereof.EQUIVALENTS

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

Claims

CLAIMS1. An engineered cell comprising a non-endogenous expression vector that includes a nucleic acid sequence encoding a Glucose Transporter 5 (GLUT5) amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the engineered cell is a fibroblast, myoblast, osteocyte, chondrocyte, adipocyte, epithelial cell, or mesenchymal stem cell.

2. The engineered cell of claim 1, wherein the nucleic acid sequence is any one of SEQ ID NOs: 3-5.

3. The engineered cell of claim 1 or 2, wherein the non-endogenous expression vector including the GLUT5 nucleic acid sequence is a plasmid, a cosmid, a bacmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, or a retroviral vector.

4. The engineered cell of any one of claims 1 to 3, wherein the GLUT5 nucleic acid sequence is operably linked to an expression control sequence.

5. The engineered cell of claim 4, wherein the expression control sequence is an inducible promoter, a constitutive promoter, a native GLUT5 promoter, or a heterologous promoter.

6. The engineered cell of any one of claims 1 to 5, wherein the engineered cell is derived from an autologous donor or an allogenic donor.

7. A composition comprising an effective amount of the engineered cell of any one of claims 1-6 and a pharmaceutically acceptable carrier.

8. A method of preparing cells for regenerative therapy comprising: isolating cells from a donor subject, wherein the cells are fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells; and transducing the isolated cells with a non-endogenous expression vector that includes a nucleic acid sequence encoding a Glucose Transporter 5 (GLUT5) amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the nucleic acid sequence is any one of SEQ ID NOs: 3-5.

9. A method of treatment, comprising: isolating cells from a donor subject, wherein the cells are fibroblasts, myoblasts, osteocytes, chondrocytes, adipocytes, epithelial cells, or mesenchymal stem cells;transducing the isolated cells with a non-endogenous expression vector that includes a nucleic acid sequence encoding a Glucose Transporter 5 (GLUT5) amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the nucleic acid sequence is any one of SEQ ID NOs: 3-5; and administering the transduced cells to a recipient subject.

10. The method of claim 9, wherein the donor subject and the recipient subject are the same.

11. The method of claim 9, wherein the donor subject and the recipient subject are different.

12. The method of any one of claims 9-11, wherein the transduced mesenchymal stem cells differentiate into cartilage, bone and the fat.

13. A method for treating damaged or diseased tissue in a subject in need thereof comprising administering to the subject an effective amount of the engineered cell of any one of claims of 1-6 or the composition of claim 7.

14. The method of claim 13, wherein the damaged or diseased tissue comprises skin tissue.

15. The method of claim 14, wherein the damaged or diseased skin tissue is caused by wounds, ulcers, cuts, crush injuries, punctures, surgical intervention, burns, infections, or compromised skin grafts.

16. The method of claim 14 or 15, wherein the engineered cell or the composition is a fibroblast, an epithelial cell, or a mesenchymal stem cell.

17. The method of claim 13, wherein the damaged or diseased tissue comprises bone tissue.

18. The method of claim 17, wherein the damaged or diseased bone tissue is caused by wounds, crush injuries, breaks, fractures, osteoporosis, osteopenia, Paget’s disease, osteogenesis imperfecta, osteonecrosis, osteoarthritis, osteomyelitis, fibrous dysplasia, bone cancers, osteomalacia, rickets, rheumatoid arthritis, achondroplasia, hypocalcemia, hypercalcemia, hypochondroplasia, various dysplasias other than fibrous, hypophosphatasia, bone spurs, or brachydactyly.

19. The method of claims 17 or 18, wherein the engineered cell or the composition is an osteocyte or a mesenchymal stem cell.

20. The method of claim 13, wherein the damaged or diseased tissue comprises muscle tissue.

21. The method of claim 20, wherein the damaged or diseased muscle tissue is caused by diabetes, cardiac ischemia, atherosclerosis, heart disease, wounds, crush injuries, puncture wounds, burns, infections, sarcopenia, muscular dystrophy, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, malnutrition, aging, or coma.

22. The method of claims 20 or 21, wherein the engineered cell or the composition is a myocyte or a mesenchymal stem cell.

23. The method of any one of claims 13 to 22, wherein the engineered cell is administered pleurally, intravenously, subcutaneously, intranodally, intramuscularly, topically, intradermally, intrathecally, intrapleurally, intraosseously, or intraperitoneally.

24. The method of any one of claims 13 to 22, further comprising sequentially, separately, or simultaneously administering to the subject at least one additional therapy.

25. The method of claim 24, wherein the at least one additional therapy comprises one or more of verteporfin, growth factors (e.g., FGFs, BMPs, VEGF, PDGF), adhesamine, 8- Bromoadenosine 3', 5 '-cyclic monophosphate (8-Br-cAMP), N6-Benzoyladenosine-3', 5'- cyclic monophosphate (6-Bnz-cAMP), forskolin, SB216763, Valproic acid, CHIR99021, Repsox, Y-27632, peptidomimetics of the N-cadherin HAVD motif, dimethyloxalylglycine, trimebutine, phenamil, tesolvin El, kartogenin, duloxetine, phenelzine sulfate, tacrine, ethinyl estradiol, crotamiton, honokiol, trimebutine-maleate, piceid, bioceramics (e.g., hydroxyapatite, tricalcium phosphate, bisphosphonates, bioactive glass, akermanite), statins, strontium and vanadium compounds, flavonoids, Calcium silicate, curcumin, and pyrintegrin.

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