Variants of activin a

Abstract

The present invention provides compositions and methods related to Activin A variants. In particular, the present disclosure provides Activin A variants which have modulated receptor binding and / or receptor activation as compared to wild-type Activin A.

Description

BIOTN-43683.601VARIANTS OF ACTIVIN AFIELD

[0001] The present invention provides compositions and methods related to Activin A variants. In particular, the present disclosure provides Activin A variants which have modulated receptor binding and / or receptor activation as compared to wild-type Activin A.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 701,944, filedOctober 1, 2024, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled BIOTN-43638-601.xml (Size: 2,275 bytes; and Date of Creation: October 1, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0004] Activin A enhances endoderm differentiation in human embryonic stem cells (hESCs) and participates in maintaining the pluripotency of human induced pluripotent stem (hiPS) cells. Moreover, Activin A promotes the differentiation of human ES cells into pancreatic P cells and endoderm. Differentiation of iPSCs to endoderm lineages can be plagued by inefficiencies of complete differentiation, contamination by non-endoderm lineages, and time-consuming protocols. Some of this inconsistency may be due to the variability in Activin A activity, which significantly varies and largely depends on the quality of the purification process.SUMMARY

[0005] Embodiments of the present disclosure include an engineered Activin A comprising an amino acid sequence having at least one amino acid substitution, addition, or deletion as compared to SEQ ID NO: 1. In some embodiments, the engineered Activin A comprises any amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%) identity to SEQ ID NO: 1.

[0006] In some embodiments, when dimerized the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is less than about 60 A. InBIOTN-43683.601 some embodiments, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is about 50 A.

[0007] In some embodiments, the engineered Activin A comprises at least one amino acid substitution at positions 318, 325, 368, 403, and 417 as compared to SEQ ID NO: 1. In some embodiments, the engineered Activin A comprises an V318F, Q325F, F368A, Y403W, and / or N417Q amino acid substitution with reference to SEQ ID NO: 1.

[0008] In some embodiments, the engineered Activin A is a hyperactive variant. In some embodiments, the engineered Activin A has increased receptor binding and / or increased receptor activation as compared to an Activin A without the at least one amino acid substitution, addition, or deletion, wherein the receptor comprises two type I and two type II activin receptors. In some embodiments, the engineered Activin A increases the rate of differentiation of stem or precursor cells to endodermal lineages.

[0009] In some embodiments, when dimerized the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is greater than about 70 A. In some embodiments, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is about 75 A.

[0010] In some embodiments, the engineered Activin A comprises at least one amino acid substitution at positions 346, 362, 364, 389, and 415 as compared to SEQ ID NO: 1. In some embodiments, the engineered Activin A comprises an H346I, S362D, S364Q, S389V, and / or I415N amino acid substitution with reference to SEQ ID NO: 1.

[0011] In some embodiments, the engineered Activin A is a hypoactive variant. In some embodiments, the engineered Activin A has decreased receptor binding and / or decreased receptor activation as compared to an Activin A without the at least one amino acid substitution, addition, or deletion, wherein the receptor comprises two type I and two type II activin receptors. In some embodiments, the engineered Activin A decreases the rate of differentiation of stem or precursor cells to endodermal lineages.

[0012] Embodiments of the present disclosure also include nucleic acids encoding an engineered Activin A as disclosed herein, and vectors comprising a nucleic acid encoding an engineered Activin A. In some embodiments, the vectors further comprise a promoter operatively linked to the nucleic acid.BIOTN-43683.601

[0013] Embodiments of the present disclosure further include compositions comprising an engineered Activin A as disclosed herein or a nucleic acid or vector encoding the engineered Activin A. In some embodiments, the composition further comprises a carrier.

[0014] Embodiments of the present disclosure additionally include cells comprising an engineered Activin A as disclosed herein or a nucleic acid or vector encoding the engineered Activin A.

[0015] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A-1C: Ribbon diagrams showing the evolution of the Activin A 3D structure over the course of a 100ns molecular dynamic simulation (FIG. 1A. S364Q Activin A, FIG. IB. wild-type Activin A, and FIG. 1C. F368A Activin A). The F368A mutation stabilizes the dimer structure, conferring hyperactivity, whereas the S364Q mutation destabilizes the dimer structure, resulting in hypoactivity.

[0017] FIG. 2: Activity of recombinant human Activin A (wild-type Activin A and F368A Activin A) to induce cytotoxicity of MPC-11 cells as measured by a fluorometric assay using the redox-sensitive dye.

[0018] FIG. 3: Signal transduction response of recombinant human Activin A (wild-type Activin A and F368A Activin A) as measured by a reporter for Firefly luciferase controlled by SMAD-responsive elements (SMAD binding elements, SBE).

[0019] FIG. 4: F368A Activin A (Activin A Hyperactive) promotes higher levels of pSMAD2 / 3 signaling in differentiating iPSCs than wild-type Activin A. iPSCs were differentiated for 24h using CHIR 99021, a potent and highly selective inhibitor of glycogen synthase kinase 3, and wildtype Activin A and F368A Activin A at two concentrations to examine pSMAD2 / 3 as a readout of signaling activity. Untreated and CHIR-treated iPSCs were used as controls. A negative control condition with TGF-P Type I Receptor inhibitor SB-431542, which prevents phosphorylation of SMAD2 / 3, was included. Cells were assessed by flow cytometry for pSMAD2 / 3 activity. At 24h, F368A Activin A showed higher pSMAD2 / 3 levels compared to wild-type.

[0020] FIG. 5: F368A Activin A (Activin A Hyperactive) promotes higher levels of markers of endoderm differentiation than wild-type Activin A. iPSCs were differentiated for 72h into definitive endoderm using wild-type Activin A and F368A Activin A at two concentrations,BIOTN-43683.601 alongside untreated and CHIR-treated only controls. At 72h of differentiation, flow cytometry was performed to quantify the percentage of cells expressing both markers of endoderm, SOX17 and CXCR4. F368A Activin A promoted efficient endoderm differentiation in iPSC cells.

[0021] FIG. 6: F368A Activin A (Activin A Hyperactive) produces high percentage of PDX1+ / NKX6.1+ pancreatic progenitor cells. iPSCs were differentiated into pancreatic progenitors with wild-type Activin A and F368A Activin A for 13 days. Other supplemental proteins and small molecules included Recombinant Human FGF-2, FGF-10, Wnt-3a, EGF (Catalog # 236-EG), SANT-1, Dorsomorphin, Retinoic Acid, and LDN-193189. On day 13, the cells were fixed and stained for flow cytometric analysis. Successful differentiation into pancreatic progenitors was assessed by measuring PDXland NKX6.1 co-expression. Efficient derivation of pancreatic progenitors (>80% PDX1+ / NKX6.1+) was achieved with F368A Activin A.DETAILED DESCRIPTION

[0022] Disclosed herein are Activin A variants which have modulated receptor binding and / or receptor activation as compared to wild-type Activin A. The disclosed Activin A variants have at least one amino acid substitution, addition, or deletion which modulates receptor binding and / or receptor activation as compared to an Activin A without the at least one amino acid substitution, addition, or deletion. Hyperactive Activin A variants stabilize the dimer and show increased receptor binding and / or increased receptor activation. Hypoactive Activin A variants destabilize the dimer and show decreased receptor binding and / or decreased receptor activation.

[0023] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Definitions

[0024] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of cell and tissue culture, molecular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.BIOTN-43683.601Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0025] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0026] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0027] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), norvaline (found in the antifungal peptide of Bacillus subtilis), and N- formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” amino acids are non-proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include P-amino acids (P3and p2), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring- substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-BIOTN-43683.601 amino acids, alpha-methyl amino acids, and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. According to certain embodiments, a peptide inhibitor comprises an intramolecular bond between two amino acid residues present in the peptide inhibitor. It is understood that the amino acid residues that form the bond will be altered somewhat when bonded to each other as compared to when not bonded to each other. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state. For example, the amino acid residue homoSerine (hSer) in its unbonded form may take the form of 2-aminobutyric acid (Abu) when participating in an intramolecular bond according to the present invention.

[0028] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader.

[0029] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc ), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, or °F for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the peptide.

[0030] In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four- character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N- methylglycine), Aib (a-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3- diaminopropanoic acid), y-Glu (y-glutamic acid), Gaba (y-aminobutanoic acid), P-ProBIOTN-43683.601(pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), hPro (P-homoproline), phPhe (P-homophenylalanine) and Bip ( , diphenylalanine), and Ida (Iminodiacetic acid).

[0031] Natural amino acids can be described by their chemical properties (e.g., charged, nonpolar, polar, hydrophobic, hydrophilic). For example, naturally occurring amino acids classified as positively charged include lysine, arginine, and histidine whereas those classified as negatively charged include aspartic acid (aspartate) and glutamic acid (glutamate). Polar, uncharged naturally occurring amino acids include, for example, serine, proline, threonine, tyrosine, asparagine, and glutamine. Non-polar naturally occurring amino acids include, for example, glycine, cysteine, alanine, valine, leucine, isoleucine, methionine, tryptophan, and phenylalanine.

[0032] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. The peptide or polypeptide may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide,” “oligopeptide,” “protein,” and “peptide” are used interchangeably herein. The peptide may be produced by recombinant genetic technology or chemical synthesis. The peptide may be isolated and purified by any number of standard methods including, but not limited to, differential solubility (e.g., precipitation), centrifugation, chromatography (e.g., affinity, ion exchange, and size exclusion), or by any other standard techniques known in the art.

[0033] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholinoBIOTN-43683.601 nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595- 8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or nonnucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0034] Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (e.g., that are identical) as between the sequence of interest and the reference sequence divided by the length of the longest sequence (e.g., the length of either the sequence of interest or the reference sequence, whichever is longer). A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(fQ): 3770- 3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).BIOTN-43683.601

[0035] The terms “non -naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which it is naturally associated in nature and as found in nature, and / or the nucleic acid molecule or the polypeptide is associated with at least one other component with which it is not naturally associated in nature and / or that there is one or more changes in nucleic acid or amino acid sequence as compared with such sequence as it is found in nature.

[0036] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.

[0037] The term “operatively linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence.

[0038] A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or commonBIOTN-43683.601 ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0039] The term “stem cell” refers to a cell that retains the ability to renew itself through mitotic cell division and that can differentiate into a diverse range of specialized cell types. Mammalian stem cells can be divided into three broad categories: embryonic stem cells, which are derived from blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Multipotent stem cells can produce only cells of a closely related family of cells (e.g., hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of selfrenewal, which distinguishes them from non-stem cells. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be derived, for example, from adult somatic cells such as peripheral blood mononuclear cells, fibroblasts, keratinocytes, epithelial cells, endothelial progenitor cells, mesenchymal stem cells, adipose derived stem cells, leukocytes, hematopoietic stem cells, bone marrow cells, or hepatocytes. Methods for preparing and culturing stem and precursor cells, specifically, and cells in general can be found in standard textbooks and reviews in cell biology, tissue culture, and embryology.

[0040] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Activin A variants

[0041] Disclosed herein are engineered Activin A proteins which have modified receptor binding and / or receptor activation as compared to wild-type Activin A. The engineered Activin ABIOTN-43683.601 proteins comprise an amino acid sequence having at least one amino acid substitution, addition, or deletion as compared to wild-type Activin A (SEQ ID NO: 1).

[0042] An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He ), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (D or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0043] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same subgroup. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.BIOTN-43683.601

[0044] The engineered Activin A proteins comprise a sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 1. In accordance with these embodiments, the engineered Activin A disclosed herein also have less than 100% identity to SEQ ID NO: 1. In some embodiments, the engineered Activin A of the present disclosure comprise a sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%) to SEQ ID NO: 1. In some embodiments, the engineered Activin A comprises a sequence having at least 80% identity an amino acid sequence to SEQ ID NO: 1. In some embodiments, the engineered Activin A comprises a sequence having at least 90% identity an amino acid sequence to SEQ ID NO: 1.

[0045] In some embodiments, the engineered Activin A stabilizes an Activin A dimer. Stabilization of the dimer can be measured by the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer. The shorter the distance the more stable the monomer association. In some embodiments, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is less than about 60 A. In some embodiments, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is between about 40 A and about 60 A. In some embodiments, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is about 40 A, about 41 A, about 42 A, about 43 A, about 44 A, about 45 A, about 46 A, about 47 A, about 48 A, about 49 A, about 50 A, about 51 A, about 52 A, about 53 A, about 54 A, about 55 A, about 56 A, about 57 A, about 58 A, about 59 A, or about 60 A.

[0046] Stabilization can also be measured using an average correlation value for the motion of the Activin A dimer from a molecular dynamics simulation, when compared to wild-type. In some embodiments, the average correlation value is greater than 0.

[0047] In some embodiments, the one or more amino acid deletions, addition, or substitutions in the amino acid sequence of the engineered Activin A results in a hyperactive variant of Activin A. In some embodiments, the one or more amino acid deletions, addition, or substitutions in the amino acid sequence of the engineered Activin A result in increased receptor binding and / or increased receptor activation as compared to an Activin A without the at least one amino acidBIOTN-43683.601 substitution, addition, or deletion. In some embodiments, the Activin A receptor comprises two type I and two type II receptors.

[0048] Embryonic stem cell-derived endoderm progenitors offer a remarkable potential for the treatment of major diseases effecting the pancreas, liver, lungs, bladder, and prostate and are useful in the study of congenital diseases, gene discovery, toxicology screening, and drug development. Activin A has been considered a potential developmental morphogen, shown to control endoderm induction alongside other factors such as nodal, TGFp, BMP, and Wnt. In some cases, the one or more amino acid deletions, addition, or substitutions in the amino acid sequence of the engineered Activin A increases the rate of differentiation of stem or precursor cells to endodermal lineages, as compared to an Activin A without the at least one amino acid substitution, addition, or deletion. The progress to endodermal lineages can be measured by the expression of a variant of known endodermal markers.

[0049] In some embodiments, the engineered Activin A comprises at least one amino acid substitution at positions 318, 325, 368, 403, and 417 as compared to SEQ ID NO: 1. In some embodiments, the engineered Activin A comprises an V318F, Q325F, F368A, Y403W, and / or N417Q amino acid substitution with reference to SEQ ID NO: 1.

[0050] In some embodiments, the engineered Activin A destabilizes an Activin A dimer. In some embodiments, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is greater than about 70 A. In some embodiments, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is between about 70 A and about 85 A. In some embodiments, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is about 70 A, about 71 A, about 72 A, about 73 A, about 74 A, about 75 A, about 76 A, about 77 A, about 78 A, about 79 A, about 80 A, about 81 A, about 82 A, about 83 A, about 84 A, or about 85 A.

[0051] In some embodiments, the one or more amino acid deletions, additions, or substitutions in the amino acid sequence of the engineered Activin A results in a hypoactive variant of Activin A. In some embodiments, the one or more amino acid deletions, additions, or substitutions in the amino acid sequence of the engineered Activin A result in decreased receptor binding and / or decreased receptor activation as compared to an Activin A without the at least one amino acidBIOTN-43683.601 substitution, addition, or deletion. In some embodiments, the Activin A receptor comprises two type I and two type II activin receptors.

[0052] In some cases, the one or more amino acid deletions, additions, or substitutions in the amino acid sequence of the engineered Activin A decreases the rate of differentiation of stem or precursor cells to endodermal lineages.

[0053] In some embodiments, the engineered Activin A comprises at least one amino acid substitution at positions 346, 362, 364, 389, and 415 as compared to SEQ ID NO: 1. In some embodiments, the engineered Activin A comprises an H346I, S362D, S364Q, S389V, and / or I415N amino acid substitution with reference to SEQ ID NO: 1.

[0054] In some embodiments, the engineered Activin A further comprises a localization or signal sequence, a sequence tag (e.g., a tag for detection, purification, and / or monitoring expression), a protein transduction domain sequence, or a combination thereof. Sequence tags may include, but are not limited to: 3xFLAG tag, an HA tag, a Myc tag, a poly-histidine tag, a SNAP- tag, a CLIP -tag, and the like. The tags may be at the N-terminus, a C-terminus, or embedded in the sequence of the engineered Activin A.Nucleic Acids

[0055] The present disclosure also provides for nucleic acids encoding the engineered Activin A or vectors containing these nucleic acids. The nucleic acids may be DNA, RNA, or combinations thereof. In some embodiments, the nucleic acids comprise one or more vectors. The vectors may be used to propagate the nucleic acid in an appropriate cell and / or to allow expression from the nucleic acid (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0056] In certain embodiments, the nucleic acids are engineered for codon-optimization. It will be appreciated altering codons to those most frequently used in the cells or subject of interest allows for maximum expression. Such modified nucleic acid sequences are commonly described in the art as “codon-optimized.” In some embodiments, the nucleic acid sequence is considered codon-optimized if at least about 60% (e.g., about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98%) of the codons encoded therein are preferred codons to the subject of interest.

[0057] To construct cells that express the disclosed engineered Activin A, expression vectors for stable or transient expression of the disclosed engineered Activin A may be constructed viaBIOTN-43683.601 conventional methods as described herein and introduced into host cells. For example, nucleic acids encoding the engineered Activin A may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter.

[0058] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in prokaryotic cells. Promoters that may be used include T7 RNA polymerase promoters, constitutive E. coli promoters, and promoters that could be broadly recognized by transcriptional machinery in a wide range of bacterial organisms.

[0059] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6: 187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0060] Vectors of the present disclosure can comprise any number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue-specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human betaactin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can beBIOTN-43683.601 used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EFl -a) promoter with or without the EFl -a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[0061] Moreover, inducible and tissue specific expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence. Examples of tissue specific or inducible promoter / regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence capable of driving expression of the desired protein operably linked thereto.

[0062] The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in aBIOTN-43683.601 region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[0063] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5’-and 3 ’-untranslated regions for mRNA stability and translation efficiency from highly-expressed genes like a-globin or P-globin; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.

[0064] When introduced into the cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[0065] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate coprecipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0066] Any of the vectors comprising a nucleic acid sequence that encodes an engineered Activin A is also within the scope of the present disclosure. Such a vector may be delivered intoBIOTN-43683.601 host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to deliver DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA 110(6): 2082-2087(2013) incorporated herein by reference); or viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the engineered Activin A can be delivered by any method appropriate for introducing nucleic acids into a cell.

[0067] Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding an engineered Activin A into cells. Such methods can be used to administer nucleic acids encoding an engineered Activin A to cells in culture. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), nucleic acids, and nucleic acids complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

[0068] In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that any or all of the engineered Activin A may be removed from the cells under certain conditions. For example, this may allow for DNA integration by transforming bacteria of interest, but then being left with engineered strains that have no memory of the plasmids or vectors used for the integration.

[0069] A variety of viral constructs may be used to deliver the engineered Activin A to the targeted cells. Nonlimiting examples of such recombinant viruses include recombinant adeno- associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(l):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.BIOTN-43683.601

[0070] Tn some embodiments, the vector or the nucleic acid encoding the engineered Activin A is a DNA molecule. In some embodiments, the nucleic acid encoding the engineered Activin A is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding the engineered Activin A is an RNA molecule, which may be electroporated to cells. Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include ribonucleoprotein (RNP) complexes, lipid- based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics.

[0071] In one embodiment, a DNA segment encoding an engineered Activin A is contained in a plasmid vector that allows expression of the protein(s) and subsequent isolation and purification produced by the recombinant vector. Accordingly, the engineered Activin A proteins disclosed herein can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods.

[0072] Accordingly, also disclosed herein are cells comprising an engineered Activin A or a nucleic acid or vector encoding the engineered Activin A. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.Compositions

[0073] Also disclosed herein are compositions comprising an engineered Activin A as described herein or a nucleic acid molecule comprising a sequence encoding the engineered Activin A as described herein.

[0074] In some embodiments, the composition is a cell culture medium. Cell culture medium refers to any media for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc.

[0075] In some embodiments, the compositions further comprise a carrier, or a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable,” as used in connection with compositions and / or cells of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably,BIOTN-43683.601 as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions and / or cells to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.

[0076] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0077] In some embodiments, the carrier is a carrier protein. Exemplary carrier proteins include, for example, albumin, gelatin, elastin (including topoelastin) or elastin-derived polypeptides (e.g., a-elastin and elastin-like polypeptides (ELPs)), gliadin, legumin, zein, soy protein (e.g., soy protein isolate (SPI)), milk protein (e.g., [3-lactoglobulin (BLG) and casein), or whey protein (e.g., whey protein concentrates (WPC) and whey protein isolates (WPI)). In preferred embodiments, the carrier protein is albumin. In select embodiments, the albumin is bovine serum albumin (BSA).Methods

[0078] The disclosure also provides methods for promoting cell proliferation, cell differentiation, cell survival, cell maintenance, or cell survival in vitro or ex vivo. The methods described herein can be used to maintain the proliferative potential of precursor cells (e.g., iPSCs) and promote differentiation (e.g., of embryonic stem cells) into endoderm and pancreatic B cells.

[0079] “Differentiation” refers to a process by which cells differentiate from one cell type (e.g., a multipotent, totipotent, or pluripotent differentiable cell) to another cell type such as a target differentiated cell. Oftentimes, as a result of differentiation, the potency or proliferation of a cell is decrease or the cell is moved to a more developmentally restricted state. “Proliferation” refersBIOTN-43683.601 to increasing cell division, either symmetric or asymmetric division of cells. “Expansion” refers to increasing the number of cells as compared to the number originally present, for example, as the outcome of cell division and cell death. “Stimulation” or “activation” refers to inducing a change in the biologic state of a cell which can result in expression of activation markers, production of cytokines, increases in autophagy, proliferation, and / or cytotoxicity to target cells.

[0080] The methods comprise contacting a cell, or a precursor or progenitor cell with an engineered Activin A or a composition comprising thereof, as described herein. The cell or precursor or progenitor cell may be genetically modified.

[0081] In some embodiments, the methods comprise adding the engineered Activin A, or composition comprising thereof, to a buffer or cell culture medium containing the cell or precursor or progenitor cell. Alternatively, or in addition, in some embodiments, the methods comprise adding a buffer or cell culture medium comprising the engineered Activin A to a population of cells or a population of precursor or progenitor cells.

[0082] A vessel used for the contacting and subsequent culturing can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, CellSTACK® Chambers, G-Rex® culture vessels, culture bag, and roller bottle, as long as it is capable of culturing the cells therein. The cells may be cultured in any volume (e.g., at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein) depending on the needs of the culture. The vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment. The vessel may be part of an open or closed system. The length of time in culture, as well as other components of the cell culture medium and cell culture conditions (e.g., temperature, CO2) are those which promote cell growth and allow for the desired degree of differentiation, stimulation, and / or expansion.

[0083] Precursor or progenitor cells are less differentiated cells or undifferentiated cells that have the potential to differentiate into the cell type of interest. Depending on cell differentiation, precursor cells could be multipotent, pluripotent, and totipotent, whereas progenitor cells can be unipotent or oligopotent.

[0084] Precursor or progenitor cells include stem cells (e.g., embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs)), and cells which are more differentiatedBIOTN-43683.601 and specified as compared to stem cells (e g., descendants of stem cells). In certain embodiments, the stem or progenitor cells may be selected from embryonic stem cells, hematopoietic stem or progenitor cells, cells isolated from bone marrow, cord blood, peripheral blood, thymus, or the progenitor cells may have been differentiated from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) in vitro. Stem or progenitor cells herein may be, but are not limited to, ESCs, iPSCs, or tissue stem cells (also called tissue-specific stem cell, or somatic stem cell). Stem or progenitor cells from primary tissue or ESCs or iPSCs may be from human or non-human animals (e.g., mouse) in origin.

[0085] Embryonic stem cells (ESCs) are pluripotent stem cells derived from early embryos. Tissue stem cells are present at particular locations in tissues and have an undifferentiated intracellular structure. Therefore, the pluripotency of tissue stem cells is typically low. Tissue stem cells are separated into categories, based on the sites from which the cells are derived, such as the dermal system, the digestive system, the bone marrow system, the nervous system, and the like. Tissue stem cells in the bone marrow system include hematopoietic stem cells, mesenchymal stem cells, and the like.

[0086] Hematopoietic stem or progenitor cells are cells that are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation and include hematopoietic stem cells, multipotential hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells).

[0087] Induced pluripotent stem cells (iPS cells or iPSCs) are pluripotent stem cells artificially prepared from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing certain factors, referred to as reprogramming factors.

[0088] The methods described herein can be used for disease modeling. Accordingly, the cell or precursor or progenitor cell may be a diseased cell or a diseased precursor or progenitor cell or may be derived from or include cells derived from a diseased tissue, a subject having or suspected of having a disease or disorder, or genetically engineered to have features similar to those found with the disease.BIOTN-43683.601Kits

[0089] Also within the scope of the present disclosure are kits that include the engineered Activin A, nucleic acids, compositions, or components thereof as disclosed herein. The kit may include instructions for use of engineered Activin A.

[0090] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. A kit may have a sterile access port (for example, a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

[0091] The packaging may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and / or alleviating a disease or disorder in a subject.

[0092] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0093] The kit will typically be provided with its various components in one or more packages, e.g., a fiber-based, a cardboard, polymeric, or a Styrofoam box. The enclosure(s) can be configured so as to maintain a temperature differential between the interior and the exterior, for example, to provide insulating properties to keep the reagents at a preselected temperature for a preselected time. The packaging can be air-tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.SequencesWild-type Activin A (SEQ ID NO: 1)MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVK KHILNMLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELM EQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQ QKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDARKSTWHVFPVS S SIQRLLDQGKS S LDVRIACEQCQESGASLVLLGKKKKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLML QARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGEBIOTN-43683.601CP SHIAGT SGS SLSFHST VINHYRMRGHSPF ANLK SC C VPTKLRPMSML YYDDGQNIIKK DIQNMIVEECGCSExamples

[0094] The following are examples of the present invention and are not to be construed as limiting.Example 1

[0095] Through modeling of the Activin A homodimer structural stability, two separate single point mutations were identified which confer an increase, or decrease of activity, as measured by a proliferation assay and an inhibition assay. The F368A mutation provides an improvement of activity, whereas the S364Q mutation decreases the activity (See Example 2). The changes in activity, as compared to the wild-type sequence, are a result of changes in the structural stability of the Activin A homodimer (FIG. 1). Structural stability is defined as the “wingspan” distance between amino acids Gly97 of chain A and Gin 214 of chain B of the Activin A homodimer. The wingspan distances are approximately 50A, 60A, and 75A for the F368A mutant, wild-type, and S365Q mutant, respectively.

[0096] A prediction metric has been developed based on the calculation of the correlation matrix for the motion of the Activin A dimer from a molecular dynamics simulation. For each single-point mutant dimer of Activin A, a 1 ns molecular dynamic simulation was performed under vacuum conditions, and snapshots of the atomic coordinates were recorded at 1 ps intervals. The Pearson’s correlation matrix for atomic coordinates (R) was calculated for each monomermonomer chain pairs, i.e. RAA, R^e, and R^B, along the entire simulation, and the arithmetic mean was calculated to determine the average correlation value for each of the single-point mutants. The average correlation value for every mutant was offset by a constant value in order to set the average correlation value for the wild-type to be equal to zero. All positive average correlation values indicate an increase in dimer stabilization relative to wild-type, and all negative values indicate a decrease in dimer stabilization relative to wild-type.

[0097] The average correlation value is computed to estimate the degree of compactness of the dimer. A positive value indicates that the atoms are moving in a correlated motion, therefore the dimer disrupts the receptor less, leading to a higher activity level. On the other hand, a negative correlation value indicates that the dimer is moving in a less correlated motion, therefore the dimer disrupts the receptor more, leading to a lower activity level. Table 1 shows the average correlationBIOTN-43683.601 values and the relative activity to WT for the F368A and S365Q Activin A variants. Similarly to the “wingspan” distance, the correlation values indicate an increase in dimer stabilization and activity of the F368A Activin A variant compared to wild-type and a decrease in dimer stabilization and activity of the S365Q Activin A variant.Table 1.

[0098] Thermodynamic integration simulations were preformed to calculate relative binding free energy (rBFE) of the variant monomer against ACVR1B and ACVR2B receptors as compared to wild-type Activin A (Table 2). Molecular dynamic simulations to calculate the Activin A and respective receptor complex structural stability / flexibility .Table 2.Example 2

[0099] Recombinant wild-type and F368A Activin A were measured for their ability to induce cytotoxicity of MPC-11 cells. MPC-11 cells were treated with various concentrations of wild-type and F368A Activin A for 48 hours (as shown in FIG. 2). Induced cytotoxicity was assessed by a fluorometric assay using the redox-sensitive dye Alamar Blue (Resazurin; R&D Systems, Catalog # AR002). Potency was defined as the effective concentration of the growth factor at which cell death is at 50% of maximum. The EC50 for Wild Type Activin A was 13.7 ng / mL and the EC50 for the F368A Activin A was 1.64 ng / mL. Thus, the hyperactive F368A Activin A increased the activity over 8-fold.

[0100] To evaluate the signal transduction response triggered by wild-type and F368A Activin A, a TGF[3 / Activin A-Responsive Reporter HEK293 cell line was utilized. The TGFp / Activin A- Responsive Reporter HEK293 cell line expressed a Firefly luciferase reporter under the control of SMAD-responsive elements (SMAD binding elements, SBE) to monitor the activity of the TGFp (transforming growth factor beta) / SMAD signaling pathway. Cells were treated with the variousBIOTN-43683.601 concentrations of wild-type and F368A Activin A for 48 hours (as shown in FIG. 3). The hyperactive F368A Activin A had a lower EC50 and higher maximal signaling as indicated by the higher luciferase RLUs.

[0101] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0102] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

BIOTN-43683.601CL IMSWhat is claimed is:

1. An engineered Activin A comprising an amino acid sequence having at least one amino acid substitution, addition, or deletion as compared to SEQ ID NO: 1, wherein when dimerized, the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is less than about 60 A.

2. The engineered Activin A of claim 1, wherein the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is about 50 A.

3. The engineered Activin A of claim 1 or 2, wherein the engineered Activin A comprises any amino acid sequence having at least 70% identity to SEQ ID NO: 1.

4. The engineered Activin A of any one of claims 1-3, wherein the engineered Activin A comprises any amino acid sequence having at least 80% identity to SEQ ID NO: 1.

5. The engineered Activin A of any one of claims 1-4, wherein the engineered Activin A comprises any amino acid sequence having at least 90% identity to SEQ ID NO: 1.

6. The engineered Activin A of any one of claims 1-5, wherein the engineered Activin A comprises at least one amino acid substitution at positions 368, 318, 325, 403, and 417 as compared to SEQ ID NO: 1.

7. The engineered Activin A of any one of claims 1-6, wherein the engineered Activin A comprises an F368A, V318F, Q325F, Y403W, and / or N417Q amino acid substitution with reference to SEQ ID NO: 1.

8. The engineered Activin A of any one of claims 1-7, wherein the engineered Activin A is a hyperactive variant.BIOTN-43683.6019. The engineered Activin A of any one of claims 1-8, wherein the engineered Activin A has increased receptor binding and / or increased receptor activation as compared to an Activin A without the at least one amino acid substitution, addition, or deletion, wherein the receptor comprises two type I and two type II activin receptors.

10. The engineered Activin A of any one of claims 1-9, wherein the engineered Activin A increases the rate of differentiation of stem or precursor cells to endodermal lineages.

11. An engineered Activin A comprising an amino acid sequence having at least one amino acid substitution, addition, or deletion as compared to SEQ ID NO: 1, wherein when dimerized the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is greater than about 70 A.

12. The engineered Activin A of claim 11, wherein the distance between amino acids Gly97 of a first Activin A monomer and Gln214 of a second Activin A monomer is about 75 A.

13. The engineered Activin A of claim 11 or 12, wherein the engineered Activin A comprises any amino acid sequence having at least 70% identity to SEQ ID NO: 1.

14. The engineered Activin A of any one of claims 11-13, wherein the engineered Activin A comprises any amino acid sequence having at least 80% identity to SEQ ID NO: 1.

15. The engineered Activin A of any one of claims 11-14, wherein the engineered Activin A comprises any amino acid sequence having at least 90% identity to SEQ ID NO: 1.

16. The engineered Activin A of any one of claims 11-15, wherein the engineered Activin A comprises at least one amino acid substitution at positions 346, 362, 364, 389, and 415 as compared to SEQ ID NO: 1.BIOTN-43683.60117. The engineered Activin A of any one of claims 1 1-16, wherein the engineered Activin A comprises an H346I, S362D, S364Q, S389V, and / or I415N amino acid substitution with reference to SEQ ID NO: 1.

18. The engineered Activin A of any one of claims 11-17, wherein the engineered Activin A is a hypoactive variant.

19. The engineered Activin A of any one of claims 11-18, wherein the engineered Activin A has decreased receptor binding and / or decreased receptor activation as compared to an Activin A without the at least one amino acid substitution, addition, or deletion, wherein the receptor comprises two type I and two type II activin receptors.

20. The engineered Activin A of any one of claims 11-19, wherein the engineered Activin A decreases the rate of differentiation of stem or precursor cells to endodermal lineages.

21. A nucleic acid comprising a sequence encoding an engineered Activin A of any one of claims 1-20.

22. A vector comprising the nucleic acid of claim 21.

23. The vector of claim 22, further comprising a promoter operatively linked to the nucleic acid.

24. A composition comprising an engineered Activin A of any one of claims 1-20 or a nucleic acid or vector encoding the engineered Activin A.

25. The composition of claim 24, wherein the composition further comprises a carrier.

26. A cell comprising an engineered Activin A of any one of claims 1-20 or a nucleic acid or vector encoding the engineered Activin A.

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