WO2024154138A1

WO2024154138A1 - Chimeric polypeptides comprising cbd and extracellular matrix (ecm) proteins or motifs thereof

Info

Publication number: WO2024154138A1
Application number: PCT/IL2024/050076
Authority: WO
Inventors: Oded Shoseyov; Amit Yaari; Yonatan ERAN; Efrat AVIGAD LARON
Original assignee: Biobetter Ltd.; Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Priority date: 2023-01-19
Filing date: 2024-01-18
Publication date: 2024-07-25

Abstract

The present invention relates to a chimeric polypeptide for use in in-vitro tissue engineering, the chimeric polypeptide comprising a cellulose binding domain (CBD); at least one extracellular matrix (ECM) module; and optionally at least one linker, wherein the at least one ECM module comprises an ECM protein selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, small integrin-binding ligand N-linked glycoproteins (SIBLING), and a functional portion thereof; and/or an ECM motif selected from RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR.

Description

CHIMERIC POLYPEPTIDES COMPRISING CBD AND EXTRACELLULAR MATRIX (ECM) PROTEINS OR MOTIFS THEREOF

FIELD OF THE INVENTION

The present disclosure is generally directed to novel growth inducing/manipulating scaffolds for production of isotropic and anisotropic tissues, in particular cultured meat. Specifically, the invention relates to recombinant cellulose binding domain (CBD) fused to extracellular matrix (ECM) proteins or peptide motifs thereof.

BACKGROUND OF THE INVENTION

Extracellular matrix (ECM), a three-dimensional network consisting of extracellular macromolecules, small molecules, effector proteins and minerals, is an important regulator of cellular growth, proliferation and differentiation. In addition, it contributes to tissue order and direction ensuring that a well-functioning tissue is generated. The composition of ECM varies between multicellular structures, while cell adhesion, cell-to-cell communication and differentiation are common functions of the ECM.

Different types of proteoglycan are found within the ECM, including heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid; proteins found within the ECM include collagen (divided into the following families: fibrillar (Type I, II, III, V, XI), facit (Type IX, XII, XIV), short chain (Type VIII, X), basement membrane (Type IV), and other (Type VI, VII, XIII)), and elastin; and cell adhesion proteins include fibronectin and laminin.

Still, there are no available techniques allowing cost-effective mimicking of the ECM environment and of tissue generation in- vitro. Among the recent technologies in the field of tissue engineering, the use of scaffolds is a key component. However, the existing technologies lack the ability to provide a complete ECM-like environment. Many types of scaffolds are available, including porous, fibrous, hydrogel, microsphere and acellular scaffolds. However, they require complicated manipulation for each type of cell, are associated with reduced cell viability, and at times generate acidic byproducts.

There thus remains a need for a tissue engineering scaffold enabling efficient generation of well-ordered and functional tissue, truly mimicking tissue generation by nature.

SUMMARY OF INVENTION

The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

The present invention is directed to constructs for attaching extracellular matrix (ECM) proteins and motifs to scaffolds, or specifically to cellulose-based scaffolds, in order to functionalize the scaffolds to facilitate adhesion, differentiation and proliferation of cells, by linking the ECM proteins or motifs to a carbohydrate binding motif (CBM) or specifically to a cellulose binding domain (CBD).

In some embodiments, there is provided a chimeric polypeptide comprising a CBM or a CBD, linked optionally by at least one linker to at least one extracellular matrix (ECM) protein or motif, as well as system and method utilizing same, for in-vitro tissue engineering.

In some embodiments, the present invention provides a chimeric polypeptide for use in in- vitro tissue engineering, the chimeric polypeptide comprising: a. a cellulose binding domain (CBD); b. a first extracellular matrix (ECM) module; and c. optionally a linker, wherein the first ECM module comprises a first ECM protein selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, small integrin -binding ligand N-linked glycoproteins (SIBLING), and a functional portion thereof.

In some embodiments, the first ECM module comprises vitronectin or a functional portion thereof. In some embodiments, the vitronectin has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments, the present invention provides a chimeric polypeptide for use in in- vitro tissue engineering, the chimeric polypeptide comprising: a. a cellulose binding domain (CBD); b. a first extracellular matrix (ECM) module; and c. optionally a linker, wherein the first ECM module comprises a first ECM motif selected from GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR.

In some embodiments, the linker links the CBD to the ECM module. In some embodiments, the linker has a length of about 10-50 amino acids.

In some embodiments, the CBD has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the chimeric polypeptide further comprises at least an additional ECM module comprising an additional ECM protein or a functional portion thereof; an additional ECM motif; and/or a growth factor (GF)-related sequence.

In some embodiments, the additional ECM protein is selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, and SIBLING.

In some embodiments, the additional ECM motif is selected from a fibronectin motif, a collagen motif, a laminin motif, a vitronectin motif, an elastin motif, an E-cadherin motif, a tenascin-C, a perlecan motif, and a SIBLING motif.

In some embodiments, the additional ECM motif is selected from a fibronectin motif comprising RGD, GRGDSP, and/or PHSRN; a collagen motif comprising GFOGER; and a laminin motif comprising IKVAV and/or YIGSR.

In some embodiments, the chimeric polypeptide further comprises at least an additional linker linking at least one of the additional ECM modules to the CBD, to the first ECM module, and/or to another one of the additional ECM modules. In some embodiments, at least one of the additional ECM modules is not identical to the first ECM module.

In some embodiments, the first ECM module and/or the additional ECM module comprise more than one identical ECM motif, and the chimeric polypeptide comprises at least about 0.4% total ECM motifs, i.e., at least about 1 ECM motif per 250 amino acids.

In some embodiments, the first ECM module and the at least additional ECM module both comprise ECM motifs independently selected from a fibronectin motif, a laminin motif, a collagen motif, a vitronectin motif, an elastin motif, an E-cadherin motif, a tenascin-C, a perlecan motif, a SIBLING motif, RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR.

In some embodiments, the additional ECM motif comprises at least one RGD motif. In some embodiments, the at least one RGD motif comprises at least two RGD motifs.

In some embodiments, the first ECM module comprises vitronectin, or a functional portion thereof, and the at least additional ECM module comprises an ECM motif.

In some embodiments, the additional ECM module comprises at least one RGD motif.

In some embodiments, the first ECM module comprises at least one of GRGDSP and PHSRN, and the at least additional ECM module comprises at least the other one of GRGDSP and PHSRN.

In some embodiments, the first ECM module and the at least additional ECM module flank the CBD. In some embodiments, the first ECM module and the at least additional ECM module are linked to each other on one side of the CBD. In some embodiments, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the chimeric polypeptide disclosed herein.

In some embodiments, the present invention provides a vector comprising the nucleic acid molecule disclosed herein.

In some embodiments, the present invention provides a transgenic plant comprising the nucleic acid molecule disclosed herein or the vector of disclosed herein, and/or expressing the chimeric polypeptide disclosed herein.

In some embodiments, the plant is a tobacco plant.

In some embodiments, the present invention provides a tissue engineering system comprising the chimeric polypeptide disclosed herein bound to a cellulose-based scaffold via the cellulose binding domain (CBD).

In some embodiments, the tissue engineering system further comprises precursor cells which adhere to the scaffold by attaching to the ECM module.

In some embodiments, the present invention provides use of the chimeric polypeptide disclosed herein, or the tissue engineering system disclosed herein, for generating an engineered tissue.

In some embodiments, the present invention provides an in vitro method of generating an engineered tissue, comprising: a. adding the chimeric polypeptide disclosed herein to a cellulose-based scaffold with a medium; b. allowing the chimeric polypeptide to bind to the cellulose-based scaffold; c. seeding precursor cells onto the scaffold -bound chimeric polypeptide; and d. growing the cells until an engineered tissue is formed.

In some embodiments, the chimeric polypeptide comprising the ECM module is added to the medium at a concentration that is at least 10% lower than a concentration of the ECM module not being linked to a CBD when added to a medium comprising a CBD and a cellulose-based scaffold, in a method for generating an engineered tissue.

In some embodiments, growing in step (d) involves application of a mechanical stress to the scaffold.

In some embodiments, the mechanical stress is in form of compression or tensile stress. In some embodiments, the compression or tensile stress is applied at specific strains or forces and at specific frequencies to mimic the natural environment required for growth and/or differentiation of the tissue.

In some embodiments, the tissue is a muscle tissue or a connective tissue. In some embodiments, the chimeric polypeptide is prepared by transforming a plant with the nucleic acid molecule disclosed herein or the vector disclosed herein, and purifying the chimeric polypeptide from the plant.

In some embodiments, the present invention provides an engineered tissue generated by the method disclosed herein.

Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more technical advantages may be readily apparent to those skilled in the art from the descriptions and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some or none of the enumerated advantages.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by study of the following detailed descriptions.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

The term "a" and "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The term "about" when referring to a measurable value such as an amount, a ratio, and the like, is meant to encompass variations of ±10% of the indicated value, as such variations are also suitable to perform the disclosed invention. Any numerical values appearing in the application are intended to be construed as if preceded by “about”, unless indicated otherwise.

The term “aa” is short for amino acid.

The present invention is directed to generating improved tissue engineering scaffolds closely mimicking the natural environment in which cells are embedded in an extracellular matrix (ECM), which provides cues for their growth and differentiation. To this end, the inventors have developed chimeric polypeptides comprising ECM proteins or motifs that are linked to carbohydrate-binding modules (CBMs), and specifically to cellulose binding domain (CBD).

Advantageously, linking CBD to an ECM protein or motif may increase the stability to the protein or motif and allow using smaller amounts of, e.g., recombinant ECM proteins or motifs. In addition, the CBD links the ECM protein or motif to the cellulose-based scaffold (mimicking natural ECM), which in turn promotes cell adhesion and facilitates anisotropic growth and development of the tissue, such as fibrous muscle for example or multi-directional tissue to give a desired 3D structure, suitable with cultured meat.

Importantly, linking an ECM protein or motif to the scaffold facilitates binding of cells to the immobilized ECM proteins or motifs, similar to the natural cellular environment. This way, mechanical stress such as stiffness can be directly transferred from the scaffold to the cells, thereby aiding in differentiation of the cells. For example, a hard scaffold (having a high Young modulus) may be used to generate hard tissues, such as bone, while a soft scaffold may be used to generate soft tissues, such as muscle and fat. Additionally, a scaffold that stretches and contracts (e.g. by outside means) may be used to generate connective tissue, e.g., tendons, ligaments, and skin. The transfer of mechanical stress such as stiffness from the scaffold to the cells is facilitated by the ECM proteins or motifs being directly linked to the CBD, which is directly linked to the cellulose- based scaffold.

In some embodiments, there is provided a chimeric polypeptide including a CBM linked to an ECM protein or peptide, optionally through a linker. The herein disclosed chimeric polypeptide is advantageously configured to provide a tailormade environment offering the required specificity, order, direction and functionality, required to generate a desired tissue.

In some embodiments, the present invention provides a chimeric polypeptide for use in in- vitro tissue engineering, the chimeric polypeptide comprising: a CBD; a first ECM module; and optionally a linker.

In some embodiments, the present invention provides a chimeric polypeptide for use in in- vitro tissue engineering, comprising a cellulose binding domain (CBD); at least one extracellular matrix (ECM) module; and optionally at least one linker, wherein the at least one ECM module comprises an ECM protein selected from vitronectin, fibronectin, laminin, collagen, elastin, E- cadherin, tenascin-C, perlecan, small integrin-binding ligand N-linked glycoproteins (SIBLING), and a functional portion thereof, and/or an ECM motif selected from RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR. As used herein, the term “ECM module” relates to a sequence capable of carrying an ECM-related function (or ECM function). ECM modules suitable for the present invention include ECM proteins, ECM motifs, and growth factor (GF)-related sequences, all further discussed below.

In some embodiments, the first ECM module includes a first ECM protein selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, small integrin-binding ligand N-linked glycoproteins (SIBLING), and a functional portion thereof; or a first ECM motif selected from GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR.

As used herein, the term “chimeric”, with reference to a polypeptide, refers to a polypeptide containing amino acid sequences originating in at least two separate proteins. The chimeric polypeptide may include any combination of complete proteins and partial sequences (such as peptide sequences of domains, or motifs) thereof. The chimeric polypeptide of the invention is a single polypeptide carrying functional properties derived from at least two of the original proteins and/or partial sequences comprised in it.

Carbohydrate-binding modules (CBMs) are protein domains found in enzymes which modulate carbohydrates, such as glycosidases, usually having carbohydrate -binding activity. Some of these domains were found on cellulose scaffold-related proteins. CBMs are classified into many families, based on amino acid sequence similarity.

Cellulose Binding Domain (CBD) is a type of a CBM which specifically binds to cellulose (see the Carbohydrate-Active enZYmes Database at http://www.cazy.org/). CBD strongly binds to cellulose, and forms a stable but reversible linkage capable of withstanding shear forces and mild changes in pH (pH 4 to 10) and salinity (lOmM to saturated NaCl). CBDs may be isolated from various species, one example being Clostridium cellulovorans.

In some embodiments, the CBM is CBD.

In some embodiments, the amino acid sequence of the CBD has at least 70%, 80%, 85%, 90%, or 95%, or about 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CBD is encoded by a nucleotide sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with a nucleotide sequence encoding the CBD of SEQ ID NO: 1. In some embodiments, the CBD has the amino acid sequence set forth in SEQ ID NO: 1: QLNLKVEFYNSQPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDH AAIIGSQGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKND WSQYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEPGGSVVPSTQPVTTPPATTKP PATTIPPS. In some embodiments, the present invention provides a chimeric polypeptide for use in in- vitro tissue engineering, the chimeric polypeptide comprising: a CBD; a first ECM module; and optionally a linker, wherein the first ECM module comprises a first ECM protein selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, SIBLING, and a functional portion thereof.

In some embodiments, the chimeric polypeptide comprises a linker. In some embodiments, the chimeric polypeptide does not comprises a linker.

The ECM is a network including various macromolecules that provide structural and biochemical support to surrounding cells, and facilitate communication between the cells. Due to different requirements of different tissues, the composition of ECM varies between multicellular structures. Functions of the ECM include cell adhesion, communication and differentiation.

The term “ECM protein” encompasses proteins commonly found in ECM of living organisms and which participate in ECM functions such as cellular adhesion and communication between cells. More specifically, ECM proteins include functions such as modulating cell adhesion and/or activating signal transduction pathways that mediate cellular signals such as cell cycle regulation and intracellular cytoskeleton organization.

The term “a functional portion” means a portion of the protein, having an ECM function. In some embodiments, “a functional portion” means a subunit of the protein, having an ECM function. In some embodiments, “a functional portion” means a domain of the protein, having an ECM function. In some embodiments, “a functional portion” means a partial sequence of the protein, having an ECM function. In some embodiments, “a functional portion” means a partial sequence of at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the protein sequence, having an ECM function.

In some embodiments, the term “ECM function” means a function related to (or mediating) cellular adhesion, cellular migration, cellular differentiation, and/or a cellular communication.

Examples for ECM proteins suitable for use in the present invention include vitronectin, fibronectin, laminin, collagen, elastin, fibrin, E-cadherin, tenascin-C, perlecan, SIBLING, a cell chemoattractant, a heparin binding protein (e.g., HBP, azurocidin or cationic antimicrobial protein of 37 KDa), a heparan sulfate binding protein, a chondroitin sulfate binding protein, a dermatan sulfate binding protein, a keratan sulfate binding protein, a hyaluronic acid binding protein, and a Tri Calcium Phosphate /hydroxyapatite (TCP-HA) binding protein. Example for chemoattractants are low-molecular- weight peptides, such as 4 kDa to 16 kDa peptides, derived from the degradation of extracellular matrix proteins or glycoproteins. In some embodiments, the first ECM protein is vitronectin.

Vitronectin is a glycoprotein of the hemopexin family which is abundantly found in serum, the extracellular matrix, and bone. Vitronectin binds to integrin alpha-V beta-3 and thus promotes cell adhesion and spreading. It also inhibits the membrane-damaging effect of the terminal cytolytic complement pathway and binds to several serpins (serine protease inhibitors). It is a 54kDa secreted protein and exists in either a single chain form or a clipped, two chain form held together by a disulfide bond.

In some embodiments, the vitronectin amino acid sequence has at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence of bovine vitronectin set forth in SEQ ID NO: 2. In some embodiments, the vitronectin is encoded by a nucleotide sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with a nucleotide sequence encoding the amino acid sequence of bovine vitronectin set forth in SEQ ID NO: 2. In some embodiments, the vitronectin has an amino acid sequence according to SEQ ID NO: 2: DQESCKGRCTEGFNATRKCQCDELCSYYQSCCADFMAECKPQVTRGDVFHLPEDEYGF HDYSDAQTVNSNVEAQPESTTEAPVEQAQTEETPVQAPVENPEKEVPPSGRGDSEPGMG TSDLGTSESPAEEETCSGKPFDAFTDLKNGSLFAFRGLYCYELDEKAVRPGYPKLIRDVW GIEGPIDAAFTRVNCQGKTYLFKGSQYWRFQDGVLEPDFPRNISDGFGGIPDDVDAALA LPAHNFNGRERVYFFKGNHYWEYVFQQQPSQEDCEGSSLPAAFKHFALMQRDSWVDIF RLLFWGGSYGGAGQPQLISRNWFGLPGRLDAAMAGHIYVSGSAPSFPRAKMTKSARRH RKRYRSLRSRGRGRGRNQNPYRRSRSAFLSWLSSEELGLGANNYDSFEMDWLVPATCE PIQSVYFFSEDKYYRVNLRTRRVDAVIPPYPRSIAQYWLGCPVPGQA.

In some embodiments, the first ECM protein is a functional portion of vitronectin. In some embodiments, the functional portion of vitronectin comprises a vitronectin domain selected from a hemopexin-like domain (also known as a somatomedin B domain) and a heparin-binding domain. In some embodiments, the functional portion of vitronectin comprises at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the vitronectin sequence, and having an ECM function.

In some embodiments, the first ECM module is linked to the CBD on the N-terminal side of the CBD. In some embodiments, the first ECM module is linked to the CBD on the C-terminal side of the CBD.

In some embodiments, the present invention provides a chimeric polypeptide for use in in- vitro tissue engineering, the chimeric polypeptide comprising: a CBD; a first ECM module; and optionally a linker, wherein the first ECM module comprises a first ECM motif selected from GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR. Definitions and embodiments mentioned above and which may be relevant to the chimeric polypeptides including an ECM motif, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

The term “ECM motif’ as used herein refers to an amino acid sequence of at least several amino acids, such as about 3-100 amino acids, derived from an ECM protein or peptide and having a defined ECM function, such as attaching to other ECM molecules or to molecules presented on cells. ECM motifs relevant to the present application include any sequence having a defined ECM function derived from any ECM protein as defined hereinabove. Non-limiting examples for ECM motifs include fibronectin motif, a laminin motif, a collagen motif, a vitronectin motif, an elastin motif, an E-cadherin motif, a tenascin-C, a perlecan motif, a SIBLING motif, RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGS, and repeats or combinations thereof.

In some embodiments, the first ECM module comprises more than one identical ECM motif (e.g., more than one RGD sequence, or more than one GRGDSP sequence). In some embodiments, when an ECM module comprises more than one identical ECM motif, the ECM motifs of the ECM module may be embedded in the sequence of the chimeric polypeptide. For example, the chimeric polypeptide may include identical ECM motifs linked to the C-terminus and/or to the N-terminus of the CBD, and/or embedded inside the CBD sequence.

In some embodiments, the chimeric polypeptide comprises at least about 0.4% total ECM motifs, i.e., at least about 1, 2, 3, or 4 total ECM motifs per 250 amino acids. In some embodiments, the chimeric polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, or 8 ECM motifs embedded in the chimeric polypeptide.

Examples for additional ECM motifs that may be used in the invention include: a cell chemoattractant peptide, a heparin binding peptide, a heparan sulfate binding protein, a chondroitin sulfate binding protein, a dermatan sulfate binding protein, a keratan sulfate binding protein, a hyaluronic acid binding peptide, and a Tri Calcium Phosphate /hydroxyapatite (TCP- 14 A) binding peptide.

Further examples for specific motif/peptide sequences include motifs which appear in Bomkamp et al., Adv. Sci. 2022, 9, 2102908; Ishihara et al., Nat. Commun. 2018, 9:2163; Dayem et al., Stem Cell Research 2020, 43, 101700; Huettner et al., Trends in Biotechnology 2018, 36(4):372; Hellmund and Koksch, Frontiers in Chemistry 2019, 7:172, as well as peptide motifs appearing in the Kollodis Biosciences web site at https://www.kollodis.com/prod/prod_l.html. The linker described herein is a short peptide, as further defined below, which links the CBM or CBD to the ECM module in the chimeric polypeptide. In some embodiments, the linker is configured to ensure that the CBD, when conjugated to the ECM module, maintains its correct folding and 3D structure, thereby allowing it to function properly in attaching the chimeric polypeptide to a cellulose matrix. In some embodiments, the linker is further configured to ensure that the ECM module, when conjugated to the CBD, maintains its correct folding and 3D structure, thereby allowing it to function properly with respect to its ECM functions. In some embodiments, the linker is configured to be cleaved by a specific or a non-specific protease or a peptidase (such as pepsin or papain), for example, by including a bulge that becomes exposed to proteolytic enzymes.

In some embodiments, the linker links the CBD to the first ECM module.

The size of the linker has several potential effects, including the stiffness or flexibility of the molecule, and the accessibility or lack thereof of certain domains. The length of the linker should be sufficient to allow for proper folding of any protein or motif comprised in the chimeric polypeptide.

In some embodiments, the linker has a length of about 5-50, 5-40, 5-30, 5-25, 5-20, 10-50, 10-40, 10-30, 10-25, 20-50, or 20-40 amino acids, or any other suitable length within the range of 5-50 amino acids.

In some embodiments, the linker has a sequence rich in proline and threonine amino acids, to allow adequate space between the CBD and the additional protein or motif, to facilitate proper folding of the chimeric polypeptide, with the CBD and any protein or motif linked to it, being functional.

In some embodiments, the linker has at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3: (GAGGGSGGGSGGGSAGGG). In some embodiments, the linker has a sequence according to SEQ ID NO: 3. In some embodiments, the linker is encoded by a nucleotide sequence that has at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or 99% identity to a nucleotide sequence encoding SEQ ID NO: 3.

In some embodiments, the chimeric polypeptide further comprises at least an additional ECM module comprising an additional ECM protein, an additional ECM motif, and/or a growth factor (GF)-related sequence. The at least additional ECM module, additional ECM protein, and additional ECM motif are defined in the same way as the first ECM module, first ECM protein and first ECM motif above. However, unlike the first ECM motif, the additional ECM motif may be an RGD motif.

In some embodiments, at least an additional ECM module comprises an ECM protein.

In some embodiments, the additional ECM protein is selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, SIBLING, a functional portion thereof, and combinations thereof.

In some embodiments, at least an additional ECM module comprises an ECM motif.

In some embodiments, the additional ECM motif is selected from a fibronectin motif, a collagen motif, a laminin motif, a vitronectin motif, an elastin motif, an E-cadherin motif, a tenascin-C, a perlecan motif, a SIBLING motif, and combinations thereof.

In some embodiments, the additional ECM motif is selected from a fibronectin motif comprising RGD, GRGDSP (SEQ ID NO: 4), and/or PHSRN (SEQ ID NO: 5); a collagen motif comprising GFOGER (SEQ ID NO: 6); a laminin motif comprising IKVAV (SEQ ID NO: 7) and/or YIGSR (SEQ ID NO: 8), and combinations thereof. In some embodiments, the additional ECM motif is selected from GRGDSP, PHSRN, GFOGER, KVAV, YIGSR, and combinations thereof.

In some embodiments, the additional ECM module comprises more than one identical ECM motif (e.g., more than one RGD sequence, or more than one GRGDSP sequence). In some embodiments, when an ECM module comprises more than one identical ECM motif, the ECM motifs of the ECM module may be embedded in the sequence of the chimeric polypeptide. For example, the chimeric polypeptide may include identical ECM motifs linked to the C-terminus and/or to the N-terminus of the CBD, and/or inserted inside the CBD sequence.

In some embodiments, the first ECM module comprises GRGDSP and the additional ECM module comprises PHSRN. In some embodiments, the first ECM module comprises PHSRN and the additional ECM module comprises GRGDSP. In some embodiments, the chimeric polypeptide comprises at least one PHSRN motif and at least one GRGDSP motif. In some embodiments, the GRGDSP and the PHSRN flank the CBD, i.e., one of the GRGDSP and the PHSRN is linked to the N-terminal side of the CBD and the other is linked to the C-terminal side of the CBD. In some embodiments, the GRGDSP and/or the PHSRN are embedded in the CBD. In some embodiments, the GRGDSP and/or the PHSRN are embedded in the CBD, wherein one of the GRGDSP and the PHSRN is embedded close to the N-terminal side of the CBD and the other is embedded close to the C-terminal side of the CBD or is linked to the C-terminal side of the CBD. In some embodiments, the GRGDSP and/or the PHSRN are embedded in the CBD, wherein one of the GRGDSP and the PHSRN is embedded close to the C-terminal side of the CBD and the other is embedded close to the N-terminal side of the CBD or is linked to the N-terminal side of the CBD. In some embodiments, the GRGDSP and the PHSRN are at a molar ratio of about 1:1.

In some embodiments, at least an additional ECM module comprises a GF-related sequence.

The term “growth factor (GF)-related sequence” encompasses GF proteins, GF protein motifs, and ECM GF-binding domains, as further detailed below.

In some embodiments, the GF-related sequence is a GF protein.

In some embodiments, the GF protein is selected from FGF2, IGF1, TGFpi, EGF, EIF, Activin A, NRG1, PDGF, IE6, IE 13 or any combination thereof. Other serum proteins such as insulin or transferrin are also suitable as GF proteins of the invention.

In some embodiments, the GF protein is an isoform of bovine FGF2 having at least about 70%, 80%, 85%, 90%, 95% or 99% sequence identity to the amino acid sequence of NCBI accession No. NP_776481 or of UniProt accession No. P03969. In some embodiments, the GF protein is encoded by a nucleotide sequence having at least about 60%, 65%, 70%, 80%, 85%, 90%, 95% or 99% sequence identity to a nucleotide sequence encoding the amino acid sequence of NCBI accession No. NP_776481 or of UniProt accession No. P03969. In some embodiments, the GF protein has an amino acid sequence as defined in NCBI accession No. NP_776481 or in UniProt accession No. P03969.

In some embodiments, the GF-related sequence is a GF protein motif.

The term “GF protein motif’ as used herein refers to a sequence of at least several amino acids, such as about 3-100 amino acids, derived from a GF protein or peptide and having a defined function. GF motifs relevant to the present application include any sequence having a defined function derived from any GF protein as defined hereinabove. A non-limiting example for such a peptide is an FGF2-derived peptide, positions 16-26 of NP_776481.

In some embodiments, the GF protein motif is selected from a functional peptide or motif of a growth factor. In some embodiments, the GF protein motif is selected from a functional peptide or motif of a growth factor protein selected from FGF2, IGF1, TGFpi, EGF, LIF, Activin A, NRG1, PDGF, IL6, IL 13 or any combination thereof.

In some embodiments, the GF-related sequence is an ECM GF-binding domain.

The term “ECM GF binding domain” as used herein relates to a sequence of at least several amino acids, such as about 3-100 amino acids, derived from an ECM protein and having a function of binding to a growth factor. Examples for such GF binding domains are heparin-binding domains (HBDs) of ECM glycoproteins, such as fibronectin, tenascin-C, and fibrinogen. GF- binding sites are often located in close proximity to integrin-binding sites in some ECM glycoprotein chains (e.g. in fibronectin and tenascin-C). Some specific examples for lamininderived peptides capable of binding to both heparin and growth factors include, but are not limited to, LAMA3: 2932-2951, 3031-3043, 3043-3067, LAMA4: 1408-1434, 1521-1543, LAMA5: 3300-3330, 3417-3436, 3539-3550. Additional relevant peptides include the NCAM2 Fibronectin type-III domains and peptides derived therefrom.

In some embodiments, the chimeric polypeptide further comprises at least an additional linker linking at least one of the additional ECM modules to the CBD, to the first ECM module, and/or to another one of the additional ECM modules.

In some embodiments, the additional linker is defined as per the above description of the linker. In some embodiments, the additional linker is the same as the (first) linker.

In some embodiments, the first ECM module and the at least additional ECM module flank the CBD, i.e. the first ECM module is on the N-terminal side of the CBD, and the at least additional ECM module is on the C-terminal side of the CBD, or the first ECM module is on the C-terminal side of the CBD, and the at least additional ECM module is on the N-terminal side of the CBD.

As noted above, when the first ECM module and/or the additional ECM module comprise an ECM motif - then the ECM motif may be on either side of the CBD or may be embedded in the CBD. When the first ECM module and/or the additional ECM module comprise more than one identical ECM motif - then the ECM motifs may flank and/or may be embedded in the CBD.

When the first ECM module and the at least additional ECM module flank the CBD, then both the first ECM module and the at least additional ECM module are linked to the CBD .

In some embodiments, the first ECM module and the at least additional ECM module are on the same side of the CBD. In some embodiments, the first ECM module and the at least additional ECM module are on the N-terminal side of the CBD. In some embodiments, the first ECM module and the at least additional ECM module are on the C-terminal side of the CBD.

When the first ECM module and the at least additional ECM module are on the same side of the CBD, then one of the first ECM module and the at least additional ECM module is bound to the CBD, and other ECM modules are bound to one that is bound to the CBD, and/or to each other.

In some embodiments, at least one of the additional ECM modules is not identical to the first ECM module. In some embodiments, at least one of the additional ECM modules is identical to the first ECM module. In some embodiments, the first ECM module comprises a first ECM motif and the at least additional ECM module comprises an additional ECM motif, and the first ECM motif and additional ECM motif are different motifs, each selected from a fibronectin motif, a laminin motif, a collagen motif, a vitronectin motif, an elastin motif, an E-cadherin motif, a tenascin-C, a perlecan motif, a SIBLING motif, RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR.

In some embodiments, the first ECM motif and the additional ECM motif flank the CBD, i.e., one is linked to the N-terminal side of the CBD and the other is linked to the C-terminal side of the CBD. In some embodiments, at least one of the first ECM motif and the additional ECM motif is embedded in the CBD. In some embodiments, the first ECM motif and/or the additional ECM motif are embedded in the CBD, wherein one is embedded close to the N-terminal side of the CBD and the other is embedded close to the C-terminal side of the CBD. In some embodiments, the first ECM motif and the additional ECM motif are close together, i.e., having no more than about 20 aa, 10 aa, or 5 aa between them. In some embodiments, the first ECM motif and the additional ECM motif are far apart, i.e., having at least about 50 aa, 60 aa, 70 aa, 80 aa, 90 aa, or 100 aa between them.

In some embodiments, the at least additional ECM module comprises at least one RGD motif. In some embodiments, the at least additional ECM module comprises more than one RGD motif.

In some embodiments, the additional ECM module comprises more than one RGD. In some embodiments, when the additional ECM module comprises more than RGD, the RGD may be embedded in the sequence of the chimeric polypeptide, and optionally embedded within the CBD. For example, the chimeric polypeptide may include RGD motifs linked to the C-terminus and/or to the N-terminus of the CBD, and/or inserted inside the CBD sequence.

In some embodiments, the chimeric polypeptide comprises at least about 0.4% RGD motifs, i.e., at least 1, 2, 3, or 4 RGD motifs per 250 amino acids. In some embodiments, the chimeric polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, or 8 RGD motifs embedded within the chimeric polypeptide.

In some embodiments, the first ECM module comprises an ECM protein or a functional portion thereof, and the at least additional ECM module comprises an ECM motif. In some embodiments, the first ECM module comprises an ECM motif and the at least additional ECM module comprises an ECM protein or a functional portion thereof.

In some embodiments, the first ECM module comprises vitronectin, or a functional portion thereof, and the at least additional ECM module comprises an ECM motif. In some embodiments, the first ECM module comprises vitronectin, or a functional portion thereof, and the at least additional ECM module comprises an RGD motif.

In some embodiments, the first ECM module comprises vitronectin, or a functional portion thereof, and the at least additional ECM module comprises an at least two RGD motifs. In some embodiments, the first ECM module comprises vitronectin, or a functional portion thereof, and the chimeric polypeptide comprises at least about 0.4% RGD motifs, i.e., at least about 1, 2, 3, or 4 RGD motifs per 250 amino acids, as part of the additional ECM module.

In some embodiments, both the vitronectin and the RGD are linked to the CBD.

In some embodiments, only the vitronectin is linked to the CBD and the RGD is linked to the vitronectin. In some embodiments, only the RGD is linked to the CBD and the vitronectin is linked to the RGD.

In some embodiments, the vitronectin is on the N-terminal side of the CBD and RGD is on the C-terminal side of the CBD. In some embodiments, the vitronectin is on the C-terminal side of the CBD and RGD is on the N-terminal side of the CBD.

In some embodiments, the vitronectin and the RGD are both on the N-terminal side of the CBD. In some embodiments, the vitronectin and the RGD are both on the C-terminal side of the CBD.

In some embodiments, the vitronectin is on the N-terminal or on the C-terminal side of the CBD, and at least one RGD motif is embedded in the CBD.

In some embodiments, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the chimeric polypeptide described herein.

Definitions and embodiments mentioned above and which may be relevant to the nucleic acid molecule, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

In some embodiments, the nucleotide sequence comprises a CBD sequence encoding the CBD described herein; at least one ECM protein and/or ECM motif sequence encoding an ECM protein (or a functional portion thereof) or ECM motif as described herein, the ECM protein being selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, SIBLING, and a functional portion thereof, and the ECM motif being selected from a fibronectin motif, a collagen motif, a laminin motif, a vitronectin motif, an elastin motif, an E-cadherin motif, a tenascin-C, a perlecan motif, a SIBLING motif, RGD, GRGDSP, PHSRN, GFOGER, IKVAV, YIGSR, and combinations thereof; and optionally at least one linker sequence encoding the optional linker described herein. In some embodiments, the ECM protein sequence comprises a sequence encoding vitronectin or a functional portion thereof. In some embodiments, the sequence encoding the vitronectin has at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the nucleotide sequence of bovine vitronectin set forth in SEQ ID NO: 9. In some embodiments, the sequence encoding vitronectin is SEQ ID NO: 9: GATCAAGAATCTTGTAAGGGAAGATGTACTGAAGGATTTAATGCTACTAGAAAGTG TCAATGTGATGAACTTTGTTCTTATTATCAATCTTGTTGTGCTGATTTTATGGCTGAA TGTAAGCCACAAGTTACTAGAGGAGATGTTTTTCATCTTCCTGAAGATGAATATGGA TTTCATGATTATTCTGATGCTCAAACTGTTAATTCTAATGTTGAAGCTCAACCTGAAT CTACTACTCTTGCTCCTGTTCTTCAAGCTCAAACTCTTGAAACTCCTGTTCAAGCTCC TGTTCTTAATCCTGAAAAGGAAGTTCCACCATCTGGAAGAGGAGATTCTGAACCTGG AATGGGAACTTCTGATCTTGGAACTTCTGAATCTCCTGCTGAAGAGGAAACTTGTTC TGGAAAGCCATTTGATGCTTTTACTGATCTTAAGAATGGATCTCTTTTTGCTTTTAGA GGACTTTATTGTTATGAACTTGATGAAAAGGCTGTTAGACCTGGATATCCAAAACTT ATTAGAGATGTTTGGGGAATTGAAGGACCAATTGATGCTGCTTTTACTAGAGTTAAT TGTCAAGGAAAGACTTATCTTTTTAAGGGATCTCAATATTGGAGATTTCAAGATGGA GTTCTTGAACCTGATTTTCCAAGAAATATTTCTGATGGATTTGGAGGTATTCCTGATG ATGTTGATGCTGCTCTTGCTCTTCCTGCTCATAATTTTAATGGAAGGGAGAGAGTTTA _{TTTTTTTAAGGGAAATCATTATTGGGAATATGTTTTTCAACAGCAACCATCTCAAGA} AGATTGTGAAGGATCTTCTCTTCCTGCTGCTTTTAAGCATTTTGCTCTTATGCAAAGA GATTCTTGGGTTGATATTTTTAGACTTCTTTTTTGGGGAGGATCTTATGGAGGAGCTG GACAACCACAACTTATTTCAAGAAATTGGTTTGGACTTCCTGGAAGACTTGATGCTG CTATGGCTGGACATATTTATGTTTCTGGATCTGCTCCATCTTTTCCAAGAGCTAAGAT GACTAAGTCTGCTAGAAGACATAGAAAGAGATATAGATCACTTAGGTCAAGAGGAA GAGGAAGAGGAAGAAATCAAAATCCATATAGAAGGTCTAGGTCTGCTTTTCTTTCTT GGCTTTCTTCTGAAGAACTTGGACTTGGAGCTAATAATTATGATTCTTTTGAAATGG ATTGGCTTGTTCCTGCTACTTGTGAACCTATTCAATCTGTGTATTTTTTTTCAGAAGA CAAGTATTATAGGGTTAATCTTAGAACTAGAAGAGTTGATGCTGTTATTCCACCATA TCCAAGGTCTATTGCTCAATATTGGCTTGGATGTCCTGTTCCTGGACAGGCT.

In some embodiment, the ECM motif sequence comprises a sequence encoding an RGD motif.

In some embodiments, the nucleotide sequence comprises a sequence encoding vitronectin and a sequenced encoding RGD. In some embodiments, the nucleic acid sequence encoding the chimeric polypeptide further encodes an endoplasmic reticulum (ER) retention signal.

In some embodiments, there is provided a vector comprising the nucleic acid sequence described herein. In some embodiments, the vector is a (pCAMBIA vector). In some embodiments, the vector is suitable for expression of the chimeric polypeptide in plants, including a promoter suitable for expression in plants, such as the CaMV promoter from cauliflower mosaic virus, or the duplicated variant thereof.

Protein purification by expressing in plants carry great advantages over other expression systems. Among these advantages are low cost of production, potential for large-scale cultivation, inherent safety reflecting the inability of human pathogens to replicate in plants, as well being a superior system for producing glycoproteins. A specific example for such a purification system would be a tobacco plant.

In some embodiments, the chimeric polypeptide may be produced by expression and purification in plants. In some embodiments, the chimeric polypeptide is produced by transforming plants with an expression construct suitable for expression in plants, encoding the chimeric polypeptide (such as the nucleic acid or vector mentioned above) under the regulation of a suitable promoter, such as cauliflower mosaic virus (CaMV) 35S promoter, or the duplicated CaMV 35S promoter. The chimeric polypeptide may then be purified from the plant by any suitable method. As described in the examples, in some embodiments, cellulose affinity to the CBD may be used to purify the chimeric polypeptide.

In sone embodiment, the plant may be tobacco, soybean, or corn. In some embodiment, the chimeric polypeptide is expressed and purified from a non-plant organism such as duckweed, yeast, bacteria, fungus, or algae.

Therefore, in some embodiments, there is provided a transgenic plant genetically modified to express the chimeric polypeptide described herein. In some embodiments, the plant is a tobacco plant. Expression of the chimeric polypeptide in the plant maybe transient or stable.

In some embodiments, the present application provides a transgenic plant comprising the nucleic acid or the vector disclosed herein. In some embodiments, the present application provides a transgenic plant expressing the chimeric polypeptide disclosed herein.

Definitions and embodiments mentioned above and which may be relevant to the transgenic plants, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. In some embodiments, the chimeric polypeptide of the invention is bound, through the CBM, to a carbohydrate -based scaffold, suitable for tissue growth. In some embodiments, the CBM is a CBD, and the carbohydrate is cellulose.

In some embodiments, there is provided an in-vitro tissue engineering system comprising the chimeric polypeptides disclosed herein bound to the cellulose-based scaffold via the CBD.

Definitions and embodiments mentioned above and which may be relevant to the tissue engineering system, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

The term “cellulose-based scaffold” is used interchangeably with the term “cellulose- containing scaffold” and refers to a scaffold which contains cellulose, to which the CBD part of the chimeric polypeptide of the invention binds. Non-limiting examples include scaffolds including 1.25% cellulose, 3:1 CNC:bamboo, (CNC: cellulose nanocrystals) crosslinked with citric acid. Additional examples for suitable scaffolds may be found, for example, in Han Seah et al., 2021, Critical reviewed in Biotechnology 42(2):1-13, and at the Good Food institute (GFI) web site at https://gfi.org/solutions/plant-based-scaffolds-nutrition.

In some embodiments, the scaffold is a three-dimensional (3D) scaffold. In some embodiments, the scaffold includes more than one surface, or region, which is defined by a certain position in the scaffold. In some embodiments, different scaffold surfaces (or regions) include different chimeric polypeptides. Such different chimeric polypeptides may differ in their ECM modules. As a result of these differences, different scaffold surfaces may induce differentiation of different cell types, enabling the complete scaffold to induce differentiation of a complete tissue or organ. It is noted that the term “scaffold surface” relates both to internal and external scaffold surfaces.

In some embodiments, the scaffold includes at least two domains. In some embodiments, each of the at least two domains is associated with chimeric polypeptides comprising different ECM modules or a different combinations of ECM modules.

In some embodiments, the scaffold is a knitted, a woven, or a non-woven, textile.

The scaffold may be, for example, plant-based, algae-based, tunicate -based, bacteria- based, or a synthetic scaffold. In some embodiments, the scaffold is made from decellularized plant, seaweed, tunicate tissue, or bacterial pellicle.

In some embodiments, the cellulose-based scaffold comprises at least one surface to which a population of the chimeric polypeptides disclosed herein is attached.

In some embodiments, the present invention provides at least one cellulose-based scaffold surface to which a population of chimeric polypeptides is attached, each chimeric polypeptide comprising a CBM, which may be a CBD; at least one ECM module selected from an ECM motif and an ECM protein; and optionally a linker, wherein at least two chimeric polypeptides comprise non-identical ECM modules.

In some embodiments, the cellulose-based scaffold comprises at least one surface to which are attached: a population of the chimeric polypeptides disclosed herein, and chimeric polypeptides comprising a CBM, which may be a CBD; at least one GF-related sequence as defined herein; and optionally at least one linker.

In some embodiments, the present invention provides a combination of the chimeric polypeptides disclosed herein.

In some embodiments, the present invention provides a combination of the chimeric polypeptides, wherein each chimeric polypeptide comprises a CBM, which may be a CBD; at least one ECM module selected from an ECM motif or an ECM protein or a functional portion thereof; and optionally at least one linker, wherein at least two chimeric polypeptides comprise nonidentical ECM modules.

In some embodiments, the combination of the chimeric polypeptides comprises at least one chimeric polypeptide comprising vitronectin or a functional portion thereof as part of the ECM module, and at least one chimeric polypeptide comprising RGD as part of the ECM module.

In some embodiments, the combination of the chimeric polypeptides comprises at least one chimeric polypeptide comprising GRGDSP as part of the ECM module, and at least one chimeric polypeptide comprising PHSRN as part of the ECM module.

In some embodiments, the present invention provides a combination comprising the chimeric polypeptides disclosed herein, and chimeric polypeptides comprising a CBM, which may be a CBD; at least one GF-related sequence; and optionally at least one linker.

In some embodiments, at least one of the chimeric polypeptides comprised in the combination or attached to the scaffold, comprises at least two ECM modules.

In some embodiments, at least one of the chimeric polypeptides comprised in the combination or attached to the scaffold, comprises at least one ECM module selected from an ECM protein, or a functional portion thereof, and an ECM motif, and at least one GF-related sequence.

In some embodiments, the tissue engineering system further comprises precursor cells, which adhere to the scaffold by attaching to the ECM module, and may differentiate into a desired tissue.

Non-limiting examples for precursor cells suitable for the invention include any type of stem cells, such as embryonic stem cells, totipotent stem cells, pluripotent stem cells, induced pluripotent stem cells, and tissue-specific stem cells such as mesenchymal stem cells. Additionally, certain types of adult stem cells are most applicable to cultivated meat production. Among them, there are three major progenitor/stem cell types present in the muscle tissue environment: muscle satellite cells, mesenchymal stem/stromal cells (MSCs), and fibro/adipogenic progenitors (FAPs). These progenitor cells have the ability to differentiate into one or more key mature cell types, namely, skeletal myocytes, adipocytes, chondrocytes, and fibroblasts. Precursor cells may be from mammalian, avian, fish, or shellfish such as crustacean origin.

In some embodiments, the chimeric polypeptides disclosed herein, or the tissue engineering system disclosed herein, are used for generating an engineered tissue.

In some embodiments, the tissue is a muscle tissue such as a skeletal muscle tissue. In some embodiments, the tissue is a fat tissue, connective tissue, or blood vessel tissue.

In some embodiments, the precursor cells are MSCs, satellite cells, or FAPs, and the engineered tissue is skeletal muscle tissue or smooth muscle tissue.

In some embodiments, the present application provides an in vitro method of generating an engineered tissue, comprising: a. adding the chimeric polypeptide disclosed herein to a cellulose-based scaffold with a suitable medium; b. allowing the chimeric polypeptide to bind to the cellulose-based scaffold; c. seeding precursor cells onto the scaffold-bound chimeric polypeptide; and d. growing the cells until an engineered tissue is formed.

Definitions and embodiments mentioned above and which may be relevant to the methods, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated.

Preparation of a desired tissue according to the present invention involves seeding precursor cells on a scaffold as described herein in the presence of a medium containing suitable ingredients, such as nutrients and growth factors. The precursor cells grow on the scaffold and differentiate into the desired tissue.

The precursor cells are the same as disclosed above.

One of the advantages of using a chimeric polypeptide, in which the ECM module is conjugated or linked to the CBD, is that by concentrating and fixing the ECM module to the scaffold, this allows to use a lower amount of the ECM module than would be needed if the ECM module was provided separately from the CBD. Accordingly, in some embodiments, the chimeric polypeptide comprising the ECM module is added to the medium at a concentration that is at least 10% lower than a concentration of the ECM module not being linked to a CBD when added to a medium comprising a CBM or CBD and a scaffold (such as a cellulose-based scaffold), in a method for generating an engineered tissue.

In some embodiments, the concentration is at least 20%, 30%, 40%, 50%, or 50% lower than a concentration of the ECM module not being linked to a CBD when added to a medium comprising a CBM or CBD and a scaffold, in a method for generating an engineered tissue.

During growth and differentiation of the cells on the scaffold, mechanical stress may be applied to the scaffold to direct differentiation into certain tissue types. As explained above, another advantage of the present invention is that since the cells are directly attached to the scaffold through the unique chimeric polypeptide described herein, this mechanical stress transfers to the cells and affects their differentiation path. The mechanical stress may be in form of compression or tensile stress, and is advantageously applied at specific strains or forces and at specific frequencies, which mimic the natural environment required for growth and/or differentiation of the desired tissue. This process is required, e.g., for generation of tendons, ligaments, muscle, and skin tissue. Just as an example, skin may experience up to 20% strain at relatively low frequencies 0.1-10 Hertz.

Accordingly, in some embodiments, the growing in step (d) involves application of a mechanical stress to the scaffold. In some embodiments, the mechanical stress is in form of compression or tensile stress. In some embodiments, the compression or tensile stress is applied at specific strains or forces and at specific frequencies to mimic the natural environment required for growth and/or differentiation of the tissue. In some embodiments, the tissue is a muscle tissue. In some embodiments, the tissue is a skeletal muscle tissue.

In some embodiments, the chimeric polypeptide is prepared by transforming a plant with the nucleic acid molecule disclosed herein or the vector disclosed herein, and purifying the chimeric polypeptide from the plant, as described above.

In some embodiments, the present invention provides an engineered tissue generated by the methods disclosed herein.

Definitions and embodiments mentioned above and which may be relevant to the engineered tissue, also apply here, and vice versa. Some particularly relevant embodiments may be pointed out or explicitly repeated. In some embodiments, there is provided a tissue generated by seeding precursor cells in the cellulose-based scaffold disclosed herein, to which chimeric polypeptides as disclosed herein are attached, in some embodiments, generating the tissue involves application of a mechanical stress to the scaffold. In some embodiments, the mechanical stress is in form of compression or tensile stress. In some embodiments, the compression or tensile stress is applied at specific strains or forces and specific frequencies to mimic the natural environment required for growth and/or differentiation of the tissue.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims which follow.

EXAMPLES

Example 1: Purification of CBD-Conjugated ECM proteins or peptides from tobacco Leaves

A vector comprising a chimeric cellulose-binding domain (CBD)-vitronectin construct, encoding CBD conjugated to vitronectin, is synthesized and transformed into tobacco plants, and expressed either transiently or stably under the control of a robust promoter such as the duplicated CaMV 35S promoter. Once a sufficient biomass of plants is produced, the chimeric CBD- vitronectin protein is purified from the plant leaves by employing cellulose affinity purification method described in publication No. WO/2018/178991. Briefly, freshly harvested leaves are shredded in a proteolysis-inhibiting extraction buffer. Subsequently, the leaf crude extract undergoes filtration using a 0.2 Micron Hollow Fiber (HF) filter, and the filtrate is incubated for two hours with nano-cellulose to facilitate binding of CBD to the cellulose fibers. The cellulose- bound CBD-vitronectin complex is subjected to multiple washes to release nonspecific binding of plant material. To release the cleaned CBD-vitronectin from the cellulose fibers, the complex undergoes a 20-minute incubation under alkaline conditions. Additional filtration steps are then employed to separate the soluble CBD-vitronectin from insoluble, large cellulose fibers and to concentrate the CBD-vitronectin soluble fraction. Finally, the purified CBD-vitronectin is dried into a powder by lyophilization, and the resulting powder is kept in -20°C until use.

The same process is used for purification of additional chimeric polypeptides including CBD conjugated to other extracellular matrix (ECM) proteins such as fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, and small integrin-binding ligand N-linked glycoproteins (SIBLING), or to peptides comprising ECM motifs such as RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR, or any combination of ECM proteins and peptides.

Example 2: Preparation of a cellulose scaffold coated with CBD-ECM protein/peptide

Cellulose scaffolds containing 1.25% cellulose, at a ratio of 3:1 CNC: bamboo (CNC: cellulose nanocrystals), crosslinked with citric acid, are coated under sterile conditions with the CBD-vitronectin conjugate. Briefly, the CBD-vitronectin powder is dissolved in a serum-free DMEM medium, with serial dilutions performed for each preparation. As controls, serial dilutions of a protein preparation from wild-type tobacco and serum-free DMEM are utilized. UV-sterilized cellulose scaffolds are placed at the bottom of wells in 48-well polystyrene plates. Thirty-five microliters of the dissolved and diluted CBD-vitronectin and the control solution are applied on top of the scaffold. The plates are then incubated at 20°C for 2 hours to facilitate proper binding of CBD to the cellulose.

Cellulose scaffolds coated with additional chimeric polypeptides including CBD conjugated to other extracellular matrix (ECM) proteins (complete proteins, a subunit, a domain, or a peptide thereof) such as fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, and small integrin-binding ligand N-linked glycoproteins (SIBLING), or to peptides comprising ECM motifs such as RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR, or any combination of ECM proteins and peptides, are prepared by the same process.

Example 3: 3T3-L1 MBX fibroblasts adhesion and proliferation Assays

3T3-L1 MBX (ATCC) is a mouse embryo fibroblast cell line which responds to externally added insulin, IGF-1, FGF2 or other growth factors by differentiation into adipocytes or by an enhanced proliferation rate. For assessing cell adhesion and proliferation of the fibroblasts to the CBD-vitronectin coated scaffold, a resazurin fluorometric assay, which measures the metabolic capacity of live cells, is used. 3T3 Ll-MBX fibroblasts are seeded on freshly prepared CBD- vitronectin coated scaffolds or control scaffolds (without CBD-vitronectin) at a density of 1.5 X 10⁴ cells per well. The plates are incubated at 37°C in a 5% CO2, 95% humidified air atmosphere for 1 hour to enable cell attachment to the scaffold. Two hours after the addition of cells, the wells are washed with PBS, and DMEM with 10% FBS is added. Resazurin viability assay of the adsorbed cells is conducted according to the manufacturer’s instructions (ABCAM, USA, cat. # abl29732), for assessing cell adhesion after 2 hours and proliferation at 24, 48 hours, and 7 days post cell seeding. The results are calculated from at least three separate assays, each performed in triplicate.

Testing of fibroblasts adhesion and proliferation on cellulose scaffolds coated with additional chimeric polypeptides including CBD conjugated to ECM proteins (complete proteins, a subunit, a domain, or a peptide thereof) such as fibronectin, laminin, collagen, elastin, E- cadherin, tenascin-C, perlecan, and small integrin-binding ligand N-linked glycoproteins (SIBLING), or to peptides comprising ECM motifs such as RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR, or any combination of ECM proteins and peptides, are performed by the same process.

Claims

1. A chimeric polypeptide for use in in-vitro tissue engineering, the chimeric polypeptide comprising: a. a cellulose binding domain (CBD); b. a first extracellular matrix (ECM) module; and c. optionally a linker, wherein the first ECM module comprises a first ECM protein selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, small integrin-binding ligand N-linked glycoproteins (SIBLING), and a functional portion thereof.

2. The chimeric polypeptide of claim 1, wherein the first ECM module comprises vitronectin or a functional portion thereof.

3. The chimeric polypeptide of claim 2, wherein the vitronectin has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2.

4. A chimeric polypeptide for use in in-vitro tissue engineering, the chimeric polypeptide comprising: a. a cellulose binding domain (CBD); b. a first extracellular matrix (ECM) module; and c. optionally a linker, wherein the first ECM module comprises a first ECM motif selected from GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR.

5. The chimeric polypeptide of any one of claims 1-4, wherein the linker links the CBD to the ECM module.

6. The chimeric polypeptide of any one of claims 1-5, wherein the linker has a length of about 10-50 amino acids.

7. The chimeric polypeptide of any one of claims 1-6, wherein the CBD has at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

8. The chimeric polypeptide of any one of claims 1-7, further comprising at least an additional ECM module comprising an additional ECM protein or a functional portion thereof; an additional ECM motif; and/or a growth factor (GF)-related sequence.

9. The chimeric polypeptide of claim 8, wherein the additional ECM protein is selected from vitronectin, fibronectin, laminin, collagen, elastin, E-cadherin, tenascin-C, perlecan, and SIBLING.

10. The chimeric polypeptide of claim 8 or 9, wherein the additional ECM motif is selected from a fibronectin motif, a collagen motif, a laminin motif, a vitronectin motif, an elastin motif, an E-cadherin motif, a tenascin-C, a perlecan motif, and a SIBLING motif.

11. The chimeric polypeptide of any one of claims 8-10, wherein the additional ECM motif is selected from a fibronectin motif comprising RGD, GRGDSP, and/or PHSRN; a collagen motif comprising GFOGER; and a laminin motif comprising IKVAV and/or YIGSR.

12. The chimeric polypeptide of any one of claims 8-11, further comprising at least an additional linker linking at least one of the additional ECM modules to the CBD, to the first ECM module, and/or to another one of the additional ECM modules.

13. The chimeric polypeptide of any one of claims 8-12, wherein at least one of the additional ECM modules is not identical to the first ECM module.

14. The chimeric polypeptide of any one of claims 8-13, wherein the first ECM module and/or the additional ECM module comprise more than one identical ECM motif, and the chimeric polypeptide comprises at least about 0.4% total ECM motifs, i.e., at least about 1 ECM motif per 250 amino acids.

15. The chimeric polypeptide of any one of claims 8-14, wherein the first ECM module and the at least additional ECM module both comprise ECM motifs independently selected from a fibronectin motif, a laminin motif, a collagen motif, a vitronectin motif, an elastin motif, an E- cadherin motif, a tenascin-C, a perlecan motif, a SIBLING motif, RGD, GRGDSP, PHSRN, GFOGER, IKVAV, and YIGSR.

16. The chimeric polypeptide of any one of claims 8-15, wherein the additional ECM motif comprises at least one RGD motif.

17. The chimeric polypeptide of claim 16, wherein the at least one RGD motif comprises at least two RGD motifs.

18. The chimeric polypeptide of any one of claims 8-14, wherein the first ECM module comprises vitronectin, or a functional portion thereof, and the at least additional ECM module comprises an ECM motif.

19. The chimeric polypeptide of claim 18, wherein the additional ECM module comprises at least one RGD motif.

20. The chimeric polypeptide of claim 15, wherein the first ECM module comprises at least one of GRGDSP and PHSRN, and the at least additional ECM module comprises at least the other one of GRGDSP and PHSRN.

21. The chimeric polypeptide of any one of claims 8-20, wherein the first ECM module and the at least additional ECM module flank the CBD.

22. The chimeric polypeptide of any one of claims 8-20, wherein the first ECM module and the at least additional ECM module are linked to each other on one side of the CBD.

23. A nucleic acid molecule comprising a nucleotide sequence encoding the chimeric polypeptide of any one of claims 1-22.

24. A vector comprising the nucleic acid molecule of claim 23.

25. A transgenic plant comprising the nucleic acid molecule of claim 23 or the vector of claim 24, and/or expressing the chimeric polypeptide of any one of claims 1-22.

26. The transgenic plant of claim 25, wherein the plant is a tobacco plant.

27. A tissue engineering system comprising the chimeric polypeptide of any one of claims 1-22 bound to a cellulose-based scaffold via the cellulose binding domain (CBD).

28. The tissue engineering system of claim 27, further comprising precursor cells which adhere to the scaffold by attaching to the ECM module.

29. Use of the chimeric polypeptide of any one of claims 1-22, or the tissue engineering system of claim 27 or 28, for generating an engineered tissue.

30. An in vitro method of generating an engineered tissue, comprising: a. adding the chimeric polypeptide of any one of claims 1-22 to a cellulose-based scaffold with a medium; b. allowing the chimeric polypeptide to bind to the cellulose-based scaffold; c. seeding precursor cells onto the scaffold-bound chimeric polypeptide; and d. growing the cells until an engineered tissue is formed.

31. The method of claim 30, wherein the chimeric polypeptide comprising the ECM module is added to the medium at a concentration that is at least 10% lower than a concentration of the ECM module not being linked to a CBD when added to a medium comprising a CBD and a cellulose-based scaffold, in a method for generating an engineered tissue.

32. The method of claim 31, wherein growing in step (d) involves application of a mechanical stress to the scaffold.

33. The method of claim 32, wherein the mechanical stress is in form of compression or tensile stress.

34. The method of claim 33, wherein the compression or tensile stress is applied at specific strains or forces and at specific frequencies to mimic the natural environment required for growth and/or differentiation of the tissue.

35. The method of any one of claims 32-34, wherein the tissue is a muscle tissue or a connective tissue.

36. The method of any one of claims 30-35, wherein the chimeric polypeptide is prepared by transforming a plant with the nucleic acid molecule of claim 23 or the vector of claim 24, and purifying the chimeric polypeptide from the plant.

37. An engineered tissue generated by the method of any one of claims 30-36.