Patent Application
20200061124
This invention generally relates to medicine and drug screening. In alternative embodiments, provided are methods for making highly dedifferentiated and stem-like human cells from human umbilical vein endothelial cells (HUVECs) ectopically expressing integrin β3 (or ITGB3). In alternative embodiments, provided are methods for reprogramming endothelial cells into a dedifferentiated state and creating an induced pluripotent stem cell (iPSCs) by inducing αvβ3 clustering. In alternative embodiments, provided are methods for inducing αvβ3 clustering, and to accelerate or facilitate angiogenesis, tissue remodeling or repair, or wound healing, for example, to accelerate healing after an infarction.
The standardized approach to reprogram somatic cells requires the introduction of 4 genes known as the Yamanaka factors (Oct-4, Sox-2, Klf4 and c-Myc) into target cells using retroviruses. Following stable introduction of the Yamanaka factors into somatic cells (6 days), cellular reprogramming to induced pluripotent stem cells (iPSCs) takes approximately 30 days. Alternatively, the Yamanaka factors (Oct-4, Sox-2, Klf4 and c-Myc) and NANOG are master transcription factors associates with pluripotency, although expression of the Yamanaka factors is sufficient to drive pluripotency. Expression of the Yamanaka factors is associated with a more aggressive cancer phenotype, including more self-renewal, tumor initiation, anchorage-independence and drug resistance.
Integrin β3 (or ITGB3, also called CD61) is expressed on angiogenic endothelial cells and invasive tumor cells, and ligands binding to ITGB3 drives diverse cell signaling pathways. ITGB3 also can promote cell anchorage-independence, and can contribute to oncogenicity and metastatic potential and pregnancy-associated breast cancer. ITGB3 is enriched in metastatic cells, and may be involved in cancer cell remodeling by driving stemness and drug resistance through an αvβ3-KRAS-RalB complex. The integrin αvβ3 is preferentially expressed on tumor cell blood vessels. Tumor angiogenesis leads to chronic vascular remodeling.
Integrin β3 expression is absent on terminally differentiated endothelial cells (ECs) in vivo; integrin β3 is absent in quiescence. Angiogenic blood vessels in tumors or during development are highly positive for integrin β3 expression. Integrin β3 is highly expressed on proliferating ECs growing in full serum. Thick basement membrane, a terminal signal, initiates EC differentiation and the cells become αvβ3 (avb3) negative.
In alternative embodiments, provided are methods for:
comprising ectopically expressing integrin β3 in the somatic cell or the human endothelial cell, or expressing or overexpressing in the somatic cell or the human endothelial cell a heterologous integrin β3,
wherein optionally the human endothelial cell is a human umbilical vein endothelial cell (HUVEC),
and optionally the pluripotency gene is a NANOG, OCT4, SOX2, and/or KLF4 gene,
and optionally the dedifferentiated or reprogrammed human endothelial cell loses its endothelial identity, optionally comprising loss of CD31, VWF, VE-cadherin, and VEGFR2, and gain of pluripotency markers such as NANOG, OCT4, SOX2, and KLF4,
and optionally ectopic expression of the integrin β3 in the somatic cell or the human endothelial cell is by transduction of a vector or a virus, optionally an adenovirus or a lentivirus (having contained therein an integrin β3-expressing nucleic acid), and expressing the integrin β3.
In alternative embodiments, the methods further comprise inducing the dedifferentiated or reprogrammed human somatic or endothelial cell, or the induced pluripotent stem cell (iPSC), to express lineages markers associated with all three germ layers ectoderm, mesoderm and endoderm, and/or inducing the dedifferentiated human somatic or endothelial cell to differentiate to one or all three germ layers ectoderm, mesoderm and endoderm, optionally comprising placing or incubating the dedifferentiated human somatic or endothelial cell in spheroid forming conditions.
In alternative embodiments, provided are methods for:
comprising: clustering of cell surface αvβ3,
and optionally the clustering of cell surface αvβ3 is by use of a multivalent ligand that binds to either integrin αv, integrin β3 or integrin αvβ3 (exposure of the cell surface to the multivalent ligand),
and optionally the clustering of cell surface αvβ3 is by (comprises) use of, or the clustering of cell surface αvβ3 comprises administration to an individual in need thereof:
In alternative embodiments of the methods, the antibody or antibodies that can bind integrin αvβ3 and cluster αvβ3 on the cell surface, or the multivalent compound capable of clustering cell surface αvβ3, is/are:
In alternative embodiments, the methods further comprise inducing the dedifferentiated or reprogrammed human somatic or endothelial cell, or the induced pluripotent stem cell (iPSC), to express lineages markers associated with all three germ layers ectoderm, mesoderm and endoderm, and/or inducing the dedifferentiated human somatic or endothelial cell to differentiate to one or all three germ layers ectoderm, mesoderm and endoderm, optionally comprising placing or incubating the dedifferentiated human somatic or endothelial cell in spheroid forming conditions.
In alternative embodiments, provided are method for:
comprising ectopically expressing integrin 133 (HUVEC 133+) or expressing in the human somatic cell or the human endothelial cell a heterologous integrin 133 (HUVEC 133+) to generate a conditioned or altered media, and culturing or exposing the human somatic cell or the human endothelial cell to the conditioned or altered media,
wherein optionally the human endothelial cell is a human umbilical vein endothelial cell (HUVEC),
and optionally the pluripotency gene is a NANOG, OCT4, SOX2, and/or KLF4 gene,
and optionally the dedifferentiated or reprogrammed human endothelial cell loses its endothelial identity, optionally comprising loss of CD31, VWF, VE-cadherin, and VEGFR2, and gain of pluripotency markers such as NANOG, OCT4, SOX2, and KLF4,
and optionally ectopic expression of the integrin 133 (HUVEC 133+) in the human somatic cell or the human endothelial cell is by transduction of a vector or a virus, optionally a lentivirus, expressing the integrin β3 (having contained therein integrin 133 (HUVEC 133+)-expressing nucleic acid).
In alternative embodiments, the methods further comprise inducing the dedifferentiated or reprogrammed human somatic or endothelial cell, or the induced pluripotent stem cell (iPSC), to express lineages markers associated with all three germ layers ectoderm, mesoderm and endoderm, and/or inducing the dedifferentiated human somatic or endothelial cell to differentiate to one or all three germ layers ectoderm, mesoderm and endoderm, optionally comprising placing or incubating the dedifferentiated human somatic or endothelial cell in spheroid forming conditions.
In alternative embodiments, provided are Uses of a human somatic cell or a human endothelial cell ectopically expressing an integrin β3 or expressing a heterologous integrin β3 to:
In alternative embodiments, provided are Uses of a human somatic cell or a human endothelial cell ectopically expressing an integrin 133 (HUVEC 133+) or expressing a heterologous integrin 133 (HUVEC 133+) to
In alternative embodiments, provided are compositions capable of clustering cell surface αvβ3, optionally a multivalent ligand that binds to either integrin αv, integrin β3 or integrin αvβ3 and is capable of clustering cell surface αvβ3, for use in:
and optionally the clustering of cell surface αvβ3 is by (comprises) use of, or the clustering of cell surface αvβ3 comprises administration to an individual in need thereof:
In alternative embodiments, provided are Uses of a composition capable of clustering cell surface αvβ3, optionally use of a multivalent ligand that binds to either integrin αv, integrin β3 or integrin αvβ3 and is capable of clustering cell surface αvβ3, in the manufacture of a medicament for:
and optionally the clustering of cell surface αvβ3 is by (comprises) use of, or the clustering of cell surface αvβ3 comprises administration to an individual in need thereof:
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Like reference symbols in the various drawings indicate like elements.
Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.
In alternative embodiments, provided are methods for making highly dedifferentiated and stem-like human cells from human umbilical vein endothelial cells (HUVECs) ectopically expressing integrin β3 (or ITGB3, also called CD61). In alternative embodiments, provided are methods for inducing undifferentiation and stemness by increasing expression of or ectopically expressing integrin β3, and for differentiating cells by decreasing expression of integrin β3. In alternative embodiments, provided are methods comprising ectopic expression of integrin β3 to: drive NANOG and OCT4 expression; reprogram and transform endothelial cells (ECs), which then lose endothelial markers and/or gain stem markers; generate IPS-like (induced pluripotent stem cell (iPSC)) colony formation. Ectopic expression of integrin β3 in HUVECs can convert these cells to pluripotent stem cells. In alternative embodiments, provided are methods comprising ectopic expression of integrin β3 followed by further differentiating the cells to a particular lineage, e.g., to a neural-ectodermal fate, e.g., differentiation to a neuron. While the invention is not limited by any particular mechanism of action, re-programming by ectopic expression of integrin β3 may be driven by a RAS complex. In alternative embodiments, the integrin β3 is ectopically expressing in a cell by use of a vector, e.g., a lentiviral vector.
Also provided are methods for making ectoderm, mesoderm, and endoderm cells from HUVECs ectopically expressing integrin β3.
Also provided are methods for making neural cells, or cells having neuronal-like morphology, from HUVECs ectopically expressing integrin β3.
Provided are methods for making cardiomyocytes, or cells having cardiomyocyte-like morphology, from HUVECs ectopically expressing integrin β3.
Provided are methods for the production of pluripotent or multipotent stem cells comprising expressing integrin β3 (or b3) in primary human endothelial cells. In alternative embodiments, provided are methods using a single gene, integrin β3, to reprogram somatic cells.
In alternative embodiments, provided are methods for reprogramming endothelial cells into a dedifferentiated state and creating an induced pluripotent stem cell (iPSCs) by inducing αvβ3 clustering. In alternative embodiments, provided are methods for promoting or initiating endothelial remodeling by inducing αvβ3 clustering, where the inducing of αvβ3 (avb3) clustering can lead to increased or enhanced tissue remodeling and/or repair following, e.g., myocardial infarction, stroke, diabetic ulcers, injury (e.g., traumatic or surgical) and other ischemic conditions or diseases. In alternative embodiments, provided are methods for αvβ3 (avb3) clustering comprising administration of e.g., multivalent antibodies or other multivalent ligands that bind to can cause clustering of αvβ3, including multivalent peptides that bind to can cause clustering of αvβ3 or αvβ3 polypeptide ligands, lectins or viral coat proteins that bind to can cause clustering of αvβ3, hydrogels or other polymers that can cause clustering of αvβ3, extracellular matrix (ECM) polymers or polypeptides that can cause clustering of αvβ3, and the like. In alternative embodiments, compositions that can cause clustering of αvβ3 used to practice embodiments provided herein include tri-, quad- or penton (5)-comprising (or more) tripeptide Arg-Gly-Asp (RGD) sites capable of binding αvβ3; polymers, e.g., hydrogels or ECM proteins, comprising multi-RGD peptide sites for clustering of αvβ3. In alternative embodiments, compositions that can cause clustering of αvβ3 used to practice embodiments provided herein include polysaccharides, lectins such as galectin-3, and extracellular matrix (ECM) proteins, some of which are involved in wound healing, including e.g., vitronectin, fibrinogen, and fibronectin.
While the invention is not limited by any particular mechanism of action, the clustering of αvβ3 causes stem-like reprogramming of endothelial cells, which increases endothelial cell plasticity during angiogenesis, which leads to increased or enhanced vascularization and tissue modeling and/or repair following, e.g., myocardial infarction, stroke, diabetic ulcers, injury (e.g., traumatic or surgical) and other ischemic conditions or diseases.
We have demonstrated that αvβ3 (avb3) is the endothelial cell receptor that mediates vascular remodeling on intact heart-derived extracellular matrix (ECM) (hydrogel); and that when compositions capable of clustering αvβ3 (avb3) (e.g., compositions that are multivalent ligands, or greater than bivalent ligands, to αvβ3 (avb3)) are injected into the heart, after vascular injury such as a myocardial infarction (MI), this promotes an improved post-MI outcome and results in less heart damage due to increased neovascularization. Accordingly, in alternative embodiments, by promoting αvβ3 (avb3) multivalent clustering with one or more of agents, e.g., by administration of multivalent ligands, or greater than bivalent ligands, to αvβ3 (avb3)), provided are methods for improving or accelerating angiogenesis in a tissue (e.g., the heart, brain) following an ischemic injury, thereby improving tissue remodeling and minimizing long term tissue injury or damage. In alternative embodiments, methods as provided herein are applied (administered) to patients with injuries, stroke or myocardial infarction (MI), e.g., provided herein are methods for improving tissue remodeling and/or minimizing tissue injury or damage due to e.g., strokes, MIs or other tissue injuries causing ischemia or ameliorated by improving tissue remodeling and accelerating vascularization. In alternative embodiments, methods as provided herein are applied (administered) to individuals, e.g., patients, to enhance or accelerate wound healing.
Provided herein are data showing: that a specific amount or level of ectopic integrin β3 expression is required for optimal “clustering” of αvβ3 on the cell surface (see e.g.,
In alternative embodiments, methods as provided herein, e.g., for initiating or accelerating tissue regeneration, enhancing or accelerating wound healing, improving tissue remodeling and/or minimizing tissue injury or damage due to e.g., strokes, MIs or other tissue injuries causing ischemia or ameliorated by improving or enhancing tissue remodeling and accelerating vascularization comprise use of ECMs such as tissue-derived ECMs, ECM-derived hydrogels or equivalents, e.g., using decellularized myocardial matrix hydrogels, e.g., from an individual or an animal, e.g., from a human or a porcine source. In alternative embodiments, the decellularized myocardial matrix hydrogels create a microenvironment for cardiac regeneration; in vivo experiments have demonstrated improved ventricular function, increased cardiac muscle, and cellular recruitment after myocardial infarction.
In alternative embodiments, a decellularized myocardial matrix hydrogel used to practice embodiments as provided herein comprises isolation of animal or human heart tissue, generating decellularized myocardium, lyophilizing and milling extracellular matrix material, and then creating a hydrogel; e.g., as described in Wang et al (2016, Jan. 15) Adv Drug Deliv Rev.; vol 96:77-82.
In alternative embodiments, provided are methods for reprogramming and dedifferentiating normal HUVEC to a pluripotent state by exposing or incubating these cells with conditioned media from HUVEC ectopically expressing integrin 133.
In alternative embodiments, provided are methods comprising administration to an individual in need thereof a multivalent composition capable of clustering αvβ3 on a cell, e.g., an endothelial cell, surface, wherein exemplary multivalent compositions comprise Extracellular Matrix Materials (ECMs), including as tissue derived-ECM, decellularized ECMs. In alternative embodiments, ECMs, or ECM-comprising compositions or materials used to practice methods as provided herein comprise individual αvβ3-clustering components of ECMs or mixtures thereof, including e.g., vitronectin, fibrinogen and/or fibronectin, also including vitronectin, fibrinogen or fibronectin or fragments thereof, which can be derived from recombinant, synthetic, or tissue or organ sources.
In alternative embodiments, ECMs used to practice methods as provided herein, or methods for making or using the ECMs, include ECMs and methods as described, e.g., in U.S. Pat. Nos. 9,801,983; 9,801,976 and 9,801,975; 9,801,910 (describing methods of making tissue-derived ECM derived from decellularized tissue); 9,795,713 (describing methods of manufacturing bioactive gels from ECM); 9,789,224 (describing digested, decellularized extracellular matrix derived from cardiac tissue); 9,788,821 (describing modified ECMs); 9,744,265 (describing cardiac fibroblast-derived 3-dimensional ECMs); 9,623,051 and 9,572,911 (describing methods of making decellularized ECMs); 8,691,276 (describing solubilized ECMs useful as cell growth scaffolds); or U.S. Pat App Pub nos: 20170312394 (describing an emulsified or injectable ECM); 20170173217 (describing methods for preparing sterilized, gelled, solubilized ECMs); 20170128624, 20170020927 and 20160279170 (describing methods of making decellularized ECMs); 20160354447 (describing compositions and methods using engineered cardiac fibroblast-derived 3-dimensional ECMs); 20160166735 (describing making injectable ECMs).
In alternative embodiments, provided are methods comprising administration to an individual in need thereof a multivalent composition capable of clustering αvβ3 on a cell, e.g., an endothelial cell, surface, wherein exemplary multivalent compositions comprise hydrogels such as tissue derived-ECM hydrogels, decellularized tissue hydrogels, and the like.
In alternative embodiments, any hydrogel capable of clustering αvβ3 on a cell, e.g., an endothelial cell, surface can be used to practice methods as provided herein, including for example, hydrogels complexed with multi-RGD peptides or anti-αvβ3 antibodies.
In alternative embodiments, hydrogels used to practice methods as provided herein, or methods for making or using the hydrogels, include hydrogels and methods as described, e.g., in the publications WO/2014/008400, WO/2015/136370, WO/2015/138514 and WO/2017/120092, describing for example PuraStat™ or PuraMatrix™ hydrogels, and/or U.S. Pat. Nos. 9,831,010; 9,814,779; 9,782,490; 9,763,968; 9,688,741; 9,364,412; 8,546,338; or 7,884,185; or U.S. Pat App Pub nos: 20170333304; 20170327813; 20170326275; 20170312368; 20170307598; 20170304499; 20170281781; 20170274082; 20160280827; 20130338084.
In alternative embodiments, provided are methods comprising the in vitro or in vivo clustering of cell surface αvβ3 by use of an antibody or antibodies that can bind integrin αvβ3 and cluster αvβ3 on the cell surface, or by use of a first antibody that can bind integrin αvβ3 and a second antibody that can bind to the first antibody such that the antibody binding clusters αvβ3 on the cell surface. In alternative embodiments, the antibody or antigen binding fragment thereof can bind to any portion of the integrin αvβ3 protein, whether it be to integrin αv alone, β3 alone, or to a conformational integrin αvβ3 immunogen.
In alternative embodiments, an antibody for practicing methods as provided herein can comprise a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, e.g., αvβ3, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. In alternative aspects, an antibody for practicing methods as provided herein includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.”
Methods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N Y (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
In alternative embodiments, methods as provided herein use “humanized” antibodies, including forms of non-human (e.g., murine) antibodies that are chimeric antibodies comprising minimal sequence (e.g., the antigen binding fragment) derived from non-human immunoglobulin. In alternative embodiments, humanized antibodies are human immunoglobulins in which residues from a hypervariable region (HVR) of a recipient (e.g., a human antibody sequence) are replaced by residues from a hypervariable region (HVR) of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In alternative embodiments, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues to improve antigen binding affinity.
In alternative embodiments, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to improve antibody affinity or functional activity. In alternative embodiments, the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of Ab framework regions are those of a human immunoglobulin sequence.
In alternative embodiments, a humanized antibody used to practice this invention can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of or derived from a human immunoglobulin.
However, in alternative embodiments, completely human antibodies also can be used to practice this invention, including human antibodies comprising amino acid sequence which corresponds to that of an antibody produced by a human. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.
In alternative embodiments, antibodies used to practice methods as provided herein comprise “affinity matured” antibodies, e.g., antibodies comprising with one or more alterations in one or more hypervariable regions which result in an improvement in the affinity of the antibody for antigen; e.g., αvβ3, compared to a parent antibody which does not possess those alteration(s). In alternative embodiments, antibodies used to practice methods as provided herein are matured antibodies having nanomolar or even picomolar affinities for the target antigen, e.g., αvβ3. Affinity matured antibodies can be produced by procedures known in the art.
In alternative embodiments, provided are pharmaceutical compositions and formulations for practicing methods as provided herein, e.g., methods for inducing αvβ3 clustering in an individual in need thereof, e.g., to accelerate or facilitate angiogenesis, tissue remodeling or repair, or wound healing, for example, to accelerate healing after an infarction. In alternative embodiments, pharmaceutical compositions and formulations for practicing methods as provided herein comprise, e.g., an antibody or antibodies that can bind integrin αvβ3 and cluster αvβ3 on the cell surface, or a multivalent (greater than bi-valent) compound capable of clustering cell surface αvβ3.
In alternative embodiments, compositions used to practice the methods as provided herein are formulated with a pharmaceutically acceptable carrier. In alternative embodiments, the pharmaceutical compositions used to practice the methods as provided herein can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa. (“Remington's”).
Therapeutic agents used to practice the methods as provided herein can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Formulations of the compositions used to practice the methods as provided herein include those suitable for oral/nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
Pharmaceutical formulations used to practice the methods as provided herein can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
Aqueous suspensions can contain an active agent (e.g., a composition used to practice the methods as provided herein) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.
In practicing methods provided herein, the pharmaceutical compounds can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
In practicing methods provided herein, the pharmaceutical compounds can also be delivered as nanoparticles or microspheres for regulated, e.g., fast or slow release in the body. For example, nanoparticles or microspheres can be administered via intradermal injection of the desired composition, which slowly releases subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674. Nanoparticles can also be given intravenously, for example nanoparticles with linkage to biological molecules as address tags could be targeted to specific tissues or organs.
In practicing methods provided herein, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
The pharmaceutical compounds and formulations used to practice the methods as provided herein can be lyophilized. Provided are a stable lyophilized formulation comprising a composition as provided herein, which can be made by lyophilizing a solution comprising a pharmaceutical as provided herein and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. patent app. no. 20040028670.
The compositions and formulations used to practice the methods as provided herein can be delivered by the use of liposomes or nanoliposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, e.g., liver cells, or are otherwise preferentially directed to a specific organ, e.g., liver, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587.
Also provided are nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds used to practice the methods as provided herein, e.g., to deliver compositions used to practice methods as provided herein (e.g., an antibody or antibodies that can bind integrin αvβ3 and cluster αvβ3 on the cell surface, or a multivalent (greater than bi-valent) compound capable of clustering cell surface αvβ3) to mammalian, e.g., heart, brain, skin, tenon or other tissues or organs, in vivo, in vitro or ex vivo. In alternative embodiments, these compositions are designed to target specific molecules, including biologic molecules, such as polypeptides, including cell surface polypeptides, e.g., for targeting a desired cell type, e.g., heart, brain, skin, tenon or other tissues or organs.
Provided are multilayered liposomes comprising compounds used to practice methods as provided herein, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice methods as provided herein.
Liposomes can be made using any method, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., an antibody or antibodies that can bind integrin αvβ3 and cluster αvβ3 on the cell surface, or a multivalent (greater than bi-valent) compound capable of clustering cell surface αvβ3), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
In one embodiment, liposome compositions used to practice methods as provided herein comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a compound (e.g., an antibody or antibodies that can bind integrin αvβ3 and cluster αvβ3 on the cell surface, or a multivalent (greater than bi-valent) compound capable of clustering cell surface αvβ3) used to practice methods as provided herein to a desired cell type (e.g., a liver endothelial cell, a liver sinusidal cell, or any liver tissue in need thereof), as described e.g., in U.S. Pat. Pub. No. 20070110798.
Provided are nanoparticles comprising compounds (e.g., an antibody or antibodies that can bind integrin αvβ3 and cluster αvβ3 on the cell surface, or a multivalent (greater than bi-valent) compound capable of clustering cell surface αvβ3) used to practice methods as provided herein in the form of active agent-containing nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment, provided are nanoparticles comprising a fat-soluble active agent used to practice a method as provided herein or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.
In one embodiment, solid lipid suspensions can be used to formulate and to deliver compositions used to practice methods as provided herein to e.g., mammalian heart, brain, skin, tenon or other tissues or organs in vivo, in vitro or ex vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.
Provided are products of manufacture and kits for practicing methods as provided herein, e.g., methods for inducing αvβ3 clustering in an individual in need thereof, e.g., to accelerate or facilitate angiogenesis, tissue remodeling or repair, or wound healing, for example, to accelerate healing after an infarction. In alternative embodiment, products of manufacture and kits include instructions for practicing methods as provided herein. In alternative embodiment, products of manufacture and kits comprise compositions for practicing methods as provided herein, e.g., an antibody or antibodies that can bind integrin αvβ3 and cluster αvβ3 on the cell surface, or a multivalent (greater than bi-valent) compound capable of clustering cell surface αvβ3.
Ectopic Expression of Integrin β3 in HUVECs Drives the Up-Regulation of Pluripotency Genes and Converts these Cells into a Highly Dedifferentiated and Stem-Like State
We discovered that ectopic expression of integrin β3 (or ITGB3) in HUVECs drives the up-regulation of several known pluripotency genes (NANOG, OCT4, SOX2, and KLF4), and, based on these finding, provided are methods for making highly dedifferentiated and stem-like human cells by ectopic expression of integrin β3 in human endothelial cells to convert these cells into a highly dedifferentiated and stem-like state. That HUVECs with ectopic β3 are converted to a dedifferentiated state is evidenced by their loss of several distinguishing markers associated with endothelial identity, including CD31, VWF, VE-cadherin, and VEGFR2, and the gain of pluripotency markers such as NANOG, OCT4, SOX2, and KLF4.
Similar to induced pluripotent stem cells, HUVEC with ectopic integrin β3 (or ITGB3) expression underwent both spontaneous and directed differentiation to other cell types. When pluripotent stem cells are placed in spheroid forming conditions, they spontaneously express lineages markers associated with all three germ layers (ectoderm, mesoderm, and endoderm). Growing HUVEC β3 cells under these same conditions produced the spontaneous induction of lineage markers associated with all three germlines. Thus, provided are methods for making ectoderm, mesoderm, and endoderm cells from HUVECs comprising ectopically expressing β3 in HUVECs and growing or incubating the cells in spheroid forming conditions, thereby inducing all three germlines.
To further test whether HUVEC β3 could be directly differentiated toward another cell type, we cultured our cells under neuronal differentiating conditions. By day 12, cells acquired a neuronal-like morphology and began to express early neuro-ectodermal lineage markers, and lost expression of pluripotency genes NANOG and OCT4. Furthermore, by day 22 cells began to express mature neuronal markers. Thus, provided are methods for making neural cells, or cells having neuronal-like morphology, from HUVECs ectopically expressing integrin β3 (or ITGB3).
In a second experiment, we cultured cells in cardiomyocyte differentiating conditions, and by day 14, the cells began to express cardiomyocyte markers. Thus, provided are methods for making cardiomyocytes, or cells having cardiomyocyte-like morphology, from HUVECs ectopically expressing integrin β3 (or ITGB3).
In alternative embodiments, provided are methods for the production of pluripotent or multipotent stem cells comprising expressing integrin β3 (or b3) in primary human endothelial cells, e.g., by using transduction of a vector or a virus or the like, e.g., a lentivirus transduction, upon which the primary endothelial cells become reprogrammed into stem-like cells after 12-15 days. Ectopic expression of ITGB3 reprograms human umbilical vein endothelial cells (HUVEC) into a dedifferentiated stem cell-like state. This is characterized by the expression of pluripotency genes NANOG, OCT4, SOX2, KLF4 and a loss of endothelial marker expression (CD31, VWF, VE-Cadherin, and VEGFR2). These cells have been demonstrated to differentiate into neuronal and cardiomyocyte cells.
In alternative embodiments, provided are methods improving on a standardized approach to reprogram somatic cells requiring the introduction of 4 genes known as the Yamanaka factors (Oct-4, Sox-2, Klf4 and c-Myc) into target cells using retroviruses; following stable introduction of the Yamanaka factors into somatic cells (6 days), cellular reprogramming to induced pluripotent stem cells (iPSCs) takes approximately 30 days. Provided herein are methods for reprogramming somatic cells, e.g., reprogramming endothelial cells, with the introduction of a single gene, ITGB3 and which in some embodiments can be done in a shorter period of time, for example, between about 12 to 20 days versus (vs.) 30 days. Provided are data demonstrating that ITGB3 strongly induces Oct-4, Sox-2 and Klf4, three of the four Yamanaka factors, and this observation supports this invention's finding that high levels of ITGB3, or increasing integrin β3 (or ITGB3) in a cell, are sufficient to reprogram endothelial cells.
Additionally, the cultivation of iPSCs requires expensive cell growth media and feeder cells, and in terms of costs, an alternative embodiment provided herein requires less expensive cell growth media, e.g., standard endothelial vs. stem cell media, such that costs are reduced by approximately 2 fold. Provided herein are methods comprising the ectopic expression of one gene ITGB3 (versus the overexpression of four genes Oct-4, Sox-2, Klf4 and c-Myc) to reprogram endothelial cells. In summary, in alternative embodiments, provided are methods requiring less time and expenses with the use of a single gene to reprogram somatic cells.
Finding that ITGB3 strongly induces Oct-4, Sox-2, Nanog and Klf4, all potent reprogramming factors, we discovered that a high threshold level of ITGB3 facilitates reprogramming in endothelial cells. We have documented clustering and induction of activated Ras by ITGB3 overexpression in endothelial cells. Downstream of Ras, we observed a strong activation of Akt and the suppression of activated extracellular signal-regulated kinase (ERK). It has been reported that ERK activation impairs cellular reprogramming while Akt promotes it. Akt is known to phosphorylate and stabilize Oct-4. Once stabilized, Oct-4 then cooperates with Sox-2 to maintain a stem phenotype by inducing Nanog. We found that high ITGB3 levels facilitate the clustering of Ras family members, that then activates Akt to stabilize the pluripotency regulator Oct-4.
In alternative embodiments, provided are methods that are a more effective and efficient means for the generation of pluripotent or multipotent stem cells. In alternative embodiments, patient-derived primary cells are used to create personalized therapeutics, to test drug responsiveness on patient-derived cells such as neurons or cardiomyocytes, or for transplantation.
Clustering of Integrin αvβ3 Drives the Up-Regulation of Pluripotency Genes, Reprogram Endothelial Cells into a Dedifferentiated State, and Creates an Induced Pluripotent Stem Cell
We have discovered that clustering of integrin αvβ3 (through an integrin cross-linking assay) drives the up-regulation of several known pluripotency genes (NANOG, OCT4, SOX2, and KLF4) in Human Umbilical Vein Endothelial Cells (HUVEC) (see figure), and that αvβ3 clustering alone is sufficient to reprogram endothelial cells into a dedifferentiated state, and create an induced pluripotent stem cell (iPSCs). In alternative embodiments, clustering of αvβ3 yields (makes, generates) iPSCs with similar biologic properties to what is achieved by current technologies that drive overexpression of pluripotency genes (OCT-4, SOX2, KLF4, and C-myc). In alternative embodiments, provided are methods that circumvent the known caveats of malignant transformation associated with current gene transduction technologies.
In alternative embodiments, these processes can be facilitated by any multivalent ligand that binds to either integrin αv or β3, and clusters them on the cell surface. In alternative embodiments, several ways of achieving this are using: an antibody integrin cross-linking assay (both adherent and in suspension); a pentavalent molecule such as a lectin, e.g., Galectin-3, to cluster αvβ3; a mimetic RGD peptide (αvβ3 binding motif) to bind and cluster αvβ3; manganese cations (Mn2+), which are known to activate and cluster αvβ3.
In alternative embodiments, provided are methods for the production of pluripotent or multipotent stem cells from readily accessible sources of HUVEC and/or adult somatic cells. Briefly, while the invention is not limited by any particular mechanism of action, αvβ3 integrin clustering drives the up-regulation of pluripotent stem genes (NANOG, OCT4, SOX2, KLF4) which will reprogram somatic cells into a dedifferentiated state. From this state, provided are methods that differentiate cells using appropriate conditions. For example, provided are methods to differentiate HUVECs to neurons and cardiomyocytes.
In alternative embodiments, provided are methods that improve on known standardized approaches to reprogram somatic cells which require the introduction of 4 genes known as the Yamanaka factors (Oct-4, Sox-2, Klf4 and c-Myc) into target cells using retroviruses; where following stable introduction of the Yamanaka factors into somatic cells (6 days), cellular reprogramming to induced pluripotent stem cells (iPSCs) takes approximately 30 days. In alternative embodiments, provided are methods that can reprogram endothelial cells with the simple manipulation of an integrin αvβ3 on the cell surface.
Current methods for stem cell reprogramming require some form of genetic manipulation, and this comes with many caveats, including genetic instability and the possibility of tumor formation. In alternative embodiments, provided are methods that completely αvoid this possibility. Current methods for the cultivation of iPSCs require expensive cell growth media and feeder cells. In alternative embodiments, provided are methods that require less expensive cell growth media (standard endothelial vs stem cell media) such that costs are reduced by approximately 2 fold. In summary, in alternative embodiments, provided are methods for the creation of pluripotent stem cells that involved no genetic manipulation or special and expensive media conditions.
In alternative embodiments, provided are methods that cluster avb3 (versus genetic manipulation by over expressing four genes (oct-4, Sox-2, Klf4 and c-Myc) used by all current methods). In alternative embodiments, provided are methods that can create stem cells that do not require genetic manipulation, which has many potential adverse side effects. In alternative embodiments, provided are methods comprising only temporary clustering of avb3 on the cell surface; this exemplary process can αvoid all the pitfalls associated with the currents methods for stem cell production. In alternative embodiments, provided are methods that are more effective and efficient way for the generation of pluripotent or multipotent stem cells. In alternative embodiments, provided are methods for using patient-derived primary cells to create personalized therapeutics, to test drug responsiveness on patient-derived cells such as neurons or cardiomyocytes, or for transplantation.
Conditioned Media from HUVEC Ectopically Expressing Integrin 133 is Capable of Reprogramming and Dedifferentiating Normal HUVEC to a Pluripotent State
We have discovered that Human umbilical vein endothelial cells (HUVEC) ectopically expressing integrin 133 (HUVEC 133+) conditions its media. This altered or conditioned media (CM), when cultured with normal HUVEC, is capable of reprogramming and dedifferentiating these cells to a pluripotent state, resulting in the loss of endothelial markers (CD31, VWF, and CD34), and the gain pluripotency gene expression (OCT4, NANOG, SOX2, KLF4) (see attached figure). HUVEC 133 CM alone is sufficient to reprogram endothelial cells into a dedifferentiated state creating an induced pluripotent stem cell (iPSCs). In alternative embodiments, provided are methods for making and using HUVEC 133 CM to yield iPSCs having similar biological properties achieved with conventional stem cell technologies that drive overexpression of pluripotency genes (OCT4, SOX2, KLF4, and C-MYC).
In alternative embodiments, provided are methods that require no genetic manipulation of our target cells, circumventing the known caveats of malignant transformation associated with current gene transduction technologies.
In alternative embodiments, provided are methods for the production of pluripotent or multipotent stem cells from readily accessible sources of Human Umbilical Vein Endothelial Cells (HUVEC). In alternative embodiments, provided are methods comprising collection of conditioned media (CM) from HUVEC ectopically expressing integrin 133, and culturing them with normal HUVEC to drive the up-regulation of pluripotent stem genes (NANOG, OCT4, SOX2, KLF4), and loss of endothelial markers (CD31, VWF, and CD34). In alternative embodiments, this treatment reprograms HUVEC into a dedifferentiated, pluripotent state, and these cells can then be differentiated using appropriate conditions; for example, dedifferentiated HUVECs can be induced to differentiate to neurons and cardiomyocytes.
In alternative embodiments, provided are methods that improve on the standardized approach to reprogram somatic cells which requires the introduction of 4 genes known as the Yamanaka factors (Oct-4, Sox-2, Klf4 and c-Myc) into target cells using retroviruses, and following stable introduction of the Yamanaka factors into somatic cells (6 days), cellular reprogramming to induced pluripotent stem cells (iPSCs) takes approximately 30 days. In alternative embodiments, provided are methods that can reprogram endothelial cells with a simple exposure to conditioned media. All other methods for stem cell reprogramming require some form of genetic manipulation, which comes with many caveats, including genetic instability and the possibility of tumor formation. In alternative embodiments, provided are methods that completely αvoid this possibility.
Known methods for the cultivation of iPSCs require expensive cell growth media and feeder cells. In alternative embodiments, provided are methods requiring less expensive cell growth media (standard endothelial vs stem cell media) such that costs are reduced by approximately 2 fold. In summary, in alternative embodiments, provided are methods for the creation of pluripotent stem cells that involve no genetic manipulation or special and expensive media conditions. In alternative embodiments, provided are methods that involve exposing HUVEC to conditioned media versus genetic manipulation by over expressing four genes (oct-4, Sox-2, Klf4 and c-Myc), as used by all current methods.
While the invention is not limited by any particular mechanism of action, given that conditioned media actively induces the expression of pluripotency genes Oct-4, Nanog, Sox-2, and Klf-4, all potent reprogramming factors, conditioned media from HUVEC 3 cells facilitates endothelial reprogramming. In alternative embodiments, provided are methods that involve exposing HUVEC to conditioned media for a limited time. In alternative embodiments, provided are methods that are an effective and efficient way to generate pluripotent or multipotent stem cells. In alternative embodiments, provided are methods using patient-derived primary cells to create personalized therapeutics, to test drug responsiveness on patient-derived cells such as neurons or cardiomyocytes, or for transplantation.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/430,777, Dec. 6, 2016. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.
This invention was made with government support under National Institutes of Health (NIH) grant no. T32 CA121938. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/064250 | 12/1/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62430777 | Dec 2016 | US |
