Genetically reprogrammed extracellular matrix

Abstract

Provided is the use of artificial sequences that can be transfected into cells for the purpose of identifying the cells themselves, as well as any extracellular matrix (ECM) material tagged with a unique artificial peptide generated by the artificial sequences.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 296,272, filed January 4, 2022, which is expressly incorporated by reference in its entirety.

[0002] Inventive embodiments of the present disclosure relate generally to the use of artificial sequences that can be transfected into cells for the purpose of identifying the cells themselves, as well as any extracellular matrix (ECM) material tagged with a unique artificial peptide generated by the artificial sequence. [Background technology]

[0003] Modifications to DNA sequences in cells can be made to confer some advantageous characteristic to the cell or to produce a peptide or protein. For example, cells can be transfected using a plasmid vector or mRNA to express a protein of interest in cultured cells (or in an animal model). Protein expression in eukaryotic cells allows the production of recombinant proteins with the proper folding and post-translational modifications required for their function. Generally, transfection is the process of artificially introducing nucleic acids (e.g., DNA or RNA) into cells using means other than viral infection. The introduction of such foreign nucleic acids using various chemical, biological, or physical methods can result in changes in the properties of the cell, allowing the study of gene function and protein expression in the cellular context. In transfection, the introduced nucleic acid can be transiently present in the cell and expressed for a limited period of time but not replicated, or it can be stably integrated into the recipient genome and replicated during the replication of the host genome. Summary of the Invention [Problem to be solved by the invention]

[0004] However, there remains a need in the art for genetically modified cells that can be stably transfected with one or more sequences for identification purposes and produce unique artificial peptides that can be used as unique tags to identify ECM materials formed from the genetically modified cells. [Means for solving the problem]

[0005] One or more embodiments of the present invention may address one or more of the problems described above. Certain embodiments according to the present invention provide transfected cells comprising SEQ ID NO:1, SEQ ID NO:2, or both. The present invention also provides a method of tagging a cell lineage of interest, comprising providing a cell lineage of interest and transfecting cells of the cell lineage of interest with a vector comprising SEQ ID NO:1, SEQ ID NO:2, or both. In another aspect, the present invention provides an extracellular matrix (ECM) material comprising SEQ ID NO:3.

[0006] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the present invention are shown. Indeed, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein but rather these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows SEQ ID NO:1. [Diagram 2] FIG. 2 shows SEQ ID NO:2. [Diagram 3] FIG. 3 shows SEQ ID NO:3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The present invention will now be described in more detail below with reference to the accompanying drawings, in which some, but not all, embodiments of the present invention are shown. Indeed, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0009] The disclosed invention generally relates to the use of SEQ ID NO:1 (FIG. 1) and / or SEQ ID NO:2 (FIG. 2) for identification purposes. For example, when a new cell line is created or a new protein is created in a laboratory environment, it may be beneficial to be able to identify the source from which the cell line or protein originates. According to certain embodiments of the invention, the invention utilizes an artificial gene sequence to stably transfect our subject cells and to identify or brand the stably transfected cells produced or the stably transfected cells associated with a given source or location (e.g., a particular laboratory) for quality control and reference purposes.

[0010] In particular, SEQ ID NO:1 (i.e., 5'GCAGTTTTACGAACCTTT3') and its complementary sequence SEQ ID NO:2 (i.e., 5'AAAGGTTCGTAAAACTGC3') can be stably transfected into cells for identification purposes. According to certain embodiments of the invention, if either sequence is successfully translated by the source cell, an artificial peptide sequence of SEQ ID NO:3 (i.e., RQNAWK) is present on the extracellular matrix secreted by the cell or source, where "R"=Arg=arginine, "Q"=Gln=glutamine, "N"=Asn=asparagine, "A"=Ala=alanine, "W"=Trp=tryptophan, and "K"=Lys=lysine. According to certain embodiments of the invention, SEQ ID NO:1 and / or SEQ ID NO:2 can be embedded in, for example, the sequence of collagen type 1 for identification purposes. The purpose of transfecting cells with this sequence is to allow for the identification of all genetically modified cells which produce a unique identification tag on the collagen fibers within any acellular material produced. This allows users to identify and cross-reference materials for related products. For example, this method of identification allows users to keep track of both the cells and acellular materials produced in their lab (or another source) and to reference and cross-reference them when producing new genetically modified products.

[0011] SEQ ID NO:1 and SEQ ID NO:2 do not naturally occur in nature. These sequences were synthesized to generate a similarly non-naturally occurring peptide (i.e., SEQ ID NO:3). This particular peptide (i.e., SEQ ID NO:3) is a tag corresponding to the filing company's name for the purposes of barcoding and / or branding cells grown using any of the filing company's platform technologies.

[0012] According to certain embodiments of the present invention, various cells can be stably transfected with SEQ ID NO:1 and / or SEQ ID NO:2 using non-viral vectors, such as lipofection, electroporation, sonication, or virally transduced into various cells using viral vectors, such as adenovirus, adeno-associated virus, lentivirus, or any other viral vector, to deliver the plasmid construct to the nucleus of the cellular host.

[0013] Thus, certain embodiments of the present invention provide custom molecular tags that are generated by cells, with the goal of providing a method of tracking and referencing both cells and acellular materials produced in a particular laboratory. Additionally, certain embodiments of the present invention facilitate audit production of cellular products, allowing for branding or tagging of new cell lines or molecular products produced for use by applicant companies.

[0014] According to certain embodiments of the invention, for example, the invention provides transfected cells comprising SEQ ID NO:1, SEQ ID NO:2, or both.

[0015] According to certain embodiments of the invention, the transfected cells may comprise human stem cells such as human Wharton's jelly cells (MSCs), human bone marrow-derived mesenchymal stem cells (MSCs), human adipose-derived mesenchymal stem cells (MSCs), human skin-derived induced pluripotent stem cells (iPSCs), human blood cell-derived induced pluripotent stem cells (iPSCs), human CD4+ T cells, or human CD8+ T cells. Additionally or alternatively, the transfected cells may include primary mammalian cells such as HepG2 cells (liver cancer cells), human adult dermal fibroblasts (primary cells), human neonatal dermal fibroblasts (primary cells), human adult keratinocytes (primary cells), mouse dorsal root ganglion (primary neural cells), bovine cardiomyocytes (primary cell line), primary porcine hepatocytes, porcine chondrocytes, porcine bone cells, equine muscle-derived stem cells (primary MSCs), primary snail cells, human macrophages, and any primary cells that can be isolated from tissue taken from a human, animal, or plant. Additionally or alternatively, the transfected cells may include immortalized mammalian cell lines such as UB-OC2 cells (mouse cochlear epithelium), human myoblastoma (muscle tumor), PC3 (prostate cancer), CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), SHSY5Y (neural tumor), PANC-1 (human pancreatic cancer), HeLa (cervical cancer), A549 (lung cancer), A673 (muscle cancer), and (4) primary plant cells such as parenchymal cells, sclerenchymal cells, sclerenchymal cells, xylem cells, phloem cells, meristematic cells, epidermal cells, etc.

[0016] In another aspect, the present invention provides a method of tagging a cell line of interest, which may include providing a cell line of interest and transfecting cells of the cell line of interest with a vector comprising SEQ ID NO:1, SEQ ID NO:2, or both. According to certain embodiments of the present invention, the vector may include non-viral vectors, such as lipofection, calcium phosphate, cationic lipids, dextran, magnetofection, electroporation, microinjection, ballistic transfer, optical / laser transfection, and sonication, or viral vectors, such as adenovirus, adeno-associated virus, and retrovirus.

[0017] In another aspect, the invention provides an extracellular matrix (ECM) material comprising SEQ ID NO:3. According to certain embodiments of the invention, the ECM material is formed from cells transfected with SEQ ID NO:1, SEQ ID NO:2, or both. For example, the ECM can include one or more tissue components tagged with SEQ ID NO:3. For example, the one or more tissue components can include glycosaminoglycans (GAGs), proteoglycans, hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), collagen types (e.g., fibrillar types (I, II, III, V, XI), facitin types (IX, XII, XIV), short chain (VIII, X), basement membrane (IV), and others (VI, VII, XIII), elasmolytic enzymes (ES) and / or endothelial cells (ES). stin, DNA, RNA, fibronectin and glycoproteins, laminin-111 (laminin-1), laminin-211 (laminin-2), laminin-121 (laminin-3), laminin-221 (laminin-4), laminin-332 / laminin-3A32 (laminin-5 / laminin-5A), laminin-3B32 (laminin-5B), laminin-311 / laminin-3A11 (laminin-6 / laminin-7), laminin-3B32 (laminin-5C), laminin-3C32 (laminin-3D), laminin-3D11 (laminin-3E), laminin-3D21 (laminin-3F), laminin-3D11 (laminin-3G), laminin-3D11 (laminin-3H), laminin-3D21 (laminin-3I), laminin-3D11 (laminin-3IH ... laminin-6A), laminin-321 / laminin-3A21 (laminin-7 / laminin-7A), laminin-411 (laminin-8), laminin-421 (laminin-9), laminin-511 (laminin-10), laminin-521 (laminin-11), laminin-213 (laminin-12), laminin-423 (laminin-14), laminin-522, laminin-523 (laminin-15), peptidase The ECM components may include cytoskeletal components, such as phosphates, lipids and phospholipids, carbohydrates, phosphates, saturated fats, unsaturated fats, trans fats, cholesterol, organelles (e.g., mitochondria, smooth endoplasmic reticulum, rough endoplasmic reticulum, Golgi apparatus / apparatus, lysosomes, endosomes, vesicles, peroxisomes, nucleoli, nuclei, and ribosomes), and cytoskeletal components (e.g., actin, myosin).

[0018] Those skilled in the art will appreciate that these and other modifications and variations to the present invention may be made without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. It should further be understood that aspects of the various embodiments may be interchanged in whole or in part. Moreover, those skilled in the art will appreciate that the foregoing description is for illustrative purposes only and is not intended to limit the invention as further described in such appended claims. Accordingly, the spirit and scope of the appended claims should not be limited to the exemplary descriptions of the interpretations contained herein.

Claims

1. A transfected cell comprising SEQ ID NO:1, SEQ ID NO:2, or both.

2. 2. The transfected cell of claim 1, wherein the transfected cell comprises a human stem cell, such as human Wharton's jelly cells (MSCs), human bone marrow-derived mesenchymal stem cells (MSCs), human adipose-derived mesenchymal stem cells (MSCs), human skin-derived induced pluripotent stem cells (iPSCs), human blood cell-derived induced pluripotent stem cells (iPSCs), human CD4+ T cells, or human CD8+ T cells.

3. 2. The transfected cells of claim 1, comprising primary mammalian cells such as HepG2 cells (liver cancer cells), human adult dermal fibroblasts (primary cells), human neonatal dermal fibroblasts (primary cells), human adult keratinocytes (primary cells), mouse dorsal root ganglia (primary nerve cells), bovine cardiac muscle cells (primary cell line), primary porcine hepatocytes, porcine chondrocytes, porcine osteocytes, equine muscle-derived stem cells (primary MSCs), primary snail cells, human macrophages, or any primary cells that can be isolated from tissues taken from humans, animals, or plants.

4. The transfected cells of claim 1, comprising immortalized mammalian cell lines such as UB-OC2 cells (mouse cochlear epithelium), human myoblastoma (muscle tumor), PC3 (prostate cancer), CHO (Chinese Hamster Ovary Cells), HEK293 (human embryonic kidney cells), SHSY5Y (neural tumor), PANC-1 (human pancreatic cancer), HeLa (cervical cancer), A549 (lung cancer), A673 (muscle cancer), and (4) primary plant cells such as parenchymal cells, sclerenchymal cells, sclerenchymal cells, xylem cells, phloem cells, meristematic cells, epidermal cells.

5. 1. A method for tagging a cell lineage of interest, comprising: providing the cell lineage of interest; and transfecting cells of the cell lineage of interest with a vector comprising SEQ ID NO:1, SEQ ID NO:2, or both.

6. 6. The method of claim 5, wherein the vector comprises a non-viral vector, such as lipofection, calcium phosphate, cationic lipids, dextran, magnetofection electroporation, microinjection, gene gun transfer, optical / laser transfection, and sonication.

7. The method of claim 5, wherein the vector comprises a viral vector such as an adenovirus, an adeno-associated virus, and a retrovirus such as a lentivirus or HIV-1.

8. An extracellular matrix (ECM) material comprising sequence number 3.

9. The ECM material of claim 5 , wherein the ECM comprises one or more tissue components tagged with SEQ ID NO:

3.

10. The one or more tissue components may be glycosaminoglycans (GAGs), proteoglycans, hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), collagen types (e.g., fibrous types (I, II, III, V, XI), facitin types (IX, XII, XIV), short chains (VIII, X), basement membrane (IV), and others (VI, VII, XIII), ellagocytes, and the like). Stem, DNA, RNA, fibronectin and glycoproteins, laminin-111 (laminin-1), laminin-211 (laminin-2), laminin-121 (laminin-3), laminin-221 (laminin-4), laminin-332 / laminin-3A32 (laminin-5 / laminin-5A), laminin-3B32 (laminin-5B), laminin-311 / laminin-3A11 (laminin-6 / laminin-6A), laminin-321 / laminin-3A21 (laminin-7 / laminin-7A), laminin-411 (laminin-8), laminin-421 (laminin-9), laminin-511 (laminin-10), laminin-521 (laminin-11), laminin-213 (laminin-12), laminin-423 (laminin-14), laminin-522, laminin-523 (laminin-15), 10. The ECM of claim 9, comprising peptides, lipids and phospholipids, carbohydrates, phosphates, saturated fats, unsaturated fats, trans fats, cholesterol, organelles (e.g., mitochondria, smooth endoplasmic reticulum, rough endoplasmic reticulum, Golgi apparatus / apparatus, lysosomes, endosomes, vesicles, peroxisomes, nucleoli, nuclei, and ribosomes), and cytoskeletal components (e.g., actin).

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