Genetically reprogrammed extracellular matrix

Abstract

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 unique artificial peptides generated by the artificial sequences, is provided. [Solution] An extracellular matrix (ECM) material containing a specific sequence is provided. The ECM comprises one or more tissue components tagged with the specific sequence.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 296,272, filed on January 4, 2022, which is hereby incorporated by reference in its entirety.

[0002] Embodiments of the invention disclosed herein generally relate to the use of artificial sequences that can be transfected into cells for the purpose of identifying the cells themselves, and any extracellular matrix (ECM) material tagged with unique artificial peptides generated by the artificial sequences.

Background Art

[0003] Modifications to DNA sequences in cells can be made to confer some advantageous feature to the cells or to produce peptides or proteins. For example, cells can be transfected using plasmid vectors or mRNA to express a target protein in cultured cells (or animal models). By expressing proteins in eukaryotic cells, it becomes possible to produce recombinant proteins with the appropriate folding and post - translational modifications required for their function. Generally, transfection is a process of artificially introducing nucleic acids (DNA or RNA) into cells using means other than viral infection. Since the introduction of such foreign nucleic acids using various chemical, biological, or physical methods can bring about changes in cell characteristics, it becomes possible to study gene function and protein expression in the context of cells. In transfection, the introduced nucleic acid can exist transiently within the cell and be expressed only for a limited period without being replicated or be stably integrated into the recipient's genome and replicated during replication of the host genome.

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, in this field of technology, there is still a need for genetically modified cells that can stably transfect 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 genetically modified cells. [Means for solving the problem]

[0005] One or more embodiments of the present invention can address one or more of the above-described problems. A particular embodiment of the present invention provides transfected cells containing SEQ ID NO: 1, SEQ ID NO: 2, or both. The present invention also provides a method for tagging a target cell lineage, comprising preparing the target cell lineage and transfecting the cells of the target cell lineage with a vector containing SEQ ID NO: 1, SEQ ID NO: 2, or both. In another embodiment, the present invention provides an extracellular matrix (ECM) material containing SEQ ID NO: 3.

[0006] Next, the present invention will be described in more detail below with reference to the accompanying drawings. The accompanying drawings show some, but not all, embodiments of the present invention. In fact, the present invention can be carried out in many different forms and should not be construed as being limited to the embodiments shown herein, but rather these embodiments are provided so that this disclosure satisfies such legal requirements. Throughout, the same numbers refer to the same elements. [Brief explanation of the drawing]

[0007] [Figure 1] This is a diagram showing sequence number 1. [Figure 2] This is a diagram showing sequence number 2. [Figure 3] This is a diagram showing sequence number 3. [Modes for carrying out the invention]

[0008] Next, the present invention will be described in more detail below with reference to the accompanying drawings. The accompanying drawings show some, but not all, embodiments of the present invention. In fact, the present invention can be carried out in many different forms and should not be construed as being limited to the embodiments shown herein, but rather these embodiments are provided so that this disclosure satisfies such legal requirements. Where used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise explicitly stated in the context.

[0009] The inventions of this disclosure generally relate to the use of Sequence ID No. 1 (Figure 1) and / or Sequence ID No. 2 (Figure 2) for identification purposes. For example, when a novel cell lineage or a novel protein is created in a laboratory setting, it may be beneficial to be able to identify the origin from which the cell lineage or protein is derived. According to certain embodiments of the present invention, the present invention uses an artificial gene sequence to stably transfect the inventors' target cells, etc., 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 specific laboratory) for quality control and reference purposes.

[0010] In particular, Sequence ID No. 1 (i.e., 5'GCAGTTTTACGAACCTTT3') and its complementary sequence, Sequence ID No. 2 (i.e., 5'AAAGGTTCGTAAAACTGC3'), can be stably transfected into cells for the purpose of identification. According to a particular embodiment of the present invention, if either sequence is successfully translated by the source cell, the artificial peptide sequence of Sequence ID No. 3 (i.e., RQNAWK) is present on the extracellular matrix secreted by the cell or source. Here, "R" = Arg = arginine, "Q" = Gln = glutamine, "N" = Asn = asparagine, "A" = Ala = alanine, "W" = Trp = tryptophan, and "K" = Lys = lysine. According to a particular embodiment of the present invention, Sequence ID No. 1 and / or Sequence ID No. 2 can be embedded, for example, within a sequence of collagen type 1 for the purpose of identification. The purpose of transfecting cells with this sequence is to enable the identification of all genetically modified cells that generate unique identification tags on collagen fibers within any cell-free material produced. This allows users to identify and cross-reference materials related to relevant products. For example, this identification method allows users to track the progress of both cells and cell-free materials produced in their own laboratory (or another source) and perform reference and cross-referencing when producing new genetically modified products.

[0011] Sequence IDs 1 and 2 do not exist naturally. These sequences were synthesized to produce a peptide (i.e., Sequence ID 3) that also does not exist naturally. This particular peptide (i.e., Sequence ID 3) is a tag corresponding to the applicant company's name, for the purpose of barcoding and / or trademarking cells grown using any of the applicant company's platform technologies.

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

[0013] Accordingly, certain embodiments of the present invention provide custom molecular tags generated by cells, with the aim of providing a method for tracking and referencing both cells and cell-free materials produced in a specific laboratory. Furthermore, certain embodiments of the present invention facilitate audit production of cell products and enable trademarking or tagging of novel cell lines or molecular products produced for use by applicant companies.

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

[0015] According to certain embodiments of the present invention, the transfected cells may include 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, transfected cells may include 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 cardiomyocytes (primary cell lineage), primary porcine hepatocytes, porcine chondrocytes, porcine osteocytes, equine muscle-derived stem cells (primary MSCs), primary snail cells, human macrophages, and other primary mammalian cells, as well as any primary cells that can be isolated from tissues taken from humans, animals, or plants. Additionally or alternatively, 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 fetal kidney cells), SHSY5Y (neuroma), PANC-1 (human pancreatic cancer), HeLa (cervical cancer), A549 (lung cancer), A673 (muscle cancer), and (4) primary plant cells such as parenchymal cells, plaque cells, sclerocysts, xylem cells, phloem cells, meristem cells, and epidermal cells.

[0016] In another embodiment, the present invention provides a method for tagging a target cell lineage. This method may include preparing the target cell lineage and transfecting the cells of the target cell lineage with a vector containing SEQ ID NO: 1, SEQ ID NO: 2, or both. According to certain embodiments of the present invention, the vector may include nonviral vectors such as lipofection, calcium phosphate, cationic lipids, dextran, magnetofection electroporation, microinjection, ballistic transfer, optical / laser transfection, and sonication, or viral vectors such as adenoviruses, adeno-associated viruses, and retroviruses.

[0017] In another embodiment, the present invention provides an extracellular matrix (ECM) material comprising SEQ ID NO: 3. According to a particular embodiment of the present invention, the ECM material is formed from cells transfected with SEQ ID NO: 1, SEQ ID NO: 2, or both. For example, the ECM may comprise one or more tissue components tagged with SEQ ID NO: 3. For example, one or more tissue components may include glycosaminoglycans (GAGs), proteoglycans, hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), types of collagen (e.g., fibrous type (I, II, III, V, XI), facilitate type (IX, XII, XIV), short-chain type (VIII, X), basement membrane (IV), and others (VI, VII, XIII)), gills. 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-5A 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), Pep It may contain ECM components including nitrates, 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, nucleolus, nucleus, and ribosomes), and cytoskeletal components (e.g., actin, myosin).

[0018] Those skilled in the art will be able to make these modifications and variations, as well as other modifications and variations, to the present invention without departing from the spirit and scope of the invention, which are further described in the appended claims. Furthermore, it should be understood that the various embodiments can be replaced in whole or in part. Furthermore, those skilled in the art will understand 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 illustrative description of interpretation contained herein.

Claims

1. Transfected cells containing SEQ ID NO: 1, SEQ ID NO: 2, or both.

2. The transfected cells according to claim 1, wherein the transfected cells include 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.

3. The transfected cells according to claim 1 include 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 cardiomyocytes (primary cell lineage), 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 collected from humans, animals, or plants.

4. The transfected cells according to claim 1, wherein the transfected cells 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 fetal kidney cells), SHSY5Y (neuroma), PANC-1 (human pancreatic cancer), HeLa (cervical cancer), A549 (lung cancer), A673 (muscle cancer), and (4) primary plant cells such as parenchymal cells, plaque cells, plaque-walled cells, xylem cells, phloem cells, meristem cells, and epidermal cells.

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

6. The method according to claim 5, wherein the vector includes nonviral vectors such as lipofection, calcium phosphate, cationic lipids, dextran, magnetofection electroporation, microinjection, gene gun delivery, optical / laser transfection, and sonication.

7. The method according to claim 5, wherein the vector includes viral vectors such as adenoviruses, adeno-associated viruses, and retroviruses such as lentiviruses and HIV-1.

8. Extracellular matrix (ECM) material containing SEQ ID NO:

3.

9. The ECM material according to claim 5, wherein the ECM comprises one or more tissue components tagged with Sequence ID No.

3.

10. The one or more tissue components mentioned above include glycosaminoglycans (GAGs), proteoglycans, hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), types of collagen (e.g., fibrous type (I, II, III, V, XI), fascic type (IX, XII, XIV), short chain (VIII, X), basement membrane (IV), and others (VI, VII, XIII)), and gills. 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-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), The ECM according to 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).

Images