INHIBITION OF FOLLISTATIN
20210207135 ยท 2021-07-08
Inventors
Cpc classification
C12N2750/14143
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
G01N2800/52
PHYSICS
A61K31/713
HUMAN NECESSITIES
International classification
C12N15/113
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
G01N33/50
PHYSICS
Abstract
Provided herein are methods for modulating follistatin, such as inhibiting follistatin, suppressing the production of follistatin, reducing the level of follistatin, inhibiting the function of follistatin, or a combination thereof. The method can include administration of a compound that acts to modulate follistatin. In one embodiment, the compound is administered to a patient having or at risk or having a disease or condition selected from diabetes, pre-diabetes, metabolic syndrome, insulin resistance, dementia, and obesity, and optionally the disease or condition is prevented, treated, ameliorated, or a combination thereof.
Claims
1. A method for inhibiting follistatin comprising delivering to a patient an effective amount of a polynucleotide that suppresses the production of follistatin.
2. A method for inhibiting follistatin comprising delivering to a patient in need thereof a therapeutically-effective amount of a polynucleotide that suppresses the production of follistatin.
3. The method of claim 1 or 2 wherein the polynucleotide comprises an siRNA molecule.
4. The method of claim 1 or 2 wherein the polynucleotide further comprises a targeting agent.
5. The method of claim 4 wherein the targeting agent comprises an N-acetylgalactosamine (GalNAc) moiety.
6. The method of claim 2 wherein the patient has or is at risk for having a disease or condition selected from diabetes, pre-diabetes, metabolic syndrome, insulin resistance, dementia, and obesity.
7. The method of claim 6 wherein disease is prevented, treated, or ameliorated.
8. The method of claim 1 or 2 wherein the polynucleotide is delivered systemically.
9. The method of claim 7 wherein the polynucleotide is delivered intravenously.
10. The method of claim 1 wherein the polynucleotide comprises a DNA molecule encoding an siRNA molecule.
11. A method for delivering to a patient an effective amount of a compound that inhibits follistatin.
12. The method of claim 11, wherein the compound is selected from a group consisting of at least one of the following: an antibody, antibody fragment, FAb fragment, FAb fragment, nanobody, small molecule, polynucleotide, RNAi, siRNA.
13. A method for inhibiting follistatin comprising delivering to a patient an effective amount of a compound that suppresses the production of follistatin.
14. A method for inhibiting follistatin comprising delivering to a patient an effective amount of a compound that reduces the levels of follistatin.
15. A method for inhibiting follistatin comprising delivering to a patient an effective amount of a compound that inhibits the function of follistatin.
16. A method for treating a patient comprising delivering to a patient an effective amount of a compound that suppresses the production of follistatin.
17. A method for treating a patient comprising delivering to a patient an effective amount of a compound that inhibits follistatin.
18. A method of determining whether a compound is an inhibitor of Fst, comprising: providing a Test Cell which overproduces Fst and exhibits an increase in binding of Fst to a protein, relative to a Control cell which produces Fst at a lower level, and which exhibits a lesser amount of binding of Fst to the protein; exposing the Test Cell to the compound; and measuring the amount of Fst bound to the protein.
19. The method of claim 18 wherein the compound which binds to Fst comprises an anti-Fst antibody or activin.
20. The method of claim 18 or 19 wherein the control cell does not produce a detectable level of Fst.
21. A method of identifying a compound capable of reducing the level of expression from an Fst promoter in a mammalian cell, comprising: providing a Test Cell which contains the Fst promoter operably linked to a reporter gene such that increased expression of the Fst promoter sequence using a substance known to upregulate an endogenous Fst gene results in an increase in reporter protein levels; exposing the Test Cell to the compound; and determining whether an increase in reporter protein level in the Test Cell has occurred.
22. A method for identifying a compound that interferes with the ability of a Fst protein to promote insulin resistance, comprising: providing a Test Cell which expresses IRS1 and the p110 catalytic subunit of PIK3 ; exposing the Test Cell to serum comprising the Fst protein; exposing the Test Cell to the compound; and determining whether an increase in the interaction of the IRS1 with the p110 catalytic subunit has occurred.
23. A method for determining whether a compound is an inhibitor of Fst, comprising: providing a Test Cell which expresses AKT; exposing the Test Cell to serum comprising the Fst protein; exposing the Test Cell to the compound; and determining whether an increase in the phosphorylation of the AKT has occurred.
24. A method for determining whether a compound is an inhibitor of Fst, comprising: providing a Test Cell which expresses hormone-sensitive lipase (HSL); exposing the Test Cell to serum comprising the Fst protein; exposing the Test Cell to the compound; and determining whether a decrease in the phosphorylation of the HSL has occurred.
25. The method of any one of claims 22-24 wherein the Test Cell is exposed to the serum after exposure to the compound.
26. The method of any one of claims 22-25 wherein the serum is from an insulin resistant LDKO-mouse.
27. The method of any one of claims 22-26 wherein the Test Cell is a differentiated 3T3L1-adipocyte.
28. The method of any one of claims 18-27 wherein the compound is a small molecule
29. The method of any one of claims 18-27 wherein the compound comprises a protein.
30. The method of any one of claims 18-29 wherein the protein comprises an antibody.
31. A method of identifying a polynucleotide that reduces expression of Fst, comprising: providing a Test Cell which produces Fst; exposing the Test Cell to the polynucleotide; and measuring the amount of the Fst in the cell.
32. The method of claim 31 wherein the polynucleotide comprises a double-stranded RNA molecule.
33. The method of claim 31 wherein the polynucleotide comprises a single-stranded RNA molecule.
34. The method of any one of claims 31-33 wherein the RNA molecule comprises at least 19 consecutive nucleotides that are complementary to a coding region encoding the Fst protein.
35. The method of any one of claims 18-34 wherein the Test Cell is a mammalian cell.
36. A method of restoring or enhancing insulin sensitivity in a cell comprising reducing or inhibiting Fst function.
37. The method of claim 36 wherein the cell is in vitro.
38. A method of treating a disease characterized by increased expression or activity of Fst, comprising reducing in a subject expression or activity of the Fst.
39. The method of claim 38 wherein the disease is a metabolic disease, diabetes, obesity, or a combination thereof.
40. The method of any one of claims 18-39 wherein the Fst is Fst288, Fst303, or Fst315.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0088]
[0089]
[0090]
DETAILED DESCRIPTION
[0091] This disclosure pertains to generalized methods of preventing, curing or inducing durable long-term remissions in patients with diabetes, metabolic disorders, central nervous system diseases, obesity, fertility and other human disorders in which an inappropriate level or functional activity of one or more follistatin variants contributes to the disease state. The disclosure is particularly concerned with follistatin and modulation of the activity of follistatin-mediated cellular signaling pathways as a mechanism for treating human disease due to its excessive production in the body and secretion into the circulation under certain conditions.
[0092] The disclosure is based on the recognition that the follistatin branch of the insulin/IGF signaling system coordinates important biochemical reactions and signaling pathways needed for proper function of peripheral insulin sensitive tissues and cells (especially in muscle and fat).
[0093] Experiments in genetically altered mice that lack follistatin or overexpress Fst reveal the essential role for Fst in peripheral insulin action and the role of Fst in the function, growth and survival of the organism. Dysregulation of Fst signaling, especially excess Fst expression or function at peripheral insulin sensitive tissues, including WAT and liver causes insulin resistance, excess hepatic glucose production and systemic glucose intolerance. Conversely, inhibition of the biological functioning of Fst or reducing its signaling potential, or blocking pathways that produce the protein or activating pathways that promote its degradation correct these problems.
[0094] Accordingly, the disclosure is directed to a general method for the treatment, cure, or prevention of various metabolic and related disorders, including diabetes, by reducing the level or functional activity of follistatin in a mammal in need thereof.
[0095] In one embodiment, the disclosure is directed to restoring or enhancing insulin sensitivity in a cell by reducing follistatin levels or activity. According to the disclosure, a disease or disorder characterized by elevated levels of follistatin can be treated by reducing follistatin levels or activity (or both). Such diseases include, but are not limited to metabolic disease, diabetes, dyslipidemia, obesity, female infertility, central nervous system disorders, Alzheimer's disease, and disorders of angiogenesis.
[0096] In another embodiment of the disclosure, upregulation of IRS2 function (Housey and White; 2003; Housey and Balash; 2014) can reduce Fst and improve WAT and peripheral insulin sensitivity. This would include activation of IRS2 or a complex that includes IRS2. Upregulation of IRS2 function is also accomplished by inhibition of phosphorylation of carboxy terminal serine residues of IRS2. Upregulation of IRS2 function can be accomplished by enhanced expression of IRS2 or by inhibition of degradation of IRS2. Increasing the expression and/or function of IRS2 will lead to a reduction in hepatic Fst levels and a concomitant reduction in the amount of hepatic Fst secreted into the circulation, which will thus improve WAT and peripheral insulin sensitivity and metabolic regulation.
[0097] In another embodiment, the disclosure is directed to a method of determining whether a compound is an inhibitor of Fst. In a cell-based assay, a Test Cell is provided which overproduces Fst and exhibits an increase in binding of an Fst-binding protein to Fstincluding a specific antibody that binds to Fstrelative to a Control cell which produces Fst at a lower level, or does not produce Fst at all, and which exhibits a lesser amount of binding of said protein to Fst. Small molecules that inhibit Fst are identified by measuring the amount of the Fst binding protein bound to Fst.
[0098] In another embodiment, the disclosure is directed to a method of identifying a compound capable of reducing the level of expression from an Fst promoter in a mammalian cell. In one such embodiment, a Test Cell is constructed which contains a construct comprising an Fst promoter operably linked to a reporter gene such that increased expression of the Fst promoter sequence using a substance known to be capable of upregulating the endogenous Fst gene results in an increase in a measurable characteristic of the Test cell resulting from increased expression of the reporter gene (and a corresponding increase in production of the reporter protein. Small molecules that inhibit Fst expression are identified by detecting a decrease in reporter gene activity (reporter protein production).
[0099] In another embodiment, the disclosure is directed to a method of identifying a compound capable of interfering with the function of Fst protein to promote WAT insulin resistance. In one such embodiment, a Test Cellfor example a differentiated 3T3L1-adipocyteis employed to screen for compounds that reverse the effect of serum from insulin-resistant mice containing Fst to promote insulin resistance. An ideal source of Fst-containing serum would be the insulin resistant LDKO-mice, which specifically lack hepatic Irs1 and Irs2. Alternatively, serum from insulin resistant mice overexpressing Fst in the liver can be used. In one embodiment the interaction of IRS1 with the p110 catalytic subunit of PI3K is measured in 3T3-L1 adipocytes exposed to the mouse serum from LDKO-mice. Compounds added to insulin stimulated 3T3-L1 adipocytes incubated with serum from LDKO-mice that increase the association between IRS1 and p110that is form more IRS1p110 complex during insulin stimulationwill be identified as compounds that inhibit Fst function.
[0100] In another embodiment an increase in insulin-stimulated phosphorylation of AKT is used to identify molecules that inhibit the function of Fst in 3T3-L1 adipocytes exposed to serum from insulin resistant LDKO-mice and lead to better insulin sensitivity through IRS1/IRS2.fwdarw.PI3K.fwdarw.AKT cascade. In another embodiment insulin stimulated dephosphorylation of hormone-sensitive lipase (HSL) is used to identify molecules that inhibit the function of Fst and lead to better insulin sensitivity through IRS1/IRS2.fwdarw.PI3K.fwdarw.AKT.fwdarw.PDE cascade in 3T3-L1 adipocytes incubated with serum from insulin resistant LDKO mice or other mice specifically designed to express and secrete hepatic Fst. Thus compounds that promote IRS1p110 complex formation, AKT phosphorylation, and/or HSL dephosphorylation in 3T3-L1 adipocytes or other cells incubated with serum from insulin resistant LDKO-mice with elevated circulating Fst reveal inhibitors of Fst function. Compounds identified by these embodiments of this disclosure are insulin sensitizing molecules for the treatment of metabolic disease, diabetes and its related disorders.
[0101] For the purposes of this disclosure, the following terms are defined as given below.
[0102] Follistatin, follistatin, Fst, an Fst polypeptide and an Fst protein refer to any isoform of a follistatin protein. Fst proteins are described herein. As used herein, the term follistatin or fst: refers to the secretory or membrane retained protein that binds activin or other TGF superfamily ligands. Follistatin includes Fst, Fst288, Fst303, Fst315, Fst317, Fst344, or any other form generated from alternative splicing of the Fst gene that retains function in a mammal.
[0103] Fst 16665837 or Fst gene or Fst mRNA refer to a nucleotide sequence encoding the follistatin (Fst) protein,
[0104] As used herein, the terms inhibitor and antagonist of Fst are used interchangeably, wherein Fst and Fst protein are identical.
[0105] An inhibitor of follistatin, which is identical to an inhibitor of Fst, is meant to include a compound that binds to Fst alone and reduces the level of Fst or inhibits the function of Fst, or a compound that binds to a complex comprising Fst and other Fst binding partner(s) (Fst binding partners include proteins such as myostatin, activin, and other non-proteinaceous molecules that bind to Fst) and wherein said compound cannot bind to the non-Fst binding partner(s) in the absence of Fst.
[0106] An inhibitor of follistatin expression, which is identical to an inhibitor of Fst expression is meant to include a compound that inhibits the expression of the Fst gene by any mechanism, including interference with the production of functional Fst mRNA or enhancing degradation of Fst mRNA.
[0107] For this disclosure, to state that a substance inhibit(s) follistatin means:the substance can bind to follistatin and reduce follistatin's activity in a cell, a tissue, the blood, or presence in the body; the substance can reduce or eliminate follistatin's functioning; the substance can reduce the amount or level of follistatin; and/or the substance can reduce the expression or production of follistatin. In order for a compound to inhibit follistatin or inhibit Fst, said compound must be either an inhibitor of Fst or an inhibitor of Fst expression.
[0108] Unless explicitly stated otherwise, an inhibitor, an antagonist and an inhibitor of follistatin are also synonymous. The inhibition by an inhibitor may be partial or complete. The terms bind(s), binding, and binds to have their ordinary meanings in the field of biochemistry in terms of describing the interaction between two substances (e.g., enzyme-substrate, protein-DNA, receptor-ligand etc.). As used herein, the term binds to is synonymous with interacts with in the context of discussing the relationship between a substance and its corresponding target protein or nucleic acid.
[0109] As used herein, the terms compound and substance are used interchangeably, and both terms refer to chemical agents and biological agents.
[0110] As used herein, the term chemical agent refers to substances that have a molecular weight up to, but not including, 2000 atomic mass units (Daltons). Such substances are sometimes referred to as small molecules.
[0111] As used herein, biological agents, are molecules which include proteins, polypeptides, and nucleic acids, and have molecular weights equal to or greater than 2000 atomic mass units (amu or Daltons), but not to exceed 990,000 amu.
[0112] As used herein, the term antibody refers to a protein or immunoglobulin produced in response to an antigen and can specifically bind the antigen. An antibody that specifically binds an antigen is one that interacts only with the epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope. An antibody that specifically binds to an epitope will, under the appropriate conditions, interact with the epitope even in the presence of a diversity of potential binding targets.
[0113] As used herein, the term antigen refers to the protein or peptide target having the epitope to which an antibody specifically binds.
[0114] As used herein, the term fragment refers to a portion of a polypeptide or polynucleotide. In one embodiment, a fragment retains the activity of the polypeptide or polynucleotide.
[0115] The term and/or means one or all of the listed elements or a combination of any two or more of the listed elements.
[0116] The words preferred and preferably refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.
[0117] The terms comprises and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
[0118] It is understood that wherever embodiments are described herein with the language include, includes, or including, and the like, otherwise analogous embodiments described in terms of consisting of and/or consisting essentially of are also provided.
[0119] Unless otherwise specified, a, an, the, and at least one are used interchangeably and mean one or more than one.
[0120] Conditions that are suitable for an event to occur, or suitable conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.
[0121] As used herein, providing in the context of a composition, an antibody, a nucleic acid, or a small molecule means making the composition, antibody, nucleic acid, or small molecule, purchasing the composition, antibody, nucleic acid, or small molecule, or otherwise obtaining the composition, antibody, nucleic acid, or small molecule.
[0122] Reference throughout this specification to one embodiment, an embodiment, certain embodiments, or some embodiments, etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.
[0123] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
[0124] According to the present disclosure, a therapeutically effective amount of one or more compounds/substances that inhibit, for instance, the function or level of expression of follistatin protein (Fst) is administered to a mammal in need thereof. The term mammal as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets and farm animals.
Fst Proteins
[0125] The human FST gene includes six exons spanning 5329 bp on chromosome 5q11.2 and gives rise to two main transcripts of 1122 bp (transcript variant FST344) and 1386 bp (transcript variant FST317) (Grusch, M., 2010). The first exon encodes the signal peptide, the second exon the N-terminal domain and exons 3-5 each code for a follistatin module. Alternative splicing leads to use of exon 6A, which codes for an acidic region in FST344, or exon 6B, which contains two bases of the stop codon of FST317 (Shimasaki, S. et al., 1988).
[0126] Mature secreted follistatin protein exists in three main forms consisting of 288, 303, and 315 amino acids (Sugino, K. et al., 1993). The FST344 transcript gives rise to a protein precursor of 344 amino acids, which results in the mature 315 amino acid form (Fst315) after removal of the signal peptide. A fraction of Fst315 is further converted to the 303 amino acid form (Fst303) by proteolytic cleavage at the C-terminus. Signal peptide removal of FST317 leads to the mature 288 amino acid form of follistatin (Fst288). All forms of follistatin contain three follistatin domains (FSD) characterized by a conserved arrangement of 10 cysteine residues. The N-terminal subdomains of the FSD have similarity with EGF-like modules, whereas the C-terminal regions resemble the Kazal domains found in multiple serine protease inhibitors.
[0127] In one embodiment, the FST that is modulated is Fst315. An example of a mature human Fst315 protein is as follows:
TABLE-US-00001 (SEQIDNO:1) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVN DNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNK PRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPE LEVQYQGRCKKTCRDVFCPGSSTCWDQTNNAYCVTCNRICPE PASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCI KAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEP VCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTE EEEEDEDQDYSFPISSILEW. (aminoacids30-344ofGenbankaccession numberP19883.2)
[0128] An example of a follistatin precursor is available at Genbank accession number AAA35851.
[0129] An example of polynucleotide sequence encoding a mature human Fst315 protein is available at Genbank accession number AH001463.
Production of Fst Inhibitors
[0130] Inhibitors of the disclosure are prepared using a variety of approaches which are standard in the field and known to the skilled practitioner. Following the creation of such inhibitors, testing of the inhibitor for potential therapeutic efficacy may be performed using the detailed methods and insights described below, or by variations that are apparent to one of ordinary skill in the art. One approach to testing such inhibitors is the cell-based assay system described below. Other methods may be utilized. No limitation is intended with respect to how an Fst inhibitor is tested for therapeutic efficacy.
[0131] Polyclonal Antibodies (Abs). Polyclonal antibodies are prepared by immunizing an animal, such as a mouse, rat, hamster, guinea pig, rabbit, goat, sheep, chicken, or horse with a specific polypeptide or peptide fragments of Fst. Routes of administration for the immunization may include, but are not limited to intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, footpad, intranodal, or intrasplenic. To increase antigenicity, the peptide fragments might be linked to a carrier protein such as albumin or keyhole limpet hemocyanin. In a preferred embodiment, 30 ug of antigenic peptide fragment are used for immunization. After one or more immunizations of the recipient animal, sera is obtained and tested for the presence of antibodies to human follistatin using an enzyme-linked immunosorbent assay (ELISA). Antibodies which bind to follistatin may be used directly or, more preferably, purified to enhance utility using the cognate peptide immobilized on argaose-based resins. See, for example, Milstein Nature 266 (1977) 550; Kohler & Milstein Nature 256 (1975) 495; Antibodies A Lab Manual 1989; Rasmussen Biotechnol Lett 29 (2007) 845; Delahaut Methods 116 (2017) 4-11; Hanly ILAR 27 (1995) 93; Newcombe C, Newcombe A R J Chromatogr B Analyt Technol Biomed Life Sci 848 (2007) 2; Murphy Antibody Tech J 6 (2016)
[0132] Polyclonal Abs are then tested for potential therapeutic efficacy as discussed below. The animal's blood is collected, and a variety of techniques such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (MA), an immunohistochemical staining, etc. can be used to measure the polyclonal antibody's titer in antiserum. Polyclonal antibodies can be purified from the complex mixtures in the serum using chromatographic or non-chromatographic techniques. Using chromatography-based methods, antibodies can be separated by passing them through a solid phase (eg, silica resin or beads, monolithic columns, or cellulose membranes) and allowing the antibodies to bind or pass through depending on which chromatographic methods are being utilized. These methodologies include different separation techniques, such as affinity-tag binding, ion-exchange, size-exclusion chromatography, or immunoaffinity chromatography. Using non-chromatography-based approaches, precipitation, flocculation, crystallization, filtration, aqueous two-phase partitioning techniques, and any combination thereof can be employed.
[0133] Anti-Fst Monoclonal Abs. Monoclonal antibodies (mAbs) are prepared using standard methodologies. Briefly, animals are immunized as given above for polyclonal Ab preparation. After verification that the immunized animal is producing relevant antibodies according to the assays described above lymphocytes are harvested from the Ab-producing animal (such as a mouse) and fused with myeloma cells according to the method of Kohler and Milstein (1975). See also Kunert Appl Microbiol Biotechnol 100 (2016) 3451; Roque Biotechnol Prog 20 (2004) 639; Maynard Annu Rev Bio Eng 2 (2000) 339.
[0134] Clones producing individual mAbs are then tested using the assay methods described below for therapeutic efficacy.
[0135] Bi-specific Antibodies that target both Fst and an Fst-binding protein such as myostatin (mst), activin, or bone morphogenetic protein (bmp) may be prepared. The method of Roque Biotechnol Prog 20 (2004) 639, is instructive.
[0136] Humanized antibodies. It is preferable to humanize the mAbs prepared by any of the above-referenced approaches (or by another appropriate method) by replacement of their constant regions with the Fc domains of human antibodies. Such a replacement has been shown to generate more clinically useful Abs with a lower likelihood of inducing side effects such as the development of neutralizing Abs in the recipient which may render the therapeutic mAb less effective or ineffective. Humanization of mAbs is well-described. See, for example, Roque Biotechnol Prog 20 (2004) 639, Kipriyanov Mol Biotech 26 (2004) 39, and Maynard Annu Rev Bio Eng 2 (2000) 339.
[0137] Often starting with monoclonal antibodies from rodent origin, the DNA segments encoding the rodent's variable regions that are specific for the target antigen are joined to the segments of DNA encoding a human constant region. By exchanging the variable regions of the human antibody heavy and light chain genes for those derived from the rodent monoclonal, the resulting chimeric (humanized) antibodies are 60-70% human. For murine monoclonal antibodies, the epitope or antigenic determinant region is contained only in the complementarity determining regions. Each domain, the heavy and light chains, have three of these regions surrounded by framework regions. To construct a monoclonal antibody that is 90-95% recognized as human, the complementarity determining regions of the murine monoclonal that were selected for a desired antigen can be adjoined to human framework regions.
[0138] A monoclonal antibody that is essentially 100% human can be obtained by genetically engineering the immune system of an animal, often a mouse using standard procedures.
[0139] Synthetic Abs. Synthetic antibodies are another approach to antibody creation. Such antibodies are created from synthetic libraries and may also be utilized to generate fully humanized, high affinity, high specificity antibodies for therapeutic use. The approach is analogous to the methods described above in terms of Ab functional activity See Shim BMB Reports 48 (2015) 489, Bradbury Nat Biotech 29 (2011) 245.
[0140] FAb fragments are prepared against Fst from anti-Fst mAbs according to standard methods. In addition, antigen binding fragments/F(ab) fragments, Variable fragments (Fv fragments), Single chain variable fragments (scFv fragments), and the like may also be prepared according to the methods of Hust BMC Biotech 7 (2007); Skerra Curr Opin Immunol 5 (1993) 256; Roque Biotechnol Prog 20 (2004) 639; Skerra-Pluckthun Science 240 (1988) 1038; Kipriyanov Mol Biotech 26 (2004) 39.
[0141] Antibody fragments are often produced in bacterial systems since they are small in size and can be produced in large quantities while maintaining function. Antigen binding fragments/F(ab) fragments may be prepared in recombinant systems as well. Variable fragments include both the heavy and light chains of the variable region on the antibody fragment that contain the antigen binding site. The antibodies or fragments are often expressed in the same bacterial cell, e.g., E. coli, and are secreted together into the periplasm of the bacteria. Using approximately equivalent amounts of each of the chains and secreting them essentially at the same time allows proper folding and assembly of a functional antibody fragment. Eukaryotic systems, such as yeast, insect, and mammalian cells, are also viable systems for the production of variable antibody (Fv) fragments.
[0142] For single chain variable fragments, the variable part of heavy chain and the variable part of the light chain of the antibody fragment that contain the antigen binding site of the whole antibody connected by a peptide linker are expressed in the same bacterial cell, such as an E. coli, and are secreted together into the periplasm of the bacteria.
[0143] Nanobodies. Nanobodies against Fst are prepared according to the methods as previously described. See, for example, Liu Mol Immunol 96 (2018) 37; Steeland Drug Discov Today 21 (July 2016) 1076; Angew Chem Int Ed Engl 57 (February 2018) 2314; Fridy Nat Methods 11 (2014) 1253; and Goldman Front Immunol July 2017.
[0144] Nanobodies are commonly obtained from any of the following created librariesimmune libraries, nave libraries, or semi-synthetic/synthetic libraries. For immune libraries, antigen specific heavy chain antibodies undergo affinity maturation following immunization of animals most commonly from the Camelidae family. mRNA is obtained from peripheral blood lymphocytes and cDNA is synthesized by reverse transcription. Nanobodies are selected by screening the library using established techniques such as phage display, cell surface display, mRNA/cDNA display, HTS DNA sequencing and mass spec identification, biotinylated nanobody screening, or a bacterial-two-hybrid system.
[0145] For nave libraries, phage display and ribosome display are common techniques used to select nanobodies generated from the mRNA obtained and cDNA synthesized from peripheral bloo lymphocytes collected from non-immunized animals.
[0146] For the semi-synthetic/synthetic libraries, the complementarity-determining regions of the nanobody are randomly changed in length and by sequence, while the framework regions are conserved. This allows for expansion of the library as well as for the generation of diversity within it.
[0147] Small molecule inhibitors of Fst. Compounds that (i) inhibit Fst binding to one or more of its binding proteins, including MST, BMP, or Activin, (ii) inhibit expression of a Fst gene, or (iii) enhance degradation of Fst may be identified using standard in-vitro cell-free radioligand or fluorescent-ligand binding assays, or their equivalent. The sources for small molecule inhibitors include, but are not limited to, for instance, chemical compound libraries, fermentation media of Streptomycetes, other bacteria and fungi, and cell extracts of plants and other vegetations. Small molecule libraries are available, and include AMRI library, AnalytiCon, BioFocus DPI Library, Chem-XInfinity, ChemBridge Library, ChemDiv Library, Enamine Library, The Greenpharma Natural Compound Library, Life Chemicals Library, LOPAC1280, MicroSource Spectrum Collection, Pharmakon, The Prestwick Chemical Library, SPECS, NIH Clinical Collection, Chiral Centers Diversity Library.
[0148] Gene Silencing using short interfering RNA (siRNA) and related approaches using RNA interference (RNAi). It is now well-established that RNAi play an important role in post-transcriptional gene silencing through molecules such as siRNAs. siRNAs are 19-22 nucleotide (nt) duplex RNA (dsRNA) molecules capable of reducing or silencing the translation of messenger RNAs (mRNAs) in a sequence specific fashion. See Walton et al., 2010; Sibley et al., 2010.
[0149] Polynucleotides can be used to reduce expression of specific genes. Such inhibitory polynucleotides include RNA interference (RNAi), mediated by double-stranded small interfering RNA (siRNA), which silences a gene with a high degree of specificity. A siRNA includes a sequence that is complementary to a protein coding messenger RNA (mRNA) and causes the degradation of the mRNA. One of ordinary skill in the art can design and synthesize siRNA molecules that are able to inhibit follistatin, as shown in the example given by Gao et al (2010). siRNA molecules for inhibition of follistatin are also commercially available (e.g., Dharmacon, Lafayette, Colo.). Automated synthesis of nucleic acids is well established, and includes modifications at numerous positions on the nucleoside and ribose/deoxyribose ring systems (Sibley et al., 2010; Walton et al., 2010).
[0150] Another type of inhibitory nucleotide includes antisense RNA, single stranded RNA complementary to a protein coding mRNA with which it hybridizes, and thereby blocks its translation into protein. A siRNA used in the methods herein has the ability to reduce expression of Fst315. RNA interference methods represent a useful approach for molecularly targeted therapy. Thus, in another embodiment, siRNAs or another RNAi methodology is utilized. siRNAs are synthesized and tested for their ability to reduce circulating Fst in a therapeutically effective manner in a mammal. Oligonucleotide synthetic methods of manufacturing siRNAs are well established. No limitation is intended with respect to the type of RNAi that may be utilized to reduce Fst levels in a mammal, including RNA-DNA chimeras, tandem hairpin RNAs, tandem siRNAs, tRNA-shRNAs, and the like (Sibley Mol Ther 18 (2010) 466).
[0151] In one embodiment, a polynucleotide useful herein include a double stranded RNA (dsRNA) polynucleotide. The sequence of a polynucleotide includes one strand, referred to herein as the sense strand, of 16 to 30 nucleotides, for instance, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The sense strand is substantially identical, preferably, identical, to a target mRNA, e.g., an mRNA that encodes Fst315. As used herein, the term identical means the nucleotide sequence of the sense strand has the same nucleotide sequence as a portion of the target mRNA. As used herein, the term substantially identical means the sequence of the sense strand differs from the sequence of a target mRNA at 1, 2, or 3 nucleotides, preferably 1 nucleotide, and the remaining nucleotides are identical to the sequence of the mRNA. These 1, 2, or 3 nucleotides of the sense strand are referred to as non-complementary nucleotides. When a polynucleotide includes a sense strand that is substantially identical to a target mRNA, the 1, 2, or 3 non-complementary nucleotides are preferably located in the middle of the sense strand. For instance, if the sense strand is 21 nucleotides in length, the non-complementary nucleotides are typically at nucleotides 9, 10, 11, or 12, preferably nucleotides 10 or 11. The other strand of a dsRNA polynucleotide, referred to herein as the anti-sense strand, is complementary to the sense strand.
[0152] The sense and anti-sense strands of a dsRNA polynucleotide may also be covalently attached, typically by a spacer made up of nucleotides. Such a polynucleotide is often referred to in the art as a short hairpin RNA (shRNA). Upon base pairing of the sense and anti-sense strands, the spacer region forms a loop. The number of nucleotides making up the loop can vary, and loops between 3 and 23 nucleotides have been reported (Sui et al., Proc. Nat'l. Acad. Sci. USA, 99, 5515-5520 (2002), and Jacque et al., Nature, 418, 435-438 (2002)).
[0153] In one embodiment, a polynucleotide useful herein includes single stranded RNA (ssRNA) polynucleotides. The sequence of a polynucleotide includes one strand, referred to herein as the anti-sense strand, of at least 16 nucleotides. The anti-sense strand is substantially complementary, preferably, complementary, to a target mRNA, e.g., an mRNA that encodes Fst315. In one embodiment, a polynucleotide for decreasing expression of a coding region in a cell includes substantially all of a coding region, or in some cases, an entire coding region. An antisense strand is substantially complementary, preferably, complementary, to a target coding region or a target mRNA. As used herein, the term substantially complementary means that at least 1, 2, or 3 of the nucleotides of the antisense strand are not complementary to a nucleotide sequence of a target mRNA.
[0154] Polynucleotides of the present disclosure are preferably biologically active. A biologically active polynucleotide causes the post-transcriptional inhibition of expression, also referred to as silencing, of a target coding region. Without intending to be limited by theory, after introduction into a cell a polynucleotide of the present invention will hybridize with a target mRNA and signal cellular endonucleases to cleave the target mRNA. The result is the inhibition of expression of the polypeptide encoded by the mRNA. Whether the expression of a target coding region is inhibited can be determined by, for instance, measuring a decrease in the amount of the target mRNA in the cell, measuring a decrease in the amount of polypeptide encoded by the mRNA, or by measuring a decrease in the activity of the polypeptide encoded by the mRNA.
[0155] A polynucleotide of the present disclosure may include additional nucleotides. For instance, with respect to the sense strand, the 5 end, the 3 end, or both ends can include additional nucleotides, provided the additional nucleotides are identical to the appropriate target mRNA and the overall length of the sense strand is not greater than 30 nucleotides.
[0156] A polynucleotide may be modified. Such modifications can be useful to increase stability of the polynucleotide in certain environments. Modifications can include a nucleic acid sugar, base, or backbone, or any combination thereof. The modifications can be synthetic, naturally occurring, or non-naturally occurring. A polynucleotide can include modifications at one or more of the nucleic acids present in the polynucleotide. Examples of backbone modifications include, but are not limited to, phosphonoacetates, thiophosphonoacetates, phosphorothioates, phosphorodithioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids. Examples of nucleic acid base modifications include, but are not limited to, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), or propyne modifications. Examples of nucleic acid sugar modifications include, but are not limited to, 2-sugar modification, e.g., 2-O-methyl nucleotides, 2-deoxy-2-fluoro nucleotides, 2-deoxy-2-fluoroarabino, 2-O-methoxyethyl nucleotides, 2-O-trifluoromethyl nucleotides, 2-O-ethyl-trifluoromethoxy nucleotides, 2-O-difluoromethoxy-ethoxy nucleotides, or 2-deoxy nucleotides. Polynucleotides can be obtained commercially synthesized to include such modifications (for instance, Dharmacon Inc., Lafayette, Colo.).
[0157] In one embodiment it is preferable to target compounds of the invention to the liver of the human being or other organism for which treatment with an Fst inhibitor is desired. As used herein, to target means to chemically modify a compound for the purpose of increasing the amount of the compound that enters the liver rather than other organs in the body. This is because Fst is produced in the liver of a mammal. Therefore, targeting a therapeutic compound to the liver will increase the efficacy of the compound for inhibition of follistatin.
[0158] Methods of targeting a compound to hepatocytes (a specific type of liver cell) are well known in the literature, and include addition of a targeting agent to a compound described herein, such as a polynucleotide, including a siRNA. One approach is to chemically conjugate targeting agent to a compound to a compound. An example of a targeting agent is an N-acetylgalactosamine (GalNAc) moiety (Nair et. al., 2014; Rajeev et al, 2015; Matsuda et. al., 2015). In one embodiment, a GalNAc moiety is conjugated to a nucleic acid sequence, such as an anti-sense oligonucleotide or an siRNA (Lee and Sinko, 2006; Willoughby et al. 2018). Since an asialoglycoprotein receptor (ASGPR) is expressed specifically on hepatocytes, and because GalNAc is a known ligand for the ASGPR, addition of a GalNAc moiety to a compound such as an siRNA results in a GalNAc-siRNA conjugate molecule that is rapidly cleared from the blood through binding to the ASGPR followed by subsequent internalization of the complex into clathrin-coated endosomes (Springer and Dowdy, 2018). In one embodiment, one or more
[0159] GalNAc moiety is conjugated to the 5 end of the sense strand of the siRNA (Kumar et al., 2019; Willoughby et al. 2018, Wang et. al., 2017).
[0160] Other targeting agents are known that are capable of targeting compounds to receptors that are expressed in a tissue-specific manner such as on hepatocytes (in the liver), glial cells (nerves), adipocytes (fat), myocytes (muscle), and the like (Lee et. al., 2012). No limitation is intended on the nature of the targeting approach that may be utilized. For the purposes of this invention directed toward the inhibition of follistatin, targeting cells in the liver, and particularly hepatocytes, is preferable.
[0161] A polynucleotide useful in a method described herein can be administered directly to a patient. In those embodiments where the polynucleotide includes RNA, the RNA can be supplied indirectly by introducing a vector that encodes the RNA. For instance, when siRNA is the desired polynucleotide, the siRNA can be supplied indirectly by administering one or more vectors that encode both single strands of a dsRNA.
[0162] Gene Therapy. In another embodiment, viral vector-based gene therapy approaches may be utilized to reduce Fst expression or production in a mammal. No limitation is intended with respect to the type of gene therapy approach that may be utilized. In one preferred embodiment which has shown clinical efficacy with other targets, a viral vector system may be utilized to introduce anti-sense nucleic acids into Fst-producing organs such as the liver. Such methods are well-known in the art. In one preferred embodiment, Adeno-Associated Viruses (AAV) are utilized. One or more coding or non-coding anti-sense segments encoding a Fst protein are utilized in an AAV vector system for introduction into the liver of an afflicted mammal. See, for example, Naso BioDrugs 31 (2017) 317; Ojala Neuroscientist 21 (2015) 84; Hanna Health Policy 122 (2018) 217; Mendell NEJM 377 (2017) 1713.
[0163] Genome Editing. The Crispr/cas9 system as well as other genomic editing techniques may be utilized to endogenously modify cells in the liver or other tissue to reduce the expression of Fst. Reduced expression of Fst will result in lower levels of Fst protein and a concomitant improvement in insulin sensitivity in the periphery. See for example, Franco-Tormo et al., 2018; Li et al., 2018; and the standard methods disclosed therein.
[0164] Fst-binding polypeptides. Using standard approaches, peptide fragments selected from Fst, or alternatively from one of follistatin's known binding partners such as myostatin, bone morphogenetic protein, activin, etc. (see above) may be used to generate a peptide capable of blocking the interaction between Fst and a known binding partner. Soluble binding assays using radioligands, ELISA techniques, or fluorescently tagged ligands or antibodies are well known in the art. See, for example, (Horowitz, A. D. et al., 1981; Knudsen, L. et al., 2012)
[0165] No limitation is intended on the method by which a particular Fst binding compound is identified or enriched.
Testing for Potential Therapeutic Efficacy: Cell Based Assay for Identifying Effective Fst Binding Compounds
[0166] A potential Fst inhibitor compound(s) is/are prepared from one or more of the methods described above and then tested for the ability to restore insulin signaling in an isolated animal or human adipocyte or 3T3L1 adipocyte, or isolated human or animal hepatocytes. The animal or cell is incubated with serum from LDKO-mice or another comparable source of Fst by assaying the relative increase in binding of PI3K to IRS1 under insulin stimulation. 3T3-L1 adipocytes are preferred for use in this cell-based assay as previously shown (Tao, R. et al., 2018). The formation and concentration of IRS1p110 complex is quantified using an XMAP binding assay on the Luminex platform.
[0167] 3T3-L1 pre-adipocytes obtained from a mycoplasma-free stock are cultured in DMEM/F12 with 10% BCS in 5% CO.sub.2. Two days post-confluence, cells are exposed to DMEM/10% FBS with isobutylmethylxanthine (0.5 mM), dexamethasone (1 M) and insulin (5 g/ml). After 2 days, cells are maintained in DMEM/10% FBS until ready for treatment at day 7. On day 9, cells are treated with insulin (10 nM) for 3 min after being maintained in DMEM/5% mouse serum from insulin resistance mice for 24 hours. Mouse serum from insulin resistant mice is useful as it provides a source of Fst and Fst targets that contribute to WAT insulin resistance (Tao, R. et al., 2018).
[0168] As described previously (Hancer et. al., 2014; Copps et. al., 2016), the IRS1 capture antibody (rabbit monoclonal antibody 58-10C-31, Millipore catalog number 05-784R) is coupled to magnetic carboxylated microspheres. The p110 subunit of PI3K associated with captured IRS1 is detected with antibodies from Cell Signaling Technology (CST #4249). For Luminex assays, cell lysates (10 g) or mouse tissue lysates (80 g) are diluted with Irs1 capture beads (4000 beads/well) in a total volume of 50 l of phosphoprotein detection wash buffer (Bio-rad) and incubated overnight in 96-well round bottom plates. After washing twice with the same buffer, the beads are incubated with 50 l of detection antibody for 1 h on a rotary plate shaker (80 rpm). After removal of the biotinylated detection antibody, the beads can be incubated with shaking in 25 l of 1 g/ml streptavidin-phycoerythrin (Prozyme) for 15 min. All solutions are then removed, and beads are suspended in PBS-BN (Sigma) for analysis in a Luminex FlexMap 3D instrument.
[0169] Alternatively, another assay for identifying potentially therapeutic mAbs is to measure the degree of AKT phosphorylation following insulin stimulation in the presence or absence of selected anti-Fst Abs using cells exposed to serum from insulin-resistant LDKO mice. Tissue or 3T3-L1 adipocytes incubated with serum from insulin resistant LDKO-mice are homogenized in the lysis buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl.sub.2, 1 mm EGTA, 10 mm sodium pyrophosphate, 100 mm sodium fluoride, and freshly added protease inhibitor cocktail and phosphatase inhibitor cocktail). Protein extracts are resolved on an SDS-PAGE gel and transferred to nitrocellulose membrane (Bio-Rad). Detection of proteins is carried out by incubations with HRP-conjugated secondary antibodies targeted against regulatory phosphorylation sites in AKTincluding T308 or S473followed by ECL detection reagents.
[0170] The skilled person may design other assay systems that measure increases in insulin signaling of anti-follistatin Abs or other Fst inhibitor compounds under the conditions given aboveincluding the use of an XMAP assay to quantify AKT phosphorylation. Moreover, other downstream targets can be selectedincluding reduced HSL phosphorylation; reduced FOXO1 phosphorylation; increased S6K phosphorylation; or increased RPS6 phosphorylation No limitation is intended on how the compounds of the disclosure may be characterized for their ability to enhance these and other insulin signaling responses in an assay that measures insulin signaling and its release from inhibitory resistance owing to Fst.
[0171] For example, insulin normally promotes dephosphorylation of HSL in 3T3-L1 adipocytes. In this assay 3T3-L1 adipocytes incubated with 5% serum from insulin resistant LDKO-mice are stimulated with insulin for a few minutes. The cells are homogenized in the lysis buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl.sub.2, 1 mm EGTA, 10 mm sodium pyrophosphate, 100 mm sodium fluoride, and freshly added protease inhibitor cocktail and phosphatase inhibitor cocktail). Protein extracts are resolved on an SDS-PAGE gel and transferred to nitrocellulose membrane (Bio-Rad). Detection of phosphorylated HSL at pS660.sup.Hsl using phospho-HSL (Ser660) (Antibody #4126, Cell Signaling Technology) is carried out by incubations with HRP-conjugated secondary antibodies targeted against antibodies that bind to the regulatory phosphorylation sites in HSLfollowed by ECL detection reagents.
[0172] Once effective mAbs or other effective compounds of the disclosure are identified in the aforementioned cellular assays, the compounds or Abs that score positively in the one of the assays given above may be further tested for in-vivo efficacy. Liver-specific Irs1 and Irs2 double knockout mice (LDKO) are preferably bred as previously described (27, 28). Alternatively, C57BL6 mice (Stock No. 000664), ob/ob mice (Stock No. 000632), B6.129S2-I16tm1Kopf/J mice (Stock No. 002650) can be purchased from The Jackson Lab (Bar Harbor, Me.). These mice are placed on the high fat diet to induce insulin resistance and diabetes between 4-16 weeks of age. Preferably, all mice are housed in plastic cages on a 12:12 h light-dark cycle with free access to water and food in an appropriate facility.
[0173] The hyperinsulinemic euglycemic clamp in conscious and unrestrained mice is used to assess the efficacy of the Fst inhibitors to inhibit Fst's ability to induce insulin resistance. Prior to the clamp experiment, one catheter is inserted into the right jugular vein for infusions. After 5-7 days of recovery, mice that lose less than 10% of their preoperative weight are subjected to the hyperinsulinemic euglycemic clamp.
[0174] The day before the experiment the mice are treated with the Fst binding protein or antibody at concentrations determined in the cell-based assays of the previous section. On the day of the experiment, mice are deprived of food for 3.5 hours at 8:00 am and then infused continuously with D-[3-.sup.3H]-glucose (PerkinElmer) (0.05 Ci/min) at a rate of 1 l/min for 1.5 h. After basal sampling from the tail vein, a 140 min hyperinsulinemic euglycemic clamp is conducted with a primed-continuous infusion of human regular insulin (4 mU/kg/min, Humulin, Eli Lilly) at a rate of 2 l/min and continuously with D-[3-.sup.3H]-glucose (PerkinElmer) (0.1 Ci/min) at a rate of 2 ul/min throughout the clamp experiment. The insulin solutions are prepared with 3% BSA in 0.9% saline. 20% glucose was infused at variable rates as needed to maintain plasma glucose at 130 mg/dl (except in
[0175] The D-[3-.sup.3H]-glucose and 2-deoxy-D-[1-.sup.14C] glucose concentrations in plasma are measured according to the procedure of GLUCOSE CLAMPING THE CONSCIOUS MOUSE from the Vanderbilt-NIDDK Mouse Metabolic Phenotyping Center with some modifications. Briefly, 6 l of plasma sample mixed with 14 l saline is treated with 100 ul 3N Ba(OH).sub.2 and ZnSO.sub.4 (add Ba(OH).sub.2 prior to ZnSO.sub.4) and 100 l of supernatant is pipetted into a scintillation vial and dried in an oven overnight; 8 ml of scintillation fluid are added to the dried vial, or to 50 l non-dry supernatant for measuring radioactivity in a liquid scintillation counter. For measuring 2-deoxy-D-[1-.sup.14C] glucose, lysates of adipose tissue and skeletal muscle are processed using a perchloric acid Ba(OH).sub.2/ZnSO.sub.4 precipitation (Ferre, P. et al., 1985). Glucose uptake into WAT, BAT and skeletal muscle in vivo may be calculated based on 2-deoxy-D-[1-.sup.14C]-glucose 6-phosphate accumulation and specific activity of 2-deoxy-D-[1-.sup.14C]-glucose in serum.
[0176] Fst binding proteins or specific antibodies that promote insulin-suppression of hepatic glucose production are selected as biologically active candidates for the enhancement of insulin action by neuralizing the effect of Fst to promote insulin resistance.
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[0329] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
[0330] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term about. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
[0331] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
[0332] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.